Stem Cells: The Revolution of Blood Transfusion. By Srinand Sundaram. Grade awarded: Pass with Merit

Transcription

1 Stem Cells: The Revolution of Blood Transfusion By Srinand Sundaram Grade awarded: Pass with Merit Research Paper Based on Pathology Lectures At Medlink, December 2014

2 Abstract For people around the world who rely on donated blood for survival, the current process of collection and transfusion of erythrocytes is suboptimal. This paper examines the potential of stem cells to reduce our reliance on blood donation by creating cultures of erythrocytes that can be grown in the laboratory. These cultures could be produced either from adult or from embryonic stem cells, each producing slightly different results, and with different associated ethical and practical issues. This paper addresses the issues that researchers would come up against in trying to establish a practice of mass-producing erythrocytes, in vitro, for transfusion. I believe that, while these issues are serious and will be difficult to overcome, it will be possible to achieve such a practice in the near future. Introduction Hospitals around the world rely heavily on donated blood in order to keep patients alive. Patients may require blood transfusions for many reasons, including large blood loss due to trauma or surgery, a severe form of anaemia, or an inherited blood disorder such as thalassaemia 1. Any one of these conditions would be fatal but for the possibility of blood transfusion. However, there are many issues associated with the use of the collected blood. Donated blood is in short supply In the USA alone, more than 41,000 units of blood are required every day 2 - this equates to close to 15 million per year. Although an average of 15.7 million blood donations are collected each year 2, there are many times during the year in which demand can outweigh the supply. In addition, the requirement for blood type matching means that many people may not have the type of blood they need easily available to them (see below). For example, despite the large numbers of units of American blood collected each year, New York City currently relies on Europe for 25% of its blood donations. 3 The problem of shortage of donated blood looks set only to increase in the USA- with an ageing population, and increase in the number of surgical procedures requiring blood transfusions, demand for donated blood is increasing by 6-8%, compared to a 2-3% increase in blood donations made annually. 3 2

3 Patients can only receive carefully matched blood transfusions The four blood types- A, B, AB and O- refer to the presence or absence of certain antigens on the surface of a person s erythrocytes. Everyone can be said to have one of these blood groups, as shown in Figure 1. Antigens present Blood Type Antibodies present Extra notes A A Anti-B B B Anti-A A and B AB None Universal acceptor None O Anti-A and Anti-B Universal donor Figure 1 As shown in the table above, people with a certain blood type develop certain antibodies, which target the antigens not present on their own red blood cells. This poses a problem for patients who require a blood donation, as there is a possibility that they will reject the donated blood. In addition to these A and B antigens, the rhesus (Rh) antigen also contributes to a person s blood type. Red blood cells either have this antigen (and so are Rh+) or do not (Rh-). As one might expect, Rhpeople produce the anti-rh antibody, whereas those who are Rh+ do not. It is for this reason that donated blood must be carefully categorised according to the antigens present, and then matched to a patient of a compatible blood type. While this may not seem difficult, it is often not possible to determine the blood type of a patient before they are given a transfusion- especially if they have suffered major trauma, and so urgently need to be given blood. In situations such as this, the only option available to doctors is to transfuse Type O- red blood cells. As shown in Figure 1, erythrocytes of this type have no ABO or Rh antigens on their surface, and so can be transfused to any patient (hence the term universal donor ). The complexity involved in blood typing means that while a seemingly adequate amount of blood may be collected each year, hospitals may still not have access to the particular type of blood required for a patient. Blood transfusions can spread diseases The potential of transfused blood to become infected makes it a less than ideal treatment for disease, particularly in poorer countries with higher infection risks, and less stringent health and safety procedures than ours. For example, Choudhury and Phadke (2001) 4 estimated that 119, 700 new cases of post transplant hepatitis occur in India each year, where 7 million units of blood are transfused annually. Although blood transfusion in the UK is more strictly regulated, spread of infection is still a concern. For example, in 2002 the British government spent $50 million to buy a private American firm to supply blood plasma to the NHS, amid fears that British plasma donations may be contaminated with the BSE virus. 5 Among the many measures taken in the UK and USA to prevent the spread of infection by blood transfusion is the keeping of donated blood for only a certain amount of time- in the case of red cells, 3

