Expansion of CD34+kdr+ cells in cord blood after culture with tpo, FLT-3l, scf, and vegf

Abstract

CD34+ cells that co-express KDR (a VEGF receptor) have been identified in umbilical cord blood (CB) and shown to be rich in primitive stem cell activity. We studied the production of CD34+KDR+ cells in stem cell “expansion” cultures. CD34+ cells were isolated from CB and cultured for 2, 4, and 6 weeks in serum-free medium supplemented with 5% CB plasma in the presence of rhSCF (20 ng/ml), rhTPO, rhFLT-3L and ± rhVEGF (50 ng/ml). By flow cytometry, the fraction of CD34-selected cells that were also KDR+ was 1.6 ± 0.3% prior to culture. After 2 weeks in culture, in the presence of rhSCF, rhTPO, and rhFLT-3L, the percent of CD34+KDR+ cells increased to 5.7 ± 3.2% with a CD34+KDR+ increase of 112-fold and all overall increase of 43-fold in total cell number. After 4 weeks in culture (± VEGF), CD34+KDR+ cells had increased >200-fold. Total cell expansion at week 6 was: 121-fold and 110-fold (with or without VEGF added, respectively). VEGF supplemented cultures had slightly greater numbers of CFU-GM and HPP-CFC. In addition, after 2 weeks in culture (without VEGF), we observed that 7–26% of the KDR+CD34+ were CD38− while cultures with VEGF contained 13–38% CD34+KDR+CD38− cells, possibly indicating the presence of a more primitive stem cell subset in these cultures.