Blood Sci Ov
Content
HARVARD STEM CELL INSTITUTE
APRIL 2008
Stem Cell Science:
Overviews of Selected Disease Areas
Stem Cells and Blood Diseases
HARVARD STEM CELL INSTITUTE
Executive Director Brock C. Reeve, MPhil, MBA Scientific Directors Douglas A. Melton, PhD David T. Scadden, MD Editor Beverly Freeman Writing Tristan Davies Amy Greenwood, PhD Sarah Opitz Lisa Girard The Harvard Stem Cell Institute (HSCI) is a scientific collaborative established in 2004 to fulfill the promise of stem cell biology as the basis for the cure and treatment of a wide range of chronic diseases and medical conditions. HSCI’s unique effort unites experts across the disciplines, schools and departments of Harvard University and all its affiliated research hospitals. HSCI also sponsors public education programs concerning the scientific, legal and ethical implications of stem cell research, conducts a summer research program for college students, and helps educate high school teachers about stem cell science. HSCI depends upon the vision and generosity of private individuals, foundation and corporate donors to carry on its work, due to current U.S. restrictions on federal funding of embryonic stem cell research.
© 2007 President and Fellows of Harvard College
HARVARD STEM CELL INSTITUTE
Introduction
T
he following scientific overview focuses on the use of stem cells-both in research and potential therapeutic applications - to address one of the most challenging, life-changing diseases and conditions of our time. This overview along with its companion pieces has two educational objectives: to make clear the opportunities and promise inherent in the basic science and to provide you with a picture of the research avenues that must be pursued to reach clinical applications. The overviews point to the areas where fundamental questions exist, where therapies need improvement, and where funding for research is urgently required. Immense hope and scientific effort at institutions like the Harvard Stem Cell Institute have been invested in stem cells. Researchers seek the fullest understanding of how cells’ fates are determined. They want to know how embryonic stem (ESC) cells recognize and respond to the signals that move them to become the full range of mature cells in an animal. They want to know how adult stem cells, whose fates are more restricted, contribute to the repair and regeneration of organs and tissues. Studying both types of cells in parallel will provide us information pertinent to developing stem cell therapies. With each experiment and clinical trial, researchers and clinicians move closer to these goals. This set of papers summarizes the current state of stem cell science in several key disease areas. Stem cell research adds its own unique challenges to those faced by any early stage science. In fact, there are many cells, along with their behavior, that are still being discovered. In some cases the benefit, whether detailed fundamental knowledge, development of new drugs, or transplantable cells, is likely to be far in the future; in other cases, progress will come amazingly quickly. In stem cell research, as in few other contemporary scientific enterprises, success depends on supportive collaboration among diverse constituents: scientists and clinicians; local, state, and federal governments; universities and industry; and patients and their advocates. As a cross-institutional collaborative research and educational organization, HSCI is committed to meeting this challenge. We hope these disease overviews will help you better understand the research areas that matter to you. I would welcome your thoughts about these overviews, your questions, and concerns. Contact me at [email protected].
Brock Reeve, MPhil, MBA Executive Director
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HARVARD STEM CELL INSTITUTE
Stem Cells and Blood Diseases
Introduction
Blood consists of red blood cells, white blood cells and platelets. It makes up 7 percent of human body weight and a few drops of blood hold about a billion red blood cells. It accounts for almost 150 different types of cells and to date, there are almost as many known blood disorders. Blood diseases include leukemias, autoimmune disorders, genetic diseases, and bone marrow failure; the mechanisms for many are still unclear. Treatments for blood diseases are often time-consuming, debilitating and costly. Currently, blood stem cell transplantation is thought to be the most successful long-term method for replacing damaged or lost blood cells with healthy blood cells. Although transfusions and transplants have been attempted and used for decades, we are still hindered by the lack of healthy viable material for transplantation.
[Adult blood stem cells] are rare, 1 in every 10,000 bone marrow cells and 1 in every 100,000 circulating blood cells, yet because HSCs are easily accessible from the circulating blood…they are the best understood.
Understanding Blood
Blood has been a subject of fascination since 2500 B.C., when Egyptians used blood-letting as therapy; it wasn’t until at least the 17th century that the properties of the heart and circulatory system were properly described. In the early 20th century, when the different blood types were discovered and the practices of “matching” donors with recipients and blood banking were initiated, the practice of blood transfusion accelerated along with the desire for more knowledge about the blood’s properties. The timeline of discovery and characterization of the blood system has been punctuated with large gaps throughout early history that have narrowed as technology has advanced. As we understood more about the blood system and its ability to replenish itself, bone marrow transplantation (BMT) to treat blood disorders became a tantalizing possibility. The first clinical BMT was reported by E. Donnell Thomas in 1957, a breakthrough for which he won the Nobel Prize. Since then, this field has grown to the point that BMT is now the only curative treatment regimen for many blood diseases (see Table 1). We now understand that the success of BMT is due to a specialized type of cell found within the bone marrow—the hematopoietic stem cell.
What Is a Hematopoietic Stem Cell?
The life of most blood cells is short: Red blood cells live for 120 days; platelets live for six days; and some white blood cells live for one day. Therefore, the blood must have the capacity to replenish itself throughout a person’s life. Enter the hematopoietic stem cell (HSC). HSCs are adult blood stem cells that have the ability to self-renew (duplicate) and the ability to generate all the mature cells of the blood and immune systems (see Figure 1). They are rare,
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with 1 in every 10,000 bone marrow cells and 1 in every 100,000 circulating blood cells, yet because HSCs are easily accessible from the circulating blood, bone marrow or umbilical cord they are the best understood adult stem cell to date.
