Single-Cell Transcriptomics Reveals the Molecular Basis of Human iPS Cell Differentiation into Ectodermal Ocular Lineages

The generation of a self-formed, ectodermal, autonomous multi-zone (SEAM) from human induced pluripotent stem cells (hiPSCs) offers a unique perspective to study the dynamics of ocular cell differentiation over time. Here, by utilising single-cell transcriptomics, we have (i) identified, (ii) molecularly characterised and (iii) ascertained the developmental trajectories of ectodermally-derived ocular cell populations which emerge within SEAMs as they form. Our analysis reveals interdependency between tissues of the early eye and delineates the sequential formation and maturation of distinct cell types over a 12-week period. We demonstrate a progression from pluripotency through to tissue specification and differentiation which encompasses both surface ectodermal and neuroectodermal ocular lineages and the generation of iPSC-derived components of the developing cornea, conjunctiva, lens, and retina. Our findings not only advance the understanding of ocular development in a stem cell-based system of human origin, but also establish a robust methodological paradigm for exploring cellular and molecular dynamics during SEAM formation at single-cell resolution and highlight the potential of hiPSC-derived systems as powerful platforms for modelling human eye development and disease. Overall design: Human iPSC cells (clone 201B7, RIKEN BioResource Center, Tsukuba, Japan) were maintained on LN511E8 (Nippi, Tokyo, Japan) coated culture dishes in serum-free StemFit medium (Ajinomoto, Tokyo, Japan) for at least 2 cell passages for stabilisation. Differentiation was initiated by culture in differentiation medium (DM) following established protocols. After culture for 4 weeks, DM was substituted for corneal DM (CDM) containing growth factors (KGF, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and the ROCK inhibitor Y-27623 (FUJIFILM Wako Pure Chemical Corporation) to promote epithelial cell growth and survival. After an additional 4 weeks of CDM culture (8 weeks in total), the medium was changed to corneal epithelial maintenance medium (CEM) consisting of DMEM/F12 (1:1; Life Technologies, Carlsbad, CA, USA) containing KGF, Y-27623 and B-27 supplement (Life Technologies). Cells were harvested at successive timepoints spanning 0 – 12 weeks and single cell suspensions were prepared according to current 10x Genomics guidelines.