The body consists of many types of specialised cells, from gametes to blood cells, each different cell type having a specific function. In contrast to specialised cells are unspecialised cells, known as stem cells. Because of the unique abilities of stem cells as opposed to a typical somatic cell, they are currently the target of ongoing research. Multiple areas of research are looking into how stem cells can offer new ways of treating disease, such as diabetes or heart disease – a field of medicine known as cell-based therapy or regenerative medicine.

Stem cells have a unique ability; they are able to self renew limitlessly allowing them to replenish themselves as well as other cells.  Another ability of stem cells is that they are able to differentiate to any cell type. A stem cell does not differentiate directly to a specialised cell however; there are often multiple intermediate stages. A stem cell will firstly differentiate to a progenitor cell – a progenitor cell is similar to a stem cell, although they are limited in the number of times they can replicate and they are also restricted in which cells they can further differentiate to.

Types of Stem Cells

Not all stem cells are the same, for instance, the stem cells of an embryo are quite different to those of an adult. The potency of a stem cell is indicated in its nomenclature:

• Totipotent stem cells – Capable of differentiating into all cell types found in the embryo and placenta i.e. the single celled zygote following fertilisation
• Pluripotent stem cells – Capable of differentiating into all cell types found in the embryo
• Multipotent stem cells – Capable of differentiating into a wide range of cell types
• Unipotent stem cells – Capable of differentiating into only one cell type

Embryonic Stem Cells

Embryonic stem cells are those found in the pre-implantation embryo. Due to their pluripotent nature, they are highly useful for research, but there clear ethical reasons against their collection. Embryonic stem cells used in research are thus primarily derived from eggs which have been fertilised in vitro at a fertilisation clinic and then donated to research by the donor.

The main differences between embryonic stem cells and adult, somatic stem cells are:

• Embryonic stem cells are pluripotent i.e. can differentiate into all cell types found in the embryo
• Embryonic stem cells are immortal and can continue to replicate indefinitely

Adult Stem Cells

Adult (somatic) stem cells are undifferentiated cells found throughout the adult body; the main difference from embryonic stem cells is that these somatic stem cells are not pluripotent. Instead somatic stem cells are multipotent. Instead of a single cell being able to differentiate in to all cell types, in the adult there are a number of different multipotent stem cell types. For example, one population of multipotent somatic stem cells are the haematopoietic stem cells – these stem cells are able to differentiate in to all types of blood cell however, they could not differentiate in to a nerve cell for instance.

Not all somatic stem cells differentiate in to such defined populations of cell types however, for example mesenchymal stem cells can differentiate in to bone, cartilage and fat cells – still not as potent as the pluripotent embryonic stem cells however.

It is believed that somatic stem cells can be found in almost every organ and tissue type, although they exist in very limited numbers and are often in a quiescent state – only becoming active when there is a need to replenish cell counts to maintain the tissue. It is also thought that within each tissue or organ, there is a specific area in which the stem cells reside, this is known as the ‘stem cell niche’.

Some examples of somatic stem cells:

• Haematopoietic stem cells – differentiate into all blood cell types
• Mesenchymal stem cells – differentiate into various bone, cartilage, fat and other connective tissue types
• Neural stem cells – differentiate into nerve cells, astrocytes and oligodendrocytes
• Epithelial stem cells –differentiate into a number of cells which line organs such as the stomach or digestive tract
• Skin stem cells – differentiate into cells of the epidermis
• Follicular stem cells  – differentiate into either cells of the epidermis or hair follicle cells

Under normal circumstances, a somatic stem cell will differentiate only to the specific cell types such as in the examples above, there have been incidences where his has not been the case. Known as transdifferentiation, this is where a stem cell differentiates into an unexpected cell type for example a haematopoietic stem cell differentiating into a nerve cell. Whilst this has been demonstrated in experiments and is seen in some vertebrates, it is unclear whether transdifferentiation occurs in humans.

Isolating Stem Cells

For stem cells to be successfully used in research, they must be isolated from their source. However, when trying to isolate adult somatic stem cells, there are so few cells it is quite difficult to locate and extract them.  For example, only around 1 in every 10,000 bone marrow cells is a stem cell. Also, somatic stem cells have a limited life span when removed from the body (unlike embryonic stem cells). Somatic stem cells isolated from older donors have an even shorter life span as stem cell senescence correlates to the telomere length – teleomeres are the regions of protective DNA repeats at the end of a chromosome which decrease in length with age.

