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

Benjamin E Mead,Marc H Wadsworth,Prerna Bhargava,Xiaolei Yin,Lauren E Levy,James J Collins,Melanie A Macmullan,Robert Langer,Jeffrey M Karp,Jose Ordovas-Montanes,Dustin A Ammendolia,Rushdy Ahmad,Steven A Carr,Alex K Shalek,Alexandra P Braun,Seth Rakoff-Nahoum,Matthew J Szucs,Travis K Hughes

doi:10.1186/s12915-018-0527-2

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.

Highlights

Single-cell genomic methods provide unprecedented resolution for characterizing the component cell types and states of tissues such as the epithelial subsets of the gastrointestinal tract
Using the Paneth cell (PC) to benchmark cell type representation of conventional organoids against their in vivo counterparts Conventional intestinal organoids produced from the spontaneous differentiation of intestinal stem cells (ISCs) have been used to study PCs in vitro in multiple contexts [23, 24]. These in vitro PCs exist as part of a heterogeneous system, yet to be rigorously benchmarked against their in vivo counterparts
To better understand the composition of PCs within conventional organoids and how well those PCs approximate their in vivo counterparts, we sought to globally compare the conventional organoid-derived PCs and their in vivo counterparts through a single-cell transcriptomic approach (Fig. 1a)

Summary

Introduction

Single-cell genomic methods provide unprecedented resolution for characterizing the component cell types and states of tissues such as the epithelial subsets of the gastrointestinal tract. The application of organoid models to the study of complex disease, such as polygenic inflammatory disease, has been limited In such instances, the subtler phenotypes, such as those present in inflammatory bowel disease (IBD), may not manifest if the originating cell state present in vivo is not accurately represented within an organoid model. The subtler phenotypes, such as those present in inflammatory bowel disease (IBD), may not manifest if the originating cell state present in vivo is not accurately represented within an organoid model This challenge is clear in IBD [11], where loci identified through genome wide association studies (GWAS) have proven difficult to efficiently examine through the use of in vivo animal models

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Discussion

Conclusion