技术资料

Maternally inherited mitochondrial (mt)DNA mutations can cause fatal or severely debilitating syndromes in children,with disease severity dependent on the specific gene mutation and the ratio of mutant to wild-type mtDNA (heteroplasmy) in each cell and tissue. Pathogenic mtDNA mutations are relatively common,with an estimated 778 affected children born each year in the United States. Mitochondrial replacement therapies or techniques (MRT) circumventing mother-to-child mtDNA disease transmission involve replacement of oocyte maternal mtDNA. Here we report MRT outcomes in several families with common mtDNA syndromes. The mother's oocytes were of normal quality and mutation levels correlated with those in existing children. Efficient replacement of oocyte mutant mtDNA was performed by spindle transfer,resulting in embryos containing<99% donor mtDNA. Donor mtDNA was stably maintained in embryonic stem cells (ES cells) derived from most embryos. However,some ES cell lines demonstrated gradual loss of donor mtDNA and reversal to the maternal haplotype. In evaluating donor-to-maternal mtDNA interactions,it seems that compatibility relates to mtDNA replication efficiency rather than to mismatch or oxidative phosphorylation dysfunction. We identify a polymorphism within the conserved sequence box II region of the D-loop as a plausible cause of preferential replication of specific mtDNA haplotypes. In addition,some haplotypes confer proliferative and growth advantages to cells. Hence,we propose a matching paradigm for selecting compatible donor mtDNA for MRT. View Publication

Myocardial ischemia-reperfusion (I/R) injury is unavoidable during cardioplegic arrest and open-heart surgery. Danshen is one of the most popular traditional herbal medicines in China,which has entered the Food and Drug Administration-approved phase III clinical trial. This study was aimed to develop a human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) model to mimic I/R injury and evaluate the cardioprotective effect of regular cardioplegic solution with Danshen. hiPSC-CMs were cultured with the crystalloid cardioplegic solution (Thomas group) and Thomas solution with 2 or 10 µg/mL Danshen (Thomas plus Danshen groups). The cells under normoxic culture condition served as baseline group. Then,the cells were placed in a modular incubator chamber. After 45 min hypoxia and 3 h reoxygenation,hiPSC-CMs subjected to hypoxia/reoxygenation resulted in a sharp increase of reactive oxygen species (ROS) content in Thomas group versus baseline group. Compared with the Thomas group,ROS accumulation was significant suppressed in Thomas plus Danshen groups,which might result from elevating the content of glutathione and enhanced activities of superoxide dismutase and glutathione peroxidase. The enhanced L-type Ca(2+) current in hiPSC-CMs after I/R injury was also significantly decreased by Danshen,and meanwhile intracellular Ca(2+) level was reduced and calcium overload was suppressed. Thomas plus Danshen groups also presented less irregular transients and lower apoptosis rates. As a result,Danshen could improve antioxidant and calcium handling in cardiomyocytes during I/R and lead to reduced arrhythmia events and apoptosis rates. hiPSC-CMs model offered a platform for the future translational study of the cardioplegia. View Publication

Human embryonic stem cell (hESC) line chHES-468 was derived from abnormal blastocyst donated by polycystic kidney syndrome (PKD) patient after preimplantation genetic diagnosis (PGD) treatment. DNA sequencing analysis confirmed that chHES-468 cell line carried a heterozygous mutation,c.1052610527delAG,of PKD1. Characteristic tests proved that the chHES-468 cell line presented typical markers of pluripotency and had the capability to form the three germ layers both in vitro and in vivo. View Publication

Pentraxin-2 (PTX-2),also known as serum amyloid P component (SAP/APCS),is a constitutive,antiinflammatory,innate immune plasma protein whose circulating level is decreased in chronic human fibrotic diseases. Here we show that recombinant human PTX-2 (rhPTX-2) retards progression of chronic kidney disease in Col4a3 mutant mice with Alport syndrome,reducing blood markers of kidney failure,enhancing lifespan by 20%,and improving histological signs of disease. Exogenously delivered rhPTX-2 was detected in macrophages but also in tubular epithelial cells,where it counteracted macrophage activation and was cytoprotective for the epithelium. Computational analysis of genes regulated by rhPTX-2 identified the transcriptional regulator c-Jun along with its activator protein-1 (AP-1) binding partners as a central target for the function of rhPTX-2. Accordingly,PTX-2 attenuates c-Jun and AP-1 activity,and reduces expression of AP-1-dependent inflammatory genes in both monocytes and epithelium. Our studies therefore identify rhPTX-2 as a potential therapy for chronic fibrotic disease of the kidney and an important inhibitor of pathological c-Jun signaling in this setting. View Publication

