Blood Disorders

Coding Dimension ID: 
278
Coding Dimension path name: 
Blood Disorders

Self-renewal and senescence in iPS cells derived from patients with a stem cell disease

Funding Type: 
Basic Biology II
Grant Number: 
RB2-01497
ICOC Funds Committed: 
$1 430 908
Disease Focus: 
Blood Disorders
Pediatrics
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Active
Public Abstract: 
The discovery of induced pluripotent stem (iPS) cell technology promises to revolutionize our understanding of human disease and to allow the development of new cellular therapies for regenerative medicine applications. The ability to reprogram a patient's fibroblasts to iPS cells creates the opportunity to expand human cells with a specific genetic defect and to study that defect in a defined cell population, either to understand the basic biology of the disease or to study potential therapeutics. Furthermore, the genetic defects in iPS cells can be repaired and the iPS cells used as a source for cellular therapies after differentiation to specific cell lineages. Although tremendous strides have been made in recent years in treating human disease, replacing damaged tissue remains almost completely beyond our grasp. Harnessing human iPS stem cells for this purpose will open completely new areas of regenerative medicine. However, a limited understanding of iPS cell self-renewal and differentiation is a major roadblock in realizing this long-term goal. One shared characteristic of iPS cells and adult stem cells that reside in many of our tissues is the ability to self-renew. Self-renewal is the ability of a stem cell to divide and give rise to a daughter cell that is undifferentiated and capable of giving rise to all the same lineages as the parent stem cell. Senescence pathways – pathways that cause dividing cells to permanently stop dividing – represents a significant barrier in the reprogramming process to engineer new iPS cells. Understanding how iPS cells self-renew is critical for determining how to maintain these cells, how to differentiate them toward specific tissue lineages and how to expand more committed stem cells or progenitor cells in cell culture. In this proposal, we investigate the molecular mechanism of self-renewal and senescence in human iPS cells using skin cells isolated from patients with a defect in the enzyme telomerase. Telomerase is an enzyme complex expressed in embryonic stem cells, some tissue stem cells and in almost all human cancers. Most differentiated cells lack telomerase expression. Telomerase adds DNA repeats to structures at the ends of our chromosomes, termed telomeres. Telomeres are very important in protecting chromosome ends and in preventing chromosome ends from breaking down or sticking to other ends inappropriately. By maintaining telomeres, telomerase supports the ability of stem cells to divide a large number of times. People with telomerase mutations develop a stem cell disease – dykeratosis congenita. In this disease, patients have defects in skin, blood and lung – tissues that depend on tissue stem cell function to maintain these organs during life. We will reprogram skin cells from dyskeratosis patients to understand how senescence responses limit iPS cell self-renewal and differentiation to specific cell lineages.
Statement of Benefit to California: 
This proposal will benefit California and its citizen in two general ways. First, I have recruited new scientists to California from Texas and from Brazil to work on this proposal. These are new taxpayers and consumers, which will benefit local businesses. They would have been less likely to come to California in the absence of the CIRM program and its strong emphasis on human stem cell biology. Second, this novel grant will generate new intellectual property in the form of patents. These patents may in fact be licensed to California companies or be used to support the formation of new start-up companies. The growth of such companies has historically fueled much of the profound growth in California. The future of California is linked to new technologies in the stem cell, biotechnology and other technology.
Progress Report: 
  • Over the past year, we have analyzed five induced pluripotent stem (iPS) cell lines engineered from different individuals with a genetic stem cell disease. Dyskeratosis congenita is a rare disease affecting stem cells in multiple tissues. Patients with dyskeratosis congenita develop life-threatening bone marrow failure and pulmonary fibrosis, and are highly prone to cancers. In addition, they develop defects in skin, nails and many other organs. Dyskeratosis congenita is caused by mutations in an enzyme - telomerase - that is particularly important in stem cells. Telomerase elongates telomeres, caps that protect chromosome ends. If telomerase is defective, telomeres shorten and loss of the protective cap at telomeres can cause serious problems in stem cells. It has been very difficult to study this disease because isolating stem cells from dyskeratosis congenita patients is challenging. To overcome this problem, we engineered iPS cells from five patients. This is a way to change skin cells into cells that closely resemble embryonic stem cells - stem cells that can give rise to all tissues within the body. We studied these iPS cells from dyskeratosis congenita patients and found that the type of effects on telomerase were very specific and depended on the specific gene that is mutated in the patient. For example, mutations in TERT, the catalytic protein in the telomerase complex, resulted in a 50% reduction in telomerase activity in the patient's iPS cells. In contrast, mutations in the protein dyskerin, seen in the X-linked form of the disease, reduced telomerase activity by a much greater amount - 90% compared to controls. Mutations in another telomerase protein, TCAB1, left telomerase activity unaffected, but made the enzyme mislocalize within the nucleus. We studied how telomeres elongated with reprogramming of skin cells to iPSCs for each patient. Normal cells from healthy people show significant elongation of telomeres during the making of iPSCs, because telomerase is reactivated during this process. For TERT-mutant patients, elongation still happened, but elongation was significantly blunted. For dyskerin-mutant iPS cells and TCAB1-mutant iPS cells, elongation was completely blocked by the mutations and instead, telomeres shortened during this process and with passage in culture. Importantly, the much more severe telomere defect in dyskerin-mutant and TCAB1-mutant cells corresponds closely with the severity of the disease in the patients themselves. Our data show that iPS cells are a very accurate system for studying dyskeratosis congenita and revealed for the first time that the severity of the disease correlates with the severity of the telomerase defect in stem cells. These findings create new opportunities to study stem cell diseases in cell culture and to develop therapies that could specifically reverse the disease defect.
  • Over the past year, we have generated and analyzed new induced pluripotent stem (iPS) cell lines engineered from different individuals with a genetic stem cell disease. Dyskeratosis congenita is a rare disease affecting stem cells in multiple tissues. Patients with dyskeratosis congenita develop life-threatening bone marrow failure and pulmonary fibrosis, and are highly prone to cancers. In addition, they develop defects in skin, nails and many other organs. Dyskeratosis congenita is caused by mutations in an enzyme - telomerase - that is particularly important in stem cells. Telomerase elongates telomeres, caps that protect chromosome ends. If telomerase is defective, telomeres shorten and loss of the protective cap at telomeres can cause serious problems in stem cells. It has been very difficult to study this disease because isolating stem cells from dyskeratosis congenita patients is challenging. To overcome this problem, we engineered iPS cells from dyskeratosis congenita patients. This is a way to change skin cells into cells that closely resemble embryonic stem cells - stem cells that can give rise to all tissues within the body. We studied these iPS cells from dyskeratosis congenita patients and found that the type of effects on telomerase were very specific and depended on the specific gene that is mutated in the patient. Normal cells from healthy people show significant elongation of telomeres during the making of iPSCs, because telomerase is reactivated during this process. In iPS cells from patients with dyskeratosis congenita by contrast, telomere elongation during reprogramming is compromised. These findings create new opportunities to study stem cell diseases in cell culture and to develop therapies that could specifically reverse the disease defect.
  • Over the past year, we have generated and analyzed new induced pluripotent stem (iPS) cell lines engineered from individuals with a genetic stem cell disease. Dyskeratosis congenita is a rare disease affecting stem cells in multiple tissues. Patients with dyskeratosis congenita develop life-threatening bone marrow failure and pulmonary fibrosis, and are highly prone to cancers. In addition, they develop defects in skin, nails and many other organs. Dyskeratosis congenita is caused by mutations in an enzyme - telomerase - that is particularly important in stem cells. Telomerase elongates telomeres, caps that protect chromosome ends. If telomerase is defective, telomeres shorten and loss of the protective cap at telomeres can cause serious problems in stem cells. It has been very difficult to study this disease because isolating stem cells from dyskeratosis congenita patients is challenging. To overcome this problem, we engineered iPS cells from dyskeratosis congenita patients. This is a way to change skin cells into cells that closely resemble embryonic stem cells - stem cells that can give rise to all tissues within the body. We studied these iPS cells from dyskeratosis congenita patients and found that the type of effects on telomerase were very specific and depended on the specific gene that is mutated in the patient. Normal cells from healthy people show significant elongation of telomeres during the making of iPSCs, because telomerase is reactivated during this process. In iPS cells from patients with dyskeratosis congenita by contrast, telomere elongation during reprogramming is compromised. These findings create new opportunities to study stem cell diseases in cell culture and to develop therapies that could specifically reverse the disease defect.a

