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A high-throughput platform for stem cell niche co-cultures and downstream gene expression analysis

Abstract

Stem cells reside in ‘niches’, where support cells provide critical signalling for tissue renewal. Culture methods mimic niche conditions and support the growth of stem cells in vitro. However, current functional assays preclude statistically meaningful studies of clonal stem cells, stem cell–niche interactions, and genetic analysis of single cells and their organoid progeny. Here, we describe a ‘microraft array’ (MRA) that facilitates high-throughput clonogenic culture and computational identification of single intestinal stem cells (ISCs) and niche cells. We use MRAs to demonstrate that Paneth cells, a known ISC niche component, enhance organoid formation in a contact-dependent manner. MRAs facilitate retrieval of early enteroids for quantitative PCR to correlate functional properties, such as enteroid morphology, with differences in gene expression. MRAs have broad applicability to assaying stem cell–niche interactions and organoid development, and serve as a high-throughput culture platform to interrogate gene expression at early stages of stem cell fate choices.

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Figure 1: Modified MRAs are compatible with long-term culture of primary ISCs.
Figure 2: Software-assisted post hoc analysis identifies initial well contents of MRA culture.
Figure 3: Single-cell qPCR confirms PC purity.
Figure 4: Cell-to-cell contact is required for PC-influenced survival of ISCs in vitro.
Figure 5: Single cells do not form cell–cell contacts in microwells after initial plating.
Figure 6: Microraft retrieval facilitates gene expression analysis of enteroid development and morphology.
Figure 7: Culture conditions drive transcriptional changes in single Lgr5high cells.

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Acknowledgements

The authors thank the UNC flow cytometry facility (P30CA06086), especially B. Udis and N. Fisher; C. D. Collins for help with quantification of survival; D. DeBree for data verification; A. Ahmad and P. Shah for useful discussions regarding array fabrication and analysis; D. Trotier for technical assistance with enteroid retrieval and graphics support; P. K. Lund, S. Henning (SJH) and C. Dekaney for useful discussions and critical review of the manuscript. A.D.G. received partial salary support from U01 DK085541 (SJH). This work was financially supported by the National Institutes of Health R01 DK091427 (S.T.M.), R03 EB013803 (Y.W./S.T.M.), R01 EB012549 (N.L.A.), Small Business Innovation Research R43 GM106421 (S.T.M./Y.W.), U01 DK085507-01 (L.L.), University Cancer Research Fund of the University of North Carolina (S.T.M./N.L.A.), and the Center for Gastrointestinal Biology and Disease P30 DK034987 (S.T.M., Y.W., J.A.G.). L.L. is a member of the Intestinal Stem Cell Consortium, supported by NIDDK and NIAID. A.D.G. was supported by a UNC Graduate School Dissertation Completion Fellowship.

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Authors

Contributions

A.D.G. and S.T.M. conceived and designed experiments. I.A.W. developed and optimized image analysis algorithms and analysed data with M.J.J., X.F.L., R.J.L. and L.T.G. Y.W., P.J.A. and J.B. microfabricated MRAs. Y.W., C.E.S. and N.L.A. supervised microfabrication and troubleshooting of MRA development. K.C.R. analysed single-cell and enteroid gene expression data. P.J.A. conducted and interpreted COMSOL modelling with N.L.A. F.W. and L.L. developed and provided methodology for highly efficient enteroid culture. J.A.G. conducted statistical analysis of enteroid formation assays. A.D.G. and S.T.M. wrote the manuscript with critical insight and commentary from all co-authors. S.T.M. initiated and supervised the project.

Corresponding author

Correspondence to Scott T. Magness.

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Competing interests

N.L.A., C.E.S., Y.W. and S.T.M. disclose a financial interest in Cell Microsystems.

Integrated supplementary information

Supplementary Figure 3 Fabrication of glass-mounted microwell arrays.

(A) PDMS is liquid molded onto a PAA-coated glass slide on a SU-8 master mold. (B,C) PDMS/SU-8 mold assemblies are cured, and cured, solid PDMS is gently removed from the mold. (D) Polystyrene solution is added to the array and degassed. (E) Dip coating with a programmed stepper motor removes excess polystyrene. (F) Subsequent discontinuous dewetting generates isolated polystyrene pockets inside PDMS microwells. (G) Arrays are baked at 95 °C to remove solvent, resulting in solid polystyrene microrafts embedded in PDMS microwells. (H) MRAs are mounted to polycarbonate cassettes to complete the fabrication process.

