US2015140657A1PendingUtilityA1

Method of generating natural killer cells and dendritic cells from human embryonic stem cell-derived hemangioblasts

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Assignee: STEM CELL & REGENERATIVE MEDICINE INTERNATIONAL INCPriority: Dec 4, 2009Filed: Aug 21, 2014Published: May 21, 2015
Est. expiryDec 4, 2029(~3.3 yrs left)· nominal 20-yr term from priority
Inventors:Erin Anne KimbrelShi-Jiang Lu
A61P 35/00A61P 37/04C12N 2501/26C12N 2501/125C12N 2506/02C12N 2501/2304A61K 2035/124C12N 2501/23C12N 2501/22C12N 2501/2303C12N 2501/115C12N 2501/145A61K 35/15C12N 2501/165C12N 2501/155C12N 2501/2315C12N 2501/2307C12N 2501/2302A61K 40/42A61K 40/15A61K 2239/48C12N 5/0639C12N 5/0646A61K 35/17
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Claims

Abstract

This invention provides methods of generating natural killer (NK) cells and dendritic cells (DCs). The methods utilize human hemangioblasts as intermediate cells to generate the NK cells and DCs. In various embodiments, the methods do not require the use of stromal feeder layers.

Claims

exact text as granted — not AI-modified
What is claimed is: 
     
         1 . A method of generating natural killer (NK) cells comprising:
 providing hemangioblasts;   culturing the hemangioblasts on methylcellulose and a first cytokine mixture comprising IL2, IL3, IL6, IL7, IL15, SCF and FL;   harvesting the cultured cells; and   culturing the harvested cells in liquid media comprising human serum, and a second cytokine mixture comprising IL7, IL15, SCF and FL to generate NK cells.   
     
     
         2 . The method of  claim 1 , wherein the methylcellulose is H4236 methylcellulose. 
     
     
         3 . The method of  claim 1 , wherein the methylcellulose is H4536 methylcellulose. 
     
     
         4 . The method of  claim 1 , wherein the concentration of IL2 is about 5-10 ng/ml, IL3 is about 1-10 ng/ml, IL6 is about 1-10 ng/ml, IL7 is about 5-20 ng/ml, IL15 is about 5-10 ng/ml, SCF is about 10-50 ng/ml, and FL is about 10-50 ng/ml. 
     
     
         5 . The method of  claim 1 , wherein culturing the hemangioblasts is for about 6 to 8 days. 
     
     
         6 . The method of  claim 1 , wherein culturing the harvested cells is for about 14 to 21 days. 
     
     
         7 . The method of  claim 1 , further comprising weekly media changes to refresh the second cytokine mixture. 
     
     
         8 . The method of  claim 1 , wherein the hemangioblasts are differentiated from human embryonic stem cells (hESCs). 
     
     
         9 . The method of  claim 1 , wherein the hemangioblasts are differentiated from induced pluripotent (iPS) cells. 
     
     
         10 . The method of  claim 1 , wherein the NK cells are immature NK cells and are CD56+ and CD16−. 
     
     
         11 . The method of  claim 1 , wherein the NK cells are mature NK cells and are CD56− and CD16+, or CD56lo and CD16+. 
     
     
         12 . A natural killer (NK) cell generated by any of the methods of  claim 1 - 11 . 
     
     
         13 . A pharmaceutical acceptable composition comprising a quantity of the NK cell of  claim 12 . 
     
     
         14 . A method of generating natural killer (NK) cells comprising:
 providing hemangioblasts;   culturing the hemangioblasts in liquid media comprising human serum and a first cytokine mixture comprising IL2, IL3, IL6, IL7, IL15, and SCF;   harvesting the cultured cells; and   culturing harvested cells in liquid media comprising human serum and a second cytokine mixture comprising IL7, IL15, SCF and FL to generate the NKs.   
     
     
         15 . The method of  claim 14 , wherein the concentration of IL2 is about 5-10 ng/ml, IL3 is about 1-10 ng/ml, IL6 is about 1-10 ng/ml, IL7 is about 5-20 ng/ml, IL15 is about 5-10 ng/ml, SCF is about 10-50 ng/ml, and FL is about 10-50 ng/ml. 
     
     
         16 . The method of  claim 14 , wherein culturing the hemangioblasts is for about 6 to 8 days. 
     
     
         17 . The method of  claim 14 , wherein culturing the harvested cells is for about 14 to 21 days. 
     
     
         18 . The method of  claim 14 , further comprising weekly media changes to refresh the second cytokine mixture. 
     
     
         19 . The method of  claim 14 , wherein the hemangioblasts are differentiated from human embryonic stem cells (hESCs). 
     
     
         20 . The method of  claim 14 , wherein the hemangioblasts are differentiated from induced pluripotent (iPS) cells. 
     
     
         21 . The method of  claim 14 , wherein the NK cells are immature NK cells and are CD56+ and CD16−. 
     
     
         22 . The method of  claim 14 , wherein the NK cells are mature NK cells and are CD56− and CD16+, or CD56lo and CD16+. 
     
     
         23 . A natural killer (NK) cell generated by any of the methods of  claims 14 - 22 . 
     
     
         24 . A pharmaceutically acceptable composition comprising a quantity of the NK cell of  claim 24 . 
     
     
         25 . A method to generate dendritic cells (DCs), comprising:
 providing hemangioblasts;   culturing the hemangioblasts in liquid media comprising human serum, SCF, FL, IL3 and GM-CSF;   adding IL4 to the liquid media; and   further culturing the hemangioblasts to generate the DCs.   
     
     
         26 . The method of  claim 25 , wherein culturing the hemangioblasts is for about 7 to 11 days. 
     
     
         27 . The method of  claim 25 , wherein further culturing of the hemangioblast after the addition of IL4 is for about 8 to 10 days. 
     
     
         28 . The method of  claim 25 , further comprising adding a cytokine mixture comprising IL1b, TNFα and IL6 to induce maturation of the DCs. 
     
     
         29 . The method of  claim 28 , wherein the cytokine mixture is added for about 48 hours. 
     
     
         30 . The method of  claim 28 , wherein the cytokine mixture further comprises a cytokine selected from the group consisting of PGE2, IFNα2b, poly I:C, IFNγ and combinations thereof. 
     
     
         31 . The method of  claim 25 , further comprising adding LPS, IFNγ and/or S-28463 to stimulate IL12p70 production from the DCs and/or HLA-DR expression from the DCs. 
     
     
         32 . The method of  claim 25 , wherein the concentration of SCF is about 20-100 ng/ml, FL is about 10-50 ng/ml, IL3 is about 5-50 ng/ml, GM-CSF is about 50-100 ng/ml, and IL4 is about 50-100 ng/ml. 
     
     
         33 . The method of  claim 28 , wherein the concentration of IL1b is about 10 ng/ml, TNFγ is about 10 ng/ml, and IL6 is about 150 ng/ml. 
     
     
         34 . The method of  claim 30 , wherein the concentration of PGE2 is about 1 μg/ml, IFNα2b is about 3000 units/ml poly I:C is about 20 μg/ml, and IFNγ is about 20 ng/ml. 
     
     
         35 . The method of  claim 25 , wherein the DCs are mature DCs and express CD83. 
     
     
         36 . The method of  claim 25 , wherein the DCs are mature DCs and the expression of CD209, HLA DR and/or CD11c is increased. 
     
     
         37 . A dendritic cell (DC) generated by any of the methods of  claims 25 - 36 . 
     
     
         38 . A pharmaceutically acceptable composition comprising a quantity of the DC cell of  claim 37 .

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