Molecular Characterization and Functional Exploration of Hemogenic Endothelium

Funding Type: 
Basic Biology I
Grant Number: 
RB1-01328
ICOC Funds Committed: 
$1 371 477
Disease Focus: 
Blood Disorders
Stem Cell Use: 
Embryonic Stem Cell
Directly Reprogrammed Cell
oldStatus: 
Active
Public Abstract: 
Hematopoietic cells are responsible for generating all cell types present in the blood and therefore critical for the provision of oxygen and nutrients to all the tissues in the body. Blood cells are also required for defense against microorganisms and even for the recognition and elimination of tumor cells. Because blood cells have a relatively short life-span, our bone marrow is constantly producing new cells from hematopoietic progenitors and responding to the relative needs to our tissues and organs. Blood cancers (leukemias), as well as other disorders or treatments that affect the production of blood cells (such as chemotherapy or radiation therapy) can significantly jeopardized health. Transfusions are done to aid the replacement of blood cell loss, but pathogens and immunological compatibility are significant and frequent roadblocks. In this grant application, we present experiments to further understand how another cell in the body, the endothelium, located in the inside wall of all our vessels, can be coax to produce large numbers of hematopoietic cells with indistinguishable immunological properties from those in the bone marrow of each individual. Endothelial cells are easily obtained from skin biopsies or from umbilical cord and they can be expanded in Petri dishes. The experiments outlined were designed to further understand how endothelial cells are capable of generating blood cells during development. This information will be used to entice endothelial cells to generate hematopoietic cell progenitors in vitro. The impact of this research is broad because of its clinical applicability and because of its potential to decipher the mechanisms used by endothelial cells to undergo normal reprogramming and generate undifferentiated progenitor cells of a distinct lineage. Adult cell reprogramming is one of the fundamental premises of stem cell research and thus, highly relevant to the main goals identified by the CIRM program.
Statement of Benefit to California: 
Technology developed from this grant application has the potential to be translated directly to clinical settings. This technology is extremely likely to engender interest by the big pharma which can potentially license the information from the University of California or purchase the patent for the invention / technology. Naturally this will bring revenues and recognition to the state of California. Furthermore, California will remain ahead of the technological wave that takes advantage of stem cell technology and implements innovative medical treatments in the entire country and abroad. In addition, the execution of this proposal will immediately provide employment to four individuals, two of these trainees in stem cell research. Indirectly, the grant will also support salaries of employees at the university associated with research, animal care and administration.
Progress Report: 
  • During this year, we have demonstrated that hematopoietic stem cells are originated from the cells that line the inside of blood vessels, named endothelial cells. Budding of hematopoietic stem cells from endothelial cells occurs during a specific and restricted time window during development and progress has been made to elucidate the regulatory genetic networks involved in this process. We have also demonstrated that hemogenic endothelium is derived from one specific embryonic tissue (lateral plate mesoderm). This information will be used to recapitulate similar conditions in vitro and induce the growth of hematopoietic stem cells outside the body from adult endothelial cells.
  • The objective of this proposal was to identify factors that allow blood vessels to generate hematopoietic stem cells early in the embryonic stage. The process of blood generation from vessels is a normal step in development, but it is poorly understood. We predicted that precise information related to the operational factors in the embryo would allow us to reproduce this process in a petri dish and generate hematopoietic stem cells when needed (situations associated with blood transplantation or cancer).
  • In the second year of this proposal, we have made significant progress and identified critical factors that are responsible for the generation of hematopoietic stem cells from the endothelium (inner layer of blood vessels). These experiments were performed in mouse embryos, as it would be impossible do achieve this goal in human samples. The genes identified are not novel, but have not been associated with this capacity previously. To verify our findings we have independently performed additional experiments and validated the information obtained from sequencing the transcripts.
  • In addition, we developed a series of novel tools to test the biological relevance of the genes identified in vivo (using mouse embryos). Specifically, we have tested whether forced expression of these genes could induce the generation of hematopoietic stem cells. Interestingly, we found that a single manipulation was not sufficient, but multiple and specific manipulations resulted in the generation of blood from endothelium. This was a very exciting result as indicated that we are in the right track and identified factors that can reprogram blood vessels to bud blood stem cells. With this information at hand, we moved into human cells (in petri dishes).
  • The first step was to test whether human endothelial cells could offer a supportive niche for the growth of hematopoietic cells. To our surprise, we found that in the absence of any manipulation, endothelial cells could direct differentiation and support the expansion of CD34+ cells (progenitor blood cells) to a very specific blood cell type, named macrophages. These were rather unexpected results that indicated the ability of endothelial cells to offer a niche for a selective group of blood cells. The final question in the proposal was to test whether the modification of endothelial cells with the identified factors could induce the formation of blood from these cells. For this, we have generated specific reagents and are currently performing the final series of experiments.
  • In this grant application we have been able to investigate the mechanisms by which endothelial cells, the cells that line the inner aspects of the entire circulatory system, produce blood cells. This capacity, called “hemogenic” (giving rise to blood) can be extremely advantageous in pathological situations when generation of new blood cells are needed, such as during leukemia or in organ-transplantation. Although the hemogenic capacity of the endothelium is, under normal conditions, restricted development we have been able to “reprogram” this ability in endothelial cells. For this, we first investigated the genes that responsible for this hemogenic activity during development using mouse models and tissue culture cells. Using this strategy we found key transcription factors in hemogenic endothelium not present in other (non-hemogenic) endothelial cells. Subsequently, we validated that these genes were able to convey hemogenic capacity when expressed in non-hemogenic sites. Finally, using human endothelial cells, we have been able to impose expression of these key transcription factors in endothelial cells. Our data indicates that the forced expression of these factors is able to initiate a program that is likely to result in blood cell generation. The progress achieved through this grant place us in a remarkable position to carry out pre-clinical trials to evaluate the potential of this technology.

