参考文献

Functional evaluation assays using human induced pluripotent stem cell (hiPSC)-derived neurons can predict the convulsion toxicity of new drugs and the neurological effects of antiepileptic drugs. However,differences in responsiveness depending on convulsant type and antiepileptic drugs,and an evaluation index capable of comparing in vitro responses with in vivo responses are not well known. We observed the difference in synchronized burst patterns in the epileptiform activities induced by pentylentetrazole (PTZ) and 4-aminopryridine (4-AP) with different action mechanisms using multi-electrode arrays (MEAs); we also observed that 100 µM of the antiepileptic drug phenytoin suppressed epileptiform activities induced by PTZ,but increased those induced by 4-AP. To compare in vitro results with in vivo convulsive responses,frequency analysis of below 250 Hz,excluding the spike component,was performed. The in vivo convulsive firing enhancement of the high gamma$ wave and beta$ wave component were observed remarkably in in vitro hiPSC-derived neurons with astrocytes in co-culture. MEA measurement of hiPSC-derived neurons in co-culture with astrocytes and our analysis methods,including frequency analysis,appear effective for predicting convulsion toxicity,side effects,and their mechanism of action as well as the comparison of convulsions induced in vivo. View Publication

Chronic myelomonocytic leukemia (CMML) is a clinically heterogeneous neoplasm in which JAK2 inhibition has demonstrated reductions in inflammatory cytokines and promising clinical activity. We hypothesize that annotation of inflammatory cytokines may uncover mutation-independent cytokine subsets associated with novel CMML prognostic features. A Luminex cytokine profiling assay was utilized to profile cryopreserved peripheral blood plasma from 215 CMML cases from three academic centers,along with center-specific,age-matched plasma controls. Significant differences were observed between CMML patients and healthy controls in 23 out of 45 cytokines including increased cytokine levels in IL-8,IP-10,IL-1RA,TNF-alpha$,IL-6,MCP-1/CCL2,hepatocyte growth factor (HGF),M-CSF,VEGF,IL-4,and IL-2RA. Cytokine associations were identified with clinical and genetic features,and Euclidian cluster analysis identified three distinct cluster groups associated with important clinical and genetic features in CMML. CMML patients with decreased IL-10 expression had a poor overall survival when compared to CMML patients with elevated expression of IL-10 (P = 0.017),even when adjusted for ASXL1 mutation and other prognostic features. Incorporating IL-10 with the Mayo Molecular Model statistically improved the prognostic ability of the model. These established cytokines,such as IL-10,as prognostically relevant and represent the first comprehensive study exploring the clinical implications of the CMML inflammatory state. View Publication

Regeneration of the adult intestinal epithelium is mediated by a pool of cycling stem cells,which are located at the base of the crypt,that express leucine-rich-repeat-containing G-protein-coupled receptor 5 (LGR5). The Frizzled (FZD) 7 receptor (FZD7) is enriched in LGR5+ intestinal stem cells and plays a critical role in their self-renewal. Yet,drug discovery approaches and structural bases for targeting specific FZD isoforms remain poorly defined. FZD proteins interact with Wnt signaling proteins via,in part,a lipid-binding groove on the extracellular cysteine-rich domain (CRD) of the FZD receptor. Here we report the identification of a potent peptide that selectively binds to the FZD7 CRD at a previously uncharacterized site and alters the conformation of the CRD and the architecture of its lipid-binding groove. Treatment with the FZD7-binding peptide impaired Wnt signaling in cultured cells and stem cell function in intestinal organoids. Together,our data illustrate that targeting the lipid-binding groove holds promise as an approach for achieving isoform-selective FZD receptor inhibition. View Publication

Abnormal signaling pathways mediated by N-methyl-d-aspartate receptors (NMDARs) have been implicated in the pathogenesis of various CNS disorders and have been long considered as promising points of therapeutic intervention. However,few efforts have been previously described concerning evaluation of therapeutic modulators of NMDARs and their downstream pathways in human neurons with endogenous expression of NMDARs. In the present study,we assessed expression,functionality,and subunit composition of endogenous NMDARs in human induced pluripotent stem cell (hiPSC)-derived cortical neurons (iCell Neurons and iCell GlutaNeurons). We initially confirmed the expected pharmacological response of iCell Neurons and iCell GlutaNeurons to NMDA by patch-clamp recordings. Subsequent pharmacological interrogation using GluN2 subunit-selective antagonists revealed the predominance of GluN2B in both iCell Neurons and iCell GlutaNeurons. This observation was also supported by qRT-PCR and Western blot analyses of GluN2 subunit expression as well as pharmacological experiments using positive allosteric modulators with distinct GluN2 subunit selectivity. We conclude that iCell Neurons and iCell GlutaNeurons express functional GluN2B-containing NMDARs and could serve as a valuable system for development and validation of GluN2B-modulating pharmaceutical agents. View Publication

