Heart Disease

Coding Dimension ID: 
295
Coding Dimension path name: 
Heart Disease

Derivation and analysis of pluripotent stem cell lines with inherited TGF-b mediated disorders from donated IVF embryos and reprogrammed adult skin fibroblasts

Funding Type: 
New Cell Lines
Grant Number: 
RL1-00662
ICOC Funds Committed: 
$1 424 412
Disease Focus: 
Heart Disease
Stem Cell Use: 
Embryonic Stem Cell
iPS Cell
Cell Line Generation: 
Embryonic Stem Cell
iPS Cell
oldStatus: 
Closed
Public Abstract: 
The field of regenerative medicine revolves around the capacity of a subset of cells, called stem cells, to become the mature tissues of the adult human body. By studying stem cells, we hope to develop methods and reagents for treating disease. For instance, we hope to develop methods for making stem cells become cardiovascular cells in the lab which could then be used to rapidly screen large numbers drugs that may be used to treat cardiovascular disease. In another example, if we are able to create bone in the lab from stem cells, we may be able to help treat people with catastrophic skeletal injuries such as wounded soldiers. Until recently, the most flexible type of stem cell known was the embryonic stem cell. Embryonic stem cells are pluripotent, meaning they can give rise to all of the adult tissues. In contrast, stem cells found in the adult are considered only multipotent, in that they can only become a limited number of mature cells. For example, bone marrow stem cells can give rise to all of the components of the blood, but cannot make nerves for a spinal chord. Breakthroughs in the past couple of months have indicated that it is possible to "reprogram" adult skin cells and make them become pluripotent, like stem cells from an embryo. These new kind of cells ares called "induced pluripotent cells" or iPS cells for short. This has lead to great excitement within the scientific community because it raises the possibility that we may use this technology to rapidly create pluripotent stem cells from a large host of human diseases using skin from affected individuals. However, whether the new iPS cells made from skin cells and embryonic stem cells are functionally the same in all applications remains to be seen. Our lab is in the unique position to test this hypothesis. We have derived several normal embryonic stem cell lines and are in the process of deriving iPS cells from normal skin. Furthermore, we are fortunate enough to have begun deriving a new embryonic stem cell line harboring an inherited mutation that results in severe cardiovascular and bone disease that affects more than 7,500 Californians. What's more, one of our collaborators has over the past ten years assembled a cell bank of more that 50 unique adult skin cell lines with the same inherited disease. Therefore, for our proposal, we will make new normal and disease specific iPS and embryonic stem cell lines. We will use these new stem cell lines to test whether the iPS and embryonic stem cells are truly functionally the same, by comparing them after we make them become cardiovascular and bone cells. This work will allow us to advance the field of regenerative medicine on two fronts. 1. We will perform an important comparison of iPS and embryonic stem cell lines. 2. We will compare the disease specific cells with normal cells which will help us better understand cardiovascular and bone disease and pave the way for the development of new therapies.
Statement of Benefit to California: 
Our proposal compares normal and disease specific pluripotent stem cells derived from embryonic and adult skin sources. This proposal will benefit the state of California and its citizens in several specific ways. First, the specific inherited disease we are studying affects approximately one in every 5,000 people worldwide. That translates into over 7,500 Californians and over 60,000 men, women and children of every race and ethnic group in the United States. By examining the characteristics of the disease specific lines, we hope to better understand the mechanisms of the disease and create assays for screening new drugs that can be used to treat people with the disease. Second, this disease is one of a broad class of cardiovascular disease, called thoracic aortic disease. An estimated 3,700 Californians are treated for thoracic aortic disease every year. Our findings may provide insight into the mechanisms underlying these diseases and other cardiovascular diseases. Third, this disease also results in skeletal defects. By studying the mechanisms of the skeletal defects, we will better understand the mechanisms of bone development, which will lead to improved applications of stem cell therapies for individuals with bone injury and disease. Finally, by providing detailed comparisons of iPS and embryonic stem cells, our work will have important ramifications for the future direction of the entire field of stem cell research and regenerative medicine.
Progress Report: 
  • During the past year, we have used the funds from this grant to derive a new embryonic stem cell line with an inherited mutation that results in a severe cardiovascular and bone disease called Marfan syndrome that affects more than 7,500 Californians. In addition, using adult skin cell lines with the same inherited disease, we have made significant progress deriving iPS cells with Marfan syndrome. During the next year we also hope to expand our studies by recruiting patients with a disease very similar to Marfan syndrome called Loeys-Dietz syndrome, to donate skin biopsies so that we can make iPS cells to study that disease as well. Using these new stem cell lines, we are testing whether the iPS and embryonic stem cells are truly functionally the same, by comparing them after we make them become cardiovascular and bone cells.
  • One of the biggest challenges in stem cell biology is figuring out how to make the stem cells become the adult cells we want to study and not some other random adult cells. Over the past year, we have made great strides in turning our stem cells into the cell types most severely affected in people with Marfan syndrome, namely bone and cardiovascular cells. What is most exciting to us is that even with these preliminary studies, it looks like we might be seeing differences between the stem cells with Marfan syndrome and normal stem cells after they are coaxed into become the bone and cardiovascular cells. These results are still very preliminary though, and we need to take great care during the next year to rigorously repeat our experiments before we can be certain of those results. If we can reproduce the differences, these differences may be the basis for screening for new drugs to treat people with Marfan syndrome or lead to a better understanding as to what exactly is the sequence of cellular events that leads to the patient’s symptoms. What’s more, by studying how to efficiently make bone and cardiovascular cells from human embryonic stem cells and iPS cells in the dish, we hope to provide important data that could be beneficial in a wide variety of applications such as tissue engineering or cellular replacement therapies using bone or blood vessels.
  • Marfan Syndrome (MFS) is a genetic disorder that affects more than 7,500 Californians. Patients develop severe complications, affecting several parts of the body (eyes, limbs, aorta). During the last two years, we have used the funds from this grant to develop new cell lines aimed at studying MFS in a dish. These cell lines, are called pluripotent stem cells, and have been generated from: (i) an embryo that was donated for research and was known to have inherited the MFS disease (these cell lines are named human embryonic stem cells (hESCs)); and (ii) from skin biopsies of adult patients (these cell lines are named induced pluripotent stem cells (iPSCs)). These stem cell lines allow us to study MFS by differentiating the cells to adult cells (mainly bone and cardiovascular cells) and not other random adult cells. Using these new stem cell lines, we can test whether hESCs and iPSCs are functionally the same, by comparing them after we make them become cardiovascular and bone cells. We have observed that when the cells form bone or muscle cells, the stem cells with MFS are different and do not behave the same as those made with normal stem cells. We also started to use reagents that can force MFS cells to resemble and behave like normal bone cells. This is called “rescuing the disease phenotype”. For the first time, we are close to describing a stem cell-based technology not only to understand the mechanism(s) of the MFS but also to develop a screen for new drugs to treat people with MFS. However, we still need to confirm our results by repeating the experiments. Our results are very promising for understanding the bone issues in MFS, but continued efforts are also required to understand the cardiovascular issue. It is important to point out that the most important health risk associated with the disease is an aortic aneurysm that, if untreated, leads to death around 35 years old. In conclusion, we are continuing to generate data that will provide the foundation for improving our knowledge of the disease, and also will potentially assist us in developing new therapies for improving MFS patient lives.
  • The field of regenerative medicine revolves around the capacity of a subset of cells, called stem cells, to become the mature tissues of the adult human body. By studying stem cells, we hope to develop methods for treating a wide variety of diseases. For instance, we hope to develop methods for making stem cells become cardiovascular cells in the lab, which could then be used to rapidly screen large numbers of drugs that may be used to treat cardiovascular disease. We are also trying to create skeletal tissue from stem cells so that we may be able to help treat people with catastrophic skeletal injuries such as wounded soldiers.
  • Until recently, the most flexible type of stem cell known was the embryonic stem cell. Embryonic stem cells are pluripotent, meaning they can give rise to all cell types in the body. In contrast, stem cells found in the adult are considered only multipotent, in that they can only become a limited number of mature cells. Breakthroughs in the past five years have indicated that it is possible to "reprogram" adult skin cells and make them become pluripotent, like stem cells from an embryo. These new kinds of cells are called "induced pluripotent cells" or iPS cells. This has lead to great excitement within the scientific community because it raises the possibility that we may use this technology to rapidly create pluripotent stem cells from a large host of human diseases using easy to obtain tissue like skin and fat from affected individuals.
  • Our laboratory is in the unique position to test this hypothesis. We have derived several normal embryonic stem cell lines and iPS cells from normal skin. Furthermore, we have derived a new embryonic stem cell line and induced pluripotent stem cells from fibroblasts harboring an inherited mutation that results in severe cardiovascular and bone disease that affects more than 7,500 Californians, called Marfan's Syndrome.
  • We have created stem cells lines, both embryonic and induced pluripotent stem cells from cells having this disease. We have compared these cells to normal embryonic and induced pluripotent stem cells to examine exactly what makes these diseased cells behave in a way to have impaired bone formation. In addition, we have completed the differentiation, banking and full characterization of vascular cells derived from Marfan's Syndrome embryonic stem cells and Marfan’s syndrome induced pluripotent stem cells. We have seen that the cells with Marfan’s syndrome have a particular signaling pathway that has functional disregulation compared to normal, healthy cells. We have been able to explore how this disease process manipulates this pathway to cause this specific disease. Through this kind of modeling, we can use these cells to screen for treatment as well as model the disease in a way to manipulate the specific pathways this disease impacts to hopefully bring clinical treatments to patients who suffer from this disease.