In Utero Embryonic Stem Cell Transplantation to Treat Congenital Anomalies

Funding Type: 
New Faculty Physician Scientist
Grant Number: 
RN3-06532
ICOC Funds Committed: 
$2 836 742
Disease Focus: 
Blood Disorders
Pediatrics
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 
Many fetuses with congenital blood stem cell disorders such as sickle cell disease or thalassemia are prenatally diagnosed early enough in pregnancy to be treated with stem cell transplantation. The main benefit to treating these diseases before birth is that the immature fetal immune system may accept transplanted cells without needing to use immunosuppressant drugs to prevent rejection. Moreover, transplanting stem cells into the fetus—in which many stem cell types are actively multiplying and migrating—can promote similar growth and differentiation of the transplanted cells. Although this strategy works well in animal models, when applied clinically, the number of surviving cells in the blood (“engraftment”) has been too low to achieve a reliable cure. Our lab studies ways to improve engraftment, with the long-term goal of applying these strategies to treat fetuses with congenital blood disorders. In this application, we will use novel embryonic stem cells that may be better suited to differentiate into blood cells in the fetal environment. We will also test various approaches to improve the survival advantage of these stem cells in fetal organs that make blood cells. Finally, we will study the fetal immune system to determine how fetuses become tolerant to the transplanted cells. The experiments in this proposal will give us important information to design clinical trials to treat fetuses with common, currently incurable stem cell disorders.
Statement of Benefit to California: 
The long-term goal of our project is to develop safe and effective ways to perform prenatal stem cell transplantation to treat fetuses with congenital blood disorders, such as thalassemia and hemoglobin disorders. These diseases affect many California citizens. For example, hemoglobin disorders are so common that they are routinely screened for at birth (and prenatal screening is performed if there is a family history). Thalassemias are found more commonly in persons of Mediterranean or Asian descent and are therefore prevalent in our state’s population. Prenatal screening is routinely offered, especially to patients with a family history or those with an ethnic predisposition. Fetal stem cell transplantation would also benefit children with sickle cell disease, 2000 of which are born each year in the United States, and inborn errors of metabolism, which occur in 1 in 4000 births. Thus, once we develop reliable techniques to treat these disorders before birth, there will be an enormous potential to make a difference. Fetal surgery was pioneered in California and is performed only in select centers across the country. Therefore, once we have developed safe and effective therapies for fetuses with stem cell disorders, we also expect increased referrals of such patients to California. The convergence of our expertise in fetal therapies with those in stem cell biology carries great promise for finally realizing the promise of fetal stem cell transplantation.
Progress Report: 
  • Our group works on developing methods for successful transplantation of blood stem cells to treat fetuses with genetic disorders such as sickle cell disease or thalassemia. In this grant, we are using novel stem cells that will differentiate into blood-forming cells and other techniques to improve the “engraftment” of these cells. This year, we focused on using a new technique that creates “space” in the bone marrow of the recipient using an antibody (ACK2) to deplete the host’s blood stem cells. In a mouse model, we showed that this antibody is very effective is improving the engraftment of transplanted blood stem cells. In fact, the treatment is more effective in the fetal environment than the adult. These findings were recently published and we are planning to use this strategy in the monkey model as a step toward clinical applications. We are also working on transplanting human blood stem cells into immunodeficient mouse fetuses to understand whether different sources of stem cells vary in their ability to make blood cells in this setting.

Human endothelial reprogramming for hematopoietic stem cell therapy.

