Blood Disorders

Coding Dimension ID: 
278
Coding Dimension path name: 
Blood Disorders
Funding Type: 
Comprehensive Grant
Grant Number: 
RC1-00354
Investigator: 
Institution: 
Type: 
PI
ICOC Funds Committed: 
$2 636 900
Disease Focus: 
Blood Disorders
Heart Disease
Immune Disease
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
The capacity of human embryonic stem cells (hESCs) to perpetuate themselves indefinitely in culture and to differentiate to all cell types of the body has lead to numerous studies that aim to isolate therapeutically relevant cells for the benefit of patients, and also to study how genetic diseases develop. However, hESCs can cause tumors called teratomas when placed in the body and therefore, we need to separate potentially beneficial cells from hazardous hESCs. Thus, potential therapeutics cannot advance until the development of methodologies that eliminate undifferentiated cells and enrich tissue stem cells. In our proposal we hope to define the cell surface markers that are differentially expressed by committed hESC-derived stem cells and others that are expressed by teratogenic hESCs. To do this we will carry out a large screen of cell subsets that form during differentiation using a collection of unique reagents called monoclonal antibodies, many already obtained or made by us, to define the cell-surface markers that are expressed by teratogenic cells and others that detect valuable tissue stem cells. This collection, after filing for IP protection, would be available for CIRM investigators in California. We were the first to isolate mouse and human adult blood-forming stem cells, human brain stem cells, and mouse muscle stem cells, all by antibody mediated cell-sorting approaches. Antibody mediated identification of cell subsets that arise during early hESC differentiation will allow separation and characterization of defined subpopulations; we would isolate cells that are committed to the earliest lineage known to form multiple cell types in the body including bone, blood, heart and muscle. These cells would be induced to differentiate further to the blood forming and heart muscle forming lineages. Enriched, and eventually purified hESC-derived blood-forming stem cells and heart muscle stem cells will be tested for their potential capacity to engraft and improve function in animal models. Blood stem cells will be transplanted into immunodeficient mice to test their capacity to give rise to all blood cell types; and heart muscle stem cells will be transferred to mouse hearts that had an artificial coronary artery blockage, a model for heart attack damage. Finally, we will test the capacity of blood stem cell transplantation to induce transplantation tolerance towards heart muscle stem cells from the same donor cell line. Transplantation tolerance in this case means that the heart cells would be accepted as ‘self’ by the mouse that had it’s unrelated donor immune system replaced wholly or in part by blood forming stem cells from the same hESC line that gave rise to the transplantable heart stem cells, and therefore would not be rejected by it’s own immune system. This procedure would allow transplantation of beneficial tissues such as heart, insulin-producing cells, etc., without the use of immunosuppressive drugs.
Statement of Benefit to California: 
The principle objective of this proposal is to develop reagents which, in combinations, can identify and isolate tissue-regenerating stem cells derived from hESC lines. The undifferentiated hESCs are dangerous for transplantation into humans, as they cause tumors. We propose to prepare reagents that identify and can be used to delete or prospectively isolate these tumor-causing undifferentiated hESCs. HESC-derived tissue stem cells have the potential to regenerate damaged tissues and organs, and don’t cause tumors. We propose to develop reagents that can be used to identify and prospectively isolate pure human blood-forming stem cells derived from hESCs, and separately other reagents that can be used to identify and prospectively isolate pure heart-forming stem or progenitor cells. These “decontaminated” hESC-derived tissue stem cells may eventually be used to treat human tissue degenerative diseases. These reagents could also be used to isolate the same cells from somatic cell nuclear transfer (SCNT)-derived pluripotent stem cell lines from patients with genetic diseases. This procedure would enable us to analyze the effects of the genetic abnormalities on blood stem and progenitor cells in patients with genetic blood and immune system disorders, and on heart stem and progenitor cells in patients with heart disorders. The antibodies and stem cells (hESCs, tissue regenerating, etc) that will be isolated from patients with specific diseases will be invaluable tools that can be used to create model(s) for understanding the diseases and their progression. In addition, the antibodies and the stem cells generated in these studies are entities that could be patented or protected by copyright, forming an intellectual property portfolio shared by the state and the state institutions wherein the research was carried out. The funds generated from the licensing of these technologies will help pay back the state, will help support increasing faculty and staff (many of whom bring in other, out of state funds for their research), and could be used to ameliorate the costs of clinical trials. Only California businesses are likely to be able to license these antibodies and cells, to develop them into diagnostic and therapeutic entities; such businesses are the heart of the CIRM strategy to enhance the California economy. Most importantly, however, is that this research will lead to tissue stem cell therapies. Such therapies will address chronic diseases that cause considerable disability and misery, currently have no cure, and therefore lead to huge medical expenses. Because tissue stem cells renew themselves for life, stem cell therapies are one-time therapies with curative intent. We expect that California hospitals and health care entities will be first in line for trials and therapies, and for CIRM to negotiate discounts on such therapies for California taxpayers, thus California will benefit both economically and with advanced novel medical care.