Embryonic stem cells survive much better in vitro and they are also much easier to extract, however, their isolation raises ethical issues as the embryo (and thus a potential life) is destroyed.

Method of Embryonic Stem Cell Isolation

The timing of embryonic stem cell extraction is critical, for successful isolation, extraction must occur of the fifth day following fertilisation of the egg. After five days, the fertilised egg will now have formed a blastocyst – a ring like structure of cells with a pocket of embryonic stem cells within. If the isolation of the stem cells does not occur before 7 days, the germ layers begin to develop and the embryonic stem cells begin to differentiate.

Once isolated, the stem cells are placed onto a culture dish containing a nutrient broth which supports their growth. The dish contains a lining of ‘feeder cells’ along the bottom, these cells act as both a surface for the stem cells to adhere to and they also release further nutrients into the broth. The cells are allowed to grow until there is no room left in the dish at which point they are split up and placed in to multiple new dishes. Each cycle of replanting the cells on a new dish is known as a passage.

Possible Applications of Stem Cell Research

Primarily, stem cell research will aid the basic understanding of developmental biology i.e. how is that undifferentiated stem cells can become the differentiated cells which form organs and tissues? Abnormal cell division and differentiation are central aspects to major medical conditions such as cancer and certain birth defects – a greater understanding of this process in general will greatly aid the development of strategies to combat these conditions.

Drug Testing

Research into the basics of developmental biology will be an ongoing effort; a possible, more immediate application of stem cell research could be the testing of new drugs. For example, somatic stem cells could be used to generate differentiated cell types, new medications could then be tested on these cell types without the risk of causing harm to a living organism such as an animal being used for testing. Whilst it would be necessary to still test the drugs on animals and humans, the required number during initial testing would be reduced. The use of human pluripotent stem cell lines would also allow for rapid screening of efficacious new drugs during initial development.

Cell-Based Therapies

The demand for organs from willing donors far exceeds the supply, by using stem cells it may be possible to essentially grow health cells and tissue in the lab and then transplant these into a patient.  Or, as another example, it may be possible to introduce new stem cells to a damaged organ which are then able to help regenerate damaged tissue.

Another example is that of patients who suffer from type I diabetes. These patients have lost pancreatic cells to their own immune system and as such have difficulty producing insulin. Studies have suggested it may be possible to culture insulin producing cells in the lab from stem cells which could then be used in transplantation therapy for sufferers of diabetes.

Technical Barriers towards Applications of Stem Cell Research

Risk of Tumour Formation

The transplantation of stem cells in to damaged tissue has been suggested as a method to aid repair of the tissue. In these situations, embryonic stem cells would be cultured and forced to differentiate into the appropriate multipotent stem cells using specific growth factors. For instance, if a patient had damaged neuronal pathways and stem cell therapy was suggested as an option, embryonic stem cells would be cultured in a manner that forced them to differentiate into neuronal stem cells.

Problems would arise however, if these neuronal stem cells were then transplanted into the patient but some had not differentiated and thus remained as embryonic stem cells. These embryonic stem cells would then have the ability to differentiate in to any cell type, if they started to differentiate into muscle cells for instance in the nerves, this would cause problems.

Another possibility is that embryonic cells are again, transplanted with the differentiated multipotent stem cells, but instead of differentiating, the embryonic cells just continue to proliferate. As embryonic stem cells are immortal, they would be able to continually replicate causing the formation of tumours.

To ensure that this did not happen, a method must be devised to ensure that either, all embryonic stem cells are removed from a culture before transplantation or that all embryonic stem cells have differentiated correctly.

Regulation of Stem Cell Behaviour

The method by which embryonic stem cells are forced to differentiate into different multipotent stem cells is not currently 100% accurate. This means that whilst attempting to get embryonic cells to differentiate into muscle stem cells with a cocktail of growth factors believed to induce only muscle stem cells – it is possible that other types of multipotent stem cells could form, for instance neuronal stem cells. Thus when applying further growth factors to these cells in an attempt to grow muscle tissue in the lab, the mixture of multipotent stem cells would give rise to both nervous and muscle tissue.

