Cord blood is better than bone marrow for generating megakaryocytic progenitor cells

Abstract

Thrombocytopenia remains an important problem for patients post high-dose chemotherapy and hematopoietic stem cell transplantation. The study of megakaryocytes, the direct precursors of platelets, has been hampered by their relatively low frequency in hematopoietic tissues. In an attempt to obtain a large number of functional megakaryocytic cells, we established a serum-free culture system to grow megakaryocytic progenitor cells derived from normal human bone marrow (BM) and cord blood (CB). Highly purified (purity >95%) CD34+ cells were obtained using magnetic cell sorting (MACS) followed by fluorescence activated cell sorting (FACS). The cells were cultured in a serum-free culture system for 3 weeks in the presence of a single dose of MGDF (50 ng/mL). On days 0, 5, 8, 12, 14, 18, and 21 of culture, the cellularity and morphology were examined. Megakaryocytic cells were monitored by detecting the expression of GPIIIa (CD61), GPIIb/IIIa (CD41) and GPIb (CD42b), and the distribution of megakaryocyte (MK) ploidy was analyzed by two-color flow cytometry. MGDF alone induced maximal nucleated cell expansion at day 14, resulting in a 38.20+/-10.47-fold increase in cell number for CB and a 5.08+/-1.30-fold increase in cell number for BM. On day 14 of the culture, the percentage of CD41-/CD14- cells derived from CB reached 73.54%+/-6.01% giving an absolute number of CD41+/CD14- cells of 27.25+/-2.23 x 10(4)/mL (27,250-fold increase), whilst the percentage of CD41+/CD14- cells derived from BM was only 29.21%+/-5.63% with an absolute number of 1.36+/-0.26 x 10(4)/mL (680-fold increase). Increased expression of GPIIIa occurred the earliest in culture, followed by GPIIb/IIIa, and then GPIb. The majority (81.6%-92.6%) of megakaryocytes (CD41+ cells) on day 14 of culture were 2N, although we did detect some 4N, 8N and greater ploidy cells. In conclusion, CD34+ cells stimulated by MGDF alone generated highly enriched MK progenitor cells at day 14 of serum-free culture. CB stem and progenitor cells have a greater proliferative response to MGDF alone than those derived from BM and may, therefore, prove to be a better source of cells for MK expansion.