Direct induction of human retinal ganglion cell-like cells using human transcription factors

Abstract

<p indent="0mm">Direct reprogramming of somatic cell transdifferentiates somatic cells from one cell type into another, providing a new strategy to explore the pathological mechanisms and therapeutic methods for neural degeneration diseases. Glaucoma is the most prevalent retinal degenerative disease caused by the degeneration and death of retinal ganglion cells (RGC) and their axons. Our previous study showed that <italic>Ascl1</italic>,<italic> Brn3b</italic>,<italic> </italic>and <italic>Islet1</italic> transcription factor combination can efficiently reprogram mouse fibroblasts into functional RGC-like cells (induced RGCs, iRGCs). Here, we investigated whether this transcription factor combination can also achieve efficient human iRGC (hiRGC) induction. For this purpose, we used lentiviruses to introduce mouse <italic>Ascl1</italic>, <italic>Brn3b</italic>, and<italic> Islet1</italic>, or human <italic>ASCL1</italic>, <italic>BRN3B</italic>, and <italic>ISLET1</italic> cDNAs into human fibroblasts, and used the Tet-ON strategy to control the expression of the exogenous genes. At specific time points after induction, we performed immunofluorescent staining to examine the expression of the representative neuron or RGC marker genes and patch clamp to evaluate the electrophysiological membrane properties of the induced cells. The results showed that by replacing mouse <italic>Ascl1</italic>, <italic>Brn3b</italic>, and <italic>Islet1</italic> cDNAs with human <italic>ASCL1</italic>, <italic>BRN3B</italic>, and<italic> ISLET1</italic> cDNAs, human fibroblasts could also be reprogrammed into hiRGCs. Furthermore, by adding the human <italic>SOX4 </italic>gene, highly efficient hiRGCs induction was achieved. The obtained iRGCs showed mature neuronal morphology, expressed various neuron-specific proteins, and exhibited the electrophysiological characteristics of functional neurons. Our study provided a valuable cell source for studying the RGC cell fate determination mechanism and exploring translational applications for retinal diseases, such as glaucoma.