Cardiac conduction system cells and uses thereof

Abstract

Isolation and amplification of cardiac pacemaking/conduction system cells and development of a pacemaking/conduction system in vitro using the expression of surrogate expression markers. Use of markers to identify and select for clusters of pacemaking “nodes” that are functionally coupled with adjacent contracting regions and generation of cell populations displaying electrical properties characteristic of specialized pacemaking/conducting cardiac myocytes for modeling the cardiac conduction system, testing of pharmaceuticals and for transplantation.

1 . A method of identifying a population of non-contracting cardiac pacemaking cells comprising: 
 transfecting a stem cell with an expression construct comprising a GATA-6 regulatory element operably linked to a coding sequence for a surrogate marker, wherein the promoter element is selectively active in cardiomyocytes that display an I f  pacemaking current.    
     
     
         2 . The method of  claim 1 , whereby the stem cell is a mammalian stem cell.  
     
     
         3 . The method of  claim 2 , whereby the mammalian stem cell is an embryonic stem cell.  
     
     
         4 . The method of  claim 1 , whereby the surrogate marker is selected from the group consisting of: fluorescent proteins, enzymes, antibiotics, cell surface antigens, and combinations thereof.  
     
     
         5 . The method of  claim 1 , whereby the GATA-6 regulatory element comprises a nucleic acid sequence derived from a chicken GATA-6 promoter/enhancer.  
     
     
         6 . The method of  claim 5 , the GATA-6 regulatory element comprises a nucleotide sequence derived a 1.5 kb proximal enhancer region of chicken GATA6 (cGATA6) promoter/enhancer.  
     
     
         7 . A method of identifying cardiac conducting system cells in a population of pluripotent cells comprising detecting expression of a developmental stage specific protein selected from the group consisting of: GATA6, minK, and combinations thereof, in the pluripotent cells  
     
     
         8 . The method of  claim 7 , whereby the expression of the developmental stage specific protein is detected by expression of a surrogate for the developmental stage specific protein.  
     
     
         9 . The method of  claim 8 , whereby the surrogate for the developmental stage specific protein comprises a marker expressed under transcriptional control of a regulatory element derived from a native promoter for the developmental stage specific protein.  
     
     
         10 . The method of  claim 9 , whereby the marker is selected from a group consisting of: fluorescent proteins, enzymes, antibiotics, cell surface antigens, and combinations thereof.  
     
     
         11 . A method of selecting pacemaking cells having a characteristic of sinoatrial node cells comprising: 
 transfecting a stem cell with an expression construct encoding a selectable marker under the transcriptional control of a regulatory element derived from a GATA-6 promoter;    selecting a population of cells expressing a selectable marker; and    expanding the population of cells in vitro, thereby generating an enriched population cells having a characteristic of sinoatrial node cells.    
     
     
         12 . The method of  claim 11 , wherein the stem cell is a mammalian stem cell.  
     
     
         13 . The method of  claim 12 , wherein the stem cell is an embryonic stem cell.  
     
     
         14 . The method of  claim 11 , wherein the selectable marker is selected from a group consisting of: fluorescent proteins, enzymes, antibiotics, cell surface antigens, and combinations thereof.  
     
     
         15 . The method of  claim 11 , wherein the regulatory element derived from the GATA-6 promoter element comprises a nucleotide sequence derived from a 1.5 kb proximal enhancer region of a chicken GATA6 (cGATA6) promoter/enhancer.  
     
     
         16 . The method of  claim 11 , where the characteristic of the sinoatrial node cell is display of an If current.  
     
     
         17 . A model system for determining a pharmacologic effect of a compound on cells of a mammalian cardiac conducting system comprising a population of embryonic stem cells that have been selected for expression of a developmental stage specific protein selected from a group consisting of: GATA6, minK, and combinations thereof.  
     
     
         18 . The model system of  claim 17 , wherein the embryonic stem cells are murine embryonic stem cells.  
     
     
         19 . The model system of  claim 17 , wherein the embryonic stem cells are human embryonic stem cells.  
     
