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Sulfated glycosaminoglycans (GAGs),or synthetic mimetics thereof,are not favorably viewed as orally bioavailable drugs owing to their high number of anionic sulfate groups. Devising an approach for oral delivery of such highly sulfated molecules would be very useful. This work presents the concept that conjugating cholesterol to synthetic sulfated GAG mimetics enables oral delivery. A focused library of sulfated GAG mimetics was synthesized and found to inhibit the growth of a colorectal cancer cell line under spheroid conditions with a wide range of potencies ( 0.8 to 46). Specific analogues containing cholesterol,either alone or in combination with clinical utilized drugs,exhibited pronounced in vivo anticancer potential with intraperitoneal as well as oral administration,as assessed by ex vivo tertiary and quaternary spheroid growth,cancer stem cell (CSC) markers,and/or self-renewal factors. Overall,cholesterol derivatization of highly sulfated GAG mimetics affords an excellent approach for engineering oral activity. View Publication

BACKGROUND AND AIMS The intestinal mucosa undergoes a continual process of proliferation,differentiation,and apoptosis. An imbalance in this highly regimented process within the intestinal crypts is associated with several intestinal pathologies. Although metabolic changes are known to play a pivotal role in cell proliferation and differentiation,how glycolysis contributes to intestinal epithelial homeostasis remains to be defined. METHODS Small intestines were harvested from mice with specific hexokinase 2 (HK2) deletion in the intestinal epithelium or LGR5+ stem cells. Glycolysis was measured using the Seahorse XFe96 analyzer. Expression of phospho-p38 mitogen-activated protein kinase,the transcription factor atonal homolog 1,and intestinal cell differentiation markers lysozyme,mucin 2,and chromogranin A were determined by Western blot,quantitative real-time reverse transcription polymerase chain reaction,or immunofluorescence,and immunohistochemistry staining. RESULTS HK2 is a target gene of Wnt signaling in intestinal epithelium. HK2 knockout or inhibition of glycolysis resulted in increased numbers of Paneth,goblet,and enteroendocrine cells and decreased intestinal stem cell self-renewal. Mechanistically,HK2 knockout resulted in activation of p38 mitogen-activated protein kinase and increased expression of ATOH1; inhibition of p38 mitogen-activated protein kinase signaling attenuated the phenotypes induced by HK2 knockout in intestinal organoids. HK2 knockout significantly decreased glycolysis and lactate production in intestinal organoids; supplementation of lactate or pyruvate reversed the phenotypes induced by HK2 knockout. CONCLUSIONS Our results show that HK2 regulates intestinal stem cell self-renewal and differentiation through p38 mitogen-activated protein kinase/atonal homolog 1 signaling pathway. Our findings demonstrate an essential role for glycolysis in maintenance of intestinal stem cell function. View Publication

Chimerism testing after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) represents a promising tool for predicting disease relapse,although its precise role in this setting remains unclear. We investigated the predictive value of T lymphocyte chimerism analysis at 90 to 120 days after allo-HSCT in 378 patients with AML/MDS who underwent busulfan/fludarabine-based myeloablative preparative regimens. Of 265 (70%) patients with available T lymphocyte chimerism data,43% of patients in first or second complete remission (CR1/CR2) at the time of transplantation had complete (100%) donor T lymphocytes at day +90 to +120 compared with 60% of patients in the non-CR1/CR2 cohort (P = .005). In CR1/CR2 patients,donor T lymphocyte chimerism ?85% at day +90 to +120 was associated with a higher frequency of 3-year disease progression (29%; 95% confidence interval [CI],18% to 46% versus 15%; 95% CI,9% to 23%; hazard ratio [HR],2.1; P = .04). However,in the more advanced,non-CR1/CR2 cohort,mixed T lymphocyte chimerism was not associated with relapse (37%; 95% CI,20% to 66% versus 34%; 95% CI,25% to 47%; HR,1.3; P = .60). These findings demonstrate that early T lymphocyte chimerism testing at day +90 to +120 is a useful approach for predicting AML/MDS disease recurrence in patients in CR1/CR2 at the time of transplantation. View Publication

