A microengineered collagen scaffold for generating a polarized crypt-villus architecture of human small intestinal epithelium

The human small intestinal epithelium possesses a distinct crypt-villus architecture and tissue polarity in which proliferative cells reside inside crypts while differentiated cells are localized to the villi. Indirect evidence has shown that the processes of differentiation and migration are driven in part by biochemical gradients of factors that specify the polarity of these cellular compartments; however, direct evidence for gradient-driven patterning of this in vivo architecture has been hampered by limitations of the in vitro systems available. Enteroid cultures are a powerful in vitro system; nevertheless, these spheroidal structures fail to replicate the architecture and lineage compartmentalization found in vivo, and are not easily subjected to gradients of growth factors. In the current work, we report the development of a micropatterned collagen scaffold with suitable extracellular matrix and stiffness to generate an in vitro self-renewing human small intestinal epithelium that replicates key features of the in vivo small intestine: a crypt-villus architecture with appropriate cell-lineage compartmentalization and an open and accessible luminal surface. Chemical gradients applied to the crypt-villus axis promoted the creation of a stem/progenitor-cell zone and supported cell migration along the crypt-villus axis. This new approach combining microengineered scaffolds, biophysical cues and chemical gradients to control the intestinal epithelium ex vivo can serve as a physiologically relevant mimic of the human small intestinal epithelium, and is broadly applicable to model other tissues that rely on gradients for physiological function

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies

Sox9 expression marks a subset of CD24-expressing small intestine epithelial stem cells that form organoids in vitro

The inability to identify, isolate, and culture intestinal epithelial stem cells (IESCs) has been prohibitive to the study and therapeutic utilization of these cells. Using a Sox9EGFP mouse model, we demonstrate that Sox9EGFP fluorescence signatures can be used to differentiate between and enrich for progenitors (Sox9EGFPsubLo) and multipotent IESCs (Sox9EGFPlo). Sox9EGFPlo cells generate “organoids” in a recently defined culture system that mimics the native IESC niche. These organoids possess all four differentiated cell types of the small intestine epithelium, demonstrating the multipotent capacity of Sox9EGFPlo cells. Our results are consistent with the previously reported observation that single IESCs generate cryptlike units without a detectable mesenchymal cell component. A prospective search revealed that CD24 is expressed in the Sox9EGFPlo population and marks IESCs that form organoids in culture. CD24 represents the first cell surface marker that facilitates fluorescence-activated cell sorting enrichment of IESCs with widely available antibodies without requiring a specialized fluorescent reporter gene mouse model

Development of Arrayed Colonic Organoids for Screening of Secretagogues Associated with Enterotoxins

Enterotoxins increase intestinal fluid secretion through modulation of ion channels as well as activation of the enteric nervous and immune systems. Colonic organoids, also known as colonoids, are functionally and phenotypically similar to in vivo colonic epithelium and have been used to study intestinal ion transport and subsequent water flux in physiology and disease models. In conventional cultures, organoids exist as spheroids embedded within a hydrogel patty of extracellular matrix, and they form at multiple depths, impairing efficient imaging necessary to capture data from statistically relevant sample sizes. To overcome these limitations, an analytical platform with colonic organoids localized to the planar surface of a hydrogel layer was developed. The arrays of densely packed colonoids (140 μm average diameter, 4 colonoids/mm2) were generated in a 96-well plate, enabling assay of the response of hundreds of organoids so that organoid subpopulations with distinct behaviors were identifiable. Organoid cell types, monolayer polarity, and growth were similar to those embedded in hydrogel. An automated imaging and analysis platform efficiently tracked over time swelling due to forskolin and fluid movement across the cell monolayer stimulated by cholera toxin. The platform was used to screen compounds associated with the enteric nervous and immune systems for their effect on fluid movement across epithelial cells. Prostaglandin E2 promoted increased water flux in a subset of organoids that resulted in organoid swelling, confirming a role for this inflammatory mediator in diarrheal conditions but also illustrating organoid differences in response to an identical stimulus. By allowing sampling of a large number of organoids, the arrayed organoid platform permits identification of organoid subpopulations intermixed within a larger group of nonresponding organoids. This technique will enable automated, large-scale screening of the impact of drugs, toxins, and other compounds on colonic physiology

A single promoter directs both housekeeping and erythroid preferential expression of the human ferrochelatase gene.

We have isolated and characterized the 5'-flanking region of the gene for human ferrochelatase (HFC), the last enzyme of the heme biosynthetic pathway. The proximal promoter of the gene is contained within a region that structurally resembles a CpG island and is devoid of general cis elements such as TATA and CAAT boxes. Recognition sites for the ubiquitous Sp1 family of transcription factors, as well as for the erythroid-specific trans-acting factors NF-E2 and GATA-1 were found, and binding of regulatory proteins to these elements was analyzed by in vitro DNase I protection assays. The contribution of the various cis elements to both ubiquitous and erythroid preferential expression of the HFC gene was assessed by using transient transfection assays. These showed that a minimal Sp1-driven promoter devoid of the upstream erythroid-specific elements was sufficient for erythroid preferential expression of the HFC gene. However, elimination of a repressor sequence lying between the minimal promoter and the erythroid-specific elements resulted in high levels of expression in human erythroleukemic K562 cells only when the cis elements recognized by GATA-1 and NF-E2 were present, suggesting that the activity of these factors is regulated by a downstream repressor in erythroid cells

