Proliferation extent of CD34(+) cells as a key parameter to maximize megakaryocytic differentiation of umbilical cord blood-derived hematopoietic stem/progenitor cells in a two-stage culture protocol()

Co-infusion of ex-vivo generated megakaryocytic progenitors with hematopoietic stem/progenitor cells (HSC/HPC) may contribute to a faster platelet recovery upon umbilical cord blood (UCB) transplantation. A two stage protocol containing cell expansion and megakaryocyte (Mk) differentiation was established using human UCB CD34(+)-enriched cells. The expansion stage used a pre-established protocol supported by a human bone marrow mesenchymal stem cells (MSC) feeder layer and the differentiation stage used TPO (100 ng/mL) and IL-3 (10 ng/mL). 18% of culture-derived Mks had higher DNA content (>4 N) and were able to produce platelet-like particles. The proliferation extent of CD34(+) cells obtained in the expansion stage (FI-CD34(+)), rather than expansion duration, determined as a key parameter for efficient megakaryocytic differentiation. A maximum efficiency yield (EY) of 48 ± 7.7 Mks/input CD34(+) cells was obtained for a FI-CD34(+) of 17 ± 2.5, where a higher FI-CD34(+) of 42 ± 13 resulted in a less efficient megakaryocytic differentiation (EY of 22 ± 6.7 and 19 ± 4.6 %CD41).