CD146+ Human Umbilical Cord Perivascular Cells Maintain Stemness under Hypoxia and as a Cell Source for Skeletal Regeneration

Wing Pui Tsang,Kenneth Ka Ho Lee,Fengjie Zhang,Gang Li,Chao Wan,Po Lam Kwok,Mei Kuen Tang,Wai-Yee Chan,Yinglan Shu,Kai Ming Chan,Rajasingh Johnson

doi:10.1371/journal.pone.0076153

Abstract

The human umbilical cord perivascular cells (HUCPVCs) have been considered as an alternative source of mesenchymal progenitors for cell based regenerative medicine. However, the biological properties of these cells remain to be well characterized. In the present study, HUCPVCs were isolated and sorted by CD146+ pericyte marker. The purified CD146+ HUCPVCs were induced to differentiate efficiently into osteoblast, chondrocyte and adipocyte lineages in vitro. Six weeks following subcutaneous transplantation of CD146+ HUCPVCs-Gelfoam-alginate 3D complexes in severe combined immunodeficiency (SCID) mice, newly formed bone matrix with embedded osteocytes of donor origin was observed. The functional engraftment of CD146+ HUCPVCs in the new bone regenerates was further confirmed in a critical-sized bone defect model in SCID mice. Hypoxic conditions suppressed osteogenic differentiation while increased cell proliferation and colony-forming efficiency of CD146+ HUCPVCs as compared to that under normoxic conditions. Re-oxygenation restored the multi-differentiation potential of the CD146+ HUCPVCs. Western blot analysis revealed an upregulation of HIF-1α, HIF-2α, and OCT-4 protein expression in CD146+ HUCPVCs under hypoxia, while there was no remarkable change in SOX2 and NANOG expression. The gene expression profiles of stem cell transcription factors between cells treated by normoxia and hypoxic conditions were compared by PCR array analysis. Intriguingly, PPAR-γ was dramatically downregulated (20-fold) in mRNA expression under hypoxia, and was revealed to possess a putative binding site in the Hif-2α gene promoter region. Chromatin immunoprecipitation assays confirmed the binding of PPAR-γ protein to the Hif-2α promoter and the binding was suppressed by hypoxia treatment. Luciferase reporter assay showed that the Hif-2α promoter activity was suppressed by PPAR expression. Thus, PPAR-γ may involve in the regulation of HIF-2α for stemness maintenance and promoting the expansion of CD146+ HUCPVCs in response to hypoxia. CD146+ HUCPVCs may serve as a potential autologous cell source for bone regeneration.

Highlights

Adult stem cells represent a promising model for regenerative medicine and tissue engineering
We provided the evidence of the occupancy and transcriptional suppression of the Hif-2a promoter by PPARc, indicating that the expansion of CD146+ human umbilical cord perivascular cells (HUCPVCs) under hypoxia was mediated by coordinated suppression of PPARc and upregulation of hypoxia inducible factors (HIFs)-2a expression
The HUCPVCs were purified by CD146-conjugated magnetic beads

Summary

Introduction

Adult stem cells represent a promising model for regenerative medicine and tissue engineering. Current sources of MSCs require invasive procurement procedures and provide relatively low frequency of progenitors. To overcome these limitations, human umbilical cord perivascular cells (HUCPVCs) have been considered as an alternative source of mesenchymal progenitors. Transplanted HUCPVCs contribute to tissue healing and matrix generation themselves, and are able to recruit resident progenitors to rapidly repair the damaged tissues [14]. These evidences indicate that HUCPVCs may function as a potential cell source for tissue regeneration therapeutics

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Results

Conclusion