Reprogramming Human Corneal Stromal Cells With Lentivirus Vectors And Transcription Factors

Pluripotent stem cell has huge potential in regenerative medicine, tissueengineering, drug design and assessment, etc. Scientists have contrived severalapproaches to get pluripotent stem cells from a variety of sources:(1) isolating andculturing the inner cell mass of the human blastocyst,(2) somatic cell nuclear transfer,(3) fusing somatic cell with pluripotent stem cell or incubating somatic cell with cellextracts from pluripotent stem cell, etc. However, the development and translation ofthese approaches confronted severe obstacles for a number of inherent deficiencies,such as technical problems, difficulties cell source availability, immunological rejectionand controversies with regard to ethics, religion, politics and law, etc. On the contrary,there are no such limitations to iPS cells and iPS technology, given the convenience iniPS cell generation, it is regarded as a new milestone in the field of regenerativemedicine. As people know more about the molecular basis underlying stem cellmaintenance and differentiation, it is promising that all kinds of human cells can beobtained from iPS cells in vitro, opening a new epoch for cellular therapy, drugscreening and other application in regenerative medicine.Human corneal stromal cell is susceptible to culture in vitro, presenting strongproliferative capacity and capability of generating normal corneal collagen fibrilstructure. And to obtain sufficient, viable human corneal stromal cells is fundamental toresearches and therapies based on corneal stromal tissue engineering. Though one canget human corneal stromal cells from autologous and allogeneic sources, allogeneiccells often trigger severe immunological rejections which makes them unfit for clinicalapplication. Autologous cells, however, also faces many problems. Human cornealstromal cells are differentiated cells which have a limited life span in vitro, generally50-60times duplication before senescence. Therefore it will be difficult to get enoughcells from a small corneal stromal biopsy. Other autologous cell types, for example bone marrow derived mesenchymal cells, skin fibroblasts and adipose derived stem cells,cannot be fully functional as a corneal stromal cell surrogate.Human embryonic stem cells can be differentiated into any cell type within ahuman body, and the invention of iPS cells has made it possible to obtain large amountof non-immunogenic autologous pluripotent stem cells and their derivatives. ThereforeiPS cell–derived human corneal stromal cells is one of the most promising candidatesfor human corneal tissue engineering. However, due to lack of the knowledge related tohuman corneal stromal cell development and robust protocol for its differentiation,scientists have yet achieved in obtaining sufficient, purified corneal stromal cells fromhuman iPS cells. Interestingly, recent findings suggest that iPS cells maintainedepigenetic memories of the original cell type and the specific metholyme in differentiPS cell lines can influence their differentiation potential in vitro, which implies us thatwe can utilize this characteristic of iPS cells to accomplish those difficultdifferentiations.In this study, firstly we established the primary and secondary culture of humancorneal stromal after their migration from corneal tissue biopsy to the culture dish. Thecultured human corneal stromal cells have dendritic morphology, maintain the normalhuman karyotype and stain positive for vimentin. Human corneal epithelial andendothelial cells are not adulterated In vitro cultured human corneal stromal cellsThen we produced lentivirus encoding for transcription facors, Oct4、Sox2、Klf, andc-Myc, respectively. Infective efficiency assay showed that over80%of human cornealstromal cells were successfully infected under MOI=10.After infection, we trypsinizedand seeded the cells on cell culture dish preloaded with matrigel. We started theinduction by adding mouse embryonic fibroblast conditional media containing bFGF tothe cell culture. At the3rdday of induction, we observed morphological changes ofsome corneal stromal cells to a more epithelial cell-like state. And at7days postinduction we observed human embryonic stem cell likes with high nuclei to plasmaratio, which grows clonegenically. We conducted AP staining assay for pluripotency ofthese cells at day20when human embryonic stem cell likes became big and apparent. We counted the AP positives and calculated the efficiency of IPS induction as0.4%-0.8%.In summary, we reprogrammed human corneal stromal cells back to inducedpluripotent stem cell in vitro in this study. We hope that this study would be thefoundation for the future work on obtaining sufficient number of iPS cell derived humancorneal stromal cells for clinic usage by utilizing the epigenetic memory of iPS cells.Moreover, out work would shed light on the acquisition of other cell types which isdevelopmentally relative to corneal stromal cell, such as human corneal endothelial cellsand human corneal epithelial cells which are hard to get and difficult to culture in vitro,for cell therapy and mechanism study.