技术资料

The paracaspase MALT1 plays an important role in immune receptor-driven signaling pathways leading to NF-κB activation. MALT1 promotes signaling by acting as a scaffold,recruiting downstream signaling proteins,as well as by proteolytic cleavage of multiple substrates. However,the relative contributions of these two different activities to T and B cell function are not well understood. To investigate how MALT1 proteolytic activity contributes to overall immune cell regulation,we generated MALT1 protease-deficient mice (Malt1(PD/PD)) and compared their phenotype with that of MALT1 knockout animals (Malt1(-/-)). Malt1(PD/PD) mice displayed defects in multiple cell types including marginal zone B cells,B1 B cells,IL-10-producing B cells,regulatory T cells,and mature T and B cells. In general,immune defects were more pronounced in Malt1(-/-) animals. Both mouse lines showed abrogated B cell responses upon immunization with T-dependent and T-independent Ags. In vitro,inactivation of MALT1 protease activity caused reduced stimulation-induced T cell proliferation,impaired IL-2 and TNF-α production,as well as defective Th17 differentiation. Consequently,Malt1(PD/PD) mice were protected in a Th17-dependent experimental autoimmune encephalomyelitis model. Surprisingly,Malt1(PD/PD) animals developed a multiorgan inflammatory pathology,characterized by Th1 and Th2/0 responses and enhanced IgG1 and IgE levels,which was delayed by wild-type regulatory T cell reconstitution. We therefore propose that the pathology characterizing Malt1(PD/PD) animals arises from an immune imbalance featuring pathogenic Th1- and Th2/0-skewed effector responses and reduced immunosuppressive compartments. These data uncover a previously unappreciated key function of MALT1 protease activity in immune homeostasis and underline its relevance in human health and disease. View Publication

The incidence of cardiovascular disease represents a significant and growing health-care challenge to the developed and developing world. The ability of native heart muscle to regenerate in response to myocardial infarct is minimal. Tissue engineering and regenerative medicine approaches represent one promising response to this difficulty. Here,we present methods for the construction of a cell-seeded cardiac patch with the potential to promote regenerative outcomes in heart muscle with damage secondary to myocardial infarct. This method leverages iPS cells and a fibrin-based scaffold to create a simple and commercially viable tissue-engineered cardiac patch. Human-induced pluripotent stem cells (hiPSCs) can,in principle,be differentiated into cells of any lineage. However,most of the protocols used to generate hiPSC-derived endothelial cells (ECs) and cardiomyocytes (CMs) are unsatisfactory because the yield and phenotypic stability of the hiPSC-ECs are low,and the hiPSC-CMs are often purified via selection for expression of a promoter-reporter construct. In this chapter,we describe an hiPSC-EC differentiation protocol that generates large numbers of stable ECs and an hiPSC-CM differentiation protocol that does not require genetic manipulation,single-cell selection,or sorting with fluorescent dyes or other reagents. We also provide a simple but effective method that can be used to combine hiPSC-ECs and hiPSC-CMs with hiPSC-derived smooth muscle cells to engineer a contracting patch of cardiac cells. View Publication

