Transwell contact co-culture promotes growth and differentiation of sin-gle-dissociated iPSCs / 中国病理生理杂志

ABSTRACT

[ABSTRACT]AIM:ToinvestigatethepromotingroleofTranswellcontactco-culturesysteminthegrowthand 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 40μm 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 mTeSR 1 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 Tran-swell 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-dis-sociated iPSCs , but also promotes their differentiation .