Severe neutropenia and protracted thrombocytopenia remain serious clinical problems following cord blood transplantation (CBT) due to the paucity of stem and progenitor cells in the grafts. Administration of ex-vivo expanded megakaryocyte progenitor cells may facilitate platelet production. We propose a novel strategy to expand these rare cells ex-vivo, from a small portion of the cord blood (CB) unit, using fibronectin (FN), a major component of hematopoietic niches, combined with cytokines, including thrombopoietin and the hematopoietic stress-associated acetylcholinesterase readthrough peptide (ARP). Application of multiple gates and high definition flow cytometry enabled clear resolution of expanded hematopoietic stem/precursor cells (HSPC) and megakaryocyte progenitors (Mk-p) and their early subsets while eliminating positively stained non-relevant cells. FN increased viability, expansion of all CD34+ HSPC populations and Mk-p. The combination of FN + thrombopoietin + ARP maintained and expanded very early myeloid and thrombopoietic precursors, increased the proliferation of megakaryocyte, granulocyte-macrophage and multilineage colony-forming progenitors and supported Mk maturation as measured by ploidy and glycoprotein IIb/IIIa expression by quantiative reverse transcription polymerase chain reaction. This approach, which involves expanding HSPC and Mk precursors from a small portion of the CB unit, without sacrificing the coveted stem cells, may lead to improved cell therapy modalities to facilitate earlier myelopoiesis and platelet production post-CBT.
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