[SOX9 enhanced chondrogenic differentiation potential of human umbilical cord mesenchymal stem cells through cellular aggregation].

Objective: To explore the effects of transcription factor SOX9 on chondrogenic differentiation potential of human umbilical cord mesenchymal stem cells (hUC-MSCs).

Methods: hUC-MSCs were harvested from human umbilical cord and their phenotypic characteristics identified by flow cytometry. To confirm their multipotency, hUC-MSCs were induced to differentiate toward adiposity and osteogenesis. After transfection with the packaging lentivirus vectors containing SOX9 in vitro, the expression of green fluorescent protein (GFP) and the efficiency of transfection were detected by fluorescence microscopy. Their cellular proliferation capacity was detected by thiazolyl blue tetrazolium bromide (MTT) assay.hUC-MSCs modified with SOX9 were seeded into monolayer and cultured for 21 days in a defined, serum-free medium supplemented with transforming growth factor (TGF)-β1. The untransduced cells or those transduced with GFP served as the controls. Morphologic changes of hUC-MSCs were observed daily and their chondrogenic differentiation was evaluated by reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunofluorescent staining. And the accumulation of sulfated glycosaminoglycans was detected by Alcian blue staining. Meanwhile, the expressions of collagen I, X and cell adhesion molecule N-cadherin were assayed.

Results: The hUC-MSCs isolated from human umbilical cord stromas exhibited fibroblastic morphology and they were positive for CD29 (95.9%), CD44 (96.5%), CD90 (98.9%), CD105 (94.3%) and negative for hematopoietic stem cells surface markers CD34 (3.0%) and CD45 (2.6%). At Day 21, hUC-MSCs differentiated toward adiposity and osteogenesis. Both oil red O and alkaline phosphatase stains were intensely positive and it confirmed the multilineage potential of hUC-MSCs. An intense expression of GFP was observed under flourescence microscope and the transfection efficiency of cells with Lenti-GFP-SOX9 or Lenti-GFP was more than 90% respectively. SOX9 gene was over-expressed in hUC-MSCs at 48 h post-transduction. The proliferation of hUC-MSCs had no significant effect after the transfection of lentivirus vectors (P > 0.05). In vitro high-density monolayer culture of these SOX9-transfected hUC-MSCs demonstrated that spontaneous cell aggregation appeared at Day 14 of culturing and subsequently generated large cartilage nodules. However there was no phenomenon of cell aggregation occurring in the cells transducted by Lenti-GFP or untransduced vectors. The expressions of collagen II and Aggrecan were higher in SOX9 transducted cells than those in the controls. Alcian blue staining also showed abundant accumulation of sulfated glycosaminoglycans in the SOX9-induced cartilage nodules. The expression of collagen I had no difference in all groups and collagen X was inhibited in SOX9 transduced cells. N-cadherin was strongly up-regulated by SOX9 and might result in cellular aggregation and formation of large cartilage nodules.

Conclusion: SOX9 may enhance the chondrogenic differentiation potential of human umbilical cord mesenchymal stem cells through cellular aggregation.