human stem cell research essay

• Open access
• Published: 26 February 2019

Stem cells: past, present, and future

• Wojciech Zakrzewski 1 ,
• Maciej Dobrzyński 2 ,
• Maria Szymonowicz 1 &
• Zbigniew Rybak 1  

Stem Cell Research & Therapy volume  10 , Article number:  68 ( 2019 ) Cite this article

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In recent years, stem cell therapy has become a very promising and advanced scientific research topic. The development of treatment methods has evoked great expectations. This paper is a review focused on the discovery of different stem cells and the potential therapies based on these cells. The genesis of stem cells is followed by laboratory steps of controlled stem cell culturing and derivation. Quality control and teratoma formation assays are important procedures in assessing the properties of the stem cells tested. Derivation methods and the utilization of culturing media are crucial to set proper environmental conditions for controlled differentiation. Among many types of stem tissue applications, the use of graphene scaffolds and the potential of extracellular vesicle-based therapies require attention due to their versatility. The review is summarized by challenges that stem cell therapy must overcome to be accepted worldwide. A wide variety of possibilities makes this cutting edge therapy a turning point in modern medicine, providing hope for untreatable diseases.

Stem cell classification

Stem cells are unspecialized cells of the human body. They are able to differentiate into any cell of an organism and have the ability of self-renewal. Stem cells exist both in embryos and adult cells. There are several steps of specialization. Developmental potency is reduced with each step, which means that a unipotent stem cell is not able to differentiate into as many types of cells as a pluripotent one. This chapter will focus on stem cell classification to make it easier for the reader to comprehend the following chapters.

Totipotent stem cells are able to divide and differentiate into cells of the whole organism. Totipotency has the highest differentiation potential and allows cells to form both embryo and extra-embryonic structures. One example of a totipotent cell is a zygote, which is formed after a sperm fertilizes an egg. These cells can later develop either into any of the three germ layers or form a placenta. After approximately 4 days, the blastocyst’s inner cell mass becomes pluripotent. This structure is the source of pluripotent cells.

Pluripotent stem cells (PSCs) form cells of all germ layers but not extraembryonic structures, such as the placenta. Embryonic stem cells (ESCs) are an example. ESCs are derived from the inner cell mass of preimplantation embryos. Another example is induced pluripotent stem cells (iPSCs) derived from the epiblast layer of implanted embryos. Their pluripotency is a continuum, starting from completely pluripotent cells such as ESCs and iPSCs and ending on representatives with less potency—multi-, oligo- or unipotent cells. One of the methods to assess their activity and spectrum is the teratoma formation assay. iPSCs are artificially generated from somatic cells, and they function similarly to PSCs. Their culturing and utilization are very promising for present and future regenerative medicine.

Multipotent stem cells have a narrower spectrum of differentiation than PSCs, but they can specialize in discrete cells of specific cell lineages. One example is a haematopoietic stem cell, which can develop into several types of blood cells. After differentiation, a haematopoietic stem cell becomes an oligopotent cell. Its differentiation abilities are then restricted to cells of its lineage. However, some multipotent cells are capable of conversion into unrelated cell types, which suggests naming them pluripotent cells.

Oligopotent stem cells can differentiate into several cell types. A myeloid stem cell is an example that can divide into white blood cells but not red blood cells.

Unipotent stem cells are characterized by the narrowest differentiation capabilities and a special property of dividing repeatedly. Their latter feature makes them a promising candidate for therapeutic use in regenerative medicine. These cells are only able to form one cell type, e.g. dermatocytes.

Stem cell biology

A blastocyst is formed after the fusion of sperm and ovum fertilization. Its inner wall is lined with short-lived stem cells, namely, embryonic stem cells. Blastocysts are composed of two distinct cell types: the inner cell mass (ICM), which develops into epiblasts and induces the development of a foetus, and the trophectoderm (TE). Blastocysts are responsible for the regulation of the ICM microenvironment. The TE continues to develop and forms the extraembryonic support structures needed for the successful origin of the embryo, such as the placenta. As the TE begins to form a specialized support structure, the ICM cells remain undifferentiated, fully pluripotent and proliferative [ 1 ]. The pluripotency of stem cells allows them to form any cell of the organism. Human embryonic stem cells (hESCs) are derived from the ICM. During the process of embryogenesis, cells form aggregations called germ layers: endoderm, mesoderm and ectoderm (Fig.  1 ), each eventually giving rise to differentiated cells and tissues of the foetus and, later on, the adult organism [ 2 ]. After hESCs differentiate into one of the germ layers, they become multipotent stem cells, whose potency is limited to only the cells of the germ layer. This process is short in human development. After that, pluripotent stem cells occur all over the organism as undifferentiated cells, and their key abilities are proliferation by the formation of the next generation of stem cells and differentiation into specialized cells under certain physiological conditions.

Oocyte development and formation of stem cells: the blastocoel, which is formed from oocytes, consists of embryonic stem cells that later differentiate into mesodermal, ectodermal, or endodermal cells. Blastocoel develops into the gastrula

Signals that influence the stem cell specialization process can be divided into external, such as physical contact between cells or chemical secretion by surrounding tissue, and internal, which are signals controlled by genes in DNA.

Stem cells also act as internal repair systems of the body. The replenishment and formation of new cells are unlimited as long as an organism is alive. Stem cell activity depends on the organ in which they are in; for example, in bone marrow, their division is constant, although in organs such as the pancreas, division only occurs under special physiological conditions.

Stem cell functional division

Whole-body development.

During division, the presence of different stem cells depends on organism development. Somatic stem cell ESCs can be distinguished. Although the derivation of ESCs without separation from the TE is possible, such a combination has growth limits. Because proliferating actions are limited, co-culture of these is usually avoided.

ESCs are derived from the inner cell mass of the blastocyst, which is a stage of pre-implantation embryo ca. 4 days after fertilization. After that, these cells are placed in a culture dish filled with culture medium. Passage is an inefficient but popular process of sub-culturing cells to other dishes. These cells can be described as pluripotent because they are able to eventually differentiate into every cell type in the organism. Since the beginning of their studies, there have been ethical restrictions connected to the medical use of ESCs in therapies. Most embryonic stem cells are developed from eggs that have been fertilized in an in vitro clinic, not from eggs fertilized in vivo.

Somatic or adult stem cells are undifferentiated and found among differentiated cells in the whole body after development. The function of these cells is to enable the healing, growth, and replacement of cells that are lost each day. These cells have a restricted range of differentiation options. Among many types, there are the following:

Mesenchymal stem cells are present in many tissues. In bone marrow, these cells differentiate mainly into the bone, cartilage, and fat cells. As stem cells, they are an exception because they act pluripotently and can specialize in the cells of any germ layer.

Neural cells give rise to nerve cells and their supporting cells—oligodendrocytes and astrocytes.

Haematopoietic stem cells form all kinds of blood cells: red, white, and platelets.

Skin stem cells form, for example, keratinocytes, which form a protective layer of skin.

The proliferation time of somatic stem cells is longer than that of ESCs. It is possible to reprogram adult stem cells back to their pluripotent state. This can be performed by transferring the adult nucleus into the cytoplasm of an oocyte or by fusion with the pluripotent cell. The same technique was used during cloning of the famous Dolly sheep.

hESCs are involved in whole-body development. They can differentiate into pluripotent, totipotent, multipotent, and unipotent cells (Fig.  2 ) [ 2 ].

Changes in the potency of stem cells in human body development. Potency ranges from pluripotent cells of the blastocyst to unipotent cells of a specific tissue in a human body such as the skin, CNS, or bone marrow. Reversed pluripotency can be achieved by the formation of induced pluripotent stem cells using either octamer-binding transcription factor (Oct4), sex-determining region Y (Sox2), Kruppel-like factor 4 (Klf4), or the Myc gene

Pluripotent cells can be named totipotent if they can additionally form extraembryonic tissues of the embryo. Multipotent cells are restricted in differentiating to each cell type of given tissue. When tissue contains only one lineage of cells, stem cells that form them are called either called oligo- or unipotent.

iPSC quality control and recognition by morphological differences

The comparability of stem cell lines from different individuals is needed for iPSC lines to be used in therapeutics [ 3 ]. Among critical quality procedures, the following can be distinguished:

Short tandem repeat analysis—This is the comparison of specific loci on the DNA of the samples. It is used in measuring an exact number of repeating units. One unit consists of 2 to 13 nucleotides repeating many times on the DNA strand. A polymerase chain reaction is used to check the lengths of short tandem repeats. The genotyping procedure of source tissue, cells, and iPSC seed and master cell banks is recommended.

Identity analysis—The unintentional switching of lines, resulting in other stem cell line contamination, requires rigorous assay for cell line identification.

Residual vector testing—An appearance of reprogramming vectors integrated into the host genome is hazardous, and testing their presence is a mandatory procedure. It is a commonly used procedure for generating high-quality iPSC lines. An acceptable threshold in high-quality research-grade iPSC line collections is ≤ 1 plasmid copies per 100 cells. During the procedure, 2 different regions, common to all plasmids, should be used as specific targets, such as EBNA and CAG sequences [ 3 ]. To accurately represent the test reactions, a standard curve needs to be prepared in a carrier of gDNA from a well-characterized hPSC line. For calculations of plasmid copies per cell, it is crucial to incorporate internal reference gDNA sequences to allow the quantification of, for example, ribonuclease P (RNaseP) or human telomerase reverse transcriptase (hTERT).

Karyotype—A long-term culture of hESCs can accumulate culture-driven mutations [ 4 ]. Because of that, it is crucial to pay additional attention to genomic integrity. Karyotype tests can be performed by resuscitating representative aliquots and culturing them for 48–72 h before harvesting cells for karyotypic analysis. If abnormalities are found within the first 20 karyotypes, the analysis must be repeated on a fresh sample. When this situation is repeated, the line is evaluated as abnormal. Repeated abnormalities must be recorded. Although karyology is a crucial procedure in stem cell quality control, the single nucleotide polymorphism (SNP) array, discussed later, has approximately 50 times higher resolution.

Viral testing—When assessing the quality of stem cells, all tests for harmful human adventitious agents must be performed (e.g. hepatitis C or human immunodeficiency virus). This procedure must be performed in the case of non-xeno-free culture agents.

Bacteriology—Bacterial or fungal sterility tests can be divided into culture- or broth-based tests. All the procedures must be recommended by pharmacopoeia for the jurisdiction in which the work is performed.

Single nucleotide polymorphism arrays—This procedure is a type of DNA microarray that detects population polymorphisms by enabling the detection of subchromosomal changes and the copy-neutral loss of heterozygosity, as well as an indication of cellular transformation. The SNP assay consists of three components. The first is labelling fragmented nucleic acid sequences with fluorescent dyes. The second is an array that contains immobilized allele-specific oligonucleotide (ASO) probes. The last component detects, records, and eventually interprets the signal.

Flow cytometry—This is a technique that utilizes light to count and profile cells in a heterogeneous fluid mixture. It allows researchers to accurately and rapidly collect data from heterogeneous fluid mixtures with live cells. Cells are passed through a narrow channel one by one. During light illumination, sensors detect light emitted or refracted from the cells. The last step is data analysis, compilation and integration into a comprehensive picture of the sample.

Phenotypic pluripotency assays—Recognizing undifferentiated cells is crucial in successful stem cell therapy. Among other characteristics, stem cells appear to have a distinct morphology with a high nucleus to cytoplasm ratio and a prominent nucleolus. Cells appear to be flat with defined borders, in contrast to differentiating colonies, which appear as loosely located cells with rough borders [ 5 ]. It is important that images of ideal and poor quality colonies for each cell line are kept in laboratories, so whenever there is doubt about the quality of culture, it can always be checked according to the representative image. Embryoid body formation or directed differentiation of monolayer cultures to produce cell types representative of all three embryonic germ layers must be performed. It is important to note that colonies cultured under different conditions may have different morphologies [ 6 ].

Histone modification and DNA methylation—Quality control can be achieved by using epigenetic analysis tools such as histone modification or DNA methylation. When stem cells differentiate, the methylation process silences pluripotency genes, which reduces differentiation potential, although other genes may undergo demethylation to become expressed [ 7 ]. It is important to emphasize that stem cell identity, together with its morphological characteristics, is also related to its epigenetic profile [ 8 , 9 ]. According to Brindley [ 10 ], there is a relationship between epigenetic changes, pluripotency, and cell expansion conditions, which emphasizes that unmethylated regions appear to be serum-dependent.

hESC derivation and media

hESCs can be derived using a variety of methods, from classic culturing to laser-assisted methodologies or microsurgery [ 11 ]. hESC differentiation must be specified to avoid teratoma formation (see Fig.  3 ).

Spontaneous differentiation of hESCs causes the formation of a heterogeneous cell population. There is a different result, however, when commitment signals (in forms of soluble factors and culture conditions) are applied and enable the selection of progenitor cells

hESCs spontaneously differentiate into embryonic bodies (EBs) [ 12 ]. EBs can be studied instead of embryos or animals to predict their effects on early human development. There are many different methods for acquiring EBs, such as bioreactor culture [ 13 ], hanging drop culture [ 12 ], or microwell technology [ 14 , 15 ]. These methods allow specific precursors to form in vitro [ 16 ].

The essential part of these culturing procedures is a separation of inner cell mass to culture future hESCs (Fig.  4 ) [ 17 ]. Rosowski et al. [ 18 ] emphasizes that particular attention must be taken in controlling spontaneous differentiation. When the colony reaches the appropriate size, cells must be separated. The occurrence of pluripotent cells lasts for 1–2 days. Because the classical utilization of hESCs caused ethical concerns about gastrulas used during procedures, Chung et al. [ 19 ] found out that it is also possible to obtain hESCs from four cell embryos, leaving a higher probability of embryo survival. Additionally, Zhang et al. [ 20 ] used only in vitro fertilization growth-arrested cells.

Culturing of pluripotent stem cells in vitro. Three days after fertilization, totipotent cells are formed. Blastocysts with ICM are formed on the sixth day after fertilization. Pluripotent stem cells from ICM can then be successfully transmitted on a dish

Cell passaging is used to form smaller clusters of cells on a new culture surface [ 21 ]. There are four important passaging procedures.

Enzymatic dissociation is a cutting action of enzymes on proteins and adhesion domains that bind the colony. It is a gentler method than the manual passage. It is crucial to not leave hESCs alone after passaging. Solitary cells are more sensitive and can easily undergo cell death; collagenase type IV is an example [ 22 , 23 ].

Manual passage , on the other hand, focuses on using cell scratchers. The selection of certain cells is not necessary. This should be done in the early stages of cell line derivation [ 24 ].

Trypsin utilization allows a healthy, automated hESC passage. Good Manufacturing Practice (GMP)-grade recombinant trypsin is widely available in this procedure [ 24 ]. However, there is a risk of decreasing the pluripotency and viability of stem cells [ 25 ]. Trypsin utilization can be halted with an inhibitor of the protein rho-associated protein kinase (ROCK) [ 26 ].

Ethylenediaminetetraacetic acid ( EDTA ) indirectly suppresses cell-to-cell connections by chelating divalent cations. Their suppression promotes cell dissociation [ 27 ].

Stem cells require a mixture of growth factors and nutrients to differentiate and develop. The medium should be changed each day.

Traditional culture methods used for hESCs are mouse embryonic fibroblasts (MEFs) as a feeder layer and bovine serum [ 28 ] as a medium. Martin et al. [ 29 ] demonstrated that hESCs cultured in the presence of animal products express the non-human sialic acid, N -glycolylneuraminic acid (NeuGc). Feeder layers prevent uncontrolled proliferation with factors such as leukaemia inhibitory factor (LIF) [ 30 ].

First feeder layer-free culture can be supplemented with serum replacement, combined with laminin [ 31 ]. This causes stable karyotypes of stem cells and pluripotency lasting for over a year.

Initial culturing media can be serum (e.g. foetal calf serum FCS), artificial replacement such as synthetic serum substitute (SSS), knockout serum replacement (KOSR), or StemPro [ 32 ]. The simplest culture medium contains only eight essential elements: DMEM/F12 medium, selenium, NaHCO 3, l -ascorbic acid, transferrin, insulin, TGFβ1, and FGF2 [ 33 ]. It is not yet fully known whether culture systems developed for hESCs can be allowed without adaptation in iPSC cultures.

Turning point in stem cell therapy

The turning point in stem cell therapy appeared in 2006, when scientists Shinya Yamanaka, together with Kazutoshi Takahashi, discovered that it is possible to reprogram multipotent adult stem cells to the pluripotent state. This process avoided endangering the foetus’ life in the process. Retrovirus-mediated transduction of mouse fibroblasts with four transcription factors (Oct-3/4, Sox2, KLF4, and c-Myc) [ 34 ] that are mainly expressed in embryonic stem cells could induce the fibroblasts to become pluripotent (Fig.  5 ) [ 35 ]. This new form of stem cells was named iPSCs. One year later, the experiment also succeeded with human cells [ 36 ]. After this success, the method opened a new field in stem cell research with a generation of iPSC lines that can be customized and biocompatible with the patient. Recently, studies have focused on reducing carcinogenesis and improving the conduction system.

Retroviral-mediated transduction induces pluripotency in isolated patient somatic cells. Target cells lose their role as somatic cells and, once again, become pluripotent and can differentiate into any cell type of human body

The turning point was influenced by former discoveries that happened in 1962 and 1987.

The former discovery was about scientist John Gurdon successfully cloning frogs by transferring a nucleus from a frog’s somatic cells into an oocyte. This caused a complete reversion of somatic cell development [ 37 ]. The results of his experiment became an immense discovery since it was previously believed that cell differentiation is a one-way street only, but his experiment suggested the opposite and demonstrated that it is even possible for a somatic cell to again acquire pluripotency [ 38 ].

The latter was a discovery made by Davis R.L. that focused on fibroblast DNA subtraction. Three genes were found that originally appeared in myoblasts. The enforced expression of only one of the genes, named myogenic differentiation 1 (Myod1), caused the conversion of fibroblasts into myoblasts, showing that reprogramming cells is possible, and it can even be used to transform cells from one lineage to another [ 39 ].

Although pluripotency can occur naturally only in embryonic stem cells, it is possible to induce terminally differentiated cells to become pluripotent again. The process of direct reprogramming converts differentiated somatic cells into iPSC lines that can form all cell types of an organism. Reprogramming focuses on the expression of oncogenes such as Myc and Klf4 (Kruppel-like factor 4). This process is enhanced by a downregulation of genes promoting genome stability, such as p53. Additionally, cell reprogramming involves histone alteration. All these processes can cause potential mutagenic risk and later lead to an increased number of mutations. Quinlan et al. [ 40 ] checked fully pluripotent mouse iPSCs using whole genome DNA sequencing and structural variation (SV) detection algorithms. Based on those studies, it was confirmed that although there were single mutations in the non-genetic region, there were non-retrotransposon insertions. This led to the conclusion that current reprogramming methods can produce fully pluripotent iPSCs without severe genomic alterations.

During the course of development from pluripotent hESCs to differentiated somatic cells, crucial changes appear in the epigenetic structure of these cells. There is a restriction or permission of the transcription of genes relevant to each cell type. When somatic cells are being reprogrammed using transcription factors, all the epigenetic architecture has to be reconditioned to achieve iPSCs with pluripotency [ 41 ]. However, cells of each tissue undergo specific somatic genomic methylation. This influences transcription, which can further cause alterations in induced pluripotency [ 42 ].

Source of iPSCs

Because pluripotent cells can propagate indefinitely and differentiate into any kind of cell, they can be an unlimited source, either for replacing lost or diseased tissues. iPSCs bypass the need for embryos in stem cell therapy. Because they are made from the patient’s own cells, they are autologous and no longer generate any risk of immune rejection.

At first, fibroblasts were used as a source of iPSCs. Because a biopsy was needed to achieve these types of cells, the technique underwent further research. Researchers investigated whether more accessible cells could be used in the method. Further, other cells were used in the process: peripheral blood cells, keratinocytes, and renal epithelial cells found in urine. An alternative strategy to stem cell transplantation can be stimulating a patient’s endogenous stem cells to divide or differentiate, occurring naturally when skin wounds are healing. In 2008, pancreatic exocrine cells were shown to be reprogrammed to functional, insulin-producing beta cells [ 43 ].

The best stem cell source appears to be the fibroblasts, which is more tempting in the case of logistics since its stimulation can be fast and better controlled [ 44 ].

• Teratoma formation assay

The self-renewal and differentiation capabilities of iPSCs have gained significant interest and attention in regenerative medicine sciences. To study their abilities, a quality-control assay is needed, of which one of the most important is the teratoma formation assay. Teratomas are benign tumours. Teratomas are capable of rapid growth in vivo and are characteristic because of their ability to develop into tissues of all three germ layers simultaneously. Because of the high pluripotency of teratomas, this formation assay is considered an assessment of iPSC’s abilities [ 45 ].

Teratoma formation rate, for instance, was observed to be elevated in human iPSCs compared to that in hESCs [ 46 ]. This difference may be connected to different differentiation methods and cell origins. Most commonly, the teratoma assay involves an injection of examined iPSCs subcutaneously or under the testis or kidney capsule in mice, which are immune-deficient [ 47 ]. After injection, an immature but recognizable tissue can be observed, such as the kidney tubules, bone, cartilage, or neuroepithelium [ 30 ]. The injection site may have an impact on the efficiency of teratoma formation [ 48 ].

There are three groups of markers used in this assay to differentiate the cells of germ layers. For endodermal tissue, there is insulin/C-peptide and alpha-1 antitrypsin [ 49 ]. For the mesoderm, derivatives can be used, e.g. cartilage matrix protein for the bone and alcian blue for the cartilage. As ectodermal markers, class III B botulin or keratin can be used for keratinocytes.

Teratoma formation assays are considered the gold standard for demonstrating the pluripotency of human iPSCs, demonstrating their possibilities under physiological conditions. Due to their actual tissue formation, they could be used for the characterization of many cell lineages [ 50 ].

Directed differentiation

To be useful in therapy, stem cells must be converted into desired cell types as necessary or else the whole regenerative medicine process will be pointless. Differentiation of ESCs is crucial because undifferentiated ESCs can cause teratoma formation in vivo. Understanding and using signalling pathways for differentiation is an important method in successful regenerative medicine. In directed differentiation, it is likely to mimic signals that are received by cells when they undergo successive stages of development [ 51 ]. The extracellular microenvironment plays a significant role in controlling cell behaviour. By manipulating the culture conditions, it is possible to restrict specific differentiation pathways and generate cultures that are enriched in certain precursors in vitro. However, achieving a similar effect in vivo is challenging. It is crucial to develop culture conditions that will allow the promotion of homogenous and enhanced differentiation of ESCs into functional and desired tissues.

