Abstract
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-1α (Hif-1α), the master regulatory protein of hypoxia, in MSCs remain unclear, due to the limited span of Hif-1α stabilization and the complex network of hypoxic responses. In this study, to further define the significance of Hif-1α 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-1α during such long-term biological processes. Using this model, we show that the stabilization of Hif-1α 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-1α 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-1α proteins in maintaining the primitive state of primary MSCs and on the cellular heterogeneities in hypoxic responses among MSC populations.
Highlights
IntroductionMesenchymal stromal cells (MSCs) can be produced from various tissues, including bone marrow (BM), adipose tissues or placenta, ex vivo culture-derived MSCs display several common surface phenotypes, including the expression of CD90 (Thy-1), CD 166 (SB10/ALCAM), CD73 (SH3) and CD105 (SH2, endoglin), and the absence of the hematopoietic marker (CD45), HLA-DR and co-stimulatory molecules, such as B7
Establishment of human Mesenchymal stromal cells (MSCs) expressing sustained high levels of hypoxia-inducible factor-1a (Hif-1a) proteins To explore the role of Hif-1a during the long-term period of biological process of MSCs, we first compared the kinetics of protein accumulation for various forms of Hif-1a proteins
Similar differences in protein stability between the MSCs transduced with WT and Hif-1a PA mutant were observed under 5% hypoxic conditions (Supplementary Figure 1A)
Summary
MSCs can be produced from various tissues, including BM, adipose tissues or placenta, ex vivo culture-derived MSCs display several common surface phenotypes, including the expression of CD90 (Thy-1), CD 166 (SB10/ALCAM), CD73 (SH3) and CD105 (SH2, endoglin), and the absence of the hematopoietic marker (CD45), HLA-DR and co-stimulatory molecules, such as B7. Despite these common features, significant heterogeneities have been reported for cultured MSCs in terms of their morphology, proliferation and differentiation potentials.[14,15,16] heterogeneities were observed in their gene expression and differentiation potential with successive culture passages,[17,18] raising the possibility that such heterogeneities could be generated. Factors and underlying mechanisms involved in the regulation of the biological characteristics of ex vivo expanded MSCs have been of major interest in the field
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