Camilla A. Richmond

Mouse telomerase reverse transcriptase (mTert) expression marks slowly cycling intestinal stem cells

The intestinal epithelium is maintained by a population of rapidly cycling (Lgr5+) intestinal stem cells (ISCs). It has been postulated, however, that slowly cycling ISCs must also be present in the intestine to protect the genome from accumulating deleterious mutations and to allow for a response to tissue injury. Here, we identify a subpopulation of slowly cycling ISCs marked by mouse telomerase reverse transcriptase (mTert) expression that can give rise to Lgr5+ cells. mTert-expressing cells distribute in a pattern along the crypt–villus axis similar to long-term label-retaining cells (LRCs) and are resistant to tissue injury. Lineage-tracing studies demonstrate that mTert+ cells give rise to all differentiated intestinal cell types, persist long term, and contribute to the regenerative response following injury. Consistent with other highly regenerative tissues, our results demonstrate that a slowly cycling stem cell population exists within the intestine.

Reprogrammed Stomach Tissue as a Renewable Source of Functional β Cells for Blood Glucose Regulation

The gastrointestinal (GI) epithelium is a highly regenerative tissue with the potential to provide a renewable source of insulin(+) cells after undergoing cellular reprogramming. Here, we show that cells of the antral stomach have a previously unappreciated propensity for conversion into functional insulin-secreting cells. Native antral endocrine cells share a surprising degree of transcriptional similarity with pancreatic β cells, and expression of β cell reprogramming factors in vivo converts antral cells efficiently into insulin(+) cells with close molecular and functional similarity to β cells. Induced GI insulin(+) cells can suppress hyperglycemia in a diabetic mouse model for at least 6 months and regenerate rapidly after ablation. Reprogramming of antral stomach cells assembled into bioengineered mini-organs in vitro yielded transplantable units that also suppressed hyperglycemia in diabetic mice, highlighting the potential for development of engineered stomach tissues as a renewable source of functional β cells for glycemic control.

Regulation of Gene Expression in the Intestinal Epithelium

Regulation of gene expression within the intestinal epithelium is complex and controlled by various signaling pathways that regulate the balance between proliferation and differentiation. Proliferation is required both to grow and to replace cells lost through apoptosis and attrition, yet in all but a few cells, differentiation must take place to prevent uncontrolled growth (cancer) and to provide essential functions. In this chapter, we review the major signaling pathways underlying regulation of gene expression within the intestinal epithelium, based primarily on data from mouse models, as well as specific morphogens and transcription factor families that have a major role in regulating intestinal gene expression, including the Hedgehog family, Forkhead Box (FOX) factors, Homeobox (HOX) genes, ParaHox genes, GATA transcription factors, canonical Wnt/β-catenin signaling, EPH/Ephrins, Sox9, BMP signaling, PTEN/PI3K, LKB1, K-RAS, Notch pathway, HNF, and MATH1. We also briefly highlight important emerging areas of gene regulation, including microRNA (miRNA) and epigenetic regulation.

Dormant Intestinal Stem Cells Are Regulated by PTEN and Nutritional Status

The cellular and molecular mechanisms underlying adaptive changes to physiological stress within the intestinal epithelium remain poorly understood. Here, we show that PTEN, a negative regulator of the PI3K→AKT→mTORC1-signaling pathway, is an important regulator of dormant intestinal stem cells (d-ISCs). Acute nutrient deprivation leads to transient PTEN phosphorylation within d-ISCs and a corresponding increase in their number. This release of PTEN inhibition renders d-ISCs functionally poised to contribute to the regenerative response during re-feeding via cell-autonomous activation of the PI3K→AKT→mTORC1 pathway. Consistent with its role in mediating cell survival, PTEN is required for d-ISC maintenance at baseline, and intestines lacking PTEN have diminished regenerative capacity after irradiation. Our results highlight a PTEN-dependent mechanism for d-ISC maintenance and further demonstrate the role of d-ISCs in the intestinal response to stress.

Development of a primary human Small Intestine-on-a-Chip using biopsy-derived organoids

Here we describe a method for fabricating a primary human Small Intestine-on-a-Chip (Intestine Chip) containing epithelial cells isolated from healthy regions of intestinal biopsies. The primary epithelial cells are expanded as 3D organoids, dissociated, and cultured on a porous membrane within a microfluidic device with human intestinal microvascular endothelium cultured in a parallel microchannel under flow and cyclic deformation. In the Intestine Chip, the epithelium forms villi-like projections lined by polarized epithelial cells that undergo multi-lineage differentiation similar to that of intestinal organoids, however, these cells expose their apical surfaces to an open lumen and interface with endothelium. Transcriptomic analysis also indicates that the Intestine Chip more closely mimics whole human duodenum in vivo when compared to the duodenal organoids used to create the chips. Because fluids flowing through the lumen of the Intestine Chip can be collected continuously, sequential analysis of fluid samples can be used to quantify nutrient digestion, mucus secretion and establishment of intestinal barrier function over a period of multiple days in vitro. The Intestine Chip therefore may be useful as a research tool for applications where normal intestinal function is crucial, including studies of metabolism, nutrition, infection, and drug pharmacokinetics, as well as personalized medicine.

An enduring role for quiescent stem cells.

