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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 cells generate three-dimensional organoids have transformed our capabilities to study the dynamics of stem cells during mammalian organogenesis and maintenance of tissue homeostasis in a dish3. Organoids represent a good alternative to cumbersome genetically modified animal models to study these processes. Protocols for the development of organoids from tissue stem cells of many organs have now been developed3, including small intestine and colon, stomach, liver, pancreas, prostate, and mammary gland3. Additionally, the development of somatic genome-editing techniques in organoid-forming stem cells now enables to quickly interrogate the molecular and cellular mechanisms that control their biology4,5.

The primary cilium is a microtubule-based structure that is assembled at the surface of stem and/or differentiated cells of various tissues6. It is generally non-motile and is assembled as a single structure per cell7. Primary ciliogenesis is the dynamic process of assembling the primary cilium7. At the cell surface, the cilium acts as a cell signaling platform8. Thus, the primary cilium is thought to act as a key regulator of stem cell self-renewal and/or differentiation in many tissues, including the brain9,10, the mammary gland4,11, the adipose tissue12, and the olfactory epithelium13, among others. Primary ciliogenesis and/or ciliary signaling are dynamically regulated in distinct cell lineages and at different developmental stages4,13,14, but the underlying mechanisms remain to be largely determined.

Ex vivo organogenesis shows promise for the development of basic knowledge on the molecular and cellular mechanisms that control stem cell biology, including primary ciliogenesis and ciliary signaling. However, it relies on the ability to properly image whole mount organoids at the single cell level and at sub-cellular scales. We recently used a mouse mammary stem cell-derived organoid model to show that primary cilia positively control mouse mammary stem cell organoid-forming capacity4. Here we present a comprehensive protocol for the immunofluorescence staining of whole mount mouse mammary organoids (Figure 1A,B), which enables the analysis of primary cilia through light sheet microscopy during ex vivo organogenesis in three-dimension. Alternative methods were recently published for the immunofluorescence staining and imaging of organoids through confocal microscopy15,16. This protocol focuses instead on the preparation and imaging of organoids through light sheet microscopy.

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.
  2. To prepare the permeabilization buffer, dilute 1.5 µL of Triton X100 in 500 µL of PBS, to produce a 0.3% Triton X100 solution.
  3. To prepare the blocking buffer, dilute 1.5 µL of Tween-20 and 75 µL of normal goat serum in PBS, to generate a 5% goat serum-0.1% Tween 20 solution.
  4. To prepare the light sheet mounting medium, dissolve 1 g of ultrapure low melting point agarose in 100 mL of dH20 or PBS at 65 °C. Prepare 1 mL aliquots and store at room temperature.

2. Organoids recovery

  1. Transfer organoids from the culture wells to a low binding polymer 1.7 mL tube, after pipetting them up and down 3 times in the wells, with an FBS-coated tip for which the end was cut (minimal diameter of the extremity 1.5 mm).
    NOTE: The coating prevents organoids from sticking to the tip. The low-binding polymer material enables to reduce the attachment of organoids to the side of the tube during the entire staining procedure.
  2. Fill the tube with PBS, and spin down at 350 x g for 3 min. Remove the supernatant.

3. Fixation, permeabilization and blocking

  1. Resuspend the organoids in 500 µL of 4% PFA. Incubate for 30 min at room temperature. Spin down at 350 x g for 30 s. Remove the supernatant.
    NOTE: From this step onwards, resuspend organoids in the different buffers by simply adding the buffers on the pellet of organoids without touching them. Perform all washing steps at room temperature.
  2. Wash the organoids with 1 mL of PBS for 3 min. Spin down at 350 x g for 30 s. Remove the supernatant.
    PAUSE: Fixed organoids can be kept in PBS at 4 °C for at least a week.
  3. Permeabilize the organoids by resuspending them in 500 µL of PBS-Triton X100 0.3% and incubate for 30 min at room temperature. Spin down the organoids at 350 g for 30 s.
  4. Wash the organoids by resuspending them with 1 mL of PBS and incubate for 3 min. Spin down at 350 x g for 30 s. Remove the supernatant. Repeat once.
  5. Optional: Resuspend the organoids in 500 µL of ice-cold methanol. Incubate at - 20 °C for 10 min. This step may be required for the staining of specific centrosomal markers.
  6. Optional (if step 5 was performed): Wash the organoids with 1 mL of PBS for 3 min. Spin down at 350 x g for 30 s. Remove the supernatant. Repeat once.
  7. Block non-specific antibody binding sites in organoids by resuspending them with 500 µL of blocking buffer (PBS, 5% goat serum, 0.1% Tween 20). Incubate 1 h and 30 min at room temperature. Spin down at 350 g for 30 s. Remove the supernatant.
  8. Wash the organoids by resuspending them with 1 mL of PBS and incubate for 3 min. Spin down at 350 x g for 30 s. Remove the supernatant.

