Blood Disorders

Coding Dimension ID: 
278
Coding Dimension path name: 
Blood Disorders
Funding Type: 
Early Translational IV
Grant Number: 
TR4-06823
Investigator: 
Type: 
PI
ICOC Funds Committed: 
$1 815 308
Disease Focus: 
Blood Disorders
Pediatrics
Stem Cell Use: 
Adult Stem Cell
Cell Line Generation: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 

Disorders affecting the blood, including Sickle Cell Disease (SCD), are the most common genetic disorders in the world. SCD causes significant suffering and early death, despite major improvements in medical management and advances in understanding the complex disease-related biology. A bone marrow transplant (BMT) can greatly benefit patients with SCD, by providing a life-long source of normal red blood cells. However, BMT is limited by the availability of suitable donors and immune complications, especially for the more than 80% of patients who lack a matched sibling donor. An alternative treatment approach for SCD is to isolate some of the patient’s own bone marrow and then use gene therapy methods to correct the sickle gene defect in the blood stem cells before transplanting them back into the patient. The gene-corrected stem cells could make normal blood cells for the life of the patient, essentially eliminating the SCD. Such an approach would avoid the complications typically associated with transplants from non-matched donors. We will define the optimal techniques to correct the sickle gene mutation in the bone marrow stem cells to develop as a therapy for patients with SCD.

Statement of Benefit to California: 

Development of methods for regenerative medicine using stem cells will have widespread applications to improve the health and to provide novel, effective therapies for millions of Californians and tens of millions of people worldwide. Many severe medical conditions can be cured or improved by transplantation of blood-forming hematopoietic stem cells (HSC), including genetic diseases of blood cells, such as sickle cell disease and inborn errors of metabolism, cancer and leukemia, and HIV/AIDS. Precise genetic engineering of stem cells to repair inherited mutation may be the best way to correct genetic defects affecting the mature cells they produce. This project will advance methods to precisely repair the genetic defect that underlies sickle cell disease in hematopoietic stem cells, which can then be transplanted to ameliorate the disease. These advances will have direct and immediate applications to enhance current medical therapies of sickle cell disease and will more broadly help to advance the capacities for regenerative medicine. 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 people of the State of California.

Progress Report: 
  • Sickle-cell disease (SCD) is characterized by a single point mutation in the seventh codon of the beta-globin gene. Site-specific correction of the sickle mutation in adult bone marrow hematopoietic stem cells (HSCs) would allow for permanent production of normal red blood cells. Site-specific correction can be achieved using proteins called zinc-finger nucleases (ZFNs) which recognize and bind the region of the genome surrounding the sickle mutation. The ZFNs are able to create a break in the DNA which the cells repair using existing repair machinery. If, at the time of repair, a homologous donor template containing the corrective base is present, the cells' repair machinery can use this template and the resulting cell genome will contain the wild-type base instead of the sickle mutation. By doing this in hematopoietic setm cells, the cell is permanently corrected and each red blood cell (RBC) derived from this corrected stem cell will produce normal, non-sickle RBCs. In this report, we show efficient targeted cleavage by the ZFNs at the beta-globin locus with minimal off-target modification. In addition, we compare two different homologous donor templates (an integrase-defective lentiviral vector [IDLV] and a single-stranded DNA oligonucleotide [oligo]) to determine the optimal donor template. In both wild-type as well as sickle cell disease patient CD34+ HSCs, we are able to deliver the ZFN and donor templates and specifically correct the genome at rates of up to 30%. When these cells are differentiated into RBCs in vitro, we demonstrate that they are not altered in their differentiation capacity and are able to produce wild-type hemoglobin at high levels (35% of all hemoglobins) by HPLC. These results provide a strong basis for moving forward with this work as we begin our efforts to increase the number of treated cells to achieve clinical levels of corrected cells as well as characterize the ability of these cells to engraft a murine model in vivo. The progress made in this year is an exciting step towards a clinical therapy and potential treatment for sickle cell disease.
Funding Type: 
Tools and Technologies III
Grant Number: 
RT3-07848
Investigator: 
Type: 
PI
ICOC Funds Committed: 
$1 500 624
Disease Focus: 
HIV/AIDS
Blood Disorders
Stem Cell Use: 
Adult Stem Cell
Public Abstract: 

The overall goal of this proposal is to develop new methods and technologies to improve our ability to engineer hematopoietic stem cells. These are the adult stem cells found in the bone marrow that give rise to all of the components of the blood and immune systems. Being able to engineer these cells provides potential treatments for diseases of the blood including genetic diseases, such as sickle cell disease or severe immune deficiencies, as well as serious infections such as HIV/AIDS. We work with a new class of genetic engineering tools called targeted nucleases that have the potential to make genetic engineering of stem cells much more precise and therefore safer. In addition, we are exploring methods to deliver these reagents directly to the stem cells in the body, without the currently necessary steps of first removing the cells and performing the genetic engineering in a lab. Such capabilities would greatly improve the safety of human gene therapy, as well as facilitate its practical implementation. HIV/AIDS is our disease of focus, and we will use these techniques to develop new treatments that go beyond the current use of targeted nucleases in patients, where HIV’s co-receptor gene, called CCR5, is being disrupted. Our goal is to develop a next-generation of anti-HIV therapies and we expect that the techniques we develop will be broadly applicable to other disease of the blood and immune systems where stem cell therapies could be of benefit.

Statement of Benefit to California: 

HIV/AIDS is a major social, economic and health burden to California and its citizens. The numbers are sobering: California has 14% of all US cases of HIV, second only to New York, with 220,543 cases reported through June 2014, including 98,161 deaths. With the advent of improved antiretroviral drugs, mortality has significantly decreased, but so has the length of time people need to take the drugs, and the economic burden to the state is revealed by the cost of drugs representing 85% of all AIDS-related costs. Both federal and state laws require that the AIDS Drug Assistance Program be the payer of last resort for these medications, and its budget is underwritten by the General Fund. Beyond the fiscal concerns, patients live with the potential for developing side effects to the drugs or drug-resistant virus, and accessing these life-long drug regimens is a daily struggle for many. Consequently, the development of stem cell based therapies for HIV brings the potential of one-shot and long-lasting treatments that could arm a patient’s own immune system with the capability to suppress HIV in the absence of drugs. Such an outcome would provide economic returns over the long-run by reducing spending on drugs, as well as improving the quality of life for individuals with HIV/AIDS. Beyond HIV, the development of technologies to improve the efficiency, safety and implementation of hematopoietic stem cell therapies will benefit other diseases where such cells could be curative.

