Three-dimensional analysis of single molecule FISH in human colon organoids.

Manja Omerzu,Buys De Barbanson,Madelon M Maurice,Jeroen De Ridder,Nicola Fenderico,Joep Sprangers

doi:10.1242/bio.042812

Abstract

ABSTRACTThe culturing of mini-organs (organoids) in three-dimensions (3D) presents a simple and powerful tool to investigate the principles underlying human organ development and tissue self-organization in both healthy and diseased states. Applications of single molecule analysis are highly informative for a comprehensive understanding of the complexity underlying tissue and organ physiology. To fully exploit the potential of single molecule technologies, the adjustment of protocols and tools to 3D tissue culture is required. Single molecule RNA fluorescence in situ hybridization (smFISH) is a robust technique for visualizing and quantifying individual transcripts. In addition, smFISH can be employed to study splice variants, fusion transcripts as well as transcripts of multiple genes at the same time. Here, we develop a 3-day protocol and validation method to perform smFISH in 3D in whole human organoids. We provide a number of applications to exemplify the diverse possibilities for the simultaneous detection of distinct mRNA transcripts, evaluation of their spatial distribution and the identification of divergent cell lineages in 3D in organoids.

Highlights

Over the last decade, considerable advances have been made in the development of 3D tissue culture systems (Clevers, 2016)
Reliable method for the spatial evaluation of individual mRNA transcripts in 3D tissue cultures that can be implemented in labs equipped with standard wide-field and confocal microscopes
Monitoring transcriptional alterations in human colon organoids using Single molecule RNA fluorescence in situ hybridization (smFISH) we investigated the sensitivity of our smFISH method for the detection of signaling-induced alterations in transcriptional activity in 3D

Summary

Introduction

Considerable advances have been made in the development of 3D tissue culture systems (Clevers, 2016). In the first reported long-term cultured organoid model, Lgr5-positive (Lgr5+) intestinal stem cells (ISC) were isolated from murine small intestinal crypts (Sato et al, 2009). Further improvement of culture protocols allowed for the establishment of organoids derived from human healthy colon stem cells and their cancer-derived equivalents as well (Jung et al, 2011; Sato et al, 2011). Human colon organoids have been employed in several applications, including disease modelling, the generation of colorectal cancer biobanks, as well as predictors for personalized medicine applications (Dekkers et al, 2016; Drost and Clevers, 2017; Drost et al, 2015; Matano et al, 2015; van de Wetering et al, 2015). The modulation of key signaling cascades within organoids has enabled mechanistic insights into the regulation of cell plasticity, lineage specification and the formation of rare cell types (Basak et al, 2017; Drost and Clevers, 2017; Jung et al, 2011; Sato et al, 2011)

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