FIGURE 1:
HSC development. Courtesy of M. William Lensch, PhD
Clinical Relevance
Blood is accessible from several sources: the circulation (peripheral blood), the umbilical cord, and bone marrow. HSCs are capable of maintaining and replenishing the entire blood system and can be isolated from the general pool of blood cells. Scientists have now turned their attention to increasing the efficiency of transplants and generating new cell-based therapies for blood diseases. Currently, there are two types of hematopoietic stem cell transplants (HSCTs): allogeneic and autologous.
Autologous: In this case, patients use their own cells as a source of donor material. This form of treatment is still experimental, yet it has been used to treat patients with solid tumors because of the advantage that the patient will not suffer the complications of graft-versus-host
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disease (GVHD). GVHD is a post-transplant condition where donor cells (graft) recognize patient tissues (host) as foreign and attack them. Diseases that are treated by autologous HSCTs generally do not originate in the bone marrow but, with the exception of leukemia, are caused by cancerous tumors located elsewhere in the body. Examples of these diseases include some types of solid tumors, such as lymphoma, sarcoma and brain tumors, breast cancer, ovarian cancer, and Hodgkin’s disease. The chemotherapy and/or radiation that patients undergo to destroy the tumor will also destroy their healthy bone marrow, without which patients will not be able to fight infection. Combining chemotherapy and/or radiation with autologous HSCT enables a physician to “rescue” the patient’s bone marrow.
Allogeneic: In this case, the donor is someone other than the patient. He or she may be a relative, such as a sibling or parent, or the donor may be a closely matched but unrelated individual. Diseases treated by allogeneic transplants are usually genetic disorders, autoimmune diseases, bone marrow failures, some leukemias, aplastic anemia and sickle cell disease. Patients facing an allogeneic transplant may also suffer GVHD. In some cases, a supportive effect that has been recently observed and documented takes place: graft-versus-leukemia or graft-versus-tumor effect. This happens when donor cells recognize the patient’s residual cancer cells as being foreign and destroy them.
Techniques for Understanding Blood
Blood is arguably the best-understood mammalian organ system and has become a model for other organ systems in terms of the study approach, methods employed and the tools developed. The term “stem cell” was coined in the scientific literature by Ernst Haeckel in 1868. Around the same time, there was debate over whether a common progenitor cell in the blood existed. In 1896, Artur Pappenheim used the word stem cell to describe a cell that has the ability to generate both red and white blood cells. In the early 1960s, James Till and Ernest McCulloch designed the first experiment to visualize a “stem cell” in a spleen colony assay, thus retrospectively studying its function and potential. The first real evidence for the existence of a cell capable of repopulating a damaged hematopoietic compartment (the bone marrow) comes from experiments in the 1950s where researchers found they could save a mouse from death caused by radiation by shielding a bone in lead, by a bone marrow infusion, or by parabiosis (surgically joining two mice together so that they share blood systems). This finding allowed researchers to determine that the cells important for regenerating the blood system are in the bone marrow. A critical next step was the ability to identify and purify selected cell populations. By adding fluorescent tags to a specific antibody or combination of antibodies, researchers can separate the cell type(s) that they want from those they don’t want. For example, researchers can identify HSCs and purify them from the pool of whole blood using lasers and a technology
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called fluorescent-activating-cell-sorting (FACS). The combination of using cell surface marking and FACS to identify specific cells was developed in 1988 by Gerald Spangrude and Irving Weissman at Stanford University School of Medicine. They were the first group to use this method to purify a cell population from bone marrow enriched for HSCs and to reconstitute the blood and immune systems of a mouse whose own system had been destroyed by radiation. The understanding of HSCs has grown with the identification of specific growth factors necessary for their development and maintenance, with the development of robust methods for assessing the properties and function of HSCs, both in vitro and in vivo, and with the ability to transplant human HSCs into mouse models; these findings were reported in the early 1980s by Bosma et al., and Greiner et al. There are still several complicated issues standing in the way of our ability to use blood stem cells for efficient, effective and safe replacement therapies: • • • • Achieving viable self-renewal and expansion of HSCs Improving HSC transplants Improving cord blood engraftment Understanding the development of blood cells from their non-blood progenitors
Self-Renewal and Expansion
Much has been accomplished with regard to separating and culturing HSCs in vitro, but these techniques increase the proportion of HSCs—it is still not possible to isolate a homogenous HSC population. Another challenge is that HSCs do not survive long outside the body. Our inability to generate large numbers of cells in vitro hampers our ability to study the developmental biology of these cells in the laboratory. It also hampers the anticipated need to scale-up production of clinical-grade cells for therapy. The process of self-renewal of HSCs is not yet well understood. HSCs are critically important in blood for simple maintenance or turnover of blood cells, for responding to injury, cellular disorder or blood loss, and for replenishing after clinical treatments such as chemotherapy. Most HSCs (90 percent) are dormant and nondividing; yet HSCs can launch a rapid response to generate the billions of blood cells needed to repopulate the system within days. What are the molecular mechanisms that keep HSCs quiescent and what are the signals that activate them? Determining these molecular pathways will enable scientists to artificially maintain HSCs in the laboratory as well as stimulate replication. Many research groups have reported successful expansion and maintenance of HSCs in vitro, yet this is short-lived.