Funding Type: 
Research Leadership
Grant Number: 
LA1-08014
Investigator: 
Name: 
Type: 
PI
ICOC Funds Committed: 
$5 174 715
Disease Focus: 
Blood Disorders
Stem Cell Use: 
Adult Stem Cell
Public Abstract: 

Bone marrow and peripheral blood transplantation utilizing blood stem cells can provide curative treatment for patients with cancers and non-cancerous diseases of the blood and immune systems. Such treatments can be curative because the stem cells contained within the bone marrow or peripheral blood of healthy donors are capable of replacing the entirety of the patient’s blood system and providing a new immune system which can eradicate the patient’s cancer cells. The application of blood stem cell transplantation could be applied to a much larger population of patients if methods could be developed to expand blood stem cells in vitro or in vivo. This would be particularly beneficial for the broadened application of human cord blood transplantation for the many patients who lack an immune-matched sibling or unrelated donor. Furthermore, a method to expand human blood stem cells in vivo could be highly beneficial for the thousands of patients with cancer who require toxic chemotherapy which frequently results in decreased blood counts, infections and bleeding complications. A systemic treatment (i.e. a shot) which could cause blood stem cells to grow and produce more blood cells in patients could markedly improve patient’s outcomes after they receive such chemotherapy in the curative treatment of cancer. However, the development of treatments capable of inducing human blood stem cells to grow in the body has been very slow, in part due to a lack of understanding of the processes which govern blood stem cell growth in general. In my laboratory, we have developed mouse genetic models which allow us to discover new proteins produced in the bone marrow (the “soil” where blood stem cells reside) which make blood stem cells grow. We have recently discovered that 2 proteins are secreted by blood vessels within the bone marrow and cause blood stem cells to grow rapidly following damage with radiation. We are currently in the process of developing one of these into a growth factor that we can deliver to patients via injection as a means to cause their blood stem cells to grow after cord blood transplantation or following chemotherapy treatment for cancer. In this proposal, we will utilize our unique mouse models to discover the additional growth factors that make blood stem cells grow and we will perform pre-clinical studies to test whether these newly discovered growth factors can cause human blood stem cells to grow in vitro and in vivo. This proposal has the potential to generate new understanding of how human stem cells grow in vivo and to facilitate the development of new therapies which can regenerate human blood stem cells and the blood system as a whole in patients.

Statement of Benefit to California: 

My research program has both basic science and pre-clinical components which I believe will benefit California in several important ways: First, my basic research program will contribute new fundamental knowledge in stem cell biology which will benefit students, fellows and faculty. My research will also synergize with other campus laboratories and other centers in California and will lead to collaborations and accelerated translation of these discoveries for regenerative medicine. Second, my research program has the potential to directly benefit patients in California. We have already discovered two niche-derived proteins which promote hematopoietic stem cell regeneration in vivo and are focusing substantial efforts now to develop these proteins as therapeutics for Phase I clinical trials. For example, we are developing one of the HSC regenerative factors which we discovered for a Phase I clinical trial to test its efficacy as a systemic therapy to accelerate cord blood engraftment and hematologic recovery in adult cord blood transplant patients. This has literal potential benefit for patients since approximately 10% of cord blood transplant patients die from complications of graft failure or delayed hematologic recovery. In addition, patients with cancer who receive myelosuppressive chemotherapy can potentially benefit from systemic administration of [REDACTED] or other HSC regenerative factors that we discover to accelerate hematologic recovery after chemotherapy. If we are able to show that administration of such regenerative factors can accelerate hematologic recovery in patients after chemotherapy, then remission rates for cancer patients may increase via more effective delivery of curative chemotherapy on time and to completion. Third, my research will provide new intellectual property. These inventions from my laboratory will be available for licensure to biotech or pharmaceutical companies in California. I have experience with licensing inventions from my laboratory to biotech companies and am eager to see my future inventions licensed to accelerate development for regenerative medicine. Fourth, my research program will provide new jobs and professional opportunities. At present, my research program provides partial or complete funding for more than 30 employees internally and more than 30 employees at our partner institutions in academia and biotechnology. I will also bring substantial federal research funding with me to California and will be hiring new fellows, technicians and faculty promptly upon my arrival. Taken together, I am hopeful that my research program will have a major benefit for the scientific community of California, for patients who may benefit from treatments we are developing, for the biotechnology community via the development of new intellectual property and for the larger economy via the creation of many new jobs. I sincerely look forward to the opportunity to bring my program to California.

Funding Type: 
Tools and Technologies III
Grant Number: 
RT3-07683
Investigator: 
Institution: 
Type: 
PI
Institution: 
Type: 
Co-PI
ICOC Funds Committed: 
$1 452 708
Disease Focus: 
Blood Disorders
Blood Cancer
Cancer
Stem Cell Use: 
Adult Stem Cell
Public Abstract: 

A goal of stem-cell therapy is to transplant into a patient “tissue-specific” stem cells, which can regenerate a particular type of healthy tissue (e.g., heart or blood cells). A major obstacle to this goal is obtaining tissue-specific stem cells that (1) are available in sufficient numbers; and (2) will not be rejected by the recipient. One approach to these challenges is to generate tissue-specific stem cells in the lab from “pluripotent” stem cells, which can produce all types of tissue-specific stem cells. The rationale is that pluripotent stem cells that will be tolerated are easier to directly obtain than tissue-specific stem cells that will be tolerated. Furthermore, descendants of a tolerated pluripotent stem cell will also be tolerated and can be produced abundantly.