Prospective isolation of hESC-derived hematopoietic and cardiomyocyte stem cells

Funding Type: 
Comprehensive Grant
Grant Number: 
RC1-00354
ICOC Funds Committed: 
$2 636 900
Disease Focus: 
Blood Disorders
Heart Disease
Immune Disease
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
The capacity of human embryonic stem cells (hESCs) to perpetuate themselves indefinitely in culture and to differentiate to all cell types of the body has lead to numerous studies that aim to isolate therapeutically relevant cells for the benefit of patients, and also to study how genetic diseases develop. However, hESCs can cause tumors called teratomas when placed in the body and therefore, we need to separate potentially beneficial cells from hazardous hESCs. Thus, potential therapeutics cannot advance until the development of methodologies that eliminate undifferentiated cells and enrich tissue stem cells. In our proposal we hope to define the cell surface markers that are differentially expressed by committed hESC-derived stem cells and others that are expressed by teratogenic hESCs. To do this we will carry out a large screen of cell subsets that form during differentiation using a collection of unique reagents called monoclonal antibodies, many already obtained or made by us, to define the cell-surface markers that are expressed by teratogenic cells and others that detect valuable tissue stem cells. This collection, after filing for IP protection, would be available for CIRM investigators in California. We were the first to isolate mouse and human adult blood-forming stem cells, human brain stem cells, and mouse muscle stem cells, all by antibody mediated cell-sorting approaches. Antibody mediated identification of cell subsets that arise during early hESC differentiation will allow separation and characterization of defined subpopulations; we would isolate cells that are committed to the earliest lineage known to form multiple cell types in the body including bone, blood, heart and muscle. These cells would be induced to differentiate further to the blood forming and heart muscle forming lineages. Enriched, and eventually purified hESC-derived blood-forming stem cells and heart muscle stem cells will be tested for their potential capacity to engraft and improve function in animal models. Blood stem cells will be transplanted into immunodeficient mice to test their capacity to give rise to all blood cell types; and heart muscle stem cells will be transferred to mouse hearts that had an artificial coronary artery blockage, a model for heart attack damage. Finally, we will test the capacity of blood stem cell transplantation to induce transplantation tolerance towards heart muscle stem cells from the same donor cell line. Transplantation tolerance in this case means that the heart cells would be accepted as ‘self’ by the mouse that had it’s unrelated donor immune system replaced wholly or in part by blood forming stem cells from the same hESC line that gave rise to the transplantable heart stem cells, and therefore would not be rejected by it’s own immune system. This procedure would allow transplantation of beneficial tissues such as heart, insulin-producing cells, etc., without the use of immunosuppressive drugs.
Statement of Benefit to California: 
The principle objective of this proposal is to develop reagents which, in combinations, can identify and isolate tissue-regenerating stem cells derived from hESC lines. The undifferentiated hESCs are dangerous for transplantation into humans, as they cause tumors. We propose to prepare reagents that identify and can be used to delete or prospectively isolate these tumor-causing undifferentiated hESCs. HESC-derived tissue stem cells have the potential to regenerate damaged tissues and organs, and don’t cause tumors. We propose to develop reagents that can be used to identify and prospectively isolate pure human blood-forming stem cells derived from hESCs, and separately other reagents that can be used to identify and prospectively isolate pure heart-forming stem or progenitor cells. These “decontaminated” hESC-derived tissue stem cells may eventually be used to treat human tissue degenerative diseases. These reagents could also be used to isolate the same cells from somatic cell nuclear transfer (SCNT)-derived pluripotent stem cell lines from patients with genetic diseases. This procedure would enable us to analyze the effects of the genetic abnormalities on blood stem and progenitor cells in patients with genetic blood and immune system disorders, and on heart stem and progenitor cells in patients with heart disorders. The antibodies and stem cells (hESCs, tissue regenerating, etc) that will be isolated from patients with specific diseases will be invaluable tools that can be used to create model(s) for understanding the diseases and their progression. In addition, the antibodies and the stem cells generated in these studies are entities that could be patented or protected by copyright, forming an intellectual property portfolio shared by the state and the state institutions wherein the research was carried out. The funds generated from the licensing of these technologies will help pay back the state, will help support increasing faculty and staff (many of whom bring in other, out of state funds for their research), and could be used to ameliorate the costs of clinical trials. Only California businesses are likely to be able to license these antibodies and cells, to develop them into diagnostic and therapeutic entities; such businesses are the heart of the CIRM strategy to enhance the California economy. Most importantly, however, is that this research will lead to tissue stem cell therapies. Such therapies will address chronic diseases that cause considerable disability and misery, currently have no cure, and therefore lead to huge medical expenses. Because tissue stem cells renew themselves for life, stem cell therapies are one-time therapies with curative intent. We expect that California hospitals and health care entities will be first in line for trials and therapies, and for CIRM to negotiate discounts on such therapies for California taxpayers, thus California will benefit both economically and with advanced novel medical care.
Progress Report: 
  • The objectives of our proposal are the isolations of blood-forming and heart-forming stem cells from human embryonic stem cell (hESCs) cultures, and the generation of monoclonal antibodies (mAbs) that eliminate residual teratogenic cells from transplantable populations of differentiated hESCs. For isolation of progenitors, we hypothesized that precursors derived from hESCs could be identified and isolated using mAbs that label unique combinations of lineage-specific cell surface molecules. We used hundreds of defined mAbs, generated hundreds of novel anti-hESC mAbs, and used these to isolate and characterize dozens of hESC-derived populations. We discovered four precursor types from early stages of differentiating cells, each expressing genes indicative of commitment to either embryonic or extraembryonic tissues. Together, these progenitors are candidates to give rise to meso-endodermal lineages (heart, blood, pancreas, etc), and yolk sac, umbilical cord and placental tissues, respectively. Importantly, we have found that cells of the meso-endodermal population give rise to beating cardiomyocytes. We are currently enriching cardiomyocyte precursors from this population using cardiac-specific genetic markers, and are assaying the putative progenitors using electrophysiological assays and by transplantation into animal hearts (a test for restoration of heart function). In addition, we established in vitro conditions that effectively promote hESC-differentiation towards the hematopoietic (blood) lineages and isolated populations that resemble hematopoietic stem cells (HSCs) in both surface phenotype as well as lineage potentials, as determined by assays in vitro. We have generated hESC-lines that express the anti-apoptotic gene BCL2, and have found that these cells produce significantly greater amounts of hematopoietic and cardiac cells, because of their increased survival during culturing and sorting. We are currently isolating hematopoietic precursors from BCL2-hESCs and will test their ability to engraft in immunodeficient mice, to examine the capacity of hESC-derived HSCs to regenerate the blood system. Finally, we have utilized the novel mAbs that we prepared against undifferentiated hESCs, to deplete residual teratogenic cells from differentiated cultures that were transplanted into animal models. We discovered that following depletion teratoma rarely formed, and we expect to determine a final cocktail of mAbs for removal of teratogenic cells from transplantation products this year.
  • The main objective of our proposal is to isolate therapeutic stem cells and progenitors from human embryonic stem cells (hESCs) that give rise to blood and heart cells. Our approach involves isolation of differentiated precursor subset of cells using monoclonal antibodies (mAbs) and cell sorting instruments, and subsequent characterization of their respective hematopoietic and cardiomyogenic potential in culture as well as following engraftment into mouse models of disease. In addition, we aim to develop mAbs that specifically bind to undifferentiated hESCs for removal of residual teratoma-initiating cells from therapeutic cell preparations, to ensure transplantation safety.
  • We have made substantial advancement towards achieving these goals. First, we discovered that the initial differentiation of hESCs occurs through only 4-5 different progenitor types, of which one is destined to give rise to heart lineages. We purified this population using three novel cell surface markers, and found a significant enrichment of cardiomyocyte clones in colony formation assays that we developed. This subset also expressed particularly high levels of cardiac genes and was receptive to further differentiation into beating cardiomyocytes or vascular endothelial cells. When transplanted into immunodeficient mice these progenitors differentiated into ventricular myocytes and vascular endothelial cells. In the coming year we will perform transplantation experiments to evaluate whether they improve the functional outcome of heart infarction in hearts of mice. Second, we have optimized cell culture conditions and cell surface markers to sort hematopoietic progenitors derived from hESCs. We have also begun to transplant these populations into immunodeficient mouse recipients to identify blood-reconstituting hematopoietic populations. Third, we identified 5 commercial and 1 custom mAbs that are specific to human pluripotent cells (hESCs and induced pluripotent cells). We are currently testing the capacity of combinations of 3 pluripotency surface markers to remove all teratoma-initiating cells from transplanted differentiated cell populations. In summary, we expect provide functional validation of the blood and heart precursor populations that we identified from hESCs by the end term of this grant.
  • The main objective of our proposal is to isolate therapeutic stem and progenitor cells derived from human embryonic stem cells (hESCs) that can give rise to blood and heart cells. Our approach involves developing differentiation protocols to drive hematopoietic (blood) and cardiac (heart) development of hESCs, then to identify and isolate stem/progenitor cells using monoclonal antibodies (mAbs) specific to surface markers expressed on blood and heart stem/progenitor cells, and finally to characterize their functional properties in vitro and in vivo. In addition, we sought to develop mAbs that specifically bind to undifferentiated hESCs for removal of residual teratoma (tumor)-initiating cells from therapeutic preparations, to ensure transplantation safety.
  • We have made substantial progress toward achieving these goals. First, we discovered that the initial differentiation of hESCs occurs through only 4-5 different progenitor types, of which one is destined to give rise to heart lineages. We purified this population using four novel cell surface markers (ROR2, PDGFRα, KDR, and CD13), and found a significant enrichment of cardiomyocyte clones in colony formation assays that we developed. This subset also expressed particularly high levels of cardiac genes and was receptive to further differentiation into beating cardiomyocytes or vascular endothelial cells. When transplanted into immunodeficient mice these progenitors differentiated into ventricular myocytes and vascular endothelial cells. We have also successfully developed a human fetal heart xenograft model to test hESC-derived cardiomyocyte stem/progenitor cells in human heart tissue for engraftment and function.
  • Second, we have optimized cell culture conditions and cell surface markers to sort hematopoietic progenitors derived from hESCs. In doing so, we have mapped the earliest stages of hematopoietic specification and commitment from a bipotent hematoendothelial precursor. Our culture conditions drive robust hematopoietic differentiation in vitro but these hESC-derived hematopoietic progenitors do not achieve hematopoietic engraftment when transplanted in mouse models. Furthermore, we overexpressed the anti-apoptotic protein BCL2 in hESCs, and discovered a significant improvement in viability upon single cell sorting, embryoid body formation, and in cultures lacking serum replacement. Moving forward, we feel the survival advantages exhibited by this BCL2-expressing hESC line will improve our chances of engrafting hESC-derived hematopoietic stem/progenitor cells.
  • Third, we identified a cocktail of 5 commercial and 1 novel mAbs that enable specific identification of human pluripotent cells (hESCs and induced pluripotent cells). We have found combinations of 3 pluripotency surface markers that can remove all teratoma-initiating cells from differentiated hESC and induced pluripotent stem cell (iPSC) populations prior to transplant. While these combinations can vary depending on the differentiation culture, we have generated a simple, easy-to-follow protocol to remove all teratogenic cells from large-scale differentiation cultures.
  • In summary, we accomplished most of the goals stated in our original proposal. We successfully achieved cardiac engraftment of an hESC-derived cardiomyocyte progenitor using a novel human heart model of engraftment. While we unfortunately did not attain hematopoietic engraftment of hESC-derived cells, we are exploring a strategy to address this. Our research has led to four manuscripts: one on the protective effects of BCL2 expression on hESC viability and pluripotency (published in PNAS, 2011), another describing markers of pluripotency and their use in depleting teratogenic potential in differentiated PSCs (accepted for publication in Nature Biotechnology), and two submitted manuscripts, one describing a novel xenograft assay to test PSC-derived cardiomyocytes for functional engraftment and the other describing the earliest fate decisions downstream of a PSC.