Progress Report: 
  • The objectives of our proposal are the isolations of blood-forming and heart-forming stem cells from human embryonic stem cell (hESCs) cultures, and the generation of monoclonal antibodies (mAbs) that eliminate residual teratogenic cells from transplantable populations of differentiated hESCs. For isolation of progenitors, we hypothesized that precursors derived from hESCs could be identified and isolated using mAbs that label unique combinations of lineage-specific cell surface molecules. We used hundreds of defined mAbs, generated hundreds of novel anti-hESC mAbs, and used these to isolate and characterize dozens of hESC-derived populations. We discovered four precursor types from early stages of differentiating cells, each expressing genes indicative of commitment to either embryonic or extraembryonic tissues. Together, these progenitors are candidates to give rise to meso-endodermal lineages (heart, blood, pancreas, etc), and yolk sac, umbilical cord and placental tissues, respectively. Importantly, we have found that cells of the meso-endodermal population give rise to beating cardiomyocytes. We are currently enriching cardiomyocyte precursors from this population using cardiac-specific genetic markers, and are assaying the putative progenitors using electrophysiological assays and by transplantation into animal hearts (a test for restoration of heart function). In addition, we established in vitro conditions that effectively promote hESC-differentiation towards the hematopoietic (blood) lineages and isolated populations that resemble hematopoietic stem cells (HSCs) in both surface phenotype as well as lineage potentials, as determined by assays in vitro. We have generated hESC-lines that express the anti-apoptotic gene BCL2, and have found that these cells produce significantly greater amounts of hematopoietic and cardiac cells, because of their increased survival during culturing and sorting. We are currently isolating hematopoietic precursors from BCL2-hESCs and will test their ability to engraft in immunodeficient mice, to examine the capacity of hESC-derived HSCs to regenerate the blood system. Finally, we have utilized the novel mAbs that we prepared against undifferentiated hESCs, to deplete residual teratogenic cells from differentiated cultures that were transplanted into animal models. We discovered that following depletion teratoma rarely formed, and we expect to determine a final cocktail of mAbs for removal of teratogenic cells from transplantation products this year.
  • The main objective of our proposal is to isolate therapeutic stem cells and progenitors from human embryonic stem cells (hESCs) that give rise to blood and heart cells. Our approach involves isolation of differentiated precursor subset of cells using monoclonal antibodies (mAbs) and cell sorting instruments, and subsequent characterization of their respective hematopoietic and cardiomyogenic potential in culture as well as following engraftment into mouse models of disease. In addition, we aim to develop mAbs that specifically bind to undifferentiated hESCs for removal of residual teratoma-initiating cells from therapeutic cell preparations, to ensure transplantation safety.
  • We have made substantial advancement towards achieving these goals. First, we discovered that the initial differentiation of hESCs occurs through only 4-5 different progenitor types, of which one is destined to give rise to heart lineages. We purified this population using three novel cell surface markers, and found a significant enrichment of cardiomyocyte clones in colony formation assays that we developed. This subset also expressed particularly high levels of cardiac genes and was receptive to further differentiation into beating cardiomyocytes or vascular endothelial cells. When transplanted into immunodeficient mice these progenitors differentiated into ventricular myocytes and vascular endothelial cells. In the coming year we will perform transplantation experiments to evaluate whether they improve the functional outcome of heart infarction in hearts of mice. Second, we have optimized cell culture conditions and cell surface markers to sort hematopoietic progenitors derived from hESCs. We have also begun to transplant these populations into immunodeficient mouse recipients to identify blood-reconstituting hematopoietic populations. Third, we identified 5 commercial and 1 custom mAbs that are specific to human pluripotent cells (hESCs and induced pluripotent cells). We are currently testing the capacity of combinations of 3 pluripotency surface markers to remove all teratoma-initiating cells from transplanted differentiated cell populations. In summary, we expect provide functional validation of the blood and heart precursor populations that we identified from hESCs by the end term of this grant.
  • The main objective of our proposal is to isolate therapeutic stem and progenitor cells derived from human embryonic stem cells (hESCs) that can give rise to blood and heart cells. Our approach involves developing differentiation protocols to drive hematopoietic (blood) and cardiac (heart) development of hESCs, then to identify and isolate stem/progenitor cells using monoclonal antibodies (mAbs) specific to surface markers expressed on blood and heart stem/progenitor cells, and finally to characterize their functional properties in vitro and in vivo. In addition, we sought to develop mAbs that specifically bind to undifferentiated hESCs for removal of residual teratoma (tumor)-initiating cells from therapeutic preparations, to ensure transplantation safety.
  • We have made substantial progress toward achieving these goals. First, we discovered that the initial differentiation of hESCs occurs through only 4-5 different progenitor types, of which one is destined to give rise to heart lineages. We purified this population using four novel cell surface markers (ROR2, PDGFRα, KDR, and CD13), and found a significant enrichment of cardiomyocyte clones in colony formation assays that we developed. This subset also expressed particularly high levels of cardiac genes and was receptive to further differentiation into beating cardiomyocytes or vascular endothelial cells. When transplanted into immunodeficient mice these progenitors differentiated into ventricular myocytes and vascular endothelial cells. We have also successfully developed a human fetal heart xenograft model to test hESC-derived cardiomyocyte stem/progenitor cells in human heart tissue for engraftment and function.
