Predominant control of PDGF/PDGF receptor signaling in the migration and proliferation of human adipose‑derived stem cells under culture conditions with a combination of growth factors

Abstract

Human adipose-derived stem cells (hASCs) play important roles in regenerative medicine and tissue engineering. However, their clinical applications are limited because of their instability during cell culture. Platelet lysates (PLTs) contain large amounts of growth factors that are useful for manufacturing cellular products. Platelet-derived growth factor (PDGF) is a major growth factor in PLTs and a potent mitogen in hASCs. To optimize growth conditions, the effects of a combination of growth factors on the promotion of hASC proliferation were investigated. Moreover, PDGF-BB combined with vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) markedly enhanced the viability of hASCs compared with the effects of PDGF-BB alone. Neither VEGF nor HGF had any effect alone. All growth factor receptor inhibitors inhibited cell proliferation. Wound healing assays revealed that VEGF and HGF stimulated PDGF-dependent cell migration. The effects of these growth factors on the activation of their cognate receptors and signaling enzymes were assessed using immunoblotting. Phosphorylation of PDGF receptor (PDGFR)β, VEGF receptor (VEGFR)2 and MET proto-oncogene and receptor tyrosine kinase was induced by PDGF-BB treatment, and was further increased by treatment with PDGF-BB/VEGF and PDGF-BB/HGF. The levels of phospho-ERK1/2 and phospho-p38MAPK were increased by these treatments in parallel. Furthermore, the expression levels of SRY-box transcription factor 2 and peroxisome proliferator-activated receptor g were increased in PDGF-BB-treated cells, and PDGF-BB played a dominant role in spheroid formation. The findings of the present study highlighted that PDGF/PDGFR signaling played a predominant role in the proliferation and migration of hASCs, and suggested that PDGF was responsible for the efficacy of other growth factors when hASCs were cultured with PLTs.

Keywords: cell proliferation; hepatocyte growth factor; human adipose-derived stem cells; platelet-derived growth factor receptor; platelet-derived growth factor-BB; vascular endothelial growth factor.

Figure 1

Growth factor-dependent proliferation of hASCs. The cells were cultured in DMEM with or without the indicated concentrations of (A) PDGF-BB, (B) VEGF, (C) HGF, (D) PDGF-BB/VEGF and (E) PDGF-BB/HGF for 48 h. Cell viability was measured by Cell Counting Kit-8 assay. Data are shown as the mean ± SD (n=4). ^*P<0.05; ^**P<0.01 vs. control. PDGF, platelet-derived growth factor; VEGF, vascular endothelial growth factor; HGF, hepatocyte growth factor; hASCs, human adipose-derived stem cells.

Figure 2

Effect of growth factors on the proliferative ability of hASCs. (A) Representative microscopy images of immunochemical staining for Ki-67 (green)/DAPI (blue) in hASCs cultured with PDGF-BB (20 ng/ml) plus VEGF (1 ng/ml) for 24 h (scale bar, 100 µm). (B) Quantification of Ki67⁺ cells. A total of 300 cells were counted after the indicated treatment and data are expressed as the mean ± SD (n=3). ^*P<0.05; ^**P<0.01 vs. control. hASCs, human adipose-derived stem cells; PDGF, platelet-derived growth factor; VEGF, vascular endothelial growth factor; HGF, hepatocyte growth factor.

Figure 3

Pharmacological inhibition of hASC viability cultured with inhibitors of growth factor receptors. The cells were cultured in DMEM containing (A) PDGF-BB (20 ng/ml), (B) PDGF-BB (20 ng/ml)/VEGF (1 ng/ml) and (C) PDGF-BB (20 ng/ml)/HGF (1 ng/ml), without or with the indicated receptor inhibitors for 48 h. Cell viability was examined by Cell Counting Kit-8 assays. The inhibitors used were imatinib (5 µM), VEGFR tyrosine kinase inhibitor II (10 µM) and tivantinib (0.5 nM). Data are expressed as a percentage of the control value (no inhibitor) and as the mean ± SD (n=4). ^**P<0.01 vs. no inhibitor. hASCs, human adipose-derived stem cells; PDGF, platelet-derived growth factor; VEGF, vascular endothelial growth factor; HGF, hepatocyte growth factor; VEGFR, VEGF receptor.

Figure 4

Activation of growth factor receptors and signal enzymes in growth factor-treated hASCs. hASCs were cultured in DMEM containing 10% FBS, followed by starvation for 16 h. The cells were then incubated in DMEM containing PDGF-BB (20 ng/ml), VEGF (1 ng/ml), HGF (1 ng/ml), PDGF-BB (20 ng/ml)/VEGF (1 ng/ml) or PDGF-BB (20 ng/ml)/HGF (1 ng/ml) for 20 min. The cells then were washed, collected and lysed. Next, cellular proteins were analyzed by SDS-PAGE using 4-15% gels, followed by (A) immunoblotting with the indicated primary antibodies. (B) Ratio of phospho-PDGFRb versus total PDGFRb, (C) ratio of phospho-VEGFR2 versus total VEGFR2, (D) ratio of phospho-c-Met versus total c-Met, (E) ratio of phospho-ERK1/2 versus total ERK1/2 and (F) ratio of phospho-p38 versus total p38 were calculated. Data are presented as the mean ± SD (n=3). ^**P<0.01 vs. control. hASCs, human adipose-derived stem cells; PDGF, platelet-derived growth factor; VEGF, vascular endothelial growth factor; HGF, hepatocyte growth factor.

