RESEARCH PAPER BASED ON PATHOLOGY LECTURES

Transcription

1 USE OF STEM CELL THERAPY TO ACHIEVE TREATMENT OF BECKER S AND DUCHENNE S MUSCULAR DYSTROPHIES BY ANUSHKA PATHAK ARYA NAIR Grade awarded: Pass with Merit RESEARCH PAPER BASED ON PATHOLOGY LECTURES

2 2 AT MEDLINK 2014

3 ABSTRACT Muscle diseases, also commonly referred to as muscular dystrophies, are the terms that refer to the variety of diseases that impact the function of the muscles or nerves controlling those muscles. In this paper we will discuss the potential uses of both adult and embryonic stem cells in therapies designed to treat a variety of muscular diseases, as stem cells are capable of differentiating into almost any type of cell in the body. This Gedanken experiment will explore several forms of muscular diseases such as Becker s and Duchenne s, and examine the ways in which stem cells can revolutionise the effective treatments for these conditions. INTRODUCTION 1. An Overview of Stem Cells Stem cells are undifferentiated cells found in multi- cellular organisms, which are capable of reproducing to make more cells while still remaining undifferentiated, or possess the ability to differentiate into specialised cells when required to do so [1]. The enzyme telomerase, found in eukaryotes, is responsible for preserving the length of the telomeres at the ends of a strand of DNA, even after repeated cell divisions. In embryonic stem cells, this enzyme is activated in order to maintain the telomere length, and thus avoid senescence of the cells [2].If enough telomerase is present, these cells can surmount the Hayflick limit, leading to cell proliferation. These qualities, unique to stem cells, allow them to be of use in providing effective treatment of diseases, in regeneration of tissues and organs in particular. 2. Sources of Stem Cells Stem cells vary in their functions and differentiating potentials, depending on where they are found in the body. There are three main sources of stem cells: embryonic (ES), fetal and somatic stem cells. 3

4 Embryonic stem cells are obtained from an embryo in the early stages of development (3-5 days old), called a blastocyst. In this stage, the each embryonic cell has all the genes for developing into the whole organism. As a result of this, in theory embryonic stem cells can differentiate into all the different types of cells in the body such as nerves, skin, the heart, lungs and muscles. The blastocyst consists of an inner cell mass called the embryoblast, and an outer cell mass called the trophoblast (Figure 1) [3].The cells from embryoblast Figure 1: Pluripotency of ES cells differentiate to form all the different structures of the body and is where the embryonic stem cells are obtained from. Embryonic stem cells are the most ethically controversial, but also the most in demand of all stem cell types, due to their totipotency or pluripotency, depending on which stage of embryonic cell division they are undergoing (i.e. ES cells are totipotent for the first 4 days of cell division, but thereafter begin to specialise and are considered pluripotent, which is the time we can actually extract them) [4].This property allows them to be used in regenerative medicine for tissues and whole organs. However, there are concerns about this use of stem cells, as there exists the possibility of malignant tumours, known as teratocarcinomas, to be formed [5],or the possibility of immune rejection. Fetal stem cells can be either fetal proper stem cells or extra embryonic fetal stem cells derived from 10 week old fetus. Cord blood stem cells are a type of fetal stem cells, which can be taken from the umbilical cord relatively easily after birth. Cord blood contains haemopoietic (blood) stem cells, which are more commonly found in bone Figure 1: Tree of blood depicting the range of different mature blood cells that can be produced from haemopoietic cells 4

5 marrow. These cells can differentiate into all the different blood cells in the body (Figure 2) [6]. They can be used in the treatment of children with cancerous disorders such as leukaemia, and in genetic blood diseases like Fanconi anaemia. However, they have been extremely difficult to use in treating adults, although there have been a few successes. Somatic (adult) stem cells are found in the body after the embryo has developed fully, and can be found in certain body tissues like the brain, skin, liver, blood vessels and bone marrow [7]. Unlike ES stem cells, somatic stem cells are tissue specific (they can only form the cell types within that organ), meaning they are multipotent. They have the narrowest differentiation potential of all stem cells however; they can produce an unlimited number of differentiated cells specific to the group of tissues they are a part of. 3. Recent Research and Uses Ever since 1908, when the Russian histologist, Alexander Maksimov coined the term stem cells, much research has been conducted, leading to many discoveries and therapeutic uses as a result [8]. Although there are still ethical considerations and debate that surrounds their use, stem cells have proved to be highly beneficial in the treatment of conditions such as a damaged cornea or retina, various forms of anaemia, leukaemia and also for organ transplants. One of the recent developments in the use of stem cells is in their capability for organ generation and transplantation. Organs are already being transplanted successfully in many cases; however the success rates can be increased if stem cells from the patient are used to generate the organ. This is due to the fact that transplanting a patient s own cells into their body reduces greatly the risk of immune rejection by the body, meaning also that immunosuppressant drugs would not have to be taken. In 2008, Claudio Castillo became the first person to have a successful tissue- engineered airway transplant. It was 5

