Calorie Restriction Governs Intestinal Epithelial Regeneration through Cell-Autonomous Regulation of mTORC1 in Reserve Stem Cells.

Aging is a complex process associated with a decline in functionality of adult stem cells affecting tissue homeostasis and regeneration. Calorie restriction (CR) is the only experimental manipulation known to extend lifespan and reduce the incidence of age-related disorders across numerous species. These benefits are likely mediated, at least in part, through the preservation of stem cell function. Here, we show that CR enhances the regenerative capacity of the intestinal epithelium through preservation of an injury-resistant reserve intestinal stem cell (ISC) pool. Cell-autonomous activity of mechanistic target of rapamycin complex 1 (mTORC1) governs the sensitivity of reserve ISCs to injury. CR inhibits mTORC1 in these cells, protecting them against DNA damage, while mTORC1 stimulation, either genetically or through nutrient sensing, sensitizes reserve ISCs to injury, thus compromising regeneration of the epithelium. These data delineate a critical role for mTORC1 in epithelial regeneration and inform clinical strategies based on nutrient modulation.

Gut with the Program: Direct Reprogramming toward Intestinal Epithelium Realized.

Intestinal organoids offer great promise for modeling intestinal diseases; however, harvesting intestinal tissue is invasive and directed hPSC differentiation protocols are laborious and costly. In this issue of Cell Stem Cell, Miura and Suzuki (2017) describe the direct conversion of somatic cells from both mice and humans into robust intestinal epithelial tissue.

The histone variant macroH2A confers functional robustness to the intestinal stem cell compartment.

A stem cell's epigenome directs cell fate during development, homeostasis, and regeneration. Epigenetic dysregulation can lead to inappropriate cell fate decisions, aberrant cell function, and even cancer. The histone variant macroH2A has been shown to influence gene expression, guide cell fate, and safeguard against genotoxic stress. Interestingly, mice lacking functional macroH2A histones (hereafter referred to as macroH2A DKO) are viable and fertile; yet suffer from increased perinatal death and reduced weight and size compared to wildtype (WT). Here, we ask whether the ostensible reduced vigor of macroH2A DKO mice extends to intestinal stem cell (ISC) function during homeostasis, regeneration, and oncogenesis. Lgr5-eGFP-IRES-CreERT2 or Hopx-CreERT2::Rosa26-LSL-tdTomato ISC reporter mice or the C57BL/6J-Apcmin/J murine intestinal adenoma model were bred into a macroH2A DKO or strain-matched WT background and assessed for ISC functionality, regeneration and tumorigenesis. High-dose (12Gy) whole-body γ-irradiation was used as an injury model. We show that macroH2A is dispensable for intestinal homeostasis and macroH2A DKO mice have similar numbers of active crypt-base columnar ISCs (CBCs). MacroH2A DKO intestine exhibits impaired regeneration following injury, despite having significantly more putative reserve ISCs. DKO reserve ISCs disproportionately undergo apoptosis compared to WT after DNA damage infliction. Interestingly, a macroH2A DKO background does not significantly increase tumorigenesis in the Apcmin model of intestinal adenoma. We conclude that macroH2A influences reserve ISC number and function during homeostasis and regeneration. These data suggest macroH2A enhances reserve ISC survival after DNA damage and thus confers functional robustness to the intestinal epithelium.

Msi RNA-binding proteins control reserve intestinal stem cell quiescence.

Regeneration of the intestinal epithelium is driven by multiple intestinal stem cell (ISC) types, including an active, radiosensitive Wnthigh ISC that fuels turnover during homeostasis and a reserve, radioresistant Wntlow/off ISC capable of generating active Wnthigh ISCs. We examined the role of the Msi family of oncoproteins in the ISC compartment. We demonstrated that Msi proteins are dispensable for normal homeostasis and self-renewal of the active ISC, despite their being highly expressed in these cells. In contrast, Msi proteins are required specifically for activation of reserve ISCs, where Msi activity is both necessary and sufficient to drive exit from quiescence and entry into the cell cycle. Ablation of Msi activity in reserve ISCs rendered the epithelium unable to regenerate in response to injury that ablates the active stem cell compartment. These findings delineate a molecular mechanism governing reserve ISC quiescence and demonstrate a necessity for the activity of this rare stem cell population in intestinal regeneration.

