Induction of arterial-type of hemogenic endothelium (ahe) and enhancement of t cell production from pscs through overexpression of ets factors or modulating mapk/erk signalling pathways

Abstract

The present invention is a method of creating a population of hemogenic endothelial cells with arterial specification. In one embodiment, the method uses ETS transgene induction at the mesodermal stage of differentiation. In another embodiment, the method activates ERK signaling at the mesodermal stage of differentiation.

We claim: 
     
         1 . A method of enhancing arterial specification of hemogenic endothelium, the method comprising:
 (a) introducing an ETS transcription factor transgene into a mesoderm cell population; and   (b) culturing the mesoderm cells under conditions sufficient to express the ETS transcription factor transgene within the mesoderm population and differentiate the mesoderm cells to arterial hemogenic endothelium (AHE) cells.   
     
     
         2 . The method of  claim 1 , wherein step (a) comprises introducing a vector comprising the ETS transcription factor transgene into the mesoderm cell population. 
     
     
         3 . The method of  claim 2 , wherein the vector comprises an inducible promoter operably linked to the ETS transcription factor transgene. 
     
     
         4 . The method of  claim 1 , wherein the mesoderm cell population is differentiated from human pluripotent stem cells (hPSCs). 
     
     
         5 . The method of  claim 1 , wherein the mesoderm cells population expresses the ETS transcription factor for at least 2 days to differentiate to AHE cells. 
     
     
         6 . A method of enhancing arterial specification of hemogenic endothelium in differentiating hPSC, comprising the steps of
 (a) introducing an ETS transcription factor transgene into a hPSC population,   (b) culturing the hPSC cells under conditions to differentiate the hPSC into mesoderm cells at two days of differentiation, and   (b) inducing expression of the transgene at day two of differentiation, such that arterial hemogenic endothelium cells (AHE) are obtained by day four of differentiation.   
     
     
         7 . The method of  claim 6 , wherein the expression is under inducible control. 
     
     
         8 . The method of  claim 6  wherein the ETS transgene is ETS1. 
     
     
         9 . The method of  claim 6  wherein the ETS transgene is selected from the group of ETV2, ETS2 and ERG. 
     
     
         10 . The method of  claim 6 , wherein the cells are further differentiated into lympho-myeloid and erythroid cell lines. 
     
     
         11 . The method of  claim 6 , wherein a population of hemogenic endothelium cells that are CD144+CD43−CD73−DLL4+CXCR4 +/−  HE and express high level of one or more arterial markers selected from the group consisting of EFNB2, NOTCH1, NOTCH 4 and SOX17 is obtained. 
     
     
         12 . A cell population produced by the method of  claim 1 . 
     
     
         13 . A method of creating a cell population, comprising the steps of
 (a) obtaining a cell population of AHE cells,   (b) further differentiating the AHE cells into an at least 90% pure population of cells, wherein the cell type of the cell population is selected from the group consisting of T-cells, B-cells, definitive (adult-type) red blood cells, myeloid progenitors and mature myelomonocytic cells.   
     
     
         14 . A method of enhancing arterial specification of hemogenic endothelium in differentiating hPSC, comprising the steps of
 (a) culturing human mesoderm cells in defined cell culture medium comprising an effective amount of a factor capable of activating ERK signaling to differentiate the mesoderm cells into arterial hemogenic endothelium cells (AHE); and   (b) obtaining the arterial hemogenic endothelium cells.   
     
     
         15 . The method of  claim 14 , wherein the factor activates ERK signaling through the VEGF receptor pathway. 
     
     
         16 . The method of  claim 14 , wherein the factor capable of activating ERK signaling is a PI3K inhibitor. 
     
     
         17 . The method of  claim 11 , wherein the PI3K inhibitor is LY294002. 
     
     
         18 . The method of  claim 14 , wherein step (a) is performed for about 3 days. 
     
     
         19 . The method of  claim 14 , wherein the human mesoderm cells are obtained from a method comprising culturing human pluripotent stem cells in a chemically defined culture medium for about 2 to about 3 days, whereby a cell population comprising human KDR +  mesoderm cells is obtained. 
     
     
         20 . The method of  claim 14 , wherein the AHE cells are CD144+CD43-CD73−DLL4 + CXCR4 +/− . 
     
     
         21 . The method of  claim 20 , wherein the AHE cells obtained further express one or more arterial markers selected from the group consisting of EFNB2, NOTCH1, NOTCH 4 and SOX17. 
     
     
         22 . The method of  claim 14 , wherein the hPSCs are embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). 
     
     
         23 . A cell population produced by the method of  claim 14 . 
     
     
         24 . A method of creating a cell population, comprising the steps of
 (a) obtaining a cell population of AHE DLL4 +  cells,   (b) further differentiating the AHE DLL4 +  cells into an at least 90% pure population of cells, wherein the cell type of the cell population is selected from the group consisting of T-cells, B-cells, definitive (adult-type) red blood cells, myeloid progenitors and mature myelomonocytic cells.   
     
     
         25 . The method of  claim 24 , wherein the cell population is T cells and wherein step (b) comprises co-culturing the AHE DLL4 +  cells with stromal cells expressing NOTCH ligand DLL4 or DLL1 in T cell differentiating medium for an effective amount of time to differentiate the AHE cells into T-cells. 
     
     
         26 . The method of  claim 24 , wherein the derived T-cells are engineered to express an exogenous chimeric antigen receptor (CAR). 
     
     
         27 . The method of  claim 26 , wherein the CAR comprises a CD19 chimeric antigen receptor. 
     
     
         28 . A method of killing tumor cells, the method comprising contacting the tumor cells with T cells derived from the method of  claim 26  in an effective amount to kill the tumor cells. 
     
     
         29 . The method of  claim 28 , wherein the tumor cells are selected from the group consisting of lymphoma, leukemia, and myeloma cells. 
     
     
         30 . A method of enhancing arterial specification of hemogenic endothelium in mesoderm cells, the method comprising culturing the mesoderm cells in defined medium comprising an effective amount of a factor capable of activating NOTCH signaling to differentiate the mesoderm cells into arterial hemogenic endothelium (AHE) cells. 
     
     
         31 . The method of  claim 30 , wherein the factor capable of activating NOTCH signaling is selected from the group consisting of DLL4, DLL1-Fc, DLL1-expressing feeder cells, plates coated with DLL4-Fc, and plates coated with DLL1-Fc.