技术资料

Neural organoids have the potential to improve our understanding of human brain development and neurological disorders. However,it remains to be seen whether these tissues can model circuit formation with functional neuronal output. Here we have adapted air–liquid interface culture to cerebral organoids,leading to improved neuronal survival and axon outgrowth. The resulting thick axon tracts display various morphologies,including long-range projection within and away from the organoid,growth-cone turning,and decussation. Single-cell RNA sequencing reveals various cortical neuronal identities,and retrograde tracing demonstrates tract morphologies that match proper molecular identities. These cultures exhibit active neuronal networks,and subcortical projecting tracts can innervate mouse spinal cord explants and evoke contractions of adjacent muscle in a manner dependent on intact organoid-derived innervating tracts. Overall,these results reveal a remarkable self-organization of corticofugal and callosal tracts with a functional output,providing new opportunities to examine relevant aspects of human CNS development and disease. View Publication

AIM OF THE STUDY Human breast milk reduces the risk and severity of necrotizing enterocolitis (NEC). Exosomes are extracellular vesicles (EVs) found in high concentrations in milk,and they mediate intercellular communication and immune responses. The aim of this study is to compare the protective effects of exosomes that are derived from different time periods of breast milk production against intestinal injury using an ex vivo intestinal organoid model. METHODS Colostrum,transitional and mature breast milk samples from healthy lactating mothers were collected. Exosomes were isolated using serial ultracentrifugation and filtration. Exosomes' presence was confirmed using transmission electron microscopy (TEM) and western blot. To form the intestinal organoids,terminal ileum was harvested from neonatal mice pups at postnatal day 9,crypts were isolated and organoids were cultured in matrigel. Organoids were either cultured with exposure to lipopolysaccharide (LPS),or in treatment groups where both LPS and exosomes were added in the culturing medium. Inflammatory markers and organoids viability were evaluated. MAIN RESULTS Human milk-derived exosomes were successfully isolated and characterized. LPS administration reduced the size of intestinal organoids,induced inflammation through increasing TNF$\alpha$ and TLR4 expression,and stimulated intestinal regeneration. Colostrum,transitional and mature human milk-derived exosome treatment all prevented inflammatory injury,while exosomes derived from colostrum were most effective at reducing inflammatory cytokine. CONCLUSIONS Human breast milk-derived exosomes were able to protect intestine organoids against epithelial injury induced by LPS. Colostrum exosomes offer the best protective effect among the breast-milk derived exosomes. Human milk exosomes can be protective against the development of intestinal injury such as that seen in NEC. View Publication

Stem cell competition could shed light on the tissue-based quality control mechanism that prevents carcinogenesis. To quantitatively evaluate stem cell competition in vitro,we developed a two-color intestinal organoid forming system. First,we improved a protocol of culturing organoids from intestinal leucine-rich-repeat containing G-protein-coupled receptor 5 (Lgr5)- enhanced green fluorescent protein (EGFP)high stem cells directly sorted on Matrigel without embedding. The organoid-forming potential (OFP) was 25{\%} of Lgr5-EGFPhigh cells sorted at one cell per well. Using this culture protocol with lineage tracing,we established a two-color organoid culture system by mixing stem cells expressing different fluorescent colors. To analyze stem cell competition,two-color organoids were formed by mixing X-ray-irradiated and non-irradiated intestinal stem cells. In the two-color organoids,irradiated stem cells exhibited a growth disadvantage,although the OFP of irradiated cells alone did not decrease significantly from that of non-irradiated cells. These results suggest that stem cell competition can be evaluated quantitively in vitro using our new system. View Publication

Increased levels of intestinal bile acids (BAs) are a risk factor for colorectal cancer (CRC). Here,we show that the convergence of dietary factors (high-fat diet) and dysregulated WNT signaling (APC mutation) alters BA profiles to drive malignant transformations in Lgr5-expressing (Lgr5+) cancer stem cells and promote an adenoma-to-adenocarcinoma progression. Mechanistically,we show that BAs that antagonize intestinal farnesoid X receptor (FXR) function,including tauro-$\beta$-muricholic acid (T-$\beta$MCA) and deoxycholic acid (DCA),induce proliferation and DNA damage in Lgr5+ cells. Conversely,selective activation of intestinal FXR can restrict abnormal Lgr5+ cell growth and curtail CRC progression. This unexpected role for FXR in coordinating intestinal self-renewal with BA levels implicates FXR as a potential therapeutic target for CRC. View Publication