Heterogeneity in readouts of canonical wnt pathway activity within intestinal crypts.

BACKGROUND: Canonical Wnt pathway signaling is necessary for maintaining the proliferative capacity of mammalian intestinal crypt base columnar stem cells (CBCs). Furthermore, dysregulation of the Wnt pathway is a major contributor to disease, including oncogenic transformation of the intestinal epithelium. Given the critical importance of this pathway, numerous tools have been used as proxy measures for Wnt pathway activity, yet the relationship between Wnt target gene expression and reporter allele activity within individual cells at the crypt base remains unclear. RESULTS: Here, we describe a novel Axin2-CreERT2-tdTomato allele that efficiently marks both Wnt(High) CBCs and radioresistant reserve intestinal stem cells. We analyze the molecular and functional identity of Axin2-CreERT2-tdTomato-marked cells using single cell gene expression profiling and tissue regeneration assays and find that Axin2 reporter activity does not necessarily correlate with expression of Wnt target genes and, furthermore, that Wnt target genes themselves vary in their expression patterns at the crypt base. CONCLUSIONS: Wnt target genes and reporter alleles can vary greatly in their cell-type specificity, demonstrating that these proxies cannot be used interchangeably. Furthermore, Axin2-CreERT2-tdTomato is a robust marker of both active and reserve intestinal stem cells and is thus useful for understanding the intestinal stem cell compartment. Developmental Dynamics 245:822-833, 2016. © 2016 Wiley Periodicals, Inc.

Mouse Label-Retaining Cells Are Molecularly and Functionally Distinct From Reserve Intestinal Stem Cells.

BACKGROUND & AIMS: Intestinal homeostasis and regeneration after injury are controlled by 2 different types of cells: slow cycling, injury-resistant reserve intestinal stem cells (ISCs) and actively proliferative ISCs. Putative reserve ISCs have been identified using a variety of methods, including CreER insertions at Hopx or Bmi1 loci in mice and DNA label retention. Label-retaining cells (LRCs) include dormant stem cells in several tissues; in the intestine, LRCs appear to share some properties with reserve ISCs, which can be marked by reporter alleles. We investigated the relationships between these populations. METHODS: Studies were performed in Lgr5-EGFP-IRESCreERT2, Bmi1-CreERT2, Hopx-CreERT2, and TRE-H2BGFP::Hopx-CreERT2::lox-stop-lox-tdTomato mice. Intestinal epithelial cell populations were purified; we compared reporter allele-marked reserve ISCs and several LRC populations (marked by H2B-GFP retention) using histologic flow cytometry and functional and single-cell gene expression assays. RESULTS: LRCs were dynamic and their cellular composition changed with time. Short-term LRCs had properties of secretory progenitor cells undergoing commitment to the Paneth or enteroendocrine lineages, while retaining some stem cell activity. Long-term LRCs lost stem cell activity and were a homogenous population of terminally differentiated Paneth cells. Reserve ISCs marked with HopxCreER were primarily quiescent (in G0), with inactive Wnt signaling and robust stem cell activity. In contrast, most LRCs were in G1 arrest and expressed genes that are regulated by the Wnt pathway or are in the secretory lineage. CONCLUSIONS: LRCs are molecularly and functionally distinct from reporter-marked reserve ISCs. This information provides an important basis for future studies of relationships among ISC populations.

Multiphasic and Dynamic Changes in Alternative Splicing during Induction of Pluripotency Are Coordinated by Numerous RNA-Binding Proteins.