Engineering a Cardiovascular Tissue Graft from Human Embryonic Stem Cells

Funding Type: 
Comprehensive Grant
Grant Number: 
RC1-00151
ICOC Funds Committed: 
$2 618 704
Disease Focus: 
Heart Disease
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Cardiovascular disease (CVD) affects more than 71 million Americans and 1.7 million Californians. Recently, engineered cardiovascular tissue grafts, or “patches”, including one made from mouse embryonic stem cells (ESC), have shown promising results as a future therapy for CVD. Our overall goal is to extend these recent results to human ESC as follows. Aim 1: Apply mechanical stretch and electrical pacemaker-like stimulation to hESC-derived heart cells in order to make them stronger and beat at the same time. Current methods to turn hESC into heart cells do not result in the organization required to generate enough strength to support a weak heart and to avoid irregular heart beats. We will use specially engineered devices to apply mechanical stretch and electrical pacemaker-like stimulation to hESC-derived heart cells in order to strengthen them and make them beat in unison. Aim 2: Engineer a cardiovascular patch from hESC-derived heart cells in order to make a potential new therapy for heart disease. Recently, heart cells from mouse ESC, supporting structures called scaffolds, and mechanical stretch have successfully been combined to engineer a cardiovascular patch. We will combine the hESC-derived heart cells from Aim 1, scaffolds, and the same stretch and pacemaker-like stimulation as in Aim 1 to engineer a cardiovascular patch. In addition, we will add a specialized substance called VEGF to our patch so that, potentially, a blood supply will form around it after it is implanted on a diseased heart. We believe a blood supply will be necessary to keep our patch healthy, and in turn, this will allow our patch to help a damaged heart pump better. Aim 3: Assess whether our patch can remain healthy and also strengthen the heart of a rat after it has undergone a heart attack. We will first implant our cardiovascular patch in the rat aorta, the main blood vessel that supplies blood to the body, to test whether the patch remains healthy and whether it can contract and beat on its own. We will first use the aortic position because we feel it will allow us to assess the inherent function of the patch, thus facilitating our efforts to improve its design. After testing in the aortic position, we will implant the patch over damaged heart tissue in a rat that has undergone an experimentally created heart attack. Over a period of several weeks, we will use novel imaging techniques, ultrasonography, echocardiography, and electrocardiography to non-invasively test whether the patch remains healthy and whether the patch helps the damaged heart pump better. We believe the above aims will address questions relevant to hESC-based cardiovascular therapies and will provide vital information needed for safe and effective future clinical translation. As we will evaluate both federally and non-federally approved cell lines, and thus unlikely to receive federal funding, we will need to rely on the support provided by CIRM to carry out our objectives.
Statement of Benefit to California: 
Cardiovascular disease (CVD) affects more than 1.7 million Californians and 71 million Americans. The societal and financial impacts are tremendous, with CVD accounting annually for an estimated $8 billion in CA and nearly $400 billion in US health care costs. In the case of chronic illness such as CVD, the state and national health care systems may not be able to meet the needs of patients or control spiraling costs, unless the focus of therapy switches away from maintenance and toward cures. Fortunately, the passage of Proposition 71 in 2004, and the subsequent creation of the California Institute for Regenerative Medicine (CIRM), has created the funding needed to advance human embryonic stem cell (hESC) research that could lead to curative therapies that would benefit both millions of Californians and Americans. Recently, engineered cardiovascular tissue grafts, made from rat neonatal cardiomyocytes (CM) and cardiomyocytes derived from mouse ESC, have shown promising results as a future therapy for CVD. The overall goal of our proposed research is to extend these recent studies to hESC and engineer a hESC-CM based cardiovascular tissue graft as a regenerative therapy for CVD. We believe the objectives of our research will benefit the people and the state of California by addressing questions relevant to hESC-based cardiovascular regenerative therapies and will provide vital information needed for safe and efficacious future clinical translation. Development of cures for diseases such as CVD could potentially improve the California health care system by reducing the long-term health care cost burden on California. In addition, the results of our research may provide an opportunity for California to benefit from royalties, patents, and licensing fees and benefit the California economy by creating projects, jobs, and therapies that will generate millions of dollars in new tax revenues in our state. Finally, stem cell research such as ours could further advance the biotech industry in California, serving as an engine for California’s economic future. We have assembled a multidisciplinary team of experienced investigators to attack the objectives of our proposed research. At the same time, we will train and mentor a new generation of bright students and junior scientists in the areas of hESC biology, regenerative medicine, and technology development. This ensures that an essential knowledge base will be preserved and passed on to both investigators and patients within and beyond California.
Progress Report: 
  • Specific Aim 1: To electromechanically condition hESC-derived cardiomyocytes.
  • Progress: Over the past year, we have designed and constructed a computer controlled integrated stretch system and electrical pacing system for applying mechanical and electrical stimulation. This system was used in conjunction with a stretchable microelectrode array (sMEA) and shown to successfully support, stretch, and pace primary murine cardiomyocytes (CM). We also have developed a strain array device for cell culture that effectively interfaces the desirable properties of high-throughput microscale fluidic devices with macroscale user-friendly features. One challenge we have encountered with our sMEAs is maintaining electrical continuity of electrodes as cells are stretched. As an alternative to traditional electrical stimulation we have created a system that optically induces electrical activity in hESC-CM. We are now able to optically and non-invasively pace cardiomyocytes differentiated from our modified hESC line.
  • Specific Aim 2: To engineer a hESC-CM based cardiovascular tissue graft.
  • Progress: From our first attempt at engineering a cardiovascular tissue graft as we reported in Year 1, we learned that our grafts would require large populatoins of relatively pure hESC-CMs. As a result, we concentrated our efforts over the past year in developing a more efficient differentiation method for producing larger yields and quantities of hESC-CM. Our method produces hESC-CM in a directed manner under feeder-free and serum-free conditions by controlling multiple cardiomyogenic developmental pathways. Also, in a collaborative effort, we are engineering a novel method for sorting cardiomyocytes. In order to promote improved viability of hESC-CM in our tissue grafts, co-transplantation with hESC-derived endothelial cells (hESC-EC), as opposed to VEGF alone, will likely be needed as shown recently by others. Over the past year, we have shown that we can produce hESC-EC and that their survival in the heart is enhanced by activation of acetylcholine receptors that lead to activation of pro-survival and anti-apoptosis pathways. Finally, in order to control spatial orientation of hESC-CM within our tissue grafts, we have demonstrated on-demand micropatterning of matrix proteins for cell localization and stem cell fate determination. We have illustrated the utility of a cantilever-based nano-contact printing technology for cellular patterning, mESC renewal, and mESC fate specification. We are currently extending our results to undifferentiated hESC and hESC-CM.
  • Specific Aim 3: To assess tissue graft viability and function in a small animal model.
  • Progress: Over the past year, we created hESC-CM based tissue grafts in linear form. In order to quantify the loss of cardiac function between healthy and diseased hearts, we have recently developed a novel in vitro hybrid experimental/computational system to measure active force generation in ventricular slices of rodent hearts. Quantification of the loss of cardiac function will guide us in determining the numbers of hESC-CM needed for producing grafts with varying force generating capacity. Finally, as outlined in our original proposal, we will first implant our tissue grafts in rat aortas as a novel test-bed to assess the graft’s inherent function while minimizing the confounding effects of underlying cardiac contractions. Over the past year we have successfully implanted decellularized aortic patches in rat aortas and are currently working on adding hESC-CM and hESC-EC to the patches to assess their viability and function.
