Direct Reprogramming Of Fibroblasts To Neurons And Retinal Ganglion-like Cells By Defined Factors

The primary goal of regenerative medicine is to generate specific cells to replace the function of degenerated organs. In2006, the generation of induced pluripotent stem cells established the milestone in the field of both cell biology and regenerative medical research. Soon after that, an even bolder idea was successfully realized, and announced that even fully differentiated cells were free to directly convert to another differentiated cell type, skipping the pluripotent stage. Skin fibroblasts are easy to harvest, and have been successfully transdifferentiated into cardiomyocytes, hepatocyte and neural stem cells. With those reprogrammed cells being transplanted back to the patients, it makes the patient-specific therapy much more feasible. In this study, adenoviruses carrying defined transcription factors were adopted to reprogram fibroblasts into functional neurons and retinal ganglion-like cells.PART I:Induction of Fibroblasts to Neurons via Adenoviral Gene DeliveryAbstractPurpose:To directly convert mouse embryonic fibroblasts and adult ear tip fibroblasts into neurons by adenoviruses carrying neuronal transcription factors.Materials and Methods:Neuronal transcription factors Ascll, Brn2, Mytll and Ngn2were constructed into pAd/CMV/V5-DEST7.3vector, and high titer adenoviruses were obtained. Mouse embryonic fibroblasts from E13.5C57BL/6J mice and ear tip fibroblasts from6-8week old C57BL/6J mice were infected with different combinations of adenoviruses carrying ABM (Ascl1+Brn2+Myt11) or ABN (Ascll+Brn2+Ngn2).13days to30days post infection, immunostaining was applied to detect the generation of induced neurons, as well as the expression of different neural markers, and electrophysiological properties of these induced neurons were investigated as well. By PCR and Southern Blot, integration of exogenous genes carried by adenoviruses into the host genome was carefully examined. Results:Both of the two transcription factor combinations ABM and ABN could directly induced mouse embryonic fibroblasts into neurons. At13days post infection, ABN combination generated2.94%±0.73%neurons from mouse embryonic fibroblasts, while1.64%±0.59%neurons were generated in ABM combination group. Both neurons generated by ABN combination and ABM combination possessed electrophysiology properties of mature neurons. Neurons generated by ABN combination could express multiple neuronal markers, like Map2a, NeuN and Synapsin; after longer period of culture (20-25days), excitatory neuron marker vGLUT and inhibitory neuron marker GAD67were also detected in these induced neurons. Co-cultured with rat pyramid neurons, excitatory postsynaptic currents were detected in induced neurons. Ear tip fibroblasts infected with ABN combination could also generated functional neurons. PCR and Southern Blot examination showed no exogenous gene integration into host genome.Conclusion:Ascll, Brn2and Ngn2carried by adenoviruses could directly convert mouse embryonic fibroblasts and ear tip fibroblasts into functional neurons, without the risk of exogenous gene integration into host genome. Transdifferentiation mediated by adenoviruses is much safer for clinical application, and is more promising in further investigation.PART II:Direct Conversion of Fibroblasts into Retinal Ganglion-like Cells by Defined FactorsAbstract:Purpose:To directly convert mouse embryonic and adult fibroblasts into retinal ganglion-like cells by adenoviruses carrying defined transcription factors.Materials and Methods:Adenoviruses carrying retinal ganglion cell related genes were packaged. Different transcription factors were combined to screen for the most efficient combination. Multiple immunostaining and patch-clamp were applied to detect the properties of induced retinal ganglion-like cells. By PCR, integration of exogenous genes carried by adenoviruses into the host genome was carefully examined.Results:By different factor combinations, Ascl+Brn3b+Ngn2was proved to be the most effective combination that could covert mouse embryonic fibroblasts into retinal ganglion-like cells. Induced retinal ganglion-like cells could co-express multiple retinal ganglion cell specific markers, and possessed mature neuron properties as well. The conversion efficiencies indicated by different markers were quite similar, from3.58%-4.00%. Ascl+Brn3b+Ngn2could also convert mouse adult ear tip fibroblasts into retinal ganglion-like cells with mature neural properties. PCR examination proved no exogenous gene integration into host genome.Conclusion:Adenoviruses carrying Ascl, Brn3b and Ngn2could efficiently convert mouse embryonic fibroblasts and adult ear tip fibroblasts into retinal ganglion-like cells, which co-expressed multiple retinal ganglion cell specific markers and possessed mature neural electrophysiological properties. Direct reprogramming of fibroblasts into retinal ganglion-like cells provides new prospective into both the research and clinical therapy of glaucoma and other optic nerve diseases.