Coculture of hematopoietic stem cells (HSC) with primary stromal cells from HSC niches supports the maintenance and expansion of HSC and progenitors ex vivo. However, a major drawback is the availability of primary human samples for research and clinical applications. We investigated the use of in vitro derived osteoblasts as a new source of feeder cells and characterized the molecular pathways that mediate their growth-promoting activities. First, we compared the growth and differentiation modulating activities of mesenchymal stromal cells (MSC)-derived osteoblasts (M-OST) with those of their undifferentiated precursor on umbilical cord blood (UCB) progenitors. Feeder-free cultures were also included as baseline control. Cell growth and expansion of hematopoietic progenitors were significantly enhanced by both feeder cell types. However, progenitor cell growth was considerably greater with M-OST. Coculture also promoted the maintenance of immature CD34 progenitor subsets and modulated in a positive fashion the expression of several homing-related cell surface receptors, in a feeder-specific fashion. Serial transplantation experiments revealed that M-OST coculture supported the maintenance of long-term lympho-myeloid reconstituting HSC that provided engraftment levels that were generally superior to those from MSC cocultures. Mechanistically, we found that coculture with M-OST was associated with enhanced beta-catenin (β-Cat) activity in UCB cells and that abrogation of β-Cat/T-cell factor activity blunted the growth-promoting activity of the M-OST coculture. Conversely, Notch inhibition reduced UCB cell expansion, but to a much lesser extent. In conclusion, this study demonstrates that M-OST are excellent feeder cells for HSC and progenitors, and it identifies key molecular pathways that are responsible for the growth-enhancing activities of osteoblasts on UCB progenitors.

Engraftment outcomes are strongly correlated with the numbers of hematopoietic stem and progenitor cells (HSPC) infused. Expansion of umbilical cord blood (CB) HSPC has gained much interest lately since infusion of expanded HSPC can accelerate engraftment and improve clinical outcomes. Many novel protocols based on different expansion strategies of HSPC and their downstream derivatives are under development. Herein, we describe the production and properties of serum-free medium (SFM) conditioned with mesenchymal stromal cells derivedosteoblasts (OCM) for the expansion of umbilical CB cells and progenitors. After optimization of the conditioning length, we show that OCM increased the production of human CB total nucleated cells and CD34 ? cells by 1.8-fold and 1.5-fold over standard SFM, respectively. Production of immature CD34 ? subpopulations enriched in hematopoietic stem cells was also improved with a shorter conditioning period. Moreover, we show that the growth modulatory activities of OCM on progenitor expansion are regulated by both soluble factors and non-soluble cellular elements. Finally, the growth and differentiation modulatory activities of OCM were fully retained after high dose-ionizing irradiation and highly stable when OCM is stored frozen. In summary, our results suggest that OCM efficiently mimics some of the natural regulatory activities of osteoblasts on HSPC and highlight the marked expansion potentials of SFM conditioned with osteoblasts.

Blood agencies worldwide face the same recurring problem of enrolling healthy donors to produce platelet concentrates (PC) to meet the fluctuating demand. Research in stem cell biology has now made possible the production of culturederived platelets (CDP) raising the possibility of producing donor-independent platelets. The aim of this paper was to first review known key concepts about megakaryopoiesis and second to describe efforts done in our laboratory and that of others for the establishment of culture processes for the production of megakaryocytes and platelets from cord blood haematopoietic stem and progenitor cells (HSPC). Recent progresses made with induced pluripotent stem cells (iPSCs) are also presented. Expansion of HSPCs and megakaryocytes can be accomplished by combining thrombopoietin (TPO) and a wide array of other cytokines. However, expansion of HSPCs ex vivo is associated with a rapid loss of megakaryocyte differentiation potential. Optimization of cytokine combinations and concentrations can improve yields and reduce consumption of growth factors. Moreover, the development of inducible megakaryocytic cell lines that can be triggered to undergo terminal megakaryocytic differentiation offers an independent solution to some of the problems associated with megakaryocyte production ex vivo. Functional characterization of platelets produced ex vivo has so far revealed that they form a heterogeneous population with some culture-derived platelets (CDP) showing normal platelet properties and functions. In summary, functional CDPs can be produced in HSPC-and iPSC-based cultures. Remaining challenges that need to be addressed before culture-derived PCs can be used to complement blood-derived PCs are also discussed.