Dysplasia is considered a key transition state between pre-cancer and cancer in gastric carcinogenesis. However, the cellular or phenotypic heterogeneity and mechanisms of dysplasia progression have not been elucidated. We have established metaplastic and dysplastic organoid lines, derived from Mist1-Kras(G12D) mouse stomach corpus and studied distinct cellular behaviors and characteristics of metaplastic and dysplastic organoids. We also examined functional roles for Kras activation in dysplasia progression using Selumetinib, a MEK inhibitor, which is a downstream mediator of Kras signaling. Here, we report that dysplastic organoids die or show altered cellular behaviors and diminished aggressive behavior in response to MEK inhibition. However, the organoids surviving after MEK inhibition maintain cellular heterogeneity. Two dysplastic stem cell (DSC) populations are also identified in dysplastic cells, which exhibited different clonogenic potentials. Therefore, Kras activation controls cellular dynamics and progression to dysplasia, and DSCs might contribute to cellular heterogeneity in dysplastic cell lineages.
IL22 Inhibits Epithelial Stem Cell Expansion in an Ileal Organoid Model
Zwarycz B, Gracz AD, Rivera KR, Williamson IA, Samsa LA, Starmer J, Daniele MA, Salter-Cid L, Zhao Q, Magness ST
Crohn's disease is an inflammatory bowel disease that affects the ileum and is associated with increased cytokines. Although interleukin (IL)6, IL17, IL21, and IL22 are increased in Crohn's disease and are associated with disrupted epithelial regeneration, little is known about their effects on the intestinal stem cells (ISCs) that mediate tissue repair. We hypothesized that ILs may target ISCs and reduce ISC-driven epithelial renewal. METHODS: A screen of IL6, IL17, IL21, or IL22 was performed on ileal mouse organoids. Computational modeling was used to predict microenvironment cytokine concentrations. Organoid size, survival, proliferation, and differentiation were characterized by morphometrics, quantitative reverse-transcription polymerase chain reaction, and immunostaining on whole organoids or isolated ISCs. ISC function was assayed using serial passaging to single cells followed by organoid quantification. Single-cell RNA sequencing was used to assess Il22ra1 expression patterns in ISCs and transit-amplifying (TA) progenitors. An IL22-transgenic mouse was used to confirm the impact of increased IL22 on proliferative cells in vivo. RESULTS: High IL22 levels caused decreased ileal organoid survival, however, resistant organoids grew larger and showed increased proliferation over controls. Il22ra1 was expressed on only a subset of ISCs and TA progenitors. IL22-treated ISCs did not show appreciable differentiation defects, but ISC biomarker expression and self-renewal-associated pathway activity was reduced and accompanied by an inhibition of ISC expansion. In vivo, chronically increased IL22 levels, similar to predicted microenvironment levels, showed increases in proliferative cells in the TA zone with no increase in ISCs. CONCLUSIONS:
Increased IL22 limits ISC expansion in favor of increased TA progenitor cell expansion.
Quantitative Analysis of Intestinal Stem Cell Dynamics Using Microfabricated Cell Culture Arrays
Regeneration of intestinal epithelium is fueled by a heterogeneous population of rapidly proliferating stem cells (ISCs) found in the base of the small intestine and colonic crypts. ISCs populations can be enriched by fluorescence-activated cell sorting (FACS) based on expression of combinatorial cell surface markers, and fluorescent transgenes. Conventional ISC culture is performed by embedding single ISCs or whole crypt units in a matrix and culturing in conditions that stimulate or repress key pathways to recapitulate ISC niche signaling. Cultured ISCs form organoid, which are spherical, epithelial monolayers that are self-renewing, self-patterning, and demonstrate the full complement of intestinal epithelial cell lineages. However, this conventional "bulk" approach to studying ISC biology is often semiquantitative, low throughput, and masks clonal effects and ISC phenotypic heterogeneity. Our group has recently reported the construction, long-term biocompatibility, and use of microfabricated cell raft arrays (CRA) for high-throughput analysis of single ISCs and organoids. CRAs are composed of thousands of indexed and independently retrievable microwells, which in combination with time-lapse microscopy and/or gene-expression analyses are a powerful tool for studying clonal ISC dynamics and micro-niches. In this protocol, we describe how CRAs are used as an adaptable experimental platform to study the effect of exogenous factors on clonal stem cell behavior.
