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Lili Hao, Zhen Xu, Wu Luo, Youchen Yan, Hui Sun, Shuyi Chen; Direct induction of functional neurons from adult human retina derived fibroblast-like cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1360.
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© ARVO (1962-2015); The Authors (2016-present)
Resent works have shown that mouse and human fibroblasts can be directly reprogrammed into functional neurons by forced expression of neurogenic transcription factors. We recently established a culture protocol to expand fibroblast-like cells from adult human retina. In this study, we wanted to test whether the fibroblast-like cells derived from adult human retina could be directly converted into functional neurons by neurogenic transcription factor combination.
Adult human retina tissues were obtained from the Eye Bank of Guangdong Province, dissociated into single cells, and expanded in serum containing culture medium.Doxycycline-inducible lentiviruses were used to introduce and control the expression of neurogenic transcription factors to the cells.Neuronal cell induction was examined by immunostaining for a variety of neural specific proteins, such as TUJ1, MAP2, GABA, vGLUT1, and the neural functions of the induced cells were examined through patch clamp recording.
Cells expanded from adult human retinasexhibited fibroblast-like morphology and culture behavior, expressed high levels of fibroblast cell markers suggesting a fibroblast cell identity, thus was term hRFLC for human Retina derived Fibroblast Like Cells . Ascl1 alone was able to induce TUJ1/MAP2-positive neuron-like cells with immature phenotype from hRFLCs. However, Ascl1+Brn2+Mytl1+NeuroD1, the neurogenic transcription factor combination previously shown to be sufficient to induce functional neuronal cells from fetal and neonatal human fibroblasts, failed to improve the neural induction phenotype from hRFLCs than Ascl1, instead, Co-infection with Pax6 greatly increased the yield of the induce neurons with mature morphology. More importantly, most Ascl1+Pax6 induced neuronal cells exhibited active neural membrane electrophysiological activities, over half of them fired recurrent action potentials when co-cultured with mouse glia cells, and formed function synaptic connections with co-cultured mouse cortical neurons.
Fibroblast-like cells (hRFLCs) can be easily expanded from adult human retinal tissues. Ascl1+Pax6 combination is sufficient to directly reprogram hRFLCs into functional neuronal cells.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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