Regarding the self-renewal of embryonic stem cells, Hwang et al. [ 52 ] noted that the ideal culture method for hESC-based cell and tissue therapy would be a defined culture free of either the feeder layer or animal components. This is because cell and tissue therapy requires the maintenance of large quantities of undifferentiated hESCs, which does not make feeder cells suitable for such tasks.

Most directed differentiation protocols are formed to mimic the development of an inner cell mass during gastrulation. During this process, pluripotent stem cells differentiate into ectodermal, mesodermal, or endodermal progenitors. Mall molecules or growth factors induce the conversion of stem cells into appropriate progenitor cells, which will later give rise to the desired cell type. There is a variety of signal intensities and molecular families that may affect the establishment of germ layers in vivo, such as fibroblast growth factors (FGFs) [ 53 ]; the Wnt family [ 54 ] or superfamily of transforming growth factors—β(TGFβ); and bone morphogenic proteins (BMP) [ 55 ]. Each candidate factor must be tested on various concentrations and additionally applied to various durations because the precise concentrations and times during which developing cells in embryos are influenced during differentiation are unknown. For instance, molecular antagonists of endogenous BMP and Wnt signalling can be used for ESC formation of ectoderm [ 56 ]. However, transient Wnt and lower concentrations of the TGFβ family trigger mesodermal differentiation [ 57 ]. Regarding endoderm formation, a higher activin A concentration may be required [ 58 , 59 ].

There are numerous protocols about the methods of forming progenitors of cells of each of germ layers, such as cardiomyocytes [ 60 ], hepatocytes [ 61 ], renal cells [ 62 ], lung cells [ 63 , 64 ], motor neurons [ 65 ], intestinal cells [ 66 ], or chondrocytes [ 67 ].

Directed differentiation of either iPSCs or ESCs into, e.g. hepatocytes, could influence and develop the study of the molecular mechanisms in human liver development. In addition, it could also provide the possibility to form exogenous hepatocytes for drug toxicity testing [ 68 ].

Levels of concentration and duration of action with a specific signalling molecule can cause a variety of factors. Unfortunately, for now, a high cost of recombinant factors is likely to limit their use on a larger scale in medicine. The more promising technique focuses on the use of small molecules. These can be used for either activating or deactivating specific signalling pathways. They enhance reprogramming efficiency by creating cells that are compatible with the desired type of tissue. It is a cheaper and non-immunogenic method.

One of the successful examples of small-molecule cell therapies is antagonists and agonists of the Hedgehog pathway. They show to be very useful in motor neuron regeneration [ 69 ]. Endogenous small molecules with their function in embryonic development can also be used in in vitro methods to induce the differentiation of cells; for example, retinoic acid, which is responsible for patterning the nervous system in vivo [ 70 ], surprisingly induced retinal cell formation when the laboratory procedure involved hESCs [ 71 ].

The efficacy of differentiation factors depends on functional maturity, efficiency, and, finally, introducing produced cells to their in vivo equivalent. Topography, shear stress, and substrate rigidity are factors influencing the phenotype of future cells [ 72 ].

The control of biophysical and biochemical signals, the biophysical environment, and a proper guide of hESC differentiation are important factors in appropriately cultured stem cells.

Stem cell utilization and their manufacturing standards and culture systems

The European Medicines Agency and the Food and Drug Administration have set Good Manufacturing Practice (GMP) guidelines for safe and appropriate stem cell transplantation. In the past, protocols used for stem cell transplantation required animal-derived products [ 73 ].

The risk of introducing animal antigens or pathogens caused a restriction in their use. Due to such limitations, the technique required an obvious update [ 74 ]. Now, it is essential to use xeno-free equivalents when establishing cell lines that are derived from fresh embryos and cultured from human feeder cell lines [ 75 ]. In this method, it is crucial to replace any non-human materials with xeno-free equivalents [ 76 ].

NutriStem with LN-511, TeSR2 with human recombinant laminin (LN-511), and RegES with human foreskin fibroblasts (HFFs) are commonly used xeno-free culture systems [ 33 ]. There are many organizations and international initiatives, such as the National Stem Cell Bank, that provide stem cell lines for treatment or medical research [ 77 ].

Stem cell use in medicine

Stem cells have great potential to become one of the most important aspects of medicine. In addition to the fact that they play a large role in developing restorative medicine, their study reveals much information about the complex events that happen during human development.

The difference between a stem cell and a differentiated cell is reflected in the cells’ DNA. In the former cell, DNA is arranged loosely with working genes. When signals enter the cell and the differentiation process begins, genes that are no longer needed are shut down, but genes required for the specialized function will remain active. This process can be reversed, and it is known that such pluripotency can be achieved by interaction in gene sequences. Takahashi and Yamanaka [ 78 ] and Loh et al. [ 79 ] discovered that octamer-binding transcription factor 3 and 4 (Oct3/4), sex determining region Y (SRY)-box 2 and Nanog genes function as core transcription factors in maintaining pluripotency. Among them, Oct3/4 and Sox2 are essential for the generation of iPSCs.

Many serious medical conditions, such as birth defects or cancer, are caused by improper differentiation or cell division. Currently, several stem cell therapies are possible, among which are treatments for spinal cord injury, heart failure [ 80 ], retinal and macular degeneration [ 81 ], tendon ruptures, and diabetes type 1 [ 82 ]. Stem cell research can further help in better understanding stem cell physiology. This may result in finding new ways of treating currently incurable diseases.

Haematopoietic stem cell transplantation

Haematopoietic stem cells are important because they are by far the most thoroughly characterized tissue-specific stem cell; after all, they have been experimentally studied for more than 50 years. These stem cells appear to provide an accurate paradigm model system to study tissue-specific stem cells, and they have potential in regenerative medicine.

Multipotent haematopoietic stem cell (HSC) transplantation is currently the most popular stem cell therapy. Target cells are usually derived from the bone marrow, peripheral blood, or umbilical cord blood [ 83 ]. The procedure can be autologous (when the patient’s own cells are used), allogenic (when the stem cell comes from a donor), or syngeneic (from an identical twin). HSCs are responsible for the generation of all functional haematopoietic lineages in blood, including erythrocytes, leukocytes, and platelets. HSC transplantation solves problems that are caused by inappropriate functioning of the haematopoietic system, which includes diseases such as leukaemia and anaemia. However, when conventional sources of HSC are taken into consideration, there are some important limitations. First, there is a limited number of transplantable cells, and an efficient way of gathering them has not yet been found. There is also a problem with finding a fitting antigen-matched donor for transplantation, and viral contamination or any immunoreactions also cause a reduction in efficiency in conventional HSC transplantations. Haematopoietic transplantation should be reserved for patients with life-threatening diseases because it has a multifactorial character and can be a dangerous procedure. iPSC use is crucial in this procedure. The use of a patient’s own unspecialized somatic cells as stem cells provides the greatest immunological compatibility and significantly increases the success of the procedure.

Stem cells as a target for pharmacological testing

Stem cells can be used in new drug tests. Each experiment on living tissue can be performed safely on specific differentiated cells from pluripotent cells. If any undesirable effect appears, drug formulas can be changed until they reach a sufficient level of effectiveness. The drug can enter the pharmacological market without harming any live testers. However, to test the drugs properly, the conditions must be equal when comparing the effects of two drugs. To achieve this goal, researchers need to gain full control of the differentiation process to generate pure populations of differentiated cells.

Stem cells as an alternative for arthroplasty

One of the biggest fears of professional sportsmen is getting an injury, which most often signifies the end of their professional career. This applies especially to tendon injuries, which, due to current treatment options focusing either on conservative or surgical treatment, often do not provide acceptable outcomes. Problems with the tendons start with their regeneration capabilities. Instead of functionally regenerating after an injury, tendons merely heal by forming scar tissues that lack the functionality of healthy tissues. Factors that may cause this failed healing response include hypervascularization, deposition of calcific materials, pain, or swelling [ 84 ].

Additionally, in addition to problems with tendons, there is a high probability of acquiring a pathological condition of joints called osteoarthritis (OA) [ 85 ]. OA is common due to the avascular nature of articular cartilage and its low regenerative capabilities [ 86 ]. Although arthroplasty is currently a common procedure in treating OA, it is not ideal for younger patients because they can outlive the implant and will require several surgical procedures in the future. These are situations where stem cell therapy can help by stopping the onset of OA [ 87 ]. However, these procedures are not well developed, and the long-term maintenance of hyaline cartilage requires further research.

Osteonecrosis of the femoral hip (ONFH) is a refractory disease associated with the collapse of the femoral head and risk of hip arthroplasty in younger populations [ 88 ]. Although total hip arthroplasty (THA) is clinically successful, it is not ideal for young patients, mostly due to the limited lifetime of the prosthesis. An increasing number of clinical studies have evaluated the therapeutic effect of stem cells on ONFH. Most of the authors demonstrated positive outcomes, with reduced pain, improved function, or avoidance of THA [ 89 , 90 , 91 ].

Rejuvenation by cell programming

Ageing is a reversible epigenetic process. The first cell rejuvenation study was published in 2011 [ 92 ]. Cells from aged individuals have different transcriptional signatures, high levels of oxidative stress, dysfunctional mitochondria, and shorter telomeres than in young cells [ 93 ]. There is a hypothesis that when human or mouse adult somatic cells are reprogrammed to iPSCs, their epigenetic age is virtually reset to zero [ 94 ]. This was based on an epigenetic model, which explains that at the time of fertilization, all marks of parenteral ageing are erased from the zygote’s genome and its ageing clock is reset to zero [ 95 ].

In their study, Ocampo et al. [ 96 ] used Oct4, Sox2, Klf4, and C-myc genes (OSKM genes) and affected pancreas and skeletal muscle cells, which have poor regenerative capacity. Their procedure revealed that these genes can also be used for effective regenerative treatment [ 97 ]. The main challenge of their method was the need to employ an approach that does not use transgenic animals and does not require an indefinitely long application. The first clinical approach would be preventive, focused on stopping or slowing the ageing rate. Later, progressive rejuvenation of old individuals can be attempted. In the future, this method may raise some ethical issues, such as overpopulation, leading to lower availability of food and energy.

For now, it is important to learn how to implement cell reprogramming technology in non-transgenic elder animals and humans to erase marks of ageing without removing the epigenetic marks of cell identity.

Cell-based therapies

Stem cells can be induced to become a specific cell type that is required to repair damaged or destroyed tissues (Fig.  6 ). Currently, when the need for transplantable tissues and organs outweighs the possible supply, stem cells appear to be a perfect solution for the problem. The most common conditions that benefit from such therapy are macular degenerations [ 98 ], strokes [ 99 ], osteoarthritis [ 89 , 90 ], neurodegenerative diseases, and diabetes [ 100 ]. Due to this technique, it can become possible to generate healthy heart muscle cells and later transplant them to patients with heart disease.

Stem cell experiments on animals. These experiments are one of the many procedures that proved stem cells to be a crucial factor in future regenerative medicine

In the case of type 1 diabetes, insulin-producing cells in the pancreas are destroyed due to an autoimmunological reaction. As an alternative to transplantation therapy, it can be possible to induce stem cells to differentiate into insulin-producing cells [ 101 ].

Stem cells and tissue banks

iPS cells with their theoretically unlimited propagation and differentiation abilities are attractive for the present and future sciences. They can be stored in a tissue bank to be an essential source of human tissue used for medical examination. The problem with conventional differentiated tissue cells held in the laboratory is that their propagation features diminish after time. This does not occur in iPSCs.

The umbilical cord is known to be rich in mesenchymal stem cells. Due to its cryopreservation immediately after birth, its stem cells can be successfully stored and used in therapies to prevent the future life-threatening diseases of a given patient.

Stem cells of human exfoliated deciduous teeth (SHED) found in exfoliated deciduous teeth has the ability to develop into more types of body tissues than other stem cells [ 102 ] (Table  1 ). Techniques of their collection, isolation, and storage are simple and non-invasive. Among the advantages of banking, SHED cells are:

Guaranteed donor-match autologous transplant that causes no immune reaction and rejection of cells [ 103 ]

Simple and painless for both child and parent

Less than one third of the cost of cord blood storage

Not subject to the same ethical concerns as embryonic stem cells [ 104 ]

In contrast to cord blood stem cells, SHED cells are able to regenerate into solid tissues such as connective, neural, dental, or bone tissue [ 105 , 106 ]

SHED can be useful for close relatives of the donor

Fertility diseases

In 2011, two researchers, Katsuhiko Hayashi et al. [ 107 ], showed in an experiment on mice that it is possible to form sperm from iPSCs. They succeeded in delivering healthy and fertile pups in infertile mice. The experiment was also successful for female mice, where iPSCs formed fully functional eggs .

Young adults at risk of losing their spermatogonial stem cells (SSC), mostly cancer patients, are the main target group that can benefit from testicular tissue cryopreservation and autotransplantation. Effective freezing methods for adult and pre-pubertal testicular tissue are available [ 108 ].

Qiuwan et al. [ 109 ] provided important evidence that human amniotic epithelial cell (hAEC) transplantation could effectively improve ovarian function by inhibiting cell apoptosis and reducing inflammation in injured ovarian tissue of mice, and it could be a promising strategy for the management of premature ovarian failure or insufficiency in female cancer survivors.

For now, reaching successful infertility treatments in humans appears to be only a matter of time, but there are several challenges to overcome. First, the process needs to have high efficiency; second, the chances of forming tumours instead of eggs or sperm must be maximally reduced. The last barrier is how to mature human sperm and eggs in the lab without transplanting them to in vivo conditions, which could cause either a tumour risk or an invasive procedure.

Therapy for incurable neurodegenerative diseases

Thanks to stem cell therapy, it is possible not only to delay the progression of incurable neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease (AD), and Huntington disease, but also, most importantly, to remove the source of the problem. In neuroscience, the discovery of neural stem cells (NSCs) has nullified the previous idea that adult CNS were not capable of neurogenesis [ 110 , 111 ]. Neural stem cells are capable of improving cognitive function in preclinical rodent models of AD [ 112 , 113 , 114 ]. Awe et al. [ 115 ] clinically derived relevant human iPSCs from skin punch biopsies to develop a neural stem cell-based approach for treating AD. Neuronal degeneration in Parkinson’s disease (PD) is focal, and dopaminergic neurons can be efficiently generated from hESCs. PD is an ideal disease for iPSC-based cell therapy [ 116 ]. However, this therapy is still in an experimental phase ( https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4539501 /). Brain tissue from aborted foetuses was used on patients with Parkinson’s disease [ 117 ]. Although the results were not uniform, they showed that therapies with pure stem cells are an important and achievable therapy.

Stem cell use in dentistry

Teeth represent a very challenging material for regenerative medicine. They are difficult to recreate because of their function in aspects such as articulation, mastication, or aesthetics due to their complicated structure. Currently, there is a chance for stem cells to become more widely used than synthetic materials. Teeth have a large advantage of being the most natural and non-invasive source of stem cells.

For now, without the use of stem cells, the most common periodontological treatments are either growth factors, grafts, or surgery. For example, there are stem cells in periodontal ligament [ 118 , 119 ], which are capable of differentiating into osteoblasts or cementoblasts, and their functions were also assessed in neural cells [ 120 ]. Tissue engineering is a successful method for treating periodontal diseases. Stem cells of the root apical areas are able to recreate periodontal ligament. One of the possible methods of tissue engineering in periodontology is gene therapy performed using adenoviruses-containing growth factors [ 121 ].

As a result of animal studies, dentin regeneration is an effective process that results in the formation of dentin bridges [ 122 ].

Enamel is more difficult to regenerate than dentin. After the differentiation of ameloblastoma cells into the enamel, the former is destroyed, and reparation is impossible. Medical studies have succeeded in differentiating bone marrow stem cells into ameloblastoma [ 123 ].

Healthy dental tissue has a high amount of regular stem cells, although this number is reduced when tissue is either traumatized or inflamed [ 124 ]. There are several dental stem cell groups that can be isolated (Fig.  7 ).

Localization of stem cells in dental tissues. Dental pulp stem cells (DPSCs) and human deciduous teeth stem cells (SHED) are located in the dental pulp. Periodontal ligaments stem cells are located in the periodontal ligament. Apical papilla consists of stem cells from the apical papilla (SCAP)

Dental pulp stem cell (DPSC)

These were the first dental stem cells isolated from the human dental pulp, which were [ 125 ] located inside dental pulp (Table  2 ). They have osteogenic and chondrogenic potential. Mesenchymal stem cells (MSCs) of the dental pulp, when isolated, appear highly clonogenic; they can be isolated from adult tissue (e.g. bone marrow, adipose tissue) and foetal (e.g. umbilical cord) [ 126 ] tissue, and they are able to differentiate densely [ 127 ]. MSCs differentiate into odontoblast-like cells and osteoblasts to form dentin and bone. Their best source locations are the third molars [ 125 ]. DPSCs are the most useful dental source of tissue engineering due to their easy surgical accessibility, cryopreservation possibility, increased production of dentin tissues compared to non-dental stem cells, and their anti-inflammatory abilities. These cells have the potential to be a source for maxillofacial and orthopaedic reconstructions or reconstructions even beyond the oral cavity. DPSCs are able to generate all structures of the developed tooth [ 128 ]. In particular, beneficial results in the use of DPSCs may be achieved when combined with other new therapies, such as periodontal tissue photobiomodulation (laser stimulation), which is an efficient technique in the stimulation of proliferation and differentiation into distinct cell types [ 129 ]. DPSCs can be induced to form neural cells to help treat neurological deficits.

Stem cells of human exfoliated deciduous teeth (SHED) have a faster rate of proliferation than DPSCs and differentiate into an even greater number of cells, e.g. other mesenchymal and non-mesenchymal stem cell derivatives, such as neural cells [ 130 ]. These cells possess one major disadvantage: they form a non-complete dentin/pulp-like complex in vivo. SHED do not undergo the same ethical concerns as embryonic stem cells. Both DPSCs and SHED are able to form bone-like tissues in vivo [ 131 ] and can be used for periodontal, dentin, or pulp regeneration. DPSCs and SHED can be used in treating, for example, neural deficits [ 132 ]. DPSCs alone were tested and successfully applied for alveolar bone and mandible reconstruction [ 133 ].

Periodontal ligament stem cells (PDLSCs)

These cells are used in periodontal ligament or cementum tissue regeneration. They can differentiate into mesenchymal cell lineages to produce collagen-forming cells, adipocytes, cementum tissue, Sharpey’s fibres, and osteoblast-like cells in vitro. PDLSCs exist both on the root and alveolar bone surfaces; however, on the latter, these cells have better differentiation abilities than on the former [ 134 ]. PDLSCs have become the first treatment for periodontal regeneration therapy because of their safety and efficiency [ 135 , 136 ].

Stem cells from apical papilla (SCAP)

These cells are mesenchymal structures located within immature roots. They are isolated from human immature permanent apical papilla. SCAP are the source of odontoblasts and cause apexogenesis. These stem cells can be induced in vitro to form odontoblast-like cells, neuron-like cells, or adipocytes. SCAP have a higher capacity of proliferation than DPSCs, which makes them a better choice for tissue regeneration [ 137 , 138 ].

Dental follicle stem cells (DFCs)

These cells are loose connective tissues surrounding the developing tooth germ. DFCs contain cells that can differentiate into cementoblasts, osteoblasts, and periodontal ligament cells [ 139 , 140 ]. Additionally, these cells proliferate after even more than 30 passages [ 141 ]. DFCs are most commonly extracted from the sac of a third molar. When DFCs are combined with a treated dentin matrix, they can form a root-like tissue with a pulp-dentin complex and eventually form tooth roots [ 141 ]. When DFC sheets are induced by Hertwig’s epithelial root sheath cells, they can produce periodontal tissue; thus, DFCs represent a very promising material for tooth regeneration [ 142 ].

Pulp regeneration in endodontics

Dental pulp stem cells can differentiate into odontoblasts. There are few methods that enable the regeneration of the pulp.

The first is an ex vivo method. Proper stem cells are grown on a scaffold before they are implanted into the root channel [ 143 ].

The second is an in vivo method. This method focuses on injecting stem cells into disinfected root channels after the opening of the in vivo apex. Additionally, the use of a scaffold is necessary to prevent the movement of cells towards other tissues. For now, only pulp-like structures have been created successfully.

Methods of placing stem cells into the root channel constitute are either soft scaffolding [ 144 ] or the application of stem cells in apexogenesis or apexification. Immature teeth are the best source [ 145 ]. Nerve and blood vessel network regeneration are extremely vital to keep pulp tissue healthy.

The potential of dental stem cells is mainly regarding the regeneration of damaged dentin and pulp or the repair of any perforations; in the future, it appears to be even possible to generate the whole tooth. Such an immense success would lead to the gradual replacement of implant treatments. Mandibulary and maxillary defects can be one of the most complicated dental problems for stem cells to address.

Acquiring non-dental tissue cells by dental stem cell differentiation

In 2013, it was reported that it is possible to grow teeth from stem cells obtained extra-orally, e.g. from urine [ 146 ]. Pluripotent stem cells derived from human urine were induced and generated tooth-like structures. The physical properties of the structures were similar to natural ones except for hardness [ 127 ]. Nonetheless, it appears to be a very promising technique because it is non-invasive and relatively low-cost, and somatic cells can be used instead of embryonic cells. More importantly, stem cells derived from urine did not form any tumours, and the use of autologous cells reduces the chances of rejection [ 147 ].

Use of graphene in stem cell therapy

Over recent years, graphene and its derivatives have been increasingly used as scaffold materials to mediate stem cell growth and differentiation [ 148 ]. Both graphene and graphene oxide (GO) represent high in-plane stiffness [ 149 ]. Because graphene has carbon and aromatic network, it works either covalently or non-covalently with biomolecules; in addition to its superior mechanical properties, graphene offers versatile chemistry. Graphene exhibits biocompatibility with cells and their proper adhesion. It also tested positively for enhancing the proliferation or differentiation of stem cells [ 148 ]. After positive experiments, graphene revealed great potential as a scaffold and guide for specific lineages of stem cell differentiation [ 150 ]. Graphene has been successfully used in the transplantation of hMSCs and their guided differentiation to specific cells. The acceleration skills of graphene differentiation and division were also investigated. It was discovered that graphene can serve as a platform with increased adhesion for both growth factors and differentiation chemicals. It was also discovered that π-π binding was responsible for increased adhesion and played a crucial role in inducing hMSC differentiation [ 150 ].

