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

ABSTRACT: 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-seq multiplexing method to retinal organoids. We demonstrate that sci-Plex and 10x methods produce highly concordant cell type compositions and then expand sci-Plex to analyze the cell type 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 types up to six weeks later. Our data show sci-Plex’s potential to dramatically scale-up the analysis of treatment conditions on relevant human models.

ORGANISM(S): Homo sapiens

PROVIDER: GSE220661 | GEO | 2022/12/14

REPOSITORIES: GEO

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Project description:Cerebral organoids â three-dimensional cultures of human cerebral tissue derived from pluripotent stem cells â have emerged as models of human cortical development. However, the extent to which in vitro organoid systems recapitulate neural progenitor cell proliferation and neuronal differentiation programs observed in vivo remains unclear. Here we use single-cell RNA sequencing (scRNA-seq) to dissect and compare cell composition and progenitor-to-neuron lineage relationships in human cerebral organoids and fetal neocortex. Covariation network analysis using the fetal neocortex data reveals known and novel interactions among genes central to neural progenitor proliferation and neuronal differentiation. In the organoid, we detect diverse progenitors and differentiated cell types of neuronal and mesenchymal lineages, and identify cells that derived from regions resembling the fetal neocortex. We find that these organoid cortical cells use gene expression programs remarkably similar to those of the fetal tissue in order to organize into cerebral cortex-like regions. Our comparison of in vivo and in vitro cortical single cell transcriptomes illuminates the genetic features underlying human cortical development that can be studied in organoid cultures. 734 single-cell transcriptomes from human fetal neocortex or human cerebral organoids from multiple time points were analyzed in this study. All single cell samples were processed on the microfluidic Fluidigm C1 platform and contain 92 external RNA spike-ins. Fetal neocortex data were generated at 12 weeks post conception (chip 1: 81 cells; chip 2: 83 cells) and 13 weeks post conception (62 cells). Cerebral organoid data were generated from dissociated whole organoids derived from induced pluripotent stem cell line 409B2 (iPSC 409B2) at 33 days (40 cells), 35 days (68 cells), 37 days (71 cells), 41 days (74 cells), and 65 days (80 cells) after the start of embryoid body culture. Cerebral organoid data were also generated from microdissected cortical-like regions from H9 embryonic stem cell derived organoids at 53 days (region 1, 48 cells; region 2, 48 cells) or from iPSC 409B2 organoids at 58 days (region 3, 43 cells; region 4, 36 cells).

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

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