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This essay will primarily characterise the principle of cellular differentiation and how its understanding and application alongside stem cell research is proving to be advantageous in the fight towards treating cancer. By examining the extracellular factors and the signalling pathways involved in the normal development of a cell, it has been possible to assess the potential value of reprogramming a specialised cell back towards an undifferentiated state. This emerging field brings together the scientific uses of stem cell biology and developmental biology in order to understand and work towards cell-based therapies to treat diseases.
Cell differentiation has been defined by Svendsen et al (1) to be the "process by which the cells of a multicellular organism develop the specialised abilities required by each of the organism's several structures." This concept underpins the basis of how a single celled zygote divides and ultimately grows into an embryo that will constitute a complex system of tissues and organs. In mammals, only the zygote and the first cleavage blastomeres (2) have the ability to form all lineages of the organism, thus being described as totipotent. Development from here on sees the gradual reduction in differential potency of newly formed cells. The 5 day old blastocyst (3) is the first structure known to possess a lineage restriction. The inner cells of the structure, the inner cell mass (ICM), are able to develop into all lineages of the body; they however are unable to contribute to the trophoblast and are thus described as pluripotent. Cells can be further classified as either multipotent, meaning they are only able to form multiple cell types of one lineage, or unipotent, whereby cells can only form one cell type. These states of differentiation can be best understood in relation to stem cells.
Inner Cell Mass
Figure - Structure of a blastocyst depicting the Inner Cell Mass (4)
Introduction to Stem Cells
Stem cells are unspecialised somatic cells that are found residing within normal tissues. The human body provides many different sources of stem cells in areas such as bone marrow (hematopoietic stem cells), fetal tissue and even umbilical chord blood. These cells have the ability to regenerate and self-renew through cell division over a long period of time. A dividing stem cell will give rise to two daughter cells, one of which will retain the phenotype expressed by the mother, whereas the second will enter into a series of cellular divisions ultimately leading to a post-mitotic, highly differentiated state (5). The two most important categories of stem cells are embryonic stem (ES) cells and adult stem (AS) cells. In 1981, Evans and Kaufman were the first people to establish ES cells from the ICM of mouse blastocysts (6). Ever since, researchers have been looking into the molecular mechanisms that underpin how both adult and embryonic stem cells are able to self-renew and proliferate from a common progenitor, to give rise to cells of many lineages.
Figure 2 - Picture shows the potential for embryonic stem cells, derived from the ICM of a blastocyst, to differentiate into many lineages. (7)
Molecular Mechanisms involved in Cell Differentiation
Determining the molecular basis of pluripotency has led scientists to investigate the signalling and gene transcriptional pathways involved in self-renewal and differentiation. Leukemia inhibitory factor (LIF), a member of the IL-6 cytokine family (6), was found to promote self-renewal and inhibit cell differentiation by the up and down regulation of genes exclusively expressed within pluripotent cells (8). Removal of this signalling molecule saw the disappearance of the cells pluripotent potential and signs of differentiation became apparent. LIF was found to stimulate mouse embryonic stem cells (mES) via the interaction of the gp130 transmembrane domain. This can either lead to the activation of the Janus-associated tyrosine kinase (JAK) and signal transducer and activation of transcription (STAT) pathway necessary for cell self-renewal, or trigger the mitogen-activated protein kinase (MAPK) pathway, promoting cell differentiation (see figure 3). Identification of the transcription factor Oct4 and the homeobox factor Nanog (9) have been further implicated in being markers in maintaining the pluripotent state. Silva et al (9) deduced that "without Nanog, pluripotency does not develop, and the inner cell mass is trapped in a pre-pluripotent, indeterminate state".
Figure 3 - Diagram showing the JAK, STAT and MAPK signalling pathways induced by cytokines of the IL-6 family. (7)
Potential of Dedifferentiation and Nuclear Reprogramming
A landmark that has only just been discovered in the last few years is that of being able to re-establish pluripotency from a differentiated cell. New techniques are emerging that are allowing scientists to reprogram differentiated cells to an embryonic-like state (10). Researchers are using their understanding of the cellular pathways involved in differentiation to reprogram a somatic cell back towards the stage of an early progenitor. If successful in this area, research "would allow creation of patient- and disease-specific stem cells" (11). One could use this understanding and channel it towards deriving cell replacement therapies and screening for new therapeutic drugs. Two techniques that involve nuclear reprogramming and which are at the forefront of this field of medicine include the generation of induced pluripotent stem (iPS) cells, and also somatic cell nuclear transfer (SCNT).