Therapeutic potential of extracellular vesicle-based therapies

Extracellular vesicles (EVs) can be released by virtually every cell of an organism, including stem cells [ 151 ], and are involved in intercellular communication through the delivery of their mRNAs, lipids, and proteins. As Oh et al. [ 152 ] prove, stem cells, together with their paracrine factors—exosomes—can become potential therapeutics in the treatment of, e.g. skin ageing. Exosomes are small membrane vesicles secreted by most cells (30–120 nm in diameter) [ 153 ]. When endosomes fuse with the plasma membrane, they become exosomes that have messenger RNAs (mRNAs) and microRNAs (miRNAs), some classes of non-coding RNAs (IncRNAs) and several proteins that originate from the host cell [ 154 ]. IncRNAs can bind to specific loci and create epigenetic regulators, which leads to the formation of epigenetic modifications in recipient cells. Because of this feature, exosomes are believed to be implicated in cell-to-cell communication and the progression of diseases such as cancer [ 155 ]. Recently, many studies have also shown the therapeutic use of exosomes derived from stem cells, e.g. skin damage and renal or lung injuries [ 156 ].

In skin ageing, the most important factor is exposure to UV light, called “photoageing” [ 157 ], which causes extrinsic skin damage, characterized by dryness, roughness, irregular pigmentation, lesions, and skin cancers. In intrinsic skin ageing, on the other hand, the loss of elasticity is a characteristic feature. The skin dermis consists of fibroblasts, which are responsible for the synthesis of crucial skin elements, such as procollagen or elastic fibres. These elements form either basic framework extracellular matrix constituents of the skin dermis or play a major role in tissue elasticity. Fibroblast efficiency and abundance decrease with ageing [ 158 ]. Stem cells can promote the proliferation of dermal fibroblasts by secreting cytokines such as platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β), and basic fibroblast growth factor. Huh et al. [ 159 ] mentioned that a medium of human amniotic fluid-derived stem cells (hAFSC) positively affected skin regeneration after longwave UV-induced (UVA, 315–400 nm) photoageing by increasing the proliferation and migration of dermal fibroblasts. It was discovered that, in addition to the induction of fibroblast physiology, hAFSC transplantation also improved diseases in cases of renal pathology, various cancers, or stroke [ 160 , 161 ].

Oh [ 162 ] also presented another option for the treatment of skin wounds, either caused by physical damage or due to diabetic ulcers. Induced pluripotent stem cell-conditioned medium (iPSC-CM) without any animal-derived components induced dermal fibroblast proliferation and migration.

Natural cutaneous wound healing is divided into three steps: haemostasis/inflammation, proliferation, and remodelling. During the crucial step of proliferation, fibroblasts migrate and increase in number, indicating that it is a critical step in skin repair, and factors such as iPSC-CM that impact it can improve the whole cutaneous wound healing process. Paracrine actions performed by iPSCs are also important for this therapeutic effect [ 163 ]. These actions result in the secretion of cytokines such as TGF-β, interleukin (IL)-6, IL-8, monocyte chemotactic protein-1 (MCP-1), vascular endothelial growth factor (VEGF), platelet-derived growth factor-AA (PDGF-AA), and basic fibroblast growth factor (bFGF). Bae et al. [ 164 ] mentioned that TGF-β induced the migration of keratinocytes. It was also demonstrated that iPSC factors can enhance skin wound healing in vivo and in vitro when Zhou et al. [ 165 ] enhanced wound healing, even after carbon dioxide laser resurfacing in an in vivo study.

Peng et al. [ 166 ] investigated the effects of EVs derived from hESCs on in vitro cultured retinal glial, progenitor Müller cells, which are known to differentiate into retinal neurons. EVs appear heterogeneous in size and can be internalized by cultured Müller cells, and their proteins are involved in the induction and maintenance of stem cell pluripotency. These stem cell-derived vesicles were responsible for the neuronal trans-differentiation of cultured Müller cells exposed to them. However, the research article points out that the procedure was accomplished only on in vitro acquired retina.

Challenges concerning stem cell therapy

Although stem cells appear to be an ideal solution for medicine, there are still many obstacles that need to be overcome in the future. One of the first problems is ethical concern.

The most common pluripotent stem cells are ESCs. Therapies concerning their use at the beginning were, and still are, the source of ethical conflicts. The reason behind it started when, in 1998, scientists discovered the possibility of removing ESCs from human embryos. Stem cell therapy appeared to be very effective in treating many, even previously incurable, diseases. The problem was that when scientists isolated ESCs in the lab, the embryo, which had potential for becoming a human, was destroyed (Fig.  8 ). Because of this, scientists, seeing a large potential in this treatment method, focused their efforts on making it possible to isolate stem cells without endangering their source—the embryo.

Use of inner cell mass pluripotent stem cells and their stimulation to differentiate into desired cell types

For now, while hESCs still remain an ethically debatable source of cells, they are potentially powerful tools to be used for therapeutic applications of tissue regeneration. Because of the complexity of stem cell control systems, there is still much to be learned through observations in vitro. For stem cells to become a popular and widely accessible procedure, tumour risk must be assessed. The second problem is to achieve successful immunological tolerance between stem cells and the patient’s body. For now, one of the best ideas is to use the patient’s own cells and devolve them into their pluripotent stage of development.

New cells need to have the ability to fully replace lost or malfunctioning natural cells. Additionally, there is a concern about the possibility of obtaining stem cells without the risk of morbidity or pain for either the patient or the donor. Uncontrolled proliferation and differentiation of cells after implementation must also be assessed before its use in a wide variety of regenerative procedures on living patients [ 167 ].

One of the arguments that limit the use of iPSCs is their infamous role in tumourigenicity. There is a risk that the expression of oncogenes may increase when cells are being reprogrammed. In 2008, a technique was discovered that allowed scientists to remove oncogenes after a cell achieved pluripotency, although it is not efficient yet and takes a longer amount of time. The process of reprogramming may be enhanced by deletion of the tumour suppressor gene p53, but this gene also acts as a key regulator of cancer, which makes it impossible to remove in order to avoid more mutations in the reprogrammed cell. The low efficiency of the process is another problem, which is progressively becoming reduced with each year. At first, the rate of somatic cell reprogramming in Yamanaka’s study was up to 0.1%. The use of transcription factors creates a risk of genomic insertion and further mutation of the target cell genome. For now, the only ethically acceptable operation is an injection of hESCs into mouse embryos in the case of pluripotency evaluation [ 168 ].

Stem cell obstacles in the future

Pioneering scientific and medical advances always have to be carefully policed in order to make sure they are both ethical and safe. Because stem cell therapy already has a large impact on many aspects of life, it should not be treated differently.

Currently, there are several challenges concerning stem cells. First, the most important one is about fully understanding the mechanism by which stem cells function first in animal models. This step cannot be avoided. For the widespread, global acceptance of the procedure, fear of the unknown is the greatest challenge to overcome.

The efficiency of stem cell-directed differentiation must be improved to make stem cells more reliable and trustworthy for a regular patient. The scale of the procedure is another challenge. Future stem cell therapies may be a significant obstacle. Transplanting new, fully functional organs made by stem cell therapy would require the creation of millions of working and biologically accurate cooperating cells. Bringing such complicated procedures into general, widespread regenerative medicine will require interdisciplinary and international collaboration.

The identification and proper isolation of stem cells from a patient’s tissues is another challenge. Immunological rejection is a major barrier to successful stem cell transplantation. With certain types of stem cells and procedures, the immune system may recognize transplanted cells as foreign bodies, triggering an immune reaction resulting in transplant or cell rejection.

One of the ideas that can make stem cells a “failsafe” is about implementing a self-destruct option if they become dangerous. Further development and versatility of stem cells may cause reduction of treatment costs for people suffering from currently incurable diseases. When facing certain organ failure, instead of undergoing extraordinarily expensive drug treatment, the patient would be able to utilize stem cell therapy. The effect of a successful operation would be immediate, and the patient would avoid chronic pharmacological treatment and its inevitable side effects.

Although these challenges facing stem cell science can be overwhelming, the field is making great advances each day. Stem cell therapy is already available for treating several diseases and conditions. Their impact on future medicine appears to be significant.

After several decades of experiments, stem cell therapy is becoming a magnificent game changer for medicine. With each experiment, the capabilities of stem cells are growing, although there are still many obstacles to overcome. Regardless, the influence of stem cells in regenerative medicine and transplantology is immense. Currently, untreatable neurodegenerative diseases have the possibility of becoming treatable with stem cell therapy. Induced pluripotency enables the use of a patient’s own cells. Tissue banks are becoming increasingly popular, as they gather cells that are the source of regenerative medicine in a struggle against present and future diseases. With stem cell therapy and all its regenerative benefits, we are better able to prolong human life than at any time in history.

Abbreviations

Basic fibroblast growth factor

Bone morphogenic proteins

Dental follicle stem cells

Dental pulp stem cells

Embryonic bodies

Embryonic stem cells

Fibroblast growth factors

Good Manufacturing Practice

Graphene oxide

Human amniotic fluid-derived stem cells

Human embryonic stem cells

Human foreskin fibroblasts

Inner cell mass

Non-coding RNA

Induced pluripotent stem cells

In vitro fertilization

Knockout serum replacement

Leukaemia inhibitory factor

Monocyte chemotactic protein-1

Fibroblasts

Messenger RNA

Mesenchymal stem cells of dental pulp

Myogenic differentiation

Osteoarthritis

Octamer-binding transcription factor 3 and 4

Platelet-derived growth factor

Platelet-derived growth factor-AA

Periodontal ligament stem cells

Rho-associated protein kinase

Stem cells from apical papilla

Stem cells of human exfoliated deciduous teeth

Synthetic Serum Substitute

Trophectoderm

Vascular endothelial growth factor

Transforming growth factors

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Wojciech Zakrzewski, Maria Szymonowicz & Zbigniew Rybak

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Zakrzewski, W., Dobrzyński, M., Szymonowicz, M. et al. Stem cells: past, present, and future. Stem Cell Res Ther 10 , 68 (2019). https://doi.org/10.1186/s13287-019-1165-5

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Current state of stem cell-based therapies: an overview

Riham mohamed aly.

1 Department of Basic Dental Science, National Research Centre, Cairo, Egypt;

2 Stem Cell Laboratory, Center of Excellence for Advanced Sciences, National Research Centre, Cairo, Egypt

Recent research reporting successful translation of stem cell therapies to patients have enriched the hope that such regenerative strategies may one day become a treatment for a wide range of vexing diseases. In fact, the past few years witnessed, a rather exponential advancement in clinical trials revolving around stem cell-based therapies. Some of these trials resulted in remarkable impact on various diseases. In this review, the advances and challenges for the development of stem-cell-based therapies are described, with focus on the use of stem cells in dentistry in addition to the advances reached in regenerative treatment modalities in several diseases. The limitations of these treatments and ongoing challenges in the field are also discussed while shedding light on the ethical and regulatory challenges in translating autologous stem cell-based interventions, into safe and effective therapies.

Introduction

Cell-based therapy as a modality of regenerative medicine is considered one of the most promising disciplines in the fields of modern science & medicine. Such an advanced technology offers endless possibilities for transformative and potentially curative treatments for some of humanities most life threatening diseases. Regenerative medicine is rapidly becoming the next big thing in health care with the particular aim of repairing and possibly replacing diseased cells, tissues or organs and eventually retrieving normal function. Fortunately, the prospect of regenerative medicine as an alternative to conventional drug-based therapies is becoming a tangible reality by the day owing to the vigorous commitment of the research communities in studying the potential applications across a wide range of diseases like neurodegenerative diseases and diabetes, among many others ( 1 ).

Recent research reporting successful translation of stem cell therapies to patients have enriched the hope that such regenerative strategies may one day become a treatment for a wide range of vexing diseases ( 2 ). In fact, the past few years witnessed, a rather exponential advancement in clinical trials revolving around stem cell-based therapies. Some of these trials resulted in remarkable impact on various diseases ( 3 ). For example, a case of Epidermolysis Bullosa manifested signs of skin recovery after treatment with keratinocyte cultures of epidermal stem cells ( 4 ). Also, a major improvement in eyesight of patients suffering from macular degeneration was reported after transplantation of patient-derived induced pluripotent stem cells (iPSCs) that were induced to differentiate into pigment epithelial cells of the retina ( 5 ).

However, in spite of the increased amount of publications reporting successful cases of stem cell-based therapies, a major number of clinical trials have not yet acquired full regulatory approvals for validation as stem cell therapies. To date, the most established stem cell treatment is bone marrow transplants to treat blood and immune system disorders ( 1 , 6 , 7 ).

In this review, the advances and challenges for the development of stem-cell-based therapies are described, with focus on the use of stem cells in dentistry in addition to the advances reached in regenerative treatment modalities in several diseases. The limitations of these treatments and ongoing challenges in the field are also discussed while shedding light on the ethical and regulatory challenges in translating autologous stem cell-based interventions, into safe and effective therapies.

Stem cell-based therapies

Stem cell-based therapies are defined as any treatment for a disease or a medical condition that fundamentally involves the use of any type of viable human stem cells including embryonic stem cells (ESCs), iPSCs and adult stem cells for autologous and allogeneic therapies ( 8 ). Stem cells offer the perfect solution when there is a need for tissue and organ transplantation through their ability to differentiate into the specific cell types that are required for repair of diseased tissues.

However, the complexity of stem cell-based therapies often leads researchers to search for stable, safe and easily accessible stem cells source that has the potential to differentiate into several lineages. Thus, it is of utmost importance to carefully select the type of stem cells that is suitable for clinical application ( 7 , 9 ).

Stem cells hierarchy

There are mainly three types of stem cells. All three of them share the significant property of self-renewal in addition to a unique ability to differentiate. However, it should be noted that stem cells are not homogeneous, but rather exist in a developmental hierarchy ( 10 ). The most basic and undeveloped of stem cells are the totipotent stem cells. These cells are capable of developing into a complete embryo while forming the extra-embryonic tissue at the same time. This unique property is brief and starts with the fertilization of the ovum and ends when the embryo reaches the four to eight cells stage. Following that cells undergo subsequent divisions until reaching the blastocyst stage where they lose their totipotency property and assume a pluripotent identity where cells are only capable of differentiating into every embryonic germ layer (ectoderm, mesoderm and endoderm). Cells of this stage are termed “embryonic stem cells” and are obtained by isolation from the inner cell mass of the blastocyst in a process that involves the destruction of the forming embryo. After consecutive divisions, the property of pluripotency is lost and the differentiation capability becomes more lineage restricted where the cells become multipotent meaning that they can only differentiate into limited types of cells related to the tissue of origin. This is the property of “adult stem cells”, which helps create a state of homeostasis throughout the lifetime of the organism. Adult stem cells are present in a metabolically quiescent state in almost all specialized tissues of the body, which includes bone marrow and oral and dental tissues among many others ( 11 ).

Many authors consider adult stem cells the gold standard in stem cell-based therapies ( 12 , 13 ). Adult stem cells demonstrated signs of clinical success especially in hematopoietic transplants ( 14 , 15 ). In contrast to ESCs, adult stem cells are not subjected to controversial views regarding their origin. The fact that ESCs derivation involves destruction of human embryos renders them unacceptable for a significant proportion of the population for ethical and religious convictions ( 16 - 18 ).

Turning point in stem cell research

It was in 2006 when Shinya Yamanka achieved a scientific breakthrough in stem cell research by succeeding in generating cells that have the same properties and genetic profile of ESCs. This was achieved via the transient over-expression of a cocktail of four transcription factors; OCT4, SOX2, KLF4 and MYC in, fully differentiated somatic cells, namely fibroblasts ( 19 , 20 ). These cells were called iPSCs and has transformed the field of stem cell research ever since ( 21 ). The most important feature of these cells is their ability to differentiate into any of the germ layers just like ESCs precluding the ethical debate surrounding their use. The development of iPSCs technology has created an innovative way to both identify and treat diseases. Since they can be generated from the patient’s own cells, iPSCs thus present a promising potential for the production of pluripotent derived patient-matched cells that could be used for autologous transplantation. True these cells symbolize a paradigm shift since they enable researchers to directly observe and treat relevant patient cells; nevertheless, a number of challenges still need to be addressed before iPSCs-derived cells can be applied in cell therapies. Such challenges include; the detection and removal of incompletely differentiated cells, addressing the genomic and epigenetic alterations in the generated cells and overcoming the tumorigenicity of these cells that could arise on transplantation ( 22 ).

Therapeutic translation of stem cell research

With the rapid increase witnessed in stem cell basic research over the past years, the relatively new research discipline “Translational Research” has evolved significantly building up on the outcomes of basic research in order to develop new therapies. The clinical translation pathway starts after acquiring the suitable regulatory approvals. The importance of translational research lies in it’s a role as a filter to ensure that only safe and effective therapies reach the clinic ( 23 ). It bridges the gap from bench to bed. Currently, some stem cell-based therapies utilizing adult stem cells are clinically available and mainly include bone marrow transplants of hematopoietic stem cells and skin grafts for severe burns ( 23 ). To date, there are more than 3,000 trials involving the use of adult stem cells registered in WHO International Clinical Trials Registry. Additionally, initial trials involving the new and appealing iPSCs based therapies are also registered. In fact, the first clinical attempt employing iPSCs reported successful results in treating macular degeneration ( 24 ). Given the relative immaturity in the field of cellular therapy, the outcomes of such trials shall facilitate the understanding of the timeframes needed to achieve successful therapies and help in better understanding of the diseases. However, it is noteworthy that evaluation of stem cell-based therapies is not an easy task since transplantation of cells is ectopic and may result in tumor formation and other complications. This accounts for the variations in the results reported from previous reports. The following section discusses the published data of some of the most important clinical trials involving the use of different types of stem cells both in medicine and in dentistry.

Stem cell-based therapy for neurodegenerative diseases

The successful generation of neural cells from stem cells in vitro paved the way for the current stem cell-based clinical trials targeting neurodegenerative diseases ( 25 , 26 ). These therapies do not just target detaining the progression of irrecoverable neuro-degenerative diseases like Parkinson’s, Alzheimer’s, amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS), but are also focused on completely treating such disorders.

Parkinson’s disease (PD)

PD is characterized by a rapid loss of midbrain dopaminergic neurons. The first attempt for using human ESC cells to treat PD was via the generation of dopaminergic-like neurons, later human iPSCs was proposed as an alternative to overcome ESCs controversies ( 27 ). Both cells presented hope for obtaining an endless source of dopaminergic neurons instead of the previously used fetal brain tissues. Subsequently, protocols that mimicked the development of dopaminergic neurons succeeded in generating dopaminergic neurons similar to that of the midbrain which were able to survive, integrate and functionally mature in animal models of PD preclinically ( 28 ). Based on the research presented by different groups; the “Parkinson’s Global Force” was formed which aimed at guiding researchers to optimize their cell characterization and help promote the clinical progress toward successful therapy. Recently, In August 2018, Shinya Yamanka initiated the first approved clinical trial to treat PD using iPSCs. Seven patients suffering from moderate PD were recruited ( 29 ). Donor matched allogeneic cells were used to avoid any genetic influence of the disease. The strategy behind the trial involved the generation of dopaminergic progenitors followed by surgical transplantation into the brains of patients by a special device. In addition, immunosuppressant medications were given to avoid any adverse reaction. Preliminary results so far revealed the safety of the treatment.

MS is an inflammatory and neurodegenerative autoimmune disease of the central nervous system. Stem cell-based therapies are now exploring the possibility of halting the disease progression and reverse the neural damage. A registered phase 1 clinical trial was conducted by the company Celgene TM in 2014 using placental-derived mesenchymal stem cells (MSCs) infusion to treat patients suffering from MS ( 30 ). This trial was performed at 6 centers in the United States and 2 centers in Canada and included 16 patients. Results demonstrated that cellular infusions were safe with no signs of paradoxical aggravation. However, clinical responses from patients indicated that the cellular treatment did not improve the MS condition ( 31 ). For the last decade immunoablative therapy demonstrated accumulative evidence of inducing long-term remission and improvement of disability caused by MS. This approach involves the replacement of the diseased immune system through administration of high-dose immunosuppressive therapy followed by hematopoietic stem cells infusion ( 32 ). However, immunoablation strategies demonstrated several complications such as infertility and neurological disabilities. A number of randomized controlled trials are planned to address these concerns ( 32 ). Currently, new and innovative stem cell-based therapies for MS are only in the initial stages, and are based on different mechanisms exploring the possibility of replacing damaged neuronal tissue with neural cells derived from iPSCs however, the therapeutic potential of iPSCs is still under research ( 33 ).

ALS is a neurodegenerative disease that causes degeneration of the motor neurons which results in disturbance in muscle performance. The first attempt to treat ALS was through the transplantation of MSCs in a mouse model. The outcomes of this experiment were promising and resulted in a decrease of the disease manifestations and thus providing proof of principal ( 34 ). Based on these results, several planned/ongoing clinical trials are on the way. These trials mainly assess the safety of the proposed concept and have not proved clinical success to date. Notably, while pre-clinical studies have reported that cells derived from un-diseased individuals are superior to cells from ALS patients; most of the clinical trials attempted have employed autologous transplantation. This information may account for the absence of therapeutic improvement reported ( 35 ).

Spinal cord injury

Other neurologic indications for the use of stem cells are spinal cord injuries. Though the transplantation of different forms of neural stem cells and oligo-dendrocyte progenitors has led to growth in the axons in addition to neural connectivity which presents a possibility for repair ( 36 ), proof of recovered function has yet to be established in stringent clinical trials. Nevertheless, Japan has recently given approval to stem-cell treatment for spinal-cord injuries. This approval was based on clinical trials that are yet to be published and involves 13 patients, who are suffering from recent spinal-cord injury. The Japanese team discovered that injection of stem cells isolated from the patients’ bone marrow aided in regaining some lost sensation and mobility. This is the first stem cell-based therapy targeting spinal-cord injuries to gain governmental approval to offer to patients ( 37 ).

Stem cell-based therapies for ocular diseases

A huge number of the currently registered clinical trials for stem cell-based therapies target ocular diseases. This is mainly due to the fact that the eye is an immune privileged site. Most of these trials span various countries including Japan, China, Israel, Korea, UK, and USA and implement allogeneic ESC lines ( 35 , 36 ). Notably, the first clinical trial to implement the use autologous iPSCs-derived retinal cells was in Japan which followed the new regulatory laws issued in 2014 by Japan’s government to regulate regenerative medicine applications. Two patients were recruited in this trial, the first one received treatment for macular degeneration using iPSCs-generated retinal cell sheet ( 37 ). After 1 year of follow-up, there were no signs of serious complications including abnormal proliferation and systemic malignancy. Moreover, there were no signs of rejection of the transplanted retinal epithelial sheet in the second year follow-up. Most importantly, the signs of corrected visual acuity of the treated eye were reported. These results were enough to conclude that iPSCs-based autologous transplantation was safe and feasible ( 38 ). It is worthy to mention that the second patient was withdrawn from the study due to detectable genetic variations the patient’s iPSCs lines which was not originally present in the patient’s original fibroblasts. Such alterations may jeopardize the overall safety of the treatment. The fact that this decision was taken, even though the performed safety assays did not demonstrate tumorgenicity in the iPSCs-derived retinal pigment epithelium (RPE) cells, indicates that researchers in the field of iPSCs have full awareness of the importance of safety issues ( 39 ).