BACKGROUND & AIMS: The intestinal epithelium is maintained by intestinal stem cells (ISCs), which produce postmitotic absorptive and secretory epithelial cells. Initial fate specification toward enteroendocrine, goblet, and Paneth cell lineages requires the transcription factor Atoh1, which regulates differentiation of the secretory cell lineage. However, less is known about the origin of tuft cells, which participate in type II immune responses to parasite infections and appear to differentiate independently of Atoh1. We investigated the role of Sox4 in ISC differentiation. METHODS: We performed experiments in mice with intestinal epithelial-specific disruption of Sox4 (Sox4fl/fl:vilCre; SOX4 conditional knockout [cKO]) and mice without disruption of Sox4 (control mice). Crypt- and single-cell-derived organoids were used in assays to measure proliferation and ISC potency. Lineage allocation and gene expression changes were studied by immunofluorescence, real-time quantitative polymerase chain reaction, and RNA-seq analyses. Intestinal organoids were incubated with the type 2 cytokine interleukin 13 and gene expression was analyzed. Mice were infected with the helminth Nippostrongylus brasiliensis and intestinal tissues were collected 7 days later for analysis. Intestinal tissues collected from mice that express green fluorescent protein regulated by the Atoh1 promoter (Atoh1GFP mice) and single-cell RNA-seq analysis were used to identify cells that coexpress Sox4 and Atoh1. We generated SOX4-inducible intestinal organoids derived from Atoh1fl/fl:vilCreER (ATOH1 inducible knockout) mice and assessed differentiation. RESULTS: Sox4cKO mice had impaired ISC function and secretory differentiation, resulting in decreased numbers of tuft and enteroendocrine cells. In control mice, numbers of SOX4+ cells increased significantly after helminth infection, coincident with tuft cell hyperplasia. Sox4 was activated by interleukin 13 in control organoids; SOX4cKO mice had impaired tuft cell hyperplasia and parasite clearance after infection with helminths. In single-cell RNA-seq analysis, Sox4+/Atoh1- cells were enriched for ISC, progenitor, and tuft cell genes; 12.5% of Sox4-expressing cells coexpressed Atoh1 and were enriched for enteroendocrine genes. In organoids, overexpression of Sox4 was sufficient to induce differentiation of tuft and enteroendocrine cells-even in the absence of Atoh1. CONCLUSIONS: We found Sox4 promoted tuft and enteroendocrine cell lineage allocation independently of Atoh1. These results challenge the longstanding model in which Atoh1 is the sole regulator of secretory differentiation in the intestine and are relevant for understanding epithelial responses to parasitic infection.
Monoclonal Antibodies Reveal Dynamic Plasticity Between Lgr5- and Bmi1-Expressing Intestinal Cell Populations
Smith NR, Swain JR, Davies PS, Gallagher AC, Parappilly MS, Beach CZ, Streeter PR, Williamson IA, Magness ST, Wong MH
BACKGROUND & AIMS: Continual renewal of the intestinal epithelium is dependent on active- and slow-cycling stem cells that are confined to the crypt base. Tight regulation of these stem cell populations maintains homeostasis by balancing proliferation and differentiation to support critical intestinal functions. The hierarchical relation of discrete stem cell populations in homeostasis or during regenerative epithelial repair remains controversial. Although recent studies have supported a model for the active-cycling leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5)+ intestinal stem cell (ISC) functioning upstream of the slow-cycling B lymphoma Mo-MLV insertion region 1 homolog (Bmi1)-expressing cell, other studies have reported the opposite relation. Tools that facilitate simultaneous analyses of these populations are required to evaluate their coordinated function. METHODS: We used novel monoclonal antibodies (mAbs) raised against murine intestinal epithelial cells in conjunction with ISC-green fluorescent protein (GFP) reporter mice to analyze relations between ISC populations by microscopy. Ex vivo 3-dimensional cultures, flow cytometry, and quantitative reverse-transcription polymerase chain reaction analyses were performed. RESULTS: Two novel mAbs recognized distinct subpopulations of the intestinal epithelium and when used in combination permitted isolation of discrete Lgr5GFP and Bmi1GFP-enriched populations with stem activity. Growth from singly isolated Lgr5GFP ISCs gave rise to small spheroids. Spheroids did not express Lgr5GFP and instead up-regulated Bmi1GFP expression. Conversely, Bmi1-derived spheroids initiated Lgr5GFP expression as crypt domains were established. CONCLUSIONS: These data showed the functional utility of murine mAbs in the isolation and investigation of Lgr5GFP and Bmi1GFP ISC-enriched populations. Ex vivo analyses showed hierarchical plasticity between different ISC-expressing states; specifically Lgr5GFP ISCs gave rise to Bmi1GFP cells, and vice versa. These data highlight the impact of temporal and physiological context on unappreciated interactions between Lgr5GFP and Bmi1GFP cells during crypt formation.
SOX9 Maintains Reserve Stem Cells and Preserves Radio-resistance in Mouse Small Intestine.