BACKGROUND Type 2 diabetes (T2D) is a recognized risk factor for the development of cognitive impairment (CI) and/or dementia,although the exact nature of the molecular pathology of T2D-associated CI remains obscure. One link between T2D and CI might involve decreased insulin signaling in brain and/or neurons in either animal or postmortem human brains as has been reported as a feature of Alzheimer's disease (AD). Here we asked if neuronal insulin resistance is a cell autonomous phenomenon in a familial form of AD. METHODS We have applied a newly developed protocol for deriving human basal forebrain cholinergic neurons (BFCN) from skin fibroblasts via induced pluripotent stem cell (iPSC) technology. We generated wildtype and familial AD mutant PSEN2 N141I (presenilin 2) BFCNs and assessed if insulin signaling,insulin regulation of the major AD proteins Abeta$ and/or tau,and/or calcium fluxes is altered by the PSEN2 N141I mutation. RESULTS We report herein that wildtype,PSEN2 N141I and CRISPR/Cas9-corrected iPSC-derived BFCNs (and their precursors) show indistinguishable insulin signaling profiles as determined by the phosphorylation of canonical insulin signaling pathway molecules. Chronic insulin treatment of BFCNs of all genotypes led to a reduction in the Abeta$42/40 ratio. Unexpectedly,we found a CRISPR/Cas9-correctable effect of PSEN2 N141I on calcium flux,which could be prevented by chronic exposure of BFCNs to insulin. CONCLUSIONS Our studies indicate that the familial AD mutation PSEN2 N141I does not induce neuronal insulin resistance in a cell autonomous fashion. The ability of insulin to correct calcium fluxes and to lower Abeta$42/40 ratio suggests that insulin acts to oppose an AD-pathophysiology. Hence,our results are consistent with a potential physiological role for insulin as a mediator of resilience by counteracting specific metabolic and molecular features of AD. View Publication

Objectives gamma$delta$ T cells,a non-conventional innate lymphocyte subset containing cells that can be activated by lipids and phosphoantigens,are abnormally regulated in systemic sclerosis (SSc). To further evaluate the significance of this dysregulation,we compared how exposure to an autoantigenic lipid,cardiolipin (CL),during co-stimulation with an amino-bisphosphonate (zoledronate,zol),affects the activation and cytokine production of SSc and healthy control (HC) gamma$delta$ T cells. Methods Expression of CD25 on Vgamma$9+,Vdelta$1+,and total CD3+ T cells in cultured peripheral blood mononuclear cells (PBMCs),their binding of CD1d tetramers,and the effect of monoclonal antibody (mAb) blockade of CD1d were monitored by flow cytometry after 4 days of in vitro culture. Intracellular production of IFNgamma$ and IL-4 was assessed after overnight culture. Results Percentages of CD25+ among CD3+ and Vdelta$1+ T cells were elevated significantly in short-term cultured SSc PBMC compared to HC. In SSc but not HC,CL and zol,respectively,suppressed {\%}CD25+ Vgamma$9+ and Vdelta$1+ T cells but,when combined,CL + zol significantly activated both subsets in HC and partially reversed inhibition by the individual reagents in SSc. Importantly,Vdelta$1+ T cells in both SSc and HC were highly reactive with lipid presenting CD1d tetramers,and a CD1d-blocking mAb decreased CL-induced enhancement of {\%}SSc CD25+ Vdelta$1+ T cells in the presence of zol. {\%}IFNgamma$+ cells among Vgamma$9+ T cells of SSc was lower than HC cultured in medium,CL,zol,or CL + zol,whereas {\%}IFNgamma$+ Vdelta$1+ T cells was lower only in the presence of CL or CL + zol. {\%}IL-4+ T cells were similar in SSc and HC in all conditions,with the exception of being increased in SSc Vgamma$9+ T cells in the presence of CL. Conclusion Abnormal functional responses of gamma$delta$ T cell subsets to stimulation by CL and phosphoantigens in SSc may contribute to fibrosis and immunosuppression,characteristics of this disease. View Publication