Stem cell-based therapies for treatment of diabetes

Pancreatic beta cells are destructed in type 1 diabetes mellitus, because of disorders in the immune system while in type 2 insulin insufficiency is caused by failure of the beta-cell to normally produce insulin. In both cases the affected cell is the beta cell, and since the pancreas does not efficiently regenerate islets from endogenous adult stem cells, other cell sources were tested ( 38 ). Pluripotent stem cells (PSCs) are considered the cells of choice for beta cell replacement strategies ( 39 ). Currently, there are a few industry-sponsored clinical trials that are registered targeting beta cell replacement using ESCs. These trials revolve around the engraftment of insulin-producing beta cells in an encapsulating device subcutaneously to protect the cells from autoimmunity in patients with type 1 diabetes ( 40 ). The company ViaCyte TM in California recently initiated a phase I/II trial ( {"type":"clinical-trial","attrs":{"text":"NCT02239354","term_id":"NCT02239354"}} NCT02239354 ) in 2014 in collaboration with Harvard University. This trial involves 40 patients and employs two subcutaneous capsules of insulin producing beta cells generated from ESCs. The results shall be interesting due to the ease of monitoring and recovery of the transplanted cells. The preclinical studies preceding this trial demonstrated successful glycemic correction and the devices were successfully retrieved after 174 days and contained viable insulin-producing cells ( 41 ).

Stem cells in dentistry

Stem cells have been successfully isolated from human teeth and were studied to test their ability to regenerate dental structures and periodontal tissues. MSCs were reported to be successfully isolated from dental tissues like dental pulp of permanent and deciduous teeth, periodontal ligament, apical papilla and dental follicle ( 42 - 44 ). These cells were described as an excellent cell source owing to their ease of accessibility, their ability to differentiate into osteoblasts and odontoblasts and lack of ethical controversies ( 45 ). Moreover, dental stem cells demonstrated superior abilities in immunomodulation properties either through cell to cell interaction or via a paracrine effect ( 46 ). Stem cells of non-dental origin were also suggested for dental tissue and bone regeneration. Different approaches were investigated for achieving dental and periodontal regeneration ( 47 ); however, assessments of stem cells after transplantation still require extensive studying. Clinical trials have only recently begun and their results are yet to be fully evaluated. However, by carefully applying the knowledge acquired from the extensive basic research in dental and periodontal regeneration, stem cell-based dental and periodontal regeneration may soon be a readily available treatment. To date, there are more than 6,000 clinical trials involving the use of with stem cells, however only a total of 44 registered clinical trials address oral diseases worldwide ( 48 ). Stem cell-based clinical trials with reported results targeting the treatment of oral disease are discussed below.

Dental pulp regeneration

The first human clinical study using autologous dental pulp stem cells (DPSCs) for complete pulp regeneration was reported by Nakashima et al. in 2017 ( 49 ). This pilot study was based on extensive preclinical studies conducted by the same group ( 50 ). Patients with irreversible pulpitis were recruited and followed up for 6 months following DPSCs’ transplantation. Granulocyte colony-stimulating factor was administered to induce stem cell mobilization to enrich the stem cell populations. The research team reported that the use of DPSCs seeded on collagen scaffold in molars and premolars undergoing pulpectomy was safe. No adverse events or toxicity were demonstrated in the clinical and laboratory evaluations. Positive electric pulp testing was obtained after cell transplantation in all patients. Moreover, magnetic resonance imaging of the de - novo tissues formed in the root canal demonstrated similar results to normal pulp, which indicated successful pulp regeneration. A different group conducted a clinical trial that recruited patients diagnosed with necrotic pulp. Autologous stem cells from deciduous teeth were employed to induce pulp regeneration ( 51 ). Follow-up of the cases after a year from the intervention reported evidence of pulp regeneration with vascular supply and innervation. In addition, no signs of adverse effects were observed in patients receiving DPSCs transplantation. Both trials are proceeding with the next phases, however the results obtained are promising.

Periodontal tissue regeneration

Aimetti et al. performed a study which included eleven patients suffering from chronic periodontitis and have one deep intra bony defect in addition to the presence of one vital tooth that needs extraction ( 52 ). Pulp tissue was passed through 50-µm filters in presence of collagen sponge scaffold and was followed by transplantation in the bony defects caused by periodontal disease. Both clinical and radiographic evaluations confirmed the efficacy of this therapeutic intervention. Periodontal examination, attachment level, and probe depth showed improved results in addition to significant stability of the gingival margin. Moreover, radiographic analysis demonstrated bone regeneration.

Regeneration of mandibular bony defects

The first clinical study using DPSCs for oro-maxillo-facial bone regeneration was conducted in 2009 ( 53 ). Patients in this study suffered from extreme bone loss following extraction of third molars. A bio-complex composed of DPSCs cultured on collagen sponge scaffolds was applied to the affected sites. Vertical repair of the damaged area with complete restoration of the periodontal tissue was demonstrated six months after the treatment. Three years later, the same group published a report evaluating the stability and quality of the regenerated bone after DPSCs transplantation ( 54 ). Histological and advanced holotomography demonstrated that newly formed bone was uniformly vascularized. However, it was of compact type, rather than a cancellous type which is usually the type of bone in this region.

Stem cells for treatment of Sjögren’s syndrome

Sjögren’s syndrome (SS) is a systemic autoimmune disease marked by dry mouth and eyes. A novel therapeutic approach for SS. utilizing the infusion of MSCs in 24 patients was reported by Xu et al. in 2012 ( 55 ). The strategy behind this treatment was based on the immunologic regulatory functions of MSCs. Infused MSCs migrated toward the inflammatory sites in a stromal cell-derived factor-1-dependent manner. Results reported from this clinical trial demonstrated suppressed autoimmunity with subsequent restoration of salivary gland secretion in SS patients.

Stem cells and tissue banks

The ability to bank autologous stem cells at their most potent state for later use is an essential adjuvant to stem cell-based therapies. In order to be considered valid, any novel stem cell-based therapy should be as effective as the routine treatment. Thus, when appraising a type of stem cells for application in cellular therapies, issues like immune rejection must be avoided and at the same time large numbers of stem cells must be readily available before clinical implementation. iPSCs theoretically possess the ability to proliferate unlimitedly which pose them as an attractive source for use in cell-based therapies. Unlike, adult stem cells iPSCs ability to propagate does not decrease with time ( 22 ). Recently, California Institute for Regenerative Medicine (CIRM) has inaugurated an iPSCs repository to provide researchers with versatile iPSCs cell lines in order to accelerate stem cell treatments through studying genetic variation and disease modeling. Another important source for stem cells banking is the umbilical cord. Umbilical cord is immediately cryopreserved after birth; which permits stem cells to be successfully stored and ready for use in cell-based therapies for incurable diseases of a given individuals. However, stem cells of human exfoliated deciduous teeth (SHEDs) are more attractive as a source for stem cell banking. These cells have the capacity to differentiate into further cell types than the rest of the adult stem cells ( 56 ). Moreover, procedures involving the isolation and cryopreservation of these cells are un-complicated and not aggressive. The most important advantage of banking SHEDs is the insured autologous transplant which avoids the possibility of immune rejection ( 57 ). Contrary to cord blood stem cells, SHEDs have the ability to differentiate into connective tissues, neural and dental tissues ( 58 ) Finally, the ultimate goal of stem cell banking, is to establish a repository of high-quality stem cell lines derived from many individuals for future use in therapy.

Current regulatory guidelines for stem cell-based therapies

With the increased number of clinical trials employing stem cells as therapeutic approaches, the need for developing regulatory guidelines and standards to ensure patients safety is becoming more and more essential. However, the fact that stem cell therapy is rather a new domain makes it subject to scientific, ethical and legal controversies that are yet to be regulated. Leading countries in the field have devised guidelines serving that purpose. Recently, the Food and Drug Administration (FDA) has released regulatory guidelines to ensure that these treatments are safe and effective ( 59 ). These guidelines state that; treatments involving stem cells that have been minimally manipulated and are intended for homogeneous use do not require premarket approval to come into action and shall only be subjected to regulatory guidelines against disease transmission. In 2014, a radical regulatory reform in Japan occurred with the passing of two new laws that permitted conditional approval of cell-based treatments following early phase clinical trials on the condition that clinical safety data are provided from at least ten patients. These laws allow skipping most of the traditional criteria of clinical trials in what was described as “fast track approvals” and treatments were classified according to risk ( 60 ). To date, the treatments that acquired conditional approval include those targeting; spinal-cord injury, cardiac disease and limb ischemia ( 61 ). Finally, regulatory authorities are now demanding application of standardization and safety regulations protocols for cellular products, which include the use of Xeno-free culture media, recombinant growth factors in addition to “Good Manufacturing Practice” (GMP) culture supplies.

Challenges & ethical issues facing stem cell-based therapies

Stem cell-based therapies face many obstacles that need to be urgently addressed. The most persistent concern is the ethical conflict regarding the use of ESCs. As previously mentioned, ESCs are far superior regarding their potency; however, their derivation requires destruction human embryos. True, the discovery of iPSCs overcame this concern; nevertheless, iPSCs themselves currently face another ethical controversy of their own which addresses their unlimited capacity of differentiation with concerns that these cells could one day be applied in human cloning. The use of iPSCs in therapy is still considered a high-risk treatment modality, since transplantation of these cells could induce tumor formation. Such challenge is currently addressed through developing optimized protocols to ensure their safety in addition to developing global clinical-grade iPSCs cell lines before these cells are available for clinical use ( 61 ). As for MSCs, these cells have been universally considered safe, however continuous monitoring and prolonged follow-up should be the focus of future research to avoid the possibility of tumor formation after treatments ( 62 ). Finally, it could be postulated that one of the most challenging ethical issues faced in the field of stem cell-based therapies at the moment, is the increasing number of clinics offering unproven stem cell-based treatments. Researchers are thus morally obligated to ensure that ethical considerations are not undermined in pursuit of progress in clinical translation.

Conclusions

Stem cell therapy is becoming a tangible reality by the day, thanks to the mounting research conducted over the past decade. With every research conducted the possibilities of stem cells applications increased in spite of the many challenges faced. Currently, progress in the field of stem cells is very promising with reports of clinical success in treating various diseases like; neurodegenerative diseases and macular degeneration progressing rapidly. iPSCs are conquering the field of stem cells research with endless possibilities of treating diseases using patients own cells. Regeneration of dental and periodontal tissues using MSCs has made its way to the clinic and soon enough will become a valid treatment. Although, challenges might seem daunting, stem cell research is advancing rapidly and cellular therapeutics is soon to be applicable. Fortunately, there are currently tremendous efforts exerted globally towards setting up regulatory guidelines and standards to ensure patients safety. In the near future, stem cell-based therapies shall significantly impact human health.

Acknowledgments

Funding: None.

Ethical Statement: The author is accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/ .

Conflicts of Interest: The author has completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/sci-2020-001 ). The author has no conflicts of interest to declare.

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Institute for Basic Biomedical Sciences

Stem cell research at johns hopkins institute of basic biomedical sciences.

Researchers at the IBBS are studying stem cells to figure out how they can give rise to a complex body, how cells of the body can revert back to stem cells and how this knowledge can be used to develop therapies for diseases and injuries.

Stem cells are cells that don’t have an identity but have the potential to develop into many types of cells for many purposes, liking building a complete organism, healing a wound or replacing old, worn-out cells in a tissue.

Embryonic stem cells can become any of the cells in the body and can form entire animals.

Not all stem cells come from embryos, adult stem cells are found throughout the body too. These cells don’t have the ability to become any cell in the body, but can transform into many different cell types. For instance, there are stem cells in our bone marrow that can become fat cells, cartilage cells or bone cells, but they can’t become eye cells or skin cells. Researchers have also figured out how to make adult cells, like a skin cell, turn back into cells with the properties of embryonic stem cells, called induced pluripotent stem cells or iPS cells for short.

Erika Matunis ,  in the Department of Cell Biology , studies in fruit flies how testis stem cells decide to stay stem cells and not become other cell types, like sperm. She has also discovered how cells that are turning into other cell types can revert back to stem cells if the permanent reservoir of stem cells is depleted and she is exploring the mechanism of how this happens. Her research, learning more about the most fundamental aspects of stem cell biology, helps all stem cell researchers better understand the cells they work with.

Jennifer Elisseeff ,  of the Department of Biomedical Engineering , studies the differences between embryonic stem cells and adult stem cells. She has found that embryonic stem cells are better at forming new tissues, whereas adult stem cells are better at secreting therapeutic molecules that promote healing of damaged tissue. Elisseeff is particularly interested in the factors released by stem cells that can help a tissue heal. She uses this information in the development of biosynthetic (part-natural and part-man-made) materials used for therapies. One of the materials her lab has developed is a bio-adhesive—essentially a glue that can be used in the body that is made of part synthetic and part natural components. The glue is used in conjunction with stitches to help prevent leakage of blood or fluids, but it’s flexible enough to allow cells to move in and heal the incision. Also, Elisseeff is collaborating with the military to develop a treatment for soft tissue facial reconstruction for people who have suffered severe trauma. They are developing tissue blueprints that can be transplanted in the face—or any other place in the body for that matter— that would allow a person’s own cells to move into a region to heal and restructure the tissue.

Warren Grayson ,  of the Department of Biomedical Engineering , takes stem cells from fat and bone marrow as well as stem cells that have the potential to become many different cell types, known as pluripotent stem cells, and coaxes them to regenerate bone or skeletal muscle in the lab. He does this by incubating stem cells in biosynthetic structures to give the cells a structured three-dimensional volume to grow in, and then places these either in bioreactors that provide heat, nutrients, movement, mechanical stress or control of any other condition like oxygen concentration to guide the stem cell to become a specific cell type or within a defect in animals to study the regenerative process. He hopes to one day be able to take a person’s own stem cells and grow tissues, like bone or muscle, to be implanted into their body to replace damaged tissue. Using a person’s own cells and tissues will reduce the likelihood that the transplanted tissue will be rejected by the immune system.

Related Links :  Stem Cell Research at Johns Hopkins

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Ethics of Stem Cell Research

Human embryonic stem cell (HESC) research offers much hope for alleviating the human suffering brought on by the ravages of disease and injury. HESCs are characterized by their capacity for self-renewal and their ability to differentiate into all types of cells of the body. The main goal of HESC research is to identify the mechanisms that govern cell differentiation and to turn HESCs into specific cell types that can be used for treating debilitating and life-threatening diseases and injuries.

Despite the tremendous therapeutic promise of HESC research, the research has met with heated opposition because the harvesting of HESCs involves the destruction of the human embryo. HESCs are derived in vitro around the fifth day of the embryo’s development (Thomson et al . 1998). A typical day-5 human embryo consists of 200–250 cells, most of which comprise the trophoblast, which is the outermost layer of the blastocyst. HESCs are harvested from the inner cell mass of the blastocyst, which consists of 30–34 cells. The derivation of HESC cultures requires the removal of the trophoblast. This process of disaggregating the blastocyst’s cells eliminates its potential for further development. Opponents of HESC research argue that the research is morally impermissible because it involves the unjust killing of innocent human beings.

Scientists recently succeeded in converting adult human skin cells into cells that appear to have the properties of HESCs by activating four genes in the adult cells (Takahashi et al . 2007; Yu et al . 2007). The reprogrammed cells—“induced pluripotent stem cells” (iPSCs)—could ultimately eliminate the need for HESCs. However, at present, the consensus in the scientific community is that both HESC and iPSC research should be pursued, as we do not yet know whether iPSCs have the same potential as HESCs or whether it is safe to transplant them into humans. Thus, the controversies around HESC research will continue, at least in the near-term.

While the principal source of the controversy surrounding HESC research lies in competing views about the value of human embryonic life, the scope of ethical issues in HESC research is broader than the question of the ethics of destroying human embryos. It also encompasses questions about, among other things, whether researchers who use but do not derive HESCs are complicit in the destruction of embryos, whether there is a moral distinction between creating embryos for research purposes and creating them for reproductive ends, the permissibility of cloning human embryos to harvest HESCs, and the ethics of creating human/non-human chimeras. This entry provides an overview of all but the last two issues just listed; cloning and human-non-human chimeras are addressed in separate entries.

1.1 When does a human being begin to exist?

1.2 the moral status of human embryos, 1.3 the case of “doomed embryos”, 2. the ethics of using human embryonic stem cells in research, 3. the ethics of creating embryos for stem cell research and therapy, 4. stem cell-derived gametes, 5. stem cell-derived organoids, gastruloids, and synthetic embryos, cited resources, other resources, related entries, 1. the ethics of destroying human embryos for research.

The potential therapeutic benefits of HESC research provide strong grounds in favor of the research. If looked at from a strictly consequentialist perspective, it’s almost certainly the case that the potential health benefits from the research outweigh the loss of embryos involved and whatever suffering results from that loss for persons who want to protect embryos. However, most of those who oppose the research argue that the constraints against killing innocent persons to promote social utility apply to human embryos. Thus, as long as we accept non-consequentialist constraints on killing persons, those supporting HESC research must respond to the claim that those constraints apply to human embryos.

In its most basic form, the central argument supporting the claim that it is unethical to destroy human embryos goes as follows: It is morally impermissible to intentionally kill innocent human beings; the human embryo is an innocent human being; therefore it is morally impermissible to intentionally kill the human embryo. It is worth noting that this argument, if sound, would not suffice to show that all or even most HESC research is impermissible, since most investigators engaged in HESC research do not participate in the derivation of HESCs but instead use cell lines that researchers who performed the derivation have made available. To show that researchers who use but do not derive HESCs participate in an immoral activity, one would further need to establish their complicity in the destruction of embryos. We will consider this issue in section 2. But for the moment, let us address the argument that it is unethical to destroy human embryos.

A premise of the argument against killing embryos is that human embryos are human beings. The issue of when a human being begins to exist is, however, a contested one. The standard view of those who oppose HESC research is that a human being begins to exist with the emergence of the one-cell zygote at fertilization. At this stage, human embryos are said to be “whole living member[s] of the species homo sapiens … [which] possess the epigenetic primordia for self-directed growth into adulthood, with their determinateness and identity fully intact” (George & Gomez-Lobo 2002, 258). This view is sometimes challenged on the grounds that monozygotic twinning is possible until around days 14–15 of an embryo’s development (Smith & Brogaard 2003). An individual who is an identical twin cannot be numerically identical to the one-cell zygote, since both twins bear the same relationship to the zygote, and numerical identity must satisfy transitivity. That is, if the zygote, A, divides into two genetically identical cell groups that give rise to identical twins B and C, B and C cannot be the same individual as A because they are not numerically identical with each other. This shows that not all persons can correctly assert that they began their life as a zygote. However, it does not follow that the zygote is not a human being, or that it has not individuated. This would follow only if one held that a condition of an entity’s status as an individual human being is that it be impossible for it to cease to exist by dividing into two or more entities. But this seems implausible. Consider cases in which we imagine adult humans undergoing fission (for example, along the lines of Parfit’s thought experiments, where each half of the brain is implanted into a different body) (Parfit 1984). The prospect of our going out of existence through fission does not pose a threat to our current status as distinct human persons. Likewise, one might argue, the fact that a zygote may divide does not create problems for the view that the zygote is a distinct human being.

There are, however, other grounds on which some have sought to reject that the early human embryo is a human being. According to one view, the cells that comprise the early embryo are a bundle of homogeneous cells that exist in the same membrane but do not form a human organism because the cells do not function in a coordinated way to regulate and preserve a single life (Smith & Brogaard 2003, McMahan 2002). While each of the cells is alive, they only become parts of a human organism when there is substantial cell differentiation and coordination, which occurs around day-16 after fertilization. Thus, on this account, disaggregating the cells of the 5-day embryo to derive HESCs does not entail the destruction of a human being.

This account is subject to dispute on empirical grounds. That there is some intercellular coordination in the zygote is revealed by the fact that the development of the early embryo requires that some cells become part of the trophoblast while others become part of the inner cell mass. Without some coordination between the cells, there would be nothing to prevent all cells from differentiating in the same direction (Damschen, Gomez-Lobo and Schonecker 2006). The question remains, though, whether this degree of cellular interaction is sufficient to render the early human embryo a human being. Just how much intercellular coordination must exist for a group of cells to constitute a human organism cannot be resolved by scientific facts about the embryo, but is instead an open metaphysical question (McMahan 2007a).

Suppose that the 5-day human embryo is a human being. On the standard argument against HESC research, membership in the species Homo sapiens confers on the embryo a right not to be killed. This view is grounded in the assumption that human beings have the same moral status (at least with respect to possessing this right) at all stages of their lives.

Some accept that the human embryo is a human being but argue that the human embryo does not have the moral status requisite for a right to life. There is reason to think that species membership is not the property that determines a being’s moral status. We have all been presented with the relevant thought experiments, courtesy of Disney, Orwell, Kafka, and countless science fiction works. The results seem clear: we regard mice, pigs, insects, aliens, and so on, as having the moral status of persons in those possible worlds in which they exhibit the psychological and cognitive traits that we normally associate with mature human beings. This suggests that it is some higher-order mental capacity (or capacities) that grounds the right to life. While there is no consensus about the capacities that are necessary for the right to life, some of the capacities that have been proposed include reasoning, self-awareness, and agency (Kuhse & Singer 1992, Tooley 1983, Warren 1973).

The main difficulty for those who appeal to such mental capacities as the touchstone for the right to life is that early human infants lack these capacities, and do so to a greater degree than many of the nonhuman animals that most deem it acceptable to kill (Marquis 2002). This presents a challenge for those who hold that the non-consequentialist constraints on killing human children and adults apply to early human infants. Some reject that these constraints apply to infants, and allow that there may be circumstances where it is permissible to sacrifice infants for the greater good (McMahan 2007b). Others argue that, while infants do not have the intrinsic properties that ground a right to life, we should nonetheless treat them as if they have a right to life in order to promote love and concern towards them, as these attitudes have good consequences for the persons they will become (Benn 1973, Strong 1997).

Some claim that we can reconcile the ascription of a right to life to all humans with the view that higher order mental capacities ground the right to life by distinguishing between two senses of mental capacities: “immediately exercisable” capacities and “basic natural” capacities. (George and Gomez-Lobo 2002, 260). According to this view, an individual’s immediately exercisable capacity for higher mental functions is the actualization of natural capacities for higher mental functions that exist at the embryonic stage of life. Human embryos have a “rational nature,” but that nature is not fully realized until individuals are able to exercise their capacity to reason. The difference between these types of capacity is said to be a difference between degrees of development along a continuum. There is merely a quantitative difference between the mental capacities of embryos, fetuses, infants, children, and adults (as well as among infants, children, and adults). And this difference, so the argument runs, cannot justify treating some of these individuals with moral respect while denying it to others.

