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Hematopoietic stem cell (HSC) quiescence is a tightly regulated process crucial for hematopoietic regeneration,which requires a healthy and supportive microenvironmental niche within the bone marrow (BM). Here,we show that deletion of Ptpn21,a protein tyrosine phosphatase highly expressed in HSCs,induces stem cell egress from the niche due to impaired retention within the BM. Ptpn21-/- HSCs exhibit enhanced mobility,decreased quiescence,increased apoptosis,and defective reconstitution capacity. Ptpn21 deletion also decreased HSC stiffness and increased physical deformability,in part by dephosphorylating Spetin1 (Tyr246),a poorly described component of the cytoskeleton. Elevated phosphorylation of Spetin1 in Ptpn21-/- cells impaired cytoskeletal remodeling,contributed to cortical instability,and decreased cell rigidity. Collectively,these findings show that Ptpn21 maintains cellular mechanics,which is correlated with its important functions in HSC niche retention and preservation of hematopoietic regeneration capacity. View Publication

CD4 T cells from HIV-1 infected patients die at excessive rates compared to those from uninfected patients,causing immunodeficiency. We previously identified a dominant negative ligand that antagonizes the TRAIL-dependent pathway of cell death,which we called TRAILshort. Because the TRAIL pathway has been implicated in CD4 T cell death occurring during HIV-1 infection,we used short hairpin RNA knockdown,CRISPR deletion,or Abs specific for TRAILshort to determine the effect of inhibiting TRAILshort on the outcome of experimental acute HIV infection in vitro. Strikingly,all three approaches to TRAILshort deletion/inhibition enhanced HIV-induced death of both infected and uninfected human CD4 T cells. Thus,TRAILshort impacts T cell dynamics during HIV infection,and inhibiting TRAILshort causes more HIV-infected and uninfected bystander cells to die. TRAILshort is,therefore,a host-derived,host-adaptive mechanism to limit the effects of TRAIL-induced cell death. Further studies on the effects of TRAILshort in other disease states are warranted. View Publication

Immune activation may underlie the pathogenesis of irritable bowel syndrome (IBS),but the evidence is conflicting. We examined whether peripheral CD4+ T-cells from IBS patients demonstrated immune activation and changes in cytokine production. To gain mechanistic insight,we examined whether immune activation correlated with psychological stress and changing symptoms over time. IBS patients (n = 29) and healthy volunteers (HV; n = 29) completed symptom and psychological questionnaires. IBS patients had a significant increase in CD4+ T-cells expressing the gut homing marker integrin beta7 (p = 0.023) and lymphoid marker CD62L (p = 0.026) compared to HV. Furthermore,phytohaemagglutinin stimulated CD4+ T-cells from IBS-D patients demonstrated increased TNFalpha secretion when compared to HV (p = 0.044). Increased psychological scores in IBS did not correlate with TNFalpha production,while stress hormones inhibited cytokine secretion from CD4+ T-cells of HV in vitro. IBS symptoms,but not markers of immune activation,decreased over time. CD4+ T-cells from IBS-D patients exhibit immune activation,but this did not appear to correlate with psychological stress measurements or changing symptoms over time. This could suggest that immune activation is a surrogate of an initial trigger and/or ongoing parallel peripheral mechanisms. View Publication

Various preclinical models have been developed to clarify the pathophysiology of prostate cancer (PCa). Traditional PCa cell lines from clinical metastatic lesions,as exemplified by DU-145,PC-3,and LNCaP cells,are useful tools to define mechanisms underlying tumorigenesis and drug resistance. Cell line-based experiments,however,have limitations for preclinical studies because those cells are basically adapted to 2-dimensional monolayer culture conditions,in which the majority of primary PCa cells cannot survive. Recent tissue engineering enables generation of PCa patient-derived xenografts (PDXs) from both primary and metastatic lesions. Compared with fresh PCa tissue transplantation in athymic mice,co-injection of PCa tissues with extracellular matrix in highly immunodeficient mice has remarkably improved the success rate of PDX generation. PDX models have advantages to appropriately recapitulate the molecular diversity,cellular heterogeneity,and histology of original patient tumors. In contrast to PDX models,patient-derived organoid and spheroid PCa models in 3-dimensional culture are more feasible tools for in vitro studies for retaining the characteristics of patient tumors. In this article,we review PCa preclinical model cell lines and their sublines,PDXs,and patient-derived organoid and spheroid models. These PCa models will be applied to the development of new strategies for cancer precision medicine. View Publication