  • In summary, in the second year of our project we have made strong progress on all three of our specific aims. Based on our current results, we anticipate we will continue to make significant progress in engineering a robust and functional cardiovascular tissue graft.
  • Specific Aim 1: To electromechanically condition hESC-derived cardiomyocyte(CM).
  • Progress: Over the past year, we tested, validated, and published an integrated strain and electrical pacing system that we designed and constructed. As mentioned in our previous reports, one challenge we encountered with our electromechanical devices is maintaining electrical continuity of electrodes as cells are stretched. As an alternative to traditional electrical stimulation, with collaborators at Stanford, we have created a system that optically induces electrical activity in hESC-CM by introducing light activated channelrhodopsin-2 (ChR2), a cationic channel, into undifferentiated hESC. In our initial manuscript we have also demonstrated the effects of light stimulation on a whole heart computational model in which we have virtually injected light-responsive hESC-CM in various areas of the simulated heart.
  • Specific Aim 2: To engineer a hESC-CM based cardiovascular tissue graft.
  • Progress: From our first attempt at engineering a cardiovascular tissue graft as we reported in Years 1, 2, and 3 we learned that our grafts would require large populations of relatively pure hESC-CMs. As a result, we’ve continued our efforts in developing a more efficient differentiation method for producing larger yields and quantities of hESC-CM. Our method produces hESC-CM and iPSC-CM in a directed manner under feeder-free, serum-free, and monolayer conditions by controlling TGF-beta/Activin, BMP, Wnt, and FGF pathways. We have used our differentiation protocols to contribute cardiomyocytes to our collaborators, which has resulted in one published manuscript and two submitted publications. Also, with our collaborator at UC Berkeley, we have engineered a novel method for identifying CMs based on their electrical signals and have reported our technology in one accepted manuscript and one under review.
  • Specific Aim 3: To assess tissue graft viability and function in a small animal model.
  • Progress: Over the past two years, we created hESC-CM based tissue grafts in linear and circular forms and our now creating grafts that can be optically controlled (see Aim 1 above). As described in our last progress report, in order to quantify the loss of cardiac function between healthy and diseased hearts, we have reported a novel in vitro hybrid experimental/computational system to measure active force generation in healthy ventricular slices of rodent hearts. Quantification of the loss of cardiac function will guide us in determining the numbers of hESC-CM needed for producing grafts with varying force generating capacity. We have also modeled eccentric and concentric cardiac growth through sarcomerogenesis in order to give us insight into how we might terminally mature our hESC-CM grafts. Finally, we have differentiated hESC into CM for one of our collaborators at Stanford and have performed detailed calcium imaging to show engraftment of hESC-CM with human heart tissue. This has given us great insight into how 3D tissue grafts might integrate with human heart tissue.
  • In summary, in the fourth year of our project we made good progress on all three of our specific aims. Based on our current results, we anticipate we will continue to make significant progress in engineering a robust and functional cardiovascular tissue graft as we originally proposed and we will continue our efforts. Undoubtedly, with the support of the CIRM grant over the past four years, we have made great strides towards creating a 3D tissue graft and believe we will demonstrate functional integration, not only with rodent hearts, but with human tissue, all within the coming year.

Modeling Myocardial Therapy with Human Embryonic Stem Cells

Funding Type: 
Comprehensive Grant
Grant Number: 
RC1-00104
ICOC Funds Committed: 
$2 229 140
Disease Focus: 
Heart Disease
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Five million people in the U.S. suffer with heart failure, at a cost of $30 billion/year. Heart failure occurs when the heart is damaged and becomes unable to meet the demands placed on it. Unlike some tissues, heart muscle does not regenerate. Human embryonic stem cells grow and divide indefinitely while maintaining the potential to develop into many tissues of the body, including heart muscle. They provide an unprecedented opportunity to both study human heart muscle in culture in the laboratory, and advance cell-based therapy for damaged heart muscle. We have developed methods for identifying and isolating specific types of human embryonic stem cells, stimulating them to become human heart muscle cells, and delivering these into the hearts of mice that have had a heart attack. This research will identify those human embryonic stem cells that are best at repairing damaged heart muscle, thereby treating or avoiding heart failure.
Statement of Benefit to California: 
More than 90,000 people in California suffer with heart failure, at a cost of ~$540 million/year. Heart failure occurs when the heart is damaged and becomes unable to meet the demands placed on it. Unlike some tissues, heart muscle does not regenerate. This research will identify human embryonic stem cells that are able to repair damaged heart muscle, thereby treating or avoiding heart failure. The medical treatments developed as a result of these studies will not only benefit the health of Californians with heart failure, but also should result in significant savings in health care costs. This research will push the field of cardiovascular regenerative medicine forward despite the paucity of federal funds, and better prepare us to utilize these funds when they become available in the future.
Progress Report: 
  • Five million people in the U.S. suffer with heart failure, at a cost of $30 billion/year. Heart failure occurs when the heart is damaged and becomes unable to meet the demands placed on it. Unlike some tissues, heart muscle does not regenerate. Human embryonic stem cells grow and divide indefinitely while maintaining the potential to develop into many tissues of the body, including heart muscle. They provide an unprecedented opportunity to both study human heart muscle in culture in the laboratory, and advance cell-based therapy for damaged heart muscle. During the first year of CIRM support, we have developed methods for identifying and isolating specific types of human embryonic stem cells, and stimulating them to become human heart muscle cells. We are currently working to determine the best methods and timing for delivering these cells into the hearts of mice that have had a heart attack. This research will identify those human embryonic stem cells that are best at repairing damaged heart muscle, thereby treating or avoiding heart failure.
  • Five million people in the U.S. suffer with heart failure, at a cost of $30 billion/year. Heart failure occurs when the heart is damaged and becomes unable to meet the demands placed on it. Unlike some tissues, heart muscle does not regenerate. Human embryonic stem cells grow and divide indefinitely while maintaining the potential to develop into many tissues of the body, including heart muscle. They provide an unprecedented opportunity to both study human heart muscle in culture in the laboratory, and advance cell-based therapy for damaged heart muscle. During this year of CIRM support, we have developed methods for identifying and isolating specific types of human embryonic stem cells, and stimulating them to become human heart muscle cells. We are currently working to determine the best methods and timing for delivering these cells into the hearts of mice that have had a heart attack. This research will identify those human embryonic stem cells that are best at repairing damaged heart muscle, thereby treating or avoiding heart failure.
  • Five million people in the U.S. suffer with heart failure, at a cost of $30 billion/year. Heart failure occurs when the heart is damaged and becomes unable to meet the demands placed on it. Unlike some tissues, heart muscle does not regenerate. Human embryonic stem cells grow and divide indefinitely while maintaining the potential to develop into many tissues of the body, including heart muscle. They provide an unprecedented opportunity to both study human heart muscle in culture in the laboratory, and advance cell-based therapy for damaged heart muscle. During this year of CIRM support, we have developed methods for identifying and isolating specific types of human embryonic stem cells, and stimulating them to become human heart muscle cells. We are currently working to determine the best methods and timing for delivering these cells into the hearts of mice that have had a heart attack. This research will identify those human embryonic stem cells that are best at repairing damaged heart muscle, thereby treating or avoiding heart failure.