Given that a human embryo cannot reason at all, the claim that it has a rational nature has struck some as tantamount to asserting that it has the potential to become an individual that can engage in reasoning (Sagan & Singer 2007). But an entity’s having this potential does not logically entail that it has the same status as beings that have realized some or all of their potential (Feinberg 1986). Moreover, with the advent of cloning technologies, the range of entities that we can now identify as potential persons arguably creates problems for those who place great moral weight on the embryo’s potential. A single somatic cell or HESC can in principle (though not yet in practice) develop into a mature human being under the right conditions—that is, where the cell’s nucleus is transferred into an enucleated egg, the new egg is electrically stimulated to create an embryo, and the embryo is transferred to a woman’s uterus and brought to term. If the basis for protecting embryos is that they have the potential to become reasoning beings, then, some argue, we have reason to ascribe a high moral status to the trillions of cells that share this potential and to assist as many of these cells as we reasonably can to realize their potential (Sagan & Singer 2007, Savulescu 1999). Because this is a stance that we can expect nearly everyone to reject, it’s not clear that opponents of HESC research can effectively ground their position in the human embryo’s potential.

One response to this line of argument has been to claim that embryos possess a kind of potential that somatic cells and HESCs lack. An embryo has potential in the sense of having an “active disposition” and “intrinsic power” to develop into a mature human being (Lee & George 2006). An embryo can mature on its own in the absence of interference with its development. A somatic cell, on the other hand, does not have the inherent capacity or disposition to grow into a mature human being. However, some question whether this distinction is viable, especially in the HESC research context. While it is true that somatic cells can realize their potential only with the assistance of outside interventions, an embryo’s development also requires that numerous conditions external to it are satisfied. In the case of embryos that are naturally conceived, they must implant, receive nourishment, and avoid exposure to dangerous substances in utero . In the case of spare embryos created through in vitro fertilization—which are presently the source of HESCs for research—the embryos must be thawed and transferred to a willing woman’s uterus. Given the role that external factors—including technological interventions—play in an embryo’s realizing its potential, one can question whether there is a morally relevant distinction between an embryo’s and somatic cell’s potential and thus raise doubts about potentiality as a foundation for the right to life (Devolder & Harris 2007).

Some grant that human embryos lack the properties essential to a right to life, but hold that they possess an intrinsic value that calls for a measure of respect and places at least some moral constraints on their use: “The life of a single human organism commands respect and protection … no matter in what form or shape, because of the complex creative investment it represents and because of our wonder at the divine or evolutionary processes that produce new lives from old ones.” (Dworkin l992, 84). There are, however, divergent views about the level of respect embryos command and what limits exist on their use. Some opponents of HESC research hold that the treatment of human embryos as mere research tools always fails to manifest proper respect for them. Other opponents take a less absolutist view. Some, for example, deem embryos less valuable than more mature human beings but argue that the benefits of HESC research are too speculative to warrant the destruction of embryos, and that the benefits might, in any case, be achieved through the use of noncontroversial sources of stem cells (e.g., adult stem cells) (Holm 2003).

Many, if not most, who support the use of human embryos for HESC research would likely agree with opponents of the research that there are some circumstances where the use of human embryos would display a lack of appropriate respect for human life, for example, were they to be offered for consumption to contestants in a reality TV competition or destroyed for the production of cosmetics. But proponents of the research hold that the value of human embryos is not great enough to constrain the pursuit of research that may yield significant therapeutic benefits. Supporters of the research also frequently question whether most opponents of the research are consistent in their ascription of a high value to human embryos, as opponents generally display little concern about the fact that many embryos created for fertility treatment are discarded.

When spare embryos exist after fertility treatment, the individuals for whom the embryos were created typically have the option of storing for them for future reproductive use, donating them to other infertile couples, donating them to research, or discarding them. Some argue that as long as the decision to donate embryos for research is made after the decision to discard them, it is morally permissible to use them in HESC research even if we assume that they have the moral status of persons. The claim takes two different forms. One is that it is morally permissible to kill an individual who is about to be killed by someone else where killing that individual will help others (Curzer, H. 2004). The other is that researchers who derive HESCs from embryos that were slated for destruction do not cause their death. Instead, the decision to discard the embryos causes their death; research just causes the manner of their death (Green 2002).

Both versions of the argument presume that the decision to discard spare embryos prior to the decision to donate them to research entails that donated embryos are doomed to destruction when researchers receive them. There are two arguments one might marshal against this presumption. First, one who wants to donate embryos to research might first elect to discard them only because doing so is a precondition for donating them. There could be cases in which one who chooses the discard option would have donated the embryos to other couples were the research donation option not available. The fact that a decision to discard embryos is made prior to the decision to donate the embryos thus does not establish that the embryos were doomed to destruction before the decision to donate them to research was made. Second, a researcher who receives embryos could choose to rescue them, whether by continuing to store them or by donating them to infertile couples. While this would violate the law, the fact that it is within a researcher’s power to prevent the destruction of the embryos he or she receives poses problems for the claim that the decision to discard the embryos dooms them or causes their destruction.

Assume for the sake of argument that it is morally impermissible to destroy human embryos. It does not follow that all research with HESCs is impermissible, as it is sometimes permissible to benefit from moral wrongs. For example, there is nothing objectionable about transplant surgeons and patients benefiting from the organs of murder and drunken driving victims (Robertson 1988). If there are conditions under which a researcher may use HESCs without being complicit in the destruction of embryos, then those who oppose the destruction of embryos could support research with HESCs under certain circumstances.

Researchers using HESCs are clearly implicated in the destruction of embryos where they derive the cells themselves or enlist others to derive the cells. However, most investigators who conduct research with HESCs obtain them from an existing pool of cell lines and play no role in their derivation. One view is that we cannot assign causal or moral responsibility to investigators for the destruction of embryos from which the HESCs they use are derived where their “research plans had no effect on whether the original immoral derivation occurred.” (Robertson 1999). This view requires qualification. There may be cases in which HESCs are derived for the express purpose of making them widely available to HESC investigators. In such instances, it may be that no individual researcher’s plans motivated the derivation of the cells. Nonetheless, one might argue that investigators who use these cells are complicit in the destruction of the embryos from which the cells were derived because they are participants in a research enterprise that creates a demand for HESCs. For these investigators to avoid the charge of complicity in the destruction of embryos, it must be the case that the researchers who derived the HESCs would have performed the derivation in the absence of external demand for the cells (Siegel 2004).

The issue about complicity goes beyond the question of an HESC researcher’s role in the destruction of the particular human embryo(s) from which the cells he or she uses are derived. There is a further concern that research with existing HESCs will result in the future destruction of embryos: “[I]f this research leads to possible treatments, private investment in such efforts will increase greatly and the demand for many thousands of cell lines with different genetic profiles will be difficult to resist.” (U.S. Conference of Catholic Bishops 2001). This objection faces two difficulties. First, it appears to be too sweeping: research with adult stem cells and non-human animal stem cells, as well as general research in genetics, embryology, and cell biology could be implicated, since all of this research might advance our understanding of HESCs and result in increased demand for them. Yet, no one, including those who oppose HESC research, argues that we should not support these areas of research. Second, the claim about future demand for HESCs is speculative. Indeed, current HESC research could ultimately reduce or eliminate demand for the cells by providing insights into cell biology that enable the use of alternative sources of cells (Siegel 2004).

While it might thus be possible for a researcher to use HESCs without being morally responsible for the destruction of human embryos, that does not end the inquiry into complicity. Some argue that agents can be complicit in wrongful acts for which they are not morally responsible. One such form of complicity arises from an association with wrongdoing that symbolizes acquiescence in the wrongdoing (Burtchaell 1989). The failure to take appropriate measures to distance oneself from moral wrongs may give rise to “metaphysical guilt,” which produces a moral taint and for which shame is the appropriate response (May 1992). The following question thus arises: Assuming it is morally wrongful to destroy human embryos, are HESC researchers who are not morally responsible for the destruction of embryos complicit in the sense of symbolically aligning themselves with a wrongful act?

One response is that a researcher who benefits from the destruction of embryos need not sanction the act any more than the transplant surgeon who uses the organs of a murder or drunken driving victim sanctions the homicidal act (Curzer 2004). But this response is unlikely to be satisfactory to opponents of HESC research. There is arguably an important difference between the transplant case and HESC research insofar as the moral wrong associated with the latter (a) systematically devalues a particular class of human beings and (b) is largely socially accepted and legally permitted. Opponents of HESC research might suggest that the HESC research case is more analogous to the following kind of case: Imagine a society in which the practice of killing members of a particular racial or ethnic group is legally permitted and generally accepted. Suppose that biological materials obtained from these individuals subsequent to their deaths are made available for research uses. Could researchers use these materials while appropriately distancing themselves from the wrongful practice? Arguably, they could not. There is a heightened need to protest moral wrongs where those wrongs are socially and legally accepted. Attempts to benefit from the moral wrong in these circumstances may be incompatible with mounting a proper protest (Siegel 2003).

But even if we assume that HESC researchers cannot avoid the taint of metaphysical guilt, it is not clear that researchers who bear no moral responsibility for the destruction of embryos are morally obligated not to use HESCs. One might argue that there is a prima facie duty to avoid moral taint, but that this duty may be overridden for the sake of a noble cause.

Most HESCs are derived from embryos that were created for infertility treatment but that were in excess of what the infertile individual(s) ultimately needed to achieve a pregnancy. The HESCs derived from these leftover embryos offer investigators a powerful tool for understanding the mechanisms controlling cell differentiation. However, there are scientific and therapeutic reasons not to rely entirely on leftover embryos. From a research standpoint, creating embryos through cloning technologies with cells that are known to have particular genetic mutations would allow researchers to study the underpinnings of genetic diseases in vitro . From a therapeutic standpoint, the HESCs obtained from leftover IVF embryos are not genetically diverse enough to address the problem of immune rejection by recipients of stem cell transplants. (Induced pluripotent stem cells may ultimately prove sufficient for these research and therapeutic ends, since the cells can (a) be selected for specific genetic mutations and (b) provide an exact genetic match for stem cell recipients.) At present, the best way to address the therapeutic problem is through the creation of a public stem cell bank that represents a genetically diverse pool of stem cell lines (Faden et al . 2003, Lott & Savulescu 2007). This kind of stem cell bank would require the creation of embryos from gamete donors who share the same HLA-types (i.e., similar versions of the genes that mediate immune recognition and rejection).

Each of these enterprises has its own set of ethical issues. In the case of research cloning, some raise concerns, for example, that the perfection of cloning techniques for research purposes will enable the pursuit of reproductive cloning, and that efforts to obtain the thousands of eggs required for the production of cloned embryos will result in the exploitation of women who provide the eggs (President’s Council on Bioethics 2002, Norsigian 2005). With respect to stem cell banks, it is not practically possible to create a bank of HESCs that will provide a close immunological match for all recipients. This gives rise to the challenge of determining who will have biological access to stem cell therapies. We might construct the bank so that it provides matches for the greatest number of people in the population, gives everyone an equal chance of finding a match, or ensures that all ancestral/ethnic groups are fairly represented in the bank (Faden et al . 2003, Bok, Schill, & Faden 2004, Greene 2006).

There are, however, more general challenges to the creation of embryos for research and therapeutic purposes. Some argue that the creation of embryos for non-reproductive ends is morally problematic, regardless of whether they are created through cloning or in vitro fertilization. There are two related arguments that have been advanced to morally distinguish the creation of embryos for reproductive purposes from the creation of embryos for research and therapeutic purposes. First, each embryo created for procreative purposes is originally viewed as a potential child in the sense that each is a candidate for implantation and development into a mature human. In contrast, embryos created for research or therapies are viewed as mere tools from the outset (Annas, Caplan & Elias 1996, President’s Council on Bioethics 2002). Second, while embryos created for research and therapy are produced with the intent to destroy them, the destruction of embryos created for reproduction is a foreseeable but unintended consequence of their creation (FitzPatrick 2003).

One response to the first argument has been to suggest that we could, under certain conditions, view all research embryos as potential children in the relevant sense. If all research embryos were included in a lottery in which some of them were donated to individuals for reproductive purposes, all research embryos would have a chance at developing into mature humans (Devander 2005). Since those who oppose creating embryos for research would likely maintain their opposition in the research embryo lottery case, it is arguably irrelevant whether embryos are viewed as potential children when they are created. Of course, research embryos in the lottery case would be viewed as both potential children and potential research tools. But this is also true in the case of embryos created for reproductive purposes where patients are open to donating spare embryos to research.

As to the second argument, the distinction between intending and merely foreseeing harms is one to which many people attach moral significance, and it is central to the Doctrine of Double Effect. But even if one holds that this is a morally significant distinction, it is not clear that it is felicitous to characterize the destruction of spare embryos as an unintended but foreseeable side-effect of creating embryos for fertility treatment. Fertility clinics do not merely foresee that some embryos will be destroyed, as they choose to offer patients the option of discarding embryos and carry out the disposal of embryos when patients request it. Patients who elect that their embryos be discarded also do not merely foresee the embryos’ destruction; their election of that option manifests their intention that the embryos be destroyed. There is thus reason to doubt that there is a moral distinction between creating embryos for research and creating them for reproductive purposes, at least given current fertility clinic practices.

Recent scientific work suggests it is possible to derive gametes from human pluripotent stem cells. Researchers have generated sperm and eggs from mouse ESCs and iPSCs and have used these stem cell-derived gametes to produce offspring (Hayashi 2011; Hayashi 2012). While it may take several years before researchers succeed in deriving gametes from human stem cells, the research holds much promise for basic science and clinical application. For example, the research could provide important insights into the fundamental processes of gamete biology, assist in the understanding of genetic disorders, and provide otherwise infertile individuals a means of creating genetically related children. The ability to derive gametes from human stem cells could also reduce or eliminate the need for egg donors and thus help overcome concerns about exploitation of donors and the risks involved in egg retrieval. Nonetheless, the research gives rise to some controversial issues related to embryos, genetics, and assisted reproductive technologies (D. Mathews et al . 2009).

One issue arises from the fact that some research on stem cell-derived gametes requires the creation of embryos, regardless of whether one is using ESCs or iPSCs. To establish that a particular technique for deriving human gametes from stem cells produces functional sperm and eggs, it is necessary to demonstrate that the cells can produce an embryo. This entails the creation of embryos through in vitro fertilization. Since it would not be safe to implant embryos created during the early stages of the research, the likely disposition of the embryos is that they would be destroyed. In such instances, the research would implicate all of the moral issues surrounding the creation and destruction of embryos for research. However, the creation of embryos for research in this situation would not necessitate the destruction of the embryos, as it does when embryos are created to derive stem cell lines. One could in principle store them indefinitely rather than destroy them. This would still leave one subject to the objection that life is being created for instrumental purposes. But the force of the objection is questionable since it is not clear that this instrumental use is any more objectionable than the routine and widely accepted practice of creating excess IVF embryos in the reproductive context to increase the probability of generating a sufficient number of viable ones to produce a pregnancy.

Further issues emerge with the prospect of being able to produce large quantities of eggs from stem cells. As the capacity to identify disease and non-disease related alleles through preimplantation genetic diagnosis (PGD) expands, the ability to create large numbers of embryos would substantially increase the chances of finding an embryo that possesses most or all of the traits one wishes to select. This would be beneficial in preventing the birth of children with genetic diseases. But matters would become morally contentious if it were possible to select for non-disease characteristics, such as sexual orientation, height, superior intelligence, memory, and musical ability. One common argument against using PGD in this way is that it could devalue the lives of those who do not exhibit the chosen characteristics. Another concern is that employing PGD to select for non-disease traits would fail to acknowledge the “giftedness of life” by treating children as “objects of our design or products of our will or instruments of our ambition” rather accepting them as they are given to us (Sandel 2004, 56). There is additionally a concern about advances in genetics heightening inequalities where certain traits confer social and economic advantages and only the well-off have the resources to access the technology (Buchanan 1995). Of course, one can question whether the selection of non-disease traits would in fact lead to devaluing other characteristics, whether it would alter the nature of parental love, or whether it is distinct enough from currently permitted methods of gaining social and economic advantage to justify regulating the practice. Nonetheless, the capacity to produce human stem cell-derived gametes would make these issues more pressing.

There have been a number of recent scientific studies in which stem cells have, under certain in vitro culture conditions, self-organized into three-dimensional structures that resemble and recapitulate some of the functions of human organs (Lancaster & Knoblich 2014; Clevers 2016). These “organoids” have been established with human stem cells for a variety of organs, including, among others, the kidney, liver, gut, pancreas, retina, and brain. In addition to organoids, stem cells have been shown to self-organize into embryo-like structures in vitro . Human embryonic stem cells have formed structures – referred to as “gastruloids” – that bear some resemblance to embryos during gastrulation, which is the stage several days after implantation where the body plan and some tissues tissue types, including the central nervous system, start to develop (Warmflash et al. 2014; Deglincerti et al . 2016; Shahbazi 2016). Researchers have also combined mouse embryonic stem cells and trophoblast stem cells to create “synthetic embryos,” which have a structure akin to pre-implantation embryos (Rivron et al . 2018). Synthetic embryos have been shown to implant into the mouse uterus, though their potential to develop to term has not been demonstrated.

While these scientific advances offer promising avenues for better understanding human development and disease, they also raise some novel and challenging ethical issues. In the case of organoids, cerebral organoids raise the most vexing issues. Researchers have produced cerebral organoids with a degree of development similar to that of a few-months-old embryo, and have already used them to study how the Zika virus causes microcephaly in fetuses (Garcez et al . 2016). At present, there is some evidence that cerebral organoids may be able to receive afferent stimulations that produce simple sensations (Quadrato et al . 2017). However, they currently lack the kind of mature neural networks and sensory inputs and outputs essential to the development of cognition. If, through bioengineering, human cerebral organoids were to develop the capacity for cognition, that would provide grounds for ascribing an elevated moral status to them, and it would raise concomitant issues about our moral obligations towards them. In the nearer term, it is more likely that cerebral organoids will develop some degree of consciousness Assuming we have a shared understanding of consciousness (e.g., phenomenal consciousness), one challenge is to identify means of measuring the presence of consciousness, since a cerebral organoid cannot communicate its internal states (Lavazza & Massimini 2018). But even if we can verify that an organoid is conscious, there remains the question of the moral significance of consciousness (Shepherd 2018). There is debate over whether consciousness has intrinsic value (Lee 2018), and whether in some cases it is better for a conscious being to not possess it (Kahane & Savulescu 2009). Those who reject the intrinsic value and moral significance of consciousness might find the case of a conscious entity that has led a solely disembodied existence, emerges and persists in the absence of any social or cultural nexus, and lacks beliefs and desires, to be a paradigmatic case where the value of consciousness is doubtful.

With respect to gastruloids and synthetic embryos (if the latter are successfully produced with human stem cells), the central question is whether these entities are sufficiently like human embryos in their structure and functions to give rise to moral concerns about their use in research. Gastruloids do not possess all the characteristics of an embryo, as they do not form all of the embryonic tissues (e.g., they do not have the trophectoderm, which mediates the attachment to the uterus). At the same time, gastruloids may, with extra-embryonic tissues, achieve a developmental stage in which they manifest a whole body plan. Recall that one argument (discussed in Section 1.1 above) for rejecting that human embryos are human beings is that the cells that comprise the early embryo do not function in a coordinated way to regulate and preserve a single organism. Gastruloids can in principle operate with this higher level of coordination. While one may still reject that this characteristic of gastruloids confers human rights on them, their more advanced stage of development might ground reasonable claims for according them greater respect than embryos at an earlier stage. In the case of both gastruloids and human synthetic embryos, the possibility that they ultimately lack the potential to develop into mature human beings may be of significance in morally distinguishing them from normal human embryos. As noted previously (in section 1.2 above), one argument for ascribing a high moral status to human embryos and for distinguishing the potential of human embryos from the potential of somatic cells and embryonic stem cells is that embryos have an “active disposition” and “intrinsic power” to develop into mature humans on their own. If synthetic embryos and gastruloids do not possess this disposition and power, then those who oppose some forms of human embryo research might not object to the creation and use of human gastruloids and synthetic embryos for research.

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How to cite this entry . Preview the PDF version of this entry at the Friends of the SEP Society . Look up topics and thinkers related to this entry at the Internet Philosophy Ontology Project (InPhO). Enhanced bibliography for this entry at PhilPapers , with links to its database.

Other Internet Resources

• President’s Council on Bioethics, 2002, Human Cloning and Human Dignity: An Ethical Inquiry
• U.S. Conference of Catholic Bishops, 2001, Fact Sheet: President Bush’s Stem Cell Decision
• International Society for Stem Cell Research
• Stem Cell Resources from the American Association for the Advancement of Science
• Stem Cell Research and Applications , recommendations and findings from the AAAS and the Institute for Civil Society.
• Medline Plus: Stem Cells
• The Pew Forum on Religion and Public Life: Bioethics
• The Hinxton Group: An International Consortium on Stem Cell, Ethics, and Law

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Examining the ethics of embryonic stem cell research

Following the recent passage by both houses of Congress of the Stem Cell Research Enhancement Act of 2007, which would permit federal funding of research using donated surplus embryonic stem cells from fertility clinics, the president has once again threatened a veto.

Because neither the House nor the Senate had sufficient votes to override a presidential veto, it appears unlikely this new bill will be enacted into law, further stalling the pace of this research. “This bill crosses a moral line that I and others find troubling,” stated Bush, following the Senate’s vote.

SCL : What are th e main arguments for and against embryonic stem cell research? MS : Proponents argue that embryonic stem cell research holds great promise for understanding and curing diabetes, Parkinson’s disease, spinal cord injury, and other debilitating conditions. Opponents argue that the research is unethical, because deriving the stem cells destroys the blastocyst, an unimplanted human embryo at the sixth to eighth day of development. As Bush declared when he vetoed last year’s stem cell bill, the federal government should not support “the taking of innocent human life.”

It is surprising that, despite the extensive public debate—in Congress, during the 2004 and 2006 election campaigns, and on the Sunday morning talk shows—relatively little attention has been paid to the moral issue at the heart of the controversy: Are the opponents of stem cell research correct in their claim that the unimplanted human embryo is already a human being, morally equivalent to a person?

“It is important to be clear about the embryo from which stem cells are extracted. It is not implanted and growing in a woman’s uterus. It is not a fetus. It has no recognizable human features or form. It is, rather, a blastocyst, a cluster of 180 to 200 cells, growing in a petri dish, barely visible to the naked eye.”

SCL : What are the contradictions in Bush’s stance? MS : Before we address that, it is important to be clear about the embryo from which stem cells are extracted. It is not implanted and growing in a woman’s uterus. It is not a fetus. It has no recognizable human features or form.