  • Second, we have optimized cell culture conditions and cell surface markers to sort hematopoietic progenitors derived from hESCs. In doing so, we have mapped the earliest stages of hematopoietic specification and commitment from a bipotent hematoendothelial precursor. Our culture conditions drive robust hematopoietic differentiation in vitro but these hESC-derived hematopoietic progenitors do not achieve hematopoietic engraftment when transplanted in mouse models. Furthermore, we overexpressed the anti-apoptotic protein BCL2 in hESCs, and discovered a significant improvement in viability upon single cell sorting, embryoid body formation, and in cultures lacking serum replacement. Moving forward, we feel the survival advantages exhibited by this BCL2-expressing hESC line will improve our chances of engrafting hESC-derived hematopoietic stem/progenitor cells.
  • Third, we identified a cocktail of 5 commercial and 1 novel mAbs that enable specific identification of human pluripotent cells (hESCs and induced pluripotent cells). We have found combinations of 3 pluripotency surface markers that can remove all teratoma-initiating cells from differentiated hESC and induced pluripotent stem cell (iPSC) populations prior to transplant. While these combinations can vary depending on the differentiation culture, we have generated a simple, easy-to-follow protocol to remove all teratogenic cells from large-scale differentiation cultures.
  • In summary, we accomplished most of the goals stated in our original proposal. We successfully achieved cardiac engraftment of an hESC-derived cardiomyocyte progenitor using a novel human heart model of engraftment. While we unfortunately did not attain hematopoietic engraftment of hESC-derived cells, we are exploring a strategy to address this. Our research has led to four manuscripts: one on the protective effects of BCL2 expression on hESC viability and pluripotency (published in PNAS, 2011), another describing markers of pluripotency and their use in depleting teratogenic potential in differentiated PSCs (accepted for publication in Nature Biotechnology), and two submitted manuscripts, one describing a novel xenograft assay to test PSC-derived cardiomyocytes for functional engraftment and the other describing the earliest fate decisions downstream of a PSC.
Funding Type: 
SEED Grant
Grant Number: 
RS1-00420
Investigator: 
ICOC Funds Committed: 
$577 037
Disease Focus: 
Blood Disorders
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Hematopoietic stem cells (HSC) have been used successfully to cure various life-threatening blood diseases. Yet, matching HSCs are not available for every patient. Human embryonic stem cells (hESC) may provide an unlimited source of HSCs for therapeutic use. However, hESC derived hematopoietic cells do not develop properly in those culture conditions that are currently used, and thereby their function is impaired. Hematopoietic cells that are derived from human ES cells lack the ability to self-renew, which is a prerequisite for the ability to generate blood cells for the individual’s lifetime. HSCs can only develop and function normally if they receive correct signal from their microenvironment, the stem cell niche. The goal of our proposal is take advantage of our knowledge of development of hematopoietic stem cells during embryogenesis, and mimic the environments where HSCs normally develop to provide the cues for proper HSC development in culture. We will attempt to mimic physiological HSC niches by deriving stroma lines from human placentas, which we have shown to be an important site for HSC development. We will further modify these lines by adding regulatory molecules that are known to aid HSC self-renewal, or inhibit molecules that might promote premature differentiation. Alternatively, we will use placental villi as a niche where ES cell derived hematopoietic cells could develop during culture. Subsequently, hESC derived cells are tested in animal models where human hematopoietic tissues have been implanted to provide an optimal environment for human HSCs to function. These studies are expected to shed light on the mechanisms that enable human HSCs to establish and maintain self-renewal ability and multipotency, and improve the differentiation of hESCs towards functional HSCs, which could be used to treat leukemias, other cancers, and inherited disease of the blood and immune system. To ensure hESC lines derived in different conditions respond in a similar way to these developmental cues, non-federally approved lines have to be used in this study, and thus governmental funding is not attainable for this project {REDACTED}.
Statement of Benefit to California: 
We aim to develop hematopoietic stem cells (HSC) from human ES cells (hESC) for ultimate theraoutic use for blood diseases. Only up to 50% of the patients that could be cured by HSC transplantation are able to receive this treatment, as matching donors are not available for every patient. If functional HSCs could be generated from hESCs, patients in California that suffer from leukemias or other acquired or inherited diseases of the blood and immune system could be treated. We aim to develop novel approaches to differentiate HSCs from hESCs by mimicking the physiological niches where human HSCs normally develop. Through these studies, we aim to understand what the critical properties in HSC microenvironment are that signal for HSCs to preserve their functionality. Identification of the regulatory cues that alter HSC fates between self-renewal and differentiation might also lead to innovative discoveries that could be developed into biotechnological or pharmaceutical products in California, thereby improving the industry and economy in California.
Progress Report: 
  • Our goal has been to improve the microenvironment where human embryonic stem cells (hESC) differentiate in order to generate functional hematopoietic stem/progenitor cells (HS/PC) in culture, with the ultimate goal to use these HS/PCs for the treatment of leukemias and other blood diseases. We have tested various human and mouse stroma lines for their ability to support expansion of multipotential human HS/PCs as well as hematopoietic specification from hESCs. So far mouse mesenchymal stem cells (MSC) have proven to provide the best supportive ability for human hematopoiesis. By combining embryoid body differentiation and co-culture on mouse MSC stroma, we have succesfully generated HS/PCs that phenotypically resemble bona fide human HSCs (CD34+CD38-CD90+CD45+). However, so far their differentiation ability has been biased toward myeloerythroid cells, with poor ability to generate B-cells in culture. Based on microarray data that we obtained from a related project supported by the CIRM New Faculty Award, we have identified molecular programs that are defective in hES derived HS/PCs. Future efforts will be directed in modifying the culture microenvironment as well as the cell intrinsic regulatory machinery in hES derived HS/PCs in order to improve their differentiation and self-renewal potential.