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Scientists have identified a few examples of molecular candidates that may be important for maintaining the self-renewal of HSCs:
Notch: A protein involved in cellular differentiation, division, cell death, and adhesion. Wnt: A protein that is crucial for the development of many organ systems. BMP (bone morphogenetic protein): A protein that plays multiple important roles in the development and regulation of many systems. They are essential in the self-renewal of mouse embryonic stem cells, yet promote the differentiation of human embryonic stem cells and may also indirectly regulate the size of the HSC microenvironment. Bmi-1: A gene that is central to HSC replication; it represses certain genes that inhibit cell division. HoxB4: A protein that may also be an important self-regulator of HSC self-renewal. Overexpression of this transcription factor can expand HSCs both in animal models and in the laboratory without promoting the formation of cancer.
Some other groups are attempting to regulate HSC expansion in vitro by strictly defining the culture conditions, for example, by regulating the substrate these cells are grown on and by adding certain growth factors and cytokines at different time points. These methods are limited, however, in that there is variation between laboratory protocols, and they often promote the expansion of mature blood cells rather than immature progenitors needed for transplant. Although significant progress has been made in understanding how to expand HSCs in vitro, there are complexities that need to be addressed. Most methods require genetic modification, the long-term consequences of which are unknown. Also, much is still unknown about the HSC microenvironment (the niche), and recent work has demonstrated that the HSC niche plays as crucial a role as the HSCs themselves. Clearly, mouse studies have established a foundation for our knowledge, but there are significant differences between mouse and human HSCs and the transition from animal models to human models will not be easy. To complicate things further, many factors crucial for HSC expansion also play central roles in the development and progression of many cancers.
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Improving Transplants
The traditional method for BMT involves high doses of chemotherapy and/or radiation with the goal of destroying a patient’s bone marrow and thereby eradicating the disease. Unfortunately, a severe side affect is that chemotherapy and radiation also destroy the patient’s healthy bone marrow cells, so doctors follow the treatment with an HSCT to rescue the patient’s bone marrow. Until recently, this was the most effective treatment but one with significant cost. Patients whose immune systems have been compromised in preparation for an HSCT often develop infection, graft rejection, and graft-versus-host disease (GVHD). Certain mature white blood cells that are carried over from donor to recipient during an HSCT are like a double-edged sword. They can be both helpful and harmful. They allow the recipient to survive while the transplanted HSCs engraft, which can take about two to four weeks. Reconstituting immune function, however, may take several months for an autologous HSCT and up to two years for allogeneic HSCT recipients, which further increases the immediate need for functional white blood cells. In contrast, white blood cells may also increase the risk of GVHD, so they are often filtered out of the bone marrow or irradiated before transplantation. In contrast, some patients develop graft-versus-leukemia (GVL), sometimes called graft-versus-tumor effect, where donor cells recognize residual or recurring disease cells of the recipient and destroy them. Interestingly, the cells that mediate GVHD and GVL are also essential for engraftment of donor cells into a recipient and are important for fighting infection. Researchers are currently investigating methods for identifying unique mechanisms in order to control the enhancement of GVL and suppression of GVHD. Strategies aimed at treating residual disease, relapsed disease and viral infections after an HSCT include donor lymphocyte infusion (DLI), T-cell therapies, monoclonal antibody immunotherapy, vaccines and immune-stimulating factors. DLI is helpful to patients who have relapsed. Since transplant recipients have had their immune system reconstituted by a donor’s cells, they can, for the most part, tolerate a later infusion of donor cells, which then recognize and target diseased cells in the recipient for destruction. Again, however, the recipient may develop GVHD. T-cell therapies generally focus on recognizing tumor antigens. There are numerous types of T-cell therapies that use specific T-cell subsets that are either depleted, expanded in the laboratory, used in combination, or genetically modified. Therapeutic vaccines, rather than prevent a disease, treat an existing disease. They are designed to target the unique antigens present on a particular type of cancer cell, thereby exposing it to the patient’s immune system. Monoclonal antibodies target specific cancer cells and can carry radioactivity or chemicals that can destroy those cells. To date, a handful of these antibodies have been approved, with more in clinical trials. Factors that stimulate the patient’s immune system to generate more blood cells include those that inhibit cancer cell replication, those that activate other white blood cells and those that stimulate the production of young blood cells, including HSCs.
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Improving Umbilical Cord Blood-Based Transplants
Umbilical cord blood (UCB) is collected from the umbilical cord and placenta after the birth of a baby and frozen for later use. The collection procedure is not harmful to the mother or newborn and access and availability are relatively easy because of banking. Umbilical cord blood transplants (UCBTs) have been performed in children since 1988 and are a promising source of HSCs, since a newborn’s immune system is immature, providing more flexibility with matching donors and recipients. However, limitations have prevented UCBT in adults until very recently. UCB, although rich in blood stem cells, contains one-tenth the number of viable cells compared with bone marrow. Depending on the size and weight of the patient, there may not be enough cells for a successful transplant. There is no option of back-up cells from the same cord blood unit, so if the initially transplanted cells do not engraft or a patient relapses, the patient will have to be infused with a different cord blood unit or then matched with an adult donor. In addition, it takes longer for engraftment and immune reconstitution with UCBTs than with BMTs, leaving patients vulnerable to greater risk of graft failure, infection and aberrant immune reconstitution —all which lead to a higher mortality rate. Thus, research is focusing on the expansion of the numbers of stem cells in cord blood grafts, enabling temporary engraftment with other stem cell sources and reducing the intensity of pre-HSCT regimens. Efforts to expand HSCs in cord blood follow similar approaches as those taken for marrow-derived HSCs, mentioned above. In addition, new methods are being tested on cord blood HSCs, such as inhibiting the maturation of HSCs during expansion; regulating the concentration of copper, which has been implicated for maintaining immature HSCs and enabling engraftment; incorporating derivatives of vitamin A, which are involved in embryonic development and cellular differentiation and have shown signs of increasing HSC expansion; and activating the Wnt pathway, which may also enhance HSC engraftment.
