Identification and isolation of pure cardiac cells derived from human pluripotent stem cells has proven to be a difficult task. We have designed a method to genetically engineer human embryonic stem cells (hESCs) to harbor a label that is expressed during sequential maturation of cardiac cells. This will allow us to prospectively isolate cardiac cells at different stages of development for further characterization and transplantation. Using this method, we have screened proteins that are expressed on the surface of cells as markers. Using antibodies against these surface markers allows for isolation of these cells using cell sorting techniques. Thus far, we have identified two surface markers that can be used to isolate early cardiac progenitors. Using these markers, we have enriched for cardiac cells from differentiating hESCs and have characterized their properties in the dish as well as in small animals. We plan to transplant these cells in large animal models and monitor their survival, expansion and their integration into the host myocardium. Molecular imaging techniques are used to track these cells upon transplantation.
Reporting Period:
Year 2
Pluripotent stem cells (PSCs) harbor several attractive features for regenerative medicine: they are capable of self-renewal and have the capacity to differentiate to the tissue lineage of all three germ layers. The overall objective of this grant is to develop new technologies that can facilitate generation of cardiovascular progenitors that upon transplantation are capable to integrate into the host tissue and function as part of the normal myocardium with no adverse effect.
Several clinical trials of cell-based therapy have generated enthusiasm about the potential of adult stem cells to treat heart disease. However, no study has yet confirmed the delivery of a pure population of stem cells capable of robust regeneration of the injured myocardium. Furthermore, the electrical properties of the viable engrafted cells remain unknown. While adult stem cells have yet failed to convincingly regenerate myocardial tissue, human embryonic stem cells (hESCs) have proven to be a potential and unlimited source for cardiomyocyte regeneration. Most attempts to isolate transplantable cells from hESCs have aimed to isolate mature cells. Mature cardiomyocytes have passed the stage of self-renewal and may pose problems due to lack of proper integration. Cardiovascular progenitors, on the other hand, could adapt to the microenvironment for optimal integration into the host tissue and reside there for the lifetime of the patient.
We have employed gene editing technology to generate new hESC lines, in which fluorescent reporter proteins are expressed only in cardiac cells. Hence, upon differentiation of hESCs towards cardiac lineage, a distinct fluorescent color is expressed sequentially at each stage of cardiovascular development. We have isolated these cells and have performed detailed analysis to fully characterize their developmental potential. We have performed global gene expression analysis to identify novel biomarkers unique to specific cardiac populations.
An addition, we have transplanted cardiovascular progenitors (at different stages of development) and mature cardiomyocytes in animal models. We have shown engraftment of these cells into the host heart, albeit a very low efficiency. Furthermore, we have shown that transplantation of immature cardiovascular progenitors may generate cardiomyocytes in addition to supporting cells such as vascular endothelial cells and fibroblasts. These results highlight the potential benefit of progenitor cells to generate other cell types that contribute to heart regeneration.
A major challenge to clinical translation of stem cells for heart regeneration is the lack of data on the integration of the transplanted cells into the host heart. . It is possible that the transplanted cells fail to physiologically couple with the host tissue, or they may modify the substrate such that a pro-arrhythmic focus is created. As an analogy, consider the performance of a great symphony orchestra that is interrupted by several members playing in isolation. As such, grafted cells in the heart can also be an ectopic source of activities, promoting arrhythmic events. These are critical issues that need to be addressed in detail in the right animal models before any clinical application can be pursued. We plan to investigate the extent of structural and functional integration of the transplanted cells into the host heart.
Reporting Period:
Year 3
Pluripotent stem cells (PSCs) harbor several attractive features for regenerative medicine: they are capable of self-renewal and have the capacity to differentiate to the tissue lineage of all three germ layers. The overall objective of this grant is to develop new technologies that can facilitate generation of cardiovascular progenitors that upon transplantation are capable to integrate into the host tissue and function as part of the normal myocardium with no adverse effect.
Several clinical trials of cell-based therapy have generated enthusiasm about the potential of adult stem cells to treat heart disease. However, no study has yet confirmed the delivery of a pure population of stem cells capable of robust regeneration of the injured myocardium. Furthermore, the electrical properties of the viable engrafted cells remain unknown. While adult stem cells have yet failed to convincingly regenerate myocardial tissue, human embryonic stem cells (hESCs) have proven to be a potential and unlimited source for cardiomyocyte regeneration. Most attempts to isolate transplantable cells from hESCs have aimed to isolate mature cells. Mature cardiomyocytes have passed the stage of self-renewal and may pose problems due to lack of proper integration. Cardiovascular progenitors, on the other hand, could adapt to the microenvironment for optimal integration into the host tissue and reside there for the lifetime of the patient.
We have employed gene editing technology to generate new hESC lines, in which fluorescent reporter proteins are expressed only in cardiac cells. Hence, upon differentiation of hESCs towards cardiac lineage, a distinct fluorescent color is expressed sequentially at each stage of cardiovascular development. Our data shows that cell isolation by mechanical cell sorting exerts harmful effect on the cells, compromising their viability. Therefore, we have developed a novel method where cardiac cells of interest harbor an antibiotic resistant gene. Therefore, treatment of these cells with an antibiotic results in selection of the desired heart cells while eliminating all unwanted contaminant cells. We have isolated these cells and have performed detailed analysis to fully characterize their developmental potential. We have performed global gene expression analysis to identify novel biomarkers unique to specific cardiac populations.
