技术资料

In culture conditions,human induced-pluripotent stem cells (hiPSC)-derived neurons form synaptic connections with other cells and establish neuronal networks,which are expected to be an in vitro model system for drug discovery screening and toxicity testing. While early studies demonstrated effects of co-culture of hiPSC-derived neurons with astroglial cells on survival and maturation of hiPSC-derived neurons,the population spiking patterns of such hiPSC-derived neurons have not been fully characterized. In this study,we analyzed temporal spiking patterns of hiPSC-derived neurons recorded by a multi-electrode array system. We discovered that specific sets of hiPSC-derived neurons co-cultured with astrocytes showed more frequent and highly coherent non-random synchronized spike trains and more dynamic changes in overall spike patterns over time. These temporally coordinated spiking patterns are physiological signs of organized circuits of hiPSC-derived neurons and suggest benefits of co-culture of hiPSC-derived neurons with astrocytes. View Publication

Glioblastoma multiforme (GBM) is a grade IV malignant brain tumor with high mortality and has been well known to involve many molecular pathways,including G-protein coupled receptor (GPCR)-mediated signaling (such as epithelial growth factor receptor [EGFR] and platelet derived growth factor receptor [PDGFR]). G protein-coupled receptor kinases (GRK) directly regulate GPCR activity by phosphorylating activated agonist-bound receptors to desensitize signaling and internalize receptors through beta-arrestins. Recent studies in various cancers,including prostate and breast cancer,have highlighted the role of change in GRK expression to oncogenesis and tumor proliferation. In this study,we evaluated the expression of GRK5 in grade II to grade IV glioma specimens using immunohistochemistry and found that GRK5 expression levels are highly correlated with aggressiveness of glioma. We used culture conditions to selectively promote the growth of either glioblastoma cells with stem cell markers (GSC) or differentiated glioblastoma cells (DGC) from fresh GBM specimens. GSC are known to be highly invasive and mobile,and have the capacity to self-renew and are more resistant to chemotherapy and radiation compared to differentiated populations of GBM. We examined the expression of GRK5 in these two sets of culturing conditions for GBM cells and found that GRK5 expression is upregulated in GSC compared to differentiated GBM cells. To better understand the role of GRK5 in GBM-derived stem cells,we created stable GRK5 knockdown and evaluated the proliferation rate. Using an ATP chemiluminescence assay,we show,for the first time,that knocking down the expression of GRK5 decreased the proliferation rate of GSC in contrast to control. View Publication

Human corneal endothelial cells are derived from neural crest and because of postmitotic arrest lack competence to repair cell loss from trauma,aging,and degenerative disorders such as Fuchs endothelial corneal dystrophy (FECD). Herein,we identified a rapidly proliferating subpopulation of cells from the corneal endothelium of adult normal and FECD donors that exhibited features of neural crest-derived progenitor (NCDP) cells by showing absence of senescence with passaging,propensity to form spheres,and increased colony forming efficacy compared with the primary cells. The collective expression of stem cell-related genes SOX2,OCT4,LGR5,TP63 (p63),as well as neural crest marker genes PSIP1 (p75(NTR)),PAX3,SOX9,AP2B1 (AP-2β),and NES,generated a phenotypic footprint of endothelial NCDPs. NCDPs displayed multipotency by differentiating into microtubule-associated protein 2,β-III tubulin,and glial fibrillary acidic protein positive neurons and into p75(NTR)-positive human corneal endothelial cells that exhibited transendothelial resistance of functional endothelium. In conclusion,we found that mitotically incompetent ocular tissue cells contain adult NCDPs that exhibit a profile of transcription factors regulating multipotency and neural crest progenitor characteristics. Identification of normal NCDPs in FECD-affected endothelium holds promise for potential autologous cell therapies. View Publication

Improved understanding of expression of immune checkpoint receptors (ICR) on tumor-infiltrating lymphocytes (TIL) may facilitate more effective immunotherapy in head and neck cancer (HNC) patients. A higher frequency of PD-1+ TIL has been reported in human papillomavirus (HPV)+ HNC patients,despite the role of PD-1 in T-cell exhaustion. This discordance led us to hypothesize that the extent of PD-1 expression more accurately defines T-cell function and prognostic impact,because PD-1high T cells may be more exhausted than PD-1low T cells and may influence clinical outcome and response to anti-PD-1 immunotherapy. In this study,PD-1 expression was indeed upregulated on HNC patient TIL,and the frequency of these PD-1+ TIL was higher in HPV+ patients (P = 0.006),who nonetheless experienced significantly better clinical outcome. However,PD-1high CD8+ TILs were more frequent in HPV- patients and represented a more dysfunctional subset with compromised IFN-γ secretion. Moreover,HNC patients with higher frequencies of PD-1high CD8+ TIL showed significantly worse disease-free survival and higher hazard ratio for recurrence (P < 0.001),while higher fractions of PD-1low T cells associated with HPV positivity and better outcome. In a murine HPV+ HNC model,anti-PD-1 mAb therapy differentially modulated PD-1high/low populations,and tumor rejection associated with loss of dysfunctional PD-1high CD8+ T cells and a significant increase in PD-1low TIL. Thus,the extent of PD-1 expression on CD8+ TIL provides a potential biomarker for anti-PD-1-based immunotherapy. Cancer Res; 77(22); 6353-64. textcopyright2017 AACR. View Publication

