• Hugo Creeth

Stem Cells - Basics

Introduction


Stem cells were first discovered 25 years ago in mice. In the two decades that have follows the technological advances and scientific understanding of these remarkable cells has been enormous. It is now possible to isolate and grow stem cells from humans. These media labelled ‘miracle’ cells offer real promise for improving therapies and even finding cures too life threatening and/or debilitating diseases. It was only recently that a paralysed Dachshund had its ability to walk reinstated thanks to a stem cell graft from its nose3. Despite this promise they have also been masked in controversy, due to various scientific misconduct and fraudulent claims. This has consequently only added to the very real ethical issues and questions that stem cell research raises in both the scientific and public sector.


So what are stem cells?


It is well known that all cells come from cells. This is undoubtedly one of the central dogmas of cell biology and quite reasonable when considering lower organisms such as bacteria and other prokaryotes. However it is astonishing that this is also the case for multi cellular organisms such as humans who are made up of millions of different cell types and hundreds of different tissue types. So the question is how does this happen? It is stem cells that have the answer.


Stem cells are a pluripotent cells that have the ability to self renew and also crucially differentiate into multiple cell lines or a particular cell lineage. Thus stem cells essentially remain in a state of perpetual inertia before a stimulus causes them to divide in such a way that they become, for example, a new muscle cell or neuron. The fundamental reason why these cells are of extreme clinical relevance is that, if they can be controlled and manipulated in certain ways, their extraordinary ability means that they may ultimately be a renewable source of specialised cells that could be used for organ transplants too cellular regeneration and also, importantly, target validation and drug discovery.


There are two main types of naturally occurring stem cells. Firstly there is the immortal embryonic stem (ES) cell. It is these cells that are completely undifferentiated and can therefore virtually become any cell in the body of an organism4. ES cells also have the ability to grow and grow forever. A characteristic of all stem cell types brought about by (amongst other things) high telomerase activity, which prevents senescence. The second type of stem cell is the adult stem (AS) cell. These stem cells have already differentiated to a certain extent and are therefore locked into a certain cell lineage. This means that AS cells may be specific to bone marrow, or muscle and can therefore only differentiate further into more specific cell types under that lineage and not out of that cell line4. Both these types of stem cell are attractive in terms of the development of medical practices. ES cells are most powerful in terms of the sheer variety of cells they are capable of producing. Whereas AS cells are not known to exist for every organ/tissue type, thus they lack this diversity and versatility at present, although because they are already directed towards a known fate they may often be the most appropriate means of generating a specific tissue.


Stem Cell Niches


Stem cells exist in niches. These niches are the environments that stem cells are apart of. It is where it continually self renews and replicates. It is this microenvironment that also provides the external stimuli necessary to alter gene expression within the stem cell ultimately causing it to differentiate in a diagnosed way. David Scadden in Nature defines a stem cell niche as being ‘specific anatomic locations that regulate how they participate in tissue generation, maintenance and repair’. He goes on to emphasise the importance of this regulation in order to prevent exaggerated stem cell generation that may cause tumours and cancerous growths, whilst also preventing their depletion6. According to many experts it is the understanding and controlled manipulation, not just of stem cells themselves, but of their niches that is fundamental to the future of stem cell therapy.


Major Clinical Uses, Issues and Controversy


There are various clinical areas that are major targets for stem cell science. These are characterised under the headings: drug discovery, target validation and stem cell therapy. This diversity may imply that differing methods are employed by each area in order to tackle and extract knowledge surrounding closely related or associated types of human disease. However this is not the case, very often similar, or indeed the same, approaches are used in order to create the relevant stem cells. Then the differences arise in the way in which these stem cells are utilised in the pursuit of developing revolutionary treatments or cures for human maladies.


