Non-genetic Direct Reprogramming And Biomimetic Platforms In A Preliminary Study For Adipose-derived Stem Cells Into Corneal Endothelia-like Cells

Part 1 The Effects of ROCK Inhibitor Y-27632 on Injectable Spheroids of Bovine Corneal Endothelial CellsObjective: 3D spherical cultured and ROCK inhibitors Y-23672 can improve cell proliferation and viability. The objective of this study was to investigate the effect of Y-27632 on the growth and injectability of bovine corneal endothelial cells(B-CECs) maintained in vitro as spheroid cultures.Methods: Primary B-CECs were isolated from the corneal tissue of bovine eyes which were obtained at a local slaughterhouse and B-CEC spheroids were cultured by multiwall agarose micro-molds. Live/Dead assay, Ed U labeling assay and Kit-8(CCK-8) assay were used to evaluate the proliferation and viability of B-CECs and B-CEC spheroids. In vitro simulation of injectability of B-CEC spheroids combined with or without Y-27632 was performed after spheroids were injected into culture plates or biomimetic acellular corneal. Cell morphology, proliferation and migration were observed under inverted phase contrast microscope and SEM.Results: Immunofluorescence staining showed that Y-27632 did not alter the cell type specificity of B-CECs, but it significantly enhanced B-CEC spherical viability and proliferation by a Live/Dead assay, Ed U labeling assay and CCK-8 assay. The uniform B-CEC spheroids could easily form in multiwall agarose micro-molds and had a higher stemness potential than single B-CECs. Injectable B-CEC spheroids were able to form monolayer growth, and polygonal B-CECs completely covered culture plates or biomimetic acellular corneal under inverted microscopy and scanning electron microscopy(SEM). B-CEC spheroids were generated from agarose micro-wells on day 1 and then adherent culture with Y-27632 for day 5. However, small B-CEC spheroids still existed on culture plates or biomimetic acellular corneal when B-CEC spheroids were cultured in the same condition except for absence of Y-27632.Conclusion: Compared with conventional two-dimensional culture, B-CEC spheroids of three-dimensional culture had higher stemness potential in vitro. The ROCK inhibitor Y- 27632 could enhance B-CEC spherical viability, proliferation and migration. At the same time, Y-27632 did not alter important morphological features of B-CECs. Our results demonstrated that the combination of agarose micro-wells and Y-27632 is a potentially powerful tool for the generation of spheroids containing precursors and their migration in vitro simulation experiment of injectability. We believe that such a combination will have important implications for future CEC therapies.Part 2 Non-Genetic Direct Reprogramming and Biomimetic Platforms in a Preliminary Stud y for Adipose-Derived Stem Cells into Corneal Endothelia-Like CellsObjective: we investigate the effects of recombinant cell penetrating reprogramming proteins Oct4/Klf4/Sox2(PTD-OKS), small molecules(purmorphamine) and SMG bioreactor on the reprogramming of human adipose-derived stem cells(ADSCs), as well as their preliminary commitment into corneal endothelia-like cells. The goal was to understand if the combination of reprogramming PTD-OKS proteins, small molecules and biomimetic environments was able to act in synergistic concert and be used as a suitable platform for non-genetic direct reprogramming of ADSCs into corneal endothelia-like cells.Methods: ADSCs could be isolated by collagenase digestion from human lipoaspirate tissues. The combinations of reprogramming proteins PTD-OKS, small molecules Purmorphamine and SMG bioreactor were used to reprogram ADSC. And these ADSCs mixed co-cultured with CEC and corneal stromal cells in transwell insert culture system. Next, the direct reprogramming ADSC transplanted onto decellularized bovine corneal stroma for further induced differentiation. RT-PCR and Immunofluorescence were used to analysis the gene and phenotype expression of direct reprogramming ADSC.Results: The flow cytometry, adipogenic induction and osteogenic induction were used to identify the isolated ADSCs. Next, we tried the possibility to generate corneal endothelia(CE)-like cells from human adipose-derived stem cells(ADSCs) by the non-genetic direct reprogramming of recombinant cell-penetrating proteins Oct4/Klf4/Sox2(PTD-OKS) and small molecules(purmorphamine, RG108 and other reprogramming chemical reagents), as well as biomimetic platforms of simulate microgravity(SMG) bioreactor. Co-cultured with corneal cells and decellularized corneal ECM, Reprogrammed ADSCs revealed spherical growth and positively expressing Nanog for RT-PCR analysis and CD34 for immunofluorescence staining after 7 days-treatment of both purmorphamine and PTD-OKS(P-OKS) and in SMG culture. ADSCs changed to CEC polygonal morphology from spindle shape after the sequential non-genetic direct reprogramming and biomimetic platforms. At the same time, induced cells converted to weakly express CD31, AQP-1 and ZO-1.Conclusion: After treatment with small molecules combinations of reprogramming proteins and Purmorphamine under SMG bioreactor, ADSCs obtain higher differential potential. After co-culture with CEC and corneal stromal cells, the morphology of these ADSC changes from stromal spindle shape into polygonal one and positively expressed CD31, CD133, AQP-1 and ZO-1. These results suggest for the first time that SMG rotary cell culture system is able to promote the stemness reprogramming for human ADSCs and can be used as a non-genetic means to enhance direct reprogramming. Non-genetic direct reprogramming can provide an alternative strategy for engineering patient-specific multipotent cells for cellular plasticity research and future autologous CEC replacement therapy that avoids complications associated with the use of human pluripotent stem cells.