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Population of undifferentiated cells

Background

The term 'stem' cell refers to a population of undifferentiated cells that has the potential of turning into any specialised cell of the body. These cells have two special characteristics. Firstly, they must be able to self-renew. When the cell divides during cell division, one of the two daughter cells produced is a stem cell. This method allows for the maintenance of the population of the stem cells since some cells need to be preserved in order for the cycle to continue. Another characteristic is their ability to give rise to differentiated cells. When a stem cell undergoes cell division, it gives rise to two daughter cells, a stem cell and a precursor molecule. This precursor undergoes another round of division before the daughter cells produced develop into a differentiated or partially differentiated cell type.

Embryonic stem cells are derived from the embryos. They have the potential to develop into any particular cell type whereas adult stem cells have a more limited potential. Here, the stem cells from one particular tissue type can only develop into another cell in the same tissue. They are generally required to give rise to stem cells that are used to replace those that are worn out or have been damaged.

The fertilisation of an egg and a sperm gives rise to a zygote. This zygote contains all the genetic information necessary for the development of a new individual. It is said that the zygote is totipotent as every cell has the capacity of turning into a differentiated cell. The zygote then changes into a small ball of cells known as the blastocyst. The blastocyst consists of an inner mass of cells (ICM) which develops to become various cells and tissues of the young embryo while the outer trophoblast, forms the placenta. Once the trophoblast attaches itself onto the uterine wall, pregnancy is established and the inner mass of cells, or embryoblast, becomes pluripotent. Pluripotency describes a state in which a cell may advance into more than one outcome. This period however, only lasts for a short while before the embryo begins to develop. Embryonic stem cells are known as ES cells. Human ES cells have a normal karyotype. They maintain high telomerase activity and possibly may allow for abundant expansion in culture.

Stem cells are also found in adults too. Cells found in the bone marrow, skin, gut and respiratory tract constantly give new stem cells, while areas such as the liver and muscle only contribute to replace cells that have been lost through normal senescence or damage. (Bioethics briefing UOE) There is a limited turnover of stem cells in the brain and pancreas. (Multilineage differentiation from human embryonic stem cell lines) Evidence suggests the presence of dormant stem cells exists in some organs. Scientists however, claim that these dormant cells rarely become active. Adult stem cells are described as multipotent. This means that the cell may give rise to daughter cells from multiple, but still limited number of lineages. In many adult cell types, stem cell activity decreases as the age of the individual readily increases.

The Derivation of Human Embryonic Stem Cell Lines

The totipotent zygote loses its ability to produce all the necessary tissues for implantation and foetal development when it divides allowing for the formation of the pluripotent mammalian blastocyst (Lines derived from single blastomeres of 2 4-cell sage embryos). Human embryonic stem cells, or hES cells, have the full potential of differentiating into any specialised cell in the body and so to investigate this further, several experiments have been carried out in the last few years.

Studies have been carried out in order to improve culture conditions, genetic alterations and differentiation techniques to produce cells viable for transplantation or even drug testing. (Derivation, growth and application) However, the very first step would be to produce uncontaminated, healthy cells. In order for successful derivation of human embryonic stem cell lines, it is essential that there are a sufficient number of embryos that are collected. Often the cleavage- stage embryos that have previously been derived by and left over from in vitro fertilization treatment are donated with the donors consent. Majority of the cell lines that have been established have been from blastocysts between day 5 and 8 of their development. With the advanced technology, these methods are being modified and various other techniques are also being applied to produce results with increased success rates.

The hES cells are obtained from the inner mass of cells of a blastocyst. hES cell lines were successfully derived from the ICM in 1998 for the very first time by Thompson et al. Five hES cell lines were derived and according to Thompson, they fulfilled his definition of a primate ES cell. The cells were obtained from the preimplantation embryo, the cells could continue to proliferate and remain in their undifferentiated state and even after prolonged culture they were able to differentiate into all three germ layers of a human embryo namely ectoderm, mesoderm and endoderm. (Derivation and spontaneous differentiation of human embryonic stem cells). These results were confirmed two years later by Reubinoff et al (2000) when two other ES cell lines had been established. (Derivation, growth and applications).