BACKGROUND: Airway remodeling is a proposed mechanism that underlies the persistent loss of lung function associated with childhood asthma. Previous studies have demonstrated that human lung fibroblasts (HLFs) co-cultured with primary human bronchial epithelial cells (BECs) from asthmatic children exhibit greater expression of extracellular matrix (ECM) components compared to co-culture with BECs derived from healthy children. Myofibroblasts represent a population of differentiated fibroblasts that have greater synthetic activity. We hypothesized co-culture with asthmatic BECs would lead to greater fibroblast to myofibroblast transition (FMT) compared to co-culture with healthy BECs. METHODS: BECs were obtained from well-characterized asthmatic and healthy children and were proliferated and differentiated at an air-liquid interface (ALI). BEC-ALI cultures were co-cultured with HLFs for 96 hours. RT-PCR was performed in HLFs for alpha smooth muscle actin ($$-SMA) and flow cytometry was used to assay for $$-SMA antibody labeling of HLFs. RT-PCR was also preformed for the expression of tropomyosin-I as an additional marker of myofibroblast phenotype. In separate experiments,we investigated the role of TGF$$2 in BEC-HLF co-cultures using monoclonal antibody inhibition. RESULTS: Expression of $$-SMA by HLFs alone was greater than by HLFs co-cultured with healthy BECs,but not different than $$-SMA expression by HLFs co-cultured with asthmatic BECs. Flow cytometry also revealed significantly less $$-SMA expression by healthy co-co-cultures compared to asthmatic co-cultures or HLF alone. Monoclonal antibody inhibition of TGF$$2 led to similar expression of $$-SMA between healthy and asthmatic BEC-HLF co-cultures. Expression of topomyosin-I was also significantly increased in HLF co-cultured with asthmatic BECs compared to healthy BEC-HLF co-cultures or HLF cultured alone. CONCLUSION: These findings suggest dysregulation of FMT in HLF co-cultured with asthmatic as compared to healthy BECs. Our results suggest TGF$$2 may be involved in the differential regulation of FMT by asthmatic BECs. These findings further illustrate the importance of BEC-HLF cross-talk in asthmatic airway remodeling. View Publication

Fibroblast growth factors (FGFs) are essential for maintaining self-renewal in human embryonic stem cells and induced pluripotent stem cells. Recombinant basic FGF (bFGF or FGF2) is conventionally used to culture pluripotent stem cells; however,because of the instability of bFGF,repeated addition of fresh bFGF into the culture medium is required in order to maintain its concentration. In this study,we demonstrate that a heat-stable chimeric variant of FGF,termed FGFC,can be successfully used for maintaining human pluripotent stem cells. FGFC is a chimeric protein composed of human FGF1 and FGF2 domains that exhibits higher thermal stability and protease resistance than do both FGF1 and FGF2. Both human embryonic stem cells and induced pluripotent stem cells were maintained in ordinary culture medium containing FGFC instead of FGF2. Comparison of cells grown in FGFC with those grown in conventional FGF2 media showed no significant differences in terms of the expression of pluripotency markers,global gene expression,karyotype,or differentiation potential in the three germ lineages. We therefore propose that FGFC may be an effective alternative to FGF2,for maintenance of human pluripotent stem cells. View Publication

Blood flow promotes emergence of definitive hematopoietic stem cells (HSCs) in the developing embryo,yet the signals generated by hemodynamic forces that influence hematopoietic potential remain poorly defined. Here we show that fluid shear stress endows long-term multilineage engraftment potential upon early hematopoietic tissues at embryonic day 9.5,an embryonic stage not previously described to harbor HSCs. Effects on hematopoiesis are mediated in part by a cascade downstream of wall shear stress that involves calcium efflux and stimulation of the prostaglandin E2 (PGE2)-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling axis. Blockade of the PGE2-cAMP-PKA pathway in the aorta-gonad-mesonephros (AGM) abolished enhancement in hematopoietic activity. Furthermore,Ncx1 heartbeat mutants,as well as static cultures of AGM,exhibit lower levels of expression of prostaglandin synthases and reduced phosphorylation of the cAMP response element-binding protein (CREB). Similar to flow-exposed cultures,transient treatment of AGM with the synthetic analogue 16,16-dimethyl-PGE2 stimulates more robust engraftment of adult recipients and greater lymphoid reconstitution. These data provide one mechanism by which biomechanical forces induced by blood flow modulate hematopoietic potential. View Publication

