Report on a talk by Professor Paul J Fairchild
By Patrick McCubbin
On Saturday 25th March, Professor Paul J Fairchild of the Sir William Dunn School of Pathology, University of Oxford, gave an informative lecture at Abingdon School, as part of the ATOM Festival [1]. Professor Fairchild described how pioneering stem cell techniques might be used in regenerative medicine with the potential to treat many degenerative and chronic diseases. He explained the defining properties of stem cells, and the advantages and disadvantages of the main sources of stem cells. This report is based on Professor Fairchild’s talk and the question and answer session that followed it. In a couple of places, where clearly indicated, it also includes details from my own reading inspired by the talk.
Professor Fairchild began by stressing the need for new and effective treatments to combat degenerative and chronic diseases. Improvements in healthcare in the twentieth century, particularly the use of antibiotics to treat bacterial infections, has led to increased life expectancy, and ‘inverted’ the population pyramid of many developed countries. As a result of such ageing populations, there has been an increased incidence of the chronic and degenerative diseases associated with old age, such as Alzheimer’s disease and Type II diabetes. There has, therefore, been an increasing focus on researching methods of treating such diseases: cellular therapies are one of the most promising approaches. A common concern, which was indeed raised in the questions after the talk, is that regenerative medicine might only result in a further increase in life expectancy, and further degenerative and chronic diseases would emerge later on. A counterargument to this concern is that regenerative medicine principally aims to keep people healthy and more productive for longer. It is the gradual decline and loss of productivity over an extended period of time, characteristics of degenerative diseases, which need to be overcome.
The defining features of pluripotent stem cells are that firstly they can divide to produce more stem cells – so can ‘self-renew’ and are not limited to a set number of mitotic divisions. By contrast, somatic cells (cells that are not related to stem cells, germ cells or gametes) are limited in the number of divisions before they undergo apoptosis (programmed cell death). Reading about this further, I found that one theory for this is that successive damage to the telomeres at the end of the chromosomes culminates in the chromosomes becoming frayed at the end. The cell does not tolerate the resultant damage to the coding part of the chromosome and undergoes apoptosis. Since stem cells produce higher levels of the telomerase enzyme which repairs damage to telomeres, they have the potential to divide many more times [2].
Returning to the content of the talk, the other defining feature of stem cells is their ability to differentiate into other cells. In general, when a stem cell divides by mitosis to produce two daughter cells, one will tend to become another stem cell (to maintain stem cell numbers), whilst the other can either also become a stem cell (for example if more stem cells are required), or alternatively can differentiate into a different, more specialised cell type through the process of ‘asymmetric cell division’. Stem cells, as with all other cells, may undergo apoptosis, for example if the number of stem cells in a tissue is too high.
Whether a stem cell divides to produce two stem cells or a stem cell and differentiating cell is determined by the axis of cell division. If an adult stem cell divides perpendicular to the basement membrane it is attached to, the cell which is not attached to the membrane can leave the niche occupied by the stem cell and go on to differentiate. If the stem cell divides parallel to the basement membrane, both daughter cells will be attached to the membrane and remain as stem cells.
Stem cells are categorised according to their ‘potency’. Knowing their place of origin can also give further detail about them. Potency refers to the range of cell types into which a stem cell can differentiate, so more specialised cells are less potent. A zygote (fertilised egg) is described as totipotent because it can differentiate into all the cells that make up the body as well as all the extra-embryonic tissues such as the placenta; the cells of the next stage in embryonic development, the morula, are also totipotent. Pluripotent stem cells – such as the cells in the inner cell mass of the human blastocyst – are more specialised since they lack the ability to form extraembryonic tissues but still retain the ability to differentiate into all two hundred and ten different cell types that make up the mammalian body.