4 this is only 42 days 2. So clearly, preventing the infection of blood for transfusion presents its own problems, by putting added pressure on stocks of blood available for transfusion. How can stem cells help? Stem cell research has the potential to revolutionise future medical care. Stem cells are undifferentiated cells that can divide infinitely by mitosis, giving rise to more, identical cells. These cells can then go on to differentiate, forming more specialised cells. In fact, all of the specialised cells in our body, from neurones to epithelial cells to erythrocytes, originated from one stem cell- the zygote. A zygote and its daughter cells are all embryonic pluripotent stem cells- in other words, they can differentiate to form any type of specialised cell. These cells were first discovered in mice blastocysts (early embryos) by Evans and Kaufman, who published their findings in Nature in They subsequently used these findings to produce genetically modified mouse embryos, that were then implanted in surrogate mothers uteruses. This practice is still commonly used today to produce specifically genetically modified mice that can be used as models for diseases in humans, thus providing scientists with an ethically acceptable organism on which to test potential drugs and other therapies. As the embryo develops further, however, the cells produced begin to differentiate, in order to form the specialised cells needed to give rise to organs with specific functions. The more differentiated a cell becomes, the fewer options it has to differentiate further. By the time we are born, none of the original pluripotent stem cells that formed the early embryo remain- all our cells are more specialised. However, some multipotent stem cells can be found even in adult tissue- for example, haematopoietic stem cells (which give rise to the many different types of cells in the blood) exist in bone marrow. These are adult stem cells. 4 Figure 2

5 Figure 2 shows the stages of differentiation of a haematopoietic stem cell 13. As indicated, a haematopoietic stem cell can differentiate not only to form an erythrocyte, but also all types of white blood cell. This discovery in the 1960s has led to the development of bone marrow transplants as a treatment for leukaemia. This allows doctors to completely destroy patients bone marrow, in which the cancerous leukaemia cells are proliferating, and replace it with stem cells from a donor or the patient himself that will divide to produce healthy bone marrow. The next breakthrough in stem cell research was the work of James Thomson, who cultured the first human embryonic stem cell line in vitro in Since then, researchers have concentrated their efforts on using stem cells to replace damaged or dysfunctional tissues in humans, in an attempt to cure many diseases. For example, in 2010, a patient with spinal injuries was treated by the injection of oligodendrocyte (neurone) progenitor cells derived from human embryonic stem cells in the laboratory 7. The idea of the treatment was that these injected cells would divide to produce new nerve fibres, replacing those that had been lost. I believe that we should now be investigating more thoroughly how the properties of stem cells could be used in the production of red blood cells in vitro, to at least reduce our reliance on human donors. Discussion Blood is a complex tissue, containing a variety of cells with different functions. At present, artificially producing (on a large scale) blood containing all the various red and white blood cells, platelets, and other molecules would be too complicated. However, the purpose of most transfusions is to replace lost erythrocytes (red blood cells). It is these cells that contain haemoglobin, the compound that attaches to oxygen molecules. Thus, they are responsible for oxygen transportation around the body, and so are vital for survival. During episodes of extreme blood loss, it is the loss of these cells that is most damaging. It is possible to mass-produce red blood cells for transfusion from both embryonic and adult stem cells. Both processes have their own advantages and drawbacks. Using adult stem cells As mentioned in the introduction, doctors can already extract haematopoietic stem cells (blood cell precursors) and infuse them into patients to produce new granulocytes. This constitutes in important form of treatment for leukaemia. The process for obtaining the haematopoietic stem cells works as follows: The patient is injected with a chemical known as granulocyte- colony stimulating factor (G- CSF). This stimulates the production of both white blood cells and haematopoietic stem cells in the bone marrow, to such an extent that some of these cells spill over into the blood. They can then be removed simply via a catheter. The sample is processed to remove blood cells and bone fragments, before the haematopoietic stem cells are refrigerated for preservation. By using a similar process to obtain haematopoietic cells from a sample of volunteers, red blood cells could be synthesised in vitro: 5

