技术资料

Stem cells are highly resistant to viral infection compared to their differentiated progeny; however,the mechanism is mysterious. Here,we analyzed gene expression in mammalian stem cells and cells at various stages of differentiation. We find that,conserved across species,stem cells express a subset of genes previously classified as interferon (IFN) stimulated genes (ISGs) but that expression is intrinsic,as stem cells are refractory to interferon. This intrinsic ISG expression varies in a cell-type-specific manner,and many ISGs decrease upon differentiation,at which time cells become IFN responsive,allowing induction of a broad spectrum of ISGs by IFN signaling. Importantly,we show that intrinsically expressed ISGs protect stem cells against viral infection. We demonstrate the in vivo importance of intrinsic ISG expression for protecting stem cells and their differentiation potential during viral infection. These findings have intriguing implications for understanding stem cell biology and the evolution of pathogen resistance. View Publication

BACKGROUND Single-cell RNA-Seq can be a valuable and unbiased tool to dissect cellular heterogeneity,despite the transcriptome's limitations in describing higher functional phenotypes and protein events. Perhaps the most important shortfall with transcriptomic 'snapshots' of cell populations is that they risk being descriptive,only cataloging heterogeneity at one point in time,and without microenvironmental context. Studying the genetic ('nature') and environmental ('nurture') modifiers of heterogeneity,and how cell population dynamics unfold over time in response to these modifiers is key when studying highly plastic cells such as macrophages. RESULTS We introduce the programmable Polaris microfluidic lab-on-chip for single-cell sequencing,which performs live-cell imaging while controlling for the culture microenvironment of each cell. Using gene-edited macrophages we demonstrate how previously unappreciated knockout effects of SAMHD1,such as an altered oxidative stress response,have a large paracrine signaling component. Furthermore,we demonstrate single-cell pathway enrichments for cell cycle arrest and APOBEC3G degradation,both associated with the oxidative stress response and altered proteostasis. Interestingly,SAMHD1 and APOBEC3G are both HIV-1 inhibitors ('restriction factors'),with no known co-regulation. CONCLUSION As single-cell methods continue to mature,so will the ability to move beyond simple 'snapshots' of cell populations towards studying the determinants of population dynamics. By combining single-cell culture,live-cell imaging,and single-cell sequencing,we have demonstrated the ability to study cell phenotypes and microenvironmental influences. It's these microenvironmental components - ignored by standard single-cell workflows - that likely determine how macrophages,for example,react to inflammation and form treatment resistant HIV reservoirs. View Publication

Despite the high incidence of trophoblast-related diseases,the molecular mechanism of inadequate early trophoblast development is still unclear due to the lack of an appropriate cellular model in vitro. In the present study,we reprogrammed the amniotic cells to be induced pluripotent stem cells (iPSCs) via a non-virus and non-integrated method and subsequently differentiated them into trophoblast-like cells by a modified BMP4 strategy in E6 medium. Compared with the previously studied trophoblast-like cells from ESCs,the iPSCs derived trophoblast-like cells behave similarly in terms of gene expression profiles and biofunctions. Also we confirmed the differentiating tendency from iPSCs to be syncytiotrophoblasts-like cells might be caused by inappropriate differentiating oxygen condition. Additionally,we preliminarily indicated in vitro artificial" differentiation of iPSCs also undergoing a possible trophoblastic stem cell stage as witnessed in vivo. In conclusion we provided an in vitro cellular model to study early trophoblast development for specific individual by using the feasible amnion. View Publication

Induced pluripotent stem cells (iPSCs) are pluripotent cells derived from adult somatic cells. After the pioneering work by Yamanaka,who first generated iPSCs by retroviral transduction of four reprogramming factors,several alternative methods to obtain iPSCs have been developed in order to increase the yield and safety of the process. However,the question remains open on whether the different reprogramming methods can influence the pluripotency features of the derived lines. In this study,three different strategies,based on retroviral vectors,episomal vectors,and Sendai virus vectors,were applied to derive iPSCs from human fibroblasts. The reprogramming efficiency of the methods based on episomal and Sendai virus vectors was higher than that of the retroviral vector-based approach. All human iPSC clones derived with the different methods showed the typical features of pluripotent stem cells,including the expression of alkaline phosphatase and stemness maker genes,and could give rise to the three germ layer derivatives upon embryoid bodies assay. Microarray analysis confirmed the presence of typical stem cell gene expression profiles in all iPSC clones and did not identify any significant difference among reprogramming methods. In conclusion,the use of different reprogramming methods is equivalent and does not affect gene expression profile of the derived human iPSCs. View Publication