It is, rather, a blastocyst, a cluster of 180 to 200 cells, growing in a petri dish, barely visible to the naked eye. Such blastocysts are either cloned in the lab or created in fertility clinics. The bill recently passed by Congress would fund stem cell research only on excess blastocysts left over from infertility treatments.

The blastocyst represents such an early stage of embryonic development that the cells it contains have not yet differentiated, or taken on the properties of particular organs or tissues—kidneys, muscles, spinal cord, and so on. This is why the stem cells that are extracted from the blastocyst hold the promise of developing, with proper coaxing in the lab, into any kind of cell the researcher wants to study or repair.

The moral and political controversy arises from the fact that extracting the stem cells destroys the blastocyst. It is important to grasp the full force of the claim that the embryo is morally equivalent to a person, a fully developed human being.

For those who hold this view, extracting stem cells from a blastocyst is as morally abhorrent as harvesting organs from a baby to save other people’s lives. This is the position of Senator Sam Brownback, Republican of Kansas, a leading advocate of the right-to-life position. In Brownback’s view, “a human embryo . . . is a human being just like you and me; and it deserves the same respect that our laws give to us all.

If Brownback is right, then embryonic stem cell research is immoral because it amounts to killing a person to treat other people’s diseases.

SCL : What is the basis for the belief that personhood begins at conception? MS : Some base this belief on the religious conviction that the soul enters the body at the moment of conception. Others defend it without recourse to religion, by the following line of reasoning: Human beings are not things. Their lives must not be sacrificed against their will, even for the sake of good ends, like saving other people’s lives. The reason human beings must not be treated as things is that they are inviolable. At what point do humans acquire this inviolability? The answer cannot depend on the age or developmental stage of a particular human life. Infants are inviolable, and few people would countenance harvesting organs for transplantation even from a fetus.

Every human being—each one of us—began life as an embryo. Unless we can point to a definitive moment in the passage from conception to birth that marks the emergence of the human person, we must regard embryos as possessing the same inviolability as fully developed human beings.

SCL : By this line of reasoning, human embryos are inviolable and should not be used for research, even if that research might save many lives. MS : Yes, but this argument can be challenged on a number of grounds. First, it is undeniable that a human embryo is “human life” in the biological sense that it is living rather than dead, and human rather than, say, bovine.

But this biological fact does not establish that the blastocyst is a human being, or a person. Any living human cell (a skin cell, for example) is “human life” in the sense of being human rather than bovine and living rather than dead. But no one would consider a skin cell a person, or deem it inviolable. Showing that a blastocyst is a human being, or a person, requires further argument.

Some try to base such an argument on the fact that human beings develop from embryo to fetus to child. Every person was once an embryo, the argument goes, and there is no clear, non-arbitrary line between conception and adulthood that can tell us when personhood begins. Given the lack of such a line, we should regard the blastocyst as a person, as morally equivalent to a fully developed human being.

SCL : What is the flaw in this argument? MS : Consider an analogy: although every oak tree was once an acorn, it does not follow that acorns are oak trees, or that I should treat the loss of an acorn eaten by a squirrel in my front yard as the same kind of loss as the death of an oak tree felled by a storm. Despite their developmental continuity, acorns and oak trees differ. So do human embryos and human beings, and in the same way. Just as acorns are potential oaks, human embryos are potential human beings.

The distinction between a potential person and an actual one makes a moral difference. Sentient creatures make claims on us that nonsentient ones do not; beings capable of experience and consciousness make higher claims still. Human life develops by degrees.

SCL : Yet there are people who disagree that life develops by degrees, and believe that a blastocyst is a person and, therefore, morally equivalent to a fully developed human being. MS : Certainly some people hold this belief. But a reason to be skeptical of the notion that blastocysts are persons is to notice that many who invoke it do not embrace its full implications.

President Bush is a case in point. In 2001, he announced a policy that restricted federal funding to already existing stem cell lines, so that no taxpayer funds would encourage or support the destruction of embryos. And in 2006, he vetoed a bill that would have funded new embryonic stem cell research, saying that he did not want to support “the taking of innocent human life.”

“The distinction between a potential person and an actual one makes a moral difference. Sentient creatures make claims on us that nonsentient ones do not; beings capable of experience and consciousness make higher claims still. Human life develops by degrees.”

But it is a striking feature of the president’s position that, while restricting the funding of embryonic stem cell research, he has made no effort to ban it. To adapt a slogan from the Clinton administration, the Bush policy might be summarized as “don’t fund, don’t ban.” But this policy is at odds with the notion that embryos are human beings.

SCL : If Bush’s policy were consistent with his stated beliefs, how, in your opinion, would it differ from his current “don’t fund, don’t ban” policy? MS : If harvesting stem cells from a blastocyst were truly on a par with harvesting organs from a baby, then the morally responsible policy would be to ban it, not merely deny it federal funding.

If some doctors made a practice of killing children to get organs for transplantation, no one would take the position that the infanticide should be ineligible for federal funding but allowed to continue in the private sector. In fact, if we were persuaded that embryonic stem cell research were tantamount to infanticide, we would not only ban it but treat it as a grisly form of murder and subject scientists who performed it to criminal punishment.

SCL : Couldn’t it be argued, in defense of the president’s policy, that Congress would be unlikely to enact an outright ban on embryonic stem cell research? MS : Perhaps. But this does not explain why, if the president really considers embryos to be human beings, he has not at least called for such a ban, nor even called upon scientists to stop doing stem cell research that involves the destruction of embryos. In fact, Bush has cited the fact that “there is no ban on embryonic stem cell research” in touting the virtues of his “balanced approach.”

The moral oddness of the Bush “don’t fund, don’t ban” position confused even his spokesman, Tony Snow. Last year, Snow told the White House press corps that the president vetoed the stem cell bill because he considered embryonic stem cell research to be “murder,” something the federal government should not support. When the comment drew a flurry of critical press attention, the White House retreated. No, the president did not believe that destroying an embryo was murder. The press secretary retracted his statement, and apologized for having “overstated the president’s position.”

How exactly the spokesman had overstated the president’s position is unclear. If embryonic stem cell research does constitute the deliberate taking of innocent human life, it is hard to see how it differs from murder. The chastened press secretary made no attempt to parse the distinction. His errant statement that the president considered embryo destruction to be “murder” simply followed the moral logic of the notion that embryos are human beings. It was a gaffe only because the Bush policy does not follow that logic.

SCL : You have stated that the president’s refusal to ban privately funded embryonic stem cell research is not the only way in which his policies betray the principle that embryos are persons. How so? MS : In the course of treating infertility, American fertility clinics routinely discard thousands of human embryos. The bill that recently passed in the Senate would fund stem cell research only on these excess embryos, which are already bound for destruction. (This is also the position taken by former governor Mitt Romney, who supports stem cell research on embryos left over from fertility clinics.) Although Bush would ban the use of such embryos in federally funded research, he has not called for legislation to ban the creation and destruction of embryos by fertility clinics.

SCL : If embryos are morally equivalent to fully developed human beings, doesn’t it then follow that allowing fertility clinics to discard thousands of embryos is condoning mass murder? MS : It does. If embryos are human beings, to allow fertility clinics to discard them is to countenance, in effect, the widespread creation and destruction of surplus children. Those who believe that a blastocyst is morally equivalent to a baby must believe that the 400,000 excess embryos languishing in freezers in U.S. fertility clinics are like newborns left to die by exposure on a mountainside. But those who view embryos in this way should not only be opposing embryonic stem cell research; they should also be leading a campaign to shut down what they must regard as rampant infanticide in fertility clinics.

Some principled right-to-life opponents of stem cell research meet this test of moral consistency. Bush’s “don’t fund, don’t ban” policy does not. Those who fail to take seriously the belief that embryos are persons miss this point. Rather than simply complain that the president’s stem cell policy allows religion to trump science, critics should ask why the president does not pursue the full implications of the principle he invokes.

If he does not want to ban embryonic stem cell research, or prosecute stem cell scientists for murder, or ban fertility clinics from creating and discarding excess embryos, this must mean that he does not really consider human embryos as morally equivalent to fully developed human beings after all.

But if he doesn’t believe that embryos are persons, then why ban federally funded embryonic stem cell research that holds promise for curing diseases and saving lives? 

Embryonic Stem Cell Research An Ethical Dilemma

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Introduction

In November 1998, two teams of U.S. scientists confirmed successful isolation and growth of stems cells obtained from human fetuses and embryos. Since then, research that utilizes human embryonic cells has been a widely debated, controversial ethical issue. Human embryonic cells possess the ability to become stem cells, which are used in medical research due to two significant features. First, they are unspecialized cells, meaning they can undergo cell division and renew themselves even with long periods of inactivity. Secondly, stem cells are pluripotent, with the propensity to be induced to become specified tissue or any “organ-specific cells with special functions” depending on exposure to experimental or physiologic conditions, as well as undergo cell division and become cell tissue for different organs.

The origin of stem cells themselves encapsulates the controversy: embryonic stem cells, originate from the inner cell mass of a blastocyst, a 5-day pre-implantation embryo. The principal argument for embryonic stem cell research is the potential benefit of using human embryonic cells to examine or treat diseases as opposed to somatic (adult) stem cells. Thus, advocates believe embryonic stem cell research may aid in developing new, more efficient treatments for severe diseases and ease the pain and suffering of numerous people. However, those that are against embryonic stem cell research believe that the possibility of scientific benefits of research do not outweigh the immoral action of tampering with the natural progression of a fetal development and interfering with the human embryo’s right to live. In light of these two opposing views, should embryonic stem cells be used in research? It is not ethically permissible to destroy human embryonic life for medical progress.

Personhood and the Scientific Questionability of Embryonic Stem Cell Research

The ethics behind embryonic stem cell research are controversial because the criteria of ‘personhood’ is “notoriously unclear.” Personhood is defined as the status of being a person, entitled to “moral rights and legal protections” that are higher than living things that are not classified as persons. Thus, this issue touches on existential questions such as: When does life begin? and What is the moral status that an embryo possesses? There is a debate on when exactly life begins in embryonic development and when the individual receives moral status. For example, some may ascribe life starting from the moment of fertilization, others may do so after implantation or the beginning of organ function. However, since the “zygote is genetically identical to the embryo,” which is also genetically identical to the fetus, and, by extension, identical to the baby, inquiring the beginning of personhood can lead to an occurrence of the Sorites paradox, also acknowledged as “the paradox of the heap.”

The paradox of the heap arises from vague predicates in philosophy. If there is a heap of sand and a grain is taken away from that heap one by one, at what point will it no longer be considered a heap – what classifies it as a heap? The definition of life is similarly arbitrary. When, in the development of a human being, is an embryo considered a person with moral standing? The complexity of the ethics of embryonic stem cell research, like the Sorites paradox, demonstrates there is no single, correct way to approach a problem; thus, there may be multiple different solutions that are acceptable. Whereas the definition of personhood cannot be completely resolved on a scientific basis, it serves a central role in the religious, political, and ethical differences within the field of embryonic stem cell research. Some ethicists attempt to determine what or who is a person by “setting boundaries” (Baldwin & Capstick, 2007).

Utilizing a functionalist approach, supporters of embryonic stem cell research argue that to qualify as a person, the individual must possess several indicators of personhood, including capacity, self-awareness, a sense of time, curiosity, and neo-cortical function. Proponents argue that a human embryo lacks these criteria, thereby is not considered a person and thus, does not have life and cannot have a moral status. Supporters of stem cell research believe a fertilized egg is just a part of another person’s body until the cell mass can survive on its own as a viable human. They further support their argument by noting that stem cell research uses embryonic tissue before its implantation into the uterine wall. Researchers invent the term “pre-embryo” to distinguish a pre-implantation state in which the developing cell mass does not have the full respects of an embryo in later stages of embryogenesis to further support embryonic stem cell research. Based on this reductionist view of life and personhood, utilitarian advocates argue that the result of the destruction of human embryos to harvest stem cells does not extinguish a life. Further, scientists state that any harm done is outweighed by the potential alleviation of the suffering enduring by tremendous numbers of people with varying diseases. This type of reasoning, known as Bentham’s Hedonic (moral) calculus, suggests that the potential good of treating or researching new cures for ailments such as Alzheimer’s disease, Parkinson’s disease, certain cancers, etc. outweighs any costs and alleviate the suffering of persons with those aliments. Thus, the end goal of stem cell use justifies sacrificing human embryos to produce stem cells, even though expending life is tantamount to murder. Opponents of embryonic stem cell research would equate the actions done to destroy the embryos as killing. Killing, defined as depriving their victims of life, will therefore reduce their victims to mere means to their own ends. Therefore, this argument touches on the question: if through the actions of embryotic stem cell research is “morally indistinguishable from murder?” (Outka, 2013). The prohibition of murder extends to human fetuses and embryos considering they are potential human beings. And, because both are innocent, a fetus being aborted and an embryo being disaggregated are direct actions with the intention of killing. Violating the prohibition of murder is considered an intolerable end. We should not justify this evil even if it achieves good. Under the deontological approach, “whether a situation is good or bad depends on whether the action that brought it about was right or wrong,” hence the ends do not justify the means. Therefore, under this feeble utilitarian approach, stem cell research proceeds at the expense of human life than at the expense of personhood.

One can reject the asserted utilitarian approach to stem cell research as a reductionist view of life because the argument fails to raise ethical concerns regarding the destruction embryonic life for the possibility of developing treatments to end certain diseases. The utilitarian approach chooses potential benefits of stem cell research over the physical lives of embryos without regard to the rights an embryo possesses. Advocates of embryonic stem cell research claim this will cure diseases but there is a gap in literature that confirms how many diseases these cells can actually cure or treat, what diseases, and how many people will actually benefit. Thus, killing human embryos for the potentiality of benefiting sick people is not ethically not ethically permissible.

Where the argument of personhood is concerned, the development from a fertilized egg (embryo) to a baby is a continuous process. Any effort to determine when personhood begins is arbitrary. If a newborn baby is a human, then surely a fetus just before birth is a human; and, if we extend a few moments before that point, we would still have a human, and so on all the way back to the embryo and finally to the zygote. Although an embryo does not possess the physiognomies of a person, it will nonetheless become a person and must be granted the respect and dignity of a person. Thus, embryotic stem cell research violates the Principle of “Full Human Potential,” which states: “Every human being […] deserves to be valued according to the full level of human development, not according to the level of development currently achieved.” As technology advances, viability outside the womb inches ever closer to the point of inception, making the efforts to identify where life begins after fertilization ineffectual. To complicate matters, as each technological innovation arrives, stem-cell scientists will have to re-define the start of life as many times as there are new technological developments, an exhausting and never-ending process that would ultimately lead us back to moment of fertilization. Because an embryo possesses all the necessary genetic information to develop into a human being, we must categorically state that life begins at the moment of conception. There is a gap in literature that deters the formation of a clear, non-arbitrary indication of personhood between conception and adulthood. Considering the lack of a general consensus of when personhood begins, an embryo should be referred to as a person and as morally equivalent to a fully developed human being.

Having concluded that a human embryo has the moral equivalent of a fully-fledged human being, this field of research clearly violates the amiable rights of personhood, and in doing so discriminates against pre-born persons. Dr. Eckman asserts that “every human being has a right to be protected from discrimination.” Thus, every human, and by extension every embryo, has the right to life and should not be discriminated against their for “developmental immaturity.” Therefore, the field of embryonic stem cell research infringes upon the rights and moral status of human embryos.

Principle of Beneficence in Embryonic Stem Cell Research

The destruction of human embryos for research is not ethically permissible because the practice violates the principle of beneficence depicted in the Belmont Report, which outlines the basic ethical principles and guidelines owed to human subjects involved in research. Stem cell researchers demonstrate a lack of respect for the autonomy and welfare of the human embryos sacrificed in stem cell research.

While supporters of embryonic stem cell research under the utilitarian approach argue the potential benefits of the research, the utilitarian argument however violates the autonomy of the embryo and its human rights, as well as the autonomy of the embryo donors and those that are Pro-Life. Though utilitarian supporters argue on the basis of rights, they exclusively refer to the rights of sick individuals. However, they categorically ignore the rights of embryos that they destroy to obtain potential disease curing stem cells. Since an embryo is regarded as a human being with morally obligated rights, the Principle of Beneficence is violated, and the autonomy and welfare of the embryo is not respected due to the destruction of an embryo in stem cell research. Killing embryos to obtain stem cells for research fails to treat embryos as ends in an of themselves. Yet, every human ought to be regarded as autonomous with rights that are equal to every other human being. Thus, the welfare of the embryo is sacrificed due to lack of consent from the subject.

The Principle of Beneficence is violated when protecting the reproductive interests of women in infertility treatment, who are dependent on the donations of embryos to end their infertility. Due to embryonic stem cell research, these patients’ “prospects of reproductive success may be compromised” because there are fewer embryos accessible for reproductive purposes. The number of embryos necessary to become fully developed and undergo embryonic stem cell research will immensely surpass the number of available frozen embryos in fertility clinic, which also contributes to the lack of embryos available for women struggling with infertility. Therefore, the basis of this research violates women’s reproductive autonomy, thus violating the Principle of Beneficence.

It is also significant to consider the autonomy and welfare of the persons involved. The autonomous choice to donate embryos to research necessitates a fully informed, voluntary sanction of the patient(s), which poses difficulty due to the complexity of the human embryonic stem cell research. To use embryos in research, there must be a consensus of agreement from the mother and father whose egg and sperm produced the embryo. Thus, there has to be a clear indication between the partners who has the authority or custody of the embryos, as well as any “third party donors” of gametes that could have been used to produce the embryo because these parties’ intentions for those gametes may solely have been for reproductive measures only. Because the researchers holding “dispositional authority” over the embryos may exchange cell lines and its derivatives (i.e., genetic material and information) with other researchers, they may misalign interests with the persons whose gametes are encompassed within the embryo. This mismatch of intent raises complications in confidentiality and autonomy.

Lastly, more ethical complications arise in the research of embryonic stem cells because of the existence viable alternatives that to not destroy human embryos. Embryonic stem cells themselves pose as a higher health risk than adult stem cells. Embryonic stem cells have a higher risk of causing tumor development in the patient’s body once the cells are implanted due to their abilities to proliferate and differentiate. Embryonic stem cells also have a high risk of immunorejection, where a patient’s immune system rejects the stem cells. Since the embryonic stem cells are derived from embryos that underwent in vitro fertilization, when implanted in the body, the stem cell’s marker molecules will not be recognized by the patient’s body, resulting in the destruction of the stem cells as a defensive response to protect the body (Cahill, 2002). With knowledge of embryonic stem cells having higher complications than the viable adult stem cells continued use of embryonic stem cells violates the Principle of Beneficence not only for the embryos but for the health and safety of the patients treated with stem cells. Several adult stem cell lines (“undifferentiated cells found throughout the body”) exist and are widely used cell research. The use of adult stem cells represents research that does not treat human beings as means to themselves, thus, complying with the Principle of Beneficence. This preferable alternative considers the moral obligation to discover treatments, and cures for life threating diseases while avoiding embryo destruction.

It is not ethically permissible to destroy human embryonic life for medical progress due to the violations of personhood and human research tenets outlined in the Belmont Report. It is significant to understand the ethical implications of this research in order to respect the autonomy, welfare, beneficence, and basic humanity afforded to all parties involved. Although embryonic stem cell research can potentially provide new medical advancements to those in need, the harms outweigh the potential, yet ill-defined benefits. There are adult stem cell alternatives with equivalent viability that avoid sacrificing embryos. As society further progresses, humans must be cautious of compromising moral principles that human beings are naturally entitled to for scientific advancements. There are ethical boundaries that are crossed when natural processes of life are altered or manipulated. Though there are potential benefits to stem cell research, these actions are morally and ethically questionable. Thus, it is significant to uphold ethical standards when practicing research to protect the value of human life.

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Article Contents

Introduction, what are (embryonic) stem cells, potential applications of hes cells and state‐of‐the‐art, ethical exploration, the status of hes cells, instrumental use of embryos, ethics of using surplus ivf embryos as a source of hes cells, therapeutic cloning, conclusions and recommendations, acknowledgements.

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Human embryonic stem cells: research, ethics and policy

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Guido de Wert, Christine Mummery, Human embryonic stem cells: research, ethics and policy, Human Reproduction , Volume 18, Issue 4, April 2003, Pages 672–682, https://doi.org/10.1093/humrep/deg143

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The use of human embryos for research on embryonic stem (ES) cells is currently high on the ethical and political agenda in many countries. Despite the potential benefit of using human ES cells in the treatment of disease, their use remains controversial because of their derivation from early embryos. Here, we address some of the ethical issues surrounding the use of human embryos and human ES cells in the context of state‐of‐the‐art research on the development of stem cell based transplantation therapy.

Human embryonic stem cells (hES cells) are currently discussed not only by the biologists by whom they were discovered but also by the medical profession, media, ethicists, governments and politicians. There are several reasons for this. On the one hand, these ‘super cells’ have a major clinical potential in tissue repair, with their proponents believing that they represent the future relief or cure of a wide range of common disabilities; replacement of defective cells in a patient by transplantation of hES cell‐derived equivalents would restore normal function. On the other hand, the use of hES cells is highly controversial because they are derived from human pre‐implantation embryos. To date, most embryos used for the establishment of hES cell lines have been spare embryos from IVF, but the creation of embryos specifically for deriving hES cells is also under discussion. The most controversial variant of this is the transfer of a somatic cell‐nucleus from a patient to an enucleated oocyte (unfertilized egg) in order to produce hES cells genetically identical to that patient for ‘autologous’ transplantation (so‐called ‘therapeutic’ cloning); this may prevent tissue rejection.

The question ‘Can these cells be isolated and used and, if so, under what conditions and restrictions’ is presently high on the political and ethical agenda, with policies and legislation being formulated in many countries to regulate their derivation. The UK has been the first to pass a law governing the use of human embryos for stem cell research. The European Science Foundation has established a committee to make an inventory of the positions taken by governments of countries within Europe on this issue ( European Science Foundation, 2001 ).

In order to discuss the moral aspects of the isolation and use of hES cells, which is the aim of the present article, it is first essential to understand exactly what these cells are, where they come from, their intended applications and to define the ethical questions to be addressed.