6 The harvested stem cells would need to be cultured in a laboratory, in a medium that would cause them to multiply on a large scale. Some of these daughter haematopoietic cells would then need to be cultured in an environment that encourages erythropoiesis (differentiation to produce red blood cells). After trialling the process in a few laboratories, it could be rolled out to institutions worldwide, so that every hospital could culture their own haematopoietic stem cell lines. However, this form therapy would not be without its difficulties. First of all, it would be vital to ensure that all the cells that differentiate give rise to red blood cells. This is because, as shown in Figure 2, haematopoietic stem cells can also differentiate to form a number of different white blood cells, each with antigens on their membranes that would prompt an immune response if transfused into a patient. Research is already underway on this issue, and in 2005, Giarratana et al. published an article describing their large-scale production of erythrocytes through the differentiation of adult haematopoietic stem cells. 8 In their report, they explain that their process relied on mimicking the marrow environment in which erythropoiesis is stimulated. This shows that it is important to understand the mechanism of erythropoiesis in order to suggest a method to stimulate this event in the laboratory. Erythropoiesis The most important molecule for the differentiation of erythroid progenitors (cells that differentiate to form red blood cells) is the cytokine (a small glycoprotein) erythropoietin. This cytokine attaches to erythropoietin receptors on haematopoietic stem cells, which then triggers a cascade of processes on a molecular level within the cell, causing differentiation. 10 In vivo, the release of erythropoietin by the kidneys is regulated in a feedback loop, as shown below in Figure

7 Figure 3 In the body, it is obviously important to regulate erythropoietin production in this manner- a lack of erythrocytes would result in inadequate carriage of oxygen to the body's tissues, while too many erythrocytes produced at one time would cause the blood to become too viscous, thereby reducing the efficiency of transport as well as increasing risk of thrombosis. In this proposed in vitro erythropoiesis, scientists would want to be able to control the rate of differentiation of these red blood cells. They would therefore need to find a way of regulating the binding of erythropoietin to the erythroid progenitors receptors. One way of doing this would be simply to culture the donated haematopoietic stem cells in a medium with a high concentration of erythropoietin (already, this glycoprotein can be manufactured by genetically-modified bacteria 14 ). They would then need to monitor the rates of differentiation and proliferation of the stem cells- a simple mechanical system could be installed that diluted or concentrated the erythropoietin medium depending on the ratio of pluripotent stem cells to terminally differentiated erythrocytes. In order to mimic the marrow microenvironment, Giarratana et al. cultured the haematopoietic stem cells on marrow stromal cells- cells that make up the connective tissue in the marrow. They hypothesise that this provided some of the many other chemicals- mainly cytokines and growth factors- that are important in the differentiation pathway that gives rise to erythrocytes. While this is a certainly a step in the right direction to ex vivo large-scale erythropoiesis, even this is a simplified view of a complex process. In reality, erythropoiesis relies on a number of different chemicals and mechanisms that remain unknown. A lot of research is currently underway into understanding this process on a molecular level, and this knowledge will clearly be vital in the development of this procedure. Other considerations Practical considerations also need to be taken into account for this solution. The main such consideration is the sheer number of red blood cells that would need to be produced. One unit of transfused blood (450ml on average) contains approximately 3 trillion erythrocytes. 3 Given that 41,000 units of blood are needed every day in the USA alone 2, ex vivo erythropoiesis would have to occur on a massive scale if it were to replace blood donation. Unfortunately, very little is currently known about how adult stem cells could be encouraged to differentiate on a large scale in the laboratory. Giarratana et al. reported proliferation of the original cells by as much as 1.95 million times in their study. 8 While this is significant, it still means that several million haematopoietic stem cells would need to be cultured just to produce enough erythrocytes for one unit of blood. The small concentration at which haematopoietic stem cells are found in adult bone marrow would thus make finding enough donors extremely time-consuming and costly. Furthermore, as scientists have not yet been able to make adult stem cells differentiate indefinitely in vitro, more and more donor cells will be needed continuously. So although the process would considerably reduce the need for blood donors (as extracted cells could be multiplied millions of times), they do not give a potentially limitless supply of erythrocytes. With these problems in mind, it is certainly worth considering the use of embryonic stem cells instead of adult haematopoietic cells for erythropoiesis. 7