To acquire the cells from the ICM, the trophoblast layer of the blastocyst is removed mechanically or using immunosurgery and inner cell mass is then plated on mitotically inactivated mouse embryonic fibroblasts (MEFs) (Derivation, growth and applications) (Derivation and spontaneous differentiation of human embryonic stem cells. Amit M 2002) These are then cultured. Using symmetry division, the cells can continue to preserve a stable population of stem cells. They are also capable of dividing asymmetrically giving rise to two daughter cells. While one daughter cell resembles the mother, the other can potentially differentiate into any cell of the early embryo.

Using the mechanical method, the separated ICM cells either differentiate or produce cells that share similar morphological characteristics with hES cells and they test positive for alkaline phosphatase staining. They also have normal karyotype. A popular method nowadays is immunosurgery. This involves the removal of the trophectoderm cells with the aid of antibodies that have been raised from either, BeWo cell, human serum or red blood cells. The latter approach is extremely reliable for the removal of all the trophectoderm cells since in any case if there were any cells remaining, they would inhibit the ICM from forming. However an advantage the former process has is that it does not involve any antibodies, so the protocol remains relatively simple and uncontaminated from animal-derived products. (Derivation, growth and application)

It has been proven that even aneuploid zygotes can also be used as a source for the generation of normal hES cell lines and aneuploid hES cell lines. The latter could prove to be useful for any types of research. Normal zygotes carry two pronuclei, one representing each parent. Some zygotes however, that were given rise to by IVF carry one or three pronuclei. An advantage about the method that is used to obtain these cells is that they do not require the mechanical method or immunosurgery. Blastocysts that were derived from aneuploid zygotes often display an unusual morphology where it is difficult to be able to distinguish between the ICM and the trophectoderm. Hence the method required the digestion of the zona pellucida and whole blastocycts were placed on inactivated MEFs. (Derivation of a diploid human embryonic stem cell line from a mononuclear zygote 2004 Edith Suss-Toby

Human embryonic stem cell lines have the potential of being used for regenerative medicine. For this reason, it is much preferable that a technique is used that does not require the use of animal products. Nearly all of the experiments that have been carried out in order to develop the cell lines have used animal products. Immunosurgery requires the use of products such as anti-human anti-serum and guinea pig complement. Some claim that immunosurgery must be circumvented for future clinical trials since the animal derived products may contaminate the ICMs and the cell lines that are derived from there. On the other hand, the mechanical method depends on the technical skills of the individual carrying out the dissection, the chemical dissolution may damage the cells. Dissection by a non-contact laser may be the next best alternative for hatching and dissection of ICM cells from the TE cells. This approach is as efficient as the efficiencies suggested by the immunosurgery reports. (Laser-assisted derivation of human embryonic stem cell lines from IVF embryos after preimplantation genetic diagnosis T. Turetsky 2007)

Another method of the derivation of hES cells is from embryos that have been reconstructed using the somatic cell nuclear transfer techniques (SCNT). This was conducted by Hwang et al (2004). The method requires the transfer of the nucleus of a somatic cell into an enucleated donor oocyte. The cytoplasm of this oocyte would then silence all the genes of the somatic cell, and activate the genes involved with the development of the embryo. The ICM cells would then be obtained from the cloned preimplanted embryo. (Evidence of a Pluripotent Human Embryonic Stem Cell Line Derived from a Cloned Blastocyst Woo Suk Hwang 2004)

Success rates in driving hES cell lines depends on the condition of the blastocysts, the conditions they were isolated in, and the experience of the group. It is difficult to point out the most successful method since some research groups publish the number of embryos donated while others only mention the numbers of blastocysts used. Similarly, it is also very likely that the published results are not fully accurate. While some research groups may have used numerous embryos but were not able to generate any hES cell lines, others may have successfully derived the cell lines but the results were not 'deemed successfully innovative' and so results were not published. (Derivation, Growth and Application)