Embryonic Stem Cell-Derived HSCs
While embryonic stem cells (ESCs) attract much media attention and often become entangled in political posturing, these cells are crucial to accelerating work on HSCs. ESCs are derived from the inner cell mass of a pre-implantation embryo. They have the unique ability to both self-renew and to mature into all cell types of the body. They grow indefinitely in vitro, meaning that researchers are able to generate large numbers of these cells and maintain them in an undifferentiated, or immature, stage. In this state, they are a crucial tool with which to investigate the possibility of making HSCs. If researchers were able to direct the maturation of blood stem cells from ESCs, this would overcome many obstacles hampering the blood disease field and indeed many other disease areas.
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James Thomson reported the first successful derivation of human ESCs (hESCs) in 1998, and since then, several groups, Thomson’s included, have demonstrated that hESCs are able to differentiate into blood. Although some published data demonstrate the ability of hESCderived HSCs to repopulate the bone marrow, the proportion of blood cells produced remains quite low. In general, most protocols currently do not meet the high standards and criteria necessary for deriving true HSCs. Another direction some research groups have initiated is to focus on deriving specific mature blood cell types —for example, T-cells—from ESCs. Interestingly, initial reports indicate that the derived cells exhibit immature properties, more reminiscent of progenitor blood cells, rather than the desired mature blood cells. Several questions must be answered so researchers can develop new therapies and improve current therapies for blood diseases. The use of ESCs looks very promising, not only for studying normal human development, but also for studying the processes involved in disease onset and progression. A key next step is to develop the defined conditions under which stem cells can be grown in the laboratory. This is essential to properly isolate specific blood cell types that are needed and for understanding how to coax these cells into desired mature adult blood cells. In addition, scientists are working on elucidating the signals for improving engraftment and long-term repopulation of the blood system, as well as the molecular mechanisms that distinguish healthy stem cells from cancer cells. Scientists are laying the foundation for determining how to create patient-specific blood cells that will negate the need for immune-compatibility (eliminating the risk associated with the patient’s body rejecting the transplant). For more information about the HSCI Stem Cell and Blood Disease Program, please visit the HSCI Web site at www.hsci.harvard.edu.
REFERENCES: Reya R., et al. “Stem cells, cancer, and cancer stem cells.” Nature. 2001, 414:105-111. (Figure 1) Shlomchik, W.D. “Graft-versus-host disease.” Nature Reviews Immunol. 2007, 7:340-352. Sornberger, J. “Canadians Till and McCulloch proved existence of stem cells.” Stem Cell Network News Magazine. 2004, 3(1):1-6. Spangrude, G.J., et al. “Purification and characterization of mouse hematopoietic stem cells.” Science. 1988, 241(4861):58-62. Thomson, J.A., et al. “Embryonic stem cell lines derived from human blastocysts.” Science. 1988, 282(5391):1145-1147.
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Table 1
This is not an exhaustive list of blood disorders, and some may belong to multiple categories. In addition, many of the syndromes encompass additional disorders, which are not listed beneath them. Bone marrow failure syndromes and syndromes with similar presentation • Fanconi anemia • Aplastic anemia • Acquired aplastic anemia • Diamond-Blackfan anemia • Dyskeratosis congenital • Schwachman-Diamond syndrome • Paroxysmal nocturnal hemoglobinuria • Myelodysplastic syndromes • Large granular lymphocyte leukemia Hemoglobinopathies and thalassemias (congenital abnormality regarding hemoglobin) • Sickle cell disease • Thalassemia • Methemoglobinemia Anemias and acquired anemias (lack of red blood cells or hemoglobin) • Iron deficiency anemia • Megaloblastic anemia: Vitamin B12 deficiency; Folate deficiency • Hemolytic anemias (destruction of red blood cells) Decreased numbers of cells • Myelofibrosis • Neutropenia: Kostmann’s syndrome • Agranulocytosis • Glanzmann’s thrombasthenia • Thrombocytopenia Myeloproliferative disorders (increased numbers of cells) • Polycythemia vera • Leukocytosis • Thrombocytosis • Myeloproliferative disorders Hematological malignancies • Lymphomas: Hodgkin’s disease; Non-Hodgkin’s disease; Burkitt’s lymphoma; Anaplastic large cell lymphoma; Splenic marginal zone lymphoma; Hepatosplenic T-cell lymphoma; Angioimmunoblastic T-cell lymphoma • Myelomas: Multiple myeloma; Waldenström macroglobulinemia: Plasmacytoma • Leukemias: ALL; CLL; AML; CML; T-PLL; CNL; HCL; T-LGL; Aggressive NK-cell leukemia
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Table 1
( continued )
Coagulopathies • Thrombycytosis • Recurrent thrombosis • Disseminated intravascular coagulation • Disorders of clotting proteins • Disorders of platelets Hematological changes secondary to non-hematologic disorders • Anemia of chronic disease • Infectious mononucleosis • AIDS • Malaria • Leishmaniasis Miscellaneous • Haemochromatosis • Asplenia • Hypersplenism • Monoclonal gammopathy of undertermined significance
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{ Notes }
{ Notes }
HARVARD STEM CELL INSTITUTE
42 Church Street Cambridge, MA 02138
www.hsci.harvard.edu
APRIL 2008
Stem Cell Science:
Overviews of Selected Disease Areas
Stem Cells and Blood Diseases
HARVARD STEM CELL INSTITUTE
Executive Director Brock C. Reeve, MPhil, MBA Scientific Directors Douglas A. Melton, PhD David T. Scadden, MD Editor Beverly Freeman Writing Tristan Davies Amy Greenwood, PhD Sarah Opitz Lisa Girard The Harvard Stem Cell Institute (HSCI) is a scientific collaborative established in 2004 to fulfill the promise of stem cell biology as the basis for the cure and treatment of a wide range of chronic diseases and medical conditions. HSCI’s unique effort unites experts across the disciplines, schools and departments of Harvard University and all its affiliated research hospitals. HSCI also sponsors public education programs concerning the scientific, legal and ethical implications of stem cell research, conducts a summer research program for college students, and helps educate high school teachers about stem cell science. HSCI depends upon the vision and generosity of private individuals, foundation and corporate donors to carry on its work, due to current U.S. restrictions on federal funding of embryonic stem cell research.
