Table 1.
Sequences of primers used for quantitative real-time PCR.
Figure 1.
Isolation and purification of CD146+ HUCPVCs from human umbilical cord.
A. A dissected human umbilical cord vessel. B. Heterogenous population of HUCPVCs digested from the umbilical cord vessel by collagenase I. Scale bar: 100 µm. C. Homogenous population of HUCPVCs purified with CD146+ antibody using MACS system. Scale bar: 50 µm. D. Expression of CD146 in HUCPVCs. Cells were immunostained with anti-CD146 antibody and visualized by fluorescent microscopy. The nucleus was counterstained with DAPI. Scale bar: 50 µm. E. Detection of cell surface markers by flow cytometry analysis. Cells were immunostained with selected cell surface markers CD146, CD44, CD90, CD105, CD34, CD45. The percentage of cell population with positive staining was shown in each figure.
Figure 2.
Multilineage differentiation of CD146+ HUCPVCs.
Osteogenic differentiated CD146+ HUCPVCs at indicated days were fixed and processed for alkaline phosphatase (A) and Alizarin red S (B) staining. Cell morphology was captured by phase contrast microscopy. Scale bar: 100 µm. (C) Relative mRNA expression of osteogenic marker genes including Runx2, Alp, Ocn, Osx, Opn and Col1α1 was detected by quantitative real-time PCR. (D) For induction of adipogenic differentiation, the cells were incubated in adipogenic medium for indicated days and stained with Oil red O. Scale bar: 100 µm. (E) mRNA expression of adipogenic marker genes Cebpα, Ppar-γ and Fabp4 was examined by real-time PCR. (F) For chondrogenic differentiation, the cells were incubated in chondrogenic medium for indicated days and stained with Alican blue. Cell morphology was captured by phase contrast microscopy. Scale bar: 100 µm. (G) The presence of collagen fibers at day 21 of incubation was detected by scanning electron microscope Scale bar: 1 µm. (H) mRNA expression of chondrogenic marker genes Sox-9, Col2α1, Alp and Col10α1 by quantitative real-time PCR. Control: Cells without treatment. Values shown are mean ± SD (n = 3). *P<0.05; **P<0.01.
Figure 3.
Functional engraftment of CD146+ HUCPVCs in the new bone regenerates in the subcutaneous transplantation model and the critical-sized bone defect model in SCID mice.
(A) X-ray image of the ectopic bone formation at 6 weeks following CD146+ HUCPVCs transplantation subcutaneously in the back of SCID mice (n = 3). (B) H&E staining of the ectopic bone section. (C) Immunohistochemistry staining for human specific mitochondria in the newly formed ectopic bone (D) Mouse IgG isotype control. (E) X-ray image of critical sized bone defect region at 6 weeks following CD146+ HUCPVCs transplantation. The newly formed bone is indicated by the white arrow. (F) H&E staining of the new bone regenerate. (G) Immunohistochemistry staining for human specific mitochondria in the new bone regenerate in the critical-sized bone defect model. (H) Mouse IgG isotype control. Red arrow: Osteoblasts; Black arrow: Chondrocytes. All scale bar: 50 µm.
Figure 4.
Hypoxia inhibits osteogenic differentiation of CD146+ HUCPVCs while maintains their multi-differentiation potential.
Osteogenic differentiated CD146+ HUCPVCs at indicated days were fixed and subject to alkaline phosphatase (A) and Alizarin red S (B) staining respectively. Control: Cells without treatment. (C) Relative mRNA expression of osteogenic marker genes including Runx2, Alp, Ocn, Osx, Opn and Col1a1 was detected by quantitative real-time PCR. Multilineage induction of hypoxia expanded CD146+ HUCPVCs was performed using defined conditions as described in Materials and Methods part, and the cells were processed for cytochemistry staining with (D) alkaline phosphatase, (E) Alizarin Red S, (F) Oil Red O, and (G) Alican Blue. Values shown are mean ± SD (n = 3). *P<0.05; **P<0.01. Scale bar: 100 µm.
Figure 5.
Hypoxia increases colony forming efficiency and proliferation of CD146+ HUCPVCs.
(A) Cell morphology captured by phase contrast microscopy (upper) and the representative colonies for each treatment were shown (lower). The colonies in CD146+ HUCPVCs culture under normoxic or hypoxic conditions were visualized by cells staining with crystal violet (B). The total numbers of colonies as well as the average diameters of colonies was quantified in (C) and (D) respectively. (E) BrdU incorporation in CD146+ HUCPVCs after 48 hours of normoxia or hypoxia exposure. N: Normoxia; H: Hypoxia. Values shown are mean ± SD (n = 3). *P<0.05; **P<0.01. Scale bar: 100 µm.
Figure 6.
Expression profiles of stem cell transcription factors in CD146+ HUCPVCs under hypoxia and normoxia.
(A) The table summarizes the fold-change of mRNA expression of stem cell transcription factors in CD146+ HUCPVCs in response to hypoxia. (B) Hierarchical clustering analysis on the PCR array data. (C) Illustration of the mRNA fold-change of the top ten upregulated and downregulated transcription factors in CD146+ HUCPVCs in response to hypoxia.
Figure 7.
Hypoxia inhibits transcriptional suppression of Hif-2α by PPAR-γ in CD146+ HUCPVCs.
Protein expression of PPAR-γ and HIFs (A) as well as pluripotency marker proteins (B) was examined by Western blot analysis. Relative protein expression was quantified by densitometry. The results shown are representative of three independent experiments. (C) Schematic outline of Hif-2α DNA with promoter region harboring the putative binding site of PPAR-γ. Nucleotide sequence for putative binding was shown. The position of the region amplified by PCR after chromatin immunoprecipitation was shown by arrows. (D) ChIP assay. Cells were exposed to normoxic or hypoxic conditions for 24 h before chromatin DNA extraction. The percentage of DNA bound was calculated by dividing the immunoprecipitated DNA with the total amount of DNA purified from the chromatin (input DNA). N: Normoxia; H: Hypoxia. (E) Luciferase activity of HUCPVCs after co-transfection of PPAR-γ expression vector with Hif-2α-pGL3 wild-type or mutated reporter construct. Luciferase activity was measured by dual-luciferase reporter assay (Promega) and was normalized to Renilla luciferase activity. Relative luciferase activity of each sample was compared to that transfected with empty pGL3-basic vector (mock). Values shown are mean ± SD (n = 3). **P<0.01. (F) Western blot analysis of HIF-1α and HIF-2α protein expression upon PPAR-γ overexpression. Cells were transfected with an empty vector (−) or PPAR-γ-expression vector (+) before protein extraction. (G) A suggested model of PPAR-γ and HIF-2α expression in regulation of self-renewal of CD146+ HUCPVCs in response to hypoxia.