RESUMO
In optic neuropathies, including glaucoma, retinal ganglion cells (RGCs) die. Cell transplantation and endogenous regeneration offer strategies for retinal repair, however, developmental programs required for this to succeed are incompletely understood. To address this, we explored cellular reprogramming with transcription factor (TF) regulators of RGC development which were integrated into human pluripotent stem cells (PSCs) as inducible gene cassettes. When the pioneer factor NEUROG2 was combined with RGC-expressed TFs (ATOH7, ISL1, and POU4F2) some conversion was observed and when pre-patterned by BMP inhibition, RGC-like induced neurons (RGC-iNs) were generated with high efficiency in just under a week. These exhibited transcriptional profiles that were reminiscent of RGCs and exhibited electrophysiological properties, including AMPA-mediated synaptic transmission. Additionally, we demonstrated that small molecule inhibitors of DLK/LZK and GCK-IV can block neuronal death in two pharmacological axon injury models. Combining developmental patterning with RGC-specific TFs thus provided valuable insight into strategies for cell replacement and neuroprotection.
RESUMO
Pluripotent stem cells (PSCs) offer an exciting resource for probing human biology; however, gene-editing efficiency remains relatively low in many cell types, including stem cells. Gene-editing using the CRISPR-Cas9 system offers an attractive solution that improves upon previous gene-editing approaches; however, like other technologies, off-target mutagenesis remains a concern. High-fidelity Cas9 variants greatly reduce off-target mutagenesis and offer a solution to this problem. To evaluate their utility as part of a cell-based gene-editing platform, human PSC lines were generated with a high-fidelity (HF) tetracycline-inducible engineered Streptococcus pyogenes SpCas9 (HF-iCas9) integrated into the AAVS1 safe harbor locus. By engineering cells with controllable expression of Cas9, we eliminated the need to include a large Cas9-expressing plasmid during cell transfection. Delivery of genetic cargo was further optimized by packaging DNA targeting guide RNAs (gRNAs) and donor fragments into a single plasmid backbone. The potential of homology-directed repair (HDR) based gene knock-in at the CLYBL safe harbor site and endogenous SOX2 and SIX6 genes were demonstrated. Moreover, we used non-homologous end-joining (NHEJ) for gene knockout of disease-relevant alleles. These high-fidelity CRISPR tools and the resulting HF-iCas9 cell lines will facilitate the production of cell-type reporters and mutants across different genetic backgrounds.