Alternative splicing (AS) plays a critical role in cell fate transitions, development, and disease. Recent studies have shown that AS also influences pluripotency and somatic cell reprogramming. We profiled transcriptome-wide AS changes that occur during reprogramming of fibroblasts to pluripotency. This analysis revealed distinct phases of AS, including a splicing program that is unique to transgene-independent induced pluripotent stem cells (iPSCs). Changes in the expression of AS factors Zcchc24, Esrp1, Mbnl1/2, and Rbm47 were demonstrated to contribute to phase-specific AS. RNA-binding motif enrichment analysis near alternatively spliced exons provided further insight into the combinatorial regulation of AS during reprogramming by different RNA-binding proteins. Ectopic expression of Esrp1 enhanced reprogramming, in part by modulating the AS of the epithelial specific transcription factor Grhl1. These data represent a comprehensive temporal analysis of the dynamic regulation of AS during the acquisition of pluripotency.

The Msi Family of RNA-Binding Proteins Function Redundantly as Intestinal Oncoproteins.

Members of the Msi family of RNA-binding proteins have recently emerged as potent oncoproteins in a range of malignancies. MSI2 is highly expressed in hematopoietic cancers, where it is required for disease maintenance. In contrast to the hematopoietic system, colorectal cancers can express both Msi family members, MSI1 and MSI2. Here, we demonstrate that, in the intestinal epithelium, Msi1 and Msi2 have analogous oncogenic effects. Further, comparison of Msi1/2-induced gene expression programs and transcriptome-wide analyses of Msi1/2-RNA-binding targets reveal significant functional overlap, including induction of the PDK-Akt-mTORC1 axis. Ultimately, we demonstrate that concomitant loss of function of both MSI family members is sufficient to abrogate the growth of human colorectal cancer cells, and Msi gene deletion inhibits tumorigenesis in several mouse models of intestinal cancer. Our findings demonstrate that MSI1 and MSI2 act as functionally redundant oncoproteins required for the ontogeny of intestinal cancers.

Transformation of the intestinal epithelium by the MSI2 RNA-binding protein.

The MSI2 RNA-binding protein is a potent oncogene playing key roles in haematopoietic stem cell homeostasis and malignant haematopoiesis. Here we demonstrate that MSI2 is expressed in the intestinal stem cell compartment, that its expression is elevated in colorectal adenocarcinomas, and that MSI2 loss-of-function abrogates colorectal cancer cell growth. MSI2 gain-of-function in the intestinal epithelium in a drug-inducible mouse model is sufficient to phenocopy many of the morphological and molecular consequences of acute loss of the APC tumour suppressor in the intestinal epithelium in a Wnt-independent manner. Transcriptome-wide RNA-binding analysis indicates that MSI2 acts as a pleiotropic inhibitor of known intestinal tumour suppressors including Lrig1, Bmpr1a, Cdkn1a and Pten. Finally, we demonstrate that inhibition of the PDK-AKT-mTORC1 axis rescues oncogenic consequences of MSI2 induction. Taken together, our findings identify MSI2 as a central component in an unappreciated oncogenic pathway promoting intestinal transformation.

Single-cell analysis of proxy reporter allele-marked epithelial cells establishes intestinal stem cell hierarchy.

The recent development of targeted murine reporter alleles as proxies for intestinal stem cell activity has led to significant advances in our understanding of somatic stem cell hierarchies and dynamics. Analysis of these reporters has led to a model in which an indispensable reserve stem cell at the top of the hierarchy (marked by Bmi1 and Hopx reporters) gives rise to active intestinal stem cells (marked by an Lgr5 reporter). Despite these advances, controversy exists regarding the specificity and fidelity with which these alleles distinguish intestinal stem cell populations. Here, we undertake a comprehensive comparison of widely used proxy reporters including both CreERT2 and EGFP cassettes targeted to the Lgr5, Bmi1, and Hopx loci. Single-cell transcriptional profiling of these populations and their progeny reveals that reserve and active intestinal stem cells are molecularly and functionally distinct, supporting a two-stem-cell model for intestinal self-renewal.