6 created by stripping the windpipe of a deceased donor down to its solid scaffold with none of the donor s cells, and then coated with Castillo s stem cells [9]. Many people have major ethical concerns surrounding the use of embryonic stem cells for treatment of diseases, which will be touched upon later in the Discussion. Cellular de- differentiation is the process by which specialised adult cells lose their function and revert back to their unspecialised state. The cells produced by this process are called induced pluripotent stem cells (ipscs), and they are created by forcing the cells to express particular genes and factors that are crucial in preserving the nature, i.e. the pluripotency, of embryonic stem cells. ipscs were first programmed in June 2006 by Dr. Yamanaka and his team in Japan, using a set of 4 master regulators, transcription factors (Oct3/4, Sox2, c- Myc, and Klf4)in mouse embryonic or adult fibroblasts (the cells responsible for making the extra- cellular matrix and collagen) [10]. The advantages of ipscs are that they overcome the ethical opposition that surrounds the use of embryonic stem cells, as they created from reprogramming somatic cells, instead of from embryos, and that they are created from a patient s own body cells, thus removing the need for immunosuppressant drugs because there will be no immune rejection as the stem cells are genetically identical to the body cells. 4. Future Potential Despite the wide array of current uses for stem cells, scientists and researchers are constantly searching for ways to broaden this range. As all specialised cells in a human body originated from embryonic stem cells, this enables scientists to use stem cells, particularly embryonic stem cells, to regenerate damaged or dead tissues under optimal culture conditions. With the wide differentiation potential of embryonic stem cells, they are being considered for providing treatments for disorders and common degenerative diseases, such as Parkinson s disease, Alzheimer s disease, and diabetes, which were previously incurable. Alzheimer s disease, for example, is a major contributor towards Dementia where a certain 6

7 number of nerve cells in the brain die. Theoretically, scientists may consider transplanting embryonic stem cells in the brain of the Alzheimer s patient, with the possibility that they would produce new and healthy neurons to replace the damaged nerve cells. This may even be possible by placing neural stem cells (a kind of stem cell found in the brain) into the brain of the patient with the hope that the damaged neurons would be replaced with healthy neurons [11]. Stem cells have been a recent source of hope for a cure for Type 1 diabetes as Douglas Melton et al managed to make fully mature beta cells, from both human ES cells and ipscs, which respond to glucose in order to produce insulin. The work has been tested on mice, and reports have shown that the cells have effectively cured the diabetes [12]. DISCUSSION In this following section, we will be discussing two common muscular dystrophies, what current treatments are available, how stem cells could be utilised to bring about a more effective treatment for Duchenne muscular dystrophy (DMD), as well as several ethical concerns that people hold. 1. Overview of Muscular Diseases Muscular diseases (also known as muscular dystrophies) are a group of hereditary genetic conditions that cause voluntary muscle groups to weaken over time, consequentially leading to an increased level of disability in the individual. These are caused by gene mutations, which then lead to changes in the proteins synthesised that control the function and structure of certain muscles in the body. These are progressive conditions, meaning that their impact on the individual increases over time, and affects a specific muscle group, before spreading and affecting the function of other muscle groups. Additionally, muscular dystrophies become seriously life threatening when they begin to impact the function of myocardial (heart) muscles or the muscles used for breathing [13] In 1986, researchers discovered the gene that, when it is defective and/or has undergone mutation, results in the development of Becker s and Duchenne s Muscular Dystrophies. It was in 7