Funding Type: 
New Faculty Physician Scientist
Grant Number: 
RN3-06479
ICOC Funds Committed: 
$3 084 000
Disease Focus: 
Blood Disorders
Blood Cancer
Cancer
Stem Cell Use: 
Directly Reprogrammed Cell
Cell Line Generation: 
Directly Reprogrammed Cell
oldStatus: 
Active
Public Abstract: 
The current roadblocks to hematopoietic stem cell (HSC) therapies include the rarity of matched donors for bone marrow transplant, engraftment failures, common shortages of donated blood, and the inability to expand HSCs ex vivo in large numbers. These major obstacles would cease to exist if an extensive, bankable, inexhaustible, and patient-matched supply of blood were available. The recent validation of hemogenic endothelium (blood vessel cells lining the vessel wall give rise to blood stem cells) has introduced new possibilities in hematopoietic stem cell therapy. As the phenomenon of hemogenic endothelium only occurs during embryonic development, we aim to understand the requirements for the process and to re-engineer mature human endothelium (blood vessels) into once again producing blood stem cells (HSCs). The approach of re-engineering tissue specific de-differentiation will accelerate the pace of discovery and translation to human disease. Engineering endothelium into large-scale hematopoietic factories can provide substantial numbers of pure hematopoietic stem cells for clinical use. Higher numbers of cells, and the ability to grow cells from matched donors (or the patients themselves) will increase engraftment and decrease rejection of bone marrow transplantation. In addition, the ability to program mature lineage restricted cells into more primitive versions of the same cell lineage will capitalize on cell renewal properties while minimizing malignancy risk.
Statement of Benefit to California: 
Bone marrow transplantation saves the lives of millions with leukemia and other diseases including genetic or immunologic blood disorders. California has over 15 centers serving the population for bone marrow transplantation. While bone marrow transplantation can be seen as a standard to which all stem cell therapies should aspire, there still remains the difficulty of finding matched donors, complications such as graft versus host disease, and the recurrence of malignancy. While cord blood has provided another donor source of stem cells and improved engraftment, it still requires pooling from multiple donors for sufficient cell numbers to be transplanted, which may increase transplant risk. By understanding how to reprogram blood vessels (such as those in the umbilical cord) for production of blood stem cells (as it once did during human development), it could eventually be possible to bank umbilical cord vessels to provide a patient matched reproducible supply of pure blood stem cells for the entire life of the patient. Higher numbers of cells, and the ability to grow cells from matched donors (or the patients themselves) will increase engraftment and decrease rejection of bone marrow transplantation. In addition, the proposed work will introduce a new approach to engineering human cells. The ability to turn back the clock to near mature cell specific stages without going all the way back to early embryonic stem cell stages will reduce the risk of malignancy.
Progress Report: 
  • We aim to understand how blood stem cells develop from blood vessels during development. We are also interested in learning whether the blood-making program can be turned back on in blood vessel cells for blood production outside the human body. During the past year we have been able to extract and culture blood vessel cells that once had blood making capacity. We have also started experiments that will help uncover the regulation of the blood making program. In addition, we have developed tools to help the process of understanding whether iPS technology can "turn back time" in mature blood vessels and turn on the blood making program.

Antibody tools to deplete or isolate teratogenic, cardiac, and blood stem cells from hESCs

Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-02060
ICOC Funds Committed: 
$1 869 487
Disease Focus: 
Blood Disorders
Heart Disease
Liver Disease
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
oldStatus: 
Active
Public Abstract: 
Purity is as important for cell-based therapies as it is for treatments based on small-molecule drugs or biologics. Pluripotent stem cells possess two properties: they are capable of self regeneration and they can differentiate to all different tissue types (i.e. muscle, brain, heart, etc.). Despite the promise of pluripotent stem cells as a tool for regenerative medicine, these cells cannot be directly transplanted into patients. In their undifferentiated state they harbor the potential to develop into tumors. Thus, tissue-specific stem cells as they exist in the body or as derived from pluripotent cells are the true targets of stem cell-based therapeutic research, and the cell types most likely to be used clinically. Existing protocols for the generation of these target cells involve large scale differentiation cultures of pluripotent cells that often produce a mixture of different cell types, only a small fraction of which may possess therapeutic potential. Furthermore, there remains the real danger that a small number of these cells remains undifferentiated and retains tumor-forming potential. The ideal pluripotent stem cell-based therapeutic would be a pure population of tissue specific stem cells, devoid of impurities such as undifferentiated or aberrantly-differentiated cells. We propose to develop antibody-based tools and protocols to purify therapeutic stem cells from heterogeneous cultures. We offer two general strategies to achieve this goal. The first is to develop antibodies and protocols to identify undifferentiated tumor-forming cells and remove them from cultures. The second strategy is to develop antibodies that can identify and isolate heart stem cells, and blood-forming stem cells capable of engraftment from cultures of pluripotent stem cells. The biological underpinning of our approach is that each cell type can be identified by a signature surface marker expression profile. Antibodies that are specific to cell surface markers can be used to identify and isolate stem cells using flow cytometry. We can detect and isolate rare tissue stem cells by using combinations of antibodies that correspond to the surface marker signature for the given tissue stem cell. We can then functionally characterize the potential of these cells for use in regenerative medicine. Our proposal aims to speed the clinical application of therapies derived from pluripotent cell products by reducing the risk of transplanting the wrong cell type - whether it is a tumor-causing residual pluripotent cell or a cell that is not native to the site of transplant - into patients. Antibodies, which exhibit exquisitely high sensitivity and specificity to target cellular populations, are the cornerstone of our proposal. The antibodies (and other technologies and reagents) identified and generated as a result of our experiments will greatly increase the safety of pluripotent stem cell-derived cellular therapies.
Statement of Benefit to California: 
Starting with human embryonic stem cells (hESC), which are capable of generating all cell types in the body, we aim to identify and isolate two tissue-specific stem cells – those that can make the heart and the blood – and remove cells that could cause tumors. Heart disease remains the leading cause of mortality and morbidity in the West. In California, 70,000 people die annually from cardiovascular diseases, and the cost exceeded $48 billion in 2006. Despite major advancement in treatments for patients with heart failure, which is mainly due to cellular loss upon myocardial injury, the mortality rate remains high. Similarly, diseases of the blood-forming system, e.g. leukemias, remain a major health problem in our state. hESC and induced pluripotent stem cells (collectively, pluripotent stem cells, or PSC) could provide an attractive therapeutic option to treat patients with damaged or defective organs. PCS can differentiate into, and may represent a major source of regenerating, cells for these organs. However, the major issues that delay the clinical translation of PSC derivatives include lack of purification technologies for heart- or blood-specific stem cells from PSC cultures and persistence of pluripotent cells that develop into teratomas. We propose to develop reagents that can prospectively identify and isolate heart and blood stem cells, and to test their functional benefit upon engraftment in mice. We will develop reagents to identify and remove residual PSC, which give rise to teratomas. These reagents will enable us to purify patient-specific stem cells, which lack cancer-initiating potential, to replenish defective or damaged tissue. The reagents generated in these studies can be patented forming an intellectual property portfolio shared by the state and the institutions where the research is carried out. The funds generated from the licensing of these technologies will provide revenue for the state, will help increase hiring of faculty and staff (many of whom will bring in other, out-of-state funds to support their research) and could be used to ameliorate the costs of clinical trials – the final step in translation of basic science research to clinical use. Only California businesses are likely to be able to license these reagents and to develop them into diagnostic and therapeutic entities; such businesses are at the heart of the CIRM strategy to enhance the California economy. Most importantly, this research will set the platform for future stem cell-based therapies. Because tissue stem cells are capable of lifelong self-renewal, stem cell therapies have the potential to be a single, curative treatment. Such therapies will address chronic diseases with no cure that cause considerable disability, leading to substantial medical expense. We expect that California hospitals and health care entities will be first in line for trials and therapies. Thus, California will benefit economically and it will help advance novel medical care.
Progress Report: 
  • Our program is focused on improving methods that can be used to purify stem cells so that they can be used safely and effectively for therapy. A significant limitation in translating laboratory discoveries into clinical practice remains our inability to separate specific stem cells that generate one type of desired tissue from a mixture of ‘pluripotent’ stem cells, which generate various types of tissue. An ideal transplant would then consist of only tissue-specific stem cells that retain a robust regenerative potential. Pluripotent cells, on the other hand, pose the risk, when transplanted, of generating normal tissue in the wrong location, abnormal tissue, or cancer. Thus, we have concentrated our efforts to devise strategies to either make pluripotent cells develop into desired tissue-specific stem cells or to separate these desired cells from a mixture of many types of cells.
  • Our approach to separating tissue-specific stem cells from mixed cultures is based on the theory that every type of cell has a very specific set of molecules on its surface that can act as a signature. Once this signature is known, antibodies (molecules that specifically bind to these surface markers) can be used to tag all the cells of a desired type and remove them from a mixed population. To improve stem cell therapy, our aim is to identify the signature markers on: (1) the stem cells that are pluripotent or especially likely to generate tumors; and (2) the tissue-specific stem cells. By then developing antibodies to the pluripotent or tumor-causing cells, we can exclude them from a group of cells to be transplanted. By developing antibodies to the tissue-specific stem cells, we can remove them from a mixture to select them for transplantation. For the second approach, we are particularly interested in targeting stem cells that develop into heart (cardiac) tissue and cells that develop into mature blood cells. As we develop ways to isolate the desired cells, we test them by transplanting them into animals and observing how they grow.
  • Thus, the first goal of our program is to develop tools to isolate pluripotent stem cells, especially those that can generate tumors in transplant recipients. To this end, we tested an antibody aimed at a pluripotent cell marker (stage-specific embryonic antigen-5 [SSEA-5]) that we previously identified. We transplanted into animals a population of stem cells that either had the SSEA-5-expressing cells removed or did not have them removed. The animals that received the transplants lacking the SSEA-5-expressing cells developed smaller and fewer teratomas (tumors consisting of an abnormal mixture of many tissues). Approaching the problem from another angle, we analyzed teratomas in animals that had received stem cell transplants. We found SSEA-5 on a small group of cells we believe to be responsible for generating the entire tumor.
  • The second goal of the program is to develop methods to selectively culture cardiac stem cells or isolate them from mixed cultures. Thus, in the last year we tested procedures for culturing pluripotent stem cells under conditions that cause them to develop into cardiac stem cells. We also tested a combination of four markers that we hypothesized would tag cardiac stem cells for separation. When these cells were grown under the proper conditions, they began to ‘beat’ and had electrical activity similar to that seen in normal heart cells. When we transplanted the cells with the four markers into mice with normal or damaged hearts, they seemed to develop into mature heart cells. However, these (human) cells did not integrate with the native (mouse) heart cells, perhaps because of the species difference. So we varied the approach and transplanted the human heart stem cells into human heart tissue that had been previously implanted in mice. In this case, we found some evidence that the transplanted cells differentiated into mature heart cells and integrated with the surrounding human cells.
  • The third goal of our project is to culture stem cells that give rise only to blood cells and test them for transplantation. In the past year, we developed a new procedure for treating cultures of pluripotent stem cells so that they differentiate into specific stem cells that generate blood cells and blood vessels. We are now working to refine our understanding and methods so that we end up with a culture of differentiated stem cells that generates only blood cells and not vessels.
  • In summary, we have discovered markers and tested combinations of antibodies for these markers that may select unwanted cells for removal or wanted cells for inclusion in stem cell transplants. We have also begun to develop techniques for taking a group of stem cells that can generate many tissue types, and growing them under conditions that cause them to develop into tissue-specific stem cells that can be used safely for transplantation.
  • Our program is focused on improving methods to purify blood-forming and heart-forming stem cells so that they can be used safely and effectively for therapy. Current methods to identify and isolate blood-forming stem cells from bone marrow and blood are efficient. In addition, we found that if blood-forming stem cells are transplanted, they create in the recipient an immune system that will tolerate (i.e., not reject) organs, tissues, or other types of tissue stem cells (e.g. skin, brain, or heart) from the same donor. Many living or recently deceased donors often cannot contribute these stem cells, so we need, in the future, a single biological source of each of the different types of stem cells (e.g., blood and heart) to change the entire field of regenerative medicine. The ultimate reason to fund embryonic stem cell and other pluripotent stem cell research is to create safe banks of predefined pluripotent cells. Protocols to differentiate these cells to the appropriate blood-forming stem cells could then be used to induce tolerance of other tissue stem cells from the same embryonic stem cell line. However, existing protocols to differentiation stem cells often lead to pluripotent cells (cells that generate multiple types of tissue), which pose a risk of generating normal tissue in the wrong location, abnormal tissue, or cancers called teratomas. To address these problems, we have concentrated our efforts to devise strategies to (a) make pluripotent cells develop into desired tissue-specific stem cells, and (b) to separate these desired cells from all other cells, including teratoma-causing cells. In the first funding period of this grant, we succeeded in raising antibodies that identify and eliminate teratoma-causing cells.
  • In the past year, we identified surface markers of cells that can only give rise to heart tissue. First we studied the genes that were activated in these cells, further confirming that they would likely give rise to heart tissue. Using antibodies against these surface markers, we purified heart stem cells to a higher concentration than has been achieved by other purification methods. We showed that these heart stem cells can be transplanted such that they integrate into the human heart, but not mouse heart, and participate in strong and correctly timed beating.
  • In the embryo, a group of early stem cells in the developing heart give rise to (a) heart cells; (b) cells lining the inner walls of blood vessels; and (c) muscle cells surrounding blood vessels. We identified cell surface markers that could be used to separate each of these subsets from each other and from their common stem cell parents. Finally, we determined that a specific chemical in the body, fibroblast growth factor, increased the growth of a group of pluripotent stem cells that give rise to more specific stem cells that produce either blood cells or the lining of blood vessels. This chemical also prevented blood-forming stem cells from developing into specific blood cells.
  • In the very early embryo, pluripotent cells separate into three distinct categories called ‘germ layers’: the endoderm, mesoderm, and ectoderm. Each of these germ layers later gives rise to certain organs. Our studies of the precursors of mesoderm (the layer that generates the heart, blood vessels, blood, etc.) led us by exclusion to develop techniques to direct ES cell differentiation towards endoderm (the layer that gives rise to liver, pancreas, intestinal lining, etc.). A graduate student before performed most of this work before he joined in our effort to find ways to make functional blood forming stem cells from ES cells. He had identified a group of proteins that we could use to sequentially direct embryonic stem cells to develop almost exclusively into endoderm, then subsets of digestive tract cells, and finally liver stem cells. These liver stem cells derived from embryonic stem cells integrated into mouse livers and showed signs of normal liver tissue function (e.g., secretion of albumin, a major protein in the blood). Using the guidelines of the protocols that generated these liver stem cells, we have now turned our attention back to our goal of generating from mesoderm the predecessors of blood-forming stem cells, and, ultimately, blood-forming stem cells.
  • In summary, we have continued to discover signals that cause pluripotent stem cells (which can generate many types of tissue) to become tissue-specific stem cells that exclusively develop into only heart, blood cells, blood vessel lining cells, cells that line certain sections of the digestive tract, or liver cells. This work has also involved determining the distinguishing molecules on the surface of various cells that allow them to be isolated and nearly purified. These results bring us closer to being able to purify a desired type of stem cell to be transplanted safely to generate only a single type of tissue.