The Growth of Undifferentiated Human Embryonic Stem Cells

The culture of ICM cells constantly requires the feeder layers. Any cultures that take place on MEFs need to be replaced every two weeks so that they can continue to support the undifferentiated and proliferative stem cells. (Culture and Maintenance of Human Embryonic Stem Cells) Some researchers believe that the feeder layers are not the optimum medium for the expansion of the culture as there is a high probability of the contamination by cross-transfer of infectious agents. Under feeder-free conditions, there have been significant differences in the expression of some genes and telomere length. Due to these differences, it is suggested that the media is suboptimal for the differentiation of hES cell lines. (Derivation, growth and application) However, once the cultured cells have been removed from the feeder layer, they are then put into a suspension culture and the hES cells aggregate into clumps of differentiated and undifferentiated cells. These multicellular groups of cells are known as embryoid bodies, EBs.

The EBs progress through a series of steps; between day 7 and 14 of post differentiation development, they grow to become cavitated and cystic EBs. Human ES cells may develop into a trophoblast in culture and also produce a-fetoprotein and hCG into the culture medium. The culture study ultimately would produce post-mitotic terminally differentiated cell types but this would depend on the conditions they were raised in. Here it is apparent that hES cells in culture represent a live model that may be used to study placental development. (multilineage differentiation from human embryonic stem cell lines) It is claimed that human ES cell lines have an advanced and a very consistent development.

It has been observed that while the embryoid bodies were developing, the cultures showed evidence of different morphologies. This included contracting cardiomyocytes, pigmented and non-pigmented epithelial cells and developing neural cells. According to Schuldiner et al, undifferentiated ES cell and differentiated EBs expressed receptors for numerous growth factors that would effect the development of the embryo in vivo. However, none of these growth factors allowed for differentiation to one specific cell type. (multilineage differentiation from human embryonic stem cell lines)

It is not only the feeder layer that acts as a setback for large scale production of hES cell lines, another hindrance is the time period for the population of hES cells to double. It takes approximately 36 hours for the number of human embryonic cells to increase in a twofold amount. (Derivation, Growth and Application)

A study claimed that on running an array-CGH to check for genomic rearrangements, duplications and deletions were found in the blasotocyst-derived hES cell lines. A process called G-banding was then used for a detailed observation. This was done by staining condensed chromosomes in order to observe the karyotype. It was believed that the abnormalities were not due to the mother's age, in fact the results of the experiments that were carried out strongly suggested that they were acquired in culture. (Human embryonic stem cell lines derived from single blastomeres of two 4-cell stage embryos Mieke Geens) Often, the prolonged in vitro culture, leads to high differentiation rates and genomic instability. There is evidence of other cell lines exhibiting trisomy, aneuploidy and aberrant X chromosomes. (Derivation, Growth and Application)

Evidence suggests that each individual hES cell line carries a unique genotype. This unique genomic sequence can be traced to the ICM of the blastocyst and it is expected that each cell line may be determined by the distinctive genomic variants it carries. In order to see whether or not each cell line expressed signature genes, reflective of the variants carried by each cell line, an assessment took place. hES cell lines were differentiated into the endoderm, mesoderm and ectoderm and the similarities and differences of gene expression were recorded. (Unique gene expression signatures of independently-derived human embryonic stem cell lines) It was clear that even though all hES cell lines were cultured under same conditions they still did not have the same genetic profile. When Abeyta et al. (2004) were comparing three hES cell lines, it was concluded that 52% of the genes found were expressed in all cell lines and 48% of the genes was only expressed in one or two cell lines. (Derivation, Growth and Application)

The initial gene expression studies on hES cell lines suggested that the derivatives may be induced to express endoderm genes. This includes, pancreatic islet genes and insulin, somatostatin, glucagon and hepatocyte nuclear factor 3 beta. It is however, still not known if the complete differentiation into pancreatic cells will lead to the growth of a population of adult islet cells and the production of insulin. Nevertheless, the data suggests that the ES cells can activate the genes required for the development of all three EG layers in culture. (multilineage differentiation from human embryonic stem cell lines)

Therapeutic Potential of Human Embryonic Stem Cells

Although the study of hES cells is still in its developing stages, there is much hope for the success of the research. Theoretically, the study can allow the production of an inexhaustible source of cells that can easily differentiate into any other type of cell. They may be used to treat malignancies, degenerative diseases, genetic diseases, inflammations and infections and trauma.

hES cells can differentiate into cardiomyocytes from the embryoid bodies. Since the approaches that have evolved have a high yield from murine ES cells, the very same protocol is used in order to directly derive cardiomyocytes from hES cells. One such method was carried our by the use of 5-aza-2'deoxycytodine which enhances the differentiation. The myocytes produced however, are immature and are consistent with foetal myocytes in terms of their structure and growth. This suggests that the developmental pathway it that of an embryo rather than an adult.

