Transwell contact co-culture promotes growth and differentiation of single-dissociated iPSCs

AIM：To investigate the promoting role of Transwell contact co-culture system in the growth and differentiation of single-dissociated induced pluripotent stem cells (iPSCs). METHODS：Bovine corneal endothelial cells (CECs) at passage 1~2 (P1~2) were seeded on the underside of Transwell inserts placed into culture plates and were cultured in 37 ℃ and 5% CO2 for 8 h. Accutase digestion and 40 μm filter process disaggregated colony-aggregated iPSCs into single-dissociated iPSCs, and the cells were seeded on the inside of Transwell inserts with CECs in medium of mTeSR1 for 3 d and then in low-glucose DMEM supplemented with 10% FBS for 2 weeks. The characteristics and differentiation markers were evaluated by real-time fluorescence quantitative polymerase chain reaction (qPCR), immunofluorescence staining, live & dead cell staining and alkaline phosphatase (ALP) staining. The group of iPSCs cultured in conventional medium was used as control group 1. The group of single-dissociated iPSCs co-cultured with CECs was set as experimental group, while single-dissociated iPSCs without co-culture were as control group 2. RESULTS：The bovine CECs showed typical hexagonal cobblestone shape. iPSCs showed colony-like growth, while became single-dissociated cells after Transwell contact co-culture with bovine CECs for 3 d. The single-dissociated iPSCs positively expressed the undifferentiated markers, Nanog and Oct4. The mRNA expression levels of Nanog, Oct4 and Sox2 between experimental group and control group 1 were both positive and had no statistical significance difference (P>0.05). The dead cells in experimental group decreased significantly, and there was statistically significant difference compared to control group 2 (P<0.01). After 14 d of induced differentiation co-culture, the single-dissociated iPSCs showed rather uniform polygonal morphology, increased dimension and no obvious colony existence. Negative ALP staining, positive immunofluorescence staining for ZO-1, AQP1 and CD31, and negative for CD34 and CD133 were also observed. The results of qPCR showed that the mRNA expression of Oct4, Nanog and Sox2 significantly decreased, and had statistically significant difference compared with control group 1 (P<0.01). CONCLUSION：When co-cultured with bovine CECs, iPSCs morphologically changed to endothelial-like cells and expressed some markers of CECs. Transwell contact co-culture system not only enhances the growth of single-dissociated iPSCs, but also promotes their differentiation.