Methods for generating hematopoietic stem cells

Abstract

In some aspects and embodiments, the invention provides methods for making hematopoietic stem cells, including for HSCT. The method comprises providing a cell population comprising hemogenic endothelial (HE) or endothelial cells, and increasing activity or expression of DNA (cytosine-5-)-methyltransferase 3 beta (Dnmt3b) and/or GTPase IMAP Family Member 6 (Gimap6) in the HE and/or endothelial cells under conditions sufficient for stimulating formation of HSCs.

What is claimed is: 
     
         1 . A method of treating a subject in need of hematopoietic stem cell therapy or transplantation, the method comprising administering to the subject a pharmaceutical composition comprising hematopoietic stem cells (HSCs), wherein the HSCs are prepared ex vivo by a process comprising increasing the expression of Dnmt3b in a cell population comprising hemogenic endothelial cells under conditions sufficient for stimulating formation of HSCs. 
     
     
         2 . The method of  claim 1 , wherein the HSCs comprise long term hematopoietic stem cells (LT-HSCs). 
     
     
         3 . The method of  claim 1 , wherein the expression of Dnmt3b is increased by contacting the cell population with an effective amount of a Piezo1 agonist. 
     
     
         4 . The method of  claim 3 , wherein the Piezo1 agonist is Yoda1. 
     
     
         5 . The method of  claim 4 , herein the effective amount of Yoda1 is in the range of about 5 to about 500 μM. 
     
     
         6 . The method  claim 5 , wherein the effective amount of Yoda1 is in the range of about 5 to about 100 μM. 
     
     
         7 . The method of  claim 5 , wherein the effective amount of Yoda1 is in the range of about 25 to about 100 μM. 
     
     
         8 . The method of  claim 1 , wherein increasing the expression of Dnmt3b in the cell population is by introducing a Dnmt3b episome or mRNA. 
     
     
         9 . The method of  claim 1 , wherein the cell population is derived from induced pluripotent stem cells (iPSCs). 
     
     
         10 . The method of  claim 9 , wherein the iPSCs are HLA-modified cells. 
     
     
         11 . The method of  claim 1 , wherein the HSCs engraft in a hematopoietic niche and reconstitute to functional, multi-lineage adult blood. 
     
     
         12 . The method of  claim 1 , wherein the subject has malignant or non-malignant form of blood, bone marrow, metabolic or immune disease. 
     
     
         13 . The method of  claim 12 , wherein the subject has multiple myeloma; non-Hodgkin lymphoma; Hodgkin disease; acute myeloid leukemia; neuroblastoma; a germ cell tumor; or myelodysplastic syndrome. 
     
     
         14 . The method of  claim 13 , wherein the pharmaceutical composition is administered following myeloablative, non-myeloablative, or immunotoxin-based conditioning regimen. 
     
     
         15 . The method of  claim 12 , wherein the subject has aplastic anemia, pure red-cell aplasia, paroxysmal nocturnal hemoglobinuria, Fanconi anemia, thalassemia major, sickle cell anemia, severe combined immunodeficiency (SCID), Wiscott-Aldrich syndrome, or hemaphagocytic lymphohistiocytosis. 
     
     
         16 . The method of  claim 15 , wherein the subject has thalassemia major. 
     
     
         17 . The method of  claim 15 , wherein the subject has sickle cell anemia or aplastic anemia. 
     
     
         18 . The method of  claim 12 , wherein the subject has severe combined neutropenia, Shwachman-Diamond syndrome, Diamond-Blackfan anemia, or leukocyte adhesion deficiency. 
     
     
         19 . The method of  claim 1 , wherein about 100,000 to about 400,000 HSCs are administered per kilogram of the subject's weight. 
     
     
         20 . A pharmaceutical composition comprising at least 10 4  hematopoietic stem cells (HSCs) and comprising long-term hematopoietic stem cells (LS-HSCs), wherein the HSCs are prepared ex vivo by a process comprising increasing the expression of Dnmt3b in a cell population comprising hemogenic endothelial cells under conditions sufficient for stimulating formation of HSCs. 
     
     
         21 . The method of  claim 20 , wherein the expression of Dnmt3b is increased by contacting the cell population with an effective amount of a Piezo1 agonist. 
     
     
         22 . The method of  claim 21 , wherein the Piezo1 agonist is Yoda1. 
     
     
         23 . The method of  claim 22 , herein the effective amount of Yoda1 is in the range of about 5 to about 500 μM. 
     
     
         24 . The method  claim 22 , wherein the effective amount of Yoda1 is in the range of about 5 to about 100 μM. 
     
     
         25 . The method of  claim 22 , wherein the effective amount of Yoda1 is in the range of about 25 to about 100 μM. 
     
     
         26 . The method of  claim 20 , wherein increasing the expression of Dnmt3b in the cell population is by introducing a Dnmt3b episome or mRNA. 
     
     
         27 . The method of  claim 20 , wherein the cell population is derived from induced pluripotent stem cells (iPSCs). 
     
     
         28 . The method of  claim 27 , wherein the iPSCs are HLA-modified cells.