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

Abstract

BackgroundSingle-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.ResultsHere, 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 PC physiology.ConclusionsOur 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 that 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.Electronic supplementary materialThe online version of this article (10.1186/s12915-018-0527-2) contains supplementary material, which is available to authorized users.

Regulatory network analysis of Paneth cell and goblet cell enriched gut organoids using transcriptomics approaches

“…In addition, signatures of enteroendocrine cells were increased in both differentiation protocols, albeit at a lower amount than the target cell type. This finding correlates with previous investigations at both the transcriptomic and proteomic levels (25,(27)(28)(29) and is likely due to the shared differentiation pathways of these secretory cells. Nevertheless, as enteroids contain a mixed population of cell types by nature and because intercellular communication is key to a functioning epithelium (19,71), the increased proportion of non-targeted secretory lineages should not be an issue for the application of these models to research.…”

“…Whilst these methods do not present single cell type resolution, they provide useful tools to study Paneth cell and goblet cell populations in the context of the other major epithelial cell types (26). A recent study by Mead et al found that Paneth cells from enriched enteroids more closely represent their in vivo counterparts than those isolated from conventionally differentiated enteroids, based on transcriptomics, proteomics and morphologic data (27). Furthermore, we have shown that enteroids enriched for Paneth cells and goblet cells recapitulate in vivo characteristics on the proteomics level (28,29) and that they are a useful tool for the investigation of health and disease related processes in specific intestinal cell types (29).…”

Regulatory network analysis of Paneth cell and goblet cell enriched gut organoids using transcriptomics approaches

“…26 A recent study by Mead et al found that Paneth cells from enriched enteroids more closely represent their in vivo counterparts than those isolated from conventionally differentiated enteroids, based on transcriptomics, proteomics and morphologic data. 27 Furthermore, we have shown that enteroids enriched for Paneth cells and goblet cells recapitulate in vivo characteristics on the proteomics level 28,29 and that they are a useful tool for the investigation of health and disease related processes in specific intestinal cell types. 29 Nevertheless, the effect of Paneth cell and goblet cell enrichment of enteroids on key regulatory landscapes has not been extensively characterised.…”

High-throughput organoid screening enables engineering of intestinal epithelial composition

“…Spontaneous differentiation of organoids grown in ENR media can be mitigated, and the population of ISCs enriched by the addition of small molecules CHIR99021 (C), an activator of WNT signaling, and valproic acid (V), an activator of Notch signaling. These ISC-enriched cultures can then be differentiated towards Paneth cells with the small molecules (C) and DAPT (D), an inhibitor of Notch signaling, as we have previously shown (Mead et al, 2018;Yin et al, 2014). To measure changes in Paneth cell abundance or quality, we employ a previously demonstrated assay of Paneth cell-specific function and relative-abundance with a commercially-available assay for secreted lysozyme (LYZ) (Mead et al, 2018).…”

“…These ISC-enriched cultures can then be differentiated towards Paneth cells with the small molecules (C) and DAPT (D), an inhibitor of Notch signaling, as we have previously shown (Mead et al, 2018;Yin et al, 2014). To measure changes in Paneth cell abundance or quality, we employ a previously demonstrated assay of Paneth cell-specific function and relative-abundance with a commercially-available assay for secreted lysozyme (LYZ) (Mead et al, 2018). To scale this model system and measurement, we adapt conventional 3-D organoid culture into a 2.5-D pseudo-monolayer, where ISC-enriched organoids are partially embedded on the surface of a thick layer of Matrigel (at the Matrigel-media interface), rather than fully encapsulated in the Matrigel structure -an approach similar to others previously reported (Langhans, 2018).…”