Key Laboratory for Regenerative Medicine, Ministry of Education, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China ; School of Biomedical Sciences Core Laboratory, The Chinese University of Hong Kong Shenzhen Research Institute, Shenzhen, China.
PLoS One. 2013 Oct 18;8(10):e76153. doi: 10.1371/journal.pone.0076153. eCollection 2013.
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.
人脐带血管周细胞（HUCPVCs）已被认为是细胞再生医学中用于间充质祖细胞的替代来源。然而，这些细胞的生物学特性仍有待很好地描述。在本研究中，我们通过 CD146（+）周细胞标志物分离和分选 HUCPVCs。纯化的 CD146（+）HUCPVCs 在体外可有效地诱导分化为成骨细胞、软骨细胞和脂肪细胞谱系。在严重联合免疫缺陷（SCID）小鼠皮下移植 CD146（+）HUCPVCs-Gelfoam-藻酸盐 3D 复合物 6 周后，观察到供体来源的新骨基质中嵌入成骨细胞。在 SCID 小鼠的临界大小骨缺损模型中进一步证实了 CD146（+）HUCPVCs 在新骨再生中的功能植入。与常氧条件相比，缺氧条件抑制成骨分化，而增加 CD146（+）HUCPVCs 的细胞增殖和集落形成效率。再氧合恢复了 CD146（+）HUCPVCs 的多向分化潜能。Western blot 分析显示，缺氧条件下 CD146（+）HUCPVCs 中 HIF-1α、HIF-2α 和 OCT-4 蛋白表达上调，而 SOX2 和 NANOG 表达无明显变化。通过 PCR 阵列分析比较了常氧和缺氧条件下细胞间干细胞转录因子的基因表达谱。有趣的是，PPAR-γ 在 mRNA 表达中显著下调（20 倍），在 Hif-2α 基因启动子区域发现具有潜在的结合位点。染色质免疫沉淀测定证实了 PPAR-γ 蛋白与 Hif-2α 启动子的结合，并且缺氧处理抑制了这种结合。荧光素酶报告基因测定显示，Hif-2α 启动子活性受 PPAR 表达抑制。因此，PPAR-γ 可能参与调节 HIF-2α 以维持干细胞特性，并促进 CD146（+）HUCPVCs 在缺氧反应中的扩增。CD146（+）HUCPVCs 可能是骨再生的潜在自体细胞来源。
