Adam D. Gracz

Isolation and Characterization of Intestinal Stem Cells Based on Surface Marker Combinations and Colony-Formation Assay

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 difficult 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. METHODS We used immunohistochemistry, real-time reverse-transcription polymerase chain reaction, 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. 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(+)CD24(lo)CD166(+) cells, isolated 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(+)CD24(lo)CD166(+) GRP78(lo/-) putative stem cells from mouse small intestine included Lgr5-GFP(hi) and Lgr5-GFP(med/lo) cells. Incubation of these cells with the GSK inhibitor CHIR99021 and the E-cadherin stabilizer Thiazovivin resulted in colony formation by 25% to 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(+)CD24(lo)CD166(+), GRP78(lo/-), and c-Kit(-) facilitated identification of putative stem cells from the mouse small intestine and colon, respectively. CD44(+)CD24(-/lo)CD166(+) also identified putative human ISCs. These findings will facilitate functional studies of mouse and human ISCs.

Activation of two distinct Sox9-EGFP-expressing intestinal stem cell populations during crypt regeneration after irradiation.

Recent identification of intestinal epithelial stem cell (ISC) markers and development of ISC reporter mice permit visualization and isolation of regenerating ISCs after radiation to define their functional and molecular phenotypes. Previous studies in uninjured intestine of Sox9-EGFP reporter mice demonstrate that ISCs express low levels of Sox9-EGFP (Sox9-EGFP Low), whereas enteroendocrine cells (EEC) express high levels of Sox9-EGFP (Sox9-EGFP High). We hypothesized that Sox9-EGFP Low ISCs would expand after radiation, exhibit enhanced proliferative capacities, and adopt a distinct gene expression profile associated with rapid proliferation. Sox9-EGFP mice were given 14 Gy abdominal radiation and studied between days 3 and 9 postradiation. Radiation-induced changes in number, growth, and transcriptome of the different Sox9-EGFP cell populations were determined by histology, flow cytometry, in vitro culture assays, and microarray. Microarray confirmed that nonirradiated Sox9-EGFP Low cells are enriched for Lgr5 mRNA and mRNAs enriched in Lgr5-ISCs and identified additional putative ISC markers. Sox9-EGFP High cells were enriched for EEC markers, as well as Bmi1 and Hopx, which are putative markers of quiescent ISCs. Irradiation caused complete crypt loss, followed by expansion and hyperproliferation of Sox9-EGFP Low cells. From nonirradiated intestine, only Sox9-EGFP Low cells exhibited ISC characteristics of forming organoids in culture, whereas during regeneration both Sox9-EGFP Low and High cells formed organoids. Microarray demonstrated that regenerating Sox9-EGFP High cells exhibited transcriptomic changes linked to p53-signaling and ISC-like functions including DNA repair and reduced oxidative metabolism. These findings support a model in which Sox9-EGFP Low cells represent active ISCs, Sox9-EGFP High cells contain radiation-activatable cells with ISC characteristics, and both participate in crypt regeneration.

Brief report: CD24 and CD44 mark human intestinal epithelial cell populations with characteristics of active and facultative stem cells.

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 (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. STEM Cells 2013;31:2024‐2030

Defining hierarchies of stemness in the intestine: evidence from biomarkers and regulatory pathways

For decades, the rapid proliferation and well-defined cellular lineages of the small intestinal epithelium have driven an interest in the biology of the intestinal stem cells (ISCs) and progenitors that produce the functional cells of the epithelium. Recent and significant advances in ISC biomarker discovery have established the small intestinal epithelium as a powerful model system for studying general paradigms in somatic stem cell biology and facilitated elegant genetic and functional studies of stemness in the intestine. However, this newfound wealth of ISC biomarkers raises important questions of marker specificity. Furthermore, the ISC field must now begin to reconcile biomarker status with functional stemness, a challenge that is made more complex by emerging evidence that cellular hierarchies in the intestinal epithelium are more plastic than previously imagined, with some progenitor populations capable of dedifferentiating and functioning as ISCs following damage. In this review, we discuss the state of the ISC field in terms of biomarkers, tissue dynamics, and cellular hierarchies, and how these processes might be informed by earlier studies into signaling networks in the small intestine.

Sry-box (Sox) transcription factors in gastrointestinal physiology and disease.