Role of intracytoplasmic pattern recognition receptors in HSC engraftment

Funding Type: 
Basic Biology V
Grant Number: 
RB5-07379
ICOC Funds Committed: 
$615 639
Disease Focus: 
Blood Disorders
oldStatus: 
Closed
Public Abstract: 
The research performed through this project is very important for the fields of solid organ and bone marrow transplantation because it focuses on a potential new target to increase engraftment of stem cells. Currently, patients that receive stem cell transplants from a non-identical donor must take medications to suppress their immune system; otherwise the stem cells will be rejected. Stem cell trials have been extended to solid organ transplantation, where it has been shown that kidney transplants can be managed with little or no immunosuppressive medications when stem cells are given to the patient at the time of transplantation. In many cases though the stem cells are rejected and the patient must resume toxic medications. Our laboratory has been very interested in understanding ways to prevent the rejection of stem cells and has focused on a phylogenetically conserved group of cellular receptors called pattern recognition receptors. This project is focused on understanding how to prevent rejection of stem cells through modifications of these receptors. We hope to identify novel targets to prevent the rejection of stem cells in order to decrease the occurrence of graft versus host disease after bone marrow transplantation and also improve the opportunities for long-term transplant survival without the use of toxic immunosuppressive medications.
Statement of Benefit to California: 
The research we will undertake will benefit the State of California and its residents in two major ways. First it promises to define a novel targets to prevent rejection of stem cells that are transplanted into their new host. This is very important because rejection of hematopoietic stem cells is a major impediment to successful efforts at both bone marrow and solid organ transplantation. Patients needed life-saving solid organ transplants and patients that receive bone marrow transplants from donors that are not perfectly matched to them reject their grafts unless they take powerful medications to suppress their immune system. This project is focused on finding a way to help prevent the rejection of these grafts without the need for immunosuppressive medications. The second way the project will benefit the State of California is to provide new employment opportunities within the State at a large University that conducts biomedical research. This project will not only directly support the employment of three California citizens devoted to biomedical research, but the work it generates will support California-based biomedical science companies, California University personal and other local companies that employ California citizens that produce the reagents and the supplies used in the proposed studies.