Induced Pluripotent Stem (iPS) Cells
Induced pluripotent cells are a type of stem cell that were created by introducing certain embryonic genes into the nucleus of an adult somatic cell. In 2006, Yamanaka et al were the first researchers to successfully reprogram mouse embryonic and adult fibroblasts into a pluripotent state (2). They initially hypothesised as to whether the molecular factors and pathways involved in the self-renewal of ES cells would also play a key role if the process was to be reversed; so called "de-differentiation". They therefore selected 24 genes that they thought were vital in inducing pluripotency. Ultimately they deduced that by introducing the following four genes, Oct3/4, Sox2, c-Myc and Klf4 (10) into the nuclei of somatic cells, they could mimic the morphology and characteristics of embryonic stem cells; these newly created cells became known as induced pluripotent stem (iPS) cells. To confirm that these iPS cells expressed certain marker gene characteristic of ES cells, the cells were injected into nude mice. The resultant growth of a teratoma showed to contain tissues that had derived from all three germ layers (endoderm, mesoderm and ectoderm). This confirmed that these cells had been successfully reprogrammed to a pluripotent state and proved that now these cells had the same capabilities of embryonic stem cells.
Somatic Cell Nuclear Transfer (SCNT)
SCNT or "therapeutic cloning" is an in vitro procedure that aims to develop patient specific human embryonic stem cells. In 1962, John Gurdon was the first individual to show how the nucleus taken from an adult somatic cell can be directly reprogrammed back to display pluripotent potential, by transplanting it into an enucleated egg cell. The exact procedure involves the removal of the nucleus of a mature, somatic cell from a donor organism. The nucleus is then integrated into an enuncleated oocyte, which is then subjected to either an electric or chemical stimulus to cause it to divide (12). This developing technique therefore results in the creation of a clonal embryo, whereby embryonic stem cells can be harvested from the ICM of the blastocyst and a resultant stem cell line can then be derived. This method has shown to be beneficial to the forefront of regenerative medicine, as it has the potential to ultimately produce cures for currently untreatable diseases. The stem cells harvested from the embryo could be potentially engineered into differentiating into any cell type of a mature organism.
This technique has proved to be successful in deriving mouse embryonic stem cell lines. These stem cells were analysed and shown to possess the same profile of genes that are normally expressed within ES cells derived from fertilised embryos (13). Research into the mechanism behind this technique revealed that epigenetic changes occurring within the cytoplasm of the enucleated oocyte is what causes the reprogramming from differentiated to the undifferentiated state (13); a combination of DNA methylation, histone modification, chromatin remodelling and genomic imprinting are thus integral to the basis of reprogramming but detailed discussion of each of these are beyond the scope of this essay. Studies using SCNT have shown a high efficiency in deriving ES cell lines from mice and bovine embryos. However, studies have also shown that if the embryo was allowed to develop to birth, hundreds of abnormal genes were seen to be expressed in the newly born. Microarray experiments have been able to identify these abnormal genes and it is believed that any cloning-associated abnormality may arise from inadequate epigenetic reprogramming (1). Despite this, although many of the embryos do not produce live offspring, SCNT has still proved to establish pregnancies, form foetuses and even develop placentas, all of which the original differentiated somatic cell could not do.
Ethical issues surrounding SCNT
Therapeutic cloning differs from reproductive cloning in that the aim is not to generate a fully formed living offspring - a clone, but to culture an embryo in a bid to derive embryonic stem cell lines from it. Despite this, there are still a number of ethical issues that surround this topic. If this method was adopted therapeutically, the transferring of nuclear material to oocytes would require a large number of human eggs and at the moment there is a distinct shortage of eggs available. Furthermore, there are also ethical issues and laws surrounding oocyte donation, and until these are addressed, further progress into SCNT will always be hindered. Evidence is also suggesting that SCNT stem cells may never develop into a human even when implanted; therefore the resources and continual depletion of eggs, without justifiable evidence that the technique can be taken forward, seems unwarrantable. Even the clones that manage to survive to birth often develop with serious abnormalities and eventually die.