The intestines ability to recover from catastrophic injury requires quiescent intestinal stem cells (q‐ISCs). While rapidly cycling (Lgr5+) crypt base columnar (CBC) ISCs normally maintain the intestine, they are highly sensitive to pathological injuries (irradiation, inflammation) and must be restored by q‐ISCs to sustain intestinal homeostasis. Despite clear relevance to human health, virtually nothing is known regarding the factors that regulate q‐ISCs. A comprehensive understanding of these mechanisms would likely lead to targeted new therapies with profound therapeutic implications for patients with gastrointestinal conditions. We briefly review the current state of the literature, highlighting homeostatic mechanisms important for q‐ISC regulation, listing key questions in the field, and offer strategies to address them. Developmental Dynamics 245:718–726, 2016.

Factors regulating quiescent stem cells: insights from the intestine and other self‐renewing tissues

Long‐lived and self‐renewing adult stem cells (SCs) are essential for homeostasis in a wide range of tissues and can include both rapidly cycling and quiescent (q)SC populations. Rapidly cycling SCs function principally during normal tissue maintenance and are highly sensitive to stress, whereas qSCs exit from their quiescent state in response to homeostatic imbalance and regenerative pressure. The regulatory mechanisms underlying the quiescent state include factors essential for cell cycle control, stress response and survival pathways, developmental signalling pathways, and post‐transcriptional modulation. Here, we review these regulatory mechanisms citing observations from the intestine and other self‐renewing tissues.

JAK/STAT-1 Signaling Is Required for Reserve Intestinal Stem Cell Activation during Intestinal Regeneration Following Acute Inflammation

Summary The intestinal epithelium serves as an essential barrier to the outside world and is maintained by functionally distinct populations of rapidly cycling intestinal stem cells (CBC ISCs) and slowly cycling, reserve ISCs (r-ISCs). Because disruptions in the epithelial barrier can result from pathological activation of the immune system, we sought to investigate the impact of inflammation on ISC behavior during the regenerative response. In a murine model of αCD3 antibody-induced small-intestinal inflammation, r-ISCs proved highly resistant to injury, while CBC ISCs underwent apoptosis. Moreover, r-ISCs were induced to proliferate and functionally contribute to intestinal regeneration. Further analysis revealed that the inflammatory cytokines interferon gamma and tumor necrosis factor alpha led to r-ISC activation in enteroid culture, which could be blocked by the JAK/STAT inhibitor, tofacitinib. These results highlight an important role for r-ISCs in response to acute intestinal inflammation and show that JAK/STAT-1 signaling is required for the r-ISC regenerative response.

Move Over Caco-2 Cells: Human-Induced Organoids Meet Gut-on-a-Chip

he intestinal epithelium serves as both a crucial Tbarrier and a critical site of interaction between the body and the environment. As such, it comprises a large surface area, and is implicated in a wide range of diseases including inflammatory bowel disease, celiac disease, infectious diarrheas, and intestinal cancers. Architecturally, the small intestine is notable for its proliferative crypts, where stem and transit-amplifying progenitor cells reside, and villi, which consist of multiple differentiated cell types from the absorptive and secretory lineages, including enterocytes, goblet cells, Paneth cells, tuft cells, M cells, and enteroendocrine cells. The polarized epithelial monolayer is supported by a network of mesenchymal and immune cells residing beneath the basement membrane, which play an important, but poorly defined, role in establishing, maintaining, and regulating intestinal morphology. Until recently, the study of the intestinal epithelium has been limited to working with transformed intestinal cancer cell lines, such as Caco2 cells. Despite their myriad advantages, such cells fail to recapitulate the normal physiology and lineage development of the native intestinal epithelium. With the advent of intestinal organoid technology in 2009, it became possible to culture primary mouse and human intestinal epithelium as 3-dimensional organotypic miniguts in a Matrigel (Corning, Tewskbury, MA) matrix that reliably recapitulates intestinal epithelial biology. Although the ability to culture organoids has enabled enormous progress in the field, the 3-dimensional nature of these structures presents certain challenges, including difficulties with imaging, accessing the central lumen, and co-culturing with other cell types. Subsequent innovations led to organoid-derived monolayer cultures, which have begun to address a number of these challenges. An additional advance arose with the development of gut-on-a-chip technology, whereby cellular monolayers can be maintained in engineered microenvironments that allow for the addition of separate luminal and basolateral compartments, the regulation of biomimetic parameters such as flow rate and mechanical stretch, and interactions between separate organ systems. Although promising, to date, gut-on-a-chip technology has yet to be studied with primary cells, limiting its scientific and commercial applications. In this issue of Cellular and Molecular Gastroenterology and Hepatology, Workman et al showed that human intestinal organoids (HIOs), derived from induced pluripotent stem cells, can be incorporated successfully into gut-on-a-chip technology. Although more complicated to

High-dimensional immune phenotyping and transcriptional analyses reveal robust recovery of viable human immune and epithelial cells from frozen gastrointestinal tissue

Simultaneous analyses of peripheral and mucosal immune compartments can yield insight into the pathogenesis of mucosal-associated diseases. Although methods to preserve peripheral immune cells are well established, studies involving mucosal immune cells have been hampered by lack of simple storage techniques. We provide a cryopreservation protocol allowing for storage of gastrointestinal (GI) tissue with preservation of viability and functionality of both immune and epithelial cells. These methods will facilitate translational studies allowing for batch analysis of mucosal tissue to investigate disease pathogenesis, biomarker discovery and treatment responsiveness.