Rabbit Keratocytes Microgravity Culture And The Preliminary Exploration Of ADSC Transdifferentiation Reprogramming

Part1Characterizing the Effects of VPA, VC and RCCS onRabbit Keratocytes onto Decellularized Bovine CorneaObjective: To investigate the morphological and growth characteristics of rabbitkeratocytes when cultured on decellularized cornea under simulate microgravity(SMG) rotary cell culture system (RCCS) and static culture or in plastic culturesupplemented with small molecules of valproic acid (VPA) and vitamin C (VC).Methods: Bovine corneas were firstly decellularized with Triton X-100and NH4OHand through short-term freezing process. Then cell count kit-8(CCK-8) and flowcytometry were used to test the effects of VPA and VC on the proliferation, cell cycleand apoptosis of rabbit keratocytes. Reverse transcription-polymerase chain reaction(RT-PCR) and immunofluorescence were used to analysis keratocan and lumicanexpressing of keratocytes. Scanning electron microscopy (SEM) and invertedmicroscope were used to observe the morphological characteristics of keratocytesunder different conditions.Results: Hematoxylin-eosin (H&E) staining and SEM imaging showed that cellswere eliminated in the decellularized bovine corneas. The proliferation of culturedkeratocytes was promoted by VPA and VC in the cell proliferation assay. VPA andVC moderately decreased the number of apoptotic cells and obviously promotedcell-cycle entrance of keratocytes. Rabbit keratocytes in plastic displayed spindleshape and rare interconnected with or without VPA and VC. Cells revealed dendriticmorphology and reticular cellular connections when cultured on the carriers ofdecellularized corneas supplemented with VPA and VC even in the presence of10%fetal bovine serum (FBS). When cultured in RCCS supplemented with VPA, VC and10%FBS, keratocytes displayed round shape with many prominences and were moreprone to grow into the pores of carriers with aggregation. RT-PCR analysis provedthat the keratocytes cultured on decellularized bovine cornea under SMG with VPA and VC expressed keratocan and lumican. Keratocytes cultured on plastic expressedlumican but not keratocan. Immunofluorescence identification revealed that cells inall groups were positively immunostained for vimentin. Keratocytes on decellularizedbovine cornea under SMG or in static culture were positively immunostained forkeratocan and lumican.Conclusion: The combination of VPA, VC, RCCS and decellularized corneal carriersprovide a good condition for keratocytes to well grow. Keratocytes can bemanipulated to be aggregates or physiological morphological growth in vitro, whichare important for the research of corneal stem cells and corneal tissue engineering. Part2Exploration of Transdifferentiation Reprogramming ofAdipose Stem Cells into Corneal Endothelial Cell-Like CellsObjective: To explore the use of a variety of conditions to promote thetransdifferentiation reprogramming of adipose stem cells (ADSC) into cornealendothelial cell (CEC)-Like cells in order to provide new ideas to obtain stemcell-derived CEC.Methods: The adipogenic induction, osteogenic induction and flow cytometry wereexplored to identify ADSC. The combinations of reprogramming proteins(PTD-OCT4, PTD-KLF4, PTD-SOX2) and small molecules [Purmorphamine (P),RG108and Reprogramming Cocktail Set I (Kit)] were used to reprogram ADSC.Furthermore, spherical and extra cellular matrix (ECM) cultures were explored topromote ADSC reprogramming in knockout serum replacement (KSR). Then thesecells mixed co-cultured with CEC and corneal limbal stromal cells in transwell insertculture system. Glycogen synthase kinase (GSK)-3β inhibitor was used to promotethe direct reprogramming of ADSC into CEC. At last, the direct reprogrammingADSC transplanted onto decellularized bovine corneal stroma for further induced differentiation. Immunofluorescence and RT-PCR were used to analysis the GAPDH,OCT4, SOX2, KLF4and Nanog expression of direct reprogramming ADSC.Results: The adipogenic induction, osteogenic induction and flow cytometry revealedthat the primary cells we isolated were ADSC. After treated with P and combinationsof reprogramming proteins, the spherical ADSC positively expressed Nanog. ADSCpositively stained with CD34after cultured on ECM. After co-cultured with CEC andcorneal limbal stromal cells, these direct reprogramming ADSC transplanted ontodecellularized bovine corneal stroma showed polygon morphological characteristicbut negatively expressed CD31, CD133, AQP-1and ZO-1.Conclusion: After direct reprogramming treatment with small molecules P andcombinations of reprogramming proteins, ADSC obtain higher differential potential.After co-culture with CEC and corneal limbal stromal cells, the morphology of theseADSC changes from stromal spindle shape into polygonal one. Thetransdifferentiation reprogramming of ADSC into CEC can be used as a new sourcefor CEC replacement to treat CEC pathological changes and dysfunction.