The routine application of human pluripotent stem cells (hPSCs) and their derivatives in biomedicine and drug discovery will require the constant supply of high-quality cells by defined processes. Culturing hPSCs as cell-only aggregates in (three-dimensional [3D]) suspension has the potential to overcome numerous limitations of conventional surface-adherent (two-dimensional [2D]) cultivation. Utilizing single-use instrumented stirred-tank bioreactors,we showed that perfusion resulted in a more homogeneous culture environment and enabled superior cell densities of 2.85 X 10(6) cells per milliliter and 47% higher cell yields compared with conventional repeated batch cultures. Flow cytometry,quantitative reverse-transcriptase polymerase chain reaction,and global gene expression analysis revealed a high similarity across 3D suspension and 2D precultures,underscoring that matrix-free hPSC culture efficiently supports maintenance of pluripotency. Interestingly,physiological data and gene expression assessment indicated distinct changes of the cells' energy metabolism,suggesting a culture-induced switch from glycolysis to oxidative phosphorylation in the absence of hPSC differentiation. Our data highlight the plasticity of hPSCs' energy metabolism and provide clear physiological and molecular targets for process monitoring and further development. This study paves the way toward more efficient GMP-compliant cell production and underscores the enormous process development potential of hPSCs in suspension culture. SIGNIFICANCE Human pluripotent stem cells (hPSCs) are a unique source for the,in principle,unlimited production of functional human cell types in vitro,which are of high value for therapeutic and industrial applications. This study applied single-use,clinically compliant bioreactor technology to develop advanced,matrix-free,and more efficient culture conditions for the mass production of hPSCs in scalable suspension culture. Using extensive analytical tools to compare established conditions with this novel culture strategy,unexpected physiological features of hPSCs were discovered. These data allow a more rational process development,providing significant progress in the field of translational stem cell research and medicine. View Publication

BACKGROUND & AIMS Down-regulation of chloride transporter SLC26A3 or down-regulated in adenoma (DRA) in colonocytes has recently been linked to the pathogenesis of ulcerative colitis (UC). Because exaggerated immune responses are one of the hallmarks of UC,these current studies were undertaken to define the mechanisms by which loss of DRA relays signals to immune cells to increase susceptibility to inflammation. METHODS NanoString Immunology Panel,fluorescence assisted cell sorting,immunoblotting,immunofluorescence,and quantitative real-time polymerase chain reaction assays were used in wild-type and DRA knockout (KO) mice. Interleukin (IL)-33 blocking was used to determine specific changes in immune cells and co-housing/broad spectrum antibiotics administration,and ex vivo studies in colonoids were conducted to rule out the involvement of microbiota. Colonoid-derived monolayers from healthy and UC patient biopsies were analyzed for translatability. RESULTS There was a marked induction of Th2 (>2-fold),CD4+ Th2 cells (~8-fold),RORt+ Th17,and FOXP3+ regulatory T cells (Tregs). DRA KO colons also exhibited a robust induction of IL-33 (>8-fold). In vivo studies using blocking of IL-33 established that T2 immune dysregulation (alterations in ILC2,Th2,and GATA3+ iTregs) in response to loss of DRA was due to altered epithelial-immune cell crosstalk via IL-33. CONCLUSIONS Loss of DRA in colonocytes triggers the release of IL-33 to drive a type 2 immune response. These observations emphasize the critical importance of DRA in mucosal immune homeostasis and its implications in the pathogenesis of UC. View Publication

Cytokines play a crucial role in the maintenance of polyclonal naive and memory T cell populations. It has previously been shown that ex vivo,the IL-7 cytokine induces the proliferation of naive recent thymic emigrants (RTE) isolated from umbilical cord blood but not mature adult-derived naive and memory human CD4(+) T cells. We find that the combination of IL-2 and IL-7 strongly promotes the proliferation of RTE,whereas adult CD4(+) T cells remain relatively unresponsive. Immunological activity is controlled by a balance between proliferation and apoptotic cell death. However,the relative contributions of IL-2 and IL-7 in regulating these processes in the absence of MHC/peptide signals are not known. Following exposure to either IL-2 or IL-7 alone,RTE,as well as mature naive and memory CD4(+) T cells,are rendered only minimally sensitive to Fas-mediated cell death. However,in the presence of the two cytokines,Fas engagement results in a high level of caspase-dependent apoptosis in both RTE as well as naive adult CD4(+) T cells. In contrast,equivalently treated memory CD4(+) T cells are significantly less sensitive to Fas-induced cell death. The increased susceptibility of RTE and naive CD4(+) T cells to Fas-induced apoptosis correlates with a significantly higher IL-2/IL-7-induced Fas expression on these T cell subsets than on memory CD4(+) T cells. Thus,IL-2 and IL-7 regulate homeostasis by modulating the equilibrium between proliferation and apoptotic cell death in RTE and mature naive and memory T cell subsets. View Publication

The efficient generation of hepatocytes from human pluripotent stem cells (hPSCs) requires the induction of a proper endoderm population,broadly characterized by the expression of the cell surface marker CXCR4. Strategies to identify and isolate endoderm subpopulations predisposed to the liver fate do not exist. In this study,we generated mouse monoclonal antibodies against human embryonic stem cell-derived definitive endoderm with the goal of identifying cell surface markers that can be used to track the development of this germ layer and its specification to a hepatic fate. Through this approach,we identified two endoderm-specific antibodies,HDE1 and HDE2,which stain different stages of endoderm development and distinct derivative cell types. HDE1 marks a definitive endoderm population with high hepatic potential,whereas staining of HDE2 tracks with developing hepatocyte progenitors and hepatocytes. When used in combination,the staining patterns of these antibodies enable one to optimize endoderm induction and hepatic specification from any hPSC line. View Publication