  • Our goal has been to improve the microenvironment where human embryonic stem cells (hESC) differentiate in order to generate functional hematopoietic stem/progenitor cells (HS/PC) in culture, with the ultimate goal to use these HS/PCs for the treatment of leukemias and other blood diseases. We have optimized a two step differentiation protocol that combines embryoid body differentiation and subsequent stroma co-culture to generate HS/PCs that exhibit the same phenotype as HSCs obtained from human hematopoietic tissues (CD34+CD38-CD90+CD45+). However, our findings indicate that the hESC derived HS/PCs have restricted developmental potential as compared to fetal liver or cord blood derived HS/PCs, and they senesce prematurely in culture, and are unable to generate B-cells . Our functional and molecular studies suggest that hES-derived HS/PCs resemble closely lineage-restricted progenitors found early in development in human hematopoietic tissues. Our recent studies have focused on exploring the possibility that another precursor that develops in the embryoid bodies could have lymphoid potential when placed in an appropriate microenvironment. Our preliminary data suggests that development of T-lymphocytes from hESCs in vitro may be feasible. Our future work will continue to focus on generating fully functional HSCs by improving the in vitro microenvironment where HS/PCs develop, and/or programming HSC transcriptional program using inducible lentiviral vectors.
Funding Type: 
SEED Grant
Grant Number: 
RS1-00280
Investigator: 
ICOC Funds Committed: 
$538 211
Disease Focus: 
Blood Disorders
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
For many therapeutic reasons it is important to have available large numbers of blood cells. However, it is difficult to generate large numbers of specialized blood cells that have the ability to neutralize autoimmunity and response to tumor cell growth. In this study we would develop a technique that would allow the production of large numbers of different types of blood cells from human embryonic stem cells. For example, a subset of white blood cells, called dendrititc cells, is currently manipulated in the laboratory in a manner that allows them to attack cancer cells. The same cells also are altered in the laboratory to counter-act the development of autoimmune diseases. A problem with these experiments is that it is difficult to isolate large numbers of these cells, since they are relatively rare. With the technology that is described in this grant application we would be able to generate large numbers of such cells in the laboratory using as a starting point, human embryonic stem cells.
Statement of Benefit to California: 
In this study we would develop an approach that would allow the production of large numbers of different types of blood cells from human embryonic stem cells. For example, a subset of white blood cells, called dendrititc cells, is currently manipulated in the laboratory in a manner that allows them to attack cancer cells. The same cells also are altered in the laboratory to counter-act the development of autoimmune diseases. A problem with these experiments is that it is difficult to isolate large numbers of these cells, since they are relatively rare. With the technology that is described in this grant application we would be able to generate large numbers of such cells in the laboratory using as a starting point, human embryonic stem cells. The approach is novel and straightforward and could be applied immediately once it has been established.
Progress Report: 
  • A prominent subset of white blood cells, named CD4 helper T cells, are critical in modulating the immune response against viral and bacterial pathogens. During HIV infection, the CD4 compartment is selectively reduced, suppressing the activity and response of cytolytic CD8 T cells, needed to abolish cells infected with the virus. Pharmaceutical therapies have been developed but they are not consistently effective and multidrug resistant viral strains are increasingly prevalent. Similarly, in vitro manipulated human dendritic cells are now being explored to tolerize against autoimmune disease or to stimulate antitumor responses. However, the number of dendritic cells that can be isolated form patients using current technologies is small. Consequently, different approaches need to be developed to enhance T cell reconstitution. In principle, multipotent hematopoietic progenitors could be derived from hESCs without long-term in vitro culture. A drawback is that the number of human hematopoietic progenitors derived from human ES cell cultures is limited using current culture conditions. Consequently, a subset of studies involving in vitro manipulated human cells would be difficult to perform. The transduction of human progenitor cells can be achieved using a tissue culture system in which human cord blood progenitors are differentiated in the presence of stromal cells that express the Notch ligand DL-1 towards the T cell lineage. However, the efficiency by which human progenitor cells differentiate into the T lineage cells is low. In the original application we proposed to develop a novel strategy that would permit the generation of large numbers of human T cell progenitors (up to 109) from human hematopoietic stem cells. To accomplish this objective we would target a critical regulator of early hematopoieisis, named E2A. Indeed during the two years period funded by CIRM we have demonstrated that murine hematopoietic progenitors that overexpress an inhibitor of E2A, named Id2, can be grown indefinitely in culture without losing their ability to generate many different types of white blood cells in the laboratory. This strategy is unconventional since it would permit the growth and isolation of large numbers of T cell progenitors, which has not been achieved so far by conventional culture conditions. We will continue these studies and use the same strategy to establish a long-term culture of human hematopoietic progenitor cells. If successful the approach would enable clinicians to reconstitute the hematopoietic compartments of patients carrying invading pathogens, including HIV infected patients, with large numbers of T cells that either express either a wild-type TCR repertoire or TCRs with specificities directed against invading pathogens. I expect this to succeed since we have already achieved this objective using murine progenitors as demonstrated during the past two years using funds obtained form the CIRM.