Impact of short-chain galactooligosaccharides on the gut microbiome of lactose-intolerant individuals

Approximately 75% of the global human population are lactose malabsorbers. In a previous clinical trial, it was shown that feeding a high-purity galactooligosaccharide (>95% GOS) could improve symptoms of lactose-intolerant subjects, attaining lactose tolerance in a majority of subjects. To investigate the mechanism, we examined the microbiome of human subjects before and after GOS feeding. The results show a significant shift in the microbiome of responsive individuals, including lactose-fermenting microbes in their stools. The high-purity prebiotic GOS resulted in adaptive shifts in the microbiome and correlated with improvement in clinical symptoms

High-Fat Diet: Bacteria Interactions Promote Intestinal Inflammation Which Precedes and Correlates with Obesity and Insulin Resistance in Mouse

Obesity induced by high fat (HF) diet is associated with inflammation which contributes to development of insulin resistance. Most prior studies have focused on adipose tissue as the source of obesity-associated inflammation. Increasing evidence links intestinal bacteria to development of diet-induced obesity (DIO). This study tested the hypothesis that HF western diet and gut bacteria interact to promote intestinal inflammation, which contributes to the progression of obesity and insulin resistance.Conventionally raised specific-pathogen free (CONV) and germ-free (GF) mice were given HF or low fat (LF) diet for 2-16 weeks. Body weight and adiposity were measured. Intestinal inflammation was assessed by evaluation of TNF-alpha mRNA and activation of a NF-kappaB(EGFP) reporter gene. In CONV but not GF mice, HF diet induced increases in body weight and adiposity. HF diet induced ileal TNF-alpha mRNA in CONV but not GF mice and this increase preceded obesity and strongly and significantly correlated with diet induced weight gain, adiposity, plasma insulin and glucose. In CONV mice HF diet also resulted in activation of NF-kappaB(EGFP) in epithelial cells, immune cells and endothelial cells of small intestine. Further experiments demonstrated that fecal slurries from CONV mice fed HF diet are sufficient to activate NF-kappaB(EGFP) in GF NF-kappaB(EGFP) mice.Bacteria and HF diet interact to promote proinflammatory changes in the small intestine, which precede weight gain and obesity and show strong and significant associations with progression of obesity and development of insulin resistance. To our knowledge, this is the first evidence that intestinal inflammation is an early consequence of HF diet which may contribute to obesity and associated insulin resistance. Interventions which limit intestinal inflammation induced by HF diet and bacteria may protect against obesity and insulin resistance

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

Therapeutically engineered induced neural stem cells are tumour-homing and inhibit progression of glioblastoma

Transdifferentiation (TD) is a recent advancement in somatic cell reprogramming. The direct conversion of TD eliminates the pluripotent intermediate state to create cells that are ideal for personalized cell therapy. Here we provide evidence that TD-derived induced neural stem cells (iNSCs) are an efficacious therapeutic strategy for brain cancer. We find that iNSCs genetically engineered with optical reporters and tumouricidal gene products retain the capacity to differentiate and induced apoptosis in co-cultured human glioblastoma cells. Time-lapse imaging shows that iNSCs are tumouritropic, homing rapidly to co-cultured glioblastoma cells and migrating extensively to distant tumour foci in the murine brain. Multimodality imaging reveals that iNSC delivery of the anticancer molecule TRAIL decreases the growth of established solid and diffuse patient-derived orthotopic glioblastoma xenografts 230- and 20-fold, respectively, while significantly prolonging the median mouse survival. These findings establish a strategy for creating autologous cell-based therapies to treat patients with aggressive forms of brain cancer

Optimization of 3-D organotypic primary colonic cultures for organ-on-chip applications

Abstract Background New advances enable long-term organotypic culture of colonic epithelial stem cells that develop into structures known as colonoids. Colonoids represent a primary tissue source acting as a potential starting material for development of an in vitro model of the colon. Key features of colonic crypt isolation and subsequent colonoid culture have not been systematically optimized compromising efficiency and reproducibility. Here murine crypt isolation yield and quality are optimized, and colonoid culture efficiency measured in microfabricated culture devices. Results An optimal incubation time of 60 min in a chelating buffer released 280,000 ± 28,000 crypts from the stroma of a single colon with 79.3% remaining intact. Mechanical agitation using an average acceleration of 1.5 × g liberated the highest quality crypts with 86% possessing well-defined lumens. Culture in 50% Matrigel resulted in the highest colonoid formation efficiency of 33 ± 5%. Immunostaining demonstrated that colonoids isolated under these conditions possessed stem/progenitor cells and differentiated cell lineages. Microfabrication substrates (glass, polystyrene, PDMS, and epoxy photoresists: SU-8 and 1002-F) were tested for compatibility with colonoid culture. PDMS promoted formation of 3-D colonoids containing stem/progenitor cells, while other substrates promoted outgrowth of a 2-D epithelial monolayer composed of differentiated cells. Conclusion Improved crypt isolation and 3-D colonoid culture, along with an understanding of colonic epithelial cell behavior in the presence of microfabrication substrates will support development of ‘organ-on-a-chip’ approaches for studies using primary colonic epithelium