Transplantation And Bio-Mechanics Of HiPSCs-Induced Retina Organoids

Retinal degeneration(RD)is characterized by progressive loss of photoreceptor cells,which can lead to visual dysfunction and even blindness.Cell transplantation is a potential therapy method for RD.In order to investigate the differentiation process of human-induced pluripotent stem cells(hiPSCs)into retinal organoids(ROs)in vitro and its potential application in cell transplantation,we utilized 3D culture method to obtain ROs with typical retinal structure.With the combination of real-time quantitative PCR and immunohistochemistry,we characterized the differentiation process scientifically.The results showed that our ROs could simulate the development of human embryonic retina and the expression of classical molecular markers.Next,we digested ROs and subretinally injected the single-cell suspension into the cavity of Gnat1-/-mice.The survival and integration ability of the implanted cells were analyzed.Three weeks after the transplantation,cells with green fluorescence were successfully moved into the outer nucleus layer of the host retina.4-6 months later,the implanted cells expressed the functional light signal transduction protein GNAT1.These results confirmed that the transplanted ROs photoreceptor precursor cells could survive in the eyes of the recipient mice for a long time,enter the outer nuclear layer and further mature.On the other hand,in the early differentiation of adherent culture,we found the boundary between the inner and outer regions of colony becomes more and more obvious,and the differentiation of these cells has region preference definitely:the central region has high expression of neuron marker TUJ1,the external eye field(EF)was formed,which is made up of retinal progenitor cells mostly.EF cells can be divided into retina or retinal pigment epithelium differentiation.Thus,we explored the molecular mechanisms by which the fate of the internal and external cells bifurcates.Through RNA-seq and mechanics analysis,we found that there were significant differences in gene expression between the inner and outer regions.In addition,the traction field also showed that the interaction between the outer cells and the extracellular matrix was significantly enhanced.We hypothesized that mechanical force regulates cell differentiation fate and related molecular mechanismsIn conclusion,we differentiated hiPSCs into ROs,which recapitulated the development of human retina.Transplanted photoreceptor precursor cells could survive in the recipient mice’s retina over several months,effectively moved into the outer nuclear layer and progressively matured.At the same time,mechanical signals such as cell adhesion in different areas of colonies may play an important role in the fate choice of cells.After being sensed by integrin or adhesion structure,the mechanical signal can regulate the cascade of signal pathways,even the expression of genes,thereby control the cell differentiation.