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Expression of miR-143 and miR-145 is reduced in hematopoietic stem/progenitor cells (HSPCs) of myelodysplastic syndrome patients with a deletion in the long arm of chromosome 5. Here we show that mice lacking miR-143/145 have impaired HSPC activity with depletion of functional hematopoietic stem cells (HSCs),but activation of progenitor cells (HPCs). We identify components of the transforming growth factor beta$ (TGFbeta$) pathway as key targets of miR-143/145. Enforced expression of the TGFbeta$ adaptor protein and miR-145 target,Disabled-2 (DAB2),recapitulates the HSC defect seen in miR-143/145-/- mice. Despite reduced HSC activity,older miR-143/145-/- and DAB2-expressing mice show elevated leukocyte counts associated with increased HPC activity. A subset of mice develop a serially transplantable myeloid malignancy,associated with expansion of HPC. Thus,miR-143/145 play a cell context-dependent role in HSPC function through regulation of TGFbeta$/DAB2 activation,and loss of these miRNAs creates a preleukemic state. View Publication

Targeted cancer immunotherapy with irradiated,granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting,allogeneic cancer cell lines has been an effective approach to reduce tumor burden in several patients. It is generally assumed that to be effective,these cell lines need to express immunogenic antigens coexpressed in patient tumor cells,and antigen-presenting cells need to take up such antigens then present them to patient T cells. We have previously reported that,in a phase I pilot study (ClinicalTrials.gov NCT00095862),a subject with stage IV breast cancer experienced substantial regression of breast,lung,and brain lesions following inoculation with clinical formulations of SV-BR-1-GM,a GM-CSF-secreting breast tumor cell line. To identify diagnostic features permitting the prospective identification of patients likely to benefit from SV-BR-1-GM,we conducted a molecular analysis of the SV-BR-1-GM cell line and of patient-derived blood,as well as a tumor specimen. Compared to normal human breast cells,SV-BR-1-GM cells overexpress genes encoding tumor-associated antigens (TAAs) such as PRAME,a cancer/testis antigen. Curiously,despite its presumptive breast epithelial origin,the cell line expresses major histocompatibility complex (MHC) class II genes (HLA-DRA,HLA-DRB3,HLA-DMA,HLA-DMB),in addition to several other factors known to play immunostimulatory roles. These factors include MHC class I components (B2M,HLA-A,HLA-B),ADA (encoding adenosine deaminase),ADGRE5 (CD97),CD58 (LFA3),CD74 (encoding invariant chain and CLIP),CD83,CXCL8 (IL8),CXCL16,HLA-F,IL6,IL18,and KITLG. Moreover,both SV-BR-1-GM cells and the responding study subject carried an HLA-DRB3*02:02 allele,raising the question of whether SV-BR-1-GM cells can directly present endogenous antigens to T cells,thereby inducing a tumor-directed immune response. In support of this,SV-BR-1-GM cells (which also carry the HLA-DRB3*01:01 allele) treated with yellow fever virus (YFV) envelope (Env) 43-59 peptides reactivated YFV-DRB3*01:01-specific CD4+ T cells. Thus,the partial HLA allele match between SV-BR-1-GM and the clinical responder might have enabled patient T lymphocytes to directly recognize SV-BR-1-GM TAAs as presented on SV-BR-1-GM MHCs. Taken together,our findings are consistent with a potentially unique mechanism of action by which SV-BR-1-GM cells can act as APCs for previously primed CD4+ T cells. View Publication

Understanding human embryonic ventral midbrain is of major interest for Parkinson's disease. However,the cell types,their gene expression dynamics,and their relationship to commonly used rodent models remain to be defined. We performed single-cell RNA sequencing to examine ventral midbrain development in human and mouse. We found 25 molecularly defined human cell types,including five subtypes of radial glia-like cells and four progenitors. In the mouse,two mature fetal dopaminergic neuron subtypes diversified into five adult classes during postnatal development. Cell types and gene expression were generally conserved across species,but with clear differences in cell proliferation,developmental timing,and dopaminergic neuron development. Additionally,we developed a method to quantitatively assess the fidelity of dopaminergic neurons derived from human pluripotent stem cells,at a single-cell level. Thus,our study provides insight into the molecular programs controlling human midbrain development and provides a foundation for the development of cell replacement therapies. View Publication

