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It is useful to be able to grow enriched populations of stem cells in vitro. Growth of stem cells as tissue spheroids is a key methodology permitting sustainable culture of adult epithelial cells. Gastrointestinal stem cells can be propagated by using conditioned medium from a supportive cell line (L-WRN). This protocol describes how to prepare conditioned medium and how to culture stem cell–enriched epithelial spheroids from the mouse gastrointestine. These spheroids are also amenable to genetic modification with recombinant lentiviruses. This system enables many types of cell biological assays that have been performed with immortalized cell lines to be applied to spheroids. Isolation of epithelial cell units from mice takes up to 2 h, and stem cell–enriched gastrointestinal spheroids are obtained within 3 d. Genetically modified spheroids with lentiviruses can be obtained in 2 weeks.

The process of wound repair in epithelium-lined organs of mammals is complex and is influenced by numerous secreted factors including cytokines, growth factors, and chemokines. However, the cellular organizers of this process are still not understood. Recent studies of tissue regeneration in organisms with simpler development have uncovered details about the activity of stem cells in the mesenchyme (the blastema) during this process. These blastemal cells are well positioned to interpret cues from the environment and to execute decisions about the direction of wound repair. In mammalian wounds, stromal stem cells appear to be positioned to perform functions similar to those of blastemal cells, including communication with both the overlying epithelium and the inflammatory cells in the mesenchyme.

Objective The technology for the growth of human intestinal epithelial cells is rapidly progressing. An exciting possibility is that this system could serve as a platform for individualised medicine and research. However, to achieve this goal, human epithelial culture must be enhanced so that biopsies from individuals can be used to reproducibly generate cell lines in a short time frame so that multiple, functional assays can be performed (ie, barrier function and host–microbial interactions). Design We created a large panel of human gastrointestinal epithelial cell lines (n=65) from patient biopsies taken during routine upper and lower endoscopy procedures. Proliferative stem/progenitor cells were rapidly expanded using a high concentration of conditioned media containing the factors critical for growth (Wnt3a, R-spondin and Noggin). A combination of lower conditioned media concentration and Notch inhibition was used to differentiate these cells for additional assays. Results We obtained epithelial lines from all accessible tissue sites within 2 weeks of culture. The intestinal cell lines were enriched for stem cell markers and rapidly grew as spheroids that required passage at 1:3–1:4 every 3 days. Under differentiation conditions, intestinal epithelial spheroids showed region-specific development of mature epithelial lineages. These cells formed functional, polarised monolayers covered by a secreted mucus layer when grown on Transwell membranes. Using two-dimensional culture, these cells also demonstrated novel adherence phenotypes with various strains of pathogenic Escherichia coli. Conclusions This culture system will facilitate the study of interindividual, functional studies of human intestinal epithelial cells, including host–microbial interactions.

Transfusion of red blood cells (RBCs) is a standard and indispensable therapy in current clinical practice. In vitro production of RBCs offers a potential means to overcome a shortage of transfusable RBCs in some clinical situations and also to provide a source of cells free from possible infection or contamination by microorganisms. Thus, in vitro production of RBCs may become a standard procedure in the future. We previously reported the successful establishment of immortalized mouse erythroid progenitor cell lines that were able to produce mature RBCs very efficiently. Here, we have developed a reliable protocol for establishing immortalized human erythroid progenitor cell lines that are able to produce enucleated RBCs. These immortalized cell lines produce functional hemoglobin and express erythroid-specific markers, and these markers are upregulated following induction of differentiation in vitro. Most importantly, these immortalized cell lines all produce enucleated RBCs after induction of differentiation in vitro, although the efficiency of producing enucleated RBCs remains to be improved further. To the best of our knowledge, this is the first demonstration of the feasibility of using immortalized human erythroid progenitor cell lines as an ex vivo source for production of enucleated RBCs.