     
         20 . The model system of  claim 17 , wherein the embryonic stem cells have been selected for co-expression of GATA6 and minK.  
     
     
         21 . The model system of  claim 17 , wherein the embryonic stem cells have been selected for expression of GATA6.  
     
     
         22 . The model system of  claim 21 , wherein the embryonic stem cells selected for expression of GATA6 display an I f  current.  
     
     
         23 . The model system of  claim 17 , wherein the embryonic stem cells have been selected for expression of minK.  
     
     
         24 . A process for isolation and enrichment of cardiac pacemaking cells from stem cell populations comprising transforming a population of stem cells with an expression construct encoding a surrogate marker for GATA6 expression.  
     
     
         25 . The process of  claim 24 , wherein the expression construct encoding a surrogate marker for GATA6 expression comprises a GATA6 regulatory element operably linked to a coding sequence for the surrogate marker.  
     
     
         26 . The process of  claim 25 , wherein the regulatory element is selectively active in cardiomyocytes that display or differentiate into display of an I f  pacemaking current.  
     
     
         27 . The process of  claim 24 , wherein the stem cell population is an embryonic stem cell population.  
     
     
         28 . The process of  claim 24 , wherein stem cell population is co-transformed with an expression construct encoding a surrogate marker for minK expression.  
     
     
         29 . The process of  claim 28 , wherein the expression construct encoding a surrogate marker for minK expression comprises a minK regulatory element operably linked to a coding sequence for a surrogate marker that differs from the surrogate marker for GATA6 expression.  
     
     
         30 . The process of  claim 24 , wherein the surrogate marker is selected from a group consisting of: fluorescent proteins, enzymes, antibiotics, cell surface antigens, and combinations thereof.  
     
     
         31 . The process of  claim 25 , wherein the GATA6 regulatory element is derived from a chicken GATA6 promoter/enhancer.  
     
     
         32 . A population of stem cells that have been differentiated and selected for expression of a PCS protein selected from the group consisting of: GATA6, minK, connexin 40, and combinations thereof.  
     
     
         33 . The population of stem cells of  claim 32 , wherein the stem cells are selected on the basis of expression of a surrogate marker for the PCS protein.  
     
     
         34 . The population of stem cells of  claim 33 , wherein the surrogate marker is expressed following transfection of stem cells with an expression construct that comprises a regulatory element for the PCS protein operably linked to a coding sequence for the surrogate marker.  
     
     
         35 . The population of stem cells of  claim 32 , where the stem cells are derived from a population of embryonic stem cells.  
     
     
         36 . A method of treating cardiac arrhythmias comprising transplantation into a heart of a population of stem cells that have been selected for expression of a PCS protein selected from a group consisting of: GATA6, minK, connexin 40, and combinations thereof.  
     
     
         37 . The method of  claim 36 , wherein the stem cells are selected on the basis of expression of a surrogate marker for the PCS protein.  
     
     
         38 . The method of  claim 37 , wherein the surrogate marker is expressed following transfection of stem cells with an expression construct that comprises a regulatory element for the PCS  
     
     
         39 . The method of  claim 36 , where the selected population of stem cells are transplanted into either a SA or AV node.  
     
     
         40 . The method of  claim 36 , where the stem cells are derived from a population of embryonic stem cells.  
     
     
         41 . A method of inducing phenotypic differentiation in a stably maintained nodal cell population by culturing the cell population in a differentiation medium.  
     
     
         42 . The method of  claim 41  wherein the nodal cell population was originally isolated by surrogate marker selection.  
     
     
         43 . The method of  claim 42  wherein the surrogate marker selection identified transcriptional activity on an enhancer for a PCS protein selected from the group consisting of: GATA6, minK, connexin 40, and combinations thereof.  
     
     
         44 . The method of  claim 43 , wherein the enhancer is a cGATA6 enhancer  
     
     
         45 . The method of  claim 41  wherein the differentiation medium contains one or more supplements selected from the group consisting of: norepinephrine, insulin, and ascorbic acid.