Bats are natural reservoirs for several zoonotic viruses,potentially due to an enhanced capacity to control viral infection. However,the mechanisms of antiviral responses in bats are poorly defined. Here we established a Jamaican fruit bat (JFB) intestinal organoid model of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. JFB organoids were susceptible to SARS-CoV-2 infection,with increased viral RNA and subgenomic RNA detected in cell lysates and supernatants. Gene expression of type I interferons and inflammatory cytokines was induced in response to SARS-CoV-2 but not in response to TLR agonists. Interestingly,SARS-CoV-2 did not lead to cytopathic effects in JFB organoids but caused enhanced organoid growth. Proteomic analyses revealed an increase in inflammatory signaling,cell turnover,cell repair,and SARS-CoV-2 infection pathways. Collectively,our findings suggest that primary JFB intestinal epithelial cells can mount a successful antiviral interferon response and that SARS-CoV-2 infection in JFB cells induces protective regenerative pathways. View Publication

The classical model of hematopoiesis predicts a dichotomous lineage restriction of multipotent hematopoietic progenitors (MPPs) into common lymphoid progenitors (CLPs) and common myeloid progenitors (CMPs). However,this idea has been challenged by the identification of lymphoid progenitors retaining partial myeloid potential (e.g.,LMPPs),implying that granulocytes can arise within both the classical lymphoid and the myeloid branches. Here,we resolve this issue by using cell-surface CD133 expression to discriminate functional progenitor populations. We show that eosinophilic and basophilic granulocytes as well as erythrocytes and megakaryocytes derive from a common erythro-myeloid progenitor (EMP),whereas neutrophilic granulocytes arise independently within a lympho-myeloid branch with long-term progenitor function. These findings challenge the concept of a CMP and restore dichotomy to the classical hematopoietic model. View Publication

A hallmark of pancreatic ductal adenocarcinoma (PDAC) is an exuberant stroma comprised of diverse cell types that enable or suppress tumor progression. Here,we explored the role of oncogenic KRAS in protumorigenic signaling interactions between cancer cells and host cells. We show that KRAS mutation (KRAS) drives cell-autonomous expression of type I cytokine receptor complexes (IL2r?–IL4r? and IL2r?–IL13r?1) in cancer cells that in turn are capable of receiving cytokine growth signals (IL4 or IL13) provided by invading Th2 cells in the microenvironment. Early neoplastic lesions show close proximity of cancer cells harboring KRAS and Th2 cells producing IL4 and IL13. Activated IL2r?–IL4r? and IL2r?–IL13r?1 receptors signal primarily via JAK1-STAT6. Integrated transcriptomic,chromatin occupancy,and metabolomic studies identified MYC as a direct target of activated STAT6 and that MYC drives glycolysis. Thus,paracrine signaling in the tumor microenvironment plays a key role in the KRAS-driven metabolic reprogramming of PDAC. SIGNIFICANCE: Type II cytokines,secreted by Th2 cells in the tumor microenvironment,can stimulate cancer cell-intrinsic MYC transcriptional upregulation to drive glycolysis. This KRAS-driven heterotypic signaling circuit in the early and advanced tumor microenvironment enables cooperative protumorigenic interactions,providing candidate therapeutic targets in the KRAS pathway for this intractable disease. View Publication

Heartland virus (HRTV),a phlebovirus first isolated from two Missouri farmers in 2009,has been proposed to be transmitted to humans by the bite of infected Amblyomma americanum ticks. It is closely related to severe fever with thrombocytopenia syndrome virus (SFTSV) from China,another previously unrecognized phlebovirus that has subsequently been associated with hundreds of cases of severe disease in humans. To expand diagnostic capacity to detect HRTV infections,20 hybridoma clones secreting anti-HRTV murine monoclonal antibodies (MAbs) were developed using splenocytes from HRTV-inoculated AG129 alpha/beta and gamma interferon receptor-deficient mice. Nine of these MAbs were characterized herein for inclusion in future HRTV diagnostic assay development. All of the MAbs developed were found to be non-neutralizing and reactive to linear epitopes on HRTV nucleocapsid protein. MAb 2AF11 was found to be cross-reactive with SFTSV. View Publication

Human intestinal organoids (HIOs) derived from pluripotent stem cells provide a valuable model for investigating human intestinal organogenesis and physiology,but they lack the immune components required to fully recapitulate the complexity of human intestinal biology and diseases. To address this issue and to begin to decipher human intestinal-immune crosstalk during development,we generated HIOs containing immune cells by transplanting HIOs under the kidney capsule of mice with a humanized immune system. We found that human immune cells temporally migrate to the mucosa and form cellular aggregates that resemble human intestinal lymphoid follicles. Moreover,after microbial exposure,epithelial microfold cells are increased in number,leading to immune cell activation determined by the secretion of IgA antibodies in the HIO lumen. This in vivo HIO system with human immune cells provides a framework for future studies on infection- or allergen-driven intestinal diseases. View Publication