Expansion and maturation of human embryonic stem cell (hESC)-derived ventricular myocytes

Funding Type: 
Basic Biology II
Grant Number: 
RB2-01512
ICOC Funds Committed: 
$0
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
iPS Cell
Public Abstract: 
Nearly 5 million people in the US are afflicted with heart failure with an additional 550,000 new cases diagnosed each year. Despite current treatment regimens, heart failure still remains the leading cause of morbidity and mortality in the US and developed world because of the inability to adequately replace lost ventricular myocardium from myocardial infarctions/ "heart attacks." Thus, cell-based therapies to replace lost or damaged ventricular myocardium hold great potential. In addition, potential therapies for heart failure and other cardiac diseases would also benefit from an experimental human ventricular cardiomyocyte model. Progenitor cells which can differentiate into various myocardial cell types and include human embryonic stem cells (hESCs), human induced pluripotent stem cells (hiPSCs), or adult human stem cells, can potentially address both of these therapeutic needs. Although progenitor cells can be differentiated into immature myocardial cell types, the failure to expand sufficiently and fully differentiate them into mature and functional ventricular cardiomyocytes has remained a major bottleneck in realizing the potential of human pluripotent stem cell (hPSC)-derived cardiomyocytes for human cardiac regenerative repair and in vitro modeling of adult human cardiac diseases. To overcome limiting numbers of progenitor cell derived cardiomyocytes, a major thrust of investigation has been directed toward increasing the efficiency with which a pluripotent cell adopts mature cardiomyocyte cell fates. In this proposal, we will perform complementary studies to enhance the yield of mature and functional ventricular cardiomyocytes by defining factors which promote their expansion. Additionally, we will also identify factors which promote maturation of early differentiated ventricular cardiomyocytes. Because of its great therapeutic potential, we will focus on defining microRNAs which can promote either proliferation or maturation. Toward this end, we have developed a novel system that allows us to specifically monitor in real-time hESC-derived cardiomyocytes as they proliferate and mature into functional ventricular cardiomyocytes after treatment with identified microRNAs . Overall, understanding these basic mechanisms which lead to expansion of early differentiated ventricular heart cells and their maturation will ultimately provide novel approaches towards creating a safer and more functional source of ventricular myocardial replacement for injured ventricles in heart failure patients. Additionally, although we are utilizing hESCs as our model system, it is likely that these basic mechanisms that we identify will also be applicable to cardiomyocytes derived from other potential cellular sources such as hiPSCs or other progenitor cell populations.
Statement of Benefit to California: 
Nearly 5 million people in the US are afflicted with heart failure with an additional 550,000 new cases diagnosed each year. Despite current treatment regimens, heart failure still remains the leading cause of morbidity and mortality in California, US and developed world because of the inability to adequately replace lost ventricular heart cells from myocardial infarctions or "heart attacks." Thus, the goal of our experiments is to address this major roadblock by understanding the molecules that are required to instruct human stem cells to expand and become mature and functional ventricular heart cells. These studies have the real potential to not only revolutionize our understanding of creating ventricular heart cells but also provide a safer and more functional source of heart tissue replacement for the many citizens of the State of California who suffer from heart failure.
Progress Report: 
  • Cardiovascular diseases remain the major cause of death in the western world. Stem and progenitor cell-derived cardiomyocytes (SPC-CMs) hold great promise for myocardial repairs. Recent advances in reprogramming somatic cells to induced pluripotent stem cells (iPSCs) open the door for future patient-specific, cell-based therapies. However, most SPC-CMs displayed immature electrophysiological (EP) phenotypes with variable automaticity. Implanting these electrically immature cardiomyocytes (CMs) into hearts might carry arrhythmogenic risks. Human embryonic stem cell (hESC)- or human iPSC-derived cardiomyocytes (hESC-CMs or iPSC-CMs) provide a model system to study the development of CMs, in part because they are an immature population of cardiomyocytes that could continue to mature in the embryoid body (EB) environment. Elucidating cellular factors and molecular pathways governing electrical maturation of early hESC-CMs would enable engineered microenvironment to create electrophysiologically compatible hESC-CMs for a safe cell-based therapy of cardiovascular diseases.
