TGF-beta family signaling by Smads in human embryonic stem cell differentiation behavior

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: 
This research proposal is aimed at gaining a better understanding of the factors that control the differentiation of human embryonic stem cells into one or another cell type. We propose a research program that focuses on the roles of a particular class of soluble, extracellular differentiation factors, i.e. the TGF-beta family, and the signaling molecules that they activate in cells. This class of differentiation factors is known to function as key regulators of many types of cell differentiation, and to play key roles in the generation of many tissues. Remarkably, these soluble factors have the ability to redirect differentiation from one cell type to another, raising the distinct possibility that changes in signaling by these factors may redirect the differentiation potential of embryonic stem cells, allowing them to give rise to cell types with different properties. Yet, very little is known about their effects in human embryonic stem cells, and how changes in signaling by TGF-beta family proteins affect embryonic stem cell differentiation. We are exceptionally well-positioned to study the role of these factors and their signaling molecules, as this lab has been at the basis of much of the current knowledge on what roles these factors play in cell dfferentiation. In the proposed research program, we will tinker with the embryonic stem cells to introduce changes in the signaling networks that are activated by TGF-beta family proteins, and then ask how these changes affect the potential of these cells to become different cell types. Furthermore, we will study the effects of such changes on the potential of cells, derived from embryonic stem cells, to become pluripotent again and to give rise to the full spectrum of cells. Finally, we will address whether changes in the cells that form the niche for embryonic stem cells affect the ability of embryonic stem cells to become many different cell types. Taken together, these studies should provide a basis for targeted manipulations of embryonic stem cells that will then result in directed changes in the cell types that can be generated from embryonic stem cells.
Statement of Benefit to California: 
This research proposal addresses basic research questions and therefore does not directly address the potential use of human embryonic stem cells for therapeutic purposes or toward a particular disease. Nevertheless, we believe that this proposal may have important implications for future uses of embryonic stem cells, in particular for our desire to instruct the cells at will to differentiate into a particular cell type. Through our research on other cell types, e.g. fat cells, muscle cells and bone cells, we have found that we can enhance the potential of precursor cells to differentiate into one or another cell type by modifying the signaling pathways of TGF-beta family proteins. Thus, we have been able to enhance bone cell differentiation and muscle cell differentiation using such approaches. In addition, we are even able to redirect differentiation by modifying the signaling by TGF-beta proteins. For example, using this approach, we have been able to redirect pre-fat cells to become bona fide bone cells or muscle cells, thus providing a possible basis for the use of pre-fat cells, obtained through liposuction, for bone and muscle regenerative therapy. We now propose to study the role of these same TGF-beta family signaling pathways in embryonic stem cells, and to evaluate whether changes in these signaling pathways will change the capacity of the embryonic cells to differentiate into one or another cell type. If successful, this approach may have substantial implications for the derivation of various cell types from embryonic stem cells.
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