CD146+ human umbilical cord perivascular cells maintain stemness under hypoxia and as a cell source for skeletal regeneration

PLoS One. 2013 Oct 18;8(10):e76153. doi: 10.1371/journal.pone.0076153. eCollection 2013.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Basic Helix-Loop-Helix Transcription Factors / genetics
  • Basic Helix-Loop-Helix Transcription Factors / metabolism
  • Bone Regeneration*
  • CD146 Antigen / metabolism*
  • Cell Differentiation
  • Cell Hypoxia
  • Cell Lineage
  • Cell Proliferation
  • Cell Separation
  • Cluster Analysis
  • Colony-Forming Units Assay
  • Gene Expression Profiling
  • Gene Expression Regulation
  • Humans
  • Mesenchymal Stem Cell Transplantation
  • Mesenchymal Stem Cells / cytology*
  • Mesenchymal Stem Cells / metabolism*
  • Mice
  • Models, Animal
  • Osteogenesis
  • PPAR gamma / genetics
  • PPAR gamma / metabolism
  • Transcription Factors / genetics
  • Transcription Factors / metabolism
  • Transcriptome
  • Umbilical Cord / cytology*

Substances

  • Basic Helix-Loop-Helix Transcription Factors
  • CD146 Antigen
  • PPAR gamma
  • Transcription Factors
  • endothelial PAS domain-containing protein 1

Grant support

The study was partly supported by Hong Kong Research Grant Council General Research Fund (CUHK 475910), NSFC-RGC Joint Research Scheme (CUHK445/10), and The Chinese University of Hong Kong School of Biomedical Sciences Start-up Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.