8 1987 that the protein that is synthesised from this gene was named dystrophin protein [14]. Of these two similar muscular dystrophies, Becker s Muscular Dystrophy (BMD) is caused by mutations on the dystrophin gene, where a shortened and only partially functioning dystrophin protein is synthesised (only about 10-40% of the normal amount) [15]. BMD involves a gradual wasting and deteriorating of the muscles, which is mainly proximal (primarily around the central region of the body). Duchenne s Muscular Dystrophy is similar to Becker s Muscular Dystrophy in the symptoms and causes, however it is a more extreme version. DMD is, like BMD, a hereditary condition that affects the function of certain muscle groups/causes muscles to weaken, particularly those in the hips and shoulders. Both DMD and BMD are caused by mutations in the DMD gene, which carries information needed to synthesise the dystrophin protein. Duchenne s Muscular Dystrophy is caused by mutations in the DMD gene that cause no functional dystrophin protein to be synthesised [16]. Symptoms for DMD can be recognised in the early years of adolescence, but not as early as when the baby is born, and the strength of these symptoms increases with age. 2. Signs and Symptoms Commonly, BMD and DMD sufferers are males between the ages of 11 and 25and sufferers will experience a wide range of similar symptoms, some examples being muscle cramping and muscle weakness and deterioration in shoulders, thighs, pelvis and hips, and naturally the individual affected may walk with a waddling gait, or stick out their abdomen to make walking less difficult. The rate of muscle wasting that comes with BMD and DMD varies depending on the individual some men can continue walking for many years with only the aid of walking sticks, while some others will be in need of a wheelchair by the age of thirty. Due to the lack of dystrophin protein, not only the limbs are affected, but the heart can also be weakened. Because of this, sufferers often develop cardio myopathy. Research suggests that lack of dystrophin protein in the brain may lead to learning and behavioural setbacks. The main learning problems that are associated with sufferers occur in three areas: memory and verbal learning, emotional communication, and attention focusing [17]. 3. Current Treatments 8

9 Although DMD is a serious genetic condition that rarely has survivors past their late 30s and 40s, there is currently no cure for the fatal disease. There are, however, several treatments available to improve the quality of life of the sufferer. A common form of treatment in DMD sufferers is the use of glucocorticoid corticosteroid medication. These have been known to slow the rate of muscle deterioration, and even improve the performance and strength of the muscles in children who are treated from six months to two years of age [18]. The decision to continue the steroid medication as an adult can sometimes hold concerns, due to the risks and large side effects of long term steroid use (for instance, glaucoma or osteoporosis). Duchenne s Muscular Dystrophy and Becker s Muscular Dystrophy both can cause deterioration of the myocardium. Due to the life threatening nature of the disease at this point, patients will frequently have electrocardiograms and echocardiograms, to ensure their heart rhythm is not abnormal and are given medication like ACE (angiotensin- converting enzyme inhibitors) to relax their arteries and to help the heart pump blood around the body and beta blockers to manage arrhythmias [19]. Despite these managing strategies, there is a great deal of research into potential treatments for muscular dystrophies. A major topic of current research is whether the unique properties of stem cells could be utilised to regenerate muscles in patients with muscle diseases. 4. How could stem cell therapy help? Stem cell therapy involves the processes of placing stem cells into a patient, in order to replace damaged or dead cells. Stem cell therapy is a sought- after option, especially in patients with genetic disorder like muscular dystrophies, given that it has the ability to regenerate muscle and express the missing genes that have the potential to repair abnormal muscle function. The main approaches to using stem cells in therapies for muscular dystrophies are by producing new healthy muscle fibres, reducing inflammation and genetically altering the stem cells of the patient in laboratory conditions. Scientists expect that future effective treatments will involve a combination of these therapies, used in conjunction with each other. 9

10 a. Reducing Inflammation In muscular dystrophies, inflammatory chemicals are produced by the immune system cells and the damaged muscle cells, causing myositis, which make the muscular environment dangerous for muscles [20]. Existing muscle cells are killed, and new ones cannot be generated due to the hostile environment. In existing therapies, corticosteroids e.g. cortisone, prednisone and methyleprednisolone are used to reduce the inflammation [21] ; however research is also being conducted in the avenue of using somatic stem cells to decreases the muscle wasting caused by muscle inflammation. Mesenchymal stem cells (MSCs) are multipotent cells found in the bone marrow stroma and have the ability to form a variety of different cells from the ectoderm, mesoderm and endoderm (Figure 4) [22]. They are able to inhibit the release of pro- inflammatory cytokines, and thus reduce inflammation in the muscles. In addition to this, MSCs can also fuse with dystrophic muscle, genetically complementing it, and produce trophic factors that can supplement the repair from endogenous cells [23]. These stem cells may be of further application in muscular dystrophies, in that they create a healthier environment in the muscles, therefore improving the possibility of success of other stem cell therapies e.g. reduced inflammation can help to ensure the survival of other types of transplanted stem cells. Figure 2: Various types of cells produced by mesenchymal stem cells b. Producing New Healthy Muscle Fibres 10