In order to prevent this, knowledge of the basic growth mechanisms of stem cells must be understood. The biology behind the fate of cell differentiation must be investigated so ‘recipes’ of growth factors can be produced that give rise to only one multipotent stem cell type.

Immune Rejection

Embryonic stem cells can be used to grow new tissues and cells in the lab, these tissues and cells can then be transplanted into a patient. For example, insulin producing cells could be produced for stem cell therapy for type I diabetes. However, as embryonic stem cells are derived typically from a non-related embryo, there is a chance that the immune system will recognise the transplanted cells as non –self and thus destroy them rendering the therapy useless.

The patient could be given immunosuppressant drugs alongside the therapy, but these would have to be taken for essentially the duration of the patient’s life, this would have severe implications on the patient’s health as the immune system would be weakened and struggle to fight off infections.

To prevent an immune reaction, embryonic stem cells would have to be derived which were genetically identical to the patient. There are two possible ways in which this can be done, either; somatic cell nuclear transfer and cloning or reprogram the somatic cells of the patient to adopt an embryonic stem cell fate (essentially dedifferentiation of somatic stem cells).

Therapeutic Cloning

Therapeutic cloning was considered as a strategy to culture embryonic stem cells which are genetically identical to their intended recipient. It is also known as somatic cell nuclear transfer (SCNT), this strategy differs to typical reproductive cloning as no new individual is produced as a result. The method of SCNT is as follows:

• Isolate the nucleus from the cell of the patient and combine this with a donor oocyte whose nucleus has been discarded  – thus producing an oocyte genetically identical to the patient
• A mild electric shock can stimulate the development of the oocyte
• The oocyte then develops in the typical manner until it reaches the stage of a blastocyst
• The embryonic stem cells can then be cultured from the blastocyst in the manner described earlier
• The embryonic stem cells can have different growth factors applied to them allowing them to differentiate into the required cell type

Following the example from earlier, if this method was followed to produce insulin-producing cells and these cells were then transplanted into the patient, no immune rejection would occur as the cells would be genetically identical to the patient.

There are problems with SCNT however, for example there are limitations on the number of donor oocytes available. More importantly though, the process is actually very difficult to carry out and despite a number of attempts, this procedure has never been successfully used to produce human embryonic stem cells. It is believed there is a difference between the oocytes of primates and other mammals, which is why problems have arisen when attempting to perform SCNT on human oocytes. Another concern is that although the resulting cells would be genetically identical, the mitochondrial DNA is always derived from the mother i.e. the oocyte; this could pose problems in some cases.

This procedure has however been performed successfully in mice, a mouse with a blood disorder caused by a genetic defect was cured following the above method. When the stem cells were isolated, the defective gene was repaired in vitro and the stem cells cultured into bone marrow multipotent stem cells. Upon transplantation into the mouse, the stem cells differentiated to blood cells which expressed a regular copy of the gene and the mouse was cured.

Human/Animal Hybrid Embryos

It has been theorised that non-primate oocytes could be used to generate human embryonic stem cells. Even though this would raise a number of ethical issues UK legislation deemed this technique legal. Despite this, a lack of funding has prevented any further research into this technique. The study aimed to develop stem cell lines from people with genetic forms of neurodegenerative conditions, including Alzheimer’s disease, Parkinson’s disease, motor neurone disease, and spinal muscular atrophy.

The method for producing human/animal hybrid embryonic stem cells would be exactly the same as with producing human embryonic stem cells by SCNT, however the oocyte would come from a mammal such as a cow instead of a human.

Reproductive Cloning

Although similar, reproductive cloning differs from therapeutic cloning in that a new individual is produced. The method of reproductive cloning is the same as SCNT; however the oocyte of the donor (which contains the nucleus of the individual to be cloned) is only allowed to reach the 4-cell embryo stage and not the blastocyst stage i.e. development in vitro is stopped earlier. At this point, the 4-cell embryo is implanted into a surrogate mother where it continues to develop normally. If the process was successful, the surrogate mother will give birth to a genetically identical clone of individual who’s DNA was transferred into the donor oocyte.

Again, because of the difference in primate oocytes all attempts at cloning thus far have been unsuccessful.

Induction of Somatic Stem Cells to Adopt an Embryonic Stem Cell State

As an alternative to SCNT, it was thought that it may be possible for differentiated somatic stem cells to revert to an embryonic stem cell state. It was theorised that it may be possible to essentially reprogram somatic stem cells to revert to pluripotency.