Mutation of highly conserved residues in transcription factors may affect protein-protein or protein-DNA interactions,leading to gene network dysregulation and human disease. Human mutations in GATA4,a cardiogenic transcription factor,cause cardiac septal defects and cardiomyopathy. Here,iPS-derived cardiomyocytes from subjects with a heterozygous GATA4-G296S missense mutation showed impaired contractility,calcium handling,and metabolic activity. In human cardiomyocytes,GATA4 broadly co-occupied cardiac enhancers with TBX5,another transcription factor that causes septal defects when mutated. The GATA4-G296S mutation disrupted TBX5 recruitment,particularly to cardiac super-enhancers,concomitant with dysregulation of genes related to the phenotypic abnormalities,including cardiac septation. Conversely,the GATA4-G296S mutation led to failure of GATA4 and TBX5-mediated repression at non-cardiac genes and enhanced open chromatin states at endothelial/endocardial promoters. These results reveal how disease-causing missense mutations can disrupt transcriptional cooperativity,leading to aberrant chromatin states and cellular dysfunction,including those related to morphogenetic defects. View Publication

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder in which the MECP2 (methyl CpG-binding protein 2) gene is mutated. Recent studies showed that RTT-derived neurons have many cellular deficits when compared to control,such as: less synapses,lower dendritic arborization and reduced spine density. Interestingly,treatment of RTT-derived neurons with Insulin-like Growth Factor 1 (IGF1) could rescue some of these cellular phenotypes. Given the critical role of IGF1 during neurodevelopment,the present study used human induced pluripotent stem cells (iPSCs) from RTT and control individuals to investigate the gene expression profile of IGF1 and IGF1R on different developmental stages of differentiation. We found that the thyroid hormone receptor (TRalpha 3) has a differential expression profile. Thyroid hormone is critical for normal brain development. Our results showed that there is a possible link between IGF1/IGF1R and the TRalpha 3 and that over expression of IGF1R in RTT cells may be the cause of neurites improvement in neural RTT-derived neurons. View Publication

The study and application of human pluripotent stem cells (hPSCs) will be enhanced by the availability of well-characterised monoclonal antibodies (mAbs) detecting cell-surface epitopes. Here we report generation of seven new mAbs that detect cell surface proteins present on live and fixed human ES cells (hESCs) and human iPS cells (hiPSCs),confirming our previous prediction that these proteins were present on the cell surface of hPSCs. The mAbs all show a high correlation with POU5F1 (OCT4) expression and other hPSC surface markers (TRA-160 and SSEA-4) in hPSC cultures and detect rare OCT4 positive cells in differentiated cell cultures. These mAbs are immunoreactive to cell surface protein epitopes on both primed and naive state hPSCs,providing useful research tools to investigate the cellular mechanisms underlying human pluripotency and states of cellular reprogramming. In addition,we report that subsets of the seven new mAbs are also immunoreactive to human bone marrow-derived mesenchymal stem cells (MSCs),normal human breast subsets and both normal and tumorigenic colorectal cell populations. The mAbs reported here should accelerate the investigation of the nature of pluripotency,and enable development of robust cell separation and tracing technologies to enrich or deplete for hPSCs and other human stem and somatic cell types. This article is protected by copyright. All rights reserved. View Publication

Variability in induced pluripotent stem cell (iPSC) lines remains a concern for disease modeling and regenerative medicine. We have used RNA-sequencing analysis and linear mixed models to examine the sources of gene expression variability in 317 human iPSC lines from 101 individuals. We found that ∼50% of genome-wide expression variability is explained by variation across individuals and identified a set of expression quantitative trait loci that contribute to this variation. These analyses coupled with allele-specific expression show that iPSCs retain a donor-specific gene expression pattern. Network,pathway,and key driver analyses showed that Polycomb targets contribute significantly to the non-genetic variability seen within and across individuals,highlighting this chromatin regulator as a likely source of reprogramming-based variability. Our findings therefore shed light on variation between iPSC lines and illustrate the potential for our dataset and other similar large-scale analyses to identify underlying drivers relevant to iPSC applications. View Publication

Postcardiac arrest acidosis can decrease survival. Effective medications without adverse side effects are still not well characterized. We aimed to analyze whether early administration of glutamine could improve survival and protect cardiomyocytes from postcardiac arrest acidosis using animal and cell models. Forty Wistar rats with postcardiac arrest acidosis (blood pH textless 7.2) were included. They were divided into study (500 mg/kg L-alanyl-L-glutamine,n = 20) and control (normal saline,n = 20) groups. Each of the rats received resuscitation. The outcomes were compared between the two groups. In addition,cardiomyocytes derived from human induced pluripotent stem cells were exposed to HBSS with different pH levels (7.3 or 6.5) or to culture medium (control). Apoptosis-related markers and beating function were analyzed. We found that the duration of survival was significantly longer in the study group (p textless 0.05). In addition,in pH 6.5 or pH 7.3 HBSS buffer,the expression levels of cell stress (p53) and apoptosis (caspase-3,Bcl-xL) markers were significantly lower in cardiomyocytes treated with 50 mM L-glutamine than those without L-glutamine (RT-PCR). L-glutamine also increased the beating function of cardiomyocytes,especially at the lower pH level (6.5). More importantly,glutamine decreased cardiomyocyte apoptosis and increased these cells' beating function at a low pH level. View Publication

