Stem Cell Research: Past, Present and Future?

By Esme Stewart

Grade awarded: Pass

Research Paper based on

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Pathology Lectures At Medlink and Vet-Medlink 2014

ABSTRACT Stem cells are becoming evermore important within medicine. They are the world’s leading resource in medical research within disease and cures as these specialised cells provide almost limitless potential and give rise to any tissue found in the body, offering a potentially revolutionary method of treating diseases and damaged tissues. Muscular Dystrophy a rare neuromuscular condition could be among diseases most likely to benefit from stem cell treatments. Genetically inherited, it is caused by a lack of the protein dystrophin and has an aggressive deteriorating effect on muscles. Although current research seeks to prevent deterioration and promote regeneration of muscle cells there is no known cure. Currently there is no evidence to support the nature of whether these cells will be reaccepted into the human body; therefore we still have a long journey before overcoming issues of rejection of transplants and how to deliver cells in the bloodstream to reach all affected muscles.

INTRODUCTION In the UK there are approximately 2,500 people who are suffering from different forms of Muscular Dystrophy, the most common being Duchenne, Myotonic and Facioscaoulohumeral. The deterioration in muscle strength is caused by mutations in the dystrophin gene which codes for the protein dystrophin, found in muscle fibre membrane. Currently there is no known cure, however the emerging field of stem cell therapies’ may hold the answer. Stem cell research within muscular dystrophy is extremely promising because if embryonic or adult stem cells can be cultured to produce muscles containing the protein dystrophin, they could aid tissue repair and regeneration within the area of distress. Similarly if a drug can be developed to enable and encourage the defective cells to produce the protein dystrophin we could avoid the issues of rejection of transplants of new cultured muscle. However

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we still have to overcome how to deliver cells in the bloodstream to reach all affected muscles.

NEUROMUSCULAR CONDITIONS MOST LIKELY TO BENEFIT FROM STEM CELL TREATMENTS As mentioned there are many different types of neuromuscular dystrophy which may benefit from stem cell treatments. One of the most promising is Duchenne muscular dystrophy. Duchenne muscular dystrophy was first described by French neurologist Guillaume Benjamin Amand Duchenne in the 1860s. It is an inherited genetic condition which causes muscle deterioration, most prominent in males as it is an X-chromosome related disease; therefore it is unlikely that girls will inherit muscular dystrophy as they would need two faulty copies of the gene, which is rare as sufferers usually die in early adulthood and so are able to pass on the affected allele. Due to a lack of the protein Dystrophin, muscle fibres do not form properly and are therefore replaced by fat and fibrotic tissue. Symptoms are noticed considerably earlier compared to other forms of muscular dystrophy and usually appear in male children between the age of 2 and 3; stem cells could be used to regenerate this missing protein. Another form that looks like it may benefit from stem cell treatments is Myotonic muscular dystrophy, which is an autosomal dominant disease. Autosomal meaning any chromosome but not the sex chromosome and dominant refers to the expression of its allele over a second allele that will be recessive. Unlike Duchenne, patients with Myotonic don't always have a shortened life expectancy unless it exists in a severe form and affects around 1 person in every 8,000. There are two types of Myotonic muscular dystrophy, referred to as Type 1 and Type 2 as the mutation occurs on different genes, DMPK and CNBP, resulting in unstable regions and prevent muscle tissues from functioning. Developing at any age, if severe enough Type 1 can be identified from birth which is referred to as Congenital myotonic dystrophy. As well as the wasting of the muscles, Myotonic dystrophy can be characterised by cataracts, cardiac conduction defects, endocrine changes and myotonia, the difficulty and delay of being able to relax muscles after contraction. If cultured defective muscle was

Figure 1: A pedigree chart to show the inheritance of Duchenne Muscular Dystrophy.

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replaced with cultured muscle, containing the protein dystrophin, muscles would be able to function properly and prevent dystrophy.

Facioscapulohumeral muscular dystrophy affects around 1 person in every 20,000 people in the UK and can develop in childhood or adulthood but progresses relatively slowly therefore is not usually life threatening although it can compromise your lifestyle. Facioscapulohumeral is usually an autosomal dominant condition, in more than 95% of known cases, the disease is associated with contraction of the D4Z4 repeat in Chromosome 4. The name is derived from the areas it initially affects such as the skeletal muscles of the face (facio), scapula or shoulder blade (scapulo) and the upper arms (humeral).

