The hematopoietic stem cell (HSC) niche is an anatomically confined space governing HSC proliferation, differentiation and self renewal. Recent research identified distinct compartments described as stromal and vascular niches. This study was initiated to directly compare mesenchymal stromal cell (MSC) and vascular endothelial progenitor cell (EPC) contribution to niche functions in a humanized co-culture system. MSCs and EPCs were propagated under animal protein-free conditions following recently established protocols. Autologous MSC-EPC pairs were established to avoid donor variation. Purified CD34+ hematopoietic progenitor cells (HPCs) and HSCs were used as responders in a cytokine-driven (IL-3, 6ng/mL; Flt-3-L, 50ng/mL; SCF, 20ng/mL) niche cell-regulated HPC co-culture system. Four different standard media were employed to compare fully serum-free conditions with humanized cultures that were supplemented with pooled human platelet lysate (pHPL) as a source of platelet-derived growth factors present within the niche. Primary expansion culture with and without niche cell support was followed by colony forming cell (CFC) assays to determine maintenance of clonogenicity. Humanized liquid cultures supplemented with 10% pHPL without niche cell support were more efficient than parallel serum-free cultures under three of four medium conditions resulting in a mean 16–84-fold increase in nucleated cell progeny within 11 days. Both MSCs and EPCs further amplified HPC proliferation, MSC actually up to 341-fold, confirming recently published results. Interestingly, the supportive effect of MSCs or EPCs was constantly more pronounced in humanized pHPL-supplemented compared to completely serum-free cultures, indicating a peculiar role of the natural platelet-derived growth factors in this process. Surprisingly, the combination of equal cumulative numbers of MSCs + EPCs constantly resulted in the highest HPC proliferation. This produced an up to 567-fold increase indicating at least nine population doublings within only 11 days. Under all tested conditions the HPC progeny mainly comprised differentiating myeloid cells. Consecutive CFC assays revealed that liquid culture supplemented with 10%HPL were more efficient than serum-free cultures resulting in a measurable 1.2–4.5 fold CFC expansion under three of four tested medium conditions. Both MSC and EPC initiated a more than 6-fold increase of CFCs. CFC expansion through combined action of MSCs + EPCs resulted in a maximum of 23.5-fold CFC increase in pHPL-supplemented cultures. We demonstrate for the first time that HPC expansion can be modulated in a fully humanized co-culture system based on the use of pHPL as a natural source of human platelet-derived growth factors. Our data indicate that the combination of MSCs + EPCs is at least as efficient in supporting HPC proliferation and CFC amplification as either niche cell compartment alone. We speculate that human platelet-derived factors may avoid premature HSC exhaustion in this system. The humanized co-culture thus provides a novel model system for subtractive analyses of HPC and HSC-niche cell interactions. It also builds the basis for further developments towards more effective animal serum-free HSC expansion strategies.

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