In order for the myocytes to become clinically utilised, methods have to be able to yield a bigger and purified population of cells. hES cells could potentially become a source for engraftable heart cells, or other tissues. For engrafting to be possible, one specific type of cell is required. An example would be in the case of a myocardial infarction, ventricular myocytes would be required. If instead a sinus-nodal type of cell was added, it would prove to be lethal.

Ideally, the hES cells derived, would be transgenes free in order to remove any possible method of contamination that would come in the way of the experiment. However, the transgenic pathway may be used to understand the developmental pathway better and this could be helpful to develop culture conditions that would aid the research without the use of transgenes. Perhaps the use of a suicide gene would be more useful. This way in case any of the grafted hES derived cells malfunction, a drug can be administered to eradicate the grafted cells.

Hematopoietic stem cells (HSCs) are the best understood cells which have been characterised and experimented with to a great extent. These cells do not support long term transplants until recently it has been discovered that genes that enhance hox genes such as hoxB4 play a major role in promoting high blood chimerism in animals with transplants. While some researchers suggest the pathway can cause the cells to acquire properties that are characteristic to adult hematopoietic other studies suggest the pathway gives rise to a particular stem cell unpredictably during cell differentiation. However, studies must be carried out regarding how to reproduce these hematopoietic cells under specific conditions that will lead to an efficient yield. The bone marrow has been relatively easy to study since HSCs have been readily accessible. Some niches however, are not accessible, that these cause major obstacles.

It is essential that the niche of the stem cells is adequate for self-renewal, growth and differentiation. In cases such as Fanconi's anaemia, a transplant could cure the disease when the bond marrow fails due to an intrinsic defect of the hemapoietic stem cells. Any disease that would destroy the stem cell niche, for example myelofibrosis, a bone marrow transplant would not be able to fixate the damage. Other conditions would include hepatic cirrhosis and burns. Although the mentioned conditions might be able to benefit from the therapy slightly, for maximised results they will have to be accompanied with therapies that are aimed at pathophysiologic process of the cell that is being replaced.

The development of the of the central nervous system takes place in early cell differentiation, this is evident as neuronal tissue is present in the embryoid bodies. The neuronal tissue is a very specialised tissue where each type of cell has specific functions hence, it is essential that directed derivation is achieved. However, there are several hurdles that must be overcome, since the derivation was not the only problematic portion. The cells must be surgically inserted into at the appropriate site. Unlike the cells of the hematopoietic system, the cells here do not come back to their correct location via the bloodstream.

Although the synaptic connections are plastic, the system starts forming during foetal development. In order for efficient functioning to take place, the newly synthesised cells will have to integrate into the existing, and fully formed neurons. One hypothesis states that perhaps the cells could be delivered in a precursor state which will develop into a proper niche when targeted and will grow in vivo. An advantage to this method would be that the growth of the embryonic stem cells would aid in the formation of the plastic connections. The addition of adult neurons could prove to be more complicated and integration might not be able to be re-established. This could result in epileptiform and it could develop inappropriately in situ giving rise to various defects and tetratoma formations.

A major issue is that transplantation often leads to tissue rejection. The immune system often responds to any alloantigen on the transplant. This may be in the form of histocompatibility complexes and ABO blood group antigens. In an undifferentiated state, the stem cells would express a low amount of MHC-1, however, in differentiated cells in vitro there is an increase in expression by up to 2-4 fold. Although this expression is much lower than that in the somatic cells, the increase can cause the hES cell not to be a HLA match and hence, destroyed in a manner that is dependent on T cells.

