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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Sato, T. & Clevers, H. Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications. Science 340, 1190–1194 (2013).
Barker, N. et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449, 1003–1007 (2007).
Gracz, A. D., Ramalingam, S. & Magness, S. T. Sox9-expression marks a subset of CD24-expressing small intestine epithelial stem cells that form organoids in vitro. Am. J. Physiol. Gastrointest. Liver Physiol. 298, G590–G600 (2010).
Sato, T. et al. Single Lgr5 stem cells build crypt–villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009).
Farin, H. F., Van Es, J. H. & Clevers, H. Redundant sources of wnt regulate intestinal stem cells and promote formation of paneth cells. Gastroenterology 143, 1518–1529 (2012).
Sato, T. et al. Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature 469, 415–418 (2011).
Formeister, E. J. et al. Distinct SOX9 levels differentially mark stem/progenitor populations and enteroendocrine cells of the small intestine epithelium. Am. J. Physiol. Gastrointest. Liver Physiol. 296, G1108–G1118 (2009).
Snippert, H. J. et al. Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Cell 143, 134–144 (2010).
Shackleton, M. et al. Generation of a functional mammary gland from a single stem cell. Nature 439, 84–88 (2006).
Till, J. E. & McCulloch, E. A. A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat. Res. 14, 213–222 (1961).
Lecault, V. et al. High-throughput analysis of single hematopoietic stem cell proliferation in microfluidic cell culture arrays. Nat. Methods 8, 581–586 (2011).
Wang, Y. et al. Micromolded arrays for separation of adherent cells. Lab Chip 10, 2917–2924 (2010).
Vintersten, K. et al. Mouse in red: red fluorescent protein expression in mouse ES cells, embryos, and adult animals. Genesis 40, 241–246 (2004).
Wang, F. et al. Isolation and characterization of intestinal stem cells based on surface marker combinations and colony-formation assay. Gastroenterology 145, 383–395, e21 (2013).
Carpenter, A. E. et al. CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 7, R100 (2006).
Powell, A. E. et al. The pan-ErbB negative regulator Lrig1 is an intestinal stem cell marker that functions as a tumor suppressor. Cell 149, 146–158 (2012).
Gracz, A. D. et al. CD24 and CD44 mark human intestinal epithelial cell populations with characteristics of active and facultative stem cells. Stem Cells 31, 2024–2030 (2013).
Von Furstenberg, R. J. et al. Sorting mouse jejunal epithelial cells with CD24 yields a population with characteristics of intestinal stem cells. Am. J. Physiol. Gastrointest. Liver Physiol. 300, G409–G417 (2010).
Buczacki, S. J. et al. Intestinal label-retaining cells are secretory precursors expressing Lgr5. Nature 495, 65–69 (2013).
Schwitalla, S. et al. Intestinal tumorigenesis initiated by dedifferentiation and acquisition of stem-cell-like properties. Cell 152, 25–38 (2013).
Van Es, J. H. et al. Dll1(+) secretory progenitor cells revert to stem cells upon crypt damage. Nat. Cell Biol. 14, 1090–1104 (2012).
Shah, P. K., Hughes, M. R., Wang, Y., Sims, C. E. & Allbritton, N. L. Scalable synthesis of a biocompatible, transparent and superparamagnetic photoresist for microdevice fabrication. J. Micromech. Microeng. 23, 107002 (2013).
Montgomery, R. K. et al. Mouse telomerase reverse transcriptase (mTert) expression marks slowly cycling intestinal stem cells. Proc. Natl Acad. Sci. USA 108, 179–184 (2011).
Munoz, J. et al. The Lgr5 intestinal stem cell signature: robust expression of proposed quiescent ‘+4’ cell markers. EMBO J. 31, 3079–3091 (2012).
Takeda, N. et al. Interconversion between intestinal stem cell populations in distinct niches. Science 334, 1420–1424 (2011).
Yan, K. S. et al. The intestinal stem cell markers Bmi1 and Lgr5 identify two functionally distinct populations. Proc. Natl Acad. Sci. USA 109, 466–471 (2012).
Kim, T. H. et al. Broadly permissive intestinal chromatin underlies lateral inhibition and cell plasticity. Nature 506, 511–515 (2014).
Stamataki, D. et al. Delta1 expression, cell cycle exit, and commitment to a specific secretory fate coincide within a few hours in the mouse intestinal stem cell system. PLoS ONE 6, e24484 (2011).
Jensen, J. et al. Control of endodermal endocrine development by Hes-1. Nat. Genet. 24, 36–44 (2000).
Yang, Q., Bermingham, N. A., Finegold, M. J. & Zoghbi, H. Y. Requirement of Math1 for secretory cell lineage commitment in the mouse intestine. Science 294, 2155–2158 (2001).
Kanatsu-Shinohara, M. et al. Reconstitution of mouse spermatogonial stem cell niches in culture. Cell Stem Cell 11, 567–578 (2012).
Wu, A. R. et al. Quantitative assessment of single-cell RNA-sequencing methods. Nat. Methods 11, 41–46 (2014).
Gobaa, S. et al. Artificial niche microarrays for probing single stem cell fate in high throughput. Nat. Methods 8, 949–955 (2011).
Roccio, M., Gobaa, S. & Lutolf, M. P. High-throughput clonal analysis of neural stem cells in microarrayed artificial niches. Integr. Biol. (Camb.) 4, 391–400 (2012).
Burdick, J. A. & Watt, F. M. High-throughput stem-cell niches. Nat. Methods 8, 915–916 (2011).
Eiraku, M. et al. Self-organizing optic-cup morphogenesis in three-dimensional culture. Nature 472, 51–56 (2011).
Lancaster, M. A. et al. Cerebral organoids model human brain development and microcephaly. Nature 501, 373–379 (2013).
Sato, T. et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology 141, 1762–1772 (2011).
Gong, S. et al. A gene expression atlas of the central nervous system based on bacterial artificial chromosomes. Nature 425, 917–925 (2003).
Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).
Pai, J. H. et al. Photoresist with low fluorescence for bioanalytical applications. Anal. Chem. 79, 8774–8780 (2007).
Jackman, R. J., Duffy, D. C., Ostuni, E., Willmore, N. D. & Whitesides, G. M. Fabricating large arrays of microwells with arbitrary dimensions and filling them using discontinuous dewetting. Anal. Chem. 70, 2280–2287 (1998).
Gracz, A. D., Puthoff, B. J. & Magness, S. T. Identification, isolation, and culture of intestinal epithelial stem cells from murine intestine. Methods Mol. Biol. 879, 89–107 (2012).
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.
N.L.A., C.E.S., Y.W. and S.T.M. disclose a financial interest in Cell Microsystems.
Integrated supplementary information
(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.
(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).
(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.
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.
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.
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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)
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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