A group led by Japanese scientist Shinya Yamanaka tested 24 candidate genes which may have been able to induce pluripotency in the somatic cells. Experiments conducted on mice cells indicated 4 genes which were necessary to generate embryonic cell colonies in vitro. These colonies of embryonic stem cells became known as induced pluripotent cells (iPS cells). The first iPS cells were generated from fibroblasts thus showing it was possible for a differentiated cell to dedifferentiate.

The four genes required to induce pluripotency were; Oct4, Sox2, c-Myc and Klf4.

To prove that the iPS cells truly were capable of pluripotency, green fluorescent protein tagged iPS mice cells were implanted into a blastocyst. This blastocyst was then implanted into a surrogate mother mouse. If the blastocyst had used the iPS cells to develop, then when exposed to fluorescent light, the offspring would glow a green colour. When tested the mice produced the green glow proving the pluripotent capabilities of the iPS cells.

Advantages of Yamanaka’s iPS strategy over SCNT are that; it works in primates and is easier to perform. Also there is no need for human oocytes or the development of an early stage embryo (reducing ethical issues) and because all genetic information is obtained from the donor, there is no issue concerning a mismatch mitochondrial DNA that would otherwise have been adopted from the oocyte in SCNT.

Ethical Issues

Human Reproductive Cloning

Whilst no-one has yet been successful in cloning a human by reproductive cloning techniques, there are a number of reasons why it is actually quite dangerous. The cloning technique has not been perfected in non-primates yet, and often, cloned animals are born with severe medical problems such as birth defects and deformities as well as life threatening diseases – however the majority die even before birth. Giving birth to a cloned animal also puts a lot of risk on the mother and offspring which do survive have a very limited life span.

The fact that the technique has not been perfected yet is of course of great concern for those who are considering experimentation with cloning humans, any cloning of humans is effectively human experimentation and is thus morally indefensible.

Use of Embryos

Therapeutic cloning requires the creation of a viable embryo which would go on to form a human life. Instead, the embryo is dismantled and the embryonic stem cells removed – thus preventing the formation of a life, however a human life is often regarded as starting at day 14 following fertilisation, 7 days after the final chance to extract embryonic stem cells so is destruction of an oocyte actually the cessation of a human life?

Embryos from IVF programs are often used in embryonic stem cell research. These are embryos left over at the clinic following successful implantation in the mother (multiple embryos are often created to increase the probability of a successful pregnancy). Some suggest it is wrong to destroy these embryos and use them to extract embryonic stem cells; however, these cells would have been discarded or used for alternative research anyway. Whatever the outcome, the embryo would not have been used to permit another pregnancy.

The importance of the research at stake in my opinion far outweighs the cost of using these 5-7 day old embryos for research. The possible breakthroughs in medicine which could be made as a result are definitely exciting possibilities.

References

Stem Cell Basics: Introduction. In Stem Cell Information [World Wide Web site]. Bethesda, MD: National Institutes of Health, U.S. Department of Health and Human Services, 2009 [cited Monday, May 09, 2011] Available at http://stemcells.nih.gov/info/basics/basics1

Stem Cell Basics: What are embryonic stem cells? . In Stem Cell Information [World Wide Web site]. Bethesda, MD: National Institutes of Health, U.S. Department of Health and Human Services, 2010 [cited Monday, May 09, 2011] Available at http://stemcells.nih.gov/info/basics/basics3

Stem Cell Basics: What are adult stem cells? . In Stem Cell Information [World Wide Web site]. Bethesda, MD: National Institutes of Health, U.S. Department of Health and Human Services, 2010 [cited Monday, May 09, 2011] Available at http://stemcells.nih.gov/info/basics/basics4

Stem Cell Basics: What are the potential uses of human stem cells and the obstacles that must be overcome before these potential uses will be realized? . In Stem Cell Information [World Wide Web site]. Bethesda, MD: National Institutes of Health, U.S. Department of Health and Human Services,2009 [cited Monday, May 09, 2011] Available at http://stemcells.nih.gov/info/basics/basics6

Lack of funding prevents human-animal stem cell research in UK, Susan Mayor, BMJ 2009; 338:b207