The goal of the proposed project is to develop techniques for generating transplantable blood-forming stem cells from pluripotent stem cells. In pursuit of this goal, we will study how blood-forming stem cells arise during development. We will also test new methods--less toxic than current chemotherapy and radiation--for preparing recipients for transplantation of blood-forming stem cells.

Additional benefit: Successful transplantation of blood-forming stem cells allows the recipient to tolerate other tissue or organ transplants from the same donor. Thus, transplanted blood-forming stem cells could allow people to receive organs that they may otherwise reject, without taking immune-suppressing drugs.

Statement of Benefit to California: 

We aim to generate from stem cells that can produce all tissues of the body those stem cells that specifically form blood. We will also test new methods--less toxic than current chemotherapy and radiation--for pretreatment before transplantation of blood-forming stem cells. A large number of patients in California could benefit from advances in this field, primarily those with diseases affecting the production of blood and immune cells: leukemia, lymphoma, thalassemia, certain types of anemia, immune deficiency diseases, autoimmune diseases (e.g., lupus), etc. For leukemia and lymphoma alone, in 2014 in California, there will be an estimated 12,060 newly diagnosed cases, 103,400 existing cases, and 4,620 deaths (per the California Cancer Registry). The cost of these blood cancers are difficult to estimate but they account for 6% of cancers in women and 9% in men in California, where the estimated cost of cancer per year is $28.3 billion.

The reagents generated in these studies can be patented, forming an intellectual property portfolio shared by the state. The funds generated from the licensing of these technologies will provide revenue for the state, help increase hiring of faculty and staff (many of whom will bring in other, out-of-state funds to support their research) and could reduce the costs of related clinical trials. Only California businesses are likely to be able to license these reagents and to develop them into diagnostic and therapeutic entities.

Funding Type: 
New Faculty Physician Scientist
Grant Number: 
RN3-06532
Investigator: 
ICOC Funds Committed: 
$2 661 742
Disease Focus: 
Blood Disorders
Pediatrics
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Active
Public Abstract: 

Many fetuses with congenital blood stem cell disorders such as sickle cell disease or thalassemia are prenatally diagnosed early enough in pregnancy to be treated with stem cell transplantation. The main benefit to treating these diseases before birth is that the immature fetal immune system may accept transplanted cells without needing to use immunosuppressant drugs to prevent rejection. Moreover, transplanting stem cells into the fetus—in which many stem cell types are actively multiplying and migrating—can promote similar growth and differentiation of the transplanted cells. Although this strategy works well in animal models, when applied clinically, the number of surviving cells in the blood (“engraftment”) has been too low to achieve a reliable cure.

Our lab studies ways to improve engraftment, with the long-term goal of applying these strategies to treat fetuses with congenital blood disorders. In this application, we will use novel embryonic stem cells that may be better suited to differentiate into blood cells in the fetal environment. We will also test various approaches to improve the survival advantage of these stem cells in fetal organs that make blood cells. Finally, we will study the fetal immune system to determine how fetuses become tolerant to the transplanted cells. The experiments in this proposal will give us important information to design clinical trials to treat fetuses with common, currently incurable stem cell disorders.

Statement of Benefit to California: 

The long-term goal of our project is to develop safe and effective ways to perform prenatal stem cell transplantation to treat fetuses with congenital blood disorders, such as thalassemia and hemoglobin disorders. These diseases affect many California citizens. For example, hemoglobin disorders are so common that they are routinely screened for at birth (and prenatal screening is performed if there is a family history). Thalassemias are found more commonly in persons of Mediterranean or Asian descent and are therefore prevalent in our state’s population. Prenatal screening is routinely offered, especially to patients with a family history or those with an ethnic predisposition. Fetal stem cell transplantation would also benefit children with sickle cell disease, 2000 of which are born each year in the United States, and inborn errors of metabolism, which occur in 1 in 4000 births. Thus, once we develop reliable techniques to treat these disorders before birth, there will be an enormous potential to make a difference.

Fetal surgery was pioneered in California and is performed only in select centers across the country. Therefore, once we have developed safe and effective therapies for fetuses with stem cell disorders, we also expect increased referrals of such patients to California. The convergence of our expertise in fetal therapies with those in stem cell biology carries great promise for finally realizing the promise of fetal stem cell transplantation.

Progress Report: 
  • Our group works on developing methods for successful transplantation of blood stem cells to treat fetuses with genetic disorders such as sickle cell disease or thalassemia. In this grant, we are using novel stem cells that will differentiate into blood-forming cells and other techniques to improve the “engraftment” of these cells. This year, we focused on using a new technique that creates “space” in the bone marrow of the recipient using an antibody (ACK2) to deplete the host’s blood stem cells. In a mouse model, we showed that this antibody is very effective is improving the engraftment of transplanted blood stem cells. In fact, the treatment is more effective in the fetal environment than the adult. These findings were recently published and we are planning to use this strategy in the monkey model as a step toward clinical applications. We are also working on transplanting human blood stem cells into immunodeficient mouse fetuses to understand whether different sources of stem cells vary in their ability to make blood cells in this setting.
  • The goal of our grant is to optimize the strategy of in utero transplantation of hematopoietic stem cells, with the ultimate goal of treating fetuses with congenital stem cell disorders. Our project includes transplantation of HSC into both mice and non-human primates. This year, we have continued our work with in utero transplantation of human HSCs into the fetuses of an immunodeficient mouse strain. We have observed engraftment of the cells and differentiation into multiple blood lineages, including T cells, B cells, and regulatory T cells. We are working with other HSC types, such as those derived from iPS cells, to determine whether they can engraft in these mice as well. We are also testing different routes of administration of these cells, including into the placenta, which is a site of hematopoiesis. These experiments are designed with the goal of translating these discoveries to treat fetuses with genetic disorders such as thalassemia or sickle cell disease.
Funding Type: 
Strategic Partnership II
Grant Number: 
SP2-06902
Investigator: 
Type: 
PI
ICOC Funds Committed: 
$6 374 150
Disease Focus: 
Blood Disorders
Pediatrics
Stem Cell Use: 
Adult Stem Cell
oldStatus: 
Active
Public Abstract: 

β-thalassemia is a genetic disease caused by diverse mutations of the β-globin gene that lead to profoundly reduced red blood cell (RBC) development. The unmet medical need in transfusion-dependent β-thalassemia is significant, with life expectancy of only ~30-50 years despite standard of care treatment of chronic blood transfusions and iron chelation therapy. Cardiomyopathy due to iron overload is the major cause of mortality, but iron-overload induced multiorgan dysfunction, blood-borne infections, and other disease complications impose a significant physical, psychosocial and economic impact on patients and families. An allogeneic bone marrow transplant (BMT) is curative. However, this therapy is limited due to the scarcity of HLA-matched related donors (<20%) combined with the significant risk of graft-versus-host disease (GvHD) after successful transplantation of allogeneic cells.

