技术资料

Filoviruses of the genus Ebolavirus include five species with marked differences in their ability to cause disease in humans. From the highly virulent Ebola virus to the seemingly nonpathogenic Reston virus,case-fatality rates can range between 0-90{\%}. In order to understand the molecular basis of these differences it is imperative to establish disease models that recapitulate human disease as faithfully as possible. Non-human primates are the gold-standard models for filovirus pathogenesis,but comparative studies are skewed by the fact that Reston virus infection can be lethal for NHP. Here we have used HLA-A2 transgenic,NOD-scid-interleukin 2$\gamma$ receptor knockout (NSG-A2) mice reconstituted with human hematopoiesis to compare Ebola virus and Reston virus pathogenesis in a human-like environment. While significantly less pathogenic than Ebola virus,Reston virus killed 20{\%} of infected mice,a finding that was linked to exacerbated inflammation and viral replication in the liver. In addition,'humanized' mice recapitulated the case-fatality ratios of different Ebolavirus species in humans. Our findings point out at humanized mice as a putative model to test the pathogenicity of newly discovered filoviruses,and warrants further investigations on Reston virus pathogenesis in humans. View Publication

The extensive molecular characterization of human pluripotent stem cells (hPSCs),human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs) is required before they can be applied in the future for personalized medicine and drug discovery. Despite the efforts that have been made with kinome analyses,we still lack in-depth insights into the molecular signatures of receptor tyrosine kinases (RTKs) that are related to pluripotency. Here,we present the first detailed and distinct repertoire of RTK characteristic for hPSC pluripotency by determining both the expression and phosphorylation profiles of RTKs in hESCs and hiPSCs using reverse transcriptase-polymerase chain reaction with degenerate primers that target conserved tyrosine kinase domains and phospho-RTK array,respectively. Among the RTKs tested,the up-regulation of EPHA1,ERBB2,FGFR4 and VEGFR2 and the down-regulation of AXL,EPHA4,PDGFRB and TYRO3 in terms of both their expression and phosphorylation levels were predominantly related to the maintenance of hPSC pluripotency. Notably,the specific inhibition of AXL was significantly advantageous in maintaining undifferentiated hESCs and hiPSCs and for the overall efficiency and kinetics of hiPSC generation. Additionally,a global phosphoproteomic analysis showed that ∼30% of the proteins (293 of 970 phosphoproteins) showed differential phosphorylation upon AXL inhibition in undifferentiated hPSCs,revealing the potential contribution of AXL-mediated phosphorylation dynamics to pluripotency-related signaling networks. Our findings provide a novel molecular signature of AXL in pluripotency control that will complement existing pluripotency-kinome networks. View Publication

Tolerogenic dendritic cell (tolDC)-based therapies have become a promising approach for the treatment of autoimmune diseases by their potential ability to restore immune tolerance in an antigen-specific manner. However,the broad variety of protocols used to generate tolDC in vitro and their functional and phenotypical heterogeneity are evidencing the need to find robust biomarkers as a key point towards their translation into the clinic,as well as better understanding the mechanisms involved in the induction of immune tolerance. With that aim,in this study we have compared the transcriptomic profile of tolDC induced with either vitamin D3 (vitD3-tolDC),dexamethasone (dexa-tolDC) or rapamycin (rapa-tolDC) through a microarray analysis in 5 healthy donors. The results evidenced that common differentially expressed genes could not be found for the three different tolDC protocols. However,individually,CYP24A1,MUCL1 and MAP7 for vitD3-tolDC; CD163,CCL18,C1QB and C1QC for dexa-tolDC; and CNGA1 and CYP7B1 for rapa-tolDC,constituted good candidate biomarkers for each respective cellular product. In addition,a further gene set enrichment analysis of the data revealed that dexa-tolDC and vitD3-tolDC share several immune regulatory and anti-inflammatory pathways,while rapa-tolDC seem to be playing a totally different role towards tolerance induction through a strong immunosuppression of their cellular processes. View Publication