A large body of evidence suggests that B-cell lymphomas with enhanced Myc expression are associated with an aggressive phenotype and poor prognosis,which makes Myc a compelling therapeutic target. Phosphodiesterase 4B (PDE4B),a main hydrolyzer of cyclic AMP (cAMP) in B cells,was shown to be involved in cell survival and drug resistance in diffuse large B cell lymphomas (DLBCL). However,the interrelationship between Myc and PDE4B remains unclear. Here,we first demonstrate the presence of the Myc-PDE4B feed-forward loop,in which Myc and PDE4B mutually reinforce the expression of each other. Next,the combined targeting of Myc and PDE4 synergistically prevented the proliferation and survival of B lymphoma cells in vitro and in a mouse xenograft model. We finally recapitulated this combinatorial effect in Emu-myc transgenic mice; co-inhibition of Myc and PDE4 suppressed lymphomagenesis and restored B cell development to the wild type level that was associated with marked reduction in Myc levels,unveiling the critical role of the Myc-PDE4B amplification loop in the regulation of Myc expression and the pathogenesis of B cell lymphoma. These findings suggest that the disruption of the Myc-PDE4B circuitry can be exploited in the treatment of B cell malignancies. View Publication

Serrated adenocarcinoma,an alternative pathway for colorectal cancer (CRC) development,accounts for 15{\%}-30{\%} of all CRCs and is aggressive and treatment resistant. We show that the expression of atypical protein kinase C zeta (PKCzeta) and PKClambda/iota was reduced in human serrated tumors. Simultaneous inactivation of the encoding genes in the mouse intestinal epithelium resulted in spontaneous serrated tumorigenesis that progressed to advanced cancer with a strongly reactive and immunosuppressive stroma. Whereas epithelial PKClambda/iota deficiency led to immunogenic cell death and the infiltration of CD8+ T cells,which repressed tumor initiation,PKCzeta loss impaired interferon and CD8+ T cell responses,which resulted in tumorigenesis. Combined treatment with a TGF-beta receptor inhibitor plus anti-PD-L1 checkpoint blockade showed synergistic curative activity. Analysis of human samples supported the relevance of these kinases in the immunosurveillance defects of human serrated CRC. These findings provide insight into avenues for the detection and treatment of this poor-prognosis subtype of CRC. View Publication

This study aimed to determine the association between non-high-fat diet-induced obesity- (non-DIO-) associated gut microbiome dysbiosis with gut abnormalities like cellular turnover of intestinal cells,tight junctions,and mucin formation that can impact gut permeability. We used leptin-deficient (Lepob/ob) mice in comparison to C57BL/6J control mice,which are fed on identical diets,and performed comparative and correlative analyses of gut microbiome composition,gut permeability,intestinal structural changes,tight junction-mucin formation,cellular turnover,and stemness genes. We found that obesity impacted cellular turnover of the intestine with increased cell death and cell survival/proliferation gene expression with enhanced stemness,which are associated with increased intestinal permeability,changes in villi/crypt length,and decreased expression of tight junctions and mucus synthesis genes along with dysbiotic gut microbiome signature. Obesity-induced gut microbiome dysbiosis is also associated with abnormal intestinal organoid formation characterized with decreased budding and higher stemness. Results suggest that non-DIO-associated gut microbiome dysbiosis is associated with changes in the intestinal cell death versus cell proliferation homeostasis and functions to control tight junctions and mucous synthesis-regulating gut permeability. View Publication

BACKGROUND Human microRNAs (miRNAs) have diverse functions in biology,and play a role in nearly every biological process. Here we report that miR-520d-5p (520d-5p) causes undifferentiated cancer cells to adopt benign or normal status in vivo in immunodeficient mice via demethylation and P53 upregulation. Further we found that 520-5p causes normal cells to elongate cellular lifetime and mesenchymal stem cell-like status with CD105 positivity. We hypothesized that ectopic 520d-5p expression reduced mutations in undifferentiated type of hepatoma (HLF) cells through synergistic modulation of methylation-related enzymatic expression. METHODS To examine whether there were any changes in mutation status in cells treated with 520d-5p,we performed next generation sequencing (NGS) in HLF cells and human iPSC-derivative cells in pre-mesenchymal stem cell status. We analyzed the data using both genome-wide and individual gene function approaches. RESULTS 520d-5p induced a shift towards a wild type or non-malignant phenotype,which was regulated by nucleotide mutations in both HLF cells and iPSCs. Further,520d-5p reduced mutation levels in both the whole genome and genomic fragment assemblies. CONCLUSIONS Cancer cell genomic mutations cannot be repaired in most contexts. However,these findings suggest that applied development of 520d-5p would allow new approaches to cancer research and improve the quality of iPSCs used in regenerative medicine. View Publication