During infancy, gamma-globin-containing fetal hemoglobin protects β-thalassemia patients from developing disease symptoms until gamma globin is replaced by adult-type β-globin chains. The proposed therapeutic intervention combines the benefits of re-activating the gamma globin gene with the curative potential of BMT, but without the toxicities associated with acute and chronic immunosuppression and GvHD. We hypothesize that harvesting hematopoietic stem and progenitor cells (HSPCs) from a patient with β-thalassemia, using genome editing to permanently re-activate the gamma globin gene, and returning these edited HSPCs to the patient could provide transfusion independence or greatly reduce the need for chronic blood transfusions, thus decreasing the morbidity and mortality associated with iron overload. The use of a patient’s own cells avoids the need for acute and chronic immunosuppression, as there would be no risk of GvHD. Moreover, due to the self-renewing capacity of HSPCs, we anticipate a lifelong correction of this severe monogenic disease.

Statement of Benefit to California: 

Our proposed treatment for transfusion dependent β-thalassemia will benefit patients in the state by offering them a significant improvement over current standard of care. β-thalassemia is a genetic disease caused by diverse mutations of the β-globin gene that lead to profoundly reduced red blood cell (RBC) development and survival resulting in the need for chronic lifelong blood transfusions, iron chelation therapy, and important pathological sequelae (e.g., endocrinopathies, cardiomyopathies, multiorgan dysfunction, bloodborne infections, and psychosocial/economic impact). Incidence is estimated at 1 in 100,000 in the US, but is more common in the state of California (incidence estimated at 1 in 55,000 births) due to immigration patterns within the State. While there are estimated to be about 1,000-2,000 β-thalassemia patients in the US, one of our proposed clinical trial sites has the largest thalassemia program in the Western United States, with a population approaching 300 patients. Thus, the state of California stands to benefit disproportionately compared to other states from our proposed treatment for transfusion dependent β-thalassemia.

An allogeneic bone marrow transplant (BMT) is curative for β-thalassemia, but limited by the scarcity of HLA-matched related donors (<20%) combined with the significant risk of graft-versus-host disease (GvHD) after successful transplantation of allogeneic cells. Our approach is to genetically engineer the patient’s own stem cells and thus (i) solve the logistical challenge of finding an appropriate donor, as the patient now becomes his/her own donor; and (ii) make use of autologous cells abrogating the risk of GvHD and need for acute and chronic immunosuppression.

Our approach offers a compelling pharmacoeconomic benefit to the State of California and its citizens. A lifetime of chronic blood transfusions and iron chelation therapy leads to a significant cost burden; despite this, the prognosis for a transfusion dependent β-thalassemia patient is still dire, with life expectancy of only ~30-50 years. Our proposed one-time treatment aims to reduce or eliminate the need for costly chronic blood transfusions and iron chelation therapy, while potentially improving the clinical benefit to patients, including the morbidity and mortality associated with transfusion-induced iron overload.

Progress Report: 
  • Summary of progress
  • Our CIRM-funded effort aims to develop a treatment for beta-thalassemia. Beta-thalassemia is an inherited genetic disorder that is caused by mutations (changes in the DNA) in a gene called beta-globin. This gene produces a protein that forms hemoglobin in red blood cells that carry oxygen to through the body. In an individual with beta-thalassemia, beta-globin is not produced (or is made in dramatically reduced quantities), and so the person does not make enough healthy red blood cells. The treatment, which is essential for life in these patients, is repeated blood transfusions (typically once a month or more frequently). The transfusion of blood this frequently results in a dangerous condition called “iron overload,” which must be treated with costly drugs. In general, the quality of life of many people with beta-thalassemia is poor.
  • At present, there is only one cure, and that is to carry out a bone marrow transplant. This involves taking special cells from a healthy person called “hematopoietic stem cells” that give rise to blood cells for the whole of a person's life, and giving them to the patient so that they that they are now able to make healthy red blood cells for their lifetime. However, the cell donor must be an immunologic match to the patient and for many people with beta-thalassemia, such donors are not available.
  • Our approach to treating beta-thalassemia aims to genetically engineer a person’s hematopoietic stem cells (change the DNA inside the cell) to allow them to make healthy red blood cells using a technology that we have developed called "zinc finger nucleases,” or ZFNs. We plan to obtain stem cells from a beta-thalassemia patient, genetically engineer them by transiently exposing them to ZFNs, and then transplant them back into the same individual, making the patient their own donor. The genetic engineering is designed to replicate a situation observed in certain people with beta-thalassemia who have milder symptoms than others. Such patients have a much higher than average level of a “backup” form of beta-globin, called fetal globin, in their blood.
  • All people make fetal globin while in utero and after birth, but in infancy the levels of fetal globin decrease and the child begins to make adult beta-globin. It is at this stage that the symptoms of beta-thalassemia become evident. However, if person with beta-thalassemia has high level of fetal globin, they will be spared the severe effects of the disease.
  • We know that certain individuals who have an elevated level of fetal globin do so because they have a less active form of a gene called BCL11A that normally shuts down the production of fetal globin during infancy. Making use of this observation, our approach is to knock out BCL11A in a patient’s own stem cells, transplant them back into the patient to allow the production of fetal hemoglobin and, as a consequence, increase production of healthy red blood cells.
  • In order to test drugs in humans investigators must consult with the US Federal Drug Administration (FDA) and ultimately submit data about the investigational drug to various regulatory bodies including the FDA as part of Investigational New Drug (IND) application. This past year, we held a meeting with the Center for Biologics Evaluation and Research of the FDA known as a “pre-IND” and received useful guidance on issues that we should address in preparing the IND filing. We also presented our program to the Recombinant DNA Advisory Committee of the NIH (RAC); our proposed preclinical safety assessment program and plan for the phase I clinical trial received unanimous approval from the RAC.
  • Our work this year focused on two major deliverables that are necessary to achieve the goal of beginning a clinical trial of our approach. The first one relates to our ability to purify and efficiently genetically engineer a sufficient quantity of stem cells from a patient with beta-thalassemia. Working with healthy volunteers, and in a setting that is identical to the one we plan to use during our clinical trial, we have been able to consistently obtain sufficient quantities of hematopoietic stem cells to treat an individual with beta-thalassemia, and attain high levels of targeted genetic engineering in those cells.
  • As part of a preclinical safety assessment program, we have initiated and completed a series of studies to determine whether the genetic engineering we perform has any unforeseen untoward consequences in the cell. When we have completed this effort, we aim to file the IND application with the FDA before the end of the year and, pending FDA acceptance, initiate the phase 1 clinical trial in 2015.
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
  • In the final year of this grant, we developed means to introduce patient mutations into human ES cells. These patient mutations derive from 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 induced pluripotent stem (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. Introducing mutations from patients directly into human ES cells bypasses the iPS-generation step and has major advantages over the use of iPS 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.
Funding Type: 
Tools and Technologies II
Grant Number: 
RT2-02060
Investigator: 
Institution: 
Type: 
PI
ICOC Funds Committed: 
$1 869 487
Disease Focus: 
Blood Disorders
Heart Disease
Liver Disease
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
oldStatus: 
Active
Public Abstract: 