Figure 2: Defective allele on the X chromosome for Duchenne’s muscular dystrophy.

Figure 3: The front and back view of a patient demonstrating effects of facioscapulohumeral.

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A BREIF HISTORY OF STEM CELL RESEARCH In the mid 1800s the importance and function of stem cells was discovered. Despite this knowledge, it was not until recently that ground breaking research is being made. As researchers became more enticed by this unexplored field they began making new discoveries. One key event occurred in1968 when the first successful bone marrow transplant was performed, on two patients with immunodeficiency and in 1978 the first in vitro fertilisation baby was born. Sir Martin John Evans and Matthew Kaufman were the first to culture embryonic stem cells from a mouse in 1981. Research was conducted on the origin of different stem cells which led to the discovery of stem cells in human cord blood and bone marrow. Although these milestones were significant, research did not develop until 2002 when the House of Lords concluded that stem cells had great potential and so research should be conducted on both adult and embryonic stem cells. RELEVANCE TO MEDICINE Stem cells are becoming evermore important within medicine; they are the world’s leading resource in medical research within disease and cures as these specialised cells provide almost limitless potential and give rise to any tissue found in the body, offering a potentially revolutionary method of treating diseases and damaged tissues. Research within stem cells started in 1978 with the first IVF baby born after Cambridge scientists fertilised human eggs outside the body, shortly followed by the isolation of mouse embryonic stem cells by Cambridge scientists in 1981. In the UK, stem cell therapy research blossomed in March 2005 when the Government set up the UK Stem Cell Initiative and in turn later that year the UK were named as a world leader in stem cell research. In order to further discuss their relevance to medicine one must first understand the role of stem cells. Either adult or embryonic, they are introduced into a patient in order to replace and repair damaged tissues. Embryonic stem cells, produced from dividing a newly fertilised egg, have the capacity to differentiate into any cell within the patient’s body and so are referred to as totipotent. Five days after fertilisation, the cell forms a blastocyst, from which the embryonic stem cell is derived. At this stage it can be manipulated into any cell. Most embryonic stem cells are either donated or specially cultured in laboratories.

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Figure 4: Flowchart to show the specialisation of embryonic stem cells. In contrast adult stem cells are extracted from developed tissues and have a more specific and limited adaptation to the body’s cells when compared to embryonic cells. Adult stem cells have been discovered in bone marrow, the blood and umbilical chords. These stem cells replenish and rebuild damaged tissues within our bodies but cannot differentiate. The reason the UK invests so much into stem cell research is because it could revolutionise health care as we know it. Every year the NHS spends dramatically more money treating the whole country. Access to free health care is becoming increasingly difficult due to such a high demand. NHS spending increased from a value of £33.5 billion in 1998 to £76.4 billion in 2006, furthermore it reached a value of £96.4 billion in 2009, as reflected in figure 5. Investing in stem cell research may lower the resources required for long term treatments such as chemotherapy for cancer patients or dialysis for kidney failure, as well as all the various types of neuromuscular conditions that I have mentioned.

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CURRENT STEM CELL RESEARCH Most recently scientists are further exploring the development and potential of this research into culturing different cells derived from stem cells. a.) Haemopoietic stem cell transplant also known as bone marrow transplants have been used for nearly 40 years and are most commonly used to treat leukaemia and sickle cell anaemia patients who can no longer produce their own blood cells independently, after receiving chemotherapy. b.) Although bone marrow transplants are the only current methods that are clinically trialed, significantly more research is being undertaken into tissue regeneration for diseases such as Parkinson’s, retinal damage of the eye, diabetes, cardiac diseases and even strokes. c.) Researchers are experimenting with mesenchymal stem cells, found in the bone marrow, to significantly reduce inflammation improving the outcome and maintaining the environment for any transplanted stem cells. Steroids have previously undertaken this role however studies have shown that when injected into the muscle tissue of mice, these stem cells have reduced inflammation as they suppress the immune response enabling recuperation of the muscle. d.) Cells that make up the cornea are constantly damaged by blinking. A significant loss of vision is lost if the limbal stem cells of the cornea cannot replace damaged cells, either because they themselves have been damaged or diseased. Experimental stem

Figure 5: A chart to show total UK health expenditure of both public and private service.

