While umbilical cord blood provides an important source of hematopoietic stem/progenitor cells (HSPC) for allogeneic transplantation in children, its use in adults is limited by the inadequate number of primitive stem cells and megakaryocyte progenitors (Mk-p) in single or even double CB units resulting in prolonged thrombocytopenia. Thrombopoietin treatment is not effective in these patients due to the paucity of target progenitors and patients require multiple platelet transfusions until the long-term engrafting cells can support thrombopoiesis, thus new modalities to increase progenitor cell dose are needed. A new transplantation strategy could involve the infusion of ex vivo-generated Mk-p together with unmanipulated single or double CB units. While CB CD34+ cells can be expanded by several reported methods, these rare cells cannot be sacrificed from the CB units due to their limited number. We propose a novel ex-vivo strategy to facilitate HSPC and Mk-p expansion from mononuclear cells (MNC) of a small aliquot of CB using conditions that mimic the hematopoietic niche, in short term cultures. Fibronectin (FN) was considered to be a prime candidate to support proliferation because it is a major extracellular matrix (ECM) component of all bone marrow hematopoietic microenvironments which is known to enhance viability and proliferation of HSPC. Other growth stimulators added were thrombopoietin (r-hu-TPO), the major physiological stimulator of MK and the synthetic hematopoietic stress peptide ARP derived from acetylcholinesterase, shown to increase transplantable Mk-p and produce human platelets in NOD/SCID mice (Pick et al, Blood 2006, Grisaru et al, J Imm 2006). High definition flow cytometry enabled assessing expansion of the SSClow/CD34high HSPC, and the SSClow/CD45dim/neg/CD41high Mk-p, and their subpopulations on day 0 and 10 of culture. True MK expansion was assessed by gating out of granulocyte and monocytes, which acquire CD41+ adherent platelets in culture. FN alone increased viability and expansion of HSPC by 6.9 fold and MK-p by 4-fold, while r-hu-TPO alone enhanced Mk-p proliferation with an average expansion of 8.3-fold in agreement with its known activity. Combining FN with r-hu-TPO produced a 25-fold increase in the number of MK-p while adding ARP to FN and r-hu-TPO was even more powerful, doubling the number of cells with a highly significant average expansion of 59-fold (p < 0.001). To define the progenitor subpopulations that contributed to Mk-p proliferation with FN, r-hu-TPO and ARP, we further analyzed the resulting subsets of MK-p cells, which also expressed either CD34, or the early myeloid marker CD33. The CD41high/CD34high population was increased by 4 fold, while the CD41high/CD33+ Mk-p, a subset with properties similar to clonogenic GEMM progenitors that could provide both myeloid and megakaryocytic cells post-transplant, were stimulated 30–50 fold. This notion is confirmed by the stimulation of CFU-MK and CFU-GEMM obtained under these conditions. Considering that expansion of MK-p requires proliferation of the HSPC precursor, we examined the proliferation of CD34+ progenitor cells and their subpopulations; CD34high/CD33+ or CD34high/CD41low uncommitted HSPC and CD41 high committed Mk subpopulations. The addition of FN alone stimulated CD34+ HSPC expansion by 6.9-fold (p < 0.05). All cultures that contained the ARP peptide maintained a high proliferation capacity, confirming that ARP protects and drives CD34+HSPC and early myeloid cell proliferation (Deutsch et al Exp Hem 2002). The addition of r-hu-TPO and ARP to FN produced a synergistic proliferative effect on the CD34+/CD41low HSPC stimulating a dramatic 440 fold increase of these uncommitted cells. These data support the notion that FN is protective and plays an essential role in enabling HSPC and MK-p expansion driven by r-hu-TPO and ARP. These conditions also supported MK maturation, as measured by increased high ploidy cells and elevated expression of GPIIb/IIIa detected by quantitative real time PCR. We demonstrate that expansion of both very early myeloid and Mk-p from a small fraction of the CB unit in short term cultures under conditions that mimic the hematopoietic niche is feasible, easy to perform and can comply with GTP requirements. This approach may lead to the development of more effective cell therapy modalities to facilitate myelopoiesis and platelet production following CBT.

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