© 2007 President and Fellows of Harvard College
HARVARD STEM CELL INSTITUTE
Introduction
T
he following scientific overview focuses on the use of stem cells-both in research and potential therapeutic applications - to address one of the most challenging, life-changing diseases and conditions of our time. This overview along with its companion pieces has two educational objectives: to make clear the opportunities and promise inherent in the basic science and to provide you with a picture of the research avenues that must be pursued to reach clinical applications. The overviews point to the areas where fundamental questions exist, where therapies need improvement, and where funding for research is urgently required. Immense hope and scientific effort at institutions like the Harvard Stem Cell Institute have been invested in stem cells. Researchers seek the fullest understanding of how cells’ fates are determined. They want to know how embryonic stem (ESC) cells recognize and respond to the signals that move them to become the full range of mature cells in an animal. They want to know how adult stem cells, whose fates are more restricted, contribute to the repair and regeneration of organs and tissues. Studying both types of cells in parallel will provide us information pertinent to developing stem cell therapies. With each experiment and clinical trial, researchers and clinicians move closer to these goals. This set of papers summarizes the current state of stem cell science in several key disease areas. Stem cell research adds its own unique challenges to those faced by any early stage science. In fact, there are many cells, along with their behavior, that are still being discovered. In some cases the benefit, whether detailed fundamental knowledge, development of new drugs, or transplantable cells, is likely to be far in the future; in other cases, progress will come amazingly quickly. In stem cell research, as in few other contemporary scientific enterprises, success depends on supportive collaboration among diverse constituents: scientists and clinicians; local, state, and federal governments; universities and industry; and patients and their advocates. As a cross-institutional collaborative research and educational organization, HSCI is committed to meeting this challenge. We hope these disease overviews will help you better understand the research areas that matter to you. I would welcome your thoughts about these overviews, your questions, and concerns. Contact me at [email protected].
Brock Reeve, MPhil, MBA Executive Director
1
HARVARD STEM CELL INSTITUTE
Stem Cells and Blood Diseases
Introduction
Blood consists of red blood cells, white blood cells and platelets. It makes up 7 percent of human body weight and a few drops of blood hold about a billion red blood cells. It accounts for almost 150 different types of cells and to date, there are almost as many known blood disorders. Blood diseases include leukemias, autoimmune disorders, genetic diseases, and bone marrow failure; the mechanisms for many are still unclear. Treatments for blood diseases are often time-consuming, debilitating and costly. Currently, blood stem cell transplantation is thought to be the most successful long-term method for replacing damaged or lost blood cells with healthy blood cells. Although transfusions and transplants have been attempted and used for decades, we are still hindered by the lack of healthy viable material for transplantation.
[Adult blood stem cells] are rare, 1 in every 10,000 bone marrow cells and 1 in every 100,000 circulating blood cells, yet because HSCs are easily accessible from the circulating blood…they are the best understood.
Understanding Blood
Blood has been a subject of fascination since 2500 B.C., when Egyptians used blood-letting as therapy; it wasn’t until at least the 17th century that the properties of the heart and circulatory system were properly described. In the early 20th century, when the different blood types were discovered and the practices of “matching” donors with recipients and blood banking were initiated, the practice of blood transfusion accelerated along with the desire for more knowledge about the blood’s properties. The timeline of discovery and characterization of the blood system has been punctuated with large gaps throughout early history that have narrowed as technology has advanced. As we understood more about the blood system and its ability to replenish itself, bone marrow transplantation (BMT) to treat blood disorders became a tantalizing possibility. The first clinical BMT was reported by E. Donnell Thomas in 1957, a breakthrough for which he won the Nobel Prize. Since then, this field has grown to the point that BMT is now the only curative treatment regimen for many blood diseases (see Table 1). We now understand that the success of BMT is due to a specialized type of cell found within the bone marrow—the hematopoietic stem cell.
What Is a Hematopoietic Stem Cell?
The life of most blood cells is short: Red blood cells live for 120 days; platelets live for six days; and some white blood cells live for one day. Therefore, the blood must have the capacity to replenish itself throughout a person’s life. Enter the hematopoietic stem cell (HSC). HSCs are adult blood stem cells that have the ability to self-renew (duplicate) and the ability to generate all the mature cells of the blood and immune systems (see Figure 1). They are rare,
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with 1 in every 10,000 bone marrow cells and 1 in every 100,000 circulating blood cells, yet because HSCs are easily accessible from the circulating blood, bone marrow or umbilical cord they are the best understood adult stem cell to date.