Funding Type: 
Basic Biology II
Grant Number: 
RB2-01497
Investigator: 
Institution: 
Type: 
PI
ICOC Funds Committed: 
$1 430 908
Disease Focus: 
Blood Disorders
Pediatrics
Stem Cell Use: 
iPS Cell
Embryonic Stem 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
Funding Type: 
Disease Team Research I
Grant Number: 
DR1-01452
Investigator: 
Type: 
PI
Type: 
Co-PI
Type: 
Co-PI
ICOC Funds Committed: 
$9 212 365
Disease Focus: 
Blood Disorders
Pediatrics
Stem Cell Use: 
Adult Stem Cell
Cell Line Generation: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 
Sickle cell disease (SCD), which results from an inherited mutation in the hemoglobin gene that causes red blood cells to "sickle" under conditions of low oxygen, occurs with a frequency of 1/500 African-Americans, and is also common in Hispanic-Americans, who comprise up to 5% of SCD patients in California. The median survival based on 1991 national data was 42 years for males and 48 years for females. More recent data indicate that the median survival for Southern California patients with SCD is only 36 years, suggesting that serious problems exist regarding access to optimal medical care in this community. By twenty years of age, about 15% of children with SCD suffer major strokes and by 40 years of age, almost half of the patients have had central nervous system damage leading to significant cognitive dysfunction. These patients suffer recurrent damage to lungs and kidneys as well as severe chronic pain that impacts on quality of life. While current medical therapies for SCD can make an important difference in short-term effects, the progressive deterioration in organ function results in compromised quality of life and early deaths in ethnic populations who are generally adversely affected by health care disparity. Transplantation of bone marrow from a healthy donor as a source of new adult blood-forming ("hematopoietic") stem cells can benefit patients with SCD, by providing a source for life-long production of normal red blood cells. However, bone marrow transplant is limited by the availability of well-matched donors and the problems that arise from immune reactions between the cells of the donor and the patient. Thus, despite major improvements in clinical care of SCD patients, SCD continues to be a major cause of illness and early death. The stem cell therapy approach to be developed by this Disease Team will be used to treat patients with SCD by transplanting them with their own bone marrow adult hematopoietic stem cells that are genetically corrected by adding a hemoglobin gene that blocks sickling of the red blood cells. This approach has the potential to permanently cure this debilitating and common illness with significantly less toxicity than with a bone marrow transplant from another person. A clinical trial using stem cell gene therapy for patients with SCD will be developed to be performed by this Team. This multi-disciplinary Disease Team combines world-leading experts in stem cell gene therapy, clinical bone marrow transplantation and the care of patients with sickle cell disease. Successful use of stem cell gene therapy for sickle cell disease has the potential to provide a more effective and safe treatment for this disease to a larger proportion of affected patients.
Statement of Benefit to California: 
Development of methods for regenerative medicine using genetically-corrected human stem cells will result in novel, effective therapies that improve the health for millions of Californians and tens of millions of people world-wide. Sickle cell disease is an inherited disease of the red blood cells that results from a specific gene mutation. Sickle cell disease disproportionately afflicts poor minority patients in the State of California, causing severe morbidity, early mortality and high medical costs. We will develop a clinical trial to evaluate a novel treatment for patients with sickle cell disease, using their own adult blood-forming stem cells, after correcting the hemoglobin gene defect. Successful treatment of sickle cell disease using adult blood forming “hematopoietic” stem cells corrected with gene therapy may provide a clinically beneficial way to treat sickle cell disease with greater safety and wider availability than current options. The clinical trial to be developed will treat sickle cell disease patients from across the state of California through the network of institutions incorporated into this Disease Team. All scientific findings and biomedical materials produced from our studies will be publicly available to non-profit and academic organizations in California, and any intellectual property developed by this Project will be developed under the guidelines of CIRM to benefit the State of California.
Progress Report: 
  • The clinical complications of sickle cell disease are due to the inherited abnormality of the oxygen-carrying hemoglobin protein in red blood cells (RBC). The RBC are made from stem cells in the bone marrow and transplantation of stem cells from the bone marrow of a healthy donor to someone with sickle cell disease (SCD) can lead to significant improvements in their health. However, most people do not have a matched sibling donor, and transplants from unrelated donors have higher risks for complications, mainly due to immune reactions between the donor and the recipient.
  • The goal of this project is to bring to the clinic a trial of treating patients with SCD by transplanting them with their own bone marrow stem cells that have been modified in the lab by adding the gene for a version of human beta-globin that will act to inhibit sickling of the patient’s RBC (“anti-sickling” gene). This approach may provide a way to improve the health of people with SCD, with advantages over clinical treatments using transplantation of bone marrow stem cells from another person.
  • The major Year 1 Milestone was to demonstrate the feasibility of this approach, i.e. that the clinical cell product, the subject’s bone marrow stem cells modified with the anti-sickling gene, can be produced suitably for clinical transplantation and that enough of the anti-sickling hemoglobin is made to reverse sickling of RBC made from the gene-modified stem cells.