‘Stem cells’ are primitive cells with the capacity to divide and give rise to more identical stem cells or to specialize and form specific cells of somatic tissues. Broadly speaking, two types of stem cell can be distinguished: embryonic stem (ES) cells which can only be derived from pre‐implantation embryos and have a proven ability to form cells of all tissues of the adult organism (termed ‘pluripotent’), and ‘adult’ stem cells, which are found in a variety of tissues in the fetus and after birth and are, under normal conditions, more specialized (‘multipotent’) with an important function in tissue replacement and repair.

hES cells are derived from the so‐called ‘inner cell mass’ of blastocyst stage embryos that develop in culture within 5 days of fertilization of the oocyte ( Thomson et al ., 1998 ; Reubinoff et al ., 2000 ). Although hES cells can form all somatic tissues, they cannot form all of the other ‘extraembryonic’ tissues necessary for complete development, such as the placenta and membranes, so that they cannot give rise to a complete new individual. They are therefore distinct from the ‘totipotent’ fertilized oocyte and blastomere cells deriving from the first cleavage divisions. hES cells are also immortal, expressing high levels of a gene called telomerase, the protein product of which ensures that the telomere ends of the chromosomes are retained at each cell division and the cells do not undergo senescence. The only other cells with proven pluripotency similar to that of ES cells are embryonic germ (EG) cells, which as their name implies, have been derived from ‘primordial germ cells’ that would ultimately form the gametes if the fetus had not been aborted. In humans, hEG cells were first established in culture in 1998, shortly after the first hES cells, from tissue derived from an aborted fetus ( Shamblott et al ., 1998 ). Biologically, hEG cells have many properties in common with hES cells ( Shamblott et al ., 2001 ).

In the adult individual, a variety of tissues have also been found to harbour stem cell populations. Examples include the brain, skeletal muscle, bone marrow and umbilical cord blood, although the heart, by contrast, contains no stem cells after birth (reviewed in McKay 1997 ; Fuchs and Segre, 2000 ; Watt and Hogan, 2000 ; Weissman et al ., 2000 ; Blau et al ., 2001 ; Spradling et al ., 2001 ). These adult stem cells have generally been regarded as having the capacity to form only the cell types of the organ in which they are found, but recently they have been shown to exhibit an unexpected versatility ( Ferrari et al ., 1998 ; Bjornson et al ., 1999 ; Petersen et al ., 1999 ; Pittenger et al ., 1999 ; Brazelton et al ., 2000 ; Clarke et al ., 2000 ; Galli et al ., 2000 ; Lagasse et al ., 2000 ; Mezey et al ., 2000 ; Sanchez‐Ramos et al ., 2000 ; Anderson et al ., 2001 ; Jackson et al ., 2001 ; Orlic et al ., 2001 ). Evidence is strongest in animal experiments, but is increasing in humans, that adult stem cells originating in one germ layer can form a variety of other derivatives of the same germ layer (e.g. bone marrow‐to‐muscle within the mesodermal lineage), as well as transdifferentiate to derivatives of other germ layers (e.g. bone marrow‐to‐brain between the mesodermal and ectodermal lineages). To what extent transdifferentiated cells are immortal or acquire appropriate function in host tissue remains largely to be established but advances in this area are rapid, particularly for multipotent adult progenitor cells (MAPCs) of bone marrow ( Reyes and Verfaillie, 2001 ). Answers to these questions with respect to MAPCs, in particular whether they represent biological equivalents to hES and can likewise be expanded indefinitely whilst retaining their differentiation potential, are currently being addressed ( Jiang et al . 2002 ; Schwartz et al ., 2002 ; Verfaillie, 2002 ; Zhao et al ., 2002 ). For other adult stem cell types, such as those from brain, skin or intestine ( Fuchs and Segre, 2000 ), this may remain unclear for the immediate future. Although the discussion here concerns hES cells and the use of embryos, the scientific state‐of‐the‐art on other types of stem cell is important in the context of the ‘subsidiarity principle’ (see below).

In theory, hES cells could be used for many different purposes ( Keller and Snodgrass, 1999 ). Examples in fundamental research on early human development are the causes of early pregnancy loss, aspects of embryonic ageing and the failure of pregnancy in older women (where genetic defects in the oocyte appear to be important). A second category might be toxicology, more specifically research on possible toxic effects of new drugs on early embryonic cells which are often more sensitive than adult cells (drug screening). The most important potential use of hES cells is, however, clinically in transplantation medicine, where they could be used to develop cell replacement therapies. This, according to most researchers in the field represents the real ‘home run’ and it is the ethics of using embryos in this aspect of medicine that will be discussed here. Examples of diseases caused by the loss, or loss of function, of only one or a limited number of cell types and which could benefit from hES cell‐based therapies include diabetes, Parkinson’s disease, stroke, arthritis, multiple sclerosis, heart failure and spinal cord lesions. Although it is known that hES cells are capable of generating neural, cardiac, skeletal muscle, pancreas and liver cells in teratocarcinomas in vivo in immunodeficient mice as well as in tissue culture, it would be an illusion to consider that cell‐therapies will have widespread application in the short term (i.e. within a couple of years). It is unfortunate that sensational treatment in the media, which implied the generation of whole organs from hES cells, initially left this impression so that the more realistic view emerging is already a disappointment to some patient groups. Nonetheless, a proper scientific evaluation of the therapeutic potential is being carried out in countries that allow the isolation and/or use of existing hES cells. The ethical questions here then also include whether the establishment of new hES cell lines can be justified, in the realisation that eventual therapies, based on either hES or adult stem cells are long‐term perspectives.

There are, at least in theory, various sources of hES cells. In most cases to date, these have been spare IVF embryos, although IVF embryos have been specifically created for the purpose of stem cell isolation ( Lanzendorf et al ., 2001 ). In one variant of ‘embryo creation’, it has even been reported that normally organized blastocysts develop from chimeras of two morphologically non‐viable embryos ( Alikani and Willadsen, 2002 ). The most revolutionary option would be the creation of embryos specifically for the purpose of isolating stem cells via ‘nuclear transfer’ (‘therapeutic cloning’). This option is purported to be the optimal medical use of hES technology since the nuclear DNA of the cells is derived from a somatic cell of a patient to receive the transplant, reducing the chances of tissue rejection (see Barrientos et al ., 1998 ; 2000). It is of note that the oocyte in this case is not fertilized, but receives maternal and paternal genomes from the donor cell nucleus. Since by some definitions an embryo is the result of fertilization of an oocyte by sperm, there is no absolute consensus that nuclear transfer gives rise to an embryo (see below).

The establishment of embryonic cell lines is becoming increasingly efficient, with up to 50% of spare IVF embryos that develop into blastocysts after thawing at the 8‐cell stage reported to yield cell lines. There are reports of efficiencies much lower than 50%, however, the quality of the donated embryos being an important determinant of success. Growth of the cell lines over extended periods and in some cases under defined conditions ( Xu et al ., 2001 ) has also been reported, but the controlled expansion and differentiation to specific cell types is an area where considerable research will be required before cell transplantation becomes clinical practice (for review, see Passier and Mummery, 2003 ). In addition, research will be required on how to deliver cells to the appropriate site in the patient to ensure that they survive, integrate in the host tissue and adopt appropriate function. These are the current scientific challenges that will have to be overcome before cell therapy becomes clinical practice; the problems are common to both hES and adult stem cells. The efficiency of establishing embryonic stem cell lines from nuclear transfer embryos is currently unknown, but expected to be lower than from IVF embryos.

In the following section, the status of hES cells is first considered. The questions of whether it is acceptable to use pre‐implantation embryos as a source of ES cells for research on cell transplantation therapy and if so, whether embryo use should be limited to spare embryos or may also include the creation of embryos via nuclear transfer (‘therapeutic cloning’), are then addressed.

What is the ontological status of hES cells? Should they be considered equivalent to embryos or not? Let us first consider the status of the ‘naked’, isolated inner cell mass (ICM; the source for deriving hES cell lines). The ICM is as it were the ‘essence’ of the pre‐implantation embryo, the precursor of the ‘embryo proper’. The isolated ICM, however, no longer has the potential to develop into a fetus and child, as trophoblast cells, necessary for implantation and nourishment of the embryo, and extra‐embryonic endoderm, are absent. It does not necessarily follow, though, that the isolated ICM is no longer an embryo—we suggest that the whole, isolated ICM could best be qualified as a disabled, ‘non‐viable’ embryo (even though it might, at least in theory, be ‘rescued’ by enveloping the ICM with sufficient trophoblast cells).

What, then, is the status of the individual cells from the ICM once isolated, and the embryonic stem cell lines derived from them? Should we consider these cells/cell lines to be non‐viable embryos too? We would argue that when the cells of the ICM begin to spread and grow in culture, the ICM disintegrates and the non‐viable embryo perishes. Some might argue that hES cells are embryos, because, although hES cells in themselves cannot develop into a human being, they might if they were ‘built into’ a cellular background able to make extra‐embryonic tissues necessary for implantation and nutrition of the embryo. At present this is only possible by ‘embryo reconstruction’ in which the ICM of an existing embryo is replaced by ES cells ( Nagy et al ., 1993 ). Commentators who, against this background, regard hES cells as equivalent to embryos, apparently take recourse to the opinion that any cell from which a human being could in principle be created, even when high technology (micromanipulation) would be required to achieve this, should be regarded as an embryo. An absurd implication of this ‘inclusive’ definition of an embryo is that one should then also regard all somatic cells as equivalent to embryos—after all, a somatic nucleus may become an embryo after nuclear transplantation in an enucleated oocyte. It is therefore unreasonable to regard hES cells as equivalent to embryos.

Research into the development of cell‐replacement therapy requires the instrumental use of pre‐implantation embryos from which hES cells are derived since current technology requires lysis of the trophectoderm and culture of the ICM; the embryo disintegrates and is thus destroyed. As has already been discussed extensively in the embryo‐research debate, considerable differences of opinion exist with regard to the ontological and moral status of the pre‐implantation embryo ( Hursthouse, 1987 ). On one side of the spectrum are the ‘conceptionalist’ view (‘the embryo is a person’) and the ‘strong’ version of the potentiality‐argument (‘because of the potential of the embryo to develop into a person, it ought to be considered as a person’). On the other side of the spectrum we find the view that the embryo (and even the fetus) as a ‘non‐person’ ought not to be attributed any moral status at all. Between these extremes are various intermediates. Here, there is a kind of ‘overlapping consensus’: the embryo has a real, but relatively low moral value. The most important arguments are the moderate version of the potentiality argument (‘the embryo deserves some protection because of its potential to become a person’) and the argument concerning the symbolic value of the embryo (the embryo deserves to be treated with respect because it represents the beginning of human life). Differences of opinion exist on the weight of these arguments (how much protection does the embryo deserve?) and their extent (do they apply to pre‐implantation embryos?). In view of the fact that up to 14 days of development, before the primitive streak develops and three germ layers appear, embryos can split and give rise to twins or two embryos may fuse into one, it may reasonably be argued that at these early stages there is in principle no ontological individuality; this limits the moral value of an embryo.

Pre‐implantation embryos are generally regarded from the ethical point of view as representing a single class, whereas in fact ∼50–60% of these embryos are aneuploid and mostly non‐viable. For non‐viable embryos, the argument of potentiality does not of course apply. Their moral status is thus only based on their symbolic value, which is already low in ‘pre‐individualized’ pre‐implantation embryos. The precise implications of this moral difference for the regulation of the instrumental use of embryos is, however, beyond the scope of the present article.

The view that research with pre‐implantation embryos should be categorically forbidden is based on shaky premises and would be difficult to reconcile with the wide social acceptance of contraceptive intrauterine devices. The dominant view in ethics is that the instrumental use of pre‐implantation embryos, in the light of their relative moral value, can be justified under certain conditions. The international debate focuses on defining these conditions.

Possible objections are connected to the principle of proportionality, the slippery slope argument, and the principle of subsidiarity.

Proportionality

It is generally agreed that research involving embryos should be related to an important goal, sometimes formulated as ‘an important health interest’ (the principle of proportionality). Opinions differ on how this should be interpreted and made operational. In a number of countries, research on pre‐implantation embryos is permitted provided it is related to human reproduction. Internationally, however, such a limitation is being increasingly regarded as too restrictive ( De Wert et al ., 2002 ). The isolation of hES cells for research into cell‐replacement therapies operates as a catalyst for this discussion. It is difficult to argue that research into hES cells is disproportional. If embryos may be used for research into the causes or treatment of infertility, then it is inconsistent to reject research into the possible treatment of serious invalidating diseases as being not sufficiently important. The British Nuffield Council on Bioethics ( Nuffield Council on Bioethics, 2000 ) also saw no reason for making a moral distinction between research into diagnostic methods or reproduction and research into potential cell therapies.

Even if one argued that there is a difference between the two types of research, research on cell therapy would, if anything, be more defensible than research on reproduction. One (in our opinion somewhat dubious) argument is to be found in McGee and Caplan (1999 ); here the suggestion is made that in using embryos for cell therapy, no embryos are actually sacrificed: ‘In the case of embryos already slated to be discarded after IVF, the use of stem cells may actually lend permanence to the embryo. Our point here is that the sacrifice of an early human embryo, whether it involves a human person or not, is not the same as the sacrifice of an adult because life of a 100‐cell embryo is contained in its cells nuclear DNA.’ In other words, the unique characteristic of an embryo is its DNA; by transplanting cells containing this DNA to a new individual, the DNA is preserved and the embryo therefore not sacrificed—a ‘win–win’ situation for both the embryo and cell transplant recipient. The implication is thus that the use of embryos for cell transplantation purposes is ethically preferable to disposing of them or using them in other (‘truly destructive’) types of research. This extreme genetic ‘reductionism’ is highly disputable and not convincing: the fact that embryos are actually sacrificed in research into cell therapy is masked. A second, more convincing, argument, that the instrumental use of embryos is in principle easier to justify for isolation of hES cells than, for example, research directed towards improving IVF, is that it has potentially far wider clinical implications. It therefore, unquestionably meets the proportionality requirement.

Slippery slope

The slippery slope argument can be considered as having two variants, one empirical and the other logical. The empirical version involves a prediction of the future: ‘Acceptance of practice X will inevitably lead to acceptance of (undesirable) practice Y. To prevent Y, X must be banned’. The logical version concerns the presumed logical implications resulting from the moral justification of X: ‘Justification of X automatically implies acceptance of (undesirable) practice Y’. In this context the problem often lies in the lack of precise definition of X: ‘The difficulty in making a conceptual distinction between X and Y that is sharp enough to justify X without at the same time justifying Y, is a reason to disallow X.’ Both versions of the argument play a role in the debate about the isolation of hES cells for research into cell replacement therapy. An example of the logical version is that acceptance of hES cells for the development of stem cell therapy for the treatment of serious disease automatically means there is no argument against acceptance of use, for example, for cosmetic rejuvenation (Nuffield Council on Bioethics, 2000). The main difficulty is, according to these critics, the ‘grey area’ between these two extremes. One answer to this objection is to consider each case individually rather than reject all cases out of hand. One could use the same objection for example against surgery, which can equally be used for serious as well as trivial treatments.

An example of the empirical version of the slippery slope argument is that the use of hES cells for the development of cell therapy would inevitably lead to applications in germ‐line gene therapy and in therapeutic cloning, then ultimately reproductive cloning. This version of the argument is unconvincing too; even if germ line gene therapy and therapeutic cloning would be categorically unacceptable, which is not self‐evident, it does not necessarily follow from this that the use of hES cells for cell‐therapy is unacceptable. The presumed automatism in the empirical version of the slippery slope argument is disputable.

Subsidiarity

A further condition for the instrumental use of embryos is that no suitable alternatives exist that may serve the same goals of the research. This is termed ‘the principle of subsidiarity’. Critics of the use of hES cells claim that at least three such alternatives exist, which have in common that they do not require the instrumental use of embryos: (i) xenotransplantation; (ii) human embryonic germ cells (hEG cells), and (iii) adult stem cells.

The question is not whether these possible alternatives require further research (this is, at least for the latter two, largely undisputed), but whether only these alternatives should be the subject of research. Is a moratorium for isolating hES cells required, or is it preferable to carry out research on the different options, including the use of hES cells, in parallel?

The answer to this question depends on how the principle of subsidiarity ought to be applied. Although the principle of subsidiarity is meant to express concern for the (albeit limited) moral value of the embryo, it is a sign of ethical one‐dimensionality to present every alternative, which does not use embryos, as a priori superior. For the comparative ethical analysis of hES cells from pre‐implantation embryos on the one hand, and the possible alternatives mentioned on the other, a number of relevant aspects should be taken into account. These include: the burdens and/or risks of the different options for the patient and his or her environment; the chance that the alternative options have the same (probably broad) applicability as hES cells from pre‐implantation embryos; and the time‐scale in which clinically useful applications are to be expected.

A basis for initiating a comparative ethical analysis is set out below:

(i) Xenotransplantation is viewed at present as carrying a risk, albeit limited, of cross‐species infections and an accompanying threat to public health. This risk is, at least for the time being, an ethical and safety threshold for clinical trials. Apart from that, the question may be raised from a perspective of animal ethics whether it is reasonable to breed and kill animals in order to produce transplants, when at the same time spare human embryos are available which would otherwise be discarded;

(ii) In principle, the use of hEG cells from primordial germ cells of dead fetuses seems from a moral perspective to be more acceptable than the instrumental use of living pre‐implantation embryos, provided that the decision to abort was not motivated by the use of fetal material for transplantation purposes. To date, however, hEG cells have been difficult to isolate and culture, with only one research group reporting success ( Shamblott et al ., 1998 ; 2001). In addition, research in mice suggests abnormal reprogramming of these cells in culture: chimeric mice generated between mouse (m)EG cells and pre‐implantation embryos develop abnormally while chimeras using mouse (m)ES cells develop as normally as non‐chimeric mice ( Steghaus‐Kovac, 1999 ; Surani, 2001 ). This makes the outcome of eventual clinical application of these cells difficult to predict in terms of health risks for the recipient.

(iii) Analysis of the developmental potential of adult stem cells is a rapidly evolving field of research, particularly in animal model systems. Experiments carried out within the last two years have demonstrated, for example, that bone marrow cells can give rise to nerve cells in mouse brain ( Mezey et al ., 2000 ), neural cells from mouse brain can turn into blood and muscle ( Bjornson et al ., 1999 ; Galli et al ., 2000 ), and even participate in the development of chimeric mouse embryos up to mid‐gestation ( Clarke et al ., 2000 ). Although apparently spectacular in demonstrating that neural stem cells from mice can form most cell types under the appropriate conditions, it is still unclear whether true plasticity in terms of function has been demonstrated or whether the cells simply ‘piggy‐back’ with normal cells during development. Published evidence of ‘plasticity’ in adult human stem cells is more limited, but recent evidence suggests that the MAPCs from bone marrow may represent a breakthrough ( Jiang et al ., 2002 ; Schwartz et al ., 2002 ;). They are accessible. Collection is relatively non‐destructive for surrounding tissue compared, for example, with the collection of neural stem cells from adult brain, although their numbers are low: 1 in 10 8 of these cells exhibit the ability to form populations of nerve, muscle and a number of other cell types and they only become evident after several months of careful culture. Clonal analysis has provided rigorous proof of plasticity: a single haematopoietic stem cell can populate a variety of tissues when injected into lethally irradiated mice ( Krause et al ., 2001 ) or into blastocyst stage embryos to generate chimeric embryos ( Jiang et al ., 2002 ). Nonetheless, there are potential hazards to using cells that have been cultured for long periods for transplantation and although MAPCs seem to have normal chromosomes, it is important to establish that the pathways governing cell proliferation are unperturbed. This is also true for hES cells. However, the powerful performance of mES cells in restoring function in a rat model for Parkinson’s disease ( Kim et al ., 2002 ), has not yet been matched by MAPCs. Bone marrow stem cells have been shown very recently to restore function to some extent in a mouse heart damaged by coronary ligation, an experiment that mimics the conditions of the human heart soon after infarction ( Orlic et al ., 2001 ). Although clinical restoration of function in a damaged organ is usually sought rather longer after the original injury than in these experiments, which were performed before scar tissue had formed, this approach will certainly be worth pursuing. An alternative, non‐invasive, haematopoietic stem cell source is umbilical cord blood. This is used clinically for transplantation as an alternative to bone marrow in patients for whom no bone marrow match is available. Cord blood contains precursors of a number of lineages but its pluripotency, or even multipotency, is far from proven. Nevertheless, the prospect of autologous transplantation of haematopoietic stem cells of bone marrow in the long term makes this an important research area in terms of alternatives to therapeutic cloning (see below).

Although studies with adult stem cells so far have been encouraging, Galli (2000 ), author of the first adult neural stem studies and much cited by advocates of the view that adult stem cells have a proven developmental potency equal to that of ES cells, himself disagrees entirely with this viewpoint (see Editorial, 2000 ). It has even been suggested that the results from adult stem cell research are being misinterpreted for political motives and ‘hints of the versatility of the adult cells have been over interpreted, overplayed and over hyped’ ( Vastag, 2001 ). Opponents of ES cell research are now heralding Verfaillie’s adult stem cells as proof that work on hES cells is no longer needed. However the stem cell research community and Verfaillie herself ( Vastag, 2002 ) have called for more research on both adult and embryonic stem cells. ES cells that can perform as powerfully as those described by Kim et al . (2002 ) in the rat Parkinson model make it far too early in the game for them to be discounted ( Editorial, 2002 ).

The question remains, however, should a moratorium be imposed on isolating hES cells for research in cell therapy in the light of the indisputably promising results from adult stem cell research? The lack of consensus arises largely from disagreement on interpretation of the subsidiarity principle. Against the restrictive viewpoint that research on hES cells may only take place if there is proof that adult stem cells are not optimally useful, there is the more permissive viewpoint that hES cell research may, and indeed should, take place so long it is unclear whether adult stem cells are complete or even partial alternatives.

On the basis of the following arguments, a less restrictive interpretation of the subsidiarity principle is morally justified. ( Stem Cell Research, 2000 ) To begin with, the most optimistic expectation is that only in the long run will adult stem cells prove to have equal plasticity and developmental potential as hES cells (and be as broadly applicable in the clinic), and there is a reasonable chance that this will never turn out to be the case. If hES cells from pre‐implantation embryos have more potential clinical applications in the short term, then the risk of a moratorium is that patients will be deprived of benefit. This in itself is a reason to forgo a moratorium—assuming that the health interests of patients overrule the relative moral value of pre‐implantation embryos. Secondly, the simultaneous development of different research strategies is preferable, considering that research on hES cells will probably contribute to speeding up and optimising clinical applications of adult stem cells. In particular, the stimuli to drive cells in particular directions of differentiation may be common to both cell types, while methods of delivery to damaged tissue are as likely to be common as complementary. A moratorium on hES cell research would remove the driving force behind adult stem cell research.