One way of overcoming this is by creating hES cell banks however, a disadvantage is that the allele combination of the three MHC-1 genes can generate upto 11 million haplotypes and a diploid combinations. Perhaps genetic engineering could be used as a method that would allow for the formation of immunologically matched cell lines, this strategy however, is only theoretical. Therapeutic potential of embryonic stem cells. Paul H.

Ethical Issues Revolving Around Human Embryonic Stem Cells

Most of the debates that revolve around embryonic stem cells are related to whether or not it is morally right to stop embryonic development. Those who oppose the idea believe that embryos should be allowed to develop. They regard the embryo as an individual the moment the zygote is formed. Each zygote turns into a blastocyst that is implanted in the uterus, and each foetus grows to become an individual. They believe that from this moment onwards the zygote carries a unique DNA sequence that has never existed before, and it never will again. Although, the genotype is only one element of an individual, in some cases, some advocates label this as murder.

It is true that according to science a life is formed at the instant that the egg and the sperm fuse. Yet it is also true that we do not have an obligation towards the embryo at every stage of its development. Our responsibility increases as the embryo develops into an individual. So, conclusively it can be said that the price of an embryo is a much smaller price to pay than the price of an adult individual. Human embryonic stem cells -- the German debate. Engels EM.

Even though the embryo has the right to live from the very start, those who oppose human embryonic stem cell research and defend the embryos claim that 'life does not depend on any of their cognitive or bodily features'. Human embryonic stem cells -- the German debate. Engels EM Supporters believe that just because the embryo does not have the ability to reason or communicate is not a valid reason not to protect the embryo. They believe that only if the mother's health is under life threatening conditions, only then can the baby be aborted and it will be ethically justified.

Techniques such as SCNT are seen as criminal offences in countries such as Germany. The transfer of genetic information of one embryo into another embryo, foetus or individual is considered illegal since it does not act to be in favour of the protection of the embryo. Human embryonic stem cells -- the German debate. Engels EM With this opinion, whether the stem cells are left over from IVF or if they have specifically been created for the experiment also does not prove to be a valid reason for destroying an individual. Although the whole purpose of the study may be to cut down the suffering of many people, some believe that the embryos must not pay this necessary cost of life. Some people are certainly against this practical approach. UOE bioethics briefing)

There are however, a few other reasons as to why some people are against the use of embryonic stem cells. It is claimed that if stem cells are allowed to be cloned, this may lead to reproductive cloning to be allowed since it leads to the devaluation of human life. Many argue, that perhaps instead allowing research on embryonic stem cells, perhaps experiments should be carried out using adult stem cells. UOE bioethics briefing)

When stem cells are donated, there are various issues that also have to be dealt with. For example, the consent form, the availability of donors and ownership of cell lines established are only some of the problems that are involved with adult stem cells. Apart from these issues, in order to treat patients with stem cell therapy, there must at least be sufficient cells to complete the treatment. While in some cases there are no stem cells for a particular cell or tissue, in other cases they may be extremely rare. The beta pancreatic cells for example, researchers have still not been able to find the stem cells in the body. (Perspectives on Human Stem Cell Research KYU WON JUNG*)

The extraction of the stem cells from other cells may be complicated but the proliferative potential and the amount of time they remain active for is much less than that of the hES cells in vitro. Another major disadvantage is the lack of opportunities for the study of embryogenesis if adult stem cells are used. Taking all this into account, adult stem cells do not serve as a sufficient alternative. (Perspectives on Human Stem Cell Research KYU WON JUNG*)

Some fear that in case the use of 'excess' embryos are allowed, over time, the public would take this for granted and would find the desire to approve of them. On the other hand, if these embryos are viewed as occasional and unavoidable, it would maintain the symbolic value of an embryo in the society. Human embryonic stem cells -- the German debate. Engels EM

Alternatively, some may believe that since embryos in some places are not granted the option to be used, perhaps they can be imported. If ethically, the use of embryos is allowed, it would be appropriate for the import of stem cells. Equally, in places where the research on stem cells is strictly prohibited, the import could still be allowed since the damage is already done. However, this may be an incentive for the destruction of another embryo since the approval of importing stem cells could also be viewed as a moral approval for the research. If the import was allowed to take place in countries that did not approve of the derivation of the stem cells, there would be some ethical inconsistencies. Human embryonic stem cells -- the German debate. Engels EM