Proliferation of cancerous cells
Having discussed the mechanisms involved in cell differentiation, it is now important to understand how crucial this process is in the development of cancer. Cells within the body are best described to exist within two growth states (5); either in an undifferentiated or differentiated state. The distinction between these two is most clearly seen in cancerous cells. The cells tend to exhibit traits whereby differentiation is either completely or partially blocked. This is best observed using the example of chronic myelogenous leukemia (CML). This disease is characterised by the malignant overproduction of white blood cells or their immature precursors. CML is a slow developing disease that leads to the excessive accumulation of close to fully differentiated neutrophils within the blood. Despite this elevation in the number of these cells, the disease can remain for a few years without becoming life-threatening. However, after a period of 3-5 years, there is a sudden accelerated proliferation in the number of less differentiated blast cells. The mechanism leading to the normal differentiation of the cells is blocked; therefore huge numbers of these immature cells become trapped in this less differentiated state. Furthermore, it has been shown that the proliferative cells constantly dividing within the chronic phase of the disease were being constantly derived from "mutant self-renewing stem cells" (5). This evidence has highlighted the possible role of stem cells within cancer and thus has led to greater research into this area.
More studies are now being conducted into rogue stem cells and their involvement within certain types of cancer. Cancer is understood to be caused by abnormal and uncontrolled cellular division, which can result in the malignant growth of a tumour. It is now believed that stem cells may be an underlying cause to the growth of these tumours. This is due to their ability to regenerate and self-renew through cell division. Stem cells will usually divide asymmetrically giving rise to one daughter cell that will remain as a stem cell, and a second that is termed a transit-amplifying cell (see figure 4) (4).
Figure 4 - Diagram showing the assymetric division of a stem cell into two daughter cells (5)
In relation to cancer, the first daughter cell will remain quiescent within its tissue of origin, whereas the second daughter cell is what will proliferate and give rise to a number of progenitors. If these are exposed to any genetic abnormalities, as seen within a number of cancers, the resultant abnormal cells will form the bulk of the tumour. Cancer therapies such as chemotherapy and radiation aim to target these abnormal cells and ultimately destroy them. Drug therapies such as Glivec look to work in the same way by targeting the progenitor cells giving rise to the cancerous cells. These treatments are understandably effective as patients suffering from cancer go into remission for a number of years; however, later on in life, it is common for cancer to return. The best explanation for this is that, despite the number of cancer treatments available, none of these are managing to eliminate the cancer stem cells that are at the heart of disease.
To summarise the findings of this essay, we have managed to grasp an understanding of cellular differentiation and the changes that occur as a cell proliferates into being a specialised entity. By looking at the mechanisms and the mitogenic signals involved in cell differentiation, we have discovered the influence of the following four transcription factors, Oct3/4, Sox 2, c-Myc and Klf4 and their ability to re-induce pluripotency. At the moment, the idea of reprogramming is still in its early days; however the basis of therapeutic cloning and the formation of induced pluripotent stem (iPS) cells are viable. In terms of therapeutic cloning, we have found that ES cells derived from cloned embryos still have exactly the same potential for tissue repair as ES cells derived from fertilised embryos. Ultimately the purpose of this technique is to find cures and therapies for currently untreatable diseases. The positives seen within this method is that patients would receive their own stem cells; thus there would be no need for any donor match resulting in no complications through rejection. Despite this, a clear downside to the technique is that at the moment it is very inefficient (2). Most clones have died soon after implantation and the few that survive to birth have serious abnormalities and may die peri- or post-natally. Furthermore, there will always be an ongoing debate about the use and possible destruction of human embryos. The respect of an early stage human embryo is being placed against the potential benefit that this technique could offer society; the use of therapeutic cloning to derive embryonic stem cells will always necessitate the destruction of potential human life, unless other alternate methods are looked into.
Induced pluripotent stem cells can be derived from adult cells of any individual, meaning they alleviate any ethical issues that are normally associated with ES cells. Furthermore, as they are directly cultured from the cells of a donor patient, they would bypass any immunological rejection. Once again, deriving pluripotent cells that are immunologically matched to the donor would provide the best possible source of transplantable cells if needed to treat a certain disease state. Nevertheless, research into whether these iPS cells will be useful in treating human diseases is very limited. This technique has only recently been discovered, so further developments in this area could still be to come. Ultimately though, these induced cells have been obtained merely by awakening the capacity of self-repair that already existed in our genes. Such an advance in technology and understanding is really proving to be beneficial in modern medicine today, and the potential for these methods to be taken even further in the near-future is still evident.
Understanding the properties of stem cell proliferation and self-renewal to embryonic development, has allowed researchers to understand the mechanism behind abnormal cell division leading to cancer. There is a lot of promising evidence in the techniques that have been discussed, however at the moment medicine is still a long way off from being able to treat and cure cancer. Different cancers are caused by different underlying mechanisms and genetic alterations; it would be unwise to say that any one method could potentially treat all types of cancers.