  • Studies done by the Laboratory component of our Disease Team showed that the gene transfer lentiviral vector we developed to insert the anti-sickling gene into bone marrow stem cells met pre-set technical criteria for: the amount of vector that can be made, its efficiency to insert the anti-sickling gene into human bone marrow stem cells, the levels of anti-sickling beta-globin protein made by the vector in RBC made from bone marrow stem cells, and the absence of adverse effects on the stem cells or their ability to make new RBC. These successful results allow advancement to the major lab focus for Years 2-3, pre-clinical efficacy and safety studies to support an IND application.
  • The Clinical/Regulatory component of our Disease team established the proposed network of California clinical hematology sites to obtain bone marrow samples from volunteer donors with SCD for laboratory research studies on cell product development (UCLA, CHLA and CHRCO). We put into place the necessary IRB-approved protocols to collect bone marrow samples at these sites to use for the laboratory research at UCLA and USC. This network obtained its first BM sample from a SCD donor on 3/18/2010 and a total of 15 over the year. These patient-derived samples have been truly essential to the advancement of the laboratory work because bone marrow from SCD patients is needed for studies to measure expression of the anti-sickling gene and improvement in RBC sickling.
  • The Clinical Regulatory component has also produced a complete first draft of the clinical trial protocol, which defines which specific people with SCD would be eligible for participation in this study, and the exact approach of the clinical study, including how the patients will be evaluated before the procedure, the details of the bone marrow harvest, stem cell processing and transplant processes, and how the effects of the procedure will be assessed. This protocol was conceived with input from the Team of physicians and scientists with expertise in clinical and experimental hematology, bone marrow transplantation, transfusion medicine, gene therapy and cell processing laboratory methods, regulatory affairs, and biostatistics.
  • These efforts provided sufficient laboratory data and definition of the clinical approach that we could have a pre-pre-IND exchange with the FDA (on 09/30/10). This interaction provided us the opportunity to receive initial guidance for three key areas that would comprise the IND application: the draft clinical protocol, the methods to make and characterize the gene-modified stem cell product for transplant, and the planned pre-clinical safety studies. The meeting was encouraging and informative.
  • In Year 2, our laboratory work will focus on determining the functional effects of inserting the anti-sickling gene into bone marrow stem cells from SCD donors on sickling of the RBC. We will begin to define the laboratory test methods that would be used to measure the results in the clinical trial (% of stem and blood cells with the gene, the amounts of anti-sickling beta-globin made, and the effects on RBC sickling). We will continue to design the studies to formally test vector safety (Toxicology study). The major goal is to advance to a pre-IND meeting with the FDA which should provide further guidance to finalize the design of the pre-clinical toxicology study and the clinical trial design. We will then be ready to implement the toxicology study and begin regulatory reviews of the protocol by local and federal authorities.
  • The clinical complications of sickle cell disease are due to the inherited abnormality of the oxygen-carrying hemoglobin protein in red blood cells (RBC). The RBC are made from stem cells in the bone marrow and transplantation of stem cells from the bone marrow of a healthy donor to someone with sickle cell disease (SCD) can lead to significant improvements in their health. However, most people do not have a matched sibling donor, and transplants from unrelated donors have higher risks for complications, mainly due to immune reactions between the donor and the recipient.
  • The goal of this project is to bring to the clinical trial of treating patients with SCD by transplanting them with their own bone marrow stem cells that have been modified in the laboratory by adding the gene for a version of human beta-globin that will act to inhibit sickling of the patient’s RBC (“anti-sickling” gene). This approach may provide a way to improve the health of people with SCD, with advantages over clinical treatments using transplantation of bone marrow stem cells from another person.
  • In the first 2 years of this project we were able to demonstrate the feasibility of this approach, i.e. that the clinical cell product, the subject’s bone marrow stem cells modified with the anti-sickling gene, can be produced suitably for clinical transplantation and that enough of the anti-sickling hemoglobin is made to reverse sickling of RBC made from the gene-modified stem cells.
  • Studies done by the Laboratory component of our Disease Team showed that the gene transfer lentiviral vector we developed to insert the anti-sickling gene into bone marrow stem cells met pre-set technical criteria for: the amount of vector that can be made, its efficiency to insert the anti-sickling gene into human bone marrow stem cells, the levels of anti-sickling beta-globin protein made by the vector in RBC, and the absence of adverse effects on the stem cells or their ability to make new RBC. These successful results allow advancement to the major lab focus for Year 3, safety studies to support an IND application.
  • The Clinical/Regulatory component of our Disease team established the proposed network of California clinical hematology sites to obtain bone marrow samples from volunteer donors with SCD for laboratory research studies on cell product development (UCLA, CHLA and CHRCO). We put into place the necessary IRB-approved protocols to collect bone marrow samples at these sites to use for the laboratory research at UCLA and USC. This network obtained its first BM sample from a SCD donor on 3/18/2010 and a total of 29 over 2 years. These patient-derived samples have been truly essential to the advancement of the laboratory work because bone marrow from SCD patients is needed for studies to measure expression of the anti-sickling gene and improvement in RBC sickling.