Purity is as important for cell-based therapies as it is for treatments based on small-molecule drugs or biologics. Pluripotent stem cells possess two properties: they are capable of self regeneration and they can differentiate to all different tissue types (i.e. muscle, brain, heart, etc.). Despite the promise of pluripotent stem cells as a tool for regenerative medicine, these cells cannot be directly transplanted into patients. In their undifferentiated state they harbor the potential to develop into tumors. Thus, tissue-specific stem cells as they exist in the body or as derived from pluripotent cells are the true targets of stem cell-based therapeutic research, and the cell types most likely to be used clinically. Existing protocols for the generation of these target cells involve large scale differentiation cultures of pluripotent cells that often produce a mixture of different cell types, only a small fraction of which may possess therapeutic potential. Furthermore, there remains the real danger that a small number of these cells remains undifferentiated and retains tumor-forming potential. The ideal pluripotent stem cell-based therapeutic would be a pure population of tissue specific stem cells, devoid of impurities such as undifferentiated or aberrantly-differentiated cells.
We propose to develop antibody-based tools and protocols to purify therapeutic stem cells from heterogeneous cultures. We offer two general strategies to achieve this goal. The first is to develop antibodies and protocols to identify undifferentiated tumor-forming cells and remove them from cultures. The second strategy is to develop antibodies that can identify and isolate heart stem cells, and blood-forming stem cells capable of engraftment from cultures of pluripotent stem cells. The biological underpinning of our approach is that each cell type can be identified by a signature surface marker expression profile.
Antibodies that are specific to cell surface markers can be used to identify and isolate stem cells using flow cytometry. We can detect and isolate rare tissue stem cells by using combinations of antibodies that correspond to the surface marker signature for the given tissue stem cell. We can then functionally characterize the potential of these cells for use in regenerative medicine.
Our proposal aims to speed the clinical application of therapies derived from pluripotent cell products by reducing the risk of transplanting the wrong cell type - whether it is a tumor-causing residual pluripotent cell or a cell that is not native to the site of transplant - into patients. Antibodies, which exhibit exquisitely high sensitivity and specificity to target cellular populations, are the cornerstone of our proposal. The antibodies (and other technologies and reagents) identified and generated as a result of our experiments will greatly increase the safety of pluripotent stem cell-derived cellular therapies.

Statement of Benefit to California: 

Starting with human embryonic stem cells (hESC), which are capable of generating all cell types in the body, we aim to identify and isolate two tissue-specific stem cells – those that can make the heart and the blood – and remove cells that could cause tumors. Heart disease remains the leading cause of mortality and morbidity in the West. In California, 70,000 people die annually from cardiovascular diseases, and the cost exceeded $48 billion in 2006. Despite major advancement in treatments for patients with heart failure, which is mainly due to cellular loss upon myocardial injury, the mortality rate remains high. Similarly, diseases of the blood-forming system, e.g. leukemias, remain a major health problem in our state.
hESC and induced pluripotent stem cells (collectively, pluripotent stem cells, or PSC) could provide an attractive therapeutic option to treat patients with damaged or defective organs. PCS can differentiate into, and may represent a major source of regenerating, cells for these organs. However, the major issues that delay the clinical translation of PSC derivatives include lack of purification technologies for heart- or blood-specific stem cells from PSC cultures and persistence of pluripotent cells that develop into teratomas. We propose to develop reagents that can prospectively identify and isolate heart and blood stem cells, and to test their functional benefit upon engraftment in mice. We will develop reagents to identify and remove residual PSC, which give rise to teratomas. These reagents will enable us to purify patient-specific stem cells, which lack cancer-initiating potential, to replenish defective or damaged tissue.
The reagents generated in these studies can be patented forming an intellectual property portfolio shared by the state and the institutions where the research is carried out. The funds generated from the licensing of these technologies will provide revenue for the state, will help increase hiring of faculty and staff (many of whom will bring in other, out-of-state funds to support their research) and could be used to ameliorate the costs of clinical trials – the final step in translation of basic science research to clinical use. Only California businesses are likely to be able to license these reagents and to develop them into diagnostic and therapeutic entities; such businesses are at the heart of the CIRM strategy to enhance the California economy. Most importantly, this research will set the platform for future stem cell-based therapies. Because tissue stem cells are capable of lifelong self-renewal, stem cell therapies have the potential to be a single, curative treatment. Such therapies will address chronic diseases with no cure that cause considerable disability, leading to substantial medical expense. We expect that California hospitals and health care entities will be first in line for trials and therapies. Thus, California will benefit economically and it will help advance novel medical care.