Characterizing clinically relevant brain metastasis models and assessing the therapeutic efficacy in such models are fundamental for the development of novel therapies for metastatic brain cancers. In this study,we have developed an in vivo imageable breast-to-brain metastasis mouse model. Using real time in vivo imaging and subsequent composite fluorescence imaging,we show a widespread distribution of micro- and macro-metastasis in different stages of metastatic progression. We also show extravasation of tumour cells and the close association of tumour cells with blood vessels in the brain thus mimicking the multi-foci metastases observed in the clinics. Next,we explored the ability of engineered adult stem cells to track metastatic deposits in this model and show that engineered stem cells either implanted or injected via circulation efficiently home to metastatic tumour deposits in the brain. Based on the recent findings that metastatic tumour cells adopt unique mechanisms of evading apoptosis to successfully colonize in the brain,we reasoned that TNF receptor superfamily member 10A/10B apoptosis-inducing ligand (TRAIL) based pro-apoptotic therapies that induce death receptor signalling within the metastatic tumour cells might be a favourable therapeutic approach. We engineered stem cells to express a tumour selective,potent and secretable variant of a TRAIL,S-TRAIL,and show that these cells significantly suppressed metastatic tumour growth and prolonged the survival of mice bearing metastatic breast tumours. Furthermore,the incorporation of pro-drug converting enzyme,herpes simplex virus thymidine kinase,into therapeutic S-TRAIL secreting stem cells allowed their eradication post-tumour treatment. These studies are the first of their kind that provide insight into targeting brain metastasis with stem-cell mediated delivery of pro-apoptotic ligands and have important clinical implications. View Publication

Host defense peptides have recently gained much interest as novel anti-infectives owing to their ability to kill bacteria and simultaneously modulate host cell responses. The cationic host defense peptide GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE),derived from the C terminus of human thrombin,inhibits proinflammatory responses in vitro and in vivo,but the mode of action is unclear. In this study,we show that GKY25,apart from binding bacterial LPS,also interacts directly with monocytes and macrophages in vitro,ex vivo,and in vivo. Moreover,GKY25 inhibits TLR4- and TLR2-induced NF-κB activation in response to several microbe-derived agonists. Furthermore,GKY25 reduces LPS-induced phosphorylation of MAPKs p38α and JNK1/2/3. FACS and electron microscopy analyses showed that GKY25 interferes with TLR4/myeloid differentiation protein-2 dimerization. The results demonstrate a previously undisclosed activity of the host defense peptide GKY25,based on combined LPS and cell interactions leading to inhibition of TLR4 dimerization and subsequent reduction of NF-κB activity and proinflammatory cytokine production in monocytes and macrophages. View Publication

BACKGROUND: Epithelial-mesenchymal transition (EMT) is involved in important malignant features of cancer cells,like invasion,metastatic potential,anti-apoptotic and stem-cell like phenotypes. Among several transcription factors,SNAI2/SLUG is supposed to play an essential role for EMT. METHODS: Paraffin embedded tumor samples from 63 patients with metastatic non-small cell lung cancer,enrolled in a randomized phase II trial,were prospectively collected,53 samples qualified for further analysis. Automated RNA extraction from paraffin and RT-quantitative PCR was used for evaluation of SNAI2/SLUG,estrogen receptor 1 (ESR1) and matrix-metalloproteinases (MMP) mRNA expression. RESULTS: Clinical features like age,gender,performance status,histological subtype and stage were similarly distributed among SNAI2/SLUG positive and negative patients. SNAI2/SLUG was significantly,inversely correlated with ESR1 mRNA expression (p textless 0.0001). In contrast,MMP2 (p = 0.387),MMP7 (p = 0.396) and MMP9 mRNA expression (p = 0.366) did not correlate with SNAI2/SLUG. Patients with high SNAI2/SLUG expression (grouped by median expression) had a worse outcome. Median overall survival in patients with high SNAI2/SLUG expression was 5.7 months versus 11.6 months with low SNAI2/SLUG expression (p = .038). Inversely,patients with high ESR1 expression (grouped by median expression) had an improved median OS with 10.9 months vs. 5.0 months in the low expression group (p = .032). In multivariate analysis,SNAI2/SLUG2 (p = .022) and ESR1 (p = .017) separately were independent prognostic factors for survival. CONCLUSION: SNAI2/SLUG is prognostic of patients' outcome. The strong inverse correlation with ESR1 indicates a significant impact of estrogen receptor pathway regarding these malignant features. View Publication

Unintended exposure to teratogenic compounds can lead to various birth defects; however current animal-based testing is limited by time,cost and high inter-species variability. Here,we developed a human-relevant in vitro model,which recapitulated two cellular events characteristic of embryogenesis,to identify potentially teratogenic compounds. We spatially directed mesoendoderm differentiation,epithelial-mesenchymal transition and the ensuing cell migration in micropatterned human pluripotent stem cell (hPSC) colonies to collectively form an annular mesoendoderm pattern. Teratogens could disrupt the two cellular processes to alter the morphology of the mesoendoderm pattern. Image processing and statistical algorithms were developed to quantify and classify the compounds' teratogenic potential. We not only could measure dose-dependent effects but also correctly classify species-specific drug (Thalidomide) and false negative drug (D-penicillamine) in the conventional mouse embryonic stem cell test. This model offers a scalable screening platform to mitigate the risks of teratogen exposures in human. View Publication