Generation of functional cells and organs from iPSCs

Funding Type: 
Research Leadership 12
Grant Number: 
LA1_C12-06917
ICOC Funds Committed: 
$6 152 065
Disease Focus: 
Blood Disorders
Stem Cell Use: 
iPS Cell
oldStatus: 
Closed
Public Abstract: 
The development of induced pluripotent stem cell (iPSC) technology may be the most important advance in stem cell biology for the future of medicine. This technology allows one to generate a patient’s own pluripotent stem cells (PSCs) from skin or blood cells. iPSCs can then be reprogrammed to multiply and produce high quality mature cells for cell therapy. Because iPSCs are derived from a patient's own cells, therapies that use them will not stimulate unwanted immune reactions or necessitate lifelong immunosuppression. If organs can be generated from iPSCs, many patients with organ failure awaiting transplants will be helped. The goal of this project is to further develop iPSC technology to bring about personalized regenerative medicine for treating intractable diseases such as cancers, viral infections, genetic blood disorders, and organ failure. Specifically, we would like to establish three major core programs for generating from iPSCs: personalized immune cells; an unlimited supply of blood stem cells; and functional organs. First, we will generate iPSC-derived immune cells that kill viruses and cancer cells. Current immunotherapy uses immune cells that are exhausted (have limited ability to function and proliferate) after they multiply in a test tube. To supply active nonexhausted immune cells, iPSCs will be generated from a patient’s immune cells that target tumor cells and infections and then redifferentiated to mature immune cells with the same targets. Second, we aim to develop iPSC technology to generate blood stem cells that replenish all blood cells throughout life. Harvesting blood stem cells from a leukemia patient for transplantation back to the patient after chemotherapy and radiation has been challenging because few blood stem cells can be harvested and may be contaminated with cancer cells. Alternatively, transplanting blood stem cells from cord blood or another person requires genetic matching to prevent immune reactions. However, generating blood stem cells from a patient’s iPSCs may avoid contamination with cancer cells, immune reactions, and the need to find a matched donor. Furthermore, we aim to generate iPSCs from a patient with a genetic blood disease, correct the genetic defect in the iPSCs, and generate from these corrected iPSCs healthy blood stem cells that may be curative when transplanted back into the patient. Lastly, we will try to generate from iPSCs not just mature cells, but organs for transplantation, to potentially address the tremendous shortage of donated organs. In a preliminary study, we generated preclinical models that could not develop pancreases. When we injected stem cells into these models, they developed functional pancreases derived from the injected cells and survived to adulthood. We hope that within 10 years, we will be able to provide a needed organ to a patient by growing it from the patient’s own PSCs in a compatible animal.
Statement of Benefit to California: 
Cancer is the second leading cause of death, accounting for 24% of all deaths in the U.S. Nearly 55,000 people will die of the disease--about 150 people each day or one of every four deaths in California. In 2012, nearly 144,800 Californians will be diagnosed with cancer. We need effective treatment to cure cancer. End-stage organ failure is another difficult disease to treat. Transplantation of kidneys, liver, heart, lungs, pancreas, and small intestine has become an accepted treatment for organ failure. In California, more than 21,000 people are on the waiting lists at transplant centers. However, one in three of these people will die waiting for transplants because of the shortage of donated organs. While end-stage renal failure patients can survive for decades with hemodialysis treatment, they suffer from high morbidity and mortality. In addition, the high medical costs for increasing numbers of dialysis patients is a social issue. We need to find a way to increase organs that can be used for transplantation. In our proposed projects, we aim to use iPSC technology and recent discoveries to develop new methods for treating cancers, viral infections, and organ failure. More specifically, we will pursue our recent discoveries using iPSCs to: (1) multiply person’s T cells that specifically target cancers and viral infections; (2) generate normal blood-forming stem cells that can be transplanted back into a patient to correct a blood disease (3) regenerate tissues and organs from a patient’s cells for transplantation back into that patient. These projects are likely to benefit the state of California in several ways. Many of the methods, cells, and reagents generated by this research will be patentable, forming an intellectual property portfolio shared by the state and the institutions where the research is performed. The funds generated from the licensing of these technologies will provide revenue for the state, will help increase hiring of faculty and staff (many of whom will bring in other, out-of-state funds to support their research), and could be used to ameliorate the costs of clinical trials--the final step in translation of basic science research to clinical use. Most importantly, this research will set the platform for stem cell-based therapies. Because tissue stem cells are capable of lifelong self-renewal, these therapies have the potential to provide a single, curative treatment. Such therapies will address chronic diseases that have no cure and cause considerable disability, leading to substantial medical expenses and loss of work. We expect that California hospitals and health care entities will be first in line for trials and therapies. Thus, California will benefit economically and the project will help advance novel medical care.

Development of a cell and gene based therapy for hemophilia

Funding Type: 
Early Translational IV
Grant Number: 
TR4-06809
ICOC Funds Committed: 
$2 322 440
Disease Focus: 
Blood Disorders
Liver Disease
Pediatrics
Stem Cell Use: 
iPS Cell
Cell Line Generation: 
iPS Cell
oldStatus: 
Closed
Public Abstract: 
Hemophilia B is a bleeding disorder caused by the lack of FIX in the plasma and affects 1/30,000 males. Patients suffer from recurrent bleeds in soft tissues leading to physical disability in addition to life threatening bleeds. Current treatment (based on FIX infusion) is transient and plagued by increased risk for blood-borne infections (HCV, HIV), high costs and limited availability. This has fueled a search for gene/cell therapy based alternatives. Being the natural site of FIX synthesis, the liver is expected to provide immune-tolerance and easy circulatory access. Liver transplantation is a successful, long-term therapeutic option but is limited by scarcity of donor livers and chronic immunosuppression; making iPSC-based cell therapy an attractive prospect. As part of this project, we plan to generate iPSCs from hemophilic patients that will then be genetically corrected by inserting DNA capable of making FIX. After validation for correction, we will then differentiate these iPSCs into liver cells that can be transplanted into our mouse model of hemophilia that is capable of accepting human hepatocytes and allowing their proliferation. These mice exhibit disease symptoms similar to human patients and we propose that by injecting our corrected liver cells they will exhibit normal clotting as measured by various biochemical and physiological assays. If successful, this will provide a long-term cure for hemophilia and other liver diseases.
Statement of Benefit to California: 
Generation of iPSCs from adult cells unlocked the potential of tissue engineering, replacement and cell transplant therapies to cure a host of debilitating diseases without the ethical concerns of working with embryos or the practical problems of immune-rejection. We aim to develop a POC for a novel cell- and gene-therapy based approach towards the treatment of hemophilia B. In addition to the obvious and direct benefit to the affected patients and families by providing a potential long-term cure; the successful development of our proposal will serve as a POC for moving other iPSC-based therapies to the clinic. Our proposal also has the potential to treat a host of other hepatic diseases like alpha-1-antitrypsin deficiency, Wilson’s disease, hereditary hypercholesterolemia, etc. These diseases have devastating effects on the patients in addition to the huge financial drain on the State in terms of the healthcare costs. There is a pressing need to find effective solutions to such chronic health problems in the current socio-economic climate. The work proposed here seeks to redress this by developing cures for diseases that, if left untreated, require substantial, prolonged medical expenditures and cause increased suffering to patients. Being global leaders in these technologies, we are ideally suited to this task, which will establish the state of California at the forefront of medical breakthroughs and strengthen its biomedical/biotechnology industries.