Progress Report: 
  • Our program is focused on improving methods that can be used to purify stem cells so that they can be used safely and effectively for therapy. A significant limitation in translating laboratory discoveries into clinical practice remains our inability to separate specific stem cells that generate one type of desired tissue from a mixture of ‘pluripotent’ stem cells, which generate various types of tissue. An ideal transplant would then consist of only tissue-specific stem cells that retain a robust regenerative potential. Pluripotent cells, on the other hand, pose the risk, when transplanted, of generating normal tissue in the wrong location, abnormal tissue, or cancer. Thus, we have concentrated our efforts to devise strategies to either make pluripotent cells develop into desired tissue-specific stem cells or to separate these desired cells from a mixture of many types of cells.
  • Our approach to separating tissue-specific stem cells from mixed cultures is based on the theory that every type of cell has a very specific set of molecules on its surface that can act as a signature. Once this signature is known, antibodies (molecules that specifically bind to these surface markers) can be used to tag all the cells of a desired type and remove them from a mixed population. To improve stem cell therapy, our aim is to identify the signature markers on: (1) the stem cells that are pluripotent or especially likely to generate tumors; and (2) the tissue-specific stem cells. By then developing antibodies to the pluripotent or tumor-causing cells, we can exclude them from a group of cells to be transplanted. By developing antibodies to the tissue-specific stem cells, we can remove them from a mixture to select them for transplantation. For the second approach, we are particularly interested in targeting stem cells that develop into heart (cardiac) tissue and cells that develop into mature blood cells. As we develop ways to isolate the desired cells, we test them by transplanting them into animals and observing how they grow.
  • Thus, the first goal of our program is to develop tools to isolate pluripotent stem cells, especially those that can generate tumors in transplant recipients. To this end, we tested an antibody aimed at a pluripotent cell marker (stage-specific embryonic antigen-5 [SSEA-5]) that we previously identified. We transplanted into animals a population of stem cells that either had the SSEA-5-expressing cells removed or did not have them removed. The animals that received the transplants lacking the SSEA-5-expressing cells developed smaller and fewer teratomas (tumors consisting of an abnormal mixture of many tissues). Approaching the problem from another angle, we analyzed teratomas in animals that had received stem cell transplants. We found SSEA-5 on a small group of cells we believe to be responsible for generating the entire tumor.
  • The second goal of the program is to develop methods to selectively culture cardiac stem cells or isolate them from mixed cultures. Thus, in the last year we tested procedures for culturing pluripotent stem cells under conditions that cause them to develop into cardiac stem cells. We also tested a combination of four markers that we hypothesized would tag cardiac stem cells for separation. When these cells were grown under the proper conditions, they began to ‘beat’ and had electrical activity similar to that seen in normal heart cells. When we transplanted the cells with the four markers into mice with normal or damaged hearts, they seemed to develop into mature heart cells. However, these (human) cells did not integrate with the native (mouse) heart cells, perhaps because of the species difference. So we varied the approach and transplanted the human heart stem cells into human heart tissue that had been previously implanted in mice. In this case, we found some evidence that the transplanted cells differentiated into mature heart cells and integrated with the surrounding human cells.
  • The third goal of our project is to culture stem cells that give rise only to blood cells and test them for transplantation. In the past year, we developed a new procedure for treating cultures of pluripotent stem cells so that they differentiate into specific stem cells that generate blood cells and blood vessels. We are now working to refine our understanding and methods so that we end up with a culture of differentiated stem cells that generates only blood cells and not vessels.
  • In summary, we have discovered markers and tested combinations of antibodies for these markers that may select unwanted cells for removal or wanted cells for inclusion in stem cell transplants. We have also begun to develop techniques for taking a group of stem cells that can generate many tissue types, and growing them under conditions that cause them to develop into tissue-specific stem cells that can be used safely for transplantation.
  • Our program is focused on improving methods to purify blood-forming and heart-forming stem cells so that they can be used safely and effectively for therapy. Current methods to identify and isolate blood-forming stem cells from bone marrow and blood are efficient. In addition, we found that if blood-forming stem cells are transplanted, they create in the recipient an immune system that will tolerate (i.e., not reject) organs, tissues, or other types of tissue stem cells (e.g. skin, brain, or heart) from the same donor. Many living or recently deceased donors often cannot contribute these stem cells, so we need, in the future, a single biological source of each of the different types of stem cells (e.g., blood and heart) to change the entire field of regenerative medicine. The ultimate reason to fund embryonic stem cell and other pluripotent stem cell research is to create safe banks of predefined pluripotent cells. Protocols to differentiate these cells to the appropriate blood-forming stem cells could then be used to induce tolerance of other tissue stem cells from the same embryonic stem cell line. However, existing protocols to differentiation stem cells often lead to pluripotent cells (cells that generate multiple types of tissue), which pose a risk of generating normal tissue in the wrong location, abnormal tissue, or cancers called teratomas. To address these problems, we have concentrated our efforts to devise strategies to (a) make pluripotent cells develop into desired tissue-specific stem cells, and (b) to separate these desired cells from all other cells, including teratoma-causing cells. In the first funding period of this grant, we succeeded in raising antibodies that identify and eliminate teratoma-causing cells.
  • In the past year, we identified surface markers of cells that can only give rise to heart tissue. First we studied the genes that were activated in these cells, further confirming that they would likely give rise to heart tissue. Using antibodies against these surface markers, we purified heart stem cells to a higher concentration than has been achieved by other purification methods. We showed that these heart stem cells can be transplanted such that they integrate into the human heart, but not mouse heart, and participate in strong and correctly timed beating.
