Probes for INS

Adult stem cells play an essential role in adult organ physiology and tissue repair and regeneration. While much has been learnt about the property and function of various adult stem cells, the mechanisms of their development remain poorly understood in mammals. Earlier studies suggest that the formation of adult mouse intestinal stem cells takes place during the first few weeks after birth, the postembryonic period when plasma thyroid hormone (T3) levels are high. Furthermore, deficiency in T3 signaling leads to defects in adult mouse intestine, including reduced cell proliferation in the intestinal crypts, where stem cells reside. Our earlier studies have shown that protein arginine methyltransferase 1 (PRMT1), a T3 receptor coactivator, is highly expressed during intestinal maturation in mouse.We have analyzed the expression of PRMT1 by immunohistochemistry and studied the effect of tissue-specific knockout of PRMT1 in the intestinal epithelium.We show that PRMT1 is expressed highly in the proliferating transit amplifying cells and crypt base stem cells. By using a conditional knockout mouse line, we have demonstrated that the expression of PRMT1 in the intestinal epithelium is critical for the development of the adult mouse intestine. Specific removal of PRMT1 in the intestinal epithelium results in, surprisingly, more elongated adult intestinal crypts with increased cell proliferation. In addition, epithelial cell migration along the crypt-villus axis and cell death on the villus are also increased. Furthermore, there are increased Goblet cells and reduced Paneth cells in the crypt while the number of crypt base stem cells remains unchanged.Our finding that PRMT1 knockout increases cell proliferation is surprising considering the role of PRMT1 in T3-signaling and the importance of T3 for intestinal development, and suggests that PRMT1 likely regulates pathways in addition to T3-signaling to affect intestinal development and/or homeostasis, thus affecting cell proliferating and epithelial turn over in the adult.

Epithelial organoids derived from intestinal tissue, called 'enteroids', recapitulate many aspects of the organ in vitro, and can be used for biological discovery, personalized medicine, and drug development. Here, we interrogated the cell signaling environment within the developing human intestine to identify niche cues that may be important for epithelial development and homeostasis. We identify an EGF family member, EPIREGULIN (EREG), which is robustly expressed in the developing human crypt. Enteroids generated from the developing human intestine grown in standard culture conditions, which contain EGF, are dominated by stem and progenitor cells, feature little differentiation and no spatial organization. Our results demonstrate that EREG can replace EGF in vitro, and EREG leads to spatially resolved enteroids that feature budded and proliferative crypt domains and a differentiated villus-like central lumen. Multiomic (transcriptome plus epigenome) profiling of native crypts, EGF-grown and EREG-grown enteroids show that EGF-enteroids have an altered chromatin landscape that is dependent on EGF concentration, downregulate the master intestinal transcription factor CDX2, and ectopically express stomach genes, a phenomenon that is reversible. This is in contrast to EREG-grown enteroids, which remain intestine-like in culture. Thus, EREG creates a homeostatic intestinal niche in vitro, enabling interrogation of stem cell function, cellular differentiation, and disease modeling.

Non-thermal plasma (NTP) is widely used in the disinfection and surface modification of biomaterials.

Wnt and Rspondin (RSPO) signaling drives proliferation, and bone morphogenetic protein inhibitors (BMPi) impede differentiation, of intestinal stem cells (ISCs). Here, we identify the mouse ISC niche as a complex, multi-layered structure that encompasses distinct mesenchymal and smooth muscle populations. In young and adult mice, diverse sub-cryptal cells provide redundant ISC-supportive factors; few of these are restricted to single cell types. Niche functions refine during postnatal crypt morphogenesis, in part to oppose the dense aggregation of differentiation-promoting BMP+ sub-epithelial myofibroblasts at crypt-villus junctions. Muscularis mucosae, a specialized muscle layer, first appears during this period and supplements neighboring RSPO and BMPi sources. Components of this developing niche are conserved in human fetuses. The in vivo ablation of mouse postnatal smooth muscle increases BMP signaling activity, potently limiting a pre-weaning burst of crypt fission. Thus, distinct and progressively specialized mesenchymal cells together create the milieu that is required to propagate crypts during rapid organ growth and to sustain adult ISCs.

The intestine is critical for not only processing and resorbing nutrients but also protecting the organism from the environment. These functions are mainly carried out by the epithelium, which is constantly being self-renewed. Many genes and pathways can influence intestinal epithelial cell proliferation. Among them is mTORC1, whose activation increases cell proliferation. Here, we report the first intestinal epithelial cell-specific knockout ( ΔIEC ) of an amino acid transporter capable of activating mTORC1. We show that the transporter, SLC7A5, is highly expressed in mouse intestinal crypt and Slc7a5 ΔIEC reduces mTORC1 signaling. Surprisingly, Slc7a5 ΔIEC mice have increased cell proliferation but reduced secretory cells, particularly mature Paneth cells. scRNA-seq and electron microscopic analyses revealed dedifferentiation of Paneth cells in Slc7a5 ΔIEC mice, leading to markedly reduced secretory granules with little effect on Paneth cell number. We further show that Slc7a5 ΔIEC mice are prone to experimental colitis. Thus, SLC7A5 regulates secretory cell differentiation to affect stem cell niche and/or inflammatory response to regulate cell proliferation.

Gut-draining mesenteric lymph nodes (LN) provide the framework to shape intestinal adaptive immune responses. Based on the transcriptional signatures established by our previous work, the composition and immunomodulatory function of LN stromal cells (SC) vary according to location. Here, we describe the single-cell composition and development of the SC compartment within mesenteric LNs derived from postnatal to aged mice. We identify CD34+ SC and fibroblastic reticular stromal cell (FRC) progenitors as putative progenitors, both supplying the typical rapid postnatal mesenteric LN expansion. We further establish the location-specific chromatin accessibility and DNA methylation landscape of non-endothelial SCs and identify a microbiota-independent core epigenomic signature, showing characteristic differences between SCs from mesenteric and skin-draining peripheral LNs. The epigenomic landscape of SCs points to dynamic expression of Irf3 along the differentiation trajectories of FRCs. Accordingly, a mesenchymal stem cell line acquires a Cxcl9+ FRC molecular phenotype upon lentiviral overexpression of Irf3, and the relevance of Irf3 for SC biology is further underscored by the diminished proportion of Ccl19+ and Cxcl9+ FRCs in LNs of Irf3-/- mice. Together, our data constitute a comprehensive transcriptional and epigenomic map of mesenteric LNSC development in early life and dissect location-specific, microbiota-independent properties of non-endothelial SCs.

Organs are composed of diverse cell types that traverse transient states during organogenesis. To interrogate this diversity during human development, we generate a single-cell transcriptome atlas from multiple developing endodermal organs of the respiratory and gastrointestinal tract. We illuminate cell states, transcription factors, and organ-specific epithelial stem cell and mesenchyme interactions across lineages. We implement the atlas as a high-dimensional search space to benchmark human pluripotent stem cell (hPSC)-derived intestinal organoids (HIOs) under multiple culture conditions. We show that HIOs recapitulate reference cell states and use HIOs to reconstruct the molecular dynamics of intestinal epithelium and mesenchyme emergence. We show that the mesenchyme-derived niche cue NRG1 enhances intestinal stem cell maturation in vitro and that the homeobox transcription factor CDX2 is required for regionalization of intestinal epithelium and mesenchyme in humans. This work combines cell atlases and organoid technologies to understand how human organ development is orchestrated.