Three-Dimensional Imaging of Organoids to Study Primary Ciliogenesis During ex vivo Organogenesis

Summary

Stem cell-derived organoids facilitate the analysis of molecular and cellular processes that regulate stem cell self-renewal and differentiation during organogenesis in mammalian tissues. Here we present a protocol for the analysis of the biology of the primary cilium in mouse mammary organoids.

Abstract

Organoids are stem cell-derived three-dimensional structures that reproduce ex vivo the complex architecture and physiology of organs. Thus, organoids represent useful models to study the mechanisms that control stem cell self-renewal and differentiation in mammals, including primary ciliogenesis and ciliary signaling. Primary ciliogenesis is the dynamic process of assembling the primary cilium, a key cell signaling center that controls stem cell self-renewal and/or differentiation in various tissues. Here we present a comprehensive protocol for the immunofluorescence staining of cell lineage and primary cilia markers, in whole-mount mouse mammary organoids, for light sheet microscopy. We describe the microscopy imaging method and an image processing technique for the quantitative analysis of primary cilium assembly and length in organoids. This protocol enables a precise analysis of primary cilia in complex three-dimensional structures at the single cell level. This method is applicable for immunofluorescence staining and imaging of primary cilia and ciliary signaling in mammary organoids derived from normal and genetically modified stem cells, from healthy and pathological tissues, to study the biology of the primary cilium in health and disease.

Introduction

Development of multicellular organisms and the maintenance of homeostasis in their adult tissues reside in a fine-tuned regulation between self-renewal and differentiation of stem cells, which orchestrate in time and space normal tissue development and regeneration¹. Subversion of this regulation causes developmental anomalies and cancers². Thus, understanding the molecular and cellular mechanisms that orchestrate stem cell self-renewal and differentiation is of key interest in developmental and cancer biology.

Recent development of ex vivo organogenesis methods, in which tissue stem ....

Protocol

NOTE: The protocol below is recommended for the staining of organoids that were grown in 5 wells of a 96 well plate and pooled together (> 100 organoids). Organoids were derived from mouse mammary stem cells. Donor mice were housed and handled in accordance with protocols approved by the Animal Care Committee of the University of Rennes (France).

1. Reagents

1. To prepare the fixative solution, dilute 125 µL of 16% paraformaldehyde (PFA) aqueous commercial solution in 375 µL of phosphate-buffered saline (PBS) to generate a 4% PFA solution.
   CAUTION: Manipulate PFA that is a toxic substance under a chemical hood.....

Results

Ex vivo organogenesis methods are transforming our capabilities to study mammalian tissue development and maintenance of tissue homeostasis in a dish. The analysis of molecular and cellular mechanisms that regulate these processes, including primary ciliogenesis and ciliary signaling, relies on the ability to image organoids in three-dimension.

The protocol described above enables the staining of whole-mount mammary organoids. They arise from mammary stem cell-enriched basal cells tha.......

Discussion

The detailed protocol presented here enables the staining and imaging of mouse mammary organoids that grow in semi-solid medium. This protocol is presumably applicable to the staining of organoids mimicking the architecture of various tissues that grow in semi-solid and solid media. For organoids that grow in 100% Matrigel with medium on top, the recovery and fixation steps slightly differ. The culture medium must be removed from the culture well. After a quick PBS wash, the fixative solution (4% PFA) may be directly add.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anti-mouse IgG1 647	Thermo-Fisher	A21240
Anti-mouse IgG2A 488	Thermo-Fisher	A21131
Anti-rabbit 546	Thermo-Fisher	A11035
Arl13b	NeuroMab	73-287
EMS 16% Paraformaldehyde Aqueous Solution, EM Grade	Electron Microscopy Sciences	15710
FBS	Thermo-Fisher	10270106
gtubulin	Sigma-Aldrich	T5326
Hoechst 33342	Thermo-Fisher	62249
Integrin a6	Biolegend	313616
Light Sheet Capillary	Zeiss	701908
Light Sheet plunger	Zeiss	701998
Low binding Microcentrifuge tubes	BioScience	27210
Normal Goat Serum Blocking Solution	Vector labs	S-1000
PBS	Sigma-Aldrich	p3587
Slug	Cell Signaling Technology	9585
Triton-X100	Sigma-Aldrich	T9284
Tween-20	Euromedex	9005-64-5
UltraPure Low Melting Point Agarose	Thermo-Fisher	16520050