A final variant on adult stem cell sources concerns the use of embryonal carcinoma (EC) cells, a stem cell population found in tumours (teratocarcinomas) of young adult patients. These cells have properties very similar to hES cells. The results of a phase I (safety) trial using these cells in 11 stroke victims in the USA have recently been published and permission granted by the Food and Drug Administration (FDA) for a phase II trial (effectivity) ( Kondziolka et al ., 2000 ). The patients received neural cells derived from retinoic acid (vitamin A) treatment of teratocarcinoma stem cells. Although the scientific and ethical consensus is that these trials were premature in terms of potential risk of teratocarcinoma development at the transplant site, all patients survived with no obvious detrimental effects, no tumour formation and in two cases a small improvement in symptoms. After two years, the transplanted cells were still detectable by scanning ( Kondziolka et al ., 2000 ). Despite its controversial nature, this trial has nevertheless probably set a precedent for similar trials using neural derivatives of hES, the best controlled differentiation pathway of hES cells at the present time ( Reubinoff et al ., 2001 ; Zhang et al ., 2001 ). Proponents believe that such trials would be feasible even in the short term ( McKay, 1997 ). Neural differentiation of hEC cells is fairly easy to induce reproducibly but most other forms of differentiation are not; even if ultimately regarded as ‘safe’, hEC cells will not replace hES cells in terms of developmental potential and are therefore not regarded as an alternative.

In view of both the only relative moral value of pre‐implantation embryos and the uncertainties and risks of the potential alternative sources for the development of cell therapy, a moratorium for isolating human embryonic stem cells is unjustified.

Before discussing the ethical issues around ‘therapeutic cloning’, the term itself requires consideration. To avoid confusion, it has been proposed that the term ‘cloning’ be reserved for reproductive cloning and that ‘Nuclear transplantation to produce stem cells’ would be better terminology for therapeutic cloning ( NAS report, 2002 ; Vogelstein et al ., 2002 ). Others have pointed out the disadvantage of this alternative term, namely that it masks the fact that an embryo is created for instrumental use. More important in our opinion however, is that the use of the adverb ‘therapeutic’ suggests that hES cell therapy is already a reality: strictu sensu there can only be a question of therapeutic applications once clinical trials have started. In the phase before clinical trials, it is only reasonable to refer to research on nuclear transfer as ‘research cloning’ or ‘nuclear transplantation for fundamental scientific research’, aimed at future applications of therapeutic cloning.

Some consider this technology to be ethically neutral; they claim that the ‘construct’ produced is not a (pre‐implantation) embryo. Qualifications suggested for these constructs include: activated oocyte, ovasome, transnuclear oocyte cell, etc. ( Kiessling, 2001 ; Hansen, 2002 ) However, to restrict the definition of ‘embryo’ to the product of fertilization in the post‐Dolly era is a misleading anachronism. Although the purpose of therapeutic cloning is not the creation of a new individual and it is unlikely that the viability of the constructed product is equivalent to that of an embryo derived from sexual reproduction, it is not correct to say that an embryo has not been created.

The core of the problem is that here human embryos are created solely for instrumental use. Whether or not this can be morally justified—and if so, under what conditions—has already been an issue of debate for years in the context of the development of ‘assisted reproductive technologies’ (ART). Is it acceptable to create embryos for research, and if so, is therapeutic cloning morally acceptable too?

A preliminary question: is it justified to create embryos for research?

Article 18 of the European Convention on Human Rights and Biomedicine forbids the creation of embryos for all research purposes ( Council of Europe, 1996 ). However, this does not close the ethical and political debates in individual EU member states.

In the ‘classical’ normative debate on embryo research, two perspectives can be distinguished: a ‘fetalist’ perspective (focusing on the moral value of the embryo), and a ‘feminist’ perspective (with the interests of women, particularly candidate oocyte donors, playing a central role) ( Raymond, 1987 ). Both perspectives have a different outlook on the question of whether or not there is a decisive moral distinction between research with spare IVF embryos on the one hand, and creating embryos for research on the other. In other words: is the difference between these practices such that the former can be acceptable under specific conditions, and the latter absolutely not?

Fetalist perspective

Instrumentalization of the embryo is sometimes regarded as far greater and fundamentally different when it involves the creation of embryos for research purposes rather than the use of spare embryos. This difference, however, is just gradual. Not only is the embryo used completely instrumentally in both cases, the moral status is also identical. The difference is in the intention at fertilization, which, although a real difference, is relative. It is a misconception to think that in the context of regular IVF treatment every embryo is created as a ‘goal in itself’: the goal is the solution of involuntary childlessness and the loss of some embryos is a calculated risk beforehand.

Feminist perspective

From a feminist perspective, the creation of embryos for research should be evaluated critically in as far as it may require hormone treatment of a woman to obtain oocytes for research purposes: can this be morally justified when it requires unpleasant treatment of the donor with no benefit at all, or even a detrimental outcome, for her own state of health? A first objection is that women themselves become objects of instrumental use. Here, however, an analogy can be made with recruiting healthy research subjects. Relevant considerations concern whether or not the research serves an important goal, whether the burdens and risks to the subjects are proportional, and whether valid informed consent of the research subject/donor is given. The second objection is that the health risks to the women themselves are too high and the degree of discomfort disproportional. Difference of opinion exists, however, also among women, about the disproportionality of hormone treatment. There are, furthermore, several potential alternatives that do not require hormone treatment of healthy women. One involves the in‐vitro maturation (IVM) of immature oocytes after their isolation from dead donors or donors having ovaries removed for other reasons. IVM is successful in cattle and sheep (efficiency ∼40%), although it is, for the moment, much lower in humans.

In conclusion, from both a fetalist and a feminist perspective there is no overriding categorical objection against bringing pre‐implantation embryos into existence for instrumental use. If the research cannot be conducted using spare embryos and its importance for human health is beyond doubt, we believe the creation of embryos specifically for research is morally justified subject to the required oocytes being obtained in a morally sound way.

Ethics of therapeutic cloning

Can therapeutic cloning be morally acceptable? The principle of proportionality, the slippery slope, and the principle of subsidiarity enter the debate again, but in a slightly different way.

It is doubtful whether the principle of proportionality provides a convincing a‐priori objection against therapeutic cloning. If it is considered acceptable to create embryos for research aimed at improving ART (freezing of oocytes; IVM of oocytes, etc…), then it is inconsistent to reject therapeutic cloning beforehand as being disproportional. Maybe even some opponents of creating embryos for the improvement of ART can conditionally accept therapeutic cloning because of the important health interests of patients.

Slippery‐slope

A consequentialist objection (fashioned as a ‘slippery‐slope’ argument) is that therapeutic cloning will inevitably lead to reproductive cloning. This objection is not convincing; if reproductive cloning is categorically unacceptable (the debate on this issue is still ongoing), it is reasonable to prohibit this specific technology, and not to ban other, non‐reproductive, applications of cloning. A second objection that could be raised in this context is that the creation of embryos through cloning for the isolation of stem cells could in the long term be used to justify the initiation of pregnancy from these embryos and their use simply as a vehicle for generating sufficient cells of the required type for transplantation; the pregnancy would be interrupted the moment the appropriate developmental stage was reached ( Lanza et al ., 2002 ). Relevant questions here are: is this a realistic scenario in the human (or just science fiction), would it be unacceptable, and is it unavoidable?

In terms of being a realistic means of generating genetically identical (fetal) tissue for transplantation, it could theoretically be an option, but whether it would actually be useful would depend on the alternatives available at the time transplantation techniques themselves have been perfected to clinical applicability (see below).

In terms of moral acceptability, most people would consider pregnancy‐and‐abortion‐for‐transplantation to be far more difficult to justify than the creation of pre‐implantation embryos for instrumental use in vitro , firstly because of the higher moral status/symbolic value of the fetus, and secondly because of the significantly greater burden of pregnancy‐and‐abortion‐for‐transplantation for women. ( De Wert et al ., 2002 ) Even though many countries do forbid pregnancy‐for‐transplantation, it has been argued that it could be morally justified as a last resort, on the basis that sacrificing a fetus (a potential person) may be justified in order to rescue the life of a person.

Finally, in scrutinising the slippery slope argument, it is important to assess whether instrumental use of pre‐implantation embryos makes pregnancy‐for‐abortion unavoidable. Again, the apparent automatism is disputable: if we reject pregnancy‐for‐abortion as being unacceptable, we can continue its prohibition.

Taking these points for and against together, the slippery slope argument does not provide a convincing basis for banning therapeutic cloning.

Therapeutic cloning can only be morally acceptable if there are no good alternatives. It is important to note that therapeutic cloning strictu sensu is not likely to be short‐term prospect. Apart from unsolved technical difficulties with nuclear transfer itself in human oocytes ( Cibelli et al ., 2002 ), much basic research is still needed to determine whether the differentiation of hES cells can be controlled and sufficient cell numbers generated to be a useful therapy. This research can be done with spare IVF embryos. In this light, creation of embryos for therapeutic cloning is, in our opinion, premature. Although critics of this point of view could use our own argument that delay in the development of research cloning could, just as a moratorium on hES cell isolation and research, have negative consequences for patients, the evidence suggests that further optimization of the technology as such could take place in animals. We believe that the duration of any ‘delay’ in offering therapy to patients would not then be of real significance.

At the same time, research on potential alternatives for therapeutic cloning, which likewise avoid (or at least reduce) the problem of rejection but which do not involve the creation of human embryos for instrumental use, should be stimulated. For the comparative ethical analysis, it is again important to avoid the pitfall of one‐dimensionality. Possible alternative options include: (i) the use of adult cells, both stem cells and differentiated cells; (ii) making optimal use of spare embryos: embryo‐banks and immuno‐tolerance and (iii) the use of entities with an undetermined status: ‘hybrids’ and ‘parthenotes’.

Adult cells

Adult tissue is a potential source of two alternatives: stem cells, which may be induced to transdifferentiate by extracellular signals, and somatic cells (nuclei) which require direct reprogramming signals, for example from an oocyte after nuclear transfer, to adopt a new fate. Both sources will, however, require substantial research to become realistic alternatives. Until it has been shown that adult stem cells at some point re‐express ES cell markers we will never know if transdifferentiation or direct reprogramming are the same or not.

For direct reprogramming of somatic nuclei, new methods may be developed which do not require nuclear transfer to oocyte cytoplasm. Examples of current work in this area include the study of cellular hybrids derived from the fusion of (embryonic) stem cells with somatic or adult stem cells ( Surani, 2001 ; Terada et al ., 2002; Ying et al ., 2002 ). An understanding of the basic mechanisms underlying reprogramming is already being undertaken in mice, cattle and sheep and indeed, the creation of ‘Dolly’ re‐initiated a wave of research in nuclear reprogramming in mammals. The ultimate aim of this research in the context of cell transplantation therapy would be chemically‐induced nuclear re‐programming in the test‐tube to derive the required cell type, obviating the necessity for therapeutic cloning altogether. First evidence that this might be feasible demonstrated direct reprogramming of fibroblasts to neural cells and T‐cells in culture by temporary permeabilization of the fibroblasts to allow them to take up extracts of neural and T‐cells, respectively ( Hakelien et al ., 2002 ). In this sense, therapeutic cloning may be regarded, perhaps, as a temporary option; in the long term it will be replaced by a direct reprogramming alternative.

Research on direct reprogramming of adult somatic nuclei may ultimately require the creation of human embryos for instrumental use. In view of the importance of this research, both in terms of the contribution to the development of cell therapy and the potential ultimately to reduce the instrumental use of human embryos by developing an alternative for therapeutic cloning, this research would no doubt also meet the principle of proportionality.

Optimal use of spare embryos

Various strategies should be considered. Firstly, the generation of a bank of hES cell lines from a wide spectrum of genotypes is required to be able to offer a reasonable tissue match for every patient requiring a cellular transplant. Estimates of the number of independent cell lines that would actually be required for this vary greatly, from a few hundred to several thousand. Such a bank is already being discussed in the UK but could ultimately be established as a European resource. However, even very good tissue matches between donor and recipient require some degree of immunosuppressive therapy, which has long term negative side‐effects for patients, including increased risk of tumorigenesis

Secondly, there should be further development and application of ‘immunotolerance’ methodology. This may be particularly useful in combination with matching from an hES cell bank. The observation that patients receiving bone marrow transplants are more immunotolerant to other tissue transplantation from the same donor have led to the suggestion that immunotolerance may also be induced by initial injection of hES‐derived haematopoietic cells followed by the cell type of interest derived from the same hES cell line ( Kaufman et al ., 2001 ). The transplant may then be tolerated without being genetically identical, and lower doses or no immunosuppressives required. The combination of ‘near match’ with immunotolerance is probably a promising option.

For certain genetically based diseases, autologous transplantation may not always be appropriate since the transplanted tissue will bear the same genetic defect. Immunotolerance hES cell strategies may then be a particularly attractive or the only option. Should the success rates be very high, then attempts to create genetically identical transplantable tissue may become superfluous, not only for these, but for all patients. If, however, it works imperfectly or only for some patients, then therapeutic cloning may well remain an important option for the majority of all other patients.

Creating entities with an undefined status

Various alternative options raise classification problems, as the entities created to obtain cells have an undefined status. Firstly, transplanting the somatic nucleus of a patient into an enucleated animal oocyte. The logic behind this variant of therapeutic cloning is twofold: one, assuming that the ‘units’ thus created are not human embryos because only their nuclear but not mitochondrial DNA is human, advocates of this strategy argue that it circumvents the controversial issue of the instrumental use of human embryos. Two, a technical advantage of this approach would be that plenty of animal oocytes would be available; the feminist objection to creating human embryos for research would, of course, not apply.

It is not yet known whether this is a scientifically realistic option (whether hES cells can be effectively obtained following this approach). Animal research has so far been limited and not generally successful ( Barrientos et al ., 1998 ; 2001); polymorphic interspecies differences in mitochondrial DNA are thought to make such reconstructed zygotes non‐viable or prone to major developmental abnormalities. There are however, unvalidated reports of successful applications of the technique in China. The Donaldson Committee advocated a ban on this approach, but without any argumentation (Stem Cell Research, 2000). However, if this were a realistic option scientifically, then we believe that the issues involved deserve further ethical discussion. The major questions that should be addressed include: is the risk acceptable? As for xenotransplantation, there is also here the risk of cross‐species infection, although this may be extremely small, because the nuclear DNA of the animal, which may harbour viruses, is removed from the oocyte. Is it reasonable to argue that this ‘artificial combination’ should not be considered equivalent to a human embryo? Since the entire nuclear DNA is human, the reconstructed combination should, we think, be regarded as a human embryo. The procedure should thus not be presented as an ‘embryo saving’ variant of therapeutic cloning. However, only further in‐utero research with reconstructed animal embryos, for example embryos created by transplanting the somatic nucleus of a rat into an enucleated mouse oocyte, will provide a more definitive answer. Finally: in‐vitro research may well show that embryos obtained by transplanting a human somatic nucleus into an enucleated animal oocyte are non‐viable (like parthenotes, see below). The moral status of non‐viable pre‐implantation embryos, and more particularly, the question as to whether the conditions for research using non‐viable embryos may be more permissive than the conditions for using viable embryos, needs further debate (see earlier).

A second option may be the generation of parthenogenetic embryos for the isolation of hES cell lines. Here, an unfertilized (haploid) oocyte is treated chemically such that it becomes diploid, with two identical sets of the maternal chromosomes. These uniparental embryos are by definition gynogenetic and never result in viable offspring, because they fail to generate extra‐embryonic tissues. Nevertheless, in mice (see Boediono et al ., 1999 ) and in apes ( Cibelli et al ., 2002 ), parthenotes have been shown to develop to the blastocyst stage and yield cell lines with properties not distinguishable from ES cells derived from fertilized oocytes. However, in view of the fact that some genes are genomically imprinted, such that they are expressed only if inherited via the male germ line, ES cells derived from parthenotes may well be abnormal. First attempts at parthenogenesis in humans have not yielded hES cell lines ( Cibelli et al ., 2002 ). It is important to realise that such hES cell lines, if developed in humans, would only provide a tissue match for the oocyte donor, i.e. women of reproductive age. Although it has been speculated that two sets of male chromosomes could also be used in parthenotes, there is no evidence that this is a real option.

Cibelli and colleagues have referred to parthenogenesis as cloning. Whether this is correct depends on the timing of parthenogenesis: if initiated before the first complete meiotic division, then the procedure amounts to cloning (the same genotype as the female); if after the first meiotic division (ie recombination and loss of half) then it is not cloning. In this light, the experiments of Cibelli et al . (2002) would not qualify as cloning in the strict sense.

Some will certainly argue that the parthenote is not an embryo; parthenogenesis would then be classified as an ‘embryo‐saving’ strategy. As the parthenote undergoes the first divisions normally and is at these stages not distinguishable from embryos derived by normal fertilization, we would argue that it should be regarded as a non‐viable embryo. In the light of its non‐viability, the potentiality argument is not applicable. The moral status of parthenotes may therefore be regarded as very low, lower even than that of normal viable embryos at the same stage (see earlier). Thus, although not an ‘embryo‐saving alternative’, all other things being equal, parthenogenesis may be regarded as ethically preferable to the generation of viable embryos by fertilization or nuclear transfer (for instrumental use). In addressing the question of whether this research is premature given the current lack of proof that human ES cells are clinically useful as a source of transplantable cells, the lower moral status of parthenotes should be taken into account.

Regarding moral judgements as a ‘quasi stable equilibrium’ is particularly appropriate when applied to the ethics of isolating hES cells for research into cell replacement therapy. Stem cell research is highly dynamic, with many questions and ‘unknowns’. New insights into the effectiveness, risks and usefulness of the various alternatives may have immediate consequences for the ethical evaluation of the isolation of hES cells.

The status of the pre‐implantation embryo is the most sensitive and disputed point in the debate on isolation of hES cells for research. The dominant view in ethics, however, is that the moral status of the pre‐implantation embryo is relatively low and that the instrumental use of these embryos can be morally justified under some conditions.

The moral status of non‐viable pre‐implantation embryos is lower than the moral status of viable pre‐implantation embryos. The precise implications of this difference in moral status for the regulation of the instrumental use of embryos need further ethical scrutiny.

Both the principle of proportionality and a permissive interpretation of the principle of subsidiarity, make a moratorium on the isolation of hES cells unjustified.

Parallel research on alternatives is important and requires major support. Research on hES cells can provide an important impetus in this context.

The moral difference between research on surplus embryos and the creation of embryos for research is only gradual. A complete ban on creating embryos for instrumental use in research is morally unjustified.

A categorical ban on research on human therapeutic cloning is not justified, although the creation of embryos by cloning for the isolation of hES cells is, at the present time, premature. The necessary research can currently be carried out using animal embryos and surplus human IVF embryos.

Research into potential alternatives for therapeutic cloning, which does not require human embryos or which requires only the use of spare embryos, should be stimulated.

Banning the transplantation of a human somatic nucleus to an animal oocyte (as a variant of therapeutic cloning) is premature and morally unjustified.

The question whether therapeutic cloning should be allowed, becomes acute if research with spare embryos suggests that usable transplants can be obtained in vitro from hES cells and if the possible alternatives for therapeutic cloning are less promising or need more time for development than is currently expected. In that case, therapeutic cloning can be morally justified on the basis of both the principle of proportionality and the principle of subsidiarity.

We are grateful to Drs K.Lawson and J.Geraedts for comments on the manuscript.

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The Mysteries of Human Development: Researchers Decipher Key Developmental Signals

New research has revealed new insights into human embryonic development, showing that the duration of BMP signal exposure is key in determining cell fate during gastrulation, with potential applications in regenerative medicine.

A research team from Rice University , led by Aryeh Warmflash, has advanced our understanding of the mechanisms that drive human embryonic development. Their findings were recently published in the scientific journal Cell Systems .

Embryonic development, the journey from a single fertilized egg to a complex organism, is orchestrated by complex interactions between biochemical signals. But mechanisms behind how the cells interpret these signals to make crucial developmental decisions have remained elusive.

“Our paper addresses a fundamental question: How are these decisions controlled by multiple pathways simultaneously?” said Warmflash, associate professor of biosciences and bioengineering.

The team includes postdoctoral research associate and current group leader at the Andalusian Center for Developmental Biology Elena Camacho-Aguilar; Sumin Yoon, a senior majoring in cultural/medical anthropology; doctoral students Miguel A. Ortiz-Salazar and Siqi Du; and laboratory technician M. Cecilia Guerra. Together they focused their study on human gastrulation, a pivotal stage where cells differentiate into the three germ layers of the embryo: ectoderm, mesoderm, and endoderm.

Previous Studies and New Findings

While previous research identified the involvement of several signals such as bone morphogenetic protein (BMP) and wingless-related integration site (WNT) during gastrulation, the precise mechanisms underlying how cells interpret them to develop into different cell types remained unclear.

To find an answer, the researchers turned to human pluripotent stem cells (hPSCs), which mimic the state of cells just before gastrulation. They hypothesized that the duration and concentration of BMP signals might dictate cell fate and devised experiments exposing hPSCs to varied BMP signal systems.

Contrary to previous assumptions, the study revealed that the duration of BMP signal exposure, rather than its strength, plays a crucial role in determining cell fate. Pulselike exposures to high BMP concentrations prompted significant changes, particularly toward mesoderm, whereas continuous low-level signals yielded less pronounced outcomes.

Mathematical Modeling and Implications

Mathematical modeling of these processes allowed the researchers to predict the fate outcomes for any combination of BMP and WNT signals. The team constructed a comprehensive “fate map” that predicts these outcomes. Leveraging this map, the researchers devised a novel protocol optimizing mesoderm formation relevant to other fields such as regenerative medicine.

“Our findings underscore the importance of understanding signaling dynamics in guiding cell fate decisions,” Camacho-Aguilar said. “By deciphering these mechanisms, we can tailor efficient differentiation protocols that could be relevant for therapeutic applications.”

Reference: “Combinatorial interpretation of BMP and WNT controls the decision between primitive streak and extraembryonic fates” by Elena Camacho-Aguilar, Sumin T. Yoon, Miguel A. Ortiz-Salazar, Siqi Du, M. Cecilia Guerra and Aryeh Warmflash, 30 April 2024, Cell Systems . DOI: 10.1016/j.cels.2024.04.001

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• 17 March 2021

First complete model of the human embryo

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Yi Zheng is in the Department of Mechanical Engineering, University of Michigan, Ann Arbor, Michigan 48109, USA.

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Jianping Fu is in the Department of Mechanical Engineering, University of Michigan, Ann Arbor, Michigan 48109, USA, and in the Departments of Biomedical Engineering and of Cell and Developmental Biology, University of Michigan.

A proper understanding of early human development is crucial if we are to improve assisted reproductive technologies and prevent pregnancy loss and birth defects. However, studying early development is a challenge — few human embryos are available, and research is subject to considerable ethical and legal constraints. The emergence of techniques that use cells cultured in vitro to construct models of mammalian embryos therefore opens up exciting opportunities 1 . Two papers in Nature now make key advances in this field, showing that human embryonic stem cells 2 or cells reprogrammed from adult tissues 2 , 3 can be induced to self-organize in a dish, forming structures that resemble early human embryos. This is the first integrated human embryo model containing cell types related to all the founding cell lineages of the fetus and its supporting tissues.

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