Methods, kits, and compositions for stem cell self-renewal

Abstract

The present invention relates to methods and kits for expanding a stem cell population. More particularly, the invention relates, inter alia, to methods, kits, and compositions for expanding a stem cell population, particularly a hematopoietic stem cell population.

1 . An ex vivo method for expanding the number of hematopoietic stem cells (HSC) in a population of mononuclear cells (MNC) comprising culturing the population of MNCs comprising at least one HSC in an HSC expansion media for a period of time sufficient to expand the number of HSCs in the MNC population, wherein the expanded HSCs are functional with long term, multi-lineage, repopulating potential. 
     
     
         2 . The method according to  claim 1 , which provides HSCs that, upon transplant into a recipient, exhibit greater than 5% donor repopulation. 
     
     
         3 . The method according to  claim 1 , which provides HSCs that, upon transplant into a recipient, exhibit greater than 25% donor repopulation. 
     
     
         4 . The method according to  claim 1 , which provides HSCs that, upon transplant into a recipient, exhibit greater than 45% donor repopulation. 
     
     
         5 . The method according to  claim 1 , which provides HSCs that, upon transplant into a recipient, exhibit greater than 60% donor repopulation. 
     
     
         6 . The method according to  claim 1 , wherein the HSC expansion media comprises a modulator of the Wnt pathway. 
     
     
         7 . The method according to  claim 6 , wherein the modulator of the Wnt pathway down-regulates GSK-3β. 
     
     
         8 . The method according to  claim 6 , wherein the modulator of the Wnt pathway is a reversible GSK-3β inhibitor selected from the group consisting of a small molecule, a biologic, an antisense RNA, a small interfering RNA (siRNA), and combinations thereof. 
     
     
         9 . The method according to  claim 8 , wherein the reversible GSK-3β inhibitor is a small molecule. 
     
     
         10 . The method according to  claim 8 , wherein the reversible GSK-3β inhibitor is selected from the group consisting of Hymenialdisine, Flavopiridol, Kenpaullone, Alsterpaullone, Azakenpaullone, Indirubin-30-oxime, 6-Bromoindirubin-30-oxime (BIO), 6-Bromoindirubin-30-acetoxime, Aloisine A, Aloisine B, TDZD8, Compound 12, CHIR98014, CHIR99021 (CT99021), CT20026, Compound 1, SU9516, ARA014418, Staurosporine, Compound 5a, Compound 29, Compound 46, GF109203x (bisindolylmaleimide I), Ro318220 (bisindolylmaleimide IX), SB216763, SB415286, I5, CGP60474, Compound 8b, TWS119, Compound 1A, Compound 17, Lithium, Beryllium, Zinc, small molecule GSK-3β inhibitors (Vertex Pharmaceuticals), NP-12 (Neuropharma), GSK-3β inhibitors (Amphora), GSK-3β inhibitors (CrystalGenomics), SAR-502250 (Sanofi-Aventis), 3544 (Hoffmann-La Roche), GSK-3β inhibitors (Lundbeck), TDZD-8 (Cancer Center, University of Rochester), pharmaceutically acceptable salts thereof, and combinations thereof. 
     
     
         11 . The method according to  claim 10 , wherein the GSK-3β inhibitor is CHIR99021. 
     
     
         12 . The method according to  claim 6 , which provides HSCs that, upon transplant into a recipient, exhibit greater than 60% donor repopulation. 
     
     
         13 . The method according to  claim 1 , wherein the HSC is obtained from a mammalian tissue selected from the group consisting of cord blood, peripheral blood, and bone marrow. 
     
     
         14 . An expanded, substantially undifferentiated HSC population made by the method according to  claim 1 . 
     
     
         15 . An expanded, substantially undifferentiated HSC population made by the method according to  claim 6 . 
     
     
         16 . A kit for expanding, ex vivo, the number of hematopoietic stem cells (HSC) in a population of mononuclear cells (MNC), the kit comprising a GSK-3β inhibitor, and instructions for the use of the inhibitor, wherein, when used, the kit provides expanded HSCs that are functional with long term, multi-lineage, repopulating potential. 
     
     
         17 . The kit according to  claim 16 , wherein the GSK-3β inhibitor is selected from the group consisting of Hymenialdisine, Flavopiridol, Kenpaullone, Alsterpaullone, Azakenpaullone, Indirubin-30-oxime, 6-Bromoindirubin-30-oxime (BIO), 6-Bromoindirubin-30-acetoxime, Aloisine A, Aloisine B, TDZD8, Compound 12, CHIR98014, CHIR99021 (CT99021), CT20026, Compound 1, SU9516, ARA014418, Staurosporine; Compound 5a, Compound 29, Compound 46, GF109203x (bisindolylmaleimide I), Ro318220 (bisindolylmaleimide IX), SB216763, SB415286, I5, CGP60474, Compound 8b, TWS119, Compound 1A, Compound 17, Lithium, Beryllium, Zinc, small molecule GSK-3β inhibitors (Vertex Pharmaceuticals), NP-12 (Neuropharma), GSK-3β inhibitors (Amphora), GSK-3β inhibitors (CrystalGenomics), SAR-502250 (Sanofi-Aventis), 3544 (Hoffmann-La Roche), GSK-3β inhibitors (Lundbeck), TDZD-8 (Cancer Center, University of Rochester), pharmaceutically acceptable salts thereof, and combinations thereof. 
     
     
         18 . The kit according to  claim 16 , wherein the GSK-3β inhibitor is CHIR99021. 
     
     
         19 . The kit according to  claim 16 , which provides HSCs that, upon transplant into a recipient, exhibit greater than 60% donor repopulation. 
     
     
         20 . A media for carrying out ex vivo expansion of a stem cell in a population of MNCs comprising a fluid media suitable for maintaining viable stem cells and a GSK-3β inhibitor present in the media at a concentration sufficient to enable expansion of the stem cell population while maintaining a long term, multi-lineage, repopulating potential in the stem cells, wherein the stem cells, when transplanted into a recipient, exhibit greater than 5% donor repopulation. 
     
     
         21 . An ex vivo method for expanding the number of cells capable of supporting multi-lineage repopulation in a population of mononuclear cells (MNC) comprising culturing the population of MNCs comprising at least one hematopoietic stem cell (HSC) and at least one hematopoietic progenitor cell in an HSC expansion media for a period of time sufficient to expand the number of cells capable of supporting multi-lineage repopulation in the MNC population. 
     
     
         22 . The method according to  claim 10 , wherein the GSK-3β inhibitor is lithium, a pharmaceutically acceptable salt thereof, or combinations thereof. 
     
     
         23 . An expanded, substantially undifferentiated HSC population made by the method according to  claim 22 . 
     
     
         24 . The kit according to  claim 16 , wherein the GSK-3β inhibitor is lithium, a pharmaceutically acceptable salt thereof, or combinations thereof. 
     
     
         25 . The media according to  claim 20 , wherein the GSK-3β inhibitor is lithium, a pharmaceutically acceptable salt thereof, or combinations thereof. 
     
     
         26 . The method according to  claim 21 , wherein the HSC expansion media comprises a reversible GSK-3β inhibitor. 
     
     
         27 . The method according to  claim 26 , wherein the reversible GSK-3β inhibitor is lithium, a pharmaceutically acceptable salt thereof, or combinations thereof.