Effects of TGF- β 1 Overexpression on Biological Characteristics of Human Dental Pulp-derived Mesenchymal Stromal Cells

Abstract

Objective: The aim of our study was to investigate the effect of Transforming growth factor beta-1 (TGF-β1) gene therapy on the surface markers, multilineage differentiation, viability, apoptosis, cell cycle, DNA damage and senescence of human Dental Pulp-derived Mesenchymal Stromal Cells (hDPSC).

Methods: hDPSCs were isolated from human teeth, and were cultured with 20% Fetal Bovine Serum (FBS) in minimum essential media-alpha (α-MEM). TGF-β1 gene transfer into hDPSCs was performed by electroporation method after the plasmid was prepared. The transfection efficiency was achieved by using western blot and flow cytometry analyses and GFP transfection. Mesenchymal stem cell (MSC) markers, multilineage differentiation, cell proliferation, apoptosis, cell cycle, DNA damage and cellular senescence assays were performed by comparing the transfected and non-transfected cells. Statistical analyses were performed using GraphPad Prism.

Results: Strong expression of TGF-β1 in pCMV-TGF-β1-transfected hDPSCs was detected in flow cytometry analysis. TGF-β1 transfection efficiency was measured as 95%. Western blot analysis showed that TGF-β1 protein levels increased at third and sixth days in pCMV-TGF-β1-transfected hDPSCs. The continuous TGF-β1 overexpression in hDPSCs did not influence the immunophenotype and surface marker expression of MSCs. Our results showed that TGF-β1 increased osteogenic and chondrogenic differentiation, but decreased adipogenic differentiation. Overexpression of TGF-β1 increased the proliferation rate and decreased total apoptosis in hDPSCs (p<0.05). The number of cells at “S” phase was higher with TGF-β1 transfection (p＜0.05). Cellular senescence decreased in TGF-β1 transfected group (p＜0.05).

Conclusions: These results reflect that TGF-β1 has major impact on MSC differentiation. TGF-β1 transfection has positive effect on proliferation, cell cycle, and prevents cellular senescence and apoptosis.

Keywords: Apoptosis; Dental Pulp-derived Mesenchymal Stem Cells; Gene therapy; Multilineage differentiation; Proliferation; TGF-β1.

Fig. 1

Continuous overexpression of TGF-β1 in hDPSCs and morphology in monolayer culture. Morphology of non-transfected hDPSCs (A) and TGF-β1 transfected hDPSCs under inverted microscope (Leica, Germany) (B). Fluorescence microscope examination showing the efficiency after pCMV-GFP transfection with 1,200 v, 20 ms, 1pulse (C). After transfection at 48 h, flow cytometry analysis for TGFβ1 expression between non-transfected hDPSCs and TGF-β1 transfected hDPSCs. Transfection efficiency was found 95% (D). Western blot analysis for TGF-β1 protein detection after transfection. TGF-β1 protein increasing at 3 and 6 days in TGFβ1 transfected hDPSCs (E). Hygromicin-B resistant TGF-β1 transfected hDPSCs (F). Viability test between non-transfected hDPSCs and TGF-β1 transfected hDPSCs with Count & Viability assay kit (Muse, Millipore, USA), The values shown are the means±SD from three independent experiments, ***p=0.0005 (G). Agarose gel electrophoresis of pCMV-TGFβ1 plasmid DNA amplified in E.coli DH5α before transfection into hDPSCs. Red ring shown that used for transfection into hDPSCs (H). Microscope magnification are 10× and scale bar is 20μm for figure 1A and 1B. For figure 1C microscope magnification is 20×, scale bar is 50μm.

Fig. 2

Flow cytometer analysis showed that non-transfected DPSCs (A) and TGF-β1 transfected DPSCs (B) were positive for CD90, CD44, CD105 and CD73, which are MSC specific markers and were negative for CD34, CD45, CD11b and HLA-DR, which are hematopoietic stem cell.

Fig. 3

Osteogenic and chondrogenic differentiation was increased remarkable by overexpression of TGF-β1 in DPSCs. However, adipogenic differentiation was reduced significantly in TGF-β1 transfected DPSCs compare with non-transfected hDPSCs. Particularly adipogenic differentiation was impaired by TGF-β 1. Osteogenic differentiation and alizarin red staining (A), Chondrogenic differentiation and safranin-o staining (B), Graphic show adipored assay fluorimetric measurement results for adipogenic differentiation (C). Microscope magnifications are 4×. Scale bar is 100 μm. *p<0.05.

Fig. 4

MTT assays results for 5 days were shown that proliferation significantly increased in TGF-β1 transfected cells (p<0.05) (A). Early, late and total apoptosis results between TGF-β1 transfected hDPSCs and non-transfected hDPSCs at 7 days, n=3, p<0.05 (B~D).

Fig. 5

TGF-β1 gene transfection was increased the number of cells at the S phase in hDPSCs, p<0.05 (A). Electroporation performed for transfection increased DNA damage in a small amount but this was no statistically significant difference, p>0.05 (B, C). Cellular senescence decreased in TGF-β1 transfected group in a statistically significant form. p<0.05 (D).