In Vivo Molecular Magnetic Resonance Imaging of Human Embryonic Stem Cells in Murine Model of Myocardial Infarction

Funding Type: 
SEED Grant
Grant Number: 
RS1-00326
ICOC Funds Committed: 
$658 125
Disease Focus: 
Heart Disease
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Magnetic resonance imaging (MRI) has emerged as one of the predominant modalities to evaluate the effects of stem cells in restoring the injured myocardium. However, MRI does not enable assessment of a fundamental issue in cell therapy, survival of the transplanted cells. The transplanted human embryonic cells (hESC) must at the very least survive to restore the injured myocardium. This research proposal will address this specific challenge to image non-invasively both the survival of the transplanted hESC and the resultant restoration of the myocardium through sensitive detection of the molecular events indicating hESC survival and rapid imaging of myocardial function. In order to achieve this dual capability, there are 2 primary considerations: 1) amplification of molecular signals and 2) high spatial and temporal resolution imaging of the myocardium. No single imaging modality will fulfill all needs of non-invasive molecular imaging in the heart. Only an imaging modality that optimizes the 2 technical specifications will provide physiologically relevant meaning of the molecular signal of the transplanted hESC. The molecular signal will be useful if some correlation between hESC survival and functional restoration can be established. In order to address these critical issues, this proposal will describe efforts to implement molecular MRI to image both the survival of transplanted hESC and restoration of cardiac function using mouse model of myocardial infarction. This research proposes an integrated, multidisciplinary approach to converge innovative approaches in MRI and stem cell biology to address a fundamental yet very critical issue in cardiac restoration: survival of hESC following transplantation into the injured myocardium. This proposal combines novel molecular techniques with the high resolution capabilities of MRI. Upon conclusion of this research, an integrated MRI platform will be developed to allow dual evaluation of the survival of transplanted hESC and their effects on myocardial function. Maturation of this imaging technology will ultimately enable accurate assessment of the survival of hESC and restoration of recipient tissue in all human organs.
Statement of Benefit to California: 
Coronary artery disease (CAD) continues to be the leading cause of death in the United States. Recent advances in cardiovascular therapy have improved immediate survival following an acute myocardial infarction (MI). The persistence of high overall mortality of CAD despite improved treatment is due to a shift in the disease process. Studies have demonstrated a critical role of the infarcted myocardium in the development of congestive heart failure (CHF). The incidence of CHF is now reaching epidemic proportions. Today, there is higher number of deaths from patients developing CHF rather than those sustaining acute MI. CHF is the leading cause of hospital admissions resulting in approximately 300,000 deaths annually. There are nearly 5 million Americans who are suffering from this illness with 550,000 new cases reported each year. Over the last several decades, advances in biomedical technology provided significant improvement in morbidity and mortality. However, the average 5-year survival today still remains around a dismal 50%, creating a major public health concern. Heart transplantation is an established treatment for end-stage CHF. Yet, this definitive therapy is limited to only 2000 donor hearts per year. Thus, a strong mandate exists for an alternative therapeutic option. Human embryonic stem cells (hESC) have demonstrated the ability to differentiate into cardiac cells, representing a potential application of cell therapy to restore the injured myocardium. The public health impact of CHF in California is representative of the emerging trend seen across the United States. As the most populous State in the nation, CHF has resulted in equivalent burden to California’s health care cost, morbidity and mortality. The State of California stands to benefit tremendously with accurate MRI-guided monitoring of the therapeutic efficacy of hESC in an effort to advance the treatment for CHF.
Progress Report: 
  • Magnetic resonance imaging (MRI) has emerged as one of the predominant modalities to evaluate the effects of stem cells in restoring the injured heart. However, MRI does not enable assessment of a fundamental issue in cell therapy, survival of the transplanted cells. The transplanted human embryonic cells (hESCs) must at the very least survive to restore the injured heart. In order to address this issue, this research has conducted the fundamental work to develop a reporter gene as outlined in the proposal and developed a reliable system to evaluate the survival of the transplanted hESCs.
  • First, using a commercially available genetic construct, the reporter gene was designed to generate specific cell surface tags as a signal of cell survival. Molecular assays demonstrated proper characteristics of the reporter gene and the construct has been inserted into human embryonic kidney cells to demonstrate proof of concept. MRI signal was generated from these cells and this result has been validated by flow cytometry confirming the expression of cell surface tags by the reporter gene. Second, the metabolic effects of the contrast agent, iron-oxide, used to magnetically activate the antibodies have been evaluated. The results demonstrated that the iron-oxide has no toxic effects to the cell metabolism. Finally, preliminary MRI of the iron-oxide labeled hESC injected directly into the mouse heart was obtained.
  • Based on above results, the molecular signal was further refined to generate optical signal of cell survival as an additional validation tool. Robust molecular signal of hESC survival was generated following transplantation of the reporter gene transduced hESC into the mouse myocardium. During the no cost extenstion period, correlation between hESC survival and functional restoration of the injured heart will be assessed. Using MRI, cell survival and functional restoration of the heart will be imaged non-invasively in order to obtain longitudinal information regarding survival of transplanted hESC and restoration of heart function.
  • Magnetic resonance imaging (MRI) has emerged as one of the predominant modalities to evaluate the effects of stem cells in restoring the injured heart. However, MRI does not assess a fundamental issue in cell therapy, survival of the transplanted cells. The transplanted human embryonic cells (hESCs) must at the very least survive to restore the injured heart. In order to address this issue, this research has conducted the fundamental work to develop a reporter gene as outlined in the proposal and developed a reliable system to evaluate the survival and teratoma formation of the transplanted hESCs.
  • Using a commercially available genetic construct, the reporter gene was designed to generate specific cell surface tags as a signal of cell survival. Molecular assays demonstrated proper characteristics of the reporter gene and the construct has been inserted into human embryonic stem cells. MRI signal was generated from these cells and this result has been validated by flow cytometry confirming the expression of cell surface tags by the reporter gene in viable human embryonic stem cells. The viable cells expressing this reporter gene were transplanted into mouse heart and MRI signal was generated from the heart of a live mouse.
  • Based on the above results, the molecular signal was further refined to generate optical signal of cell survival as an additional validation tool. Robust molecular signal of hESC survival was generated following transplantation of the reporter gene transduced hESC into the mouse myocardium. During the no cost extenstion period, detection of hESC survival, proliferation, and early teratoma formation was studied. These biological properties of the transplanted hESCs were monitored accurately. This information will be used to correlate hESC survival/proliferation/teratoma formation with functional restoration of the injured heart.