  • In the embryo, a group of early stem cells in the developing heart give rise to (a) heart cells; (b) cells lining the inner walls of blood vessels; and (c) muscle cells surrounding blood vessels. We identified cell surface markers that could be used to separate each of these subsets from each other and from their common stem cell parents. Finally, we determined that a specific chemical in the body, fibroblast growth factor, increased the growth of a group of pluripotent stem cells that give rise to more specific stem cells that produce either blood cells or the lining of blood vessels. This chemical also prevented blood-forming stem cells from developing into specific blood cells.
  • In the very early embryo, pluripotent cells separate into three distinct categories called ‘germ layers’: the endoderm, mesoderm, and ectoderm. Each of these germ layers later gives rise to certain organs. Our studies of the precursors of mesoderm (the layer that generates the heart, blood vessels, blood, etc.) led us by exclusion to develop techniques to direct ES cell differentiation towards endoderm (the layer that gives rise to liver, pancreas, intestinal lining, etc.). A graduate student before performed most of this work before he joined in our effort to find ways to make functional blood forming stem cells from ES cells. He had identified a group of proteins that we could use to sequentially direct embryonic stem cells to develop almost exclusively into endoderm, then subsets of digestive tract cells, and finally liver stem cells. These liver stem cells derived from embryonic stem cells integrated into mouse livers and showed signs of normal liver tissue function (e.g., secretion of albumin, a major protein in the blood). Using the guidelines of the protocols that generated these liver stem cells, we have now turned our attention back to our goal of generating from mesoderm the predecessors of blood-forming stem cells, and, ultimately, blood-forming stem cells.
  • In summary, we have continued to discover signals that cause pluripotent stem cells (which can generate many types of tissue) to become tissue-specific stem cells that exclusively develop into only heart, blood cells, blood vessel lining cells, cells that line certain sections of the digestive tract, or liver cells. This work has also involved determining the distinguishing molecules on the surface of various cells that allow them to be isolated and nearly purified. These results bring us closer to being able to purify a desired type of stem cell to be transplanted safely to generate only a single type of tissue.
  • The main focus of our program is to improve methods to generate pure populations of tissue-specific stem cells that form only heart tissue or blood. Such tissue-specific stem cells are necessary for developing safe and effective therapies. If injected into patients with heart damage, heart-forming stem cells might be used to regenerate healthy heart tissue. Blood-forming stem cells are capable of regenerating the blood-forming system after cancer therapy and replacing a defective blood forming-system. We showed that blood-forming stem cells from a given donor induce in the recipient permanent transplant tolerance of all organs, tissues, or other tissue stem cells from the same donor. Therefore, having a single biological source of each of the different types of stem cells (e.g., blood and heart) would revolutionize regenerative medicine.
  • Our projects involve generating tissue-specific stem cells from pluripotent stem cells (PSCs), the latter of which are stem cells that can form all tissues of the body. PSCs (which include embryonic stem cells and induced pluripotent stem cells) can turn into all types of more specialized cells in a process known as “differentiation.” Because PSCs can be grown to very large numbers, differentiating PSCs into tissue-specific stem cells could lead to banks of defined tissue stem cells for transplantation into patients—the ultimate reason to conduct PSC research.
  • However, current methods to differentiate PSCs often generate mixtures of various cell types that are unsafe for injection into patients. Therefore, generating a pure population of a desired cell type from PSC is pivotal for regenerative medicine—purity is a key concern for cell therapy as it is with medications.
  • We have invented technologies to purify desired types of cells from complex cell populations, allowing us to precisely isolate a pure population of tissue-specific stem cells from differentiating PSCs for cell therapy. For instance, in our work on heart-forming cells, we developed labels for cells that progressively give rise to heart cells. We used these labeled cells to clarify the natural, stepwise, differentiation process that leads from PSCs to heart-forming stem cells, and finally to different cells within the heart. Exploiting these technologies to isolate desired cell types, we have completed the first step of turning human PSCs into heart-forming stem cells. In the laboratory, when we transplanted these heart-forming stem cells into a human heart, they integrated with the surrounding tissue and participated in correctly timed beating. In the future we hope to deliver heart-forming stem cells into the damaged heart to regenerate healthy tissue.
  • We have also attempted to turn PSCs into blood-forming stem cells by understanding the complex process of blood formation in the early embryo. As mentioned above, if blood-forming stem cells are transplanted into patients, they create in the recipient an immune system that will tolerate (i.e., not reject) other tissues and types of tissue stem cells (e.g., for skin or heart) from the same donor. Thus, turning PSCs into blood-forming stem cells will provide the basis for all regenerative medicine, whereby the blood-forming stem cells and the needed other tissue stem cells can be generated from the same pluripotent cell line and be transplanted together.
  • In parallel studies to those above, we have turned PSCs into liver-forming stem cells. In the embryo, the liver emerges from a cell type known as endoderm, whereas the blood and heart emerge from a different cell type known as mesoderm. We learned that PSCs could only be steered to form endoderm (and subsequently, liver) by diverting them away from the path that leads to mesoderm. Through this approach, we could turn human PSCs into endoderm cells (at >99% purity) and then into liver-forming stem cells that, when injected into the mouse liver, gave rise to human liver cells. This could be of therapeutic importance for human patients with liver damage.
  • Finally, we have developed methods to deplete PSCs from mixtures of cells differentiated from PSCs, because residual PSCs in these mixtures can form tumors (known as teratomas). These methods should increase the safety of PSC-derived tissue stem cell therapy.
  • In summary, we have developed methods to turn PSCs to tissue-specific stem cells that exclusively develop into only heart, blood cells, or liver cells. This work has involved determining the distinguishing molecules on the surface of various cells that allow them to be isolated and nearly purified. These results bring us closer to being able to purify a desired type of stem cell to be transplanted safely to generate only a single type of tissue.
Funding Type: 
Basic Biology V
Grant Number: 
RB5-07379
Investigator: 
Type: 
PI
ICOC Funds Committed: 
$615 639
Disease Focus: 
Blood Disorders
oldStatus: 
Closed
Public Abstract: 