We report that SHIP(-/-) mice,compared to SHIP(+/+) mice,are Th2 skewed with elevated serum IgE and twice as many splenic CD4(+) Th2 cells that,when stimulated with anti-CD3,produce more IL-4 and less IFN-$\gamma$. Exploring the reason for this Th2 skewing,we found that freshly isolated SHIP(-/-) splenic and bone marrow basophils are present in elevated numbers and secrete far more IL-4 in response to IL-3 or to Fc$\epsilon$RI stimulation than do WT basophils. These SHIP(-/-) basophils markedly skew wild-type macrophage colony stimulating factor-derived macrophages toward an M2 phenotype,stimulate OT-II CD4(+) Th cells to differentiate into Th2 cells,and trigger SHIP(+/+) B cells to become IgE-producing cells. All these effects are completely abrogated with neutralizing anti-IL-4 Ab. Exploring the cell signaling pathways responsible for hyperproduction of IL-4 by SHIP(-/-) basophils,we found that IL-3-induced activation of the PI3K pathway is significantly enhanced and that PI3K inhibitors,especially a p110$\alpha$ inhibitor,dramatically suppresses IL-4 production from these cells. In vivo studies,in which basophils were depleted from mast cell-deficient SHIP(+/+) and SHIP(-/-) mice,confirmed the central role that basophils play in the Th2 skewing of naive SHIP-deficient mice. Taken together,these studies demonstrate that SHIP is a potent negative regulator of IL-4 production from basophils and thus may be a novel therapeutic target for Th1- and Th2-related diseases. View Publication

Long-lived multipotent stem cells (ISCs) at the base of intestinal crypts adjust their phenotypes to accommodate normal maintenance and post-injury regeneration of the epithelium. Their long life,lineage plasticity,and proliferative potential underlie the necessity for tight homeostatic regulation of the ISC compartment. In that context,the guanylate cyclase C (GUCY2C) receptor and its paracrine ligands regulate intestinal epithelial homeostasis,including proliferation,lineage commitment,and DNA damage repair. However,a role for this axis in maintaining ISCs remains unknown. Transgenic mice enabling analysis of ISCs (Lgr5-GFP) in the context of GUCY2C elimination (Gucy2c -/- ) were combined with immunodetection techniques and pharmacological treatments to define the role of the GUCY2C signaling axis in supporting ISCs. ISCs were reduced inGucy2c -/- mice,associated with loss of active Lgr5+cells but a reciprocal increase in reserve Bmi1+cells. GUCY2C was expressed in crypt base Lgr5+cells in which it mediates canonical cyclic (c) GMP-dependent signaling. Endoplasmic reticulum (ER) stress,typically absent from ISCs,was elevated throughout the crypt base inGucy2c -/- mice. The chemical chaperone tauroursodeoxycholic acid resolved this ER stress and restored the balance of ISCs,an effect mimicked by the GUCY2C effector 8Br-cGMP. Reduced ISCs inGucy2c -/- mice was associated with greater epithelial injury and impaired regeneration following sub-lethal doses of irradiation. These observations suggest that GUCY2C provides homeostatic signals that modulate ER stress and cell vulnerability as part of the machinery contributing to the integrity of ISCs. View Publication

Traumatic brain injury (TBI) is soon predicted to become the third leading cause of death and disability worldwide. After the primary injury,a complex set of secondary injuries develops hours and days later with prolonged neuroinflammation playing a key role. TBI and other inflammatory conditions are currently being treated in preclinical and clinical trials by a number of cellular therapies. Mesenchymal stem cells (MSC) are of great interest due to their widespread usage,safety,and relative ease to isolate and culture. However,there has been a wide range in efficacy reported using MSC clinically and in preclinical models,likely due to differences in cell preparations and a significant amount of donor variability. In this study,we seek to find a correlation between in vitro activity and in vivo efficacy. We designed assays to explore the responsiveness of MSC to immunological cues to address the immunomodulatory properties of MSC,one of their primary modes of therapeutic activity in TBI. Our results showed intrinsic differences in the immunomodulatory capacity of MSC preparations from different bone marrow and amniotic fluid donors. This difference mirrored the therapeutic capacity of the MSC in an experimental model of TBI,an effect confirmed using siRNA knockdown of COX2 followed by overexpressing COX2. Among the immunomodulatory factors assessed,the therapeutic benefit correlated with the secretion of prostaglandin E2 (PGE2 ) by MSC prior to treatment,suggesting that measurement of PGE2 could be a very useful potency marker to create an index of predicted efficacy for preparations of MSC to treat TBI. Stem Cells 2017;35:1416-1430. View Publication