AIMS: Circular RNA (circRNA) is a newly validated class of single-stranded RNA,ubiquitously expressed in mammalian tissues and possessing key functions including acting as microRNA sponges and as transcriptional regulators by binding to RNA-binding proteins. While independent studies confirm the expression of circRNA in various tissue types,genome-wide circRNA expression in the heart has yet to be described in detail. METHODS AND RESULTS: We performed deep RNA-sequencing on ribosomal-depleted RNA isolated from 12 human hearts,25 mouse hearts and across a 28-day differentiation time-course of human embryonic stem cell-derived cardiomyocytes. Using purpose-designed bioinformatics tools,we uncovered a total of 15 318 and 3017 cardiac circRNA within human and mouse,respectively. Their abundance generally correlates with the abundance of their cognate linear RNA,but selected circRNAs exist at disproportionately higher abundance. Top highly expressed circRNA corresponded to key cardiac genes including Titin (TTN),RYR2,and DMD. The most abundant cardiac-expressed circRNA is a cytoplasmic localized single-exon circSLC8A1-1. The longest human transcript TTN alone generates up to 415 different exonic circRNA isoforms,the majority (83%) of which originates from the I-band domain. Finally,we confirmed the expression of selected cardiac circRNA by RT-PCR,Sanger sequencing and single molecule RNA-fluorescence in situ hybridization. CONCLUSIONS: Our data provide a detailed circRNA expression landscape in hearts. There is a high-abundance of specific cardiac-expressed circRNA. These findings open up a new avenue for future investigation into this emerging class of RNA. View Publication

Gaucher disease (GD) is caused by insufficient activity of acid $\$-glucosidase (GCase) resulting from mutations in GBA1. To understand the pathogenesis of the neuronopathic GD,induced pluripotent stem cells (iPSCs) were generated from fibroblasts isolated from three GD type 2 (GD2) and 2 unaffected (normal and GD carrier) individuals. The iPSCs were converted to neural precursor cells (NPCs) which were further differentiated into neurons. Parental GD2 fibroblasts as well as iPSCs,NPCs,and neurons had similar degrees of GCase deficiency. Lipid analyses showed increases of glucosylsphingosine and glucosylceramide in the GD2 cells. In addition,GD2 neurons showed increased $\$-synuclein protein compared to control neurons. Whole cell patch-clamping of the GD2 and control iPSCs-derived neurons demonstrated excitation characteristics of neurons,but intriguingly,those from GD2 exhibited consistently less negative resting membrane potentials with various degree of reduction in action potential amplitudes,sodium and potassium currents. Culture of control neurons in the presence of the GCase inhibitor (conduritol B epoxide) recapitulated these findings,providing a functional link between decreased GCase activity in GD and abnormal neuronal electrophysiological properties. To our knowledge,this study is first to report abnormal electrophysiological properties in GD2 iPSC-derived neurons that may underlie the neuropathic phenotype in Gaucher disease. View Publication

Neutrophils are the most abundant WBCs and have an essential role in the clearance of pathogens. Tight regulation of neutrophil numbers and their recruitment to sites of inflammation is critical in maintaining a balanced immune response. In various inflammatory conditions,such as rheumatoid arthritis,vasculitis,cystic fibrosis,and inflammatory bowel disease,increased serum G-CSF correlates with neutrophilia and enhanced neutrophil infiltration into inflamed tissues. We describe a fully human therapeutic anti-G-CSFR antibody (CSL324) that is safe and well tolerated when administered via i.v. infusion to cynomolgus macaques. CSL324 was effective in controlling G-CSF-mediated neutrophilia when administered either before or after G-CSF. A single ascending-dose study showed CSL324 did not alter steady-state neutrophil numbers,even at doses sufficient to completely prevent G-CSF-mediated neutrophilia. Weekly infusions of CSL324 (%10 mg/kg) for 3 wk completely neutralized G-CSF-mediated pSTAT3 phosphorylation without neutropenia. Moreover,repeat dosing up to 100 mg/kg for 12 wk did not result in neutropenia at any point,including the 12-wk follow-up after the last infusion. In addition,CSL324 had no observable effect on basic neutrophil functions,such as phagocytosis and oxidative burst. These data suggest that targeting G-CSFR may provide a safe and effective means of controlling G-CSF-mediated neutrophilia as observed in various inflammatory diseases. View Publication

Human induced pluripotent stem cells (hiPSCs) are being considered for use in understanding haematopoietic disorders and as a potential source of in vitro manufactured red cells. Here,we show that hiPSCs are able to recapitulate various stages of developmental erythropoiesis. We show that primitive erythroblasts arise first,express CD31(+) with CD235a(+),embryonic globins and red cell markers,but fail to express the hallmark red cell transcripts of adult erythropoiesis. When hiPSC-derived CD45(+) CD235a(-) haematopoietic progenitors are isolated on day 12 and further differentiated on OP9 stroma,they selectively express CD36(+) and CD235a(+),adult erythroid transcripts for transcription factors (e.g.,BCL11A,KLF1) and fetal/adult globins (HBG1/2,HBB). Importantly,hiPSC- and cord-derived CD36(+) CD235a(+) erythroblasts show a striking homology by transcriptome array profiling (only 306 transcripts with a 2Log fold change<1textperiodcentered5- or 2textperiodcentered8-fold). Phenotypic and transcriptome profiling of CD45(+) CD117(+) CD235a(+) pro-erythroblasts and terminally differentiated erythroblasts is also provided,including evidence of a HbF (fetal) to HbA (adult) haemoglobin switch and enucleation,that mirrors their definitive erythroblast cord-derived counterparts. These findings provide a molecular roadmap of developmental erythropoiesis from hiPSC sources at several critical stages,but also helps to inform on their use for clinical applications and modelling human haematopoietic disease. View Publication

