Single-cell sequencing of individual retinal organoids reveals determinants of cell-fate heterogeneity

With a critical need for more complete in vitro models of human development and disease, organoids hold immense potential. Their complex cellular composition makes single-cell sequencing of great utility; however, the limitation of current technologies to a handful of treatment conditions restricts their use in screens or studies of organoid heterogeneity. Here, we apply sci-Plex, a single-cell combinatorial indexing (sci)-based RNA sequencing (RNA-seq) multiplexing method to retinal organoids. We demonstrate that sci-Plex and 103 methods produce highly concordant cell-class compositions and then expand sci-Plex to analyze the cell-class composition of 410 organoids upon modulation of critical developmental pathways. Leveraging individual organoid data, we develop a method to measure organoid heterogeneity, and we identify that activation of Wnt signaling early in retinal organoid cultures increases retinal cell classes up to 6 weeks later. Our data show sci-Plex's potential to dramatically scale up the analysis of treatment conditions on relevant human models.