Nonsense mutations are present in 10{\%} of patients with CF,produce a premature termination codon in CFTR mRNA causing early termination of translation,and lead to lack of CFTR function. There are no currently available animal models which contain a nonsense mutation in the endogenous Cftr locus that can be utilized to test nonsense mutation therapies. In this study,we create a CF mouse model carrying the G542X nonsense mutation in Cftr using CRISPR/Cas9 gene editing. The G542X mouse model has reduced Cftr mRNA levels,demonstrates absence of CFTR function,and displays characteristic manifestations of CF mice such as reduced growth and intestinal obstruction. Importantly,CFTR restoration is observed in G542X intestinal organoids treated with G418,an aminoglycoside with translational readthrough capabilities. The G542X mouse model provides an invaluable resource for the identification of potential therapies of CF nonsense mutations as well as the assessment of in vivo effectiveness of these potential therapies targeting nonsense mutations. View Publication

$\beta$-hemoglobinopathies such as sickle cell disease (SCD) and $\beta$-thalassemia result from mutations in the adult HBB ($\beta$-globin) gene. Reactivating the developmentally silenced fetal HBG1 and HBG2 ($\gamma$-globin) genes is a therapeutic goal for treating SCD and $\beta$-thalassemia 1 . Some forms of hereditary persistence of fetal hemoglobin (HPFH),a rare benign condition in which individuals express the $\gamma$-globin gene throughout adulthood,are caused by point mutations in the $\gamma$-globin gene promoter at regions residing {\~{}}115 and 200 bp upstream of the transcription start site. We found that the major fetal globin gene repressors BCL11A and ZBTB7A (also known as LRF) directly bound to the sites at -115 and -200 bp,respectively. Furthermore,introduction of naturally occurring HPFH-associated mutations into erythroid cells by CRISPR-Cas9 disrupted repressor binding and raised $\gamma$-globin gene expression. These findings clarify how these HPFH-associated mutations operate and demonstrate that BCL11A and ZBTB7A are major direct repressors of the fetal globin gene. View Publication

Hematotoxicity is a significant issue for drug safety and can result from direct cytotoxicity toward circulating mature blood cell types as well as targeting of immature blood-forming stem cells/progenitor cells in the bone marrow. In vitro models for understanding and investigating the hematotoxicity potential of new test items/drugs are critical in early preclinical drug development. The traditional method,colony forming unit (CFU) assay,is commonly used and has been validated as a method for hematotoxicity screening. The CFU assay has multiple limitations for its application in investigative work. In this paper,we describe a detailed protocol for a liquid-culture,microplate-based in vitro hematotoxicity assay to evaluate lineage-specific (myeloid,erythroid,and megakaryocytic) hematotoxicity at different stages of differentiation. This assay has multiple advantages over the traditional CFU assay,including being suitable for high-throughput screening and flexible enough to allow inclusion of additional endpoints for mechanistic studies. Therefore,it is an extremely useful tool for scientists in pharmaceutical discovery and development. {\textcopyright} 2018 by John Wiley & Sons,Inc. View Publication

The mouse trachea is thought to contain two distinct stem cell compartments that contribute to airway repair-basal cells in the surface airway epithelium (SAE) and an unknown submucosal gland (SMG) cell type. Whether a lineage relationship exists between these two stem cell compartments remains unclear. Using lineage tracing of glandular myoepithelial cells (MECs),we demonstrate that MECs can give rise to seven cell types of the SAE and SMGs following severe airway injury. MECs progressively adopted a basal cell phenotype on the SAE and established lasting progenitors capable of further regeneration following reinjury. MECs activate Wnt-regulated transcription factors (Lef-1/TCF7) following injury and Lef-1 induction in cultured MECs promoted transition to a basal cell phenotype. Surprisingly,dose-dependent MEC conditional activation of Lef-1 in vivo promoted self-limited airway regeneration in the absence of injury. Thus,modulating the Lef-1 transcriptional program in MEC-derived progenitors may have regenerative medicine applications for lung diseases. View Publication