Long-term in vitro culture of undifferentiated human embryonic stem cells (hESCs) traditionally requires a fibroblast feeder cell layer. Using feeder cells in hESC cultures is highly laborious and limits large-scale hESC production for potential application in regenerative medicine. Replacing feeder cells with defined human extracellular matrix (ECM) components or synthetic biomaterials would be ideal for large-scale production of clinical-grade hESCs. We tested and compared different feeder cell-free hESC culture methods based on different human ECM proteins,human and animal sera matrices,and a Matrigel matrix. Also selected biomaterials were tested for feeder cell-free propagation of undifferentiated hESCs. The matrices were tested together with conventional and modified hESC culture media,human foreskin fibroblast-conditioned culture medium,chemically defined medium,TeSR1,and modified TeSR1 media. The results showed the undefined,xenogeneic Matrigel to be a superior matrix for hESC culture compared with the purified human ECM proteins,serum matrices,and the biomaterials tested. A long-term,feeder cell-free culture system was successful on Matrigel in combination with mTeSR1 culture medium,but a xeno-free,fully defined,and reproducible feeder cell-free hESC culture method still remains to be developed. View Publication

Type 1 diabetes mellitus (T1DM) is the most common type of diabetes in children and adolescents. Diabetic subjects are more likely to experience a myocardial infarction compared to nondiabetic subjects. In recent years,induced pluripotent stem cells (iPSCs) have received increasing attention from basic scientists and clinicians and hold promise for myocardial regeneration due to their unlimited proliferation potential and differentiation capacity. However,cardiomyogenesis of type 1 diabetic donor-derived iPSCs (T1DM-iPSCs) has not been investigated yet. The aim of the study was to comparatively analyze cardiomyocyte (CM) differentiation capacity of nondiabetic donor-derived iPSCs (N-iPSCs) and T1DM-iPSCs. The differentiated CMs were confirmed by both expression of cardiac-specific markers and presence of cardiac action potential. Since mitochondrial bioenergetics is vital to every aspect of CM function,extracellular acidification rates and oxygen consumption rates were measured using Seahorse extracellular flux analyzer. The results showed that N-iPSCs and T1DMiPSCs demonstrated similar capacity of differentiation into spontaneously contracting CMs exhibiting nodal-,atrial-,or ventricular-like action potentials. Differentiation efficiency was up to 90%. In addition,the CMs differentiated from N-iPSCs and T1DM-iPSCs (N-iPSC-CMs and T1DM-iPSC-CMs,respectively) showed 1) well-regulated glucose utilization at the level of glycolysis and mitochondrial oxidative phosphorylation and 2) the ability to switch metabolic pathways independent of extracellular glucose concentration. Collectively,we demonstrate for the first time that T1DM-iPSCs can differentiate into functional CMs with well-regulated glucose utilization as shown in N-iPSCs,suggesting that T1DM-iPSC-CMs might be a promising autologous cell source for myocardial regeneration in type 1 diabetes patients. View Publication

Multiple myeloma is an incurable malignancy of plasma cells resulting from impaired terminal B cell development. Almost all patients with multiple myeloma eventually have a relapse. Many studies have demonstrated the importance of the various genomic mutations that characterize multiple myeloma as a complex heterogeneous disease. In recent years,next-generation sequencing has been used to identify the genomic mutation landscape and clonal heterogeneity of multiple myeloma. This is the first study,a prospective observational study,to identify somatic mutations in plasma cell disorders in the Thai population using targeted next-generation sequencing. Twenty-seven patients with plasma cell disorders were enrolled comprising 17 cases of newly diagnosed multiple myeloma,5 cases of relapsed/refractory multiple myeloma,and 5 cases of other plasma cell disorders. The pathogenic mutations were found in 17 of 27 patients. Seventy percent of those who had a mutation (12/17 patients) habored a single mutation,whereas the others had more than one mutation. Fifteen pathogenic mutation genes were identified: ATM,BRAF,CYLD,DIS3,DNMT3A,FBXW7,FLT3,GNA13,IRF4,KMT2A,NRAS,SAMHD1,TENT5C,TP53,and TRAF3. Most have previously been reported to be involved in the RAS/MAPK pathway,the nuclear factor kappa B pathway,the DNA-repair pathway,the CRBN pathway,tumor suppressor gene mutation,or an epigenetic mutation. However,the current study also identified mutations that had not been reported to be related to myeloma: GNA13 and FBXW7. Therefore,a deep understanding of molecular genomics would inevitably improve the clinical management of plasma cell disorder patients,and the increased knowledge would ultimately result in better outcomes for the patients. View Publication

