Specification of retinal cell types

Molecular profiling of stem cell-derived retinal pigment epithelial cell differentiation established for clinical translation

“…To identify populations during RPE induction, we obtained enriched genes by cluster and cross-referenced the literature, revealing a mixture of intermediate cell states resembling those described in rostral neural tube patterning and eye development (see supplemental experimental procedures ). We identified seven groups (pluripotent-like, endodermal-like, lateral neural fold-like, pre-placodal epithelium-like, cranial neural crest-like, mesenchymal, and retinal progenitor) and confirmed these annotations using several literature-supported marker genes ( Figures 2 A, 2B, and S2 A; Table S2 ; supplemental experimental procedures ) ( Begbie, 2013 ; Bosze et al., 2020 ). This highlighted differences between lines: KARO1 retained pluripotent-like cells at initial time points, HS980 generated more pre-placodal-like cells, and E1C3 initiated an endodermal-like population while also establishing the largest percentage of retinal progenitors ( Figures 2 C and 2D).…”

“…EB cultures also continued to harbor more diversity at D28: while 2D cultures were largely defined by various stages of RPE maturation, in the 3D setting other retinal and brain-related cell types were present, including neural retina, caudal neuroblasts, and glial cells ( Figures 3 D, 3E, and S3 D) ( Brodie-Kommit et al., 2021 ; La Manno et al., 2021 ). This evidence, in conjunction with the presence of distinct TBX2 + dorsal optic cup-like and VAX2 + ventral optic cup-/stalk-like progenitor populations, strongly suggests that a wider set of morphogenetic events are recapitulated during the EB protocol ( Bosze et al., 2020 ).…”

Molecular profiling of retinal pigment epithelial cell differentiation for therapeutic use

“…To determine the identity of the 24 cell clusters found during pigmentation induction (D7, D14, D30), we analyzed enriched genes by population, cross-referenced the literature and annotated each cluster with a primary (group) and secondary (cluster) category. This process revealed a mixture of intermediate cell states resembling those described in the context of rostral embryo patterning and eye development (Bosze et al, 2020;Sarkar et al, 2020).…”

“…The observation of in vitro MesCh is interesting because periocular mesenchyme expresses inductive signals in vivo that promote RPE fate, a role carried out by Activin A in the present protocol (Bosze et al, 2020 cells obtained using 42 well-characterized marker genes (6 pluripotency, 20 retinal progenitor, and 16 RPE genes). (D) Signature scores constructed from 42 marker genes (see Methods).…”

“…PCA, UMAP, and Louvain clustering were performed with standard parameters in scanpy and velocyto using spliced counts (2,000 selected genes, 20 PCs, 50 nearest neighbors). Global dimensionality reduction of the entire dataset was performed using a profile of pluripotency (6 genes: LIN28A, NANOG, POU5F1, SALL4, SOX2, ZFP42), retinal progenitor (20 genes: BMP4, FGF3, FGF8, LHX2, NCAM1, OPTC, OTX1, OTX2, PAX2, PAX6, RAX, SIX3, SIX6, TBX2, TBX3, TBX5, VAX2, VSX2, ZIC1, ZIC2) and RPE (16 genes: BEST1, ELN, IGFBP5, MITF, PMEL, RGR, RLBP1, RPE65, SERPINF1, SLC7A8, TMEFF2, TRPM1, TRPM3, TTR, TYR, TYRP1) marker genes, and the first two principal components derived from normalized spliced counts were visualized (Bosze et al, 2020;Fuhrmann, 2010;Schmitt et al, 2009). Signature score for apoptosis and stress response were computed using the following 37 genes: CASP8, CASP10, E2F5, CASP2, PTEN, CASP7, CDKN2A, E2F3, E2F4, CASP6, TP53, E2F1, CDKN1B, CDKN1A, E2F2, CASP3, FHIT, CASP9, HSPH1 GPX3, SOCS3, FOS, HSPA1A, SOD2, SOD1, HSF1, SOD3, HSPA1B, HSPA4, CASP6, GPX4, CAT, TNF, IL6, HSPA1L, NR3C1, HSPB1.…”