  • The Clinical Regulatory component has also produced a complete first draft of the clinical trial protocol, which defines which specific people with SCD would be eligible for participation in this study, and the exact approach of the clinical study, including how the patients will be evaluated before the procedure, the details of the bone marrow harvest, stem cell processing and transplant processes, and how the effects of the procedure will be assessed. This protocol was conceived with input from the Team of physicians and scientists with expertise in clinical and experimental hematology, bone marrow transplantation, transfusion medicine, gene therapy and cell processing laboratory methods, regulatory affairs, and biostatistics.
  • These efforts provided sufficient laboratory data and definition of the clinical approach that we could have a pre-IND meeting with the FDA (on 08/22/11). This interaction provided us the opportunity to receive guidance for three key areas that would comprise the IND application: the draft clinical protocol, the methods to make and characterize the gene-modified stem cell product for transplant, and the planned pre-clinical safety studies. The meeting was encouraging and informative.
  • In Year 3, our laboratory work will focus on performing pre-clinical safety studies (Toxicology study), qualifying end point assays and finalizing stem cell processing.
  • The clinical complications of sickle cell disease are due to the inherited abnormality of the oxygen-carrying hemoglobin protein in red blood cells (RBC). The RBC are made from stem cells in the bone marrow and transplantation of stem cells from the bone marrow of a healthy donor to someone with sickle cell disease (SCD) can lead to significant improvements in their health. However, most people do not have a matched sibling donor, and transplants from unrelated donors have higher risks for complications, mainly due to immune reactions between the donor and the recipient.
  • The goal of this project is to develop a clinical trial to treat patients with SCD by transplanting them with their own bone marrow stem cells that have been modified in the laboratory by adding the gene for a version of human beta-globin that will act to inhibit sickling of the patient’s RBC (“anti-sickling” gene). This approach may provide a way to improve the health of people with SCD, with advantages over clinical treatments using transplantation of bone marrow stem cells from another person.
  • In the first 2 years of this project we demonstrated the feasibility of this approach, i.e. that the clinical cell product, the subject’s bone marrow stem cells modified with the anti-sickling gene, can be produced suitably for clinical transplantation and that enough of the anti-sickling hemoglobin is made to reverse sickling of RBC made from the gene-modified stem cells. The Clinical/Regulatory component of our Disease Team established the proposed network of California clinical hematology sites to obtain bone marrow samples from volunteer donors with SCD for laboratory research studies on cell product development (UCLA, CHLA and CHRCO). We put into place the necessary IRB-approved protocols to collect bone marrow samples at these sites to use for the laboratory research at UCLA and USC. This network obtained its first BM sample from a SCD donor on 3/18/2010 and a total of 45 over 3 years. These patient-derived samples have been truly essential to the advancement of the laboratory work because bone marrow from SCD patients is needed for studies to measure expression of the anti-sickling gene and improvement in RBC sickling. The Clinical Regulatory component has also produced the clinical trial protocol, which defines which specific people with SCD would be eligible for participation in this study, and the exact approach of the clinical study, including how the patients will be evaluated before the procedure, the details of the bone marrow harvest, stem cell processing and transplant processes, and how the effects of the procedure will be assessed. This protocol was conceived with input from the Team of physicians and scientists with expertise in clinical and experimental hematology, bone marrow transplantation, transfusion medicine, gene therapy and cell processing laboratory methods, regulatory affairs, and biostatistics.
  • During the third year the Clinical Gene Therapy Laboratory component of the Team has demonstrated the feasibility of the stem cell processing procedure. Mimicking the future clinical scenario, the Lab was able to isolate stem cells from a largescale bone marrow harvest, insert the anti-sickling gene in adequate amount and recover the needed amount of stem cells that would be transplanted into the patient. The Clinical/Regulatory component of our Disease Team is focusing on validating all the assays that will be used during the clinical trial i.e. to characterize the final cell product and also the end-point assays to analyze the efficacy of this approach in patients. Another major focus during the third year has been safety and toxicology studies in a murine model of bone marrow transplant; the studies are still ongoing and will be completed in the next year. These successful results allow advancement to support an IND application in year 4.
  • CIRM DR1-01452 - Stem Cell Gene Therapy for Sickle Cell Disease
  • Scientific Progress in Year 4
  • The clinical complications of sickle cell disease are due to the inherited abnormality of the oxygen-carrying hemoglobin protein in red blood cells (RBC). The RBC are made from stem cells in the bone marrow and transplantation of stem cells from the bone marrow of a healthy donor to someone with sickle cell disease (SCD) can lead to significant improvements in their health. However, most people do not have a matched sibling donor, and transplants from unrelated donors have higher risks for complications, mainly due to immune reactions between the donor and the recipient.
  • The goal of this project is to develop a clinical trial to treat patients with SCD by transplanting them with their own bone marrow stem cells that have been modified in the laboratory by adding the gene for a version of human beta-globin that will act to inhibit sickling of the patient’s RBC (“anti-sickling” gene). This approach may provide a way to improve the health of people with SCD, with advantages over clinical treatments using transplantation of bone marrow stem cells from another person.