Mesenchymal stromal cells (MSCs) are multipotent stem cells that participate in tissue repair and posses considerable immunomodulatory potential. MSCs have been shown to promote allograft survival,yet the mechanisms behind this phenomenon have not been fully defined. Here,we investigate the capacity of MSCs to suppress the allogeneic immune response by secreting the pleiotropic molecule hepatocyte growth factor (HGF). Using an in vivo mouse model of corneal transplantation,we report that MSCs promote graft survival in an HGF-dependent manner. Moreover,our data indicate that topically administered recombinant HGF (1) suppresses antigen-presenting cell maturation in draining lymphoid tissue,(2) limits T-helper type-1 cell generation,(3) decreases inflammatory cell infiltration into grafted tissue,and (4) is itself sufficient to promote transplant survival. These findings have potential translational implications for the development of HGF-based therapeutics. Stem Cells Translational Medicine 2019. View Publication

Antagonists of the 5-hydroxytryptamine (serotonin) 3 receptor (5-HT3R) have anti-inflammatory and anti-apoptotic activities,but the detailed,underlying mechanisms are not well understood. We focused on anti-apoptotic activities via 5-HT3R signaling to clarify the underlying mechanisms. Mice were administered 5-fluorouracil (5-FU),which induced apoptosis in intestinal epithelial cells. Coadministration with 5-HT3R antagonists or agonists tended to decrease or increase the number of apoptotic cells,respectively. In serotonin 3A receptor (5-HT3AR) null (HTR3A-/-) mice,the number of apoptotic cells induced by 5-FU was decreased compared with that in wild-type (WT) mice. Bone marrow (BM) transplantation was performed to determine if BM-derived immune cells regulated 5-FU-induced apoptosis,but they were found to be unrelated to this process. Data from 5-HT3AR/enhanced green fluorescent protein reporter mice revealed that 50{\%} of enterochromaffin (EC) cells expressed 5-HT3AR,but the number of apoptotic cells induced by 5-FU in the intestinal crypt organoids of HTR3A-/- mice was not altered compared with WT mice. In contrast,plasma 5-HT concentrations in WT mice but not in HTR3A-/- mice administered 5-FU were increased significantly. In conclusion,5-HT3R signaling may enhance 5-HT release,possibly from EC cells intravascularly,or paracrine,resulting in increases in plasma 5-HT concentration,which in turn,enhances apoptotic activities induced by 5-FU.-Mikawa,S.,Kondo,M.,Kaji,N.,Mihara,T.,Yoshitake,R.,Nakagawa,T.,Takamoto,M.,Nishimura,R.,Shimada,S.,Ozaki,H.,Hori,M. Serotonin 3 receptor signaling regulates 5-fluorouracil-mediated apoptosis indirectly via TNF-alpha production by enhancing serotonin release from enterochromaffin cells. View Publication

Metabolic health depends on the capacity of adipose tissue progenitor cells to undergo de novo adipogenesis. The cellular hierarchy and mechanisms governing adipocyte progenitor differentiation are incompletely understood. Through single-cell RNA sequence analyses,we show that the lineage hierarchy of adipocyte progenitors consists of distinct mesenchymal cell types that are present in both mouse and human adipose tissues. Cells marked by dipeptidyl peptidase-4 (DPP4)/CD26 expression are highly proliferative,multipotent progenitors. During the development of subcutaneous adipose tissue in mice,these progenitor cells give rise to intercellular adhesion molecule-1 (ICAM1)/CD54-expressing (CD54+) committed preadipocytes and a related adipogenic cell population marked by Clec11a and F3/CD142 expression. Transforming growth factor-beta maintains DPP4+ cell identity and inhibits adipogenic commitment of DPP4+ and CD142+ cells. Notably,DPP4+ progenitors reside in the reticular interstitium,a recently appreciated fluid-filled space within and between tissues,including adipose depots. View Publication

The complex process of platelet formation originates with the hematopoietic stem cell,which differentiates through the myeloid lineage,matures,and releases proplatelets into the BM sinusoids. How formed platelets maintain a low basal activation state in the circulation remains unknown. We identify Lepr+ stromal cells lining the BM sinusoids as important contributors to sustaining low platelet activation. Ablation of murine Lepr+ cells led to a decreased number of platelets in the circulation with an increased activation state. We developed a potentially novel culture system for supporting platelet formation in vitro using a unique population of CD51+PDGFRalpha+ perivascular cells,derived from human umbilical cord tissue,which display numerous mesenchymal stem cell (MSC) properties. Megakaryocytes cocultured with MSCs had altered LAT and Rap1b gene expression,yielding platelets that are functional with low basal activation levels,a critical consideration for developing a transfusion product. Identification of a regulatory cell that maintains low baseline platelet activation during thrombopoiesis opens up new avenues for improving blood product production ex vivo. View Publication