Mitochondria are a major target for aging and are instrumental in the age-dependent deterioration of the human brain,but studying mitochondria in aging human neurons has been challenging. Direct fibroblast-to-induced neuron (iN) conversion yields functional neurons that retain important signs of aging,in contrast to iPSC differentiation. Here,we analyzed mitochondrial features in iNs from individuals of different ages. iNs from old donors display decreased oxidative phosphorylation (OXPHOS)-related gene expression,impaired axonal mitochondrial morphologies,lower mitochondrial membrane potentials,reduced energy production,and increased oxidized proteins levels. In contrast,the fibroblasts from which iNs were generated show only mild age-dependent changes,consistent with a metabolic shift from glycolysis-dependent fibroblasts to OXPHOS-dependent iNs. Indeed,OXPHOS-induced old fibroblasts show increased mitochondrial aging features similar to iNs. Our data indicate that iNs are a valuable tool for studying mitochondrial aging and support a bioenergetic explanation for the high susceptibility of the brain to aging. View Publication

Enteric pathogens including Salmonella enteric serovar Typhimurium can breach the epithelial barrier of the host and spread to systemic tissues. In response to infection,the host activates innate immune receptors via the signaling molecule MyD88,which induces protective inflammatory and antimicrobial responses. Most of these innate immune responses have been studied in hematopoietic cells,but the role of MyD88 signaling in other cell types remains poorly understood. Surprisingly,we found that Dermo1-Cre;Myd88fl/fl mice with mesenchymal cell-specific deficiency of MyD88 were less susceptible to orogastric and i.p. STyphimurium infection than their Myd88fl/fl littermates. The reduced susceptibility of Dermo1-Cre;Myd88fl/fl mice to infection was associated with lower loads of S. Typhimurium in the liver and spleen. Mutant analyses revealed that S. Typhimurium employs its virulence type III secretion system 2 to promote its growth through MyD88 signaling pathways in mesenchymal cells. Inflammatory monocytes function as a major cell population for systemic dissemination of S. Typhimurium Mechanistically,mesenchymal cell-specific MyD88 signaling promoted CCL2 production in the liver and spleen and recruitment of inflammatory monocytes to systemic organs in response to STyphimurium infection. Consistently,MyD88 signaling in mesenchymal cells enhanced the number of phagocytes including Ly6ChiLy6G- inflammatory monocytes harboring STyphimurium in the liver. These results suggest that S. Typhimurium promotes its systemic growth and dissemination through MyD88 signaling pathways in mesenchymal cells. View Publication

We hypothesized that the neonatal rat heart would bring transplanted human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to maturity as it grows to adult size. In neonatal rat heart,engrafted hiPSC derivatives developed partially matured myofibrils after 3 months,with increasing cell size and sarcomere length. There was no difference between grafts from hiPSC-CMs or hiPSC-derived cardiac progenitors (hiPSC-CPs) at 3 months,nor was maturation influenced by infarction. Interestingly,the infarcted adult heart induced greater human cardiomyocyte hypertrophy and induction of cardiac troponin I expression than the neonatal heart. Although human cardiomyocytes at all time points were significantly smaller than the host rat cardiomyocytes,transplanted neonatal rat cardiomyocytes reached adult size and structure by 3 months. Thus,the adult rat heart induces faster maturation than the neonatal heart,and human cardiomyocytes mature more slowly than rat cardiomyocytes. The slower maturation of human cardiomyocytes could be related to environmental mismatch or cell-autonomous factors. View Publication

The airways are lined by secretory and multiciliated cells which function together to remove particles and debris from the respiratory tract. The transcriptome of multiciliated cells has been extensively studied,but the function of many of the genes identified is unknown. We have established an assay to test the ability of over-expressed transcripts to promote multiciliated cell differentiation in mouse embryonic tracheal explants. Overexpression data indicated that Fibronectin type 3 and ankyrin repeat domains 1 (Fank1) and JAZF zinc finger 1 (Jazf1) promoted multiciliated cell differentiation alone,and cooperatively with the canonical multiciliated cell transcription factor Foxj1. Moreover,knock-down of Fank1 or Jazf1 in adult mouse airway epithelial cultures demonstrated that these factors are both required for ciliated cell differentiation in vitro This analysis identifies Fank1 and Jazf1 as novel regulators of multiciliated cell differentiation. Moreover,we show that they are likely to function downstream of IL6 signalling and upstream of Foxj1 activity in the process of ciliated cell differentiation. In addition,our in vitro explant assay provides a convenient method for preliminary investigation of over-expression phenotypes in the developing mouse airways.This article has an associated First Person interview with the first author of the paper. View Publication