Technology for hESC-Derived Cardiomyocyte Differentiation and Optimization of Graft-Host Integration in Adult Myocardium

Funding Type: 
SEED Grant
Grant Number: 
RS1-00242
ICOC Funds Committed: 
$634 287
Disease Focus: 
Heart Disease
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Stem cells therapies hold great promise in the treatment of cardiac diseases such as coronary heart disease or congestive heart failure. Thanks to their ability to transform into almost any kind of tissue, engrafted stem cells can potentially replace damaged heart tissues with healthy tissues, effectively restoring the heart’s original functions. While initial studies demonstrated the potential benefits of stem cell injection for repairing heart damage, they told researchers little about exactly how improvements were made to the heart and how the improvement might be enhanced. Also, there is concern that the stem cells could negatively impact some aspects of heart function and lead to disturbances of heart rhythm and future attacks. In light of this, we propose to develop a model to study the detailed interaction of stem cells and healthy heart tissue in the laboratory, where events within the cells and between the cells can be measured accurately and many experiments can be done to increase our understanding, without the use of human subjects. Specifically, we plan to focus on two main goals. The first goal is to develop a platform to better understand the gradual transition that stem cell lines make as they mature into heart cells, process known as differentiation. We will record the electrical activity arising from newly formed heart cells to determine when exactly they form and how the behave in response to electrical stimuli or drugs as they mature. This will tell us more about the behavior of the cells that could be injected into the heart so that we know how they will respond when they merge with the heart and when is the best time to introduce them. The second goal, building on the first one, is to observe how the stem cells make contact with the heart cells, including how they grow together mechanically and how they begin to communicate electrically as a repaired tissue. This will be carried out by growing the stem cells and heart cells separately and then allowing them to grow together, just as they would in the heart. Simultaneous recording of electrical activity at numerous locations in the culture will let us map the activity across the culture and evaluate the communication between heart cells (host) and stem cells (graft). Understanding the microscopic nature of integration of stem cells into healthy tissue will lead to a greater knowledge of what can happen when stem cells are injected into the heart and begin to replace the non-functional tissue and connect to healthy tissue. Insights gained with such model should lead to a better understanding of the repair process and highlight strategies for making stem cell-based therapies safer and more effective. This model will also allow testing and development of chemical or electrical manipulations that would increase the yield and reliability of the differentiation process, paving the way for the ultimate scale-up of stem cell therapies for clinical use.
Statement of Benefit to California: 
There is currently no cure for heart damage caused by heart attack, and stem cells offer a very promising solution to this problem that affects millions of Americans. We feel that addressing possible solutions to this pervasive problem is a very constructive and meaningful way to utilize some of the financial resources allocated for stem cell research in California. Within (and outside) the CIRM community, we also have the important goal of making currently unavailable electronic, microfabrication and signal processing technologies available in the form our proposed research platforms. With our planned outreach efforts, we will freely share our methods and equipment, hopefully enhancing the work of many other research groups. By using CIRM funds, we could make such systems available for use with non-registered (as well as registered) cell lines. The outcome of this research stands to impact not only citizens of California, but also the nation and the world. We aim to make considerable progress with research paid for by the citizens of California, demonstrating the degree to which we, as a people, are committed to solving problems in medicine and health care and improving the lives of others. This work will also benefit our State and taxpayers through the training of post-doctoral and graduate students with a clear mindset of leadership, creativity and compassion. Through publication and presentations at local, national and international forums, we hope to disseminate the knowledge gained and encourage further advances.
Progress Report: 
  • The success of cardiac cell grafts for repair of infarcts or congestive heart failure has been moderate to date. While graft cells may survive transplantation, their contribution to conduction and force generation is neither well-defined nor understood. Also, there is concern that the stem cells could negatively impact some aspects of heart function and lead to disturbances of heart rhythm. In light of this, we proposed to develop a model to study the detailed interaction of stem cells and healthy heart tissue in the laboratory, focusing on two main thrusts.
  • The first part of this project had seen the successful development of a platform to better understand the transition that stem cells make as they mature into heart cells, a process known as differentiation. Using arrays of microelectrodes, recording of electrical activity from maturing stem cells was demonstrated. Impact of electrical stimulation on the differentiation process had been probed. Investigation of the interaction between stem cells and heart cells had also been initiated. The second part focused mainly on the latter aspect – functional coupling of stem cells in the heart tissue. New analysis tools for the quantification of the conduction of the electrical activity across a heart tissue were developed. Studies with mixed co-cultures of cardiac cells and fibroblasts revealed a high sensitivity of the conduction properties to the presence of non-conductive cells (fibroblasts), and provide a model for assessing conduction in stem cell grafts of varying homogeneity. Co-cultures of heart cells (host) and stem cells (graft), first grown separately then allowed them to merge, highlighted issues of conduction mismatch at the interface between the host and graft tissue, as well as the dependence of this conduction on the maturity and purity of the grafts used. Most importantly, these studies demonstrated the value of the model developed under this grant for the investigation of electrical coupling and conduction in stem cell grafts, issues that are vital to the safe, effective and successful use of stem cell therapy.

Micro Platform for Controlled Cardiac Myocyte Differentiation

Funding Type: 
SEED Grant
Grant Number: 
RS1-00239-A
ICOC Funds Committed: 
$363 707
Disease Focus: 
Heart Disease
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
Congestive heart failure, the inability of the heart to continue to pump effectively due to damage of its muscle cells, affects approximately 4.8 million Americans and is a leading cause of mortality. Causes of the irreversible damage to the cardiomyocytes that results in congestive heart failure include hypertension, heart attacks, and coronary disease. Because the cadiomyocytes in the adult heart tissue are terminally differentiated and thus cannot regenerate themselves, once they are damaged, they are irreversibly damaged. As a consequence, despite the advances in medical devices and pharmaceuticals, still more than 50% of congestive heart failure patients die within 5 years of initial diagnosis. The goal therefore must be to restore the heart cells’ functions. This is possible by transplanting fetal and neonatal cardiomyocytes which can then integrate into the host tissue. This approach has demonstrated success in improving heart function. However, the limited availability of fetal donors has prevented its adoption as a viable therapeutic approach. Embryonic stem cells can overcome this challenge as they proliferate continuously in vitro and can be furthermore stimulated to differentiate. Embryoid bodies are three-dimensional clusters of heterogenous stem cells, some of which contain cardiac myocytes, which demonstrate characteristic spontaneous contractions. Controlled and efficient differentiation of the stem cells into cardiomyocytes and an effective way to characterize/verify these cells is critical. Ensuring a pure population of cardiac myocytes is essential because otherwise there is a high-likelihood of tumor formation when transplanted. Previous studies have shown that a low percentage of all embryoid bodies spontaneously form cardiomyocytes. Our goal is to therefore develop a self-contained system to grow and controllably differentiate the human embryonic stem cells into cardiomyocytes in high-yields. Few studies have characterized the types of cardiac myocytes in the differentiating human EBs. Our strategy is to use electrical and chemical cues to induce the high-yield differentiation of stem cells into cardiomyocytes and to monitor this process over time both electrically and optically.
Statement of Benefit to California: 
Improvements in differentiating stem cells into homogenous populations of specific cell types are much needed for transplantation therapy in general—and for congestive heart failure patients in particular. The benefits associated with the development of this micro platform have even broader reaching implications beyond biomedical research. After this system is developed, it will serve as a first platform of its kind that can be later commercialized, which would help spur industry growth. To vitalize and enable high-tech/biotech companies to this {REDACTED} area {REDACTED}, engaging industry involvement to this area is necessary. Supporting such activities would furthermore foster the opportunity for student internships with industry and well as afford the students opportunities in entrepreneurship. Our institution is a Hispanic-serving undergraduate institute with almost 50% minority students. Such a proposed system is vital for promoting both the diversity and research culture {REDACTED} and will be leveraged extensively in outreach programs to encourage underrepresented minorities in science education and training. By actively reaching out to specific students who would particularly benefit from our proposed undergraduate internship program, we can attract at-risk students to engage them in research to promote their retention.