There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support,but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study,five separate laboratories,each with experience in human embryonic stem cell culture,used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods,with propagation in the presence of Knockout Serum Replacer,FGF-2,and mouse embryonic fibroblast feeder cell layers serving as a positive control. The cultures were assessed for up to ten passages for attachment,death,and differentiated morphology by phase contrast microscopy,for growth by serial cell counts,and for maintenance of stem cell surface marker expression by flow cytometry. Of the eight culture systems,only the control and those based on two commercial media,mTeSR1 and STEMPRO,supported maintenance of most cell lines for ten passages. Cultures grown in the remaining media failed before this point due to lack of attachment,cell death,or overt cell differentiation. Possible explanations for relative success of the commercial formulations in this study,and the lack of success with other formulations from academic groups compared to previously published results,include: the complex combination of growth factors present in the commercial preparations; improved development,manufacture,and quality control in the commercial products; differences in epigenetic adaptation to culture in vitro between different ES cell lines grown in different laboratories. View Publication

Electronic cigarettes (EC) and refill fluids are distributed with little information on their pre- and postnatal health effects. This study compares the cytotoxicity of EC refill fluids using embryonic and adult cells and examines the chemical characteristics of refill fluids using HPLC. Refill solutions were tested on human embryonic stem cells (hESC),mouse neural stem cells (mNSC),and human pulmonary fibroblasts (hPF) using the MTT assay,and NOAELs and IC50s were determined from dose-response curves. Spectral analysis was performed when products of the same flavor had different MTT outcomes. hESC and mNSC were generally more sensitive to refill solutions than hPF. All products from one company were cytotoxic to hESC and mNSC,but non-cytotoxic to hPF. Cytotoxicity was not due to nicotine,but was correlated with the number and concentration of chemicals used to flavor fluids. Additional studies are needed to fully assess the prenatal effect of refill fluids. ?? 2012 Elsevier Inc. View Publication

AbstractIntroduction
Preeclampsia and other placental pathologies are characterized by a lack of spiral artery remodeling associated with insufficient invasion by extravillous trophoblast cells (EVT). Because trophoblast invasion occurs in early pregnancy when access to human placental tissue is limited,there is a need for model systems for the study of trophoblast differentiation and invasion. Human embryonic stem cells (hESC) treated with BMP4- differentiate to trophoblast,and express HLA-G,a marker of EVT. The goals of the present study were to further characterize the HLA-G+ cells derived from BMP4-treated hESC,and determine their suitability as a model.
Methods
HESC were treated with BMP4 under 4% or 20% oxygen and tested in Matrigel invasion chambers. Both BMP4-treated hESC and primary human placental cells were separated into HLA-G+ and HLA-G−/TACSTD2+ populations with immunomagnetic beads and expression profiles analyzed by microarray.
Results
There was a 10-fold increase in invasion when hESC were BMP4-treated. There was also an independent,stimulatory effect of oxygen on this process. Invasive cells expressed trophoblast marker KRT7,and the majority were also HLA-G+. Gene expression profiles revealed that HLA-G+,BMP4-treated hESC were similar to,but distinct from,HLA-G+ cells isolated from first trimester placentas. Whereas HLA-G+ and HLA-G− cells from first trimester placentas had highly divergent gene expression profiles,HLA-G+ and HLA-G− cells from BMP4-treated hESC had somewhat similar profiles,and both expressed genes characteristic of early trophoblast development.
Conclusions
We conclude that hESC treated with BMP4 provide a model for studying transition to the EVT lineage. View Publication

