EXTRINSIC REGULATION OF INTESTINAL STEM CELL PROLIFERATION AND DIFFERENTIATION BY NICHE COMPONENTS

The small intestinal epithelium facilitates the absorption of nutrients and provides a barrier against damaging toxins, indigestible contents, and microbes in the intestinal lumen. The epithelium is maintained by a pool of intestinal stem cells (ISCs) that reside at the base of the crypt in a supportive niche environment, made up of both cellular and non-cellular components. Niche cells, including epithelial Paneth cells, subepithelial myofibroblasts, and immune cells, along with the non-cellular extracellular matrix (ECM) provide cues that promote ISC proliferation and differentiation. The niche environment is complex and dynamic, with various cell types present that secrete different growth factors and cytokines in response to intestinal damage, inflammation, and regeneration. Extrinsic niche factors are integral for the survival and proliferation of ISCs; therefore, understanding the influence of individual niche components on ISC behavior is essential for the development of therapeutics for patient health. Here, two components of the ISC niche are investigated for their influence on ISC proliferation and differentiation: cytokines secreted from local immune cells and the underlying ECM scaffold. Through a screen of inflammatory bowel disease-related cytokines, Interleukin 22 demonstrated a concentration-dependent effect on ileal organoid size and survival in vitro. Elevated levels of Interleukin 22 limited ISC expansion in favor of increased progenitor cell differentiation and proliferation, resulting in increased organoids size and expression of antimicrobial gene products. ISC cultures rely on the use of non-intestinal based ECM components for the survival of ISCs in vitro. Using a natural, acellular intestinal scaffold provides a more physiologically relevant substrate for use both in vitro culture systems and for tissue engineering applications. By optimizing decellularization techniques, an acellular porcine small intestinal scaffold was created that retained mucosal architecture, preserved key ECM components, and supported the proliferation and differentiation of mouse small intestinal epithelium. Together, these to findings further the understanding of how extrinsic factors from the niche influence ISCs, which is of particular importance when developing therapies for intestinal disease.Doctor of Philosoph

Orphan Gpr182 suppresses ERK-mediated intestinal proliferation during regeneration and adenoma formation

Orphan GPCRs provide an opportunity to identify potential pharmacological targets, yet their expression patterns and physiological functions remain challenging to elucidate. Here, we have used a genetically engineered knockin reporter mouse to map the expression pattern of the Gpr182 during development and adulthood. We observed that Gpr182 is expressed at the crypt base throughout the small intestine, where it is enriched in crypt base columnar stem cells, one of the most active stem cell populations in the body. Gpr182 knockdown had no effect on homeostatic intestinal proliferation in vivo, but led to marked increases in proliferation during intestinal regeneration following irradiation-induced injury. In the ApcMin mouse model, which forms spontaneous intestinal adenomas, reductions in Gpr182 led to more adenomas and decreased survival. Loss of Gpr182 enhanced organoid growth efficiency ex vivo in an EGF-dependent manner. Gpr182 reduction led to increased activation of ERK1/2 in basal and challenge models, demonstrating a potential role for this orphan GPCR in regulating the proliferative capacity of the intestine. Importantly, GPR182 expression was profoundly reduced in numerous human carcinomas, including colon adenocarcinoma. Together, these results implicate Gpr182 as a negative regulator of intestinal MAPK signaling–induced proliferation, particularly during regeneration and adenoma formation

Bioengineered Systems and Designer Matrices That Recapitulate the Intestinal Stem Cell Niche