Adipose tissue stem cells (ADSCs) would be an attractive autologous cell source. However,ADSCs require invasive procedures,and has potential complications. Recently,urine stem cells (USCs) have been proposed as an alternative stem cell source. In this study,we compared USCs and ADSCs collected from the same patients on stem cell characteristics and capacity to differentiate into various cell lineages to provide a useful guideline for selecting the appropriate type of cell source for use in clinical application. The urine samples were collected via urethral catheterization,and adipose tissue was obtained from subcutaneous fat tissue during elective laparoscopic kidney surgery from the same patient (n = 10). Both cells were plated for primary culture. Cell proliferation,colony formation,cell surface markers,immune modulation,chromosome stability and multi-lineage differentiation were analyzed for each USCs and ADSCs at cell passage 3,5,and 7. USCs showed high cell proliferation rate,enhanced colony forming ability,strong positive for stem cell markers expression,high efficiency for inhibition of immune cell activation compared to ADSCs at cell passage 3,5,and 7. In chromosome stability analysis,both cells showed normal karyotype through all passages. In analysis of multi-lineage capability,USCs showed higher myogenic,neurogenic,and endogenic differentiation rate,and lower osteogenic,adipogenic,and chondrogenic differentiation rate compared to ADSCs. Therefore,we expect that USC can be an alternative autologous stem cell source for muscle,neuron and endothelial tissue reconstruction instead of ADSCs. View Publication

Droplet single-cell RNA-sequencing (dscRNA-seq) has enabled rapid,massively parallel profiling of transcriptomes. However,assessing differential expression across multiple individuals has been hampered by inefficient sample processing and technical batch effects. Here we describe a computational tool,demuxlet,that harnesses natural genetic variation to determine the sample identity of each droplet containing a single cell (singlet) and detect droplets containing two cells (doublets). These capabilities enable multiplexed dscRNA-seq experiments in which cells from unrelated individuals are pooled and captured at higher throughput than in standard workflows. Using simulated data,we show that 50 single-nucleotide polymorphisms (SNPs) per cell are sufficient to assign 97% of singlets and identify 92% of doublets in pools of up to 64 individuals. Given genotyping data for each of eight pooled samples,demuxlet correctly recovers the sample identity of<99% of singlets and identifies doublets at rates consistent with previous estimates. We apply demuxlet to assess cell-type-specific changes in gene expression in 8 pooled lupus patient samples treated with interferon (IFN)-β and perform eQTL analysis on 23 pooled samples. View Publication

IL2 is an immunostimulatory cytokine for key immune cells including T cells and natural killer (NK) cells. Systemic IL2 supplementation could enhance NK-mediated immunity in a variety of diseases ranging from neoplasms to viral infection. However,its systemic use is restricted by its serious side effects and limited efficacy due to activation of T regulatory cells (Tregs). IL2 signaling is mediated through interactions with a multi-subunit receptor complex containing IL2Rα,IL2Rβ,and IL2Rγ. Adult natural killer (NK) cells express only IL2Rβ and IL2Rγ subunits and are therefore relatively insensitive to IL2. To overcome these limitations,we created a novel chimeric IL2-IL2Rβ fusion protein of IL2 and its receptor IL2Rβ joined via a peptide linker (CIRB). NK92 cells expressing CIRB (NK92CIRB) were highly activated and expanded indefinitely without exogenous IL2. When compared with an IL2-secreting NK92 cell line,NK92CIRB were more activated,cytotoxic,and resistant to growth inhibition. Direct contact with cancer cells enhanced the cytotoxic character of NK92CIRB cells,which displayed superior in vivo antitumor effects in mice. Overall,our results showed how tethering IL2 to its receptor IL2Rβ eliminates the need for IL2Rα and IL2Rβ,offering a new tool to selectively activate and empower immune therapy. Cancer Res; 77(21); 5938-51. textcopyright2017 AACR. View Publication

Efforts to improve the efficacy of adoptive T-cell therapies and immune checkpoint therapies in myelogenous leukemia are desired. In this study,we evaluated the antileukemia activity of adoptively transferred polyclonal cancer antigen-reactive T cells deficient in the regulator diacylglycerol kinase zeta (DGKζ) with or without PD-1/PD-L1 blockade. In the C1498 mouse model of myeloid leukemia,we showed that leukemia was eradicated more effectively in DGKζ-deficient (DGKζ-/-) mice than wild-type mice. T cells transferred from DGKζ-deficient mice to wild-type tumor-bearing recipients conferred this benefit. Leukemia clearance was similar to mice treated with anti-PD-L1. Strikingly,we found that the activity of adoptively transferred DGKζ-/- T cells relied partly on induction of sustainable host T-cell immunity. Transferring DGKζ-deficient T cells increased the levels of IFNγ and other cytokines in recipient mice,especially with coadministration of anti-PD-L1. Overall,our results offered evidence that targeting DGKζ may leverage the efficacy of adoptive T-cell and immune checkpoint therapies in leukemia treatment. Furthermore,they suggest that DGKζ targeting might decrease risks of antigen escape or resistance to immune checkpoint blockade. Cancer Res; 77(20); 5676-86. textcopyright2017 AACR. View Publication

