Wnt/GSK3 as a general regulator of protein half-life in human embryonic stem cells

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: 
Human embryonic stem cells (hESCs) have the remarkable capacity of limitless self-renewal. This property is known to be controlled by signaling of a growth factor called Wnt. This proposal investigates the molecular mechanism by which Wnt causes self-renewal. Current conversational wisdom is that Wnt only prolongs the half-life of a protein called β-Catenin. Here we propose the hypothesis that Wnt regulates the stability of a multitude of proteins, all of them characterized by receiving phosphates from an enzyme called Glycogen Synthase Kinase 3 (GSK3). In this view, Wnt would be a general metabolic signal that instructs cells to slow down protein degradation by inhibiting GSK3 activity. How is GSK3 inhibition achieved? This is a key unanswered question in the Wnt signaling field. We propose a new cellular mechanism by which the GSK3 enzyme is sequestered inside intracellular vesicular organelles (called multivesicular bodies) after Wnt signaling. If this GSK3 sequestration hypothesis can be proven, it would constitute an important contribution to stem cell research. Human embryonic stem cells are essential for these investigations because they naturally have very high levels of Wnt signaling. In addition, they have asymmetric mitotic divisions. We will develop methods to investigate why some hESCs differentiate, losing their astonishing self-renewal potential. The experiments proposed will help understand how Wnt signaling maintains the pluripotent state in hESCs. By investigating the hypothesis that Wnt signaling functions as a general regulator of protein stability, we hope to significantly advance the field of human embryonic stem cell research and regenerative medicine.
Statement of Benefit to California: 
The State of California benefits from the proposed basic research by strengthening its leadership role in worldwide stem cell research. This project would provide employment for five full-time researchers, who will obtain training in cutting-edge molecular and cell biology research. It has been our experience that trainees leave our laboratory always for better high-tech jobs. Many go into the biotech industry and others into teaching at the college level. Funding this project will benefit California by producing an improved and highly trained workforce. It will also strengthen our already excellent university research system.
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