Multipotent stem cells are more specialised, each stem cell capable of differentiating into only a limited range of fully specialised somatic cells. They are responsible for maintaining tissue integrity and activity in adults. In some species, such as the salamander, division and differentiation of multipotent stem cells are capable of completely regenerating whole tissues, and even a lost limb. Human multipotent stem cells do not regenerate whole organs, but play a vital role in replenishing many cell types. Haematopoietic stem cells in the bone marrow are responsible for generating all the different cells of the blood. Bone marrow transplants are now routinely used, for example to treat anaemia or after cancer treatment since stem cells are particularly affected by chemotherapy drugs targeting dividing cells. Therefore a transplant is often needed to replenish stem cell numbers. There have also been some promising developments for using neural stem cells to treat paralysis. Despite the established success and obvious potential for the use of multipotent stem cells in regenerative medicine, there are a few important limitations that have driven research towards other stem cell types: multipotent stems cells have limited availability and accessibility (for example there are obvious dangers in trying to extract neural stem cells from the brain) and a limited lifespan in vitro of often only a couple of days. Further disadvantages include their intrinsic lack of potency and the need for a donor, which limits the scaling up of treatment. The donor must also be a close genetic match for the patient in order to minimise the risk of rejection or graft versus host disease, where the transplanted bone marrow cells reject the recipient and so attack it. Reading about the need for genetic matching further, I found that it is the genes coding for the major histocompatibility complex (MHC) that must be closely matched, since it is these molecules on cell surface membranes that often act as antigens that trigger an immune response [2].
Given the disadvantages of multipotent stem cells, the focus of the talk returned to pluripotent stem cells. Planarian flatworms are known as the masters of tissue regeneration. It was shown by Wagner et al. [3] that clonogenic neoblasts, a type of adult planarian pluripotent stem cell, were responsible for this regenerative ability. Indeed flatworms that had been lethally irradiated survived if a single clonogenic neoblast was transplanted into their tissue. In humans, there are no known adult stem cells that have retained pluripotency. Instead, the common source of pluripotent stem cells is the inner cell mass of the blastocyst, the early embryo (the part excluding the outer circumference of cells, the trophectoderm, which goes on to form the placenta). These can be maintained long term in vitro, and potentially indefinitely. Thus they are scalable for clinical application.
An example of a technique using pluripotent stem cells is the development of chimeric mice, which contain tissues from two pairs of p
arents.
These are formed from the transplantation of pluripotent stem cells from one blastocyst into the inner cell mass of another, and allowing it to develop. The different tissues of the chimeric mouse do not reject each other because the immune system develops much later on in development.
Professor Fairchild emphasised that in humans, pluripotent stem cells have a broad remit for regenerative medicine. Their pluripotency is their key advantage. Human embryonic stem cells have been used in animal models in cell replacement therapy, and their use is being investigated in various human clinical trials, including stem cells engineered to differentiate into retinal pigmented epithelial (RPE) cells of the eye to treat macular degeneration, nerve cells to repair spinal cord injury, and heart muscle cells to help recovery from myocardial infarction.
Macular degeneration is commonly the first disease that a new regenerative stem cell technique is trialled on. Firstly this is because of its accessibility – often stem cells are transplanted by injection, and the retina is easily accessed in this way. Secondly this is because, given the capacity for pluripotent stem cells to divide indefinitely, they are only one or two mutations away from becoming cancerous. Cells in the eye are easily monitored, and if a tumour develops it is quickly removed whilst it is small and benign. Stem cells can be made to differentiate into (for example) RPE cells in vitro, provided that the cell matrix, nutrients and factors in the cell culture are maintained at the correct level.
However, there are still further technical problems when using embryonic stem cells. The issue of rejection of the transplanted cells can be overcome through a technique called Somatic Cell Nuclear Transfer (also referred to as therapeutic cloning). The transfer of the nucleus from a somatic cell (fully differentiated adult cell of the patient) into an unfertilised and enucleated egg results in a cell that is essentially analogous to a fertilised egg (an electric pulse is used to help the two fuse and to start the mitotic division process which culminates in the formation of a blastocyst). The embryonic stem cells derived from the resulting blastocyst have the same genes as the patient and so immune rejection is less of a problem. The embryonic stem cells can be harvested and then forced to differentiate into a specific tissue or organ that could be transplanted into the patient. It should be noted that there are concerns surrounding the application of similar technology in reproductive cloning whereby the resultant embryo is instead transplanted into the womb of a surrogate mother. The reproductive cloning of humans is currently banned throughout the world[4].
