Irradiated mesenchymal stem cells improve the ex vivo expansion of hematopoietic progenitors by partly mimicking the bone marrow endosteal environment

Mesenchymal Stem Cells (MSCs) regulate the growth and differentiation of Hematopoietic Progenitor cells (HPCs) through the release of soluble factors or through their differentiation into osteoblasts. We recently demonstrated that expansion of megakaryocyte (MK) progenitors ex vivo had reached a plateau when CD34+ cells were grown with two optimized cytokine cocktails developed for the growth of MK. Hence, we sought to determine whether co-culture of CD34+ cells with Bone Marrow (BM) MSCs could further increase the expansion of myeloid and MK progenitors. First, we tested the impact of cell–cell contact and pre-irradiation treatment of the MSCs to identify the condition that best supports HPC expansion. This screen revealed that HPC expansions were generally greater in the non-contact conditions, and that pre-irradiation of the MSCs appeared to be of added benefits. Improved expansion of both myeloid and MK progenitors in co-culture with irradiated MSCs without contact was subsequently confirmed. Next, cytokine array profiling was carried out to investigate why irradiation promoted progenitor expansion. This revealed that the levels of as many as 33 factors were potentially altered. ELISA confirmed the significant up regulation of NT-3 and IGFBP-2. Since, these factors are known to be released by and important for osteogenic and endothelial cells, we investigated and confirmed that irradiation of MSCs induced their rapid differentiation into osteogenic-like cells, but not into endothelial-like cells. Supporting this finding, expansions of myeloid and MK progenitors were increased when CD34+ cells were co-culture with MSCs-derived osteoblasts. Altogether, these results indicate that the improved expansion of HPCs obtained with irradiated MSCs is due in part to their differentiation into osteoblast-like cells, thereby recreating an endosteal-like environment that provides improved support for HPCs expansion.

► Improved expansion of UCB myeloid and megakaryocyte progenitors is obtained when MSCs are irradiated prior to co-culture.
► Irradiation treatment of MSCs modulated the secretion of up to 33 candidate growth factors.
► NT-3 and IGFBP-2 levels were significantly increased in conditioned media from irradiated MSCs.
► Irradiation of MSCs was shown to induce their rapid differentiation into osteogenic-like cells.
► We confirmed that co-culture with osteoblasts improved expansions of hematopoietic progenitors in co-culture.