8 Using embryonic stem cells Current research is focusing primarily on the use of human embryonic stem cells (hescs) for the task. The main reason for this is that, unlike adult stem cells, their embryonic counterparts retain the ability to divide indefinitely, giving rise to new pluripotent stem cells, in vitro. So embryonic stem cells give a much greater hope of producing an unlimited supply of erythrocytes, without the need for donors. The most promising study on the subject to date was published by Lu et al. (2008) 11. It outlined a process in which embryonic stem cells could be encouraged to divide and differentiate, with 100% erythrocyte terminal differentiation. The medium which the scientists used to culture the hescs contained a combination of cytokines, growth factors and other substances known to be important for erythropoiesis, the most significant of which were erythropoietin, stem cell factor, and methylcellulose (the main source of energy for the dividing cells). In most other respects, the process used to culture the cells was identical to that used by the Giarratana et al. in the study mentioned earlier in this paper. Unlike the other study, however, the rate of proliferation and differentiation of the stem cells was much greater, with erythrocytes produced per six wells (small colony of haematopoietic cells). Furthermore, simply scaling up the quantities of chemicals used in this method should, theoretically, scale up the numbers of erythrocytes produced. In addition, this process does not rely on donors cells to start a culture, so would cause no problems in this respect. A further advantage to this procedure is that it would be possible to genetically modify the embryonic stem cells to ensure that they give rise to O- (universal donor) erythrocytes. It would be much more difficult to carry out this modification on every donated adult haematopoietic stem cell, and so for the method described previously, donors of all blood types would have been required to ensure that all patients would have blood of their required type available. Having said this, considerable research needs to be undertaken to carry out this genetic modification even in embryonic stem cells. Ethical issues also add to the difficulty of accepting this procedure (see below). Ethical issues Practically, embryonic stem cells may seem like the obvious solution to this problem of a shortage of blood donations. However, the scientific community has an obligation to consider the ethical implications of its actions. This is currently the main barrier to embryonic stem cell research. To be of use, these embryonic stem cells must be pluripotent. As shown in the introduction, this would require removing them from an embryo at a very early stage in development- the blastocyst stage. Unfortunately, removing a cell from a blastocyst would prevent the embryo from developing. Thus, many people would regard the use of these cells as a violation of the sanctity of life, rendering it unacceptable. Those who argue this say that life begins at conception, as any zygote has the potential to become a human being, and therefore preventing it from doing so can be described as killing it. On the other hand, some would say that an embryo is simply a collection of cells until it becomes autonomous, and so we should have no qualms with carrying out these experiments. Of course, these contrasting viewpoints are ends of a wide spectrum of opinions on the real question that underpins this body of research- at what point in development is an embryo really a human being? Recent scientific research has tried to answer this question, and many now agree that life begins at approximately fourteen days after conception. For until this stage, it is possible that an embryo could 8