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cell research on repair of the cornea has been very successful, with the use of adult eye stem cells. Repairing retina damage is less successful due to difficulty to enable them to detect light. e.) Within the University of Pittsburgh there has been promising abilities of stimulating the development of beta cells using the genes ‘cdk’ and ‘cyclin d’ via a virus. For patients with type 1 diabetes, who lack and subsequently cannot develop these insulin-producing cells in the pancreas, this could be life changing. Stem cells are used in the body as an inbuilt repair system and are contained in many tissues, including muscles. They are useful because of their replicating ability which is triggered by chemical signals from muscle fibres, signalling satellite cells to produce new muscle fibres. However in patients with forms of muscular dystrophy, a prevailing field I find particularly interesting, satellite cells within muscles lack the ability to replicate and over time muscles become less able to repair themselves which is why the deterioration occurs. Currently the main areas of research using stem cells investigates the production of new healthy muscle fibres and also reducing inflammation. It has been shown that mesoangioblast cells, a type of stem cell found in the walls of blood vessels, are able to form muscle fibres which produce the protein dystrophin. Most importantly these cells are able to be injected and transported through the blood stream and delivered to all distressed muscles, due to their ability to cross blood vessel walls. There is evidence to suggest that the likelihood of these transplanted mesoangioblasts being rejected as an immune response, will be significantly lower than using other forms of stem cells in the same procedure. Extraction and culture of our own mesoangioblast or those of a relative, with genetic modification could develop a viable method in ability to restore the production of dystrophin. Pluriopotent cells have an extremely useful role in stem cell research as they are able to divide into any cell in the body. A method to turn induced pluripotent stem cells into mesoangioblasts has been discovered by Franesco Tedesco, a London- based researcher. Extracted from a patient’s skin cells, after modification these cells act like mesoangioblasts and when injected into mice, observation in strengthening of muscles occurred. This procedure has not been trialed in humans yet so it is not known whether it is effective or not, however they promote promising results if not rejected by the body as they are easier to develop in a lab in comparison to cells from a donor and possess the ability to independently replicate an unlimited number of times.

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Research on Duchenne Muscular Dystrophy, the most common type, has recently been conducted by scientists at Duke University, USA. This technique used gene editing, CRIPSR/Cas9, which involves cultured muscle cells to restore their ability to produce dystrophin. This procedure works by manipulating a protein, present in bacteria, that acts as molecular scissors as a defence mechanism against invading viruses. For Duchenne muscular dystrophy, this protein is utilised to splice out the region of the gene between exons 45 and 55, typically where the majority of mutations occur. However the patient will still suffer from muscular dystrophy but a milder form referred to as Becker muscular dystrophy, characterised by the shortened dystrophin gene. There is no evidence to suggest this technique is effective in humans therefore there is no known cure in the present day. In some cases Muscular Dystrophy (MD) can be extremely severe as many types of MD are multi-system disorders, meaning they affect body systems such as the heart, gastrointestinal system, nervous system, endocrine glands, eyes and brain. Usually MD begins by affecting a certain area of muscles before it begins to spread to others, interfering with their ability to function, which overtime causes increased disability. Unfortunately this can be life threatening as we see muscles around the heart become defective and these essential systems seize, resulting in death. For people with Duchenne muscular dystrophy, heart failure is but one of the major concerns. A clinical study this year lead by Dr Subha Raman, a cardiologist and professor at Ohio State University, has highlighted the successful use of a drug called eplerenone, traditionally used for high blood pressure and late stage heart failure, on reducing the deterioration rate of the heart in Duchenne muscular dystrophy. Illustrated in a new 12 month trial 42 participants with Duchenne muscular dystrophy were either given one dose a day for a year of eplerenone or a placebo, an inactive substance to resemble eplerenone, ruling out the possibility of inaccuracies and whether the drug is effective or not. Eventually, if modified, the drug eplerenone could sustain the fibres that contribute to strengthening the muscle preventing early death and allowing sufferers to lead a normal life. Although there are many developing fields within the research for a cure, or even just a treatment for muscular dystrophy, barriers against progressing these treatments to clinical trials have been identified due to the small population affected by such a rare disease. This creates difficulty in securing funding for such developments and research. If legalised, pharmaceutical companies will charge extortionate rates for new found drugs in order to recuperate the costs for