4. Labelling

  1. Resuspend the organoids with 200 µL of blocking buffer with diluted primary antibodies and incubate overnight at 4 °C with mild shaking (60 rpm on a horizontal shaker). Place the tubes with a 45° angle with the horizontal plan of the shaker. It will maintain the organoids at the bottom of the tubes in the staining buffer.
  2. Wash the organoids by resuspending them with 1 mL of PBS and incubate for 5 min. Spin down at 350 x g for 30 s. Remove the supernatant. Repeat twice.
    NOTE: Some organoids in the last step may stick to the side of the tube, resulting in organoid loss during aspiration of the supernatant after centrifugation, adding 0.2% (w/v) bovine serum albumin (BSA) to the PBS in the washing steps may reduce organoid loss.
  3. Resuspend the organoids with 200 µL of blocking buffer with secondary antibodies and incubate for 1 h and 30 min with mild shaking (60 rpm on a horizontal shaker). Place the tubes with a 45° angle with the horizontal plan of the shaker. It will maintain the organoids at the bottom of the tubes in the staining buffer.
    NOTE: Hoechst (or other nuclear dyes, such as DAPI or DRAQ5) can be added to the buffer with secondary antibodies.
  4. Wash the organoids by resuspending them with 1 mL of PBS and incubate for 5 min. Spin down at 350 x g for 30 s. Remove the supernatant. Repeat twice.
    ​NOTE: Some organoids in the last step may stick to the side of the tube, resulting in organoid loss during aspiration of the supernatant after centrifugation, adding 0.2% (w/v) BSA to the PBS in the washing steps may reduce organoid loss.

5. Preparation of the agarose sample for imaging

  1. Melt light sheet mounting medium by incubating it at 65 °C. Once the medium has melted, incubate it at 37 °C for 5 min.
  2. Resuspend the organoids in 100 µL of mounting medium using a 200 µL tip, with the extremity of the tip cut (minimal size of the extremity: 1.5 mm), by pipetting up and down twice.
  3. Suck the mounting medium with the organoids in a glass capillary (green capillary, inner diameter: 1.5 mm) using a plunger. Incubate the capillary at room temperature for 5 min for the mounting medium to solidify.
    PAUSE: Capillary can be stored in PBS at 4 °C for a week before imaging.

6. Imaging

  1. Using a light sheet microscope (e.g., ZEISS Lightsheet Z.1), image the organoids with 20x or 40x water immersion objectives.
    1. Place the glass capillary in the observation chamber. Locate the capillary with the front camera and place the tip of the glass capillary at the upper limit of the detection objective.
    2. Select the sample and set the optimal focus using the tip of the glass capillary in brightfield illumination. Push out the agarose sample slowly from the capillary and locate organoids within the solidified agarose.
    3. Keep the organoids to be imaged close to the tip of the capillary, to reduce movements of the agarose sample in the PBS. Rotate the capillary to set the optimal sample orientation and define the desired zoom.
    4. Select acquisition mode. Define the channels to image, set proper orientations of the light sheet, set the laser power for each channel (to reduce photobleaching, use low laser power), set the size of the image and the desired illumination side(s). Example of imaging parameters: Image size 1920 x 1920, illumination: dual side.
    5. Define the Z-stack to image by setting the Z-extremities of the stack using the Z-stack module and set the Z-step size to optimal.
  2. Process the output file using the image processing module of the microscope software to navigate in the sample, obtain a Z-projection and 3D-representation.
  3. Turn the sample and acquire a new image from a different angle or image other organoids.
  4. Analyze images with an interactive microscopy image analysis software (e.g., Imaris) enabling (i) the visualization of the sample in 3D (ii) the segmentation of objects (iii) identification and quantitative analysis of objects.

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...

Disclosures

The authors declare no conflict of interest.

