Co-culture Of Human IPS Cells With Rabbit Corneal Endothelial Cells To Induce Differentiation Of IPS Cells

Part1Application of quantum dots labeling on induced pluripotent stemcellsAIM: To investigate the feasibility of labeling the induced pluripotent stem cells withCdSe/ZnS nucleus/shell structural quantum dots and the intensity and stability of thefluorescence.METHODS: The human iPS cell lines were cultured and amplified on Matrigel in afeeder-free method and their characteristics were evaluated by western blot andimmunofluorescence detection. The differentiated cells spontaneously were eliminated bypicking handwork combined with a one-off transfusion tube for exsuction. The quantumdots with CdSe/ZnS nuclear shell structure of different concentration (5nmol/L,7.5nmol/Land10nmol/L) were chosen to label the iPS cells after passage and proliferation.The effect after labeling was observed with a three-dimensional deconvolution real-timeliving cells imaging system. The iPS cells labeled with QDs were continuously cultivated,and their adhesion, growth, proliferation and morphology were observed and then thechanges of the fluorescence were examined at day7after the first and the second passageof iPS cells.RESULTS: iPS cells were observed to grow in a cloning form under the invertedmicroscopy. The small clones combined bigger in a condition that the differentiated cellsspontaneously were eliminated manually. OCT4and Nanog, two pluripotency proteinswere expressed positively by western blot and immunofluorescence detection. It isfeasible to label iPS cells with5nmol/L,7.5nmol/L and10nmol/L QDs. The cellslabeled with10nmol/L QDs showed the brightest fluorescence of jacinth. iPS cellslabeled with QDs displayed normal adhesion, growth, proliferation and morphology. Thelabeled cells still showed faint positive fluorescence at day7even after the secondpassage but they were weaker than before.CONCLUSION: QDs could be used to label and trace iPS cells and the labeling effecthad a positive correlation with the concentration of QDs within10nmol/L range. Thefluorescence of QDs in this experiment condition could last2weeks at least. This experiment confirms that labeling iPS with CdSe/ZnS nucleus/shell structural quantumdots is feasible, which can be used to label and trace iPS. Part2Co-culture of human iPS cells with rabbit corneal endothelialcells to induce differentiation of iPS cellsAIM: To investigate the morphology and protein expression of human inducedpluripotent stem cells co-cultured with rabbit corneal endothelial cells (CECs) andprovide the experimental basis for further study of the potentiality of differentiation of iPScells into CECs.METHODS: Alizarin red staining was made on corneal endothelial cells of tissue fromthe New Zealand rabbits. Primary rabbit CECs were isolated with trypsin passagednormally. The human iPS cell lines were cultured and amplified with feeder-free methodand subcultured at a state of90%combination.1/2,1/3and1/4of suspending iPS cellswere put into rabbit CECs for direct mix co-culture. The optimum proportion of the twokinds of cells was confirmed under converted microscopy through their coexistence andinteraction. The changes of iPS morphology after one week of direct co-culture withCECs were evaluated by AFM and inverted microscopy. iPS cells labeled with quantumdots of appropriate concentration were used and protein expression of CD31, CD34,CD133and AQP1of iPS cells were tested by immunofluorescence after2weeks of mixco-culture. On the other hand, iPS cells were digested and inoculated into transwell insertchamber at1:4,1:5and1:6proportion, and then inserts put onto6-well plates which hadthe first passage rabbit CECs in60%confluence, from which an indirect co-culturesystem was established. The expression of CD31, CD34, CD133and AQP1of iPS cellswere also tested by immunofluorescence after2weeks of indirect co-culture.RESULTS: The rabbit CECs on cornea tissue were positively stained with Alizarin redand showed hexagonal shape with tight cellular connection. The cultivating rabbit CECsalso had a hexagonal and cobblestone appearance.1/4Suspension of iPS cells labeledwith10nmol/L quantum dots and60%confluence of rabbit CECs made best coexistenceand interaction in direct co-culture condition for both cells． Under atomic force microscopy and inverted microscopy, the volume of iPS cells became bigger and thenuclear-cytoplasm ratio was decreased after7days of direct co-culture. The indentation ofcellular membrane increased, deepened and widened, while some membrane protrusionsincreased and looked like the fingers. The roughness of cellular membrane increased.These morphological changes were closer to CECs. The best indirect co-culture resultcould be set up when iPS cells subcultured at1:5and rabbit CECs grew in60%confluence. IPS cells displayed multifarious shapes and irregular growth when cultured inindirect co-culture condition. Immunofluorescence test showed negative expressions ofCD34, CD133, CD31and AQP1before co-culture and after indirect co-culture, but after2weeks of direct co-culture, AQP1could be positively expressed.CONCLUSION: iPS cells changed morphologically to corneal endothelial-like cells andexpressed marker protein AQP1of CECs after mix co-cultured with rabbit CECs. Thecellular density, co-culture period, communication of cytokine and the cell-to-cell contactwere important factors for iPS cells differentiation. This study lays the foundation for thework of the differentiation of iPS cells into CECs and provides a useful experimentalbasis.