Micro Platform for Controlled Cardiac Myocyte Differentiation

Funding Type: 
SEED Grant
Grant Number: 
RS1-00239-B
ICOC Funds Committed: 
$363 707
Disease Focus: 
Heart Disease
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Congestive heart failure, the inability of the heart to continue to pump effectively due to damage of its muscle cells, affects approximately 4.8 million Americans and is a leading cause of mortality. Causes of the irreversible damage to the cardiomyocytes that results in congestive heart failure include hypertension, heart attacks, and coronary disease. Because the cadiomyocytes in the adult heart tissue are terminally differentiated and thus cannot regenerate themselves, once they are damaged, they are irreversibly damaged. As a consequence, despite the advances in medical devices and pharmaceuticals, still more than 50% of congestive heart failure patients die within 5 years of initial diagnosis. The goal therefore must be to restore the heart cells’ functions. This is possible by transplanting fetal and neonatal cardiomyocytes which can then integrate into the host tissue. This approach has demonstrated success in improving heart function. However, the limited availability of fetal donors has prevented its adoption as a viable therapeutic approach. Embryonic stem cells can overcome this challenge as they proliferate continuously in vitro and can be furthermore stimulated to differentiate. Embryoid bodies are three-dimensional clusters of heterogenous stem cells, some of which contain cardiac myocytes, which demonstrate characteristic spontaneous contractions. Controlled and efficient differentiation of the stem cells into cardiomyocytes and an effective way to characterize/verify these cells is critical. Ensuring a pure population of cardiac myocytes is essential because otherwise there is a high-likelihood of tumor formation when transplanted. Previous studies have shown that a low percentage of all embryoid bodies spontaneously form cardiomyocytes. Our goal is to therefore develop a self-contained system to grow and controllably differentiate the human embryonic stem cells into cardiomyocytes in high-yields. Few studies have characterized the types of cardiac myocytes in the differentiating human EBs. Our strategy is to use electrical and chemical cues to induce the high-yield differentiation of stem cells into cardiomyocytes and to monitor this process over time both electrically and optically.
Statement of Benefit to California: 
Improvements in differentiating stem cells into homogenous populations of specific cell types are much needed for transplantation therapy in general—and for congestive heart failure patients in particular. The benefits associated with the development of this micro platform have even broader reaching implications beyond biomedical research. After this system is developed, it will serve as a first platform of its kind that can be later commercialized, which would help spur industry growth. To vitalize and enable high-tech/biotech companies to this {REDACTED} area {REDACTED}, engaging industry involvement to this area is necessary. Supporting such activities would furthermore foster the opportunity for student internships with industry and well as afford the students opportunities in entrepreneurship. Our institution is a Hispanic-serving undergraduate institute with almost 50% minority students. Such a proposed system is vital for promoting both the diversity and research culture {REDACTED} and will be leveraged extensively in outreach programs to encourage underrepresented minorities in science education and training. By actively reaching out to specific students who would particularly benefit from our proposed undergraduate internship program, we can attract at-risk students to engage them in research to promote their retention.
Progress Report: 
  • This year, we have made quite some progress in developing the microtechnology platform. We have developed a new way to form and culture human embryonic stem cells into uniform embryoid bodies in a high throughput fashion. Instead of using the laborious ‘hanging drop method’ or the complicated ‘spinning flask method’, we have developed a way for researchers to easily pipette their cells into standard well plates and increase their throughput by almost 1000x. This is achieved by placing inserts with rounded-bottom microwells into standard well plates. Each one of these inserts that can fit into a standard 24 or 96 well plate can have up to 1000 wells and therefore can create 1000 embryoid bodies, all of uniform size. We can even create wells of various sizes such that we can induce embryoid bodies of predefined sizes and numbers of cells. Many recent publications have demonstrated that the initial size of the embryoid bodies affect differentiation. We have observed this as well. Moreover, this new platform allows researchers to perform real-time microscopy of the cells during this whole process.
  • In addition to developing this new chip, we have also electrically stimulated at different stages during differentiation. The different stages of differentiation include: 1) during embryoid body development 2) when transferred to gelatin coated dishes 3) after about a week on gelatin and 4) isolated beating areas. Electrical stimulation was accomplished using a C-PACE voltage pulsing device at a 1 Hz frequency, 4.5 V (2.5 V/cm), and a 1 ms duration. Unfortunately, none of the electrical stimulation yielded any exhibited increased expression of cardiac markers. Future studies will examine pacing of differentiated cardiac cells for synchronization and will employ more markers using a PCR super microarray.
  • We have also worked on custom software development that allows us to automatically identify and track individual cells within the microplatform.
  • There were a number of factors that caused some unexpected delays in scientific progress this year. Most notably, the PI Michelle Khine and her lab moved to a new university. Therefore, this took quite some time to take down and then re-establish the lab at its new location. Now at UC Irvine, she finally has the ideal infrastructure to make progress quickly on this project. This one year extension to finish this project is therefore much needed and greatly appreciated.
  • To uniformly control the differentiation of embryoid bodies (EBs), we have developed a very simple to use culture platform the create homogenous-sized EBs.
  • We have made quite some progress with the EB array culture plate development, described in detail in the last progress report. Since then, we have developed a way to 1) translate to a more transparent material with lower autofluorescence (cyclic olefin copolymer, COC) to be compatible with optical imaging (Figure 1, c) and then 2) mated the microwells to the bottom of 24 well plates for ease of handling. While we have not had success with applying electric fields to induce cardiomyocyte differentiation, we are now working with !) optimizing the EB size to yield the most cardiomyocytes and then 2)perfusing the EBs with soluble factors.

Specification of Ventricular Myocyte and Pacemaker Lineages Utilizing Human Embryonic Stem Cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00198
ICOC Funds Committed: 
$609 999
Disease Focus: 
Heart Disease
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
Heart failure is a leading cause of mortality in California and the United States. Currently, there are no “cures” for heart failure.Other life threatening forms of heart disease include dysfunction of cardiac pacemaker cells, necessitating implantation of mechanical pacemakers. Although mechanical pacemakers can be efficacious, there are potential associated problems, including infection, limited battery half-life, and lack of responsiveness to normal biological cues. Our research with human embryonic stem cells will be aimed at developing therapies for heart failure, and cardiac pacemaker dysfunction. In each of these disease settings, one might effect a “cure” by replacing worn out or dysfunctional cardiac cells with new ones. In the case of heart failure, the cells that need to be replaced are heart muscle cells, which do the majority of the work in the heart. In the case of pacemaker dysfunction, the cells that need to be replaced are pacemaker cells, a highly specialized type of heart muscle cell. To replace these cells, we need to find cells that can become heart muscle or cardiac pacemaker cells, understand how to generate fairly large numbers of them, and how to persuade them to become either heart muscle or cardiac pacemaker cells. Potential cardiac progenitor cells may come from a number of different sources, either from patients themselves, or from extrinsic sources. Regardless of the source,we need to define factors which will make the cells multiply and will make them become the cell type that we need for repair. The biology of human heart cells is likely to be distinctive from that of heart cells from other animals. For example, a human heart has to function for multiple decades, unlike hearts of other animals who live in general for shorter periods of time. The size, required function, and rhythm of the human heart are also distinct from that of other animals. For these reasons, for repair of human heart, it is important to study human cardiac progenitors and to define pathways required to grow them and to differentiate them utilizing human cells as a model experimental system. Our proposed research will utilize human embryonic stem cells as a source of cardiac progenitors. As human embryonic stem cells can turn into many different kinds of cells, we will create special lines of human embryonic stem cells that will become fluorescent when they adopt the cardiac progenitor, heart muscle, or pacemaker state. These lines will then be treated with a large number of small molecules to find small molecules which amplify cells the number of fluorescent cells in each of these states. The small molecules activate known biochemical pathways, so we can then use the small molecules themselves, or activate identified pathways to achieve the goal of obtaining sufficient numbers of specific cardiac cell types for cardiac therapy.
Statement of Benefit to California: 
More Californians die each year of cardiovascular disease than from the next four leading causes of death combined. Californians continue to die or be disabled as a direct result of cardiovascular disease. Although advances in medical treatment have improved post-infarct survival, heart failure is an increasingly abundant manifestation of cardiovascular disease. A secondary complication of heart failure, and other cardiac diseases, is cardiac pacemaker dysfunction, a potentially fatal condition which is currently ameliorated by mechanical pacemakers. However, mechanical pacemakers have many associated complications,particularly for pediatric patients. For both heart failure and pacemaker dysfunction, replacement of heart muscle cells or biological pacemaker cells offers the hope of improving upon current medical practice. Our research is aimed toward developing new therapies which will allow for the replacement of these critical cell types in diseased heart.
Progress Report: 
  • In the current reporting period, we have worked on the generation of human embryonic stem cell derived marker cell lines for different steps of cardiomyocyte differentiation. The cell lines are designed to express fluorescent proteins under the control of gene promoters that mark cardiac progenitor cells, cardiomyocytes, or cardiac conduction system cells.
  • We tested several HUES cell lines for this purpose and chose cell lines that can differentiate into cardiomyocytes efficiently but are easy to expand and appear stable over several passages in culture. We generated several BAC transgenic cell lines that specifically express green fluorescent protein in cardiomyocytes. Further cell lines are being generated. The cell lines will be used in high-throughput screens to identify molecules and mechanisms that direct the efficient in vitro differentiation into different cardiac cells.