A Treatment For Beta-thalassemia via High-Efficiency Targeted Genome Editing of Hematopoietic Stem Cells

Funding Type: 
Strategic Partnership II
Grant Number: 
SP2-06902
ICOC Funds Committed: 
$6 374 150
Disease Focus: 
Blood Disorders
Pediatrics
Stem Cell Use: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 
β-thalassemia is a genetic disease caused by diverse mutations of the β-globin gene that lead to profoundly reduced red blood cell (RBC) development. The unmet medical need in transfusion-dependent β-thalassemia is significant, with life expectancy of only ~30-50 years despite standard of care treatment of chronic blood transfusions and iron chelation therapy. Cardiomyopathy due to iron overload is the major cause of mortality, but iron-overload induced multiorgan dysfunction, blood-borne infections, and other disease complications impose a significant physical, psychosocial and economic impact on patients and families. An allogeneic bone marrow transplant (BMT) is curative. However, this therapy is limited due to the scarcity of HLA-matched related donors (<20%) combined with the significant risk of graft-versus-host disease (GvHD) after successful transplantation of allogeneic cells. During infancy, gamma-globin-containing fetal hemoglobin protects β-thalassemia patients from developing disease symptoms until gamma globin is replaced by adult-type β-globin chains. The proposed therapeutic intervention combines the benefits of re-activating the gamma globin gene with the curative potential of BMT, but without the toxicities associated with acute and chronic immunosuppression and GvHD. We hypothesize that harvesting hematopoietic stem and progenitor cells (HSPCs) from a patient with β-thalassemia, using genome editing to permanently re-activate the gamma globin gene, and returning these edited HSPCs to the patient could provide transfusion independence or greatly reduce the need for chronic blood transfusions, thus decreasing the morbidity and mortality associated with iron overload. The use of a patient’s own cells avoids the need for acute and chronic immunosuppression, as there would be no risk of GvHD. Moreover, due to the self-renewing capacity of HSPCs, we anticipate a lifelong correction of this severe monogenic disease.
Statement of Benefit to California: 
Our proposed treatment for transfusion dependent β-thalassemia will benefit patients in the state by offering them a significant improvement over current standard of care. β-thalassemia is a genetic disease caused by diverse mutations of the β-globin gene that lead to profoundly reduced red blood cell (RBC) development and survival resulting in the need for chronic lifelong blood transfusions, iron chelation therapy, and important pathological sequelae (e.g., endocrinopathies, cardiomyopathies, multiorgan dysfunction, bloodborne infections, and psychosocial/economic impact). Incidence is estimated at 1 in 100,000 in the US, but is more common in the state of California (incidence estimated at 1 in 55,000 births) due to immigration patterns within the State. While there are estimated to be about 1,000-2,000 β-thalassemia patients in the US, one of our proposed clinical trial sites has the largest thalassemia program in the Western United States, with a population approaching 300 patients. Thus, the state of California stands to benefit disproportionately compared to other states from our proposed treatment for transfusion dependent β-thalassemia. An allogeneic bone marrow transplant (BMT) is curative for β-thalassemia, but limited by the scarcity of HLA-matched related donors (<20%) combined with the significant risk of graft-versus-host disease (GvHD) after successful transplantation of allogeneic cells. Our approach is to genetically engineer the patient’s own stem cells and thus (i) solve the logistical challenge of finding an appropriate donor, as the patient now becomes his/her own donor; and (ii) make use of autologous cells abrogating the risk of GvHD and need for acute and chronic immunosuppression. Our approach offers a compelling pharmacoeconomic benefit to the State of California and its citizens. A lifetime of chronic blood transfusions and iron chelation therapy leads to a significant cost burden; despite this, the prognosis for a transfusion dependent β-thalassemia patient is still dire, with life expectancy of only ~30-50 years. Our proposed one-time treatment aims to reduce or eliminate the need for costly chronic blood transfusions and iron chelation therapy, while potentially improving the clinical benefit to patients, including the morbidity and mortality associated with transfusion-induced iron overload.

Differentiation of Human Hematopoietic Stem Cells into iNKT Cells

Funding Type: 
Basic Biology V
Grant Number: 
RB5-07089
ICOC Funds Committed: 
$614 400
Disease Focus: 
Blood Disorders
oldStatus: 
Active
Public Abstract: 
Blood stem cells living in the bone marrow of adult humans give rise to all of the cells in our blood, including the red blood cells that carry oxygen to supply our body, and the white blood cells such as T and B lymphocytes that fight infections and keep us healthy. Among the T lymphocytes there is a small population called invariant natural killer T (iNKT) cells. Despite their low frequency in humans (~0.001-1% in blood), iNKT cells have the remarkable capacity to mount immediate and potent responses when stimulated, and have been suggested to play important roles in regulating multiple human diseases including infections, allergies, cancer, and autoimmunity (such as Type I diabetes and multiple sclerosis). However, successful clinical interventions with iNKT cells have been greatly hindered by our limited knowledge on how these cells are produced by blood stem cells, largely due to the lack of tools to track these cells in humans. We therefore propose a novel model system to overcome this research bottleneck by transplanting human blood stem cells into a mouse and genetically programming these cells to develop into iNKT cells. This “humanized” mouse model will allow us to directly track the differentiation of human blood stem cells into iNKT cells in a living animal. From this study, we will address some critical unanswered questions for iNKT cell development, and shed light on developing stem-cell based iNKT cell therapies.
Statement of Benefit to California: 
Allergies, cancer and autoimmunity are leading health hazards in California. These diseases affect millions of Californians, impairing their life quality and creating huge economic burdens for the State of California. This proposal intends to study invariant natural killer (iNKT) T cells, a special population of T lymphocytes that have been suggested to play important roles in regulating these diseases. To date, clinical applications of iNKT cells have been greatly limited by their low frequency in humans and their high variability between individuals (~0.001-1% in blood). Thus, an improved understanding of how these cells are naturally generated is important for their use clinically. Like all other cells in blood, iNKT cells are descendants of the blood stem cells that live in the bone marrow of adult humans. Our goal is to study how human blood stem cells give rise to iNKT cells. If successful, our results can be exploited to develop stem cell-based iNKT cell therapies to treat allergies, cancer and autoimmunity, and therefore may benefit the millions of Californians currently suffering from these diseases. In addition, the knowledge and reagents generated from this proposed study will be shared freely with non-profit and academic organizations in California, and any new intellectual property derived from this study will be developed under the guidance of CIRM to benefit the State of California.

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