技术资料

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

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

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

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

Alzheimer's disease (AD) is emerging as a synaptopathology driven by metaplasticity. Indeed,reminiscent of metaplasticity,oligomeric forms of the amyloid-beta$ peptide (oAbeta$) prevent induction of long-term potentiation (LTP) via the prior activation of GluN2B-containing NMDA receptors (NMDARs). However,the downstream Ca2+-dependent signaling molecules that mediate aberrant metaplasticity are unknown. In this study,we show that oAbeta$ promotes the activation of Ca2+/calmodulin-dependent kinase II (CaMKII) via GluN2B-containing NMDARs. Importantly,we find that CaMKII inhibition rescues both the LTP impairment and the dendritic spine loss mediated by oAbeta$. Mechanistically resembling metaplasticity,oAbeta$ prevents subsequent rounds of plasticity from inducing CaMKII T286 autophosphorylation,as well as the associated anchoring and accumulation of synaptic AMPA receptors (AMPARs). Finally,prolonged oAbeta$ treatment-induced CaMKII misactivation leads to dendritic spine loss via the destabilization of surface AMPARs. Thus,our study demonstrates that oAbeta$ engages synaptic metaplasticity via aberrant CaMKII activation. View Publication

Advancing chimeric antigen receptor (CAR)-engineered adoptive T cells for the treatment of solid cancers is a major focus in the field of immunotherapy,given impressive recent clinical responses in hematological malignancies. Prostate cancer may be amenable to T cell-based immunotherapy since several tumor antigens,including prostate stem-cell antigen (PSCA),are widely over-expressed in metastatic disease. While antigen selectivity of CARs for solid cancers is crucial,it is problematic due to the absence of truly restricted tumor antigen expression and potential safety concerns with on-target off-tumor" activity. Here View Publication

Induced pluripotent stem cells (iPSCs) were generated from peripheral blood mononuclear cells (PBMCs) isolated from the whole blood of a 43-year-old male with hypertrophic cardiomyopathy (HCM) who carries the pathogenic variant p.Val698Ala in beta-myosin heavy chain (MYH7). Patient-derived PBMCs were reprogrammed using non-integrative episomal vectors containing reprogramming factors OCT4,SOX2,LIN28,KLF4 and L-MYC. iPSCs were shown to express pluripotent markers,have trilineage differentiation potential,carry the pathogenic MYH7 variant p.Val698Ala,have a normal karyotype and no longer carry the episomal reprogramming vector. This line is useful for studying the link between variants in MYH7 and the pathogenesis of HCM. View Publication

Lgr5-expressing intestinal stem cells (ISCs) maintain continuous and rapid generation of the intestinal epithelium. Here we present evidence that dedifferentiation of committed enteroendocrine cells (EECs) contributes to maintenance of the epithelium under both basal conditions and in response to injury. Lineage tracing studies identified a subset of EECs that reside at +4 position for more than 2 weeks,most of which were BrdU-label-retaining cells. Under basal conditions,cells derived from these EECs grow from the bottom of the crypt to generate intestinal epithelium according to neutral drift kinetics that is consistent with dedifferentiation of mature EECs to ISCs. The lineage tracing of EECs demonstrated reserve stem cell properties in response to radiation-induced injury with the generation of reparative EEC-derived epithelial patches. Finally,the enterochromaffin (EC) cell was the predominant EEC type participating in these stem cell dynamics. These results provide novel insights into the +4 reserve ISC hypothesis,stem cell dynamics of the intestinal epithelium and novel insight in the development of EC-derived small intestinal tumors. View Publication

Availability of tumor and non-tumor patient-derived models would promote the development of more effective therapeutics for non-small cell lung cancer (NSCLC). Recently,conditionally reprogrammed cells (CRC) methodology demonstrated exceptional potential for the expansion of epithelial cells from patient tissues. However,the possibility to expand patient-derived lung cancer cells using CRC protocols is controversial. Here,we used CRC approach to expand cells from non-tumoral and tumor biopsies of patients with primary or metastatic NSCLC as well as pulmonary metastases of colorectal or breast cancers. CRC cultures were obtained from both tumor and non-malignant tissues with extraordinary high efficiency. Tumor cells were tracked in vitro through tumorigenicity assay,monitoring of tumor-specific genetic alterations and marker expression. Cultures were composed of EpCAM+ lung epithelial cells lacking tumorigenic potential. NSCLC biopsies-derived cultures rapidly lost patient-specific genetic mutations or tumor antigens. Similarly,pulmonary metastases of colon or breast cancer generated CRC cultures of lung epithelial cells. All CRC cultures examined displayed epithelial lung stem cell phenotype and function. In contrast,brain metastatic lung cancer biopsies failed to generate CRC cultures. In conclusion,patient-derived primary and metastatic lung cancer cells were negatively selected under CRC conditions,limiting the expansion to non-malignant lung epithelial stem cells from either tumor or non-tumor tissue sources. Thus,CRC approach cannot be applied for direct therapeutic testing of patient lung tumor cells,as the tumor-derived CRC cultures are composed of (non-tumoral) airway basal cells. View Publication

BACKGROUND & AIMS Inflammatory bowel diseases (IBD) increase risk for colorectal cancer. Mutations in the Mediterranean fever gene (MEFV or pyrin) are associated with hereditary autoinflammatory disease and severe IBD. Expression of MEFV,a sensor protein that the initiates assembly of the inflammasome complex,is increased in colon biopsies from patients with IBD. We investigated the role of pyrin in intestinal homeostasis in mice. METHODS Mefv-/- mice and C57/BL6 mice (controls) were given azoxymethane followed by multiple rounds of dextran sodium sulfate (DSS) to induce colitis and tumorigenesis. In some experiments,Mefv-/- mice were given injections of recombinant interleukin 18 (rIL18) or saline (control) during DSS administration. Colon tissues were collected at different time points during colitis development and analyzed by histology,immunohistochemistry,immunoblots,or ELISAs (to measure cytokines). Spleen and mesenteric lymph node were collected,processed,and analyzed by flow cytometry. Colon epithelial permeability was measured in mice with colitis by gavage of fluorescent dextran and quantification of serum levels. RESULTS MEFV was expressed in colons of control mice and expression increased during chronic and acute inflammation; high levels were detected in colon tumor and adjacent non-tumor tissues. Mefv-/- mice developed more severe colitis than control mice,with a greater extent of epithelial hyperplasia and a larger tumor burden. Levels of inflammatory cytokines (IL6) and chemokines were significantly higher in colons of Mefv-/- mice than control mice following colitis induction,whereas the level IL18,which depends on the inflammasome for maturation and release,was significantly lower in colons of Mefv-/- mice. Mefv-/- mice had increased epithelial permeability following administration of DSS than control mice,and loss of the tight junction proteins occludin and claudin-2 from intercellular junctions. STAT3 was activated (phosphorylated) in inflamed colon tissues from Mefv-/-,which also had increased expression of stem cell markers (OLFM4,BMI1,and MSI1) compared with colons from control mice. Administration of rIL18 to Mefv-/- mice reduced epithelial permeability,intestinal inflammation,the severity of colitis,and colon tumorigenesis. CONCLUSIONS In studies with DSS-induced colitis,we found that pyrin (MEFV) is required for inflammasome activation and IL18 maturation,which promote intestinal barrier integrity and prevent colon inflammation and tumorigenesis. Strategies to increase activity of MEFV or IL18 might be developed for the treatment of IBD and prevention of colitis-associated tumorigenesis. View Publication