We have started pre-clinical studies of transplanting cardiovascular progenitors (at different stages of development) and mature cardiomyocytes in animal models. We have shown engraftment of these cells into the host heart, albeit a very low efficiency. Furthermore, we have shown that transplantation of immature cardiovascular progenitors may generate cardiomyocytes in addition to supporting cells such as vascular endothelial cells and fibroblasts. These results highlight the potential benefit of progenitor cells to generate other cell types that contribute to heart regeneration. A major challenge to clinical translation of stem cells for heart regeneration is the lack of data on the integration of the transplanted cells into the host heart. . It is possible that the transplanted cells fail to physiologically couple with the host tissue, or they may modify the substrate such that a pro-arrhythmic focus is created. As an analogy, consider the performance of a great symphony orchestra that is interrupted by several members playing in isolation. As such, grafted cells in the heart can also be an ectopic source of activities, promoting arrhythmic events. These are critical issues that need to be addressed in detail in the right animal models before any clinical application can be pursued. We are in the process of developing imaging modalities to investigate the extent of structural and functional integration of the transplanted cells into the host heart.
Reporting Period:
Year 5
The human heart has a limited regenerative capacity. Therefore, severe cardiac injury leads to fibrosis and subsequent remodeling of the heart and development of heart failure. Human embryonic stem cells (hESCs) can be differentiated to heart cells and can provide a source to restore heart function after an injury. The CIRM New Faculty Award afforded us to look into this issue carefully. We successfully generated specialized heart muscle cells (referred to as cardiomyocytes) from hESCs. We performed full characterization of these cells including their electrical properties and the genes and proteins they express as they develop from a progenitor state to mature cardiomyocytes.
Considering the heart has different types of cardiomyocytes in its different compartments, we generated hESC lines that express a distinct fluorescent reporter protein upon differentiation into different types of cardiomyocytes. This strategy allowed up to isolate the different types of cardiomyocytes and assess their properties both in a dish as well as in small animal models. We have also performed transplantation studies in large animal models, using pigs since their cardiac physiology closely resembles that of human’ hearts. We transplanted approximately 400 million hESC-derived cardiomyocytes in pig’s hearts while maintaining them on immunosuppressive therapy. Eight weeks after transplantation, we noticed the engraftment of the transplanted cells with large patches of muscle that could be potentially improve the heart function.
We believe that we have made significant contribution to this field and our results are very promising. We plan to continue these studies and move onto preclinical studies by preparing a tissue-engineered scaffold that contains millions of left ventricular cardiomyocytes for transplantation into large animals after an experimental myocardial infarction. We are optimistic that our findings will change clinical practice in the near future.
Reporting Period:
NCE (Year 6)
The human heart has a limited regenerative capacity. Therefore, severe cardiac injury leads to fibrosis and subsequent remodeling of the heart and development of heart failure. Human embryonic stem cells (hESCs) can be differentiated to heart cells and can provide a source to restore heart function after an injury. The CIRM New Faculty Award afforded us to look into this issue carefully. We successfully generated specialized heart muscle cells (referred to as cardiomyocytes) from hESCs. We performed full characterization of these cells including their electrical properties and the genes and proteins they express as they develop from a progenitor state to mature cardiomyocytes.
Considering the heart has different types of cardiomyocytes in its different compartments, we generated hESC lines that express a distinct fluorescent reporter protein upon differentiation into different types of cardiomyocytes. This strategy allowed up to isolate the different types of cardiomyocytes and assess their properties both in a dish as well as in small animal models. We have also performed transplantation studies in large animal models, using pigs since their cardiac physiology closely resembles that of human’ hearts. We transplanted approximately 400 million hESC-derived cardiomyocytes in pig’s hearts while maintaining them on immunosuppressive therapy. Eight weeks after transplantation, we noticed the engraftment of the transplanted cells with large patches of muscle that could be potentially improve the heart function.
We believe that we have made significant contribution to this field and our results are very promising. We are optimistic that our findings will change clinical practice in the near future.
Grant Application Details
Application Title:
Preclinical evaluation of human embryonic stem cell-derived cardiovascular progenitors
Public Abstract:
Because the regenerative capacity of adult heart is limited, any substantial cell loss as a result of a heart attack is mostly irreversible and may lead to progressive heart failure. Human pluripotent stem cells can be differentiated to heart cells, but their properties when transplanted into an injured heart remain unresolved. We propose to perform preclinical evaluation for transplantation of pluripotent stem cell-derived cardiac cells into the injured heart of an appropriate animal model. However, an important issue that has limited the progress to clinical use is their fate upon transplantation; that is whether they are capable of integrating into their new environment or they will function in isolation at their own pace. As an analogy, the performance of a symphony can go into chaos if one member plays in isolation from all surrounding cues. Therefore, it is important to determine if the transplanted cells can beat in harmony with the rest of the heart and if these cells will provide functional benefit to the injured heart. We plan to isolate cardiac cells derived from human pluripotent stem cells, transplant them into the model’s injured heart, determine if they result in improvement of the heart function, and perform detailed electrophysiology studies to determine their integration into the host tissue. The success of the proposed project will set the platform for future clinical trails of stem cell therapy for heart disease.
Statement of Benefit to California:
Heart disease remains the leading cause of mortality and morbidity in the US with an estimated annual cost of over $300 billion. In California alone, more than 70,000 people die every year from cardiovascular diseases. 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. Human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC) could provide an attractive therapeutic option to treat patients with damaged heart. We propose to isolate heart cells from hESCs and transplant them in an injured animal model's heart and study their fate. In the process, we will develop reagents that can be highly valuable for future research and clinical studies. The reagents generated in these studies can be patented forming an intellectual property portfolio shared by the state and the institution where the research is carried out. Most importantly, the research that is proposed in this application could lead to future stem cell-based therapies that would restore heart function after a heart attack. 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.