One stem cell technology that is of great significance for all three areas, in various ways, is the production of new stem cells and stem cell lines through the process of nuclear transfer cloning (NTC). NTC is also referred to as somatic cell nuclear transfer (SCNT) and is the process whereby the nucleus of a somatic cell is inserted into an egg cell that has had its nucleus removed and then using an external stimulus, stimulating it to divide eventually forming a blastocyst from which embryonic stem cells can be extracted7. It was this method, or one that was incredibly similar, that was used to create Dolly the sheep. The resulting organisms that are produced are genetically identical to the parent organism, which is SCNTs primary aim. SCNT has two major uses these are: embryonic stem cell research and potentially regenerative medicine (therapeutic cloning). In relation to human disease however neither is currently possible, as no SCNT cell lines have been generated10. Theoretically the technique would work via the creation of genetically customised stem cells that have genes associated with or coupled too a particular disease. That is if an individual with Alzheimer’s was to provide somatic cells the resulting ES cells would carry genes that were linked to this disease meaning they could potentially be studied to gauge a better understanding of the disease and even develop drugs to combat it, something that has also been proposed for multiple sclerosis. Likewise these customised ES cell lines could be effectively used to make specific personalised cells or indeed tissues and organs that could be used for transplantation into a patient, the advantages of such a method would be that the cells would be genetically and functionally identical to the individual and thus would overcome the major problem of immune system rejection of an organ12. The difficulty faced in this sphere however, as with most ES cell research, is that it is unbelievably hard to manipulate these cells to differentiate in a specific way. This is due to their extreme plasticity and pluripotency, which results in unexpected and hard to control differentiation.


As already mentioned in relation to human SCNT cell lines, this has not yet been achieved. Yet back in 2005 a South Korean laboratory published a paper claiming to have produced the first successful human SCNT cell line from patients suffering from type 1 diabetes, spinal cord injuries and immune disease. Professor Hwang Woo-Suk, who led the team of scientists that supposedly made the discovery, has however since been shown to have lied about the data. The lines were actually generated from an organism that reproduces by parthenogenesis. This controversy has since shrouded the technique in a bad light and it is only being pursued in a small number of research bases around the world.


So despite the huge possibilities this technique may give to stem cell research and therapeutic medicine it has been somewhat superseded by novel approaches to creating stem cell lines in humans. This new direction was validated in 2007 when scientists successfully reprogrammed normal human body cells into stem cells2,13. These cells called induced pluripotent stem (iPS) cells were previously produced in mice tails through the incorporation of four genes (oct4, sox2, c-myc and klf4 – Figure 1). This snowballed and in the two years that followed one group generated iPS cells from an elderly women suffering from Lou Gehrig’s disease, while another reported iPS cells for ten varying disease types. This was an incredibly important step in stem cell technology as many of the diseases that were made into iPS cell lines are extremely hard to model in animals. This being said the efficiency of the methods for producing reprogrammed cells needed major improvement as only around 1 in 10,000 cells were being successfully reprogrammed at the time. This efficiency has since increased to around 1 in 50 but can still be as low as 1 in 1000.

The applications for this technology are much the same as those proposed for SCNT cell lines and for stem cells in general. That is they may be used in regenerative medicine (including cell therapy), target validation and drug discovery. More specifically however the possibilities of iPS cells range from the generation of autologous pluripotent stem cells, cell therapy without immune rejection, gene therapy coupled with cell therapy too patient derived disease models and drug screens.


The most attractive aspect of embryonic derived, adult and induced pluripotent stem cells is undoubtedly their potential in regenerative medicine. From their first discovery this possibility has been very much the Holy Grail of stem cell technologies. In fact iPS cells have already been used in the treatment of sickle cell anaemia in a humanized mouse model through the harvesting of tail tip fibroblasts from the sickle cell mouse and then production of iPS cells followed by specific gene targeting of sickle cell mutation and differentiation of the corrected iPS cells into embryoid bodies and insertion back into the irradiated mouse17. iPS cells have also recently been implemented in promising studies highlighting their regenerative medicinal use in the repair of myocardium following myocardinal infarction. This is due to iPS cells extraordinary ability to generate multitudes of cardiomyocytes. The group behind this study was able to produce cardiac tissue sheets from mouse iPS cells. Immunostaining showed that these so called bioengineered myocardium were 99% pure and highly functional. This promise has huge implications on the future ways heart failure may be treated in humans once satisfactory screening is available to develop this concept18. Despite this massively exciting prospect and the widely held belief within the scientific community that this practice should become available to use in humans in the very near future, ethical issues and the possible dangers of such manipulation are slowing down the process. More imminent is the use of these stem cell technologies for the drug discovery process.


The drug discovery pipeline is a sequential procedure that begins with target validation. Target validation is a vital process in biotechnological and pharmaceutical development as it is the process of identifying disease-associated pathways and then designing specific molecular treatments in order to target the causal regions of the disease. The reasons why new more accurate methods of target identification and validation are needed and are so important is that in the development of new drugs there are huge financial costs, regularly costing in excess of £400 million. Also the process can take up to 15 years before drugs are approved for human use. Therefore improved in vitro human derived models will result in better precision, more cost effective assays and safer drugs with lower attrition rates. Stem cells offer such a novel approach in the identification of disease pathways and subsequently areas for effective drug targeting (Figure 2). iPS cells can be isolated from an individual with a chosen disease and then cultured. The resulting cells and tissue can then be used as a subsystem to test drugs upon and gain vital knowledge for the mechanisms behind the disease.