9 split to form twins. 12 Furthermore, after this two-week period, a recognisable form of a nervous system begins to develop as the embryonic stem cells become increasingly specialised, leading developmental biologists to believe that this is the point at which the foetus begins to experience sensations, and to develop a sense of awareness. 12 As the blastocyst usually implants at about 8 days after fertilisation, those who agree with this view would ethically approve of the use of embryonic stem cells. Adding another dimension to the debate, even if one does deem an embryo to be a form of life, we have to address the importance of two important moral obligations- that of alleviating suffering and that of respecting human life. So even if someone might accord an embryo the status of a living being, they may still feel that sacrificing this embryo is a necessary evil to provide the treatment for another human life- one that is already being lived, and could be improved with this research. Furthermore, while the creation of embryos for the sake of research seems immoral, many researchers use surplus embryos from in vitro fertilisation procedures. On the one hand, this does sound much more acceptable- the embryos have already been created, and are likely to be discarded or stored indefinitely, so they may as well be of benefit to humanity. On the other hand, however, this brings up the need to discuss the ethical implications of abortion- again, a hotly debated topic. In the context of in vitro erythrocyte production, one could argue that the use of embryonic stem cells is even more problematic than it would be for the treatment of a disease such as Parkinson s Disease- a debilitating condition for which embryonic stem cells could provide the only cure as blood transfusions do currently exist. Furthermore, the genetic modification of embryonic stem cells (as mentioned in the previous section) adds further ethical uncertainty, raising the possibility of a slippery slope to the acceptance of this form of genetic intervention for non-medical purposes. In contrast, the current blood donation system and the use of adult stem cells are other solutions for this issue, and while they may not be as practically feasible, scientists clearly have a duty to try and circumvent the difficulties of these ethically favourable alternatives. In short, the application of embryonic stem cell research raises a huge range of ethical questions, all of which would need to be addressed if erythrocytes were to be produced in such a way. Conclusion Stem cell research offers the chance to revolutionise medicine, and the production of blood cells for transfusion is one area in which this could improve the quality of life of millions of people. The use of adult stem cells may not seem, at first, like a helpful solution, given that it would still require some donors (to provide the haematopoietic cells), and that this would not be a long-term selfsustaining process. Nevertheless, the fact that one adult stem cell could be encouraged to divide to produce well over a million red blood cells would still greatly reduce the demand for donors overall. The alternative- the use of embryonic stem cells- sounds far more attractive at first. For these pluripotent cells would be able to divide almost indefinitely, giving rise to the possibility of an infinite store of erythrocytes. However, as research in this area continues, the many ethical problems raised will need to be addressed. Thus, there is no one solution that comes without its drawbacks. For the time being, therefore, research into both areas should continue. For, despite all these problems, it seems very possible that ambitions for a world without red blood cell donation may yet be realised. 9

10 Bibliography 1Blood transfusion- NHS Choices transfusion/pages/introduction.aspx 2 Blood facts and statistics- American Red Cross 3 Blood substitutes 4N. Choudhury and S. Phadke (2001): Transfusion Transmitted Diseases. Indian Journal of Paediatrics; 68 (10): BBC News: UK buys safe blood supply for the NHS (2002) 6 M. Evans, M Kaufman (1981): Establishment in culture of pluripotential cells from mouse embryos. Nature 292 (5819): A. Coghlan (2010): First person treated in milestone stem cell trial. New Scientist; (2782) 8 M. Giarratana et al. (2005): Ex vivo generation of fully mature human red blood cells from hematopoietic stem cells. Nature Biotechnology; 23: PGCC A&P2- Blood 10

11 10 S.Middleton et al. (1999): Shared and unique determinants of the erythropoietin (EPO) receptor are important for binding EPO and EPO mimetic peptide. J. Biol. Chem. 274 (20): S. Lu et al. (2008): Biologic properties and enucleation of red blood cells from human embryonic stem cells. Blood 112 (12). 12 EuroStemCell: Human embryonic stem cell research and ethics 13 Vector: New research on blood stem cells takes root 14M. Kamionka et al. (2011): Engineering of Therapeutic Proteins Production in Escherichia coli. Current Pharmacological Biotechnology 12 (2): J. Thomson et al (1998): Embryonic Stem Cell Lines Derived from Human Blastocysts. Science 282 (5391):