  • Using hESC-CMs and an antibiotic-selection system to isolate hESC-CMs (>95% purity), we found that non-myocardial cells in EBs induced electrical maturation and ion channel expression of primitive hESC-CMs during differentiation. A novel add-back (co-culture) method was also established to enable an engineered microenvironment for controlled EP maturation of primitive hESC-CMs. With these established methods, we further studied the role of endothelial cells (ECs) and their molecular pathways in inducing EP maturation of primitive hESC-CMs. In the Year 1, our data firmly support that ECs influenced the EP maturation of primitive hESC-CMs through their paracrine pathways and various types of receptors. In particular, we found that ECs significant influenced the expression of several specific types of ion channels of early hESC-CMs via paracrine pathways. We also generated new iPSC lines from various fibroblast sources to determine if these iPSCs possess similar cardiogenic capability as H9 hESCs. We will apply information obtained from hESC-CM experiments to induce EP maturation of cardiomyocytes derived from various iPSCs. Our proposed study potentially will provide significant insights in directed ion channel maturation of primitive SPC-CMs and in improving the safety of current cell-based therapies in hearts.
  • Cardiovascular diseases remain the major cause of death in the western world. Stem and progenitor cell-derived cardiomyocytes (SPC-CMs) hold great promise for myocardial repairs. Recent advances in reprogramming somatic cells to induced pluripotent stem cells (iPSCs) open the door for future patient-specific, cell-based therapies. However, most SPC-CMs displayed immature electrophysiological (EP) phenotypes with variable automaticity. Implanting these electrically immature cardiomyocytes (CMs) into hearts might carry arrhythmogenic risks. Human embryonic stem cell (hESC)- or human iPSC-derived cardiomyocytes (hESC-CMs or iPSC-CMs) provide a model system to study the development of CMs, in part because they are an immature population of cardiomyocytes that could continue to mature in the embryoid body (EB) environment. Elucidating cellular factors and molecular pathways governing electrical maturation of early hESC-CMs would enable engineered microenvironment to create electrophysiologically compatible hESC-CMs for a safe cell-based therapy of cardiovascular diseases.
  • Using hESC-CMs and an antibiotic-selection system to isolate hESC-CMs (>95% purity), we found that non-myocardial cells in EBs induced electrical maturation and ion channel expression of primitive hESC-CMs during differentiation. A novel add-back (co-culture) method was established to enable an engineered microenvironment for controlled EP maturation of primitive hESC-CMs. With these established methods, we further studied the role of endothelial cells (ECs) and their molecular pathways in inducing EP maturation of primitive hESC-CMs. In the Year 2, our data confirmed that ECs influenced the EP maturation of primitive hESC-CMs through their paracrine pathways and various types of receptors. In particular, we found that ECs significant influenced the expression of two specific types of ion channels of early hESC-CMs via paracrine pathways. We have generated new iPSC lines from various fibroblast sources and found that fibroblast source influence the cardiogenic potentials of iPSC lines. We will elucidate the potential molecular mechanisms that may influence EP maturation of cardiomyocytes derived from various iPSCs. Our proposed study potentially will provide significant insights in directed ion channel maturation of primitive SPC-CMs and in improving the safety of current cell-based therapies in hearts.
  • Cardiovascular diseases remain the major cause of death in the western world. Stem and progenitor cell-derived cardiomyocytes (SPC-CMs) hold great promise for myocardial repairs. Recent advances in reprogramming somatic cells to induced pluripotent stem cells (iPSCs) open the door for future patient-specific, cell-based therapies. However, most SPC-CMs displayed immature electrophysiological (EP) phenotypes with variable automaticity. Implanting these electrically immature cardiomyocytes (CMs) into hearts might carry arrhythmogenic risks. Human embryonic stem cell (hESC)- or human iPSC-derived cardiomyocytes (hESC-CMs or iPSC-CMs) provide a model system to study the development of CMs, in part because they are an immature population of cardiomyocytes that could continue to mature in the embryoid body (EB) environment. Elucidating cellular factors and molecular pathways governing electrical maturation of early hESC-CMs would enable engineered microenvironment to create electrophysiologically compatible hESC-CMs for a safe cell-based therapy of cardiovascular diseases.