Related technologies have been used with recent success in the UK to prevent mitochondrial disease in a child. My description in this paragraph of mitochondrial disease and the technologies being developed to prevent it also draws on a talk I attended by Dr Andrew Greenfield in 2015 [5]. Since mitochondria from the sperm are destroyed after fertilisation with the egg, in general all mitochondria in the developing embryo are maternally derived and we inherit all our mitochondrial DNA from our mothers. Which mitochondria end up in the egg cell is subject to a ‘bottleneck effect’ as a small sample of the total number of mitochondria of the cell undergoing meiosis end up in the egg. Therefore, even if 80% of the mother’s mitochondria function properly i.e. she is perfectly healthy, the egg cell may end up with a high proportion of its mitochondria being from the faulty 20%, so the child may have debilitating mitochondrial disease. Pronuclear transfer overcomes this by transfer of the diploid nucleus from the fertilised egg into a healthy enucleated egg (i.e. an egg with mitochondria that function normally). Another technique is spindle transfer, where the haploid genetic material of the egg is transferred to a healthy egg which has had its DNA removed, prior to fertilisation with the father’s sperm. A technique transferring the polar body instead of the haploid genetic material is also in development. The main technical issue with the technique is the transfer of some dysfunctional mitochondria alongside the transferred genetic material, into the healthy egg. An important ethical issue with the technique is the fact that the child born will inherit genetic material from three ‘parents’: although their nuclear DNA will be from their mother’s egg and father’s sperm, their mitochondrial DNA will be that of the donor of the enucleated egg.
Some of the greatest challenges in the use of embryonic stem cells relate to ethical and religious objections to the use of embryos in research. Professor Fairchild explained that there is a further type of pluripotent stem cell that has great potential for use in regenerative medicine, and which does not require the use of embryos. Induced pluripotent stem cells (iPS cells) are derived in the laboratory directly from somatic cells. The somatic cells are ‘reprogrammed’ back to a pluripotent state by the insertion of key transcription factors. From my additional reading, these include Oct-4 and Sox-2 (which normally help maintain the pluripotent state of stem cells), the insertion usually using a Sendai virus as a vector [6]. The iPS cells can then be made to differentiate into the required cell types through careful control of their chemical environment and interactions with other cells. Since the iPS cells are pluripotent, they have the potential to differentiate into any required cell type. A further advantage of iPS cells is the ability to maintain an iPS cell line long term in vitro. The use of iPS cells instead of embryonic stem cells has a major technical benefit: since the iPS cells are derived from the patient’s cells (‘autologous derivation’) there are no issues over rejection of transplanted tissues by the patient.
The use of iPS cells in regenerative medicine faces many challenges before it can become a widely available treatment for degenerative and chronic disease. Aside from difficulties with the cost and timescales involved with reprogramming somatic cells to produce iPS cells, there are technical issues that need to be overcome. The ease with which the iPS cell can become cancerous is a serious problem. This is exacerbated by the lack of understanding behind the genetics of iPS cells. Whilst iPS cells behave analogously to embryonic stem cells in their developmental flexibility, their gene expression profile and epigenetic status is different to embryonic stem cells. There are also issues with iPS cells developing into the foetal version of the cells required, although similar issues are also encountered when using embryonic stem cells.
Despite the difficulties there are many uses for iPS cells beyond regenerative medicine – for example, the use of iPS cells to model disease [7]. This has the advantage that the cells used to model the disease can be derived directly from the patient. This is particularly useful for understanding monogenic diseases which are genetic diseases caused by mutation of a single gene. It has further potential in developing models for complex diseases such as autism and schizophrenia and also late onset diseases, although this is more difficult than modelling monogenic diseases since multiple genetic factors are involved.
