In Vitro Expansion of Fetal Liver Hematopoietic Stem Cells

Abstract

Fetal liver hematopoietic stem cells because of their proliferative potential have been considered appropriate for management of aplastic anemia. Bone marrow recovery was possible in some cases; engraftment potential of these cells however, was unsatisfactory, possibly due to the availability of smaller number of these cells from a single fetus. Present study was undertaken to see if fetal liver hematopoietic stem cells could be expanded in vitro. Mononuclear cells were isolated and hematopoietic stem cells were identified and analyzed by cell surface marker CD34. CD34+ cells were separated by magnetic cell sorting positive selection method using flow cytometry. Hematopoietic stem cells (CD34+) were cultured by using 5 cytokines, stem cell factor, granulocyte macrophages-colony stimulating factor, interlukin-6, Fms-related tyrosine kinase 3 and erythropoietin, in 4 different combinations along with supplements, in serum free culture media for 21 days. Cell viability continued to be greater than 90% throughout 21 days of culture. The cells expanded best in combination of media, supplements and 4 cytokines, namely SCF, Flt-3, IL6 and Epo to yield large number of total (CD34+ & CD34-) cells. Even though the total number of nucleated cells increased in culture significantly, levels of CD34 antigen expression declined steadily over this period.