Blood Disorders

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

Generation of long-term cultures of human hematopoietic multipotent progenitors from embryonic stem cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00280
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.

Stem Cell Gene Therapy for Sickle Cell Disease

Funding Type: 
Disease Team Research I
Grant Number: 
DR1-01452
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.

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.

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