Areas of a junction between two types of epithelia are known to be cancer-prone in many organ systems. However,mechanisms for preferential malignant transformation at the junction areas remain insufficiently elucidated. Here we report that inactivation of tumor suppressor genes Trp53 and Rb1 in the gastric squamous-columnar junction (SCJ) epithelium results in preferential formation of metastatic poorly differentiated neoplasms,which are similar to human gastroesophageal carcinoma. Unlike transformation-resistant antral cells,SCJ cells contain a highly proliferative pool of immature Lgr5-CD44+ cells,which are prone to transformation in organoid assays,comprise early dysplastic lesions,and constitute up to 30{\%} of all neoplastic cells. CD44 ligand osteopontin (OPN) is preferentially expressed in and promotes organoid formation ability and transformation of the SCJ glandular epithelium. OPN and CD44 overexpression correlate with the worst prognosis of human gastroesophageal carcinoma. Thus,detection and selective targeting of the active OPN-CD44 pathway may have direct clinical relevance. View Publication

Humans and other mammalian hosts have evolved mechanisms to control the bacteria colonizing their mucosal barriers to prevent invasion. While the breach of barriers by bacteria typically leads to overt infection,increasing evidence supports a role for translocation of commensal bacteria across an impaired gut barrier to extraintestinal sites in the pathogenesis of autoimmune and other chronic,non-infectious diseases. Whether gut commensal translocation is a cause or consequence of the disease is incompletely defined. Here we discuss factors that lead to translocation of live bacteria across the gut barrier. We expand upon our recently published demonstration that translocation of the gut pathobiont Enterococcus gallinarum can induce autoimmunity in susceptible hosts and postulate on the role of Enterococcus species as instigators of chronic,non-infectious diseases. View Publication

In patients with inactivating mutations in myosin Vb (Myo5B),enterocytes show large inclusions lined by microvilli. The origin of inclusions in small-intestinal enterocytes in microvillus inclusion disease is currently unclear. We postulated that inclusions in Myo5b KO mouse enterocytes form through invagination of the apical brush border membrane. 70-kD FITC-dextran added apically to Myo5b KO intestinal explants accumulated in intracellular inclusions. Live imaging of Myo5b KO-derived enteroids confirmed the formation of inclusions from the apical membrane. Treatment of intestinal explants and enteroids with Dyngo resulted in accumulation of inclusions at the apical membrane. Inclusions in Myo5b KO enterocytes contained VAMP4 and Pacsin 2 (Syndapin 2). Myo5b;Pacsin 2 double-KO mice showed a significant decrease in inclusion formation. Our results suggest that apical bulk endocytosis in Myo5b KO enterocytes resembles activity-dependent bulk endocytosis,the primary mechanism for synaptic vesicle uptake during intense neuronal stimulation. Thus,apical bulk endocytosis mediates the formation of inclusions in neonatal Myo5b KO enterocytes. View Publication

We introduce machine learning (ML) to perform classification and quantitation of images of nuclei from human blood neutrophils. Here we assessed the use of convolutional neural networks (CNNs) using free,open source software to accurately quantitate neutrophil NETosis,a recently discovered process involved in multiple human diseases. CNNs achieved {\textgreater}94{\%} in performance accuracy in differentiating NETotic from non-NETotic cells and vastly facilitated dose-response analysis and screening of the NETotic response in neutrophils from patients. Using only features learned from nuclear morphology,CNNs can distinguish between NETosis and necrosis and between distinct NETosis signaling pathways,making them a precise tool for NETosis detection. Furthermore,by using CNNs and tools to determine object dispersion,we uncovered differences in NETotic nuclei clustering between major NETosis pathways that is useful in understanding NETosis signaling events. Our study also shows that neutrophils from patients with sickle cell disease were unresponsive to one of two major NETosis pathways. Thus,we demonstrate the design,performance,and implementation of ML tools for rapid quantitative and qualitative cell analysis in basic science. View Publication