The control of uptake and utilization of necessary extracellular nutrients-glucose and glutamine-is an important aspect of B cell activation. Let-7 is a family of microRNAs known to be involved in metabolic control. Here,we employed several engineered mouse models,including B cell-specific overexpression of Lin28a or the let-7a-1/let-7d/let-7f-1 cluster (let-7adf) and knockout of individual let-7 clusters to show that let-7adf specifically inhibits T cell-independent (TI) antigen-induced immunoglobulin (Ig)M antibody production. Both overexpression and deletion of let-7 in this cluster leads to altered TI-IgM production. Mechanistically,let-7adf suppresses the acquisition and utilization of key nutrients,including glucose and glutamine,through directly targeting hexokinase 2 (Hk2) and by repressing a glutamine transporter Slc1a5 and a key degradation enzyme,glutaminase (Gls),a mechanism mediated by regulation of c-Myc. Our results suggest a novel role of let-7adf as a metabolic brake" on B cell antibody production." View Publication

BACKGROUND The increasing usage of statins (the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors) has revealed a number of unexpected beneficial effects,including a reduction in cancer risk. METHODS We investigated the direct anticancer effects of different statins approved for clinical use on human breast and brain cancer cells. We also explored the effects of statins on cancer cells using in silico simulations. RESULTS In vitro studies showed that cerivastatin,pitavastatin,and fluvastatin were the most potent anti-proliferative,autophagy inducing agents in human cancer cells including stem cell-like primary glioblastoma cell lines. Consistently,pitavastatin was more effective than fluvastatin in inhibiting U87 tumour growth in vivo. Intraperitoneal injection was much better than oral administration in delaying glioblastoma growth. Following statin treatment,tumour cells were rescued by adding mevalonate and geranylgeranyl pyrophosphate. Knockdown of geranylgeranyl pyrophosphate synthetase-1 also induced strong cell autophagy and cell death in vitro and reduced U87 tumour growth in vivo. These data demonstrate that statins main effect is via targeting the mevalonate synthesis pathway in tumour cells. CONCLUSIONS Our study demonstrates the potent anticancer effects of statins. These safe and well-tolerated drugs need to be further investigated as cancer chemotherapeutics in comprehensive clinical studies. View Publication

Surgery is an essential component in the treatment of brain tumors. However,delineating tumor from normal brain remains a major challenge. We describe the use of stimulated Raman scattering (SRS) microscopy for differentiating healthy human and mouse brain tissue from tumor-infiltrated brain based on histoarchitectural and biochemical differences. Unlike traditional histopathology,SRS is a label-free technique that can be rapidly performed in situ. SRS microscopy was able to differentiate tumor from nonneoplastic tissue in an infiltrative human glioblastoma xenograft mouse model based on their different Raman spectra. We further demonstrated a correlation between SRS and hematoxylin and eosin microscopy for detection of glioma infiltration (κ = 0.98). Finally,we applied SRS microscopy in vivo in mice during surgery to reveal tumor margins that were undetectable under standard operative conditions. By providing rapid intraoperative assessment of brain tissue,SRS microscopy may ultimately improve the safety and accuracy of surgeries where tumor boundaries are visually indistinct. View Publication

The activity of neural precursor cells in the adult hippocampus is regulated by various stimuli; however,whether these stimuli regulate the same or different precursor populations remains unknown. Here,we developed a novel cell-sorting protocol that allows the purification to homogeneity of neurosphere-forming neural precursors from the adult mouse hippocampus and examined the responsiveness of individual precursors to various stimuli using a clonal assay. We show that within the Hes5-GFP(+)/Nestin-GFP(+)/EGFR(+) cell population,which comprises the majority of neurosphere-forming precursors,there are two distinct subpopulations of quiescent precursor cells,one directly activated by high-KCl depolarization,and the other activated by norepinephrine (NE). We then demonstrate that these two populations are differentially distributed along the septotemporal axis of the hippocampus,and show that the NE-responsive precursors are selectively regulated by GABA,whereas the KCl-responsive precursors are selectively modulated by corticosterone. Finally,based on RNAseq analysis by deep sequencing,we show that the progeny generated by activating NE-responsive versus KCl-responsive quiescent precursors are molecularly different. These results demonstrate that the adult hippocampus contains phenotypically similar but stimulus-specific populations of quiescent precursors,which may give rise to neural progeny with different functional capacity. View Publication