The adenohypophysis (anterior pituitary) is a major centre for systemic hormones. At present, no efficient stem-cell culture for its generation is available, partly because of insufficient knowledge about how the pituitary primordium (Rathke’s pouch) is induced in the embryonic head ectoderm. Here we report efficient self-formation of three-dimensional adenohypophysis tissues in an aggregate culture of mouse embryonic stem (ES) cells. ES cells were stimulated to differentiate into non-neural head ectoderm and hypothalamic neuroectoderm in adjacent layers within the aggregate, and treated with hedgehog signalling. Self-organization of Rathke’s-pouch-like three-dimensional structures occurred at the interface of these two epithelia, as seen in vivo , and various endocrine cells including corticotrophs and somatotrophs were subsequently produced. The corticotrophs efficiently secreted adrenocorticotropic hormone in response to corticotrophin releasing hormone and, when grafted in vivo , these cells rescued the systemic glucocorticoid level in hypopituitary mice. Thus, functional anterior pituitary tissue self-forms in ES cell culture, recapitulating local tissue interactions. Mouse embryonic stem cells are induced to form functional anterior pituitary tissue in three-dimensional culture. Pituitary tissue grown from stem cells A three-dimensional cell culture system has been developed that produces functioning pituitary tissue from mouse embryonic stem cells. The cells differentiate into layered structures, the development of which depends on close juxtaposition of two tissue types in formations that resemble local tissue reactions seen in vivo . Endocrine cells including corticotrophs and somatotrophs are produced. The corticotrophs efficiently secreted adrenocorticotropic hormone in response to corticotrophin releasing hormone, and transplantation of the pituitary tissues into a hypopituitary mouse model rescued a lethal deficiency in adrenocorticotropin. This work opens the possibility of developing regenerative therapies for pituitary defects, a major category of endocrinological disorders that includes empty sella syndrome, Sheehan syndrome and pituitary apoplexy.

Cancer stem cell (CSC)-specific markers may be potential therapeutic targets. We previously identified that Dclk1, a tuft cell marker, marks tumor stem cells (TSCs) in mouse intestinal adenomas. Based on the analysis of mouse Dclk1⁺ tumor cells, we aimed to identify a CSC-specific cell surface marker in human colorectal cancers (hCRCs) and validate the therapeutic effect of targeting it. IL17RB was distinctively expressed by Dclk1⁺ mouse intestinal tumor cells. Using Il17rb-CreERT2-IRES-EGFP mice, we show that IL17RB marked intestinal TSCs in an IL13-dependent manner. Tuft cell-like cancer cells were detected in a subset of hCRCs. In these hCRCs, lineage-tracing experiments in CRISPR-Cas9–mediated IL17RB-CreERT2 knockin organoids and xenograft tumors revealed that IL17RB marks CSCs that expand independently of IL-13. We observed up-regulation of POU2F3, a master regulator of tuft cell differentiation, and autonomous tuft cell-like cancer cell differentiation in the hCRCs. Furthermore, long-term ablation of IL17RB-expressing CSCs strongly suppressed the tumor growth in vivo. These findings reveal insights into a CSC-specific marker IL17RB in a subset of hCRCs, and preclinically validate IL17RB⁺ CSCs as a cancer therapeutic target.

Delivery of granule contents to epithelial surfaces by secretory cells is a critical physiologic process. In the intestine, goblet cells secrete mucus that is required for homeostasis. Autophagy proteins are required for secretion in some cases, though the mechanism and cell biological basis for this requirement remain unknown. We found that in colonic goblet cells, proteins involved in initiation and elongation of autophagosomes were required for efficient mucus secretion. The autophagy protein LC3 localized to intracellular multi‐vesicular vacuoles that were consistent with a fusion of autophagosomes and endosomes. Using cultured intestinal epithelial cells, we found that NADPH oxidases localized to and enhanced the formation of these LC3‐positive vacuoles. Both autophagy proteins and endosome formation were required for maximal production of reactive oxygen species (ROS) derived from NADPH oxidases. Importantly, generation of ROS was critical to control mucin granule accumulation in colonic goblet cells. Thus, autophagy proteins can control secretory function through ROS, which is in part generated by LC3‐positive vacuole‐associated NADPH oxidases. These findings provide a novel mechanism by which autophagy proteins can control secretion. In vivo findings on mucus secretion in the colon illustrate how autophagy proteins can control secretion mechanisms via ROS generation.

