Cultured Hematopoietic Stem Cells and Method for Expansion and Analysis Thereof

Abstract

Hematopoietic stem cells and methods for ex vivo expansion of hematopoietic stem cells are provided. The methods comprise culturing the cells in a media containing an effective amount insulin-like growth factor (IGF), fibroblast growth factor (FGF), thrombopoietin (TPO), and stem cell factor (SCF), under conditions sufficient for expansion of said cells. Methods for identifying expanded hematopoeitic stem cells and kits for ex vivo expansion of hematopoietic stem cells are also provided.

1 - 22 . (canceled) 
     
     
         23 . A method for identifying hematopoietic stem cells, comprising characterizing surface phenotype of cultured cells, wherein a surface phenotype of Sca + , IGF-2 receptor + , CD31 +  and one or more of the group consisting of PrP − , Tie-2 − , CD62L − , prominin 1 −  (AC133 − ), CD34 − , CD38 − , and Lin −  is indicative of a hematopoietic stem cell. 
     
     
         24 . The method of  claim 23 , wherein a surface phenotype of Sca + , IGF-2 receptor + , CD31 +  and two or more of the group consisting of PrP − , Tie-2 − , CD62L − , prominin 1 −  (AC133 − ), CD34 − , CD38 − , and Lin −  is indicative of a hematopoietic stem cell. 
     
     
         25 . The method of  claim 23 , wherein a surface phenotype of Sca + , IGF-2 receptor + , CD31 + , PrP −  and Tie-2 −  is indicative of a hematopoietic stem cell. 
     
     
         26 . Cultured hematopoietic stem cells having a surface phenotype of Sca + , IGF-2 receptor + , CD31 +  and one or more of PrP − , Tie-2 − , CD62L − , prominin 1 −  (AC133 − ), CD34 − , CD38 − , and Lin − . 
     
     
         27 . The cultured hematopoietic stem cells of  claim 26  having a surface phenotype of Sca + , IGF-2 receptor + , CD31 +  and two or more of PrP − , Tie-2 − , CD62L − , prominin 1 −  (AC133 − ), CD34 − , CD38 − , and Lin − . 
     
     
         28 . The cultured hematopoietic stem cells of  claim 27  having a surface phenotype of Sca + , IGF-2 receptor + , CD31 + , PrP −  and Tie-2 − . 
     
     
         29 . A kit for expanding hematopoietic stem cells ex vivo comprising
 a) insulin-like growth factor-2 (IGF-2) and at least one factor selected from group consisting of SCF, TOP, and FGF-1; and   b) instructions for expanding hematopoietic stem cells ex vivo.   
     
     
         30 . The kit of  claim 29 , comprising IGF-2 and two of the factors from said group. 
     
     
         31 . The kit of  claim 29 , comprising IGF-2 and each of the factors from said group. 
     
     
         32 . The kit of  claim 30 , where the factors are provided in separate containers. 
     
     
         33 . The method of  claim 23 , wherein a surface phenotype of Sca + , IGF-2 receptor + , CD31 + , PrP − , Tie-2 − , and prominin 1 −  (AC133 − ) is indicative of a hematopoietic stem cell. 
     
     
         34 . The method of  claim 23 , wherein a surface phenotype of Lin − , Sca + , IGF2-receptor + , PrP − , and CD62L −  is indicative of a hematopoietic stem cell. 
     
     
         35 . The method of  claim 23 , wherein the hematopoietic stem cell further has a Kit +  surface marker. 
     
     
         36 . The cultured hematopoietic stem cells of  claim 26  having a surface phenotype of Sca + , IGF-2 receptor + , CD31 + , PrP − , Tie-2 − , and prominin 1 −  (AC133 − ). 
     
     
         37 . The cultured hematopoietic stem cells of  claim 26  having a surface phenotype of Lin − , Sca + , IGF2-receptor + , PrP − , and CD62L − . 
     
     
         38 . The cultured hematopoietic stem cells of  claim 26  further having a Kit +  surface marker. 
     
     
         39 . A hematopoietic stem cell from a population of ex vivo cultured hematopoietic stem cells, having a surface phenotype of Sca + , IGF-2 receptor + , CD31 +  and one or more of the group consisting of PrP − , Tie-2 − , CD62L − , prominin 1 −  (AC133 − ), CD34 − , CD38 − , and Lin − . 
     
     
         40 . The hematopoietic stem cell of  claim 39  having a surface phenotype of Sca + , IGF-2 receptor + , CD31 +  and two or more of PrP − , Tie-2 − , CD62L − , prominin 1 −  (AC133 − ), CD34 − , CD38 − , and Lin − . 
     
     
         41 . The hematopoietic stem cell of  claim 39  having a surface phenotype of Sca + , IGF-2 receptor + , CD31 + , PrP −  and Tie-2 − . 
     
     
         42 . The hematopoietic stem cell of  claim 39  having a surface phenotype of Sca + , IGF-2 receptor + , CD31 + , PrP − , Tie-2 − , and prominin 1 −  (AC133 − ). 
     
     
         43 . The hematopoietic stem cell of  claim 39  having a surface phenotype of Lin − , Sca + , IGF2-receptor + , PrP − , and CD62L − . 
     
     
         44 . The hematopoietic stem cell of  claim 39  further having a Kit +  surface marker. 
     
     
         45 . A composition for transplantation, comprising the hematopoietic stem cell of  claim 39 . 
     
     
         46 . A composition for reconstituting hematopoietic cells, comprising the hematopoietic stem cell of  claim 39 . 
     
     
         47 . A method for transplantation, comprising administrating to a patient in need thereof the hematopoietic stem cell of  claim 39 . 
     
     
         48 . A method for reconstituting hematopoietic cells, comprising administrating to a patient in need thereof the hematopoietic stem cell of  claim 39 .