Human umbilical cord and dental pulp-derived mesenchymal stem cells: biological characteristics and potential roles in vitro and in vivo

Abstract

Mesenchymal stem/stromal cells (MSCs) have a wide application in cell‑based therapies and tissue engineering. In the present study, the differentiation, survivin (SVV)‑modified effects and molecular basis of human umbilical cord‑derived MSCs (HUMSCs) and dental pulp‑derived stem cells (DPSCs) were investigated. The HUMSCs were found to differentiate into adipocytes more readily than the DPSCs and the HUMSCs and DPSCs were each able to differentiate into osteoblasts and chondroblasts. Following modification of the MSCs by SVV, the secretion of SVV in the modified HUMSCs was significantly higher compared with that in the modified DPSCs. In vivo, survival of the SVV‑modified DPSCs was observed at 4 and 14 days after intrastriatal transplantation, as was the expression of SVV and differentiation into astrocytes. The gene expression profiles of the control and modified HUMSCs and DPSCs were compared using RNA sequencing and an association was observed between gene expression and variability in cell line function. These findings provide novel information regarding the differences between HUMSCs and DPSCs and insight into optimal cell sources for therapeutic applications.

Figure 1

Phenotypic characterization of HUMSCs and DPSCs. (A) Passage 5 DPSCs were more similar to fibroblasts than the HUMSCs. Scale bar: 500 μm. (B) 96.5% of the HUMSCs were positive for CD73, CD90 and CD105, but negative for CD34, CD45, CD11b or CD14, CD19, CD79α and HLA-DR. (C) 99.2% of the DPSCs were positive for CD73, CD90 and CD105, but negative for CD34, CD45, CD11b or CD14, CD19, CD79α and HLA-DR. HUMSCs, human umbilical cord-derived mesenchymal stem cells; DPSCs, dental pulp-derived stem cells; FITC, fluorescein isothiocyanate; CD, cluster of differentiation.

Figure 2

Adipogenic, osteogenic and chondrogenic differentiation of the (A-C) HUMSCs and (D-F) DPSCs, respectively. Lipid droplets, stained using oil red O, were present in the (A) HUMSCs, but not the (D) DPSCs (Scale bar: 250 μm). Osteogenic differentiation in the (B) HUMSCs and (E) DPSCs was detected using alizarin red S staining of calcium precipitation (Scale bar, 250 μm). Aggrecan, a chondrogenic marker, was highly expressed in the (C) HUMSCs and (F) DPSCs (Scale bar, 200 μm). HUMSCs, human umbilical cord-derived mesenchymal stem cells; DPSCs, dental pulp-derived stem cells.

Figure 3

Gene transduction. The MSCs were transduced with a pLVX-IRES-ZsGreen1 vector containing an inserted full length cDNA of SVV. Scale bar, 500 μm. Expression of green fluorescent protein in the SVV-modified MSCs indicated that the efficiency of gene transduction was >90%. MSCs, mesenchymal stem cells; HUMSCs, human umbilical cord-derived MSCs; DPSCs, dental pulp-derived stem cells; SVV, survivin.

Figure 4

Protein levels of SVV are significantly increased in the SVV-modified HUMSCs and DPSCs. (A) Protein levels of SVV were measured in the cell culture supernatant using an enzyme-linked immunosorbent assay. (B) Representative western blot analysis of the control, SVV-modified HUMSCs and DPSCs. The level of GAPDH was used as an internal control. Lanes: 1, HUMSCs; 2, SVV-modified HUMSCs; 3, DPSCs; 4, SVV-modified DPSCs. (C) Quantitative analysis revealed that the modified HUMSCs and DPSCs had a significantly higher expression of SVV. (^*P<0.05, compared with the control and ^#P<0.05, compared with the SVV-modified DPSCs. HUMSCs, human umbilical cord-derived mesechymal stem cells; DPSCs, dental pulp-derived stem cells; SVV, survivin.

Figure 5

Survival of the control and DP-SVV cells in the wild-type C57B/6 mice 4 and 14 days after transplantation. Microscopic analysis of the immunofluorescence-labeled striatal sections in the cell-transplanted mice. Transplanted cells expressing GFP (green) were immunostained against SVV (red) and counterstained with DAPI (blue). Scale bar, 100 μm. DPSCs, dental pulp-derived stem cells; SVV, survivin; DP-SVV, SVV-modified DPSCs; PBS, phosphate-buffered saline; GFP, green fluorescent protein; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 6

Quantification of GFP and SVV fluorescence intensity of the transplanted control DPSCs and DP-SVV cells in the striata 4 and 14 days after transplantation. (A) GFP fluorescence intensity was not significantly different between the DPSCs and DP-SVV on day 4, but was decreased on day 14. The GFP fluorescence intensity of DP-SVV was more marked compared with that of the control DPSCs. (B) SVV fluorescence intensity of the DP-SVV cells was more marked compared with that of the control DPSCs on days 4 and 14. ^*P<0.05, compared with the control DPSCs. DPSCs, dental pulp-derived stem cells; SVV, survivin; DP-SVV, SVV-modified DPSCs; GFP, green fluorescent protein.

Figure 7

Immunohistochemical analysis of the control and DP-SVV cells using NeuN. Microscopic images revealed no GFP-labeled cells coexpressing NeuN (red) in the striatum at days 4 or 14 post-transplantation. Scale bar, 100 μm. DPSCs, dental pulp-derived stem cells; SVV, survivin; DP-SVV, SVV-modified DPSCs; PBS, phosphate-buffered saline; GFP, green fluorescent protein; NeuN, neuronal nuclei.

Figure 8

Immunohistochemical analysis of control and DP-SVV cells using GFAP. Microscopic images revealed a few GFP-labeled cells coexpressing GFAP (red) in the DP-SVV group 14 days after transplantation. Scale bar, 100 μm. DPSCs, dental pulp-derived stem cells; SVV, survivin; DP-SVV, SVV-modified DPSCs; GFAP, glial fibrillary acidic protein; PBS, phosphate-buffered saline; GFP, green fluorescent protein.

Figure 9

Heat map of group-specific highly expressed genes with RPKM values >100. (A) HUMSC-specific high expression genes. (B) HU-SVV-specific highly expressed genes. (C) DPSC-specific highly expressed genes. (D) DP-SVV-specific highly expressed genes. RPKM, reads per kilobase of exon per million mapped reads; HUMSCs, human umbilical cord-derived mesenchymal stem cells; DPSCs, dental pulp-derived stem cells; SVV, survivin; HU-SVV, SVV-modified HUMScs; DP-SVV, SVV-modified DPSCs.

Figure 10

Heat map of genes associated with stem cell differentiation and cytokine-cytokine receptor interaction. (A) Genes associated with stem cell differentiation with RPKM values >5 were compared through RPKM value analysis in the HUMSCs, HU-SVV, DPSCs and DP-SVV cells. (B) Genes associated wirth cytokine-cytokine receptor interactions with RPKM values >5 in the four cell lines. RPKM, reads per kilobase of exon per million mapped reads; HUMSCs, human umbilical cord-derived mesenchymal stem cells; DPSCs, dental pulp-derived stem cells; SVV, survivin; HU-SVV, SVV-modified HUMScs; DP-SVV, SVV-modified DPSCs; TNFRSF, tumor necrosis factor receptor superfamily; VEGF, vascular endothelial growth factor.