The High Yield Expansion and Megakaryocytic Differentiation of Human Umbilical Cord Blood CD133+ Cells

Abstract

Objective: Despite of many benefits, umbilical cord blood (UCB) hematopoietic stem cell(HSC) transplantation is associated with low number of stem cells and slow engraftment;in particular of platelets. So, expanded HSCs and co-transfusion of megakaryocyte (MK)progenitor cells can shorten this period. In this study, we evaluated the cytokine conditionsfor maximum expansion and MK differentiation of CD133+ HSCs.Materials and Methods: In this experimental study, The CD133+ cells were separatedfrom three cord blood samples by magnetic activated cell sorting (MACS) method, expandedin different cytokine combinations for a week and differentiated in thrombopoietin(TPO) for the second week. Differentiation was followed by the flow cytometry detectionof CD41 and CD61 surface markers. Colony forming unit (CFU) assay and DNA analysiswere done for colonogenic capacity and ploidy assay.Results: CD133+ cells showed maximum expansion in the stem span medium with stemcell factor (SCF) + FMS-like tyrosine kinase 3-ligand (Flt3-L) + TPO but the maximum differentiationwas seen when CD133+ cells were expanded in stem span medium with SCF+ Interleukin 3 (IL-3) + TPO for the first and in TPO for the second week. Colony FormingUnit-MK (CFU-MK) was formed in three sizes of colonies in the mega-cult medium. In theDNA analysis; 25.2 ± 6.7% of the cells had more than 2n DNA mass.Conclusion: Distinct differences in the MK progenitor cell count were observed when thecells were cultured in stem span medium with TPO, SCF, IL-3 and then the TPO in thesecond week. Such strategy could be applied for optimization of CD133+ cells expansionfollowed by MK differentiation.

Keywords

• Expansion
• Differentiation
• CD133+
• Megakaryocyte
• Cord Blood