Methods for Expansion and Analysis of Cultured Hematopoietic Stem Cells

Abstract

Methods and kits for propagating hematopoietic stem cells are provided. The methods comprise culturing cells in medium comprising one or more angiopoietin-like proteins, under conditions sufficient for expansion of HSCs. Angiopoietin-like proteins include angiopoietin-like protein 2, angiopoietin-like protein 3, angiopoietin-like protein 4, angiopoietin-like protein 5, angiopoietin-like protein 7, and microfibrillar-associated glycoprotein (Mfap4). Methods for identifying hematopoietic stem cells are provided and isolated hematopoietic stem cells are also provided.

1 - 38 . (canceled) 
     
     
         39 . A kit for propagating hematopoietic stem cells (HSCs) in vitro comprising:
 a) medium suitable for culturing one or more cells comprising an HSC,   b) at least one angiopoietin-like protein, and   c) at least one additional growth factor selected from the group consisting of insulin-like growth factor (IGF), fibroblast growth factor (FGF), thrombopoietin (TPO), and stem cell factor (SCF).   
     
     
         40 - 46 . (canceled) 
     
     
         47 . The kit of  claim 39 , wherein the angiopoietin-like protein is selected from the group consisting of angiopoietin-like protein 2, angiopoietin-like protein 3, angiopoietin-like protein 4, angiopoietin-like protein 5, angiopoietin-like protein 7, and Mfap4. 
     
     
         48 . The kit of  claim 39 , wherein when in use, the angiopoietin-like protein is present in the medium at a concentration of about 0.1 ng/mL to about 500 ng/mL. 
     
     
         49 . The kit of  claim 39 , wherein the angiopoietin-like protein is a recombinant protein. 
     
     
         50 . The kit of  claim 39 , wherein the kit comprises SCF. 
     
     
         51 . The kit of  claim 39 , wherein when in use, the additional growth factor is present in the medium at a concentration of about 0.1 ng/mL to about 500 ng/mL. 
     
     
         52 . The kit of  claim 39 , wherein the one or more cells are selected from the group consisting of bone marrow cells, umbilical cord blood cells, and fetal liver cells. 
     
     
         53 . The kit of  claim 39 , wherein the one or more cells are human cells. 
     
     
         54 . The kit of  claim 39 , wherein the one or more cells are primary cells. 
     
     
         55 . The kit of  claim 39 , further comprising an instruction to culture the one or more cells for at least five days. 
     
     
         56 . A kit for propagating hematopoietic stem cells (HSCs) in vitro comprising:
 a) medium suitable for culturing primary cells comprising an HSC;   b) at least one angiopoietin-like protein selected from the group consisting of angiopoietin-like protein 2, angiopoietin-like protein 3, angiopoietin-like protein 4, angiopoietin-like protein 5, angiopoietin-like protein 7, and Mfap4; and   c) at least one additional growth factor selected from the group consisting of insulin-like growth factor (IGF), fibroblast growth factor (FGF), thrombopoietin (TPO), and stem cell factor (SCF).   
     
     
         57 . The kit of  claim 56 , wherein when in use, the angiopoietin-like protein is present in the medium at a concentration of about 0.1 ng/mL to about 500 ng/mL 
     
     
         58 . The kit of  claim 56 , wherein the angiopoietin-like protein is a recombinant protein. 
     
     
         59 . The kit of  claim 56 , wherein the kit comprises SCF. 
     
     
         60 . The kit of  claim 56 , wherein when in use, the additional growth factor is present in the medium at a concentration of about 0.1 ng/mL to about 500 ng/mL 
     
     
         61 . The kit of  claim 56 , wherein the primary cells are selected from the group consisting of bone marrow cells, umbilical cord blood cells, and fetal liver cells. 
     
     
         62 . The kit of  claim 56 , wherein the primary cells are human cells. 
     
     
         63 . The kit of  claim 56 , further comprising an instruction to culture the one or more cells for at least five days. 
     
     
         64 . A method of propagating hematopoietic stem cells (HSCs) in vitro comprising:
 (a) providing the kit of  claim 39 ; and   (b) culturing the one or more cells in the medium comprising the angiopoietin-like protein and the additional growth factor.   
     
     
         65 . A method of propagating hematopoietic stem cells (HSCs) in vitro comprising:
 (a) providing the kit of  claim 56 ; and   (b) culturing the primary cells in the medium comprising the angiopoietin-like protein and the additional growth factor.