A further important use of iPS cells is in the screening of potential new drugs. Many potential new drugs fail at a very late stage through the drug development and clinical trial process, which is referred to as a ‘high rate of late attrition’, principally on account of their toxicity. Using iPS cells, particularly hepatocytes, podocytes and cardiomyocytes, in high throughput screening can help rule out drug candidates with high toxicity at an early stage, saving time and money in the drug development process.
Professor Fairchild concluded by explaining how iPS cells have to date shown the potential to treat degenerative disease in the same way tha
t embryonic stem cells can. The use of iPS cells to treat macular degeneration in humans has already been demonstrated. The possibility of using iPS cells to generate organs for transplant is also being explored. The technique is analogous to the techniques used to create chimeric mice (see above): blastocyst complementation. Mouse pluripotent stem cells were injected into the inner cell mass of a Pdx-1 deficient rat blastocyst, which, if permitted to develop in vivo, would not develop a pancreas. A pancreas composed of cells derived from the mouse pluripotent stem cells grew in the rat, ‘rescuing’ it from fatality. Tissues prepared from this pancreas, could then be transplanted into a diabetic mouse to cure the diabetes [8]. The pancreas is not rejected by the host rat it develops in because the immune system is exposed to the mouse cells during development, whilst the mouse does not reject its transplanted pancreas because the pluripotent stem cells were taken from it in the first place. Such a procedure could be used to grow a human pancreas in livestock (a pig or a cow) and then transplant it to cure diabetes in humans.
In summary, Professor Fairchild’s inspiring talk demonstrated the potential stem cells have to address degenerative and chronic diseases. Although there are many challenges to be overcome, recently developed techniques indicate that regenerative medicine may, in the future, become an important part of modern medicine.
References
1. Fairchild, Paul J. 2017. \”Tapping The Fountain Of Youth: Stem Cells And The Promise Of Regenerative Medicine\”. Presentation, ATOM Science Festival, Abingdon. Saturday 25 March 2017.
2. Allen, T., and Cowling, G. J. 2011. The Cell: A Very Short Introduction. 1st ed. Oxford: Oxford University Press. (ISBN 978-0-19-957875-7)
3. Wagner, D. E., Wang I. E., and Reddien P. W. 2011. \”Clonogenic Neoblasts Are Pluripotent Adult Stem Cells That Underlie Planarian Regeneration\”. Science 332 (6031): 811-816. (doi:10.1126/science.1203983). https://www.ncbi.nlm.nih.gov/pubmed/21566185 , accessed 10 April 2017
4. Australian Stem cell Centre. 2010. Fact Sheet 4: Therapeutic Cloning (Somatic Cell Nuclear Transfer). Pdf. http://www.stemcellfoundation.net.au/docs/fact-sheets/fact-sheet-4—therapeutic-cloning-(somatic-cell-nuclear-transfer).pdf?sfvrsn=5., accessed 10 April 2017
5. Greenfield, Andrew. 2015. Presentation, Biology Society, Abingdon School.
6. Winograd, C. 2016. \”Induced Pluripotent Stem Cell (Ips Cell) | Biology\”. Encyclopedia Britannica. https://www.britannica.com/topic/induced-pluripotent-stem-cell, accessed 10 April 2017.
7. Avior, Y., Sagi, I., and Benvenisty, N. 2016. \”Pluripotent Stem Cells In Disease Modelling And Drug Discovery\”. Nature Reviews Molecular Cell Biology 17 (3): 170-182. (doi:10.1038/nrm.2015.27). http://www.nature.com/nrm/journal/v17/n3/full/nrm.2015.27.html, accessed 10 April 2017
8. Yamaguchi, T. et al. 2017. \”Interspecies Organogenesis Generates Autologous Functional Islets\”. Nature 542 (7640): 191-196. (doi:10.1038/nature21070). http://www.nature.com/nature/journal/v542/n7640/full/nature21070.html, accessed 10 April 2017