The contribution of mast cells in the microenvironment of solid malignancies remains controversial. Here we functionally assess the impact of tumor-adjacent,submucosal mast cell accumulation in murine and human intestinal-type gastric cancer. We find that genetic ablation or therapeutic inactivation of mast cells suppresses accumulation of tumor-associated macrophages,reduces tumor cell proliferation and angiogenesis,and diminishes tumor burden. Mast cells are activated by interleukin (IL)-33,an alarmin produced by the tumor epithelium in response to the inflammatory cytokine IL-11,which is required for the growth of gastric cancers in mice. Accordingly,ablation of the cognate IL-33 receptor St2 limits tumor growth,and reduces mast cell-dependent production and release of the macrophage-attracting factors Csf2,Ccl3,and Il6. Conversely,genetic or therapeutic macrophage depletion reduces tumor burden without affecting mast cell abundance. Therefore,tumor-derived IL-33 sustains a mast cell and macrophage-dependent signaling cascade that is amenable for the treatment of gastric cancer. View Publication

Inflammatory bowel diseases (IBDs) are chronic and relapsing immune disorders that result,or possibly originate,from epithelial barrier defects. Intestinal organoids are a new reliable tool to investigate epithelial response in models of chronic inflammation. We produced organoids from the ulcerative colitis murine model Winnie to explore if the chronic inflammatory features observed in the parental intestine were preserved by the organoids. Furthermore,we investigated if quercetin administration to in vitro cultured organoids could suppress LPS-induced inflammation in wild-type organoids (WT-organoids) and spontaneous inflammation in ulcerative colitis organoids (UC-organoids). Our data demonstrate that small intestinal organoids obtained from Winnie mice retain the chronic intestinal inflammatory features characteristic of the parental tissue. Quercetin administration was able to suppress inflammation both in UC-organoids and in LPS-treated WT-organoids. Altogether,our data demonstrate that UC-organoids are a reliable experimental system for investigating chronic intestinal inflammation and pharmacological responses. View Publication

Post-transcriptional mechanisms have the potential to influence complex changes in gene expression,yet their role in cell fate transitions remains largely unexplored. Here,we show that suppression of the RNA helicase DDX6 endows human and mouse primed embryonic stem cells (ESCs) with a differentiation-resistant,hyper-pluripotent" state which readily reprograms to a naive state resembling the preimplantation embryo. We further demonstrate that DDX6 plays a key role in adult progenitors where it controls the balance between self-renewal and differentiation in a context-dependent manner. Mechanistically DDX6 mediates the translational suppression of target mRNAs in P-bodies. Upon loss of DDX6 activity P-bodies dissolve and release mRNAs encoding fate-instructive transcription and chromatin factors that re-enter the ribosome pool. Increased translation of these targets impacts cell fate by rewiring the enhancer heterochromatin and DNA methylation landscapes of undifferentiated cell types. Collectively our data establish a link between P-body homeostasis chromatin organization and stem cell potency." View Publication

mTORC1 regulates cellular growth and is activated by growth factors and by essential amino acids such as Leu. Leu enters cells via the Leu transporter LAT1-4F2hc (LAT1). Here we show that the Na+/K+/2Cl- cotransporter NKCC1 (SLC12A2),a known regulator of cell volume,is present in complex with LAT1. We further show that NKCC1 depletion or deletion enhances LAT1 activity,as well as activation of Akt and Erk,leading to activation of mTORC1 in cells,colonic organoids,and mouse colon. Moreover,NKCC1 depletion reduces intracellular Na+ concentration and cell volume (size) and mass and stimulates cell proliferation. NKCC1,therefore,suppresses mTORC1 by inhibiting its key activating signaling pathways. Importantly,by linking ion transport and cell volume regulation to mTORC1 function,NKCC1 provides a long-sought link connecting cell volume (size) to cell mass regulation. View Publication