  • Using hESC-CMs and an antibiotic-selection system to isolate hESC-CMs (>95% purity), we found that non-myocardial cells in EBs induced electrical maturation and ion channel expression of primitive hESC-CMs during differentiation. A novel add-back (co-culture) method was established to enable an engineered microenvironment for controlled EP maturation of primitive hESC-CMs. With these established methods, we further studied the role of endothelial cells (ECs) and their molecular pathways in inducing EP maturation of primitive hESC-CMs. In the Year 3, our data confirmed that Endothelin-1 (ET-1), secreted from endothelial cells, influenced the EP maturation of primitive hESC-CMs through mainly a subtype of the ET-1 receptors. In particular, we confirmed with patch-clamp recordings that ET-1 significant influenced the expression of two specific types of ion channels of early hESC-CMs. We also found that neuregulin affects ion channel development of primitive hESC-CMs in a different fashion from ET-1. In addition, we have generated new iPSC lines from various fibroblast sources and found that fibroblast sources influence the cardiogenic potentials of iPSC lines. We have performed microRNA profiling and found that a certain set of miRNAs might underlie the cardiogenic potentials of cardiomyocytes derived from iPSCs generated from various fibroblast sources. Our findings might provide significant insights in directed ion channel maturation of primitive SPC-CMs and in improving the safety of current cell-based therapies in hearts.
  • Cardiovascular diseases remain the major cause of death in the western world. Stem and progenitor cell-derived cardiomyocytes (SPC-CMs) hold great promise for myocardial repairs. Recent advances in reprogramming somatic cells to induced pluripotent stem cells (iPSCs) open the door for future patient-specific, cell-based therapies. However, most SPC-CMs displayed immature electrophysiological (EP) phenotypes with variable automaticity. Implanting these electrically immature cardiomyocytes (CMs) into hearts might carry arrhythmogenic risks. Human embryonic stem cell (hESC)- or human iPSC-derived cardiomyocytes (hESC-CMs or iPSC-CMs) provide a model system to study the development of CMs, in part because they are an immature population of cardiomyocytes that could continue to mature in the embryoid body (EB) environment. Elucidating cellular factors and molecular pathways governing electrical maturation of early hESC-CMs would enable engineered microenvironment to create electrophysiologically compatible hESC-CMs for a safe cell-based therapy of cardiovascular diseases.
  • Using hESC-CMs and an antibiotic-selection system to isolate hESC-CMs (>95% purity), we found that non-myocardial cells in EBs induced electrical maturation and ion channel expression of primitive hESC-CMs during differentiation. A novel add-back (co-culture) method was established to enable an engineered microenvironment for controlled EP maturation of primitive hESC-CMs. With these established methods, we further studied the role of endothelial cells (ECs) and their molecular pathways in inducing EP maturation of primitive hESC-CMs. In the no-cost extension period, our data confirmed that Endothelin-1, secreted from endothelial cells, influenced the EP maturation of primitive hESC-CMs through EC receptors. In particular, we confirmed with patch-clamp recordings that Endothelin-1 significant influenced the expression of two specific types of ion channels of early hESC-CMs. We also found that neuregulin exerts complicated effects on electrical maturation of hESC-CMs. We have generated new iPSC lines from various fibroblast sources and found that fibroblast source influence the cardiogenic potentials of iPSC lines. We have performed microRNA profiling and found that a certain subset of miRNAs may underlie the cardiogenic potentials of cardiomyocytes derived from iPSCs generated from various fibroblast sources. Our findings might provide significant insights in directed ion channel maturation of primitive SPC-CMs and in improving the safety of current cell-based therapies in hearts.

© 2013 California Institute for Regenerative Medicine