  • In the first 2 years of this project, we demonstrated the feasibility of this approach, i.e. that the clinical cell product, the subject’s bone marrow stem cells modified with the anti-sickling gene, can be produced suitably for clinical transplantation and that enough of the anti-sickling hemoglobin is made to reverse sickling of RBC made from the gene-modified stem cells. The Clinical/Regulatory component of our Disease Team established the proposed network of California clinical hematology sites to obtain bone marrow samples from volunteer donors with SCD for laboratory research studies on cell product development (UCLA, CHLA and CHRCO). We put into place the necessary IRB-approved protocols to collect bone marrow samples at these sites to use for the laboratory research at UCLA and USC. This network obtained its first BM sample from a SCD donor on 3/18/2010 and a total of 56 over 4 years. These patient-derived samples have been truly essential to the advancement of the laboratory work because bone marrow from SCD patients is needed for studies to measure expression of the anti-sickling gene and improvement in RBC sickling. The Clinical Regulatory component has also produced the clinical trial protocol, which defines which specific people with SCD would be eligible for participation in this study, and the exact approach of the clinical study, including how the patients will be evaluated before the procedure, the details of the bone marrow harvest, stem cell processing and transplant processes, and how the effects of the procedure will be assessed. This protocol was conceived with input from the Team of physicians and scientists with expertise in clinical and experimental hematology, bone marrow transplantation, transfusion medicine, gene therapy and cell processing laboratory methods, regulatory affairs, and biostatistics. It has now been approved by the UCLA Institutional Review Board and the Institutional Scientific Protocol review Committee, as well as the NIH Recombinant DNA Advisory Committee.
  • During the last 2 years the Clinical Gene Therapy Laboratory component of the Team has demonstrated the feasibility of the stem cell processing procedure. Mimicking the future clinical scenario, the Lab was able to isolate stem cells from a large scale bone marrow harvest, insert the anti-sickling gene in adequate amount and recover the needed amount of stem cells that would be transplanted into the patient. The Clinical/Regulatory component of our Disease Team validated all the assays that will be used during the clinical trial i.e. to characterize the final cell product and also the end-point assays to analyze the efficacy of this approach in patients. Another major focus during the third and fourth year has been safety and toxicology studies in a murine model of bone marrow transplant; these successful results allow advancement to support an IND application in the second quarter of 2014, with a goal of opening the trial in the third quarter of the year.
  • The clinical complications of sickle cell disease are due to the inherited abnormality of the oxygen-carrying hemoglobin protein in red blood cells (RBC). The RBC are made from stem cells in the bone marrow and transplantation of stem cells from the bone marrow of a healthy donor to someone with sickle cell disease (SCD) can lead to significant improvements in their health. However, most people do not have a matched sibling donor, and transplants from unrelated donors have higher risks for complications, mainly due to immune reactions between the donor and the recipient.
  • The goal of this project is to develop a clinical trial to treat patients with SCD by transplanting them with their own bone marrow stem cells that have been modified in the laboratory by adding the gene for a version of human beta-globin that will act to inhibit sickling of the patient’s RBC (“anti-sickling” gene). This approach may provide a way to improve the health of people with SCD, with advantages over clinical treatments using transplantation of bone marrow stem cells from another person.
  • In the first 2 years of this project, we demonstrated the feasibility of this approach, i.e. that the clinical cell product, the subject’s bone marrow stem cells modified with the anti-sickling gene, can be produced suitably for clinical transplantation and that enough of the anti-sickling hemoglobin is made to reverse sickling of RBC made from the gene-modified stem cells. The Clinical/Regulatory component of our Disease Team established the proposed network of California clinical hematology sites to obtain bone marrow samples from volunteer donors with SCD for laboratory research studies on cell product development (UCLA, CHLA and CHRCO). We put into place the necessary IRB-approved protocols to collect bone marrow samples at these sites to use for the laboratory research at UCLA and USC. This network obtained its first BM sample from a SCD donor on 3/18/2010 and a total of 58 over 4+ years. These patient-derived samples have been truly essential to the advancement of the laboratory work because bone marrow from SCD patients is needed for studies to measure expression of the anti-sickling gene and improvement in RBC sickling. The Clinical Regulatory component has also produced the clinical trial protocol, which defines which specific people with SCD would be eligible for participation in this study, and the exact approach of the clinical study, including how the patients will be evaluated before the procedure, the details of the bone marrow harvest, stem cell processing and transplant processes, and how the effects of the procedure will be assessed. This protocol was conceived with input from the Team of physicians and scientists with expertise in clinical and experimental hematology, bone marrow transplantation, transfusion medicine, gene therapy and cell processing laboratory methods, regulatory affairs, and biostatistics. It has now been approved by the UCLA Institutional Review Board and the Institutional Scientific Protocol review Committee, as well as the NIH Recombinant DNA Advisory Committee.
  • During the last 2 years the Clinical Gene Therapy Laboratory component of the Team has demonstrated the feasibility of the stem cell processing procedure. Mimicking the future clinical scenario, the Lab was able to isolate stem cells from a large scale bone marrow harvest, insert the anti-sickling gene in adequate amount and recover the needed amount of stem cells that would be transplanted into the patient. The Clinical/Regulatory component of our Disease Team validated all the assays that will be used during the clinical trial i.e. to characterize the final cell product and also the end-point assays to analyze the efficacy of this approach in patients. Another major focus during the third and fourth year has been to demonstrate the safety of this approach in a murine model of bone marrow transplant; these successful results allowed advancement to support an IND application and opening a clinical trial for gene therapy of SCD in the second quarter of 2014.
Funding Type: 
Basic Biology I
Grant Number: 
RB1-01328
Investigator: 
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.

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