CharacteriSectionzation of human ES or iPS-derived striatal neurons for Huntington Disease

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: 
The goal of our work is to understand the mechanisms and pathways that turn undifferentiated human stem or iPS cells into differentiated neurons that can be used in the brain as replacement for neurons lost in neurodegeneration. We will focus on Huntington's disease or chorea (HD), a incurable neurodegenerative disorder that manifests itself first in the middle age. HD is caused by a mutation in the gene, huntingtin, in which an expansion in the CAG coding for glutamine is expanded to above 36 repeats. Huntington’s disease is a dominantly inherited neurodegenerative disorder with massive loss of cells in the striatum- the cell type lost is medium spiny neurons. We will evaluate methods to induce cellular differentiation of ES or iPS into medium spiny neurons. We will also take iPS cells generated from HD skin fibroblasts and develop methods to correct the mutation in the gene huntingtin.
Statement of Benefit to California: 
Huntington’s disease is a dominantly inherited neurodegenerative disorder with massive loss of cells in the striatum and replacement of these cells offers a possible therapy. We will use human embryonic stem cells and induced pluripotent stem (iPS) cell technologies with established methods to differentiate stem cells into pure medium spiny neurons and we will develop methods to genetically correct Huntington’s disease iPS cells for use therapeutically in patients. The studies we perform should benefit the state of California in several ways: 1. We hope to increase the ability to generate medium spiny neurons that can be used in cell-replacement therapies for neurodegenerative diseases such as Huntington’s disease in which loss of these cells correlates with disease. This will directly benefit patients in California and elsewhere. 2. The human embryonic stem cell research we perform may bring new biotechnology jobs to California, thus increasing the state's visibility as a leader in stem cell technology. 3. New biochemical methods for genetically correcting disease iPS cells will have implications for all genetic diseases. Our discoveries could bring revenues to California due to the ability of the state to obtain licensing fees on technology generated using CIRM funds.
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