Crosstalk between adipose-derived stem cells and chondrocytes: when growth factors matter

Abstract

Adipose-derived stem cells (ASCs) and mesenchymal stem cells are promising for tissue repair because of their multilineage differentiation capacity. Our previous data confirmed that the implantation of mixed ASCs and chondrocytes into cartilage defects induced desirable in vivo healing outcomes. However, the paracrine action of ASCs on chondrocytes needs to be further elucidated. In this study, we established a co-culture system to achieve cell-to-cell and cell-to-tissue crosstalk and explored the soluble growth factors in both ASCs and chondrocytes supplemented with 1% fetal bovine serum to mimic the physiological microenvironment. In ASCs, we screened for growth factors by semi-quantitative PCR and quantitative real-time PCR and found that the expression of bone morphogenetic protein 2 (BMP-2), vascular endothelial growth factor B (VEGFB), hypoxia inducible factor-1α (HIF-1α), fibroblast growth factor-2 (FGF-2), and transforming growth factor-β1 significantly increased after co-culture in comparison with mono-culture. In chondrocytes, VEGFA was significantly enhanced after co-culture. Unexpectedly, the expression of collagen II and aggrecan was significantly down-regulated in the co-culture group compared with the mono-culture group. Meanwhile, among all the growth factors screened, we found that the BMP family members BMP-2, BMP-4, and BMP-5 were down-regulated and that VEGFB, HIF-1α, FGF-2, and PDGF were significantly decreased after co-culture. These results suggest that crosstalk between ASCs and chondrocytes is a pathway through the regulated growth factors that might have potential in cartilage repair and regeneration and could be useful for tissue engineering.

Figure 1

Cell morphologies and cell behaviors of ASCs and chondrocytes. (a) Cell morphologies and cytoskeleton staining of ASCs and chondrocytes after 7 days of mono- and co-culture. Cell morphological features are shown in phase-contrast (upper) and with immune-fluorescent staining for the cytoskeleton (lower). (b) Cell migration assay of ASCs and chondrocytes by trans-well chamber (left lane). Cell migration rates after co-culture at 24 h by RTCA confirmed the results by trans-well chamber. *P < 0.05, compared with the mono-culture control. The data were represented as the means ± SD (n = 3). (c) Cell proliferation of ASCs and chondrocytes after co-culture, detected by RTCA. No significant differences were found between the mono- and co-culture groups. NS denotes no significant difference. The data were represented as the means ± SD (n = 3).

Figure 2

Co-culture with ASCs down-regulates specific genes in chondrocytes. (a) Col II and AGC in ASCs and chondrocytes after mono- and co-culture, as detected by semi-quantitative PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-Actin were used as the internal controls. The gels shown are representative of three different experiments (n = 3). The cultured cell samples were taken at 1, 2, 3, 5, and 7 d. (b) Quantitative real-time PCR confirmed the different expression of Col II and AGC in both ASCs and chondrocytes at the fifth day. GAPDH was used as the internal control. The △Ct method was used to measure the fold changes. The data presented are the means of three different experiments (n = 3). Col II and AGC show no expression in ASCs but lower expression in chondrocytes co-cultured with ASCs. ***Represents P < 0.001. The error bar reflects the SD.

Figure 3

The BMP family is modulated in both ASCs and chondrocytes after co-culture. (a) Variation in gene expression of the BMP family members, BMP-2, BMP-4, BMP-5, BMP-6, and BMP-7, in ASCs and chondrocytes after mono- and co-culture, is revealed by semi-quantitative PCR. GAPDH and β-Actin were used as the internal controls. The gels shown are represent ative of three different experiments (n = 3). The cultured cell samples were taken from 1, 2, 3, 5, and 7 d. (b) Quantitative real-time PCR confirmed the different expression of BMPs in both ASCs and chondrocytes. GAPDH was used as the internal control. The △Ct method was used to calculate the fold changes. The data presented are the means of three different experiments (n = 3). BMP-4/-5/-7 in ASCs and BMP-6/-7 in chondrocytes showed no expression, consistent with the results of semi-quantitative PCR. The cultured cell samples were taken on the fifth day. The *, **, and *** represent P < 0.05, P < 0.01, and P < 0.000 1, respectively. The error bar reflects the SD.

Figure 4

VEGF members and vascular-related growth factors are regulated in both ASCs and chondrocytes after co-culture. (a) The VEGFs include VEGFA and VEGFB and the FGFs include FGF-1 and FGF-2. VEGFs, HIF-1α, and FGFs in ASCs and chondrocytes after mono- and co-culture were detected by semi-quantitative PCR. GAPDH and β-Actin were used as the internal controls. The gels shown are representative of three different experiments (n = 3). The cultured cell samples were taken from 1, 2, 3, 5, and 7 d. (b) Quantitative real-time PCR confirmed the differential expression of VEGFs, HIF-1α, and FGFs in both ASCs and chondrocytes on the fifth day. GAPDH was used as the internal control. The △Ct method was used to calculate the fold changes. The data presented are the means of three different experiments (n = 3). The *, **, and *** represent P < 0.05, P < 0.01, and P < 0.000 1, respectively. The error bar reflects the SD.

Figure 5

Relevant growth factors have varied expression in ASCs and chondrocytes after co-culture. (a) Semi-quantitative PCR revealed variations in IGF-1, PDGF, EGF, VE-ca, and TGF-β₁ in ASCs and chondrocytes after mono- and co-culture. GAPDH and β-Actin were used as the internal controls. The gels shown are representative of three different experiments (n = 3). The cultured cell samples were taken from 1, 2, 3, 5, and 7 d. (b) Quantitative real-time PCR confirmed the differential expressions of relevant growth factors in both ASCs and chondrocytes on the fifth day. The △Ct method was used to calculate the fold changes. The data presented are the means of three different experiments (n = 3). EGF and VE-ca showed no expression in ASCs and chondrocytes. The *, **, and *** represent P < 0.05, P < 0.01, and P < 0.000 1, respectively. The error bar reflects the SD.