技术资料

Hematopoietic stem cells (HSCs) reside in the bone marrow within niches that provide microenvironmental signals in the form of biophysical cues,bound and diffusible biomolecules,and heterotypic cell-cell interactions that influence HSC fate decisions. This study seeks to inform the development of a synthetic culture platform that promotes ex vivo HSC expansion without exhaustion. A library of methacrylamide-functionalized gelatin (GelMA) hydrogels is used to explore remodeling and crosstalk from mesenchymal stromal cells (MSCs) on the expansion and quiescence of murine HSCs. The use of a degradable GelMA hydrogel enables MSC-mediated remodeling,yielding dynamic shifts in the matrix environment over time. An initially low-diffusivity hydrogel for co-culture of hematopoietic stem and progenitor cells to MSCs facilitates maintenance of an early progenitor cell population over 7 days. Excitingly,this platform promotes retention of a quiescent HSC population compared to HSC monocultures. These studies reveal MSC-density-dependent upregulation of MMP-9 and changes in hydrogel mechanical properties ($\Delta$E = 2.61 ± 0.72) suggesting MSC-mediated matrix remodeling may contribute to a dynamic culture environment. Herein,a 3D hydrogel is reported for ex vivo HSC culture,in which HSC expansion and quiescence is sensitive to hydrogel properties,MSC co-culture,and MSC-mediated hydrogel remodeling. View Publication

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

Parasitic helminths cause significant damage as they migrate through host tissues to complete their life cycle. While chronic helminth infections are characterized by a well-described Type 2 immune response,the early,tissue-invasive stages are not well understood. Here we investigate the immune pathways activated during the early stages of Heligmosomoides polygyrus bakeri (Hpb),a natural parasitic roundworm of mice. In contrast to the Type 2 immune response present at later stages of infection,a robust Type 1 immune signature including IFNg production was dominant at the time of parasite invasion and granuloma formation. This early response was associated with an accumulation of activated Natural Killer (NK) cells,with no increase of other innate lymphoid cell populations. Parabiosis and confocal microscopy studies indicated that NK cells were recruited from circulation to the small intestine,where they surrounded parasitic larvae. NK cell recruitment required IFN$\gamma$ receptor signaling,but was independent of CXCR3 expression. The depletion of tissue-infiltrating NK cells altered neither worm burden nor parasite fitness,but increased vascular injury,suggesting a role for NK cells in mediating tissue protection. Together,these data identify an unexpected role for NK cells in promoting disease tolerance during the invasive stage of an enteric helminth infection. 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

The higher-order architecture observed in biological systems,like viruses,is very effective in nucleic acid transport. The replications of this system has been attempted with both synthetic and naturally occurring polymers with mixed results. Here we describe a peptide/siRNA quaternary complex that functions as an siRNA delivery system. The rational design of a peptide assembly is inspired by the viral capsids,but not derived from them. We selected the collagen peptide (COL) to provide the structural stability and the folding framework,and hybridize it with the cell penetrating peptide (CPP) that allows for effective penetration of biological barriers. The peptide/siRNA quaternary complex forms stoichiometric,10 nm nanoparticles,that show fast cellular uptake ({\textless}30 min),effective siRNA release,and gene silencing. The complex provides capsid-like protection for siRNA against nucleases without being immunostimulatory,or cytotoxic. Our data suggests that delivery vehicles based on synthetic quaternary structures that exhibit higher-order architecture may be effective in improving delivery and release of nucleic acid cargo. 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

Despite the clinical success of cancer immunotherapies,the majority of patients fail to respond or develop resistance through disruption of pathways that promote neo-antigen presentation on MHC I molecules. Here,we conducted a series of unbiased,genome-wide CRISPR/Cas9 screens to identify genes that limit natural killer (NK) cell anti-tumor activity. We identified that genes associated with antigen presentation and/or interferon-$\gamma$ (IFN-$\gamma$) signaling protect tumor cells from NK cell killing. Indeed,Jak1-deficient melanoma cells were sensitized to NK cell killing through attenuated NK cell-derived IFN-$\gamma$-driven transcriptional events that regulate MHC I expression. Importantly,tumor cells that became resistant to T cell killing through enrichment of MHC I-deficient clones were highly sensitive to NK cell killing. Taken together,we reveal the genes targeted by tumor cells to drive checkpoint blockade resistance but simultaneously increase their vulnerability to NK cells,unveiling NK cell-based immunotherapies as a strategy to antagonize tumor immune escape. 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

Filoviruses of the genus Ebolavirus include five species with marked differences in their ability to cause disease in humans. From the highly virulent Ebola virus to the seemingly nonpathogenic Reston virus,case-fatality rates can range between 0-90{\%}. In order to understand the molecular basis of these differences it is imperative to establish disease models that recapitulate human disease as faithfully as possible. Non-human primates are the gold-standard models for filovirus pathogenesis,but comparative studies are skewed by the fact that Reston virus infection can be lethal for NHP. Here we have used HLA-A2 transgenic,NOD-scid-interleukin 2$\gamma$ receptor knockout (NSG-A2) mice reconstituted with human hematopoiesis to compare Ebola virus and Reston virus pathogenesis in a human-like environment. While significantly less pathogenic than Ebola virus,Reston virus killed 20{\%} of infected mice,a finding that was linked to exacerbated inflammation and viral replication in the liver. In addition,'humanized' mice recapitulated the case-fatality ratios of different Ebolavirus species in humans. Our findings point out at humanized mice as a putative model to test the pathogenicity of newly discovered filoviruses,and warrants further investigations on Reston virus pathogenesis in humans. 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