Abstract

Platelet recovery following umbilical cord blood (CB) transplants can be delayed for several months. In vitro expansion and transplantation of CB megakaryocyte progenitor cells (MPC) may be one way to increase circulating platelets thereby shortening the period of thrombocytopenia. The in vitro expansion and differentiation of CB MPC derived from enriched CB CD34+ cells was studied after immuno-magnetic bead isolation. Isolated CB CD34+ cells were incubated in serum-free culture medium supplemented with a Tpo peptide agonist (TpoA), Flt-3L, Stem Cell Factor (SCF) and Vascular Endothelial Growth Factor (VEGF). At week 2, the cultures were divided and 1) maintained in serum-free medium, 2) supplemented with 5% human serum and, 3) supplemented with 5% mouse serum. Using flow cytometry and histocytochemistry, the cultured cells were evaluated for MPC using CD41a, CD36, von Willebrand factor (VWF) and also for myeloid progenitors using CD15 and CD33. The mean total percent MPC (CD41a+ and CD36+) at week 2 was 53 ±17% and 49 ±12%, respectively. After 2–4 weeks of additional culture the %MPC declined to less than 30% in cultures with or without serum supplements. However, at weeks 5–8 of additional culture the %MPC increased in the cultures supplemented with human or mouse serum. The mean %MPC was 45 ±19% (serum-free), 55 ±16% (human serum) and, 71 ±5% (mouse serum) in cultures at week 5–8 of additional culture. The remaining cells in the cultures were myeloid progenitors (CD15+ or CD33+). Total cell numbers at week 5–8 were inhibited 3 to 5x-fold in mouse serum supplemented cultures compared to serum free and human serum supplemented cultures. In addition, VWF+ cells could be detected after 5 weeks of additional culture when either human or mouse serum was present. These data help to explain the delay in platelet recovery typically associated with cord blood transplants but, indicate that additional serum factors are present to enhance megakaryocyte differentiation. These results also suggest that the transplant of cultured CB MPC may prove beneficial for the treatment of thrombocytopenic patients and normal platelet recovery.