Inflammatory bowel disease Crohn's disease, a chronic inflammation of the gut, has been linked to over thirty gene loci. Two papers in this issue focus a recent addition to that list, ATG16L1 (Atg16-like 1). Atg16 protein itself was first identified in yeast as an essential gene for the process of autophagy, a system that clears away unwanted cellular components and is involved in the pathogenesis of microbial infection, neurodegeneration and tumorigenesis. Cadwell et al . report a unique role for Atg16L1 in Paneth cells, a type of epithelial cell that secretes granules containing antimicrobial peptides into the intestines. Saitoh et al . show that ATG16L1 plays a role in the inflammatory response in isolated macrophages and in the mouse intestine, as an essential component of the autophagic machinery. This work implicates Atg16L1 in the control of inflammatory immune response and the maintenance of intestinal barrier, both of which are important for the prevention of inflammatory bowel disease. Susceptibility to Crohn’s disease, a complex inflammatory disease involving the small intestine, is controlled by over 30 loci 1 . One Crohn’s disease risk allele is in ATG16L1 , a gene homologous to the essential yeast autophagy gene ATG16 (ref. 2 ). It is not known how ATG16L1 or autophagy contributes to intestinal biology or Crohn’s disease pathogenesis. To address these questions, we generated and characterized mice that are hypomorphic for ATG16L1 protein expression, and validated conclusions on the basis of studies in these mice by analysing intestinal tissues that we collected from Crohn’s disease patients carrying the Crohn’s disease risk allele of ATG16L1 . Here we show that ATG16L1 is a bona fide autophagy protein. Within the ileal epithelium, both ATG16L1 and a second essential autophagy protein ATG5 are selectively important for the biology of the Paneth cell, a specialized epithelial cell that functions in part by secretion of granule contents containing antimicrobial peptides and other proteins that alter the intestinal environment 3 . ATG16L1- and ATG5-deficient Paneth cells exhibited notable abnormalities in the granule exocytosis pathway. In addition, transcriptional analysis revealed an unexpected gain of function specific to ATG16L1-deficient Paneth cells including increased expression of genes involved in peroxisome proliferator-activated receptor (PPAR) signalling and lipid metabolism, of acute phase reactants and of two adipocytokines, leptin and adiponectin, known to directly influence intestinal injury responses. Importantly, Crohn’s disease patients homozygous for the ATG16L1 Crohn’s disease risk allele displayed Paneth cell granule abnormalities similar to those observed in autophagy-protein-deficient mice and expressed increased levels of leptin protein. Thus, ATG16L1, and probably the process of autophagy, have a role within the intestinal epithelium of mice and Crohn’s disease patients by selective effects on the cell biology and specialized regulatory properties of Paneth cells.

Despite the clinical impact of DNMT3A mutation on acute myeloid leukaemia, the molecular mechanisms regarding how this mutation causes leukaemogenesis in vivo are largely unknown. Here we show that, in murine transplantation experiments, recipients transplanted with DNMT3A mutant-transduced cells exhibit aberrant haematopoietic stem cell (HSC) accumulation. Differentiation-associated genes are downregulated without accompanying changes in methylation status of their promoter-associated CpG islands in DNMT3A mutant-transduced stem/progenitor cells, representing a DNA methylation-independent role of mutated DNMT3A. DNMT3A R882H also promotes monoblastic transformation in vitro in combination with HOXA9. Molecularly, the DNMT3A mutant interacts with polycomb repressive complex 1 (PRC1), causing transcriptional silencing, revealing a DNA methylation-independent role of DNMT3A mutation. Suppression of PRC1 impairs aberrant HSC accumulation and monoblastic transformation. From our data, it is shown that DNMT3A mutants can block the differentiation of HSCs and leukaemic cells via PRC1. This interaction could be targetable in DNMT3A-mutated leukaemias. DNMT3A mutations are known to cause acute myeloid leukaemia. Here, Koya et al . show that DNMT3A R882H mutation causes monoblastic transformation and haematopoietic stem cell accumulation in a methylation-independent manner, by suppressing the polycomb repressive complex 1, causing transcriptional silencing.

Induced pluripotent stem (iPS) cells can be established from somatic cells. However, there is currently no established strategy to generate corneal epithelial cells from iPS cells. In this study, we investigated whether corneal epithelial cells could be differentiated from iPS cells. We tested 2 distinct sources: human adult dermal fibroblast (HDF)-derived iPS cells (253G1) and human adult corneal limbal epithelial cells (HLEC)-derived iPS cells (L1B41). We first established iPS cells from HLEC by introducing the Yamanaka 4 factors. Corneal epithelial cells were successfully induced from the iPS cells by the stromal cell-derived inducing activity (SDIA) differentiation method, as Pax6(+)/K12(+) corneal epithelial colonies were observed after prolonged differentiation culture (12 weeks or later) in both the L1B41 and 253G1 iPS cells following retinal pigment epithelial and lens cell induction. Interestingly, the corneal epithelial differentiation efficiency was higher in L1B41 than in 253G1. DNA methylation analysis revealed that a small proportion of differentially methylated regions still existed between L1B41 and 253G1 iPS cells even though no significant difference in methylation status was detected in the specific corneal epithelium-related genes such as K12, K3, and Pax6. The present study is the first to demonstrate a strategy for corneal epithelial cell differentiation from human iPS cells, and further suggests that the epigenomic status is associated with the propensity of iPS cells to differentiate into corneal epithelial cells.