INTRODUCTION: The assay of endogenous erythroid colony formation (EEC),a characteristic of polycythemia vera and essential thrombocythemia,is not standardized. In this multicentric study,we tested four semisolid,serum-free,cytokine-free media based on either methylcellulose (M1,M2) or collagen (C1,C2) commercialized for the EEC assay. MATERIALS AND METHODS: Bone marrow mononuclear cells (BMMC) from 73 individuals (62 patients with either polycythemia vera (26),essential thrombocythemia (19),secondary polyglobuly (17) or chronic myeloid leukemia (2) and 11 healthy donors) were grown in parallel in the four media without,or with 0.01 U/ml erythropoietin (EPo). RESULTS: In all four media EEC formation was specific,as it was not observed in cultures of patients with secondary polyglobuly or chronic myeloid leukemia,nor of healthy donors. Analysis of fresh or MGG-stained collagen gel cultures allowed detection of EEC formation significantly more frequently than methylcellulose-based media; addition of 0.01 U/ml of EPo had little or no effect on EEC formation. Collagen-based medium C1 gave better results than the other media tested: the 'C1' EEC assay was positive for 68.2% of polycythemia vera cultures with significantly higher median EEC numbers (6.5/10(5) BMMC for patients with one major criteria of polycythemia vera and 19 and 21/10(5) BMMC for patients with two or three major criteria,respectively). Medium C1 was also better for essential thrombocythemia cultures with 47.4% of positive results but with a low median EEC number (6.7/10(5) BMMC). When associated with the ELISA dosage of serum EPo,the 'C1' EEC assay allowed confirmation or elimination of the diagnosis of polycythemia vera for 91% (20/22) of polyglobulic patients. CONCLUSION: We propose that serum-free collagen-based culture systems be considered to standardize the EEC assay,now part of the new criteria of polycythemia vera. View Publication

Blood of both humans and mice contains 2 main monocyte subsets. Here,we investigated the extent of their similarity using a microarray approach. Approximately 270 genes in humans and 550 genes in mice were differentially expressed between subsets by 2-fold or more. More than 130 of these gene expression differences were conserved between mouse and human monocyte subsets. We confirmed numerous of these differences at the cell surface protein level. Despite overall conservation,some molecules were conversely expressed between the 2 species' subsets,including CD36,CD9,and TREM-1. Other differences included a prominent peroxisome proliferator-activated receptor gamma (PPARgamma) signature in mouse monocytes,which is absent in humans,and strikingly opposed patterns of receptors involved in uptake of apoptotic cells and other phagocytic cargo between human and mouse monocyte subsets. Thus,whereas human and mouse monocyte subsets are far more broadly conserved than currently recognized,important differences between the species deserve consideration when models of human disease are studied in mice. View Publication

Predictive in vitro hematotoxicity assays using human cells will provide estimation of tolerable level and aid considerably the development of agents with greater therapeutic activity and less toxicity. Human hematopoietic cells can be derived from three sources: human bone marrow by sternal or femoral aspiration,mobilized peripheral blood,or umbilical cord blood samples collected from placentas after deliveries. Because of the difficulties to have a continuous supply of bone marrow cells from normal human donors and the related ethical problems,we performed a study to compare the sensitivity of human bone marrow cells (h-BMC) and human cord blood cells (h-CBC) to chemicals in order to confirm if h-CBC can readily replace bone marrow cells in checking the sensitivity of GM-CFU progenitors to drugs as preliminarily reported in literature. Our results showed that the prediction of IC50 values in human model is quite similar by using h-BMC or h-CBC. On the contrary,the type of medium influenced in a significant way the ICs determination of some drugs. View Publication