FIGURE 1:
HSC development. Courtesy of M. William Lensch, PhD
Clinical Relevance
Blood is accessible from several sources: the circulation (peripheral blood), the umbilical cord, and bone marrow. HSCs are capable of maintaining and replenishing the entire blood system and can be isolated from the general pool of blood cells. Scientists have now turned their attention to increasing the efficiency of transplants and generating new cell-based therapies for blood diseases. Currently, there are two types of hematopoietic stem cell transplants (HSCTs): allogeneic and autologous.
Autologous: In this case, patients use their own cells as a source of donor material. This form of treatment is still experimental, yet it has been used to treat patients with solid tumors because of the advantage that the patient will not suffer the complications of graft-versus-host
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disease (GVHD). GVHD is a post-transplant condition where donor cells (graft) recognize patient tissues (host) as foreign and attack them. Diseases that are treated by autologous HSCTs generally do not originate in the bone marrow but, with the exception of leukemia, are caused by cancerous tumors located elsewhere in the body. Examples of these diseases include some types of solid tumors, such as lymphoma, sarcoma and brain tumors, breast cancer, ovarian cancer, and Hodgkin’s disease. The chemotherapy and/or radiation that patients undergo to destroy the tumor will also destroy their healthy bone marrow, without which patients will not be able to fight infection. Combining chemotherapy and/or radiation with autologous HSCT enables a physician to “rescue” the patient’s bone marrow.
Allogeneic: In this case, the donor is someone other than the patient. He or she may be a relative, such as a sibling or parent, or the donor may be a closely matched but unrelated individual. Diseases treated by allogeneic transplants are usually genetic disorders, autoimmune diseases, bone marrow failures, some leukemias, aplastic anemia and sickle cell disease. Patients facing an allogeneic transplant may also suffer GVHD. In some cases, a supportive effect that has been recently observed and documented takes place: graft-versus-leukemia or graft-versus-tumor effect. This happens when donor cells recognize the patient’s residual cancer cells as being foreign and destroy them.
Techniques for Understanding Blood
Blood is arguably the best-understood mammalian organ system and has become a model for other organ systems in terms of the study approach, methods employed and the tools developed. The term “stem cell” was coined in the scientific literature by Ernst Haeckel in 1868. Around the same time, there was debate over whether a common progenitor cell in the blood existed. In 1896, Artur Pappenheim used the word stem cell to describe a cell that has the ability to generate both red and white blood cells. In the early 1960s, James Till and Ernest McCulloch designed the first experiment to visualize a “stem cell” in a spleen colony assay, thus retrospectively studying its function and potential. The first real evidence for the existence of a cell capable of repopulating a damaged hematopoietic compartment (the bone marrow) comes from experiments in the 1950s where researchers found they could save a mouse from death caused by radiation by shielding a bone in lead, by a bone marrow infusion, or by parabiosis (surgically joining two mice together so that they share blood systems). This finding allowed researchers to determine that the cells important for regenerating the blood system are in the bone marrow. A critical next step was the ability to identify and purify selected cell populations. By adding fluorescent tags to a specific antibody or combination of antibodies, researchers can separate the cell type(s) that they want from those they don’t want. For example, researchers can identify HSCs and purify them from the pool of whole blood using lasers and a technology
4
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called fluorescent-activating-cell-sorting (FACS). The combination of using cell surface marking and FACS to identify specific cells was developed in 1988 by Gerald Spangrude and Irving Weissman at Stanford University School of Medicine. They were the first group to use this method to purify a cell population from bone marrow enriched for HSCs and to reconstitute the blood and immune systems of a mouse whose own system had been destroyed by radiation. The understanding of HSCs has grown with the identification of specific growth factors necessary for their development and maintenance, with the development of robust methods for assessing the properties and function of HSCs, both in vitro and in vivo, and with the ability to transplant human HSCs into mouse models; these findings were reported in the early 1980s by Bosma et al., and Greiner et al. There are still several complicated issues standing in the way of our ability to use blood stem cells for efficient, effective and safe replacement therapies: • • • • Achieving viable self-renewal and expansion of HSCs Improving HSC transplants Improving cord blood engraftment Understanding the development of blood cells from their non-blood progenitors
Self-Renewal and Expansion
Much has been accomplished with regard to separating and culturing HSCs in vitro, but these techniques increase the proportion of HSCs—it is still not possible to isolate a homogenous HSC population. Another challenge is that HSCs do not survive long outside the body. Our inability to generate large numbers of cells in vitro hampers our ability to study the developmental biology of these cells in the laboratory. It also hampers the anticipated need to scale-up production of clinical-grade cells for therapy. The process of self-renewal of HSCs is not yet well understood. HSCs are critically important in blood for simple maintenance or turnover of blood cells, for responding to injury, cellular disorder or blood loss, and for replenishing after clinical treatments such as chemotherapy. Most HSCs (90 percent) are dormant and nondividing; yet HSCs can launch a rapid response to generate the billions of blood cells needed to repopulate the system within days. What are the molecular mechanisms that keep HSCs quiescent and what are the signals that activate them? Determining these molecular pathways will enable scientists to artificially maintain HSCs in the laboratory as well as stimulate replication. Many research groups have reported successful expansion and maintenance of HSCs in vitro, yet this is short-lived.
