Constitutive stabilization of hypoxia-inducible factor alpha selectively promotes the self-renewal of mesenchymal progenitors and maintains mesenchymal stromal cells in an undifferentiated state

With the increasing use of culture-expanded mesenchymal stromal cells (MSCs) for cell therapies, factors that regulate the cellular characteristics of MSCs have been of major interest. Oxygen concentration has been shown to influence the functions of MSCs, as well as other normal and malignant stem cells. However, the underlying mechanisms of hypoxic responses and the precise role of hypoxia-inducible factor-1alpha (Hif-1alpha), the master regulatory protein of hypoxia, in MSCs remain unclear, due to the limited span of Hif-1alpha stabilization and the complex network of hypoxic responses. In this study, to further define the significance of Hif-1alpha in MSC function during their self-renewal and terminal differentiation, we established adult bone marrow (BM)-derived MSCs that are able to sustain high level expression of ubiquitin-resistant Hif-1alpha during such long-term biological processes. Using this model, we show that the stabilization of Hif-1alpha proteins exerts a selective influence on colony-forming mesenchymal progenitors promoting their self-renewal and proliferation, without affecting the proliferation of the MSC mass population. Moreover, Hif-1alpha stabilization in MSCs led to the induction of pluripotent genes (oct-4 and klf-4) and the inhibition of their terminal differentiation into osteogenic and adipogenic lineages. These results provide insights into the previously unrecognized roles of Hif-1alpha proteins in maintaining the primitive state of primary MSCs and on the cellular heterogeneities in hypoxic responses among MSC populations.

Intramarrow injection of beta-catenin-activated, but not naive mesenchymal stromal cells stimulates self-renewal of hematopoietic stem cells in bone marrow

Bone marrow mesenchymal stromal cells (MSCs) have been implicated in the microenvironmental support of hematopoietic stem cells (HSCs) and often co-transplanted with HSCs to facilitate recovery of ablated bone marrows. However, the precise effect of transplanted MSCs on HSC regeneration remains unclear because the kinetics of HSC self-renewal in vivo after co-transplantation has not been monitored. In this study, we examined the effects of intrafemoral injection of MSCs on HSC self-renewal in rigorous competitive repopulating unit (CRU) assays using congenic transplantation models in which stromal progenitors (CFU-F) were ablated by irradiation. Interestingly, naive MSCs injected into femur contributed to the reconstitution of a stromal niche in the ablated bone marrows, but did not exert a stimulatory effect on the in-vivo self-renewal of co-transplanted HSCs regardless of the transplantation methods. In contrast, HSC self-renewal was four-fold higher in bone marrows intrafemorally injected with beta-catenin-activated MSCs. These results reveal that naive MSCs lack a stimulatory effect on HSC self-renewal in-vivo and that stroma must be activated during recoveries of bone marrows. Stromal targeting of wnt/beta-catenin signals may be a strategy to activate such a stem cell niche for efficient regeneration of bone marrow HSCs.