Figure 5

In vitro hASC wound healing assay. (A) Wounds were created on hASCs seeded in 24-well plates at 90% confluence using a pipette tip, and the cells were then incubated in DMEM without or with PDGF-BB (20 ng/ml), VEGF (1 ng/ml), HGF (1 ng/ml), PDGF-BB (20 ng/ml)/VEGF (1 ng/ml) and PDGF-BB (20 ng/ml)/HGF (1 ng/ml) for 12 and 24 h. Wounds were observed under an optical microscope (magnification, x40). (B) Quantitative analysis of wound closure after 12 and 24 h incubation. Wound healing closure rates were calculated using ImageJ software (version 1.53t). Values are presented as the percentage of wound closure ± SD (n=4). ^*P<0.05; ^**P<0.01 vs. control. hASCs, human adipose-derived stem cells; PDGF, platelet-derived growth factor; VEGF, vascular endothelial growth factor; HGF, hepatocyte growth factor.

Figure 6

Gene expression of differentiation-related genes, including (A) PPARG, (B) RUNX2, (C) COL10A1 and (D) ACTA, and stem cell-related genes, including (E) SOX2 and (F) CD73 in growth factor-treated hASCs. hASCs were incubated in DMEM without or with PDGF-BB (20 ng/ml), VEGF (1 ng/ml), HGF (1 ng/ml), PDGF-BB (20 ng/ml)/VEGF (1 ng/ml) and PDGF-BB (20 ng/ml)/HGF (1 ng/ml), and the cells were cultured for 6 days. The individual medium was changed every 2 days. Total RNA in the cells was isolated and reverse transcription-quantitative PCR was performed. mRNA levels were normalized to GAPDH mRNA expression. Data are presented as the mean ± SD (n=3). ^*P<0.05 vs. control. hASCs, human adipose-derived stem cells; PPARG, peroxisome proliferator-activated receptor γ; RUNX2, RUNX family transcription factor 2; COL10A1, collagen type X α1 chain; ACTA, actin α1; SOX2, SRY-box transcription factor 2.

Figure 7

Spheroid formation of hASCs with PDGF-BB, PDGF-BB/VEGF and PDGF-BB/HGF. (A) Representative microscopy images of spheroids under the indicated conditions. Cells were seeded at 1,000 cells/well in low-attachment plates (96-wells), and cultured with DMEM containing PDGF-BB (20 ng/ml), VEGF (1 ng/ml), HGF (1 ng/ml), PDGF-BB (20 ng/ml)/VEGF (1 ng/ml) and PDGF-BB (20 ng/ml)/HGF (1 ng/ml) for 6 days. The cells were also cultured with serum-free DMEM as a control (scale bar, 1 mm). (B) Quantification of total spheroids distributed by size. The shapes of the spheroids were analyzed by ImageJ software, and spheres <20 µm were excluded. The diameter of spheroids was divided into 20-50, 50-100, 100-200 and >200 µm, and the number of spheroid in each group was shown. Data are presented as the mean ± SD (n=3). ^*P<0.05; ^**P<0.01 vs. control. hASCs, human adipose-derived stem cells; PDGF, platelet-derived growth factor; VEGF, vascular endothelial growth factor; HGF, hepatocyte growth factor.

Figure 8

Gene expression of differentiation-related genes, including (A) PPARG, (B) RUNX2, (C) COL10A1 and (D) ACTA, and stem cell-related genes, including (E) SOX2 and (F) CD73 in growth factor-treated hASC spheroids. Spheroids treated with the indicated growth factors for 6 days were collected by centrifugation at 1,000 x g for 5 min at room temperature. Total RNA in the cells was isolated and reverse transcription-quantitative PCR was performed. Data are presented as the mean ± SD (n=3). ^*P<0.05 vs. control. hASCs, human adipose-derived stem cells; PPARG, peroxisome proliferator-activated receptor γ; RUNX2, RUNX family transcription factor 2; COL10A1, collagen type X α1 chain; ACTA, actin α1; SOX2, SRY-box transcription factor 2.

Figure 9

PDGF-BB/PDGFR signal dominates the stimulation of the migration and proliferation of hASCs via activation of multiple growth factor receptors. VEGF and HGF synergistically enhanced such stimulation. hASCs, human adipose-derived stem cells; PDGF, platelet-derived growth factor; VEGF, vascular endothelial growth factor; HGF, hepatocyte growth factor; PDGFR, PDGF receptor; VEGFR, VEGF receptor; c-Met, HGF receptor; hASCs, human adipose-derived stem cells.