The Research Of The Differentiation Of IPSCs Into Corneal Endothelial-like Cells

Part 1 The promoting effects of i PSCs-derived conditioned medium on the growth of B-CECs Objective: To evaluate the promoting role of i PSCs-derived conditioned medium(i PSCs-CM) on the growth of B-CECs. Methods: The collected medium of cultured i PSCs was filtered(0.22 μm) to remove dead cells and stored at-80 as i PSCs-supernatant. The certain proportion of i PSCs-supernatant into normal endothelial medium was used as i PSCs-CM, with which was accounted for the experimental group, while without it as control group. Through the CCK-8, cell cycle, cell apoptosis, living cells count and Western blot analysis of phosphorylated Akt protein expression, we verificationed the promoting proliferation of i PSCs-CM on the growth of B-CECs. Using PCR, immunofluorescence staining, scratch experiment, fluorescein sodium penetration assays, we investigated the i PSCs-CM on the effects of CEC marker protein and mark gene expressions as well as the influence on endothelial cell function. Using ELISA analysis, we explored the main effective components in conditioned medium. Result: The result of CCK-8 assay showed that the proliferation of cultured B-CECs was the most obvious in supplement with 25% i PSCs-CM compared with other proportions. Based on the result, we used this concentration as our i PSC-CM to treat B-CECs in later study. Cells morphologically maintained a homogeneous hexagonal shape until passage 4(P4). On the contrary, B-CECs at P4 showed irregular enlarged cell shape and lost their polygonal appearance. Immunofluorescence revealed that B-CECs expressed ZO-1, Na+/K+-ATPase in both experimental group and control group. The quantitative gene expressions of Ki67, Na+/K+-ATPase, Col4 A and Col8 A as well as and the percentage of cells entering the S and G2 phases were higher in experimental group than those in control group, while the numbers of apoptotic cells decreased in experimental group, which showed statistical significant difference(P<0.05, n=3). In comparison to the control cultures, i PSCs-CM further facilitated the B-CECs migration and had a better barrier function after several passages. The mechanism of cell growth mediated by i PSCs-CM had been investigated, phosphorylation of Akt was observed in B-CECs after exposure to i PSCs-CM and showed sustaining phosphorylation up to 180 minutes. These findings indicated that i PSCs-CM may employ PI 3-kinase signaling to regulate cell cycle progression and leading to the enlargened proliferation. Conditioned medium effective component analysis found that in the experimental group Activin A expression significantly increased and if add Activin A alone also can keep B–CECs morphology similar to the conditioned medium has done. Conclusion: i PSCs-CM can effectively promote the proliferation of B-CECs and maintain the major function of CECs. Activin A is one of the effective components of conditioned medium for these roles. The mechanical involving proliferation is the continuous high expression of phosphorylated Akt.Part 2 The promoting effects of transwell contact co-culture on the growth and differentiation of single-dissociated i PSCs Objective: To investigate the promoting role of transwell contact co-culture system on the growth and differentiation of single-dissociated induced pluripotent stem cells(i PSCs). Methods: Bovine corneal endothelial cells(CECs) at passage 1-2(P1-2) seeded on the invert of the insert culture plate of transwell and cultured in 37°C, 5% CO2 for 8 h. The treatment of ACCUTASE enzymatic digestion and 40 μm filter process disaggregated clone aggregated i PSCs into single-dissociated i PSCs and seeded on the inside of the insert of transwell with CEC in medium of m Te SR1 for 3 days and then using medium of low-glucose DMEM supplemented with 10% FBS for two weeks. The characteristics and differentiation markers were evaluated by real-time quantitative polymerase chain reaction(q PCR), immunofluorescence staining, Live & Dead Cells staining. The group of i PSCs cultured in conventional medium was used as control group 1. The group of single-dissociated i PSCs co-cultured with CECs was set as experimental group, while single-dissociated i PSCs without co-culture as control group 2. Results: The bovine CECs showed typical hexagonal cobblestone shape. i PSCs were cloned growth while became single-dissociated cells after transwell contact co-culture with bovine CECs for 3 days. The single-dissociated i PSCs expressed positively the undifferentiated markers of Nanog and Oct4. The undifferentiated gene expressions of Nanog, Oct4 and Sox2 by q PCR detection between experimental group and control group 1 were both positive and had no statistical significance differences(P > 0.05). The dead cells in experimental group decreased significantly by Live & Dead Cells staining, and there was statistically significant difference compared to the control group 2(P < 0.001). After 14 days of induced differentiation co-culture, the single-dissociated i PSCs became rather uniform polygon morphology, increased dimension and there were no obvious clone existence. Immunofluorescence staining for CD31 expressed positive, while CD34 and CD133 were negative. Conclusions: When co-cultured with bovine CECs, i PSCs morphologically changed to endothelial-like cells and expressed some makers of corneal endothelial cells. transwell contact co-culture system not only enhances the growth of single-dissociated i PSCs, but also promots their differentiation.Part 3 The preliminary study for i PSCs differentiation into corneal endothelia-like cells Objective: To observe the promoting role of the combination of the contact co-culture of transwell and small molecules on the inducion i PSCs into corneal endothelial-like cells. Method: Under the condition of the transwell, i PSCs had been co-cultured with B-CECs in a contact system as above, small molecule were sequentially used to induce i PSCs into neural crest cells(1u M GSK3 beta inhibitor VI+10u M SB431542) for 10 days. Then, these cells induced into endothelial progenitor cells(1u M GSK3 beta inhibitor VI+1u M RA+5ng/ml TGF-beta2) for 4 days. Lastly, endothelial progenitor cells differentiated into corneal endothelial-like cells(1u M GSK3 beta inhibitor VI+5ng/ml TGF-beta2+1u M Y-27632) for 7 days. The morphological changes of differentiated cells were observed and photographed under inverted microscope after above inductions. The marker gene and protein expressions were detected by using PCR, immunofluorescence and alkaline phosphatase(AP) staining, the cell microscopic changes were also observed under scanning electron microscopy(SEM). i PSCs differentiation group was used as experimental group and conventional culture of i PSCs as control group. Result: After 10 days of neural crest induction, i PSCs cell volume increased obviously and displayed a long spindle shape. After 4 days of endothelial progenitor cells differentiation, the cells became irregular shape and shortened gradually. Immunofluorescence staining showed the expression of endothelial progenitor marker Pitx2. RT-PCR also revealed positive Pitx2 expression while cells in control group were negative Pitx2 expression. Finally after 7 days of endothelial cells differentiation, cells became uniform with typical hexagonal cobblestone shape appearance without cloning clumps. SEM demonstrated monolayer cells with abundant cytoplasm and microvilli on the surface as well as tight intercellular connection. AP staining was negative in experimental group while cells in control group showed positive AP staining with a lot of cloning clumps. Immunofluorescence staining revealed positive expressions of endothelial markers of AQP1, ZO-1 and Na+/K+-ATPase in experimental group while cells in control group showed weak expression of ZO-1 but negative expressions of AQP1 and Na+/K+-ATPase. q PCR displayed that the gene expressions of Nanog, Oct4 and Sox2 in experimental group decreased compared with control group, and the difference was statistically significant(P<0.001, n=3). Conclusion: The combination of the contact co-culture of transwell and small molecules can induce i PSCs sequential differentiation into corneal endothelial-like cells. Some of corneal endothelial markers can positively express after our sequential CECs differentiation. The biomimetic way of CECs development favors the induction of i PSCs differentiation into corneal endothelial-like cells.