Figure 2: Potential clinical uses for iPS cells and indeed stem cells in general if learnt to be controlled.


Target identification can also be used to discover stem cell niches via the monitoring of changes in the cell cycle of specific cells and their proliferation. As a result these findings can be used to target specific cell mechanisms or indeed lineages for treatment purposes. Tracking cells in this way can be done using various techniques, including labels and cell markers such as: BrdU labelling indices, cell cycle antigens and telomerase/TRAP assays. Direct observation is also possible using reporter transgenes or through direct lineage observation, something that has been comprehensively performed in the nematode C. elegans. Viral vectors and immunohistochemical techniques are also used to identify lineages based on stage specific antigens19,21. The advantage over this approach compared to using the widely accepted and universally used animal model approach is that, often, animal models aren’t an accurate reflection of human disease processes and can provide false positives or indeed false negatives in pre-clinical trials. Currently animal models are a prerequisite to further trials, however as already mentioned the implementation of a stem cell approach might enable a faster target validation process and thus drug discovery and development. A recent example of a drug screening process using iPS cells involves the poignant socially relevant disease, Alzheimer’s. Alzheimer’s disease is a neurodegenerative disorder, which is characterised by steady memory loss and general cognitive decline. The cause of the disease is thought to be the deposition of amyloid beta-peptides in the brain. Yahata and his colleagues designed iPS cells to act as a model for the disease and then tested three drugs on the production of amyloid beta-peptide production. They found that the drugs reduced production of the peptide, however it also reduced the production of functionally important peptides produced from the same amyloid precursor protein that forms amyloid beta-peptide. This study demonstrates the validity of using stem cell derived models to screen drugs and gain relevant information that will lead to the development of more effective treatments.


The dangers of stem cell manipulation and iPS cells are not well known. However there is a real possibility of danger to human health if they are not fully understood and stringently controlled. This includes tumour development and other somatic mutations. It has also been suggested that iPS cells lack immunogenicity. In some experts opinions however these abnormalities are in fact just a characteristic of stem cells in general and not specific to iPS cells. This does however mean that if human manipulation isn’t controlled these abnormalities may become over expressed in future patients. The general consensus nowadays about the validity of iPS cells compared to ES cells and AS cells is that they are almost indistinguishable however the as mentioned the permanent switching on of genes that have recently been linked to cancer poses ethical considerations.

Undoubtedly the discovery of stem cells has been a remarkable step forward in the development of novel drugs to treat and therapies to cure a host of diseases. They are proving equally useful in the understanding one of the hardest to treat of all human diseases, cancer. Stem cells can divide to make more stem cells in a process called symmetric division, but also can sometimes divide in an asymmetric way so that one daughter cell begins to become specialised. It has been hypothesised that cancer is a product of symmetric division that has gone wrong. This is based around various other hypotheses that have shown genes, which promote asymmetric division to also be tumour suppressor genes4. While likewise genes that express symmetric division pathways have been highlighted as potential oncogenes when over expressed. Oct4 and Sox2 are thought to be minimally oncogenic while c-myc and Klf4 are strong oncogenes. This theory now seems increasingly plausible. Tumours are made up of a vast array of cell types. This heterogeneity expressed in the tumour has given rise to the view that there exist cancer stem cells. These cancer stem cells are thought to be of two, the cancer-initiating cell and the tumour-propagating cell. Research is now being undertaken in order to identify these stem cells so that the hypothetical symmetric switch within it allowing continual division and growth can be turned off using asymmetric promoter genes, thus killing the cancer at its source.


Conclusion


So overall it is self evident that stem cells have had a dramatic impact upon the approaches to medical discoveries in the past decade. It is also evident that their technologies are causing a paradigm shift in the way in which human disease is researched and subsequently going to be treated in the future. They are pioneering the fields of regenerative medicine and cell therapy as well as offering new more secure ways of developing drugs. Their power is obvious. Their dangers are less so. It is well known that with power comes great responsibility; it is therefore vital that scientists and politicians are sensible in the use of stem cells. This is so far what is happening with scaling up approaches to the application of this exciting science into the human scenario.

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