Aging impairs the regenerative potential of hematopoietic stem cells (HSC) and skews differentiation towards the myeloid lineage. The bone marrow (BM) microenvironment has recently been suggested to influence HSC aging, however the mechanisms whereby BM stromal cells mediate this effect is unknown. Here we show that aging-associated decreased expression of CXCR4 expression on BM mesenchymal stem cells (MSC) plays a crucial role in the development of the hematopoietic stem and progenitor cells (HSPC) aging phenotype. The BM MSC from old mice was sufficient to drive a premature aging phenotype of young HSPC when cultured together ex vivo. The impaired ability of old MSC to support HSPC function is associated with reduced expression of CXCR4 on BM MSC of old mice. Deletion of the CXCR4 gene in young MSC accelerates an aging phenotype in these cells characterized by increased production of reactive oxygen species (ROS), DNA damage, senescence, and reduced proliferation. Culture of HSPC from young mice with CXCR4 deficient MSC also from young mice led to a premature aging phenotype in the young HSPC, as evidenced by reduced hematopoietic regeneration and enhanced myeloid differentiation. Mechanistically, CXCR4 signaling prevents BM MSC dysfunction by suppressing oxidative stress, as treatment of old or CXCR4 deficient MSC with N-acetyl-L-cysteine (NAC), improved their niche supporting activity, and attenuated the HSPC aging phenotype. Our studies suggest that age-associated reduction in CXCR4 expression on BM MSC impairs hematopoietic niche activity with increased ROS production, driving an HSC aging phenotype. Thus, modulation of the SDF-1/CXCR4 axis in MSC may lead to novel interventions to alleviate the age-associated decline in immune/hematopoietic function. View Publication

Both antitumor and protumor property of mesenchymal stem cells (MSCs) have been demonstrated. We hypothesize that this contradiction is due to the heterogeneity of MSC subsets and that extracellular vesicles (EVs) from distinct MSC subsets can transfer the corresponding antitumor activities. Here we evaluated the antitumor activities of two subsets of adipose-derived mesenchymal stem cells (ADSCs) and ADSC-derived EVs (ADSC-EVs) in immunocompetent syngeneic mouse models of breast cancer. We identified CD90high and CD90low ADSC subsets and demonstrated that CD90high ADSCs could be converted into CD90low ADSCs by stimulation with LPS. CD90low ADSCs and its derived EVs significantly inhibited tumor growth in tumor-bearing mice. Benefit of tumor control were associated with decreased tumor cell proliferation and migration, and enhanced tumor cell apoptosis mediated by ADSC-EVs. Antioncogenic miRNA-16-5p loaded CD90low ADSC-EVs further significantly enhanced antitumor activities. Taken together, this study represents the first attempt to apply our newly identified antitumor ADSCs and its derived EVs in preclinical treatment of breast cancer. This study also provides the evidence that EVs can serve as a novel and effective therapeutics or drug delivery vesicle. This new therapeutic approach could be potentially applicable to breast cancer and many other types of cancer. View Publication

3-dimensional (3D) bioprinting technology provides promising strategy in the fabrication of artificial tissues and organs. As the fundamental element in bioprinting process, preparation of bioink with ideal mechanical properties without sacrifice of biocompatibility is a great challenge. In this study, a supramolecular hydrogel-based bioink is prepared by polyethylene glycol (PEG) grafted chitosan, $\alpha$-cyclodextrin ($\alpha$-CD) and gelatin. It has a primary crosslinking structure through the aggregation of the pseudo-polyrotaxane-like side chains, which are formed from the host-guest interactions between $\alpha$-CD and PEG side chain. Apparent viscosity measurement shows the shear-shinning property of this bioink, which might be due to the reversibility of the physical crosslinking. Moreover, with $\beta$-glycerophosphate at different concentrations as the secondary crosslinking agent, the printed constructs demonstrate different Young's modulus (p {\textless} 0.001). They could also maintain the Young's modulus in cell culture condition for at least 21 days (p {\textless} 0.05). By co-culturing each component with fibroblasts, CCK-8 assay demonstrate cellular viability is higher than 80{\%}. After bioprinting and culturing, immunofluorescence staining with quantification indicate the expression of Ki-67, Paxillin, and N-cadherin is higher in day 14 than those in day 3 (p {\textless} 0.05). Oil red O and Nissl body specific staining reflect strength tunable bioink may have impact on the cell fate of mesenchymal stem cells (p {\textless} 0.05). This work might provide new idea for advanced bioink in the application of re-establishing complicated tissues and organs. View Publication

Gelatin coatings are effective in increasing the retention of MSCs injected into the heart and minimizing the damage from acute myocardial infarction (AMI), but early studies suffered from low fractions of the MSCs coated with gelatin. Biotinylation of the MSC surface is a critical first step in the gelatin coating process, and in this study, we evaluated the use of biotinylated cholesterol lipid insertion" anchors as a substitute for the covalent NHS-biotin anchors to the cell surface. Streptavidin-eosin molecules where eosin is our photoinitiator can then be bound to the cell surface through biotin-streptavidin affinity. The use of cholesterol anchors increased streptavidin density on the surface of MSCs further driving polymerization and allowing for an increased fraction of MSCs coated with gelatin (83{\%}) when compared to NHS-biotin (52{\%}). Additionally the cholesterol anchors increased the uniformity of the coating on the MSC surface and supported greater numbers of coated MSCs even when the streptavidin density was slightly lower than that of an NHS-biotin anchoring strategy. Critically this improvement in gelatin coating efficiency did not impact cytokine secretion and other critical MSC functions. Proper selection of the cholesterol anchor and the biotinylation conditions supports cellular function and densities of streptavidin on the MSC surface of up to {\~{}}105 streptavidin molecules/$\mu$m2 . In all these cholesterol anchors offer an effective path towards the formation of conformal coatings on the majority of MSCs to improve the retention of MSCs in the heart following AMI." 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

Both anti-tumoral and pro-tumoral effects of mesenchymal stem cells (MSCs) in preclinical treatment of ovarian cancer have been controversially demonstrated. In this study, we profiled the phenotypes of mouse compact bone-derived MSCs (CB-MSCs) and bone marrow-derived MSCs (BM-MSCs) and found that CB-MSCs expressed lower CD90 compared to BM-MSCs. We examined gene expression of immune regulating cytokines of CB-MSCs in 2D and 3D culture and under stimulation with TLR4 agonist LPS or immune activator VIC-008. Our data showed that when CB-MSCs were cultured in simulated in vivo 3D condition, CD90 expression was further decreased. Moreover, gene expressions of immune activating cytokines IL-12, IL-21, IFNgamma and a pro-inflammatory cytokine CXCL10 in CB-MSCs were increased in 3D culture whereas gene expression of anti-inflammatory cytokines IL-10 and CCL5 were downregulated. Stimulation of CB-MSCs by LPS or VIC-008 presented similar profile of the cytokine gene expressions to that in 3D culture which might benefit the anti-tumor efficacy of CD90low MSCs. The anti-tumor effects of CD90low CB-MSCs alone or in combination with VIC-008 were evaluated in a syngeneic orthotopic mouse model of ovarian cancer. Treatment that combines CB-MSCs and VIC-008 significantly decreased tumor growth and prolonged mouse survival. This was associated with the increase of activated anti-tumoral CD4+ and CD8+ T cells and the decrease of Treg cells in the tumor microenvironment. Taken together, our study demonstrates the synergistic anti-tumoral efficacy by application of CB-MSCs combined with immune activator VIC-008 and provides new insight into CD90low MSCs as a new anti-tumor arsenal. View Publication