11 Patients with DMD or BMD suffer from possessing no dystrophin protein, and a lack of dystrophin protein respectively. Due to this major problem, many scientists and researchers have been studying several potential uses of stem cells to produce new muscle fibres, such as myoblast cells. When myoblasts (cells formed from satellite (support) cells) are fused together, they can form muscle fibres. Studies have indicated that inserting these cells into the bodies of mice with similar muscular deterioration to sufferers of DMD enables the healthy myoblasts to then fuse together with the damaged muscle fibres in the mice to partly form the dystrophin muscle that the sufferer does not possess. Although theoretically this seems like a successful approach to treating patients with DMD or BMD, clinical trials and further research has identified several obstacles that prevent progress with humans. For instance, as Duchenne Muscular Dystrophy affects a majority of the body s muscles, the large number of myoblast cells needed to transplant into the patient s body, and then delivering them to the body s affected muscles would be very difficult. What poses an even bigger challenge is that in order to deliver these cells to the muscles in the body, they will need to be transported through the blood stream, but would be unable to diffuse through the blood vessel walls, and so could not travel to the surrounding muscles. Additionally, another major flaw is the fact that healthy myoblasts will not be able to survive when introduced to damaged dystrophin muscle, and so would be attacked by the body s natural immune system and inflammatory cells, resulting in rejection. Despite problems posed by myoblasts, a more successful approach is the use of mesoangioblasts (a type of stem cell found in blood vessel walls). Researchers have discovered how, when trialled with dogs and mice, healthy mesoangioblast cells have been able to form dystrophin- producing muscle fibres. Unlike with myoblasts, MBA cells are capable of diffusing across the blood vessel walls, and so can therefore reach all the damaged muscle cells that require it, though the level to which production of these new muscle fibres can undo muscle wastage varies depending on the animal(figure 5) [24].The only concern surrounding this is the need for scientists Figure 3: Use of MBA cells 11

12 to harvest the patient s own MBA cells and genetically modify them under controlled laboratory conditions, before reinserting them into the patient, to ensure there is no risk of rejection, and the cells are capable of producing normal dystrophin (this will be discussed later). Francesco Tedesco and his team learned that it is possible to transform ips cells, obtained from the patient s skin cells, into cells that behave like MBA cells. To prove this was correct, they inserted these cells into mice with muscular dystrophy, and found that the mice began to grow stronger and produce normal, healthy muscle proteins. While there are still a number of concerns towards whether this is a safe treatment for humans, and it may still be some time before human trials occur, this does provide hope towards utilising ips cells to grow MBA cells and use them for treating several muscular diseases, especially with the knowledge that ips cells can replenish themselves continuously, therefore producing an unlimited number of MBA- like cells to treat muscular dystrophies. However, although MBA cells may be effective at producing new muscle fibres for patients suffering from muscular diseases, it is not possible to treat a genetic disease purely by producing new muscle fibres. The idea is therefore to equip the satellite cells additionally with a healthy gene that repairs the defective gene and then to transfect it with the aid of a non- viral gene taxi into the muscles to be treated [25]. 5. Ethical Implications of These Therapies The above therapies being considered to treat muscular dystrophies involve the use of stem cells found in blood vessel walls (mesoangioblasts), or any somatic stem cell source, and ips cells. Although embryonic stem cells are known for being capable of differentiating into any other body cell, this trait is still not known to be of a particular advantage for treatments of muscular dystrophies, as adult stem cells are equally as efficient. This also avoids several ethical concerns and objections that many people may have involving the use of ES cells. 12