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research, which could automatically deny treatment to many. However before being legalised the procedure must be approved by a number of different associations. Allison Morgan from Prosensa, speaking about the process stated: “The MHRA is the regulatory body that approves the protocol, then it has to go to the Central Ethics Committee in the UK, then sometimes the hospital committee, then a local research unit, and then we have to negotiate the contract. All of this takes a lot of time.” ETHICAL ISSUES In 1990 The Human Fertilisation and Embryology Act was dictated to limit the use of invitro fertilisation and the creation, usage, storage and disposal of embryos. It wasn't until January 2001 that the House of Lords passed and first permitted the research on embryos in order to increase our understanding. Numerous regulations exists against the use and sourcing of embryonic stem cells; they must meet all required criteria for funding. HEFA - the Human Fertilisation and Embryology Authority regulates and authorises all embryo research within the UK and a licence is required by all researchers. Sourcing of these cells can be from: - Extracted from adult tissues - Stem-cell-rich chord blood - Specially cultured embryos - Embryos created by in vitro fertilisation The NHS has a number of organisations for donation of stem cells for research such as the Cord blood bank, shown in figure 6, which encourages the public to donate Umbilical cord blood, which would usually be discarded as medical waste. Figure 6: NHS Cord Blood Bank website which links to more pages on the importance of donating and how to help.

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Although there are regulations restricting research on embryos, on the contrary, organisations against it still exist against the whole scenario. Different organisations regard the status of embryos differently. For example the Roman Catholic Church, Orthodox and Protestant Churches believe no research should be gifted the embryo as it is regarded with the status of a human from the moment of conception. However currently at which point the embryo reaches personhood is defined 14 days after fertilisation occurs by UK law. Before these 14 days the embryo has not yet developed a central nervous system and is not defined as an individual but has increasing status as it develops. The criteria for ‘personhood’ is incredibly unclear. Any embryo that has not transferred into the woman’s uterus, cannot develop into a child, simply because it doesn't possess the psychological, emotional and physical properties associated with being a person. Therefore why not use these to benefit those patients who are already people? We need to weigh both sides and evaluate whether the damage done would out rule the benefit of potential gains of this research. In the UK we are allowed to use organs for transplants taken from brain dead patients therefore why cant we use an embryo if it isn't a person yet? A large majority of Embryonic stem cells used in in-vitro fertilisation are wasted as the chance of them developing in to full-term successful births is low as most treatments are unsuccessful and many couples have to have multiple treatments before pregnancy occurs. So ultimately should an embryo be treated like a person simply because it has the potential to become a person?

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In the United States embryonic stem cell research is even more closely controlled compared to the UK. An article released on 9th March, 2009, published the remarks of Barack Obama as he prepared to sign the Stem Cell Executive Order and Scientific Integrity Presidential Memorandum; this supported and permitted human stem cell research within America. Obama stated the importance of supporting and encouraging research: ’When the government fails to make these investments, opportunities are missed’, it was necessary if America wanted to remain a leading country in scientific research but also at the same time find a balance between faith and the want and need to ease human suffering. This is achieved by the strict regulations that are enforced rigorously. CONCLUSION There are many possible pathways within stem cell research due to their almost limitless nature. Some of these fields I have outlined in this paper are currently being used also in other countries across Europe and the world. Although we are currently limited by our resources, stem cell research will always remain at the forefront of medical research as it proposes the most potential and is the answer to many questions. Even though stem cell research has developed now more than ever, researchers still have a long way to go before being able to harness the full potential of stem cells to cure diseases and save lives. In the near future instead of having to cope with an increased demand of transplants and a lack of Organ donors, scientists will be able to culture stem cells and engineer new organs. The demand for organs will therefore decrease since they will become more accessible. There is successful evidence of more simple organs being engineered such as bladders and tracheas however if more complex organs such as the heart can be grown many more lives can be saved as the heart is the third most needed organ. These organs are built using scaffolds. Due to the complexity of this organ it’s scaffold needs the correct conditions and growth factors in order to target growth. The reason it is so complex is due to the electrical impulses that naturally occur to make the heart beat, valves and different chambers within the heart. Engineering tissues of different patients will allow observations to be made on growth. Different treatments can be tested on the tissues of cancer patients to determine the suitability and effectiveness of treatments in reducing the rate at which the cells mutate. Similarly engineering tissue samples would provide scientists with a more in-depth understanding of many rare conditions, such as the neuromuscular diseases I have outlined in this paper. As the scaffolds of tissue are