Roche KC, Gracz AD, Liu XF, Newton V, Akiyama H, Magness ST
BACKGROUND & AIMS: Reserve intestinal stem cells (rISCs) are quiescent/slowly cycling under homeostatic conditions, allowing for their identification with label-retention assays. rISCs mediate epithelial regeneration after tissue damage by converting to actively proliferating stem cells (aISCs) that self renew and demonstrate multipotency, which are defining properties of stem cells. Little is known about the genetic mechanisms that regulate the production and maintenance of rISCs. High expression levels of the transcription factor Sox9 (Sox9(high)) are associated with rISCs. This study investigates the role of SOX9 in regulating the rISC state. METHODS: We used fluorescence-activated cell sorting to isolate cells defined as aISCs (Lgr5(high)) and rISCs (Sox9(high)) from Lgr5(EGFP) and Sox9(EGFP) reporter mice. Expression of additional markers associated with active and reserve ISCs were assessed in Lgr5(high) and Sox9(high) populations by single-cell gene expression analyses. We used label-retention assays to identify whether Sox9(high) cells were label-retatining cells (LRCs). Lineage-tracing experiments were performed in Sox9-CreERT2 mice to measure the stem cell capacities and radioresistance of Sox9-expressing cells. Conditional SOX9 knockout mice and inducible-conditional SOX9 knockout mice were used to determine whether SOX9 was required to maintain LRCs and rISC function. RESULTS: Lgr5(high) and a subset of crypt-based Sox9(high) cells co-express markers of aISC and rISC (Lgr5, Bmi1, Lrig1, and Hopx). LRCs express high levels of Sox9 and are lost in SOX9-knockout mice. SOX9 is required for epithelial regeneration after high-dose irradiation. Crypts from SOX9-knockout mice have increased sensitivity to radiation, compared with control mice, which could not be attributed to impaired cell-cycle arrest or DNA repair. CONCLUSIONS: SOX9 limits proliferation in LRCs and imparts radiation resistance to rISCs in mice.
A high-throughput platform for stem cell niche co-cultures and downstream gene expression analysis
Gracz AD, Williamson IA, Roche KC, Johnston MJ, Wang F, Wang Y, Attayek PJ, Balowski J, Liu XF, Laurenza RJ, Gaynor LT, Sims CE, Galenko JA, Li L, Allbritton NL, Magness ST
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.
Isolation and Characterization of Intestinal Stem Cells Based on Surface Marker Combinations and Colony-Formation Assay
Wang F, Scoville D, He XC, Mahe M, Box A, Perry J, Smith NR, Nanye NL, Davies PS, Fuller MK, Haug JS, McClain M, Gracz AD, Ding S, Stelzner M, Dunn JCY, Magness ST, Wong MH, Martin M, Helmrath M, and Li L
Gastroenterology. 2013 Aug;145(2):383-95. Epub 2013 May 2. PMID: 23644405
BACKGROUND & AIMS: Identification of intestinal stem cells (ISCs) has relied heavily on the use of transgenic reporters in mice, but this approach is limited by mosaic expression patterns, and difficulty to directly apply to human tissues. We sought to identify reliable surface markers of ISCs and establish a robust functional assay to characterize ISCs from mouse and human tissues. We used immunohistochemistry, real-time reverse transcription PCR, and fluorescence-activated cell sorting (FACS) to analyze intestinal epithelial cells isolated from mouse and human intestinal tissues. We compared different combinations of surface markers among ISCs isolated based on expression of Lgr5-green fluorescent protein (GFP). We developed a culture protocol to facilitate the identification of functional ISCs from mice, and then tested the assay with human intestinal crypts and putative ISCs.
RESULTS: CD44+CD24loCD166+ cells, sorted by FACS from mouse small intestine and colon, expressed high levels of stem-cell associated genes. Transit amplifying cells and progenitor cells were then excluded based on expression of GRP78 or c-Kit. CD44+CD24loCD166+ GRP78 lo/- putative stem cells from mouse small intestine included Lgr5-GFPhi and Lgr5-GFPmed/lo cells. Incubation of these cells with the GSK inhibitor CHIR99021 and the E-cadherin stabilizer Thiazovivin resulted in colony formation by 25%-30% of single-sorted ISCs. CONCLUSIONS: We developed a culture protocol to identify putative ISCs from mouse and human tissues based on cell surface markers.CD44+CD24loCD166+, GRP78lo/- and c-Kit- facilitated identification of putative stem cells from the mouse small intestine and colon respectively. CD44+CD24-/loCD166+ also identified putative human ISCs. These findings will facilitate functional studies of mouse and human ISCs.
CD24 and CD44 mark human intestinal epithelial cell populations with characteristics of active and facultative stem cells
Gracz AD*, Fuller MK*, Wang F, Li L, Stelzner M, Dunn JCY, Martin M, Magness ST
Recent seminal studies have rapidly advanced the understanding of intestinal epithelial stem cell (IESC) biology in murine models. However, the lack of techniques suitable for isolation and subsequent downstream analysis of IESCs from human tissue has hindered the application of these findings toward the development of novel diagnostics and therapies with direct clinical relevance. This study demonstrates that the cluster of differentiation genes CD24 and CD44 are differentially expressed across LGR5 positive “active” stem cells as well as HOPX positive “facultative” stem cells. Fluorescence-activated cell sorting enables differential enrichment of LGR5 cells (CD24-/CD44+) and HOPX (CD24+/CD44+) cells for gene expression analysis and culture. These findings provide the fundamental methodology and basic cell surface signature necessary for isolating and studying intestinal stem cell populations in human physiology and disease.