BACKGROUND Pluripotent human embryonic stem cells (hESC) are a promising source of repopulating cardiomyocytes. We hypothesized that we could improve maturation of cardiomyocytes and facilitate electrical interconnections by creating a model that more closely resembles heart tissue; that is,containing both endothelial cells (ECs) and cardiomyocytes. METHODS We induced cardiomyocyte differentiation in the coculture of an hESC line expressing the cardiac reporter NKX2.5-green fluorescent protein (GFP),and an Akt-activated EC line (E4(+)ECs). We quantified spontaneous beating rates,synchrony,and coordination between different cardiomyocyte clusters using confocal imaging of Fura Red-detected calcium transients and computer-assisted image analysis. RESULTS After 8 days in culture,94% ± 6% of the NKX2-5GFP(+) cells were beating when hESCs embryonic bodies were plated on E4(+)ECs compared with 34% ± 12.9% for controls consisting of hESCs cultured on BD Matrigel (BD Biosciences) without ECs at Day 11 in culture. The spatial organization of beating areas in cocultures was different. The GFP(+) cardiomyocytes were close to the E4(+)ECs. The average beats/min of the cardiomyocytes in coculture was faster and closer to physiologic heart rates compared with controls (50 ± 14 [n = 13] vs 25 ± 9 [n = 8]; p < 0.05). The coculture with ECs led to synchronized beating relying on the endothelial network,as illustrated by the loss of synchronization upon the disruption of endothelial bridges. CONCLUSIONS The coculturing of differentiating cardiomyocytes with Akt-activated ECs but not EC-conditioned media results in (1) improved efficiency of the cardiomyocyte differentiation protocol and (2) increased maturity leading to better intercellular coupling with improved chronotropy and synchrony. View Publication

Mitochondrial disease is associated with a wide variety of clinical presentations,even among patients carrying heteroplasmic mitochondrial DNA (mtDNA) mutations,probably because of variations in mutant mtDNA proportions at the tissue and organ levels. Although several case reports and clinical trials have assessed the effectiveness of various types of drugs and supplements for the treatment of mitochondrial diseases,there are currently no cures for these conditions. In this study,we demonstrated for the first time that low dose resveratrol (RSV) ameliorated mitochondrial respiratory dysfunction in patient-derived fibroblasts carrying homoplasmic mtDNA mutations. Furthermore,low dose RSV also facilitated efficient cellular reprogramming of the patient-derived fibroblasts into induced pluripotent stem cells,partly due to improved cellular viability. Our results highlight the potential of RSV as a new therapeutic drug candidate for the treatment of mitochondrial diseases. View Publication

Although gene-gene interaction,or epistasis,plays a large role in complex traits in model organisms,genome-wide by genome-wide searches for two-way interaction have limited power in human studies. We thus used knowledge of a biological pathway in order to identify a contribution of epistasis to autism spectrum disorders (ASDs) in humans,a reverse-pathway genetic approach. Based on previous observation of increased ASD symptoms in Mendelian disorders of the Ras/MAPK pathway (RASopathies),we showed that common SNPs in RASopathy genes show enrichment for association signal in GWAS (P = 0.02). We then screened genome-wide for interactors with RASopathy gene SNPs and showed strong enrichment in ASD-affected individuals (P < 2.2 x 10-16),with a number of pairwise interactions meeting genome-wide criteria for significance. Finally,we utilized quantitative measures of ASD symptoms in RASopathy-affected individuals to perform modifier mapping via GWAS. One top region overlapped between these independent approaches,and we showed dysregulation of a gene in this region,GPR141,in a RASopathy neural cell line. We thus used orthogonal approaches to provide strong evidence for a contribution of epistasis to ASDs,confirm a role for the Ras/MAPK pathway in idiopathic ASDs,and to identify a convergent candidate gene that may interact with the Ras/MAPK pathway. View Publication

Endothelial progenitor cell (EPC) nomenclature remains ambiguous and there is a general lack of concordance in the stem cell field with many distinct cell subtypes continually grouped under the term EPC." It would be highly advantageous to agree on standards to confirm an endothelial progenitor phenotype and this should include detailed immunophenotyping potency assays and clear separation from hematopoietic angiogenic cells which are not endothelial progenitors. In this review we seek to discourage the indiscriminate use of "EPCs and instead propose precise terminology based on defining cellular phenotype and function. Endothelial colony forming cells and myeloid angiogenic cells are examples of two distinct and well-defined cell types that have been considered EPCs because they both promote vascular repair,albeit by completely different mechanisms of action. It is acknowledged that scientific nomenclature should be a dynamic process driven by technological and conceptual advances; ergo the ongoing EPC" nomenclature ought not to be permanent and should become more precise in the light of strong scientific evidence. This is especially important as these cells become recognized for their role in vascular repair in health and disease and in some cases progress toward use in cell therapy. Stem Cells Translational Medicine 2017;6:1316-1320. View Publication