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Harnessing single-cell genomics to improve the physiological fidelity of organoid-derived cell types

ABSTRACT: Mead BE, Ordovas-Montanes J, Braun AP, Levy LE, Bhargava P, Szucs MJ, Ammendolia DA, MacMullan MA, Zheng Y, Yin X, Hughes TK, Wadsworth MH, Ahmad R, Rakoff-Nahoum S, Carr SA, Langer R, Collins JJ, Shalek AK, Karp JM. BMC Biology 2018. Background: Single-cell genomic methods now provide unprecedented resolution for characterizing the component cell types and states of tissues, such as the epithelial subsets of the gastrointestinal tract. Nevertheless, functional studies of these subsets at scale require faithful in vitro models of identified in vivo biology. While intestinal organoids have been invaluable in providing mechanistic insights in vitro, the extent to which organoid-derived cell types recapitulate their in vivo counterparts remains formally untested, with no systematic approach for improving model fidelity. Results: Here, we present a generally applicable framework that utilizes massively-parallel single-cell RNA-seq to compare cell types and states found in vivo to those of in vitro models, such as organoids. Furthermore, we leverage identified discrepancies to improve model fidelity. Using the Paneth cell (PC), which supports the stem cell niche and produces the largest diversity of antimicrobials in the small intestine, as an exemplar, we uncover fundamental gene expression differences in lineage-defining genes between in vivo PCs and those of the current in vitro organoid model. With this information, we nominate a molecular intervention to rationally improve the physiological fidelity of our in vitro PCs. We then perform transcriptomic, cytometric, morphologic, and proteomic characterization, and demonstrate functional (antimicrobial activity, niche support) improvements in Paneth cell physiology. Conclusions: Our systematic approach provides a simple workflow for identifying the limitations of in vitro models and enhancing their physiological fidelity. Using adult stem cell-derived PCs within intestinal organoids as a model system, we successfully benchmark organoid representation relative to in vivo of a specialized cell type and use this comparison to generate a functionally improved in vitro PC population. We predict that the generation of rationally-improved cellular models will facilitate mechanistic exploration of specific disease-associated genes in their respective cell types.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Steven A. Carr

PROVIDER: MSV000081369 | MassIVE | Thu Jul 20 14:58:00 BST 2017

REPOSITORIES: MassIVE

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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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Harnessing single-cell genomics to improve the physiological fidelity of organoid-derived cell types

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