Acknowledgements

We thank Xavier Pinson for help in development of light sheet microscopy; the Biosit biotechnology center, including MRic, Arche, the Flow cytometry core facilities, and SFR Santé F. Bonamy, including the MicroPICell core facility, for technical support. This work was supported by Fondation ARC, Cancéropôle Grand Ouest, Université de Rennes 1, Fondation de France. M.D. was supported by a Graduate Fellowship from the University of Rennes. V.J.G. was supported by a Postdoctoral Fellowship from Fondation ARC.

Materials

NameCompanyCatalog NumberComments
Anti-mouse IgG1 647Thermo-FisherA21240
Anti-mouse IgG2A 488Thermo-FisherA21131
Anti-rabbit 546Thermo-FisherA11035
Arl13bNeuroMab73-287
EMS 16% Paraformaldehyde Aqueous Solution, EM GradeElectron Microscopy Sciences15710
FBSThermo-Fisher10270106
gtubulinSigma-AldrichT5326
Hoechst 33342Thermo-Fisher62249
Integrin a6Biolegend313616
Light Sheet CapillaryZeiss701908
Light Sheet plungerZeiss701998
Low binding Microcentrifuge tubesBioScience27210
Normal Goat Serum Blocking SolutionVector labsS-1000
PBSSigma-Aldrichp3587
SlugCell Signaling Technology9585
Triton-X100Sigma-AldrichT9284
Tween-20Euromedex9005-64-5
UltraPure Low Melting Point AgaroseThermo-Fisher16520050

References

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  2. Batlle, E., Clevers, H. Cancer stem cells revisited. Nature Medicine. 23 (10), 1124-1134 (2017).
  3. Clevers, H. Modeling Development and Disease with Organoids. Cell. 165 (7), 1586-1597 (2016).
  4. Guen, V. J., et al. EMT programs promote basal mammary stem cell and tumor-initiating cell stemness by inducing primary ciliogenesis and Hedgehog signaling. Proceedings of the National Academy of Sciences of the United States of America. , (2017).
  5. Hendriks, D., Clevers, H., Artegiani, B. CRISPR-Cas Tools and Their Application in Genetic Engineering of Human Stem Cells and Organoids. Cell Stem Cell. 27 (5), 705-731 (2020).
  6. Satir, P., Pedersen, L. B., Christensen, S. T. The primary cilium at a glance. Journal of Cell Science. 123, 499-503 (2010).
  7. Guen, V. J., Prigent, C. Targeting Primary Ciliogenesis with Small-Molecule Inhibitors. Cell Chemical Biology. 27 (10), 1224-1228 (2020).
  8. Goetz, S. C., Anderson, K. V. The primary cilium: a signalling centre during vertebrate development. Nature Reviews Genetics. 11 (5), 331-344 (2010).
  9. Han, Y. G., et al. Hedgehog signaling and primary cilia are required for the formation of adult neural stem cells. Nature Neuroscience. 11 (3), 277-284 (2008).
  10. Tong, C. K., et al. Primary cilia are required in a unique subpopulation of neural progenitors. Proceedings of the National Academy of Sciences of the United States of America. 111 (34), 12438-12443 (2014).
  11. Wilson, M. M., Weinberg, R. A., Lees, J. A., Guen, V. J. Emerging Mechanisms by which EMT Programs Control Stemness. Trends in Cancer. 6 (9), 775-780 (2020).
  12. Hilgendorf, K. I., et al. Omega-3 Fatty Acids Activate Ciliary FFAR4 to Control Adipogenesis. Cell. 179 (6), 1289-1305 (2019).
  13. Joiner, A. M., et al. Primary Cilia on Horizontal Basal Cells Regulate Regeneration of the Olfactory Epithelium. Journal of Neuroscience. 35 (40), 13761-13772 (2015).
  14. Bangs, F. K., Schrode, N., Hadjantonakis, A. K., Anderson, K. V. Lineage specificity of primary cilia in the mouse embryo. Nature Cell Biology. 17 (2), 113-122 (2015).
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Three dimensional ImagingOrganoidsPrimary CiliogenesisEx Vivo OrganogenesisStem Cell Self renewalCiliary SignalingImmunofluorescence StainingLight Sheet MicroscopyImage Processing TechniqueQuantitative AnalysisPrimary Cilia MarkersMammary OrganoidsSingle Cell LevelHealthy TissuesPathological Tissues

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