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from the patients the risk of rejection by the patient’s immune system would be reduced. Perhaps there will be different centres across the UK; with one centre specifically for patients with neuromuscular conditions like muscular dystrophy where they could receive rapid treatment. Once bioengineering of other organs is achieved, research may commence on targeting desirable characteristics, creating customised organs. For example a heart with more muscle for athletes or lungs with a larger capacity for swimmers and divers. Of course ethical issues will still be raised by certain groups however correct use and disposal of the stem cells and specially engineered tissues could lead to a future mutual agreement, otherwise how will we ever progress within medical research if we do not overcome these obstacles and take chances. Stem cells are the future. Writing this paper has shown me that work still needs to be done and that discoveries still need to be made to exploit stem cell research. The journey will be tough but reaching the full potential of stem cells is inevitable, even if it is not in our lifetime. REFERENCES

1.) Background and inheritance of Muscular dystrophy.

http://www.nhs.uk/conditions/Muscular-dystrophy/Pages/Introduction.aspx

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2.) The uses and specialties and importance within stem cell research. http://www.ama-

assn.org/ama/pub/physician-resources/medical-science/genetics-molecular-

medicine/related-policy-topics/stem-cell-research/basics-stem-cell-research.page?

3.) Research by conducted by Rando.T at Stanford University which identifies the default

in stem cells associated with muscular dystrophy. http://med.stanford.edu/news/all-

news/2014/12/stem-cells-faulty-in-duchenne-muscular-dystrophy.html

4.) Possible pathways of using stem cells to improve suffering of muscular dystrophy.

http://www.eurostemcell.org/factsheet/muscular-dystrophy-how-could-stem-cells-help

5.) Current research within stem cells.

http://www.medicinenet.com/stem_cells/page5.htm#what_are_some_stem_cell_therapies

_that_are_currently_available

6.) Figure 1: http://www.mda.org/publications/facts-about-genetics-and-NMDs/what-

happens-in-DMD

7.) Figure 2.

http://www.biologycorner.com/worksheets/articles/treating_genetic_disorders.html

8.) Figure 3. http://www.neurology.org/content/73/5/e24/F1.expansion

9.) http://www.ama-assn.org/ama/pub/physician-resources/medical-science/genetics-

molecular-medicine/related-policy-topics/stem-cell-research/basics-stem-cell-

research.page?

10.) http://en.wikipedia.org/wiki/Muscular_dystrophy

11.) Figure 4.

https://www.hta.gov.uk/sites/default/files/stem_cell_pack_200806170144.pdf

12.) Figure 5: http://davidstockmanscontracorner.com/when-the-state-pays-health-

spending-soars-long-term-publicprivate-comparison-for-us-uk/

13.) Figure 6. http://www.nhsbt.nhs.uk/cordblood/

14.) http://www.explorestemcells.co.uk/historystemcellresearch.html

15.) https://www.whitehouse.gov/the_press_office/Remarks-of-the-President-As-Prepared-

for-Delivery-Signing-of-Stem-Cell-Executive-Order-and-Scientific-Integrity-Presidential-

Memorandum/

16.) http://www.medicinenet.com/stem_cells/page6.htm

17.) Ethics of embryonic stem cell research.

http://unesdoc.unesco.org/images/0013/001322/132287e.pdf