Human brain endothelial cells and growth medium and method for expansion of primitive CD34+CD38- bone marrow stem cells

Abstract

A novel co-culture system using human brain endothelial cells (HUBEC) which promotes the expansion of human CD34 + CD38 − cells consistent with the PMVEC system is disclosed. HUBEC were isolated from cadaveric donors, passed in primary culture, cloned and found to be Von Willebrand Factor positive. Cultivation of purified bone marrow CD34 + cells on HUBEC monolayers supplemented with GM-CSF+IL-3+IL-6+SCF+flt-3 ligand caused a 14.5-fold increase in total cells, an 6.6-fold increase in CD34 + cells, and, most remarkably, a 440-fold increase in CD34 + CD38 − cells after 7 days. Further, CFU-GM production increased 15.1-fold, BFU-E increased 8-fold, and CFU-Mix increased 5.2-fold. Optimal generation was dependent upon the continued presence of exogenous supplied cytokines. In comparison, identically treated stroma-free suspension cultures supported a 10.2-fold expansion of total cells, a 3-fold increase in CD34 + cells and maintained the CD34 + CD38 − cell pool after 7 days of culture. Moreover, we found that non-brain human endothelial cells isolated from the same donors supported neither the expansion nor the maintenance of human CD34 + CD38 − cells. Although few steady state CD34 + CD38 − cells give rise to visible colony-forming cells in methylcellulose cultures, our FACS based cell cycle and sorting experiments demonstrated the activation of a highly clonogenic CD34 + CD38 − population (24% cloning efficiency) during ex-vivo culture on cytokine treated HUBEC. These results suggest that bone marrow CD34 + CD38 − cells require a stromal cell microenvironment for optimal expansion and that ex-vivo expanded CD34 + CD38 − cells generated in the HUBEC culture system appear to retain some degree of primitive “stemness”.

1 - 8 . (canceled) 
     
     
         9 . A method of engrafting human bone marrow CD34+CD38− hematopoietic 
       progenitor cells in a human, the method comprising the steps of:
 i) contacting the isolated CD34+CD38− hematopoietic progenitor cells with 
 
       human brain endothelial cells containing a factor or factors that expand the CD34+CD38− hematopoietic progenitor cells;
 ii) co-culturing the contacted CD34+CD38− hematopoietic progenitor cells and endothelial cells in the presence of at least one cytokine in an amount sufficient to support amplification/expansion of said CD34+CD38− hematopoietic progenitor cells; 
 iii) isolating the amplified/expanded CD34+CD38− hematopoietic progenitor cells from the culture; and 
 iv) infusing the amplified/expanded CD34+CD38− hematopoietic progenitor cells into said human. 
 
     
     
         10 . A method of engrafting human bone marrow CD34+CD38− hematopoietic progenitor cells in a human, the method comprising the steps of:
 i) isolating CD34+CD38− hematopoietic progenitor cells from human bone marrow; 
 ii) contacting the isolated CD34+CD38− hematopoietic progenitor cells with human brain endothelial cells containing a factor or factors that expand the CD34+CD38− hematopoietic progenitor cells; 
 iii) co-culturing the contacted CD34+CD38− hematopoietic progenitor cells 
 
       and endothelial cells in the presence of at least one cytokine in an amount sufficient to support amplification/expansion of said CD34+CD38− hematopoietic progenitor cells;
 iv) isolating the amplified/expanded CD34+CD38− hematopoietic progenitor cells from the culture; and 
 v) infusing the amplified/expanded CD34+CD38− hematopoietic progenitor cells into said human. 
 
     
     
         11 . The method according to  claim 9  or  10 , wherein said cells are isolated from the bone marrow of the human. 
     
     
         12 . The method according to  claim 9  or  10 , wherein said cells are isolated from the bone marrow of the human in need of said CD34+CD38− hematopoietic progenitor cells. 
     
     
         13 . The method according to  claim 9  or  10 , wherein said CD34+CD38− hematopoietic progenitor cells are isolated from the bone marrow of a donor. 
     
     
         14 - 25 . (canceled)