The relationship between intestinal stem cells (ISCs) and the surrounding niche environment is complex and dynamic. Key factors localized at the base of the crypt are necessary to promote ISC self-renewal and proliferation, to ultimately provide a constant stream of differentiated cells to maintain the epithelial barrier. These factors diminish as epithelial cells divide, migrate away from the crypt base, differentiate into the postmitotic lineages, and end their life span in approximately 7 days when they are sloughed into the intestinal lumen. To facilitate the rapid and complex physiology of ISC-driven epithelial renewal, in vivo gradients of growth factors, extracellular matrix, bacterial products, gases, and stiffness are formed along the crypt-villus axis. New bioengineered tools and platforms are available to recapitulate various gradients and support the stereotypical cellular responses associated with these gradients. Many of these technologies have been paired with primary small intestinal and colonic epithelial cells to re-create select aspects of normal physiology or disease states. These biomimetic platforms are becoming increasingly sophisticated with the rapid discovery of new niche factors and gradients. These advancements are contributing to the development of high-fidelity tissue constructs for basic science applications, drug screening, and personalized medicine applications. Here, we discuss the direct and indirect evidence for many of the important gradients found in vivo and their successful application to date in bioengineered in vitro models, including organ-on-chip and microfluidic culture devices

PAR2-dependent activation of GSK3 beta regulates the survival of colon stem/progenitor cells

Protease-activated receptors PAR1 and PAR(2) play an important role in the control of epithelial cell proliferation and migration. However, the survival of normal and tumor intestinal stem/progenitor cells promoted by proinflammatory mediators may be critical in oncogenesis. The glycogen synthase kinase-3 beta (GSK3 beta) pathway is overactivated in colon cancer cells and promotes their survival and drug resistance. We thus aimed to determine PAR(1) and PAR(2) effects on normal and tumor intestinal stem/progenitor cells and whether they involved GSK3 beta. First, PAR(1) and PAR(2) were identified in colon stem/progenitor cells by immunofluorescence. In three-dimensional cultures of murine crypt units or single tumor Caco-2 cells, PAR(2) activation decreased numbers and size of normal or cancerous spheroids, and PAR(2)-deficient spheroids showed increased proliferation, indicating that PAR(2) represses proliferation. PAR(2)-stimulated normal cells were more resistant to stress (serum starvation or spheroid passaging), suggesting prosurvival effects of PAR(2). Accordingly, active caspase-3 was strongly increased in PAR(2)-deficient normal spheroids. PAR(2) but not PAR(1) triggered GSK3 beta activation through serine-9 dephosphorylation in normal and tumor cells. The PAR(2)-triggered GSK3 beta activation implicates an arrestin/PP2A/GSK3 beta complex that is dependent on the Rho kinase activity. Loss of PAR(2) was associated with high levels of GSK3 beta nonactive form, strengthening the role of PAR2 in GSK3 beta activation. GSK3 pharmacological inhibition impaired the survival of PAR(2)-stimulated spheroids and serum-starved cells. Altogether our data identify PAR(2)/GSK3 beta as a novel pathway that plays a critical role in the regulation of stem/progenitor cell survival and proliferation in normal colon crypts and colon cancer

Orphan Gpr182 suppresses ERK-mediated intestinal proliferation during regeneration and adenoma formation

Orphan GPCRs provide an opportunity to identify potential pharmacological targets, yet their expression patterns and physiological functions remain challenging to elucidate. Here, we have used a genetically engineered knockin reporter mouse to map the expression pattern of the Gpr182 during development and adulthood. We observed that Gpr182 is expressed at the crypt base throughout the small intestine, where it is enriched in crypt base columnar stem cells, one of the most active stem cell populations in the body. Gpr182 knockdown had no effect on homeostatic intestinal proliferation in vivo, but led to marked increases in proliferation during intestinal regeneration following irradiation-induced injury. In the Apc(Min) mouse model, which forms spontaneous intestinal adenomas, reductions in Gpr182 led to more adenomas and decreased survival. Loss of Gpr182 enhanced organoid growth efficiency ex vivo in an EGF-dependent manner. Gpr182 reduction led to increased activation of ERK1/2 in basal and challenge models, demonstrating a potential role for this orphan GPCR in regulating the proliferative capacity of the intestine. Importantly, GPR182 expression was profoundly reduced in numerous human carcinomas, including colon adenocarcinoma. Together, these results implicate Gpr182 as a negative regulator of intestinal MAPK signaling–induced proliferation, particularly during regeneration and adenoma formation