However, there are plenty of arguments for the use of embryonic stem cells. Approximately 80% of human embryos do not attach themselves to the uterine wall and pregnancy is not successful. Thus, it can be said that although each embryo does carry a unique genotype, it is not certain that each embryo will eventually become an individual. Also, most scientists consider human life to begin at the appearance of the primitive streak; the point at which neural development is initiated. This development takes place at approximately day 14 of fertilization even though there is no single consensus that confirms so. (Perspectives on Human Stem Cell Research KYU WON JUNG*)

Alongside this, it is only after several rounds of cell division that the cell lines are designated to either the placenta or embryo. Even after this, the zygote might divide giving rise to twins but still maintaining the fact that that at this stage the embryo can still not be considered as an individual. In various organisms, studies have indicated that due to genetic mosaicism, it is not very often that two embryos merge together giving rise to one foetus.

In many cases, parents do allow research to be carried out on the embryos that are left over from IVF. However, if parents do not give their consent to this, the embryos would be frozen in storage and would then be discarded years later. (UOE bioethics briefing) With continued stem cell research several medical problems could be treated and even cured. Parkinson's disease, type I diabetes, spinal cord injuries, and birth defects are only some of the conditions that can be resolved. (http://www.experiment-resources.com/stem-cell-pros-and-cons.html)

Often, religion plays a major role in ones belief. For example, in 2004, when George Bush was elected president of the United Sates of America, the protestant Christian and the Republican party opposed research on stem cells. (UOE bioethics briefing) Claiming to defend human life to those embryos that were going to be discarded at fertility clinics, Bush only allowed the use of 21 stem cell lines that had been produced before his decision. However, President Obama removed this ban against the research on embryonic stem cells in 2009 since "medical miracles do not happen simply by accident," and promised that the lost ground would be made up for under his administration. (http://www.cbsnews.com/stories/2009/03/09/politics/100days/domesticissues/main4853385.shtml)

Islamic Iran is a country where the religious Grand Ayatollah, has highest religious and legislative power this is very different when compared to the other Islamic countries. Most of the country's judgement and practice is dependent on the religion. Islam describes the holiness of the embryo in the womb in the Quran. In 2002 however, the Grand Ayatollah declared that stem cell research may continue in Iran. Saudi Arabia on the other hand perform IVFs on a regular basis, however, producing embryos in order to destruct is strictly prohibited so only embryos that have been legally aborted can be utilised. The idea of using supernumerary IVF embryos for research is still debatable. (Ethical Aspects of Human Embryonic Stem Cell Researchin the Islamic World: Positions and Reflections )

Conclusion

Human embryonic stem cells have become prevalent in today's society due to their priceless properties. With their ability to self-renew and produce multilineage cell lines, these cells have the potential of carrying the name of science to a new level. Their ability to limitlessly supply have made their research field a rapidly developing one. Researchers are not only working to deriving the cells but maintaining them too. Experiments are being carried out in the hope to find answers to various diseases from paralysis to Alzheimer's disease.

While discussing ethical and legal issues, it is imperative that the science that takes place behind it is kept in mind. As science progresses, the ethical and legal system also changes. Everything has its own benefits and limits. Given that there are strict guidelines overlooking all the possible issues which may cause a problem, all researches should be able to co-exist freely. For this a world wide regulated system should be effective. One that would include rules and regulations, mentioning how many days old a blastocyst has to be in order for it not to be used anymore or if countries may import stem cells.

Since nobody is sure of the outcomes of the experiments, perhaps the research should continue and we should not stand in the way of science. All knowledge is acquired. It is difficult to say if the knowledge will be misused or not, but this risk is carried in all arenas. It should be kept in mind however, that stem cells are found in many parts of the adult body. If adult stem cells can be reprogrammed to be able to differentiate into any cell of the body, perhaps embryonic stem cells would not be required thereafter. I believe we should move forward with the values we have been given by our ancestors and while doing so, we must keep human welfare and this invaluable research tool in mind.

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