Supplementary Figure 4 Glass-mounting reduces PDMS sagging in microwell arrays.

(A,C) The elastic properties of PDMS cause standard microwell arrays to sag, preventing tile-scanned imaging in a single Z-plane. (B,D) Mounting arrays to glass slides prevents sagging and facilitates imaging. Scale bars represent 300 μm.

Supplementary Figure 5 Sox9EGFP transgenic mice facilitate high purity FACS isolation of Paneth cells.

(A) To increase population purity, we developed novel FACS criteria for Paneth cell sorting. Standard size, double, and live-dead exclusion criteria were applied to all FACS isolations. (B) We compared putative Paneth cell populations isolated using previously described methods, which define Paneth cells as CD24high:SSChigh, and (C) our newly developed method, which applies the same parameters, but excludes all Sox9EGFP expressing cells.

Supplementary Figure 6 Sox9EGFPneg :CD24high :SSChigh enriches for highly pure Paneth cell populations.

Gene expression analysis demonstrates upregulation of Paneth cell marker Lyz2, and downregulation of ISC marker Lgr5, as well as EE cell marker Chga in Paneth cell populations isolated with Sox9EGFP exclusion, when compared to populations isolated with CD24 and SSC alone (values represent three technical replicates carried out on one biological replicate per sorting strategy).

Supplementary Figure 7 Retrieval of magnetic rafts for downstream gene expression analysis.

(A) Raft retrieval. A device containing a fine needle positioned in the center of a clear plexiglass window was fitted onto a 10X objective lens. Z-plane focus was used to puncture the bottom of the PDMS liberating the raft. A magnetic wand was used to collect the magnetic raft. (B) The magnetic wand facilitates efficient retrieval of magnetic rafts (note red magnetic raft on tip of wand). (C) The raft is liberated from the magnetic wand when placed in a 96-well format dish that is positioned over a stronger magnet place on ice. (D) Time lapse image of raft retrieval (frame 1-4). The raft with enteroid depicted in frame 1 was captured by magnetic wand and placed in PBS (frame 5). Enteroid was efficiently lysed in RNA lysis buffer prior to cDNA synthesis (frame 6). (E) A large well-developed enteroid was identified in the MrA and retrieved using the magnetic wand. (F) Matrigel anchors the large enteroid to the magnetic raft for efficient capture. (G) Rafts and associated images can be ordered in a conventional 96-well format for indexing and retrospective analysis. Scale bars represent 10μm.

Supplementary Figure 8 Enteroid morphologies are correlated with gene expression analysis.

Cystic (“Cyst”) and columnar (“Col”) enteroids are analyzed at (A) 24hr and (B) 48hr by microfluidic qPCR against 33 genes. Image capture immediately prior to raft retrieval allows for matching phenotypic characteristics, such as morphology, with gene expression results. Heat map represents Ct values.

Supplementary Figure 9 dCt values reveal differences in enteroid morphology at the genetic level.

Gene expression changes in cystic and columnar enteroids at 24hr and 48hr, for all genes assayed. Violin plots represent dCt values normalized to 18s signal; n = 13 cystic and 14 columnar enteroids at 24hr; 13 cystic and 12 columnar at 48hr; different letters represent statistical significance, one-way ANOVA, p < 0.05. Exact p values presented in Supplementary Table 3. Graphs without letters do not have statistically significant differences between groups.

Supplementary Table 1 Number of individual microwells examined for each combination of initial contents.
Supplementary Table 2 Taqman probes used for qPCR.

Supplementary information

Supplementary Information

Supplementary Information (PDF 6598 kb)

Supplementary Table 3

Supplementary Information (XLSX 46 kb)

Supplementary Video 1

The movie shows an enteroid developing from two touching ISCs (Sox9EGFP+) and migrating away from a Paneth cell (DsRed+) that dies early in culture, demonstrating movement of cells within microwells. The microwell was imaged every 30min for the first 22hrs of culture. (MP4 9962 kb)

Supplementary Video 2

The movie shows several small multimers of Sox9EGFP+ ISCs merging to form an enteroid in the lower left corner of the microwell. Images were taken every 30min for the first 22hrs of culture. (MP4 9971 kb)

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Gracz, A., Williamson, I., Roche, K. et al. A high-throughput platform for stem cell niche co-cultures and downstream gene expression analysis. Nat Cell Biol 17, 340–349 (2015). https://doi.org/10.1038/ncb3104

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