13 The use of embryonic stem cells can lead to a struggle between deciding which of two moral views is better. One of these is that ES cells could be used to prevent the suffering of others e.g. those with degenerative diseases. On the other hand, there are those who believe that ES cells are a form of life, and so using them for research and in treatments is destroying that life, with its potential to become a human being. Several religious groups and other organisations such as the National Right to Life Committee in the USA, often hold this view, as they believe that human life begins at conception and therefore using ES cells for medical purposes is equivalent to murder. This strikes a controversial issue of whether stem cells should be used to save patient s lives, or whether a potential human life should be respected. There is legislation in place to protect the moral rights of embryos, for instance the Human Fertilisation and Embryology Act (1990), which states that embryos have moral rights but not to the same extent as a human being. It also states that ES cell research can only be justified if the purpose behind it has been strictly defined. Many people are also reserved about using embryonic stem cells, when alternative methods of gaining stem cells are available, such as adult stem cells and ips cells. However, scientists say that ES cells are the only way to study basic biological mechanisms, and thus are an essential part of scientific research. CONCLUSIONS 13

14 Our paper has highlighted a number of possible methods by which stem cells and their properties could be utilised to provide effective treatments for muscular dystrophies. Though there have not yet been clinical trials in this area, existing research, such as that conducted by Francesco Tedesco involving the transplantation of IPS cells into mouse models for muscle regeneration hints at promising future potential for this use of stem cells in treatment of muscle diseases. Our proposed methods of treatment are using stem cells to treat muscular diseases through: reducing inflammation, and producing new healthy muscle fibres. These, if proven successful in human trials could lead to a drastic increase in quality of life and indeed in life expectancy of the million muscular dystrophy sufferers throughout the world. Although human trials have not yet been conducted, a foreseeable obstacle that would arise from using these therapies are that they would not completely cure the dystrophy, and would only be used in the management of the disease. These would have to be used in combination with gene therapies, which involve replacing the dysfunctional dystrophin gene with a fully functional gene, in order to provide a possible cure. These treatments do however have the added advantage of the absence of use of ES cells, thus avoiding the ethical complications that arise through their use e.g. the controversial issue of whether using a human embryo is removing the potential for a life. 1) Torrance J et al. (2014), Higher Biology for CfE, UK, Hodder Gibson 2) Thomson, J. A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, (1998). 3) Stem Cell Therapy for Neuromuscular Diseases. Mirella Meregalliet al,"stem Cells in Clinic and Research", book, ISBN ) emcellsclassversatility.html 5) Teratoma Formation in Stem Cell Therapy progress/awards/prevention- and- treatment- teratoma- formation- during- stem- cell- therapy 14

15 6) Stem Cell Factsheet stem- cells- pioneers- stem- cell- research 7) Stem Cell Factsheet 8) Kumar et al, Stem cells: An overview with respect to cardiovascular and renal disease. J Nat Sci Biol Med Jul- Dec; 1(1): ) Macchiarini, Paolo et al. Clinical transplantation of a tissue- engineered airway. The Lancet. Volume 372, Issue 9655, ) Takahashi Ket al, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.cell.2006 Aug 25;126(4): ) Alzheimer s Disease How Could Stem Cells Help? disease- how- could- stem- cells- help 12) Pagliuca, F, Generation of functional human pancreatic beta cells in vitro. Cell. October 9, ) NHS Information dystrophy/pages/introduction.aspx 14) Becker s Factsheet muscular- dystrophy/causes- inheritance 15) Hanane Bellayouet al, Duchenne and Becker Muscular Dystrophy: Contribution of a Molecular and Immunohistochemical Analysis in Diagnosis in Morocco J Biomed Biotechnol. 2009; 2009: ) muscular- dystrophy/signs- and- symptoms 17) 18) Wagner KR et al, Currenttreatment of adultduchenne muscular dystrophy. Biochim Biophys Acta Feb;1772(2): ) dystrophy/pages/treatment.aspx 15

16 20) dystrophy- how- could- stem- cells- help 21) 22) Antonio Uccelli et al, Mesenchymal stem cells in health and diseasenature Reviews Immunology 8, (September 2008) 23) Ichim TEet al, Mesenchymal stem cells as anti- inflammatories: implications for treatment of Duchenne muscular dystrophy. Cell Immunology. 2010;260(2): ) Marg Aet al Humansatellitecells have regenerativecapacity and are geneticallymanipulablej Clin Invest Oct;124(10):