The research performed through this project is very important for the fields of solid organ and bone marrow transplantation because it focuses on a potential new target to increase engraftment of stem cells. Currently, patients that receive stem cell transplants from a non-identical donor must take medications to suppress their immune system; otherwise the stem cells will be rejected.

Stem cell trials have been extended to solid organ transplantation, where it has been shown that kidney transplants can be managed with little or no immunosuppressive medications when stem cells are given to the patient at the time of transplantation. In many cases though the stem cells are rejected and the patient must resume toxic medications.

Our laboratory has been very interested in understanding ways to prevent the rejection of stem cells and has focused on a phylogenetically conserved group of cellular receptors called pattern recognition receptors. This project is focused on understanding how to prevent rejection of stem cells through modifications of these receptors. We hope to identify novel targets to prevent the rejection of stem cells in order to decrease the occurrence of graft versus host disease after bone marrow transplantation and also improve the opportunities for long-term transplant survival without the use of toxic immunosuppressive medications.

Statement of Benefit to California: 

The research we will undertake will benefit the State of California and its residents in two major ways. First it promises to define a novel targets to prevent rejection of stem cells that are transplanted into their new host. This is very important because rejection of hematopoietic stem cells is a major impediment to successful efforts at both bone marrow and solid organ transplantation. Patients needed life-saving solid organ transplants and patients that receive bone marrow transplants from donors that are not perfectly matched to them reject their grafts unless they take powerful medications to suppress their immune system. This project is focused on finding a way to help prevent the rejection of these grafts without the need for immunosuppressive medications.

The second way the project will benefit the State of California is to provide new employment opportunities within the State at a large University that conducts biomedical research. This project will not only directly support the employment of three California citizens devoted to biomedical research, but the work it generates will support California-based biomedical science companies, California University personal and other local companies that employ California citizens that produce the reagents and the supplies used in the proposed studies.

Progress Report: 
  • The research performed through this project is very important for the fields of solid organ and bone marrow transplantation because it focuses on a potential new target to increase engraftment of stem cells. Currently, patients that receive stem cell transplants from a non-identical donor must take medications to suppress their immune system; otherwise the stem cells will be rejected.
  • In our first year of this project we were able to develop a reliable model to delete specific receptors which directly influence stem cell engraftment in genetically different hosts. We have also found that deletion of defined pattern recognition receptors greatly improves stem cell engraftment for up to 20 weeks after injection.
  • The plan for the next reporting period is to continue to focus our studies on characterizing the lodging and fate of the engrafted stem cells in long-lasting chimeric animals and to look into ways to improve the opportunities for long-term transplant survival without the use of toxic immunosuppressive medications.
Funding Type: 
Basic Biology V
Grant Number: 
RB5-07089
Investigator: 
Name: 
Type: 
PI
ICOC Funds Committed: 
$614 400
Disease Focus: 
Blood Disorders
oldStatus: 
Active
Public Abstract: 

Blood stem cells living in the bone marrow of adult humans give rise to all of the cells in our blood, including the red blood cells that carry oxygen to supply our body, and the white blood cells such as T and B lymphocytes that fight infections and keep us healthy. Among the T lymphocytes there is a small population called invariant natural killer T (iNKT) cells. Despite their low frequency in humans (~0.001-1% in blood), iNKT cells have the remarkable capacity to mount immediate and potent responses when stimulated, and have been suggested to play important roles in regulating multiple human diseases including infections, allergies, cancer, and autoimmunity (such as Type I diabetes and multiple sclerosis). However, successful clinical interventions with iNKT cells have been greatly hindered by our limited knowledge on how these cells are produced by blood stem cells, largely due to the lack of tools to track these cells in humans. We therefore propose a novel model system to overcome this research bottleneck by transplanting human blood stem cells into a mouse and genetically programming these cells to develop into iNKT cells. This “humanized” mouse model will allow us to directly track the differentiation of human blood stem cells into iNKT cells in a living animal. From this study, we will address some critical unanswered questions for iNKT cell development, and shed light on developing stem-cell based iNKT cell therapies.

Statement of Benefit to California: 

Allergies, cancer and autoimmunity are leading health hazards in California. These diseases affect millions of Californians, impairing their life quality and creating huge economic burdens for the State of California. This proposal intends to study invariant natural killer (iNKT) T cells, a special population of T lymphocytes that have been suggested to play important roles in regulating these diseases. To date, clinical applications of iNKT cells have been greatly limited by their low frequency in humans and their high variability between individuals (~0.001-1% in blood). Thus, an improved understanding of how these cells are naturally generated is important for their use clinically. Like all other cells in blood, iNKT cells are descendants of the blood stem cells that live in the bone marrow of adult humans. Our goal is to study how human blood stem cells give rise to iNKT cells. If successful, our results can be exploited to develop stem cell-based iNKT cell therapies to treat allergies, cancer and autoimmunity, and therefore may benefit the millions of Californians currently suffering from these diseases. In addition, the knowledge and reagents generated from this proposed study will be shared freely with non-profit and academic organizations in California, and any new intellectual property derived from this study will be developed under the guidance of CIRM to benefit the State of California.

Progress Report: 
  • Despite their small numbers (~0.001-1% in blood), invariant natural killer T (iNKT) cells in humans have been suggested to play important roles regulating multiple diseases including infections, allergies, cancer and autoimmunity. Like all other immune cells, iNKT cells are derived from the blood stem cells living in the bone marrow of adult humans. Successful clinical interventions with iNKT cells have been greatly hindered by our limited knowledge on how these cells are produced by blood stem cells, largely due to the lack of tools and track these cells in humans. Our project proposes to overcome this research bottleneck by transplanting human blood stem cells into a mouse and genetically engineer these cells to develop into human iNKT cells. This “humanized” mouse model will allow us to directly track the differentiation of human blood stem cells into iNKT cells in a living animal. In this reporting period, we have demonstrated the feasibility of this model system, and have successfully generated stem cell-engineered human iNKT cells. In the coming year, we plan to use this established model system to address some critical unanswered questions for iNKT cell development, and explore the therapeutic potential of stem-cell based iNKT cell therapies.

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