Monomeric clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated 9 (Cas9) nucleases have been widely adopted for simple and robust targeted genome editing but also have the potential to induce high-frequency off-target mutations. In principle,two orthogonal strategies for reducing off-target cleavage,truncated guide RNAs (tru-gRNAs) and dimerization-dependent RNA-guided FokI-dCas9 nucleases (RFNs),could be combined as tru-RFNs to further improve genome editing specificity. Here we identify a robust tru-RFN architecture that shows high activity in human cancer cell lines and embryonic stem cells. Additionally,we demonstrate that tru-gRNAs reduce the undesirable mutagenic effects of monomeric FokI-dCas9. Tru-RFNs combine the advantages of two orthogonal strategies for improving the specificity of CRISPR-Cas nucleases and therefore provide a highly specific platform for performing genome editing. View Publication

PURPOSE To investigate the effects and mechanisms of fasudil hydrochloride (fasudil) on and in alkali burn-induced corneal neovascularization (CNV) in mice. METHODS To observe the effect of fasudil,mice with alkali-burned corneas were treated with either fasudil eye drops or phosphate-buffered saline (PBS) four times per day for 14 consecutive days. After injury,CNV and corneal epithelial defects were measured. The production of reactive oxygen species (ROS) and heme oxygenase-1(HO-1) was measured. The infiltration of polymorphonuclear neutrophils (PMNs) and the mRNA expressions of CNV-related genes were analyzed on day 14. RESULTS The incidence of CNV was significantly lower after treatment with 100 μM and 300 μM fasudil than with PBS,especially with 100 μM fasudil. Meanwhile,the incidences of corneal epithelial defects was lower (n=15,all ptextless0.01). After treatment with 100 μM fasudil,the intensity of DHE fluorescence was reduced in the corneal epithelium and stroma than with PBS treatment (n=5,all ptextless0.01),and the number of filtrated PMNs decreased. There were significant differences between the expressions of VEGF,TNF-a,MMP-8,and MMP-9 in the 100 μM fasudil group and the PBS group (n=8,all ptextless0.05). The production of HO-1 protein in the 100 μM fasudil group was 1.52±0.34 times more than in the PBS group (n=5 sample,ptextless0.05). CONCLUSIONS 100 μM fasudil eye drops administered four times daily can significantly inhibit alkali burn-induced CNV and promote the healing of corneal epithelial defects in mice. These effects are attributed to a decrease in inflammatory cell infiltration,reduction of ROS,and upregulation of HO-1 protein after fasudil treatment. View Publication

In the CNS,oligodendrocytes act as the myelinating cells. Oligodendrocytes have been identified to be key players in several neurodegenerative disorders. This protocol describes a robust,fast and reproducible differentiation protocol to generate human oligodendrocytes from pluripotent stem cells (PSCs) using a chemically defined,growth factor-rich medium. Within 8 d,PSCs differentiate into paired box 6-positive (PAX6(+)) neural stem cells,which give rise to OLIG2(+) progenitors by day 12. Oligodendrocyte lineage transcription factor 2-positive (OLIG2(+)) cells begin to express the transcription factor NKX2.2 around day 18,followed by SRY-box 10 (SOX10) around day 40. Oligodendrocyte progenitor cells (OPCs) that are positive for the cell surface antigen recognized by the O4 antibody (O4(+)) appear around day 50 and reach,on average,43% of the cell population after 75 d of differentiation. O4(+) OPCs can be isolated by cell sorting for myelination studies,or they can be terminally differentiated to myelin basic protein-positive (MBP(+)) oligodendrocytes. This protocol also describes an alternative strategy for markedly reducing the length and the costs of the differentiation and generating ∼30% O4(+) cells after only 55 d of culture. View Publication