Discovering Potent Molecules with Human ESCs to Treat Heart Disease

Funding Type: 
SEED Grant
Grant Number: 
RS1-00169
ICOC Funds Committed: 
$714 654
Disease Focus: 
Heart Disease
Stem Cell Use: 
Embryonic Stem Cell
oldStatus: 
Closed
Public Abstract: 
This work is directly relevant to human embryonic stem cell (hESC) research because it brings new ideas about novel compounds to affect cardiomyogenesis. The work addresses an urgent need to develop new agents to treat cardiovascular disease. We will develop potent and selective drug-like molecules as cardiomyocyte differentiation agents. Heart disease is the leading cause of mortality and decline in the quality of life in the developed world. The ability of hESCs to form cardiomyocytes has spawned hope that these cells may be used to replace damaged myocardium. Despite their ability to form cardiomyocytes, efficient and controlled cardiomyogenesis in ESC cultures has not been achieved due to the unavailability of differentiation agents and an incomplete understanding of the pathways that regulate cardiac development. Success has been achieved in developing a robust and dependable high-throughput assay to study the effects of small molecules on cardiomyocyte differentiation. Powerful cell-based assays were developed and provided readouts that led to high-content results because multiple signals were probed. The assay is capable of capturing fast or long-acting biology because of the time-course readouts. Cell-based assays are superior to molecular screens because the cell-based assay delivers active compounds or “hits” that are permeable and non-cytotoxic. Moreover, refined “hits” can be used as probes to reveal novel signaling pathways and proteins that control differentiation, in a process termed chemical biology. By taking advantage of knowledge of the current “hits” we will rapidly synthesize novel drug-like compounds in a low-risk approach to. The “hits” will be refined and improved through an efficient synthetic process we use in our lab called “Dynamic Medicinal Chemistry”. Even after miniaturization and automation, screening is still expensive. A key to improve the screening process is to use pharmacologically active, drug-like compounds to provide rich target-relevant information. Intelligently designing libraries for screening by incorporating drug-like features into “lead” library design will improve the attrition rate and lead to more pharmacologically relevant compounds for future studies. This proposal is directly responsive to the California Institute for Regenerative Medicine SEED Grant Program because it provides for developing and testing new agents of use in cardiomyoenesis of hESCs. Importantly, it brings new investigators and a collaborative approach to the stem cell field. The agents discovered and developed may hold great promise as the groundwork for future medications development for a new class of damaged myocardium replacement agents. The theoretical rationale for the work is the use of high-content screening coupled with drug-like new agent discovery approaches. The work will also be of use in the elucidation of key biochemical targets and novel signaling pathways important in hESC cardiomyogenesis.
Statement of Benefit to California: 
In 2002, in the State of California, approximately 697,000 adult Californians died from heart disease. The cost as measured by loss of lifelong earnings was more than $79 billion. Setting aside the pain and suffering, the economic impact of cardiovascular disease to the State of California is staggering. Despite recent advances in cardiovascular medications development, new approaches and novel drug-like compounds are urgently needed to treat cardiovascular disease in California and elsewhere. The poor prognosis for heart disease for Californians underscores the critical need to develop alternative therapeutic strategies. The demonstrated ability of human embryonic stem cells (hESCs) to form cardiomyocytes has spawned widespread hope that these cells may be used as a source to replace damaged myocardium in humans. Despite their ability to form cardiomyocytes, efficient and controlled cardiomyogenesis in hESC culture has not been achieved due to the unavailability of differentiation agents and also because of an incomplete understanding of the pathways that regulate cardiac cell development. Using a high-throughput whole cell assay with image analysis, we have identified four small molecules that promote cardiomyogenesis in human ESCs. This proposal is directly responsive to the California Institute for Regenerative Medicine SEED Grant Program because it provides for developing and testing new agents of use in cardiomyogenesis of hESCs. It also brings new investigators and new collaborative approaches to the field. The promising agents discovered already constitute an excellent starting point and further refinement and development of these compounds may hold great promise as the groundwork for future medications development for a new class of damaged myocardium differentiation agents. The theoretical rationale for the work is the use of high-content screening coupled with drug-like new agent discovery approaches. The work will be of use in the elucidation of key biochemical targets and novel signaling pathways important in hESC cardiomyogenesis. The compounds discovered in our whole hESC-based assays thus far are not potent enough to be developed as drug candidates. But these compounds hold great promise as agents that could be refined further into drug leads. If the leads become drugs, promise of a new class of medication to treat cardiovascular disease may become a reality. Such drugs would decrease cardiovascular disease and decrease health care costs in California. This will likely have a significant economic impact to the State of California. The proposed work represents essential translational research required for new drug development.
Progress Report: 
  • The original goals of the proposal were to apply medicinal chemistry to generate more potent and drug-like analogs of small molecules that stimulate differentiation of cardiomyocytes from embryonic stem cell (ESC) and potentially other progenitor cell types found in adult human heart. During the grant period, we over-achieved each Aim and provided large numbers of drug-like small molecules for cardiomyocyte differentiation studies. In addition, other related information was gained that has considerably expanded our understanding related to developing regenerative medicines.
  • 1. Synthetic Chemistry: From an initial screen of thousands of compounds, six 'hits' were identified. Almost 1300 compounds were synthesized as analogs of these “hits” with the goal of generating more effective novel compounds as possible therapeutics for heart disease.
  • 2. Assay development and screening: Novel synthetic chemical analogs were studied in cell-based assays to evaluate potency of stimulating cardiac cell development relative to the starting 'hit' compounds. The biological data contributed to structure activity relationship (SAR) studies, and provided valuable information about parts of the molecules important for cardiomyocyte stem cell differentiation and for other important pharmaceutical properties. The iterative feedback from the biological testing helped to guide the next generation designs of new and ever more effective compounds.
  • 3. Chemical optimization. Focused structure activity relationship (SAR) studies for 4 chemical series from the ESC cardiogenesis differentiation screen were done. SAR for 2 additional chemical classes was done but those agents proved less potent. In addition to SAR, considerable information was obtained leading to improved solubility and membrane permeability of compounds in development, which became a focus of the chemical optimizations.
  • In summary, the work has already led to one or more promising drug-like compounds ready for efficacy testing in animal models and thus, efforts have greatly accelerated the timeline of getting compounds to human patients.
  • The original goals of the proposal were to apply medicinal chemistry to generate more potent and drug-like analogs of small molecules that stimulate differentiation of cardiomyocytes from embryonic stem cells (ESCs) and potentially other progenitor cell types found in adult human heart. During the grant period, we over-achieved each Aim and provided large numbers of drug-like small molecules for cardiomyocyte differentiation studies. In addition, other related information was gained that has considerably expanded our understanding related to developing regenerative medicines.
  • 1. Synthetic Chemistry: From an initial screen of thousands of compounds, six ‘hits’ were identified. Almost 1400 compounds were synthesized as analogs of these “hits” with the goal of generating more effective novel compounds as possible therapeutics for heart disease.
  • 2. Assay development and screening: Novel synthetic chemical analogs were studied in cell-based assays to evaluate potency of stimulating cardiac cell development relative to the starting ‘hit’ compounds. The biological data contributed to structure activity relationship (SAR) studies, and provided valuable information about parts of the molecules important for cardiomyocyte stem cell differentiation and for other important pharmaceutical properties. The iterative feedback from the biological testing helped to guide the next generation design of new and ever more effective compounds.
  • 3. Chemical optimization. Focused structure activity relationship (SAR) studies for 4 chemical series from the ESC cardiogenesis differentiation screen were done. SAR for 2 additional chemical classes was done but those agents proved less potent. In addition to SAR, considerable information was obtained leading to improved solubility and membrane permeability of compounds in development, which became a focus of the chemical optimizations. The most potent compounds increased stem cell differentiation to cardiomyocytes 5-10 fold. The compounds were non-toxic, reasonably tractable to make, stable and were water-soluble and hence relatively easy to handle.
  • 4. A number of biological signaling pathways were identified as affiliated with cardiomyocyte differentiation. One such pathway also is involved in anti-cancer activities. Thus, our efforts in identifying cardiomyocyte differentiation agents led us to study novel biology associated with cancer. One “hit” of this signaling pathway was chosen to do synthetic chemistry and “hit” to lead refinement. Approximately 100 compounds were synthesized and tested for inhibition of this signaling pathway.
  • In summary, the work has already led to a number of promising drug-like compounds ready for efficacy testing in animal models and thus, efforts have greatly accelerated the timeline of getting compounds to human patients. A total of 1500 compounds were synthesized to optimize the potency and properties of cardiomyocyte differentiation agents. The most potent stimulated production of human cardiomyocytes 5-10-fold compared to vehicle-stimulated cells.

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