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Scientists have identified a few examples of molecular candidates that may be important for maintaining the self-renewal of HSCs:
Notch: A protein involved in cellular differentiation, division, cell death, and adhesion. Wnt: A protein that is crucial for the development of many organ systems. BMP (bone morphogenetic protein): A protein that plays multiple important roles in the development and regulation of many systems. They are essential in the self-renewal of mouse embryonic stem cells, yet promote the differentiation of human embryonic stem cells and may also indirectly regulate the size of the HSC microenvironment. Bmi-1: A gene that is central to HSC replication; it represses certain genes that inhibit cell division. HoxB4: A protein that may also be an important self-regulator of HSC self-renewal. Overexpression of this transcription factor can expand HSCs both in animal models and in the laboratory without promoting the formation of cancer.
Some other groups are attempting to regulate HSC expansion in vitro by strictly defining the culture conditions, for example, by regulating the substrate these cells are grown on and by adding certain growth factors and cytokines at different time points. These methods are limited, however, in that there is variation between laboratory protocols, and they often promote the expansion of mature blood cells rather than immature progenitors needed for transplant. Although significant progress has been made in understanding how to expand HSCs in vitro, there are complexities that need to be addressed. Most methods require genetic modification, the long-term consequences of which are unknown. Also, much is still unknown about the HSC microenvironment (the niche), and recent work has demonstrated that the HSC niche plays as crucial a role as the HSCs themselves. Clearly, mouse studies have established a foundation for our knowledge, but there are significant differences between mouse and human HSCs and the transition from animal models to human models will not be easy. To complicate things further, many factors crucial for HSC expansion also play central roles in the development and progression of many cancers.
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Improving Transplants
The traditional method for BMT involves high doses of chemotherapy and/or radiation with the goal of destroying a patient’s bone marrow and thereby eradicating the disease. Unfortunately, a severe side affect is that chemotherapy and radiation also destroy the patient’s healthy bone marrow cells, so doctors follow the treatment with an HSCT to rescue the patient’s bone marrow. Until recently, this was the most effective treatment but one with significant cost. Patients whose immune systems have been compromised in preparation for an HSCT often develop infection, graft rejection, and graft-versus-host disease (GVHD). Certain mature white blood cells that are carried over from donor to recipient during an HSCT are like a double-edged sword. They can be both helpful and harmful. They allow the recipient to survive while the transplanted HSCs engraft, which can take about two to four weeks. Reconstituting immune function, however, may take several months for an autologous HSCT and up to two years for allogeneic HSCT recipients, which further increases the immediate need for functional white blood cells. In contrast, white blood cells may also increase the risk of GVHD, so they are often filtered out of the bone marrow or irradiated before transplantation. In contrast, some patients develop graft-versus-leukemia (GVL), sometimes called graft-versus-tumor effect, where donor cells recognize residual or recurring disease cells of the recipient and destroy them. Interestingly, the cells that mediate GVHD and GVL are also essential for engraftment of donor cells into a recipient and are important for fighting infection. Researchers are currently investigating methods for identifying unique mechanisms in order to control the enhancement of GVL and suppression of GVHD. Strategies aimed at treating residual disease, relapsed disease and viral infections after an HSCT include donor lymphocyte infusion (DLI), T-cell therapies, monoclonal antibody immunotherapy, vaccines and immune-stimulating factors. DLI is helpful to patients who have relapsed. Since transplant recipients have had their immune system reconstituted by a donor’s cells, they can, for the most part, tolerate a later infusion of donor cells, which then recognize and target diseased cells in the recipient for destruction. Again, however, the recipient may develop GVHD. T-cell therapies generally focus on recognizing tumor antigens. There are numerous types of T-cell therapies that use specific T-cell subsets that are either depleted, expanded in the laboratory, used in combination, or genetically modified. Therapeutic vaccines, rather than prevent a disease, treat an existing disease. They are designed to target the unique antigens present on a particular type of cancer cell, thereby exposing it to the patient’s immune system. Monoclonal antibodies target specific cancer cells and can carry radioactivity or chemicals that can destroy those cells. To date, a handful of these antibodies have been approved, with more in clinical trials. Factors that stimulate the patient’s immune system to generate more blood cells include those that inhibit cancer cell replication, those that activate other white blood cells and those that stimulate the production of young blood cells, including HSCs.
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Improving Umbilical Cord Blood-Based Transplants
Umbilical cord blood (UCB) is collected from the umbilical cord and placenta after the birth of a baby and frozen for later use. The collection procedure is not harmful to the mother or newborn and access and availability are relatively easy because of banking. Umbilical cord blood transplants (UCBTs) have been performed in children since 1988 and are a promising source of HSCs, since a newborn’s immune system is immature, providing more flexibility with matching donors and recipients. However, limitations have prevented UCBT in adults until very recently. UCB, although rich in blood stem cells, contains one-tenth the number of viable cells compared with bone marrow. Depending on the size and weight of the patient, there may not be enough cells for a successful transplant. There is no option of back-up cells from the same cord blood unit, so if the initially transplanted cells do not engraft or a patient relapses, the patient will have to be infused with a different cord blood unit or then matched with an adult donor. In addition, it takes longer for engraftment and immune reconstitution with UCBTs than with BMTs, leaving patients vulnerable to greater risk of graft failure, infection and aberrant immune reconstitution —all which lead to a higher mortality rate. Thus, research is focusing on the expansion of the numbers of stem cells in cord blood grafts, enabling temporary engraftment with other stem cell sources and reducing the intensity of pre-HSCT regimens. Efforts to expand HSCs in cord blood follow similar approaches as those taken for marrow-derived HSCs, mentioned above. In addition, new methods are being tested on cord blood HSCs, such as inhibiting the maturation of HSCs during expansion; regulating the concentration of copper, which has been implicated for maintaining immature HSCs and enabling engraftment; incorporating derivatives of vitamin A, which are involved in embryonic development and cellular differentiation and have shown signs of increasing HSC expansion; and activating the Wnt pathway, which may also enhance HSC engraftment.