The genetic mechanisms underlying tissue maintenance of the gastrointestinal tract are critical for the proper function of the digestive system under normal physiological stress. The identification of transcription factors and related signal transduction pathways that regulate stem cell maintenance and lineage allocation is attractive from a clinical standpoint in that it may provide targets for novel cell- or drug-based therapies. Sox [sex-determining region Y (Sry) box-containing] factors are a family of transcription factors that are emerging as potent regulators of stem cell maintenance and cell fate decisions in multiple organ systems and might provide valuable insight toward the understanding of these processes in endodermally derived tissues of the gastrointestinal tract. In this review, we focus on the known genetic functions of Sox factors and their roles in epithelial tissues of the esophagus, stomach, intestine, colon, pancreas, and liver. Additionally, we discuss pathological conditions in the gastrointestinal tract that are associated with a dysregulation of Sox factors. Further study of Sox factors and their role in gastrointestinal physiology and pathophysiology may lead to advances that facilitate control of tissue maintenance and development of advanced clinical therapies.

IL22 Inhibits Epithelial Stem Cell Expansion in an Ileal Organoid Model

Background & Aims 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.

Organoid Cultures for Assessing Intestinal Epithelial Differentiation and Function in Response to Type-2 Inflammation

During helminth infection of the gastrointestinal tract, a complex Type-2 inflammatory response involving immunological and mucosal components is mounted to clear the infection and reestablish a physiologically normal state. This response is characterized by the secretion of key interleukins, which impact epithelial lineage allocation and drive tuft and goblet cell hyperplasia to lead to eventual clearance of parasitic organisms. While there have been advances toward understanding Type-2 inflammatory responses in the intestine, detailed cellular and molecular mechanisms of epithelial responses to general inflammation and specific inflammatory cytokines remain to be explored. Intestinal organoids represent a physiologically relevant in vitro model to study how Type-2 inflammation impacts stem cell maintenance and differentiation and offer a new approach for investigators to test compounds that modulate mechanisms involved in worm clearance. The methods described in this chapter include: (1) intestinal crypt and single cell isolation; (2) organoid culture and cytokine treatment, as well as methods for downstream organoid analyses; (3) gene expression analysis by qRT-PCR; (4) protein analysis by western blot, immunohistochemistry, and florescence-activated cell sorting; and (5) organoid self-renewal by serial passaging.

SOX4 Promotes ATOH1-independent Intestinal Secretory Differentiation Toward Tuft and Enteroendocrine Fates

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.

Sox4 drives intestinal secretory differentiation toward tuft and enteroendocrine fates

Background & Aims The intestinal epithelium is maintained by intestinal stem cells (ISCs), which produce post-mitotic absorptive and secretory epithelial cells. Initial fate specification toward enteroendocrine, goblet, and Paneth cell lineages is dependent on Atoh1, a master regulator of secretory differentiation. However, the origin of tuft cells, which participate in Type II immune responses to parasitic infection, is less clear and appears to occur in an Atoh1-independent manner. Here we examine the role of Sox4 in ISC proliferation and differentiation. Methods We used mice with intestinal epithelial-specific conditional knockout of Sox4 (Sox4fl/fl:vilCre; Sox4cKO) to study the role of Sox4 in the small intestine. Crypt- and single cell-derived organoids were used to assay proliferation and ISC potency between control and Sox4cKO mice. Lineage allocation and genetic consequences of Sox4 ablation were studied by immunofluorescence, RT-qPCR, and RNA-seq. In vivo infection with helminths and in vitro cytokine treatment in primary intestinal organoids were used to assess tuft cell hyperplasia in control and Sox4cKO samples. Atoh1GFP reporter mice and single cell RNA-seq (scRNA-seq) were used to determine co-localization of SOX4 and Atoh1. Wild-type and inducible Atoh1 knockout (Atoh1fl/fl:vilCreER; Atoh1iKO) organoids carrying an inducible Sox4 overexpression vector (Sox4OE) were used to determine the role of Atoh1 in Sox4 driven secretory differentiation. Results Loss of Sox4 impairs ISC function and secretory differentiation, resulting in decreased numbers of enteroendocrine and tuft cells. In wild-type mice, SOX4+ cells are significantly upregulated following helminth infection coincident with tuft cell hyperplasia. Sox4 is activated by IL13 in vitro and Sox4cKO knockout mice demonstrate impaired tuft cell hyperplasia and parasite clearance following infection with helminths. A subset of Sox4-expressing cells colocalize with Atoh1 and enteroendocrine markers by scRNA-seq, while Sox4+/Atoh1-cells correlate strongly with tuft cell populations. Gain-of-function studies in primary organoids demonstrate that Sox4 is sufficient to drive both enteroendocrine and tuft cell differentiation, and can do so in the absence of Atoh1. Conclusion Our data demonstrate that Sox4 promotes enteroendocrine and tuft cell lineage allocation independently of Atoh1. These results challenge long-standing views of Atoh1 as the sole regulator of secretory differentiation in the intestine and are relevant for understanding host epithelial responses to parasitic infection.