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Xian-Jie Yang, Yun-Xia Leng, Xiangmei Zhang, Steve Barnes; Direct Induction of Retinal Neuron-Like Cells from Human ES Cells-Derived Progenitor Cell Lines. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6069.
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© ARVO (1962-2015); The Authors (2016-present)
In order to enable efficient and robust retinal neuron production, we aim at establishing pluripotent human ESC- or iPSC-derived cell lines that can rapidly differentiate into retinal neuron-like cells. These cell lines will be useful to study neuronal differentiation, survival, and disease etiology, and will facilitate the development of new therapeutics.
Human pluripotent stem cells are co-transduced with lentiviruses expressing the rtTA regulator and transcription factors known to play critical roles in retinal ganglion cell (RGC) development. The infected ES cells are plated at clonal density and selected for co-transduced clones under conditions with or without doxycycline (Dox). Molecular analyses are carried out for ES cell markers, virally expressed genes, and retinal neuronal markers using immunocytochemistry and RT-PCR. Upon induction with Dox, the cultures are changed to a medium that favors neuronal differentiation. In addition, functionality of differentiated neurons is assayed by whole cell patch clamping to determine the electrophysiological properties of the induced neurons.
We have established human ES cell-derived clones carrying TetO lentivirus encoding the bHLH neurogenic factors NGN2, ATOH7, or both. As previously reported, NGN2 virus-transduced human ES cell clones show a propensity to differentiate into neurons upon Dox induction, as indicated by their elaborate dendritic morphology, expression of neuronal markers such as TUJ1 and neurofilament. Electrophysiological recording has detected single spike or multiple action potentials from neurons cultured for two week after onset of NGN2 induction. Preliminary analysis indicates that cells co-transduced with NGN2 and ATOH7 viruses exhibit distinct neuronal morphology and express POU4f1/Brn3a, a transcription factor involved in RGC differentiation.
Pluripotent stem cell-derived inducible cell lines can differentiate into functional neurons in vitro. The in vitro Dox induced neuronal differentiation is rapid and robust. Upon further characterization, these cell lines can become useful tools to study neuronal differentiation, survival, axonal regeneration, and disease treatment.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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