Embryonic Stem Cell-Derived HSCs
While embryonic stem cells (ESCs) attract much media attention and often become entangled in political posturing, these cells are crucial to accelerating work on HSCs. ESCs are derived from the inner cell mass of a pre-implantation embryo. They have the unique ability to both self-renew and to mature into all cell types of the body. They grow indefinitely in vitro, meaning that researchers are able to generate large numbers of these cells and maintain them in an undifferentiated, or immature, stage. In this state, they are a crucial tool with which to investigate the possibility of making HSCs. If researchers were able to direct the maturation of blood stem cells from ESCs, this would overcome many obstacles hampering the blood disease field and indeed many other disease areas.
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James Thomson reported the first successful derivation of human ESCs (hESCs) in 1998, and since then, several groups, Thomson’s included, have demonstrated that hESCs are able to differentiate into blood. Although some published data demonstrate the ability of hESCderived HSCs to repopulate the bone marrow, the proportion of blood cells produced remains quite low. In general, most protocols currently do not meet the high standards and criteria necessary for deriving true HSCs. Another direction some research groups have initiated is to focus on deriving specific mature blood cell types —for example, T-cells—from ESCs. Interestingly, initial reports indicate that the derived cells exhibit immature properties, more reminiscent of progenitor blood cells, rather than the desired mature blood cells. Several questions must be answered so researchers can develop new therapies and improve current therapies for blood diseases. The use of ESCs looks very promising, not only for studying normal human development, but also for studying the processes involved in disease onset and progression. A key next step is to develop the defined conditions under which stem cells can be grown in the laboratory. This is essential to properly isolate specific blood cell types that are needed and for understanding how to coax these cells into desired mature adult blood cells. In addition, scientists are working on elucidating the signals for improving engraftment and long-term repopulation of the blood system, as well as the molecular mechanisms that distinguish healthy stem cells from cancer cells. Scientists are laying the foundation for determining how to create patient-specific blood cells that will negate the need for immune-compatibility (eliminating the risk associated with the patient’s body rejecting the transplant). For more information about the HSCI Stem Cell and Blood Disease Program, please visit the HSCI Web site at www.hsci.harvard.edu.
REFERENCES: Reya R., et al. “Stem cells, cancer, and cancer stem cells.” Nature. 2001, 414:105-111. (Figure 1) Shlomchik, W.D. “Graft-versus-host disease.” Nature Reviews Immunol. 2007, 7:340-352. Sornberger, J. “Canadians Till and McCulloch proved existence of stem cells.” Stem Cell Network News Magazine. 2004, 3(1):1-6. Spangrude, G.J., et al. “Purification and characterization of mouse hematopoietic stem cells.” Science. 1988, 241(4861):58-62. Thomson, J.A., et al. “Embryonic stem cell lines derived from human blastocysts.” Science. 1988, 282(5391):1145-1147.
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Table 1
This is not an exhaustive list of blood disorders, and some may belong to multiple categories. In addition, many of the syndromes encompass additional disorders, which are not listed beneath them. Bone marrow failure syndromes and syndromes with similar presentation • Fanconi anemia • Aplastic anemia • Acquired aplastic anemia • Diamond-Blackfan anemia • Dyskeratosis congenital • Schwachman-Diamond syndrome • Paroxysmal nocturnal hemoglobinuria • Myelodysplastic syndromes • Large granular lymphocyte leukemia Hemoglobinopathies and thalassemias (congenital abnormality regarding hemoglobin) • Sickle cell disease • Thalassemia • Methemoglobinemia Anemias and acquired anemias (lack of red blood cells or hemoglobin) • Iron deficiency anemia • Megaloblastic anemia: Vitamin B12 deficiency; Folate deficiency • Hemolytic anemias (destruction of red blood cells) Decreased numbers of cells • Myelofibrosis • Neutropenia: Kostmann’s syndrome • Agranulocytosis • Glanzmann’s thrombasthenia • Thrombocytopenia Myeloproliferative disorders (increased numbers of cells) • Polycythemia vera • Leukocytosis • Thrombocytosis • Myeloproliferative disorders Hematological malignancies • Lymphomas: Hodgkin’s disease; Non-Hodgkin’s disease; Burkitt’s lymphoma; Anaplastic large cell lymphoma; Splenic marginal zone lymphoma; Hepatosplenic T-cell lymphoma; Angioimmunoblastic T-cell lymphoma • Myelomas: Multiple myeloma; Waldenström macroglobulinemia: Plasmacytoma • Leukemias: ALL; CLL; AML; CML; T-PLL; CNL; HCL; T-LGL; Aggressive NK-cell leukemia
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Table 1
( continued )
Coagulopathies • Thrombycytosis • Recurrent thrombosis • Disseminated intravascular coagulation • Disorders of clotting proteins • Disorders of platelets Hematological changes secondary to non-hematologic disorders • Anemia of chronic disease • Infectious mononucleosis • AIDS • Malaria • Leishmaniasis Miscellaneous • Haemochromatosis • Asplenia • Hypersplenism • Monoclonal gammopathy of undertermined significance
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