Hypoxia Preconditioning of Human MSCs: a Direct Evidence of HIF-1α and Collagen Type XV Correlation

Abstract

Background/Aims: Mesenchymal stromal cells (MSCs) hold considerable promise in bone tissue engineering, but their poor survival and potency when in vivo implanted limits their therapeutic potential. For this reason, the study on culture conditions and cellular signals that can influence the potential therapeutic outcomes of MSCs have received considerable attention in recent years. Cell maintenance under hypoxic conditions, in particular for a short period, is beneficial for MSCs, as low O₂ tension is similar to that present in the physiologic niche, however the precise mechanism through which hypoxia preconditioning affects these cells remains unclear. Methods: In order to explore what happens during the first 48 h of hypoxia preconditioning in human MSCs (hMSCs) from bone marrow, the cells were exposed to 1.5% O₂ tension in the X3 Hypoxia Hood and Culture Combo – Xvivo System device. The expression modulation of critical genes which could be good markers of increased osteopotency has been investigated by Western blot, immunufluorescence and ELISA. Luciferase reporter assay and Chromatin immunoprecipitation was used to investigate the regulation of the expression of Collagen type XV (ColXV) gene. Results: We identified ColXV as a new low O₂ tension sensitive gene, and provided a novel mechanistic evidence that directly HIF-1α (hypoxia-inducible factor-1 alpha) mediates ColXV expression in response to hypoxia, since it was found specifically in vivo recruited at ColXV promoter, in hypoxia-preconditioned hMSCs. This finding, together the evidence that also Runx2, VEGF and FGF-2 expression increased in hypoxia preconditioned hMSCs, is consistent with the possibility that increased ColXV expression in response to hypoxia is mediated by an early network that supports the osteogenic potential of the cells. Conclusion: These results add useful information to understand the role of a still little investigated collagen such as ColXV, and identify ColXV as a marker of successful hypoxia preconditioning. As a whole, our data give further evidence that hypoxia preconditioned hMSCs have greater osteopotency than normal hMSCs, and that the effects of hypoxic regulation of hMSCs activities should be considered before they are clinically applied.

Pro-Osteogenic Properties of Violina pumpkin (Cucurbita moschata) Leaf Extracts: Data from In Vitro Human Primary Cell Cultures

“…The ability of hOBs to migrate to the wounded area was assessed using the scratch assay method [ 21 ]. Cells were seeded into 24-well plates and incubated with complete medium at 37 °C and 5% CO 2 .…”

Vadadustat, a HIF Prolyl Hydroxylase Inhibitor, Improves Immunomodulatory Properties of Human Mesenchymal Stromal Cells

“…However, MSCs are routinely cultured under 21% oxygen pressure conditions that several times exceeds the physiological level available in their natural niches (which ranges from 1% to 7% O 2 ) [6][7][8]. There are several reports confirming the beneficial effect of hypoxia and hypoxic preconditioning on migration, regenerative potential, proangiogenic activity and expanded survival of MSCs [9][10][11][12][13][14]. However, culturing cells under low oxygen conditions is demanding, has some limitations and multiplies the costs of MSCs culture.…”

Osteoarthritic Milieu Affects Adipose‐Derived Mesenchymal Stromal Cells

“…To better understand their function in the local environment, it is necessary to evaluate whether MSC also tolerate lower oxygen concentrations (<2%) . In fact, it has been demonstrated that hypoxic conditions (1% O 2 ) affect MSC function by reducing their senescence, increasing proliferation, and maintaining the differentiation properties . In addition, a hypoxic environment increases the expression of chemokine receptors such as CXCR4 and CXCR7 …”