Rapid and efficient generation of a transplantable population of functional retinal ganglion cells from fibroblasts

Abstract Glaucoma and other optic neuropathies lead to progressive and irreversible vision loss by damaging retinal ganglion cells (RGCs) and their axons. Cell replacement therapy is a potential promising treatment. However, current methods to obtain RGCs have inherent limitations, including time‐consuming procedures, inefficient yields and complex protocols, which hinder their practical application. Here, we have developed a straightforward, rapid and efficient approach for directly inducing RGCs from mouse embryonic fibroblasts (MEFs) using a combination of triple transcription factors (TFs): ASCL1, BRN3B and PAX6 (ABP). We showed that on the 6th day following ABP induction, neurons with molecular characteristics of RGCs were observed, and more than 60% of induced neurons became iRGCs (induced retinal ganglion cells) in the end. Transplanted iRGCs had the ability to survive and appropriately integrate into the RGC layer of mouse retinal explants and N‐methyl‐d‐aspartic acid (NMDA)‐damaged retinas. Moreover, they exhibited electrophysiological properties typical of RGCs, and were able to regrow dendrites and axons and form synaptic connections with host retinal cells. Together, we have established a rapid and efficient approach to acquire functional RGCs for potential cell replacement therapy to treat glaucoma and other optic neuropathies.


Figure S1 .
Figure S1.Screening of TFs for iRGC induction.(A) Positive (P) or negative (N) immunostaining results of TUJ1 and BRN3A in MEFs infected with the indicated single TF lentiviruses (A, ASCL1; B, BRN3B; D, DLX1; E, EBF1; I, ISL1; P, PAX6).Only ASCL1 could induce TUJ1-positive neurons.(B) Positive or negative immunostaining results of TUJ1 and BRN3A in MEFs co-infected with both ASCL1 and the other indicated TFs.Only AI-and ABreprogrammed neurons co-expressed TUJ1 and BRN3A.(C) Positive or negative immunostaining results of TUJ1 and BRN3A in MEFs infected with the indicated triple-TF lentiviruses.All the triple-TF combinations were able to induce TUJ1 and BRN3A coexpressing neurons.(D, E) qRT-PCR analyses showing relative expression levels of Tuj1 and Brn3a in MEFs infected with the indicated lentiviruses on D21 post induction.Data are presented as mean ± SD (n = 3).Asterisks indicate significance in one-way ANOVA test: *p < 0.001, **p < 0.0001.(F-U) Double-immunostaining for TUJ1 and BRN3A in MEFs on D21 after infection with the indicated lentiviruses.Nuclei were stained with DAPI.Arrows indicate colocalized cells.Scale bars, 40 µm.

Figure S3 .
Figure S3.Detection of a few commonly used RGC markers in the mouse retina, and

Figure S5 .
Figure S5.Global gene expression profiles determined by bulk RNA-seq analysis at four time points during the iRGC induction process.(A) Principal component analysis (PCA) of the gene expression profiles of the indicated samples.ABP(D0-21): ABPtransduced MEFs on day 0-21.(B) Heatmap showing the correlations among the indicated samples.(C-E) Volcano plots showing gene expression changes between D0 and 6 (C), D0 and 13 (D), and D0 and 21 (E) after ABP lentivirus infection.Some representative neuron

Figure S6 .
Figure S6.Induction of iRGCs by ABP without undergoing a proliferative intermediate state.(A) Schematic illustration of EdU labeling schedule during the ABP reprogramming process.(B-B'') ABP-transduced MEFs were labeled by EdU for 19 days and co-labeled by fluorescence for both TUJ1 and EdU.Scale bars: 40 µm.(C) Quantification of corresponding EdU-labeled cells in populations of TUJ1-negative and TUJ1-positive cells.Data are presented as mean ± SD (n = 4).The asterisk indicates significance in unpaired two-tailed Student's t-test: *p < 0.0001.(D-D'') ABP-transduced MEFs were labeled by EdU for 24 hours and co-labeled by fluorescence for both TUJ1 and EdU.Scale bars: 40 µm.(E) Quantification of corresponding EdU-labeled cells in populations of TUJ1-negative and TUJ1-positive cells.Data are presented as mean ± SD (n = 4).The asterisk indicates significance in unpaired two-tailed Student's t-test: *p<0.05.

Figure S7 .
Figure S7.Integrated analysis of scRNA-seq transcriptomes of ABP-induced iRGCs and ABI-induced iSG neurons.(A) t-SNE visualization of the ABP-induced iRGCs and the iSG (induced sensory ganglion) neurons induced by ABI (ASCL1 + BRN3B/3A + ISL1).The accession code of the iSG scRNA-seq dataset is PRJNA597624.(B) t-SNE plot of the 17 cell clusters generated from the integrated iRGC and iSG datasets.(C) Comparison of t-SNE plots of iRGCs and iSG neurons reveals that they form very distinct cell clusters.(D) Split t-SNE plots of iRGCs and iSG neurons colored by expression of the general neuronal marker genes Tubb3, Stmn1 and Vamp2, iRGC-specific genes Sncg, Jam2 and Myt1, and iSGspecific genes Gal, Ntrk3 and P2rx3.