Biochemical factors can help reprogram somatic cells into pluripotent stem cells,yet the role of biophysical factors during reprogramming is unknown. Here,we show that biophysical cues,in the form of parallel microgrooves on the surface of cell-adhesive substrates,can replace the effects of small-molecule epigenetic modifiers and significantly improve reprogramming efficiency. The mechanism relies on the mechanomodulation of the cells' epigenetic state. Specifically,decreased histone deacetylase activity and upregulation of the expression of WD repeat domain 5 (WDR5)—a subunit of H3 methyltranferase—by microgrooved surfaces lead to increased histone H3 acetylation and methylation. We also show that microtopography promotes a mesenchymal-to-epithelial transition in adult fibroblasts. Nanofibrous scaffolds with aligned fibre orientation produce effects similar to those produced by microgrooves,suggesting that changes in cell morphology may be responsible for modulation of the epigenetic state. These findings have important implications in cell biology and in the optimization of biomaterials for cell-engineering applications. View Publication

BACKGROUND: A few reports suggested that low levels of Wnt signaling might drive cell reprogramming,but these studies could not establish a clear relationship between Wnt signaling and self-renewal networks. There are ongoing debates as to whether and how the Wnt/β-catenin signaling is involved in the control of pluripotency gene networks. Additionally,whether physiological β-catenin signaling generates stem-like cells through interactions with other pathways is as yet unclear. The nasopharyngeal carcinoma HONE1 cells have low expression of β-catenin and wild-type expression of p53,which provided a possibility to study regulatory mechanism of stemness networks induced by physiological levels of Wnt signaling in these cells.backslashnbackslashnRESULTS: Introduction of increased β-catenin signaling,haploid expression of β-catenin under control by its natural regulators in transferred chromosome 3,resulted in activation of Wnt/β-catenin networks and dedifferentiation in HONE1 hybrid cell lines,but not in esophageal carcinoma SLMT1 hybrid cells that had high levels of endogenous β-catenin expression. HONE1 hybrid cells displayed stem cell-like properties,including enhancement of CD24(+) and CD44(+) populations and generation of spheres that were not observed in parental HONE1 cells. Signaling cascades were detected in HONE1 hybrid cells,including activation of p53- and RB1-mediated tumor suppressor pathways,up-regulation of Nanog-,Oct4-,Sox2-,and Klf4-mediated pluripotency networks,and altered E-cadherin expression in both in vitro and in vivo assays. qPCR array analyses further revealed interactions of physiological Wnt/β-catenin signaling with other pathways such as epithelial-mesenchymal transition,TGF-β,Activin,BMPR,FGFR2,and LIFR- and IL6ST-mediated cell self-renewal networks. Using β-catenin shRNA inhibitory assays,a dominant role for β-catenin in these cellular network activities was observed. The expression of cell surface markers such as CD9,CD24,CD44,CD90,and CD133 in generated spheres was progressively up-regulated compared to HONE1 hybrid cells. Thirty-four up-regulated components of the Wnt pathway were identified in these spheres.backslashnbackslashnCONCLUSIONS: Wnt/β-catenin signaling regulates self-renewal networks and plays a central role in the control of pluripotency genes,tumor suppressive pathways and expression of cancer stem cell markers. This current study provides a novel platform to investigate the interaction of physiological Wnt/β-catenin signaling with stemness transition networks. View Publication

RATIONALE: Human embryonic stem cell (hESC) derivatives are attractive candidates for therapeutic use. The engraftment and survival of hESC derivatives as xenografts or allografts require effective immunosuppression to prevent immune cell infiltration and graft destruction.backslashnbackslashnOBJECTIVE: To test the hypothesis that a short-course,dual-agent regimen of two costimulation-adhesion blockade agents can induce better engraftment of hESC derivatives compared to current immunosuppressive agents.backslashnbackslashnMETHODS AND RESULTS: We transduced hESCs with a double fusion reporter gene construct expressing firefly luciferase (Fluc) and enhanced green fluorescent protein,and differentiated these cells to endothelial cells (hESC-ECs). Reporter gene expression enabled longitudinal assessment of cell engraftment by bioluminescence imaging. Costimulation-adhesion therapy resulted in superior hESC-EC and mouse EC engraftment compared to cyclosporine therapy in a hind limb model. Costimulation-adhesion therapy also promoted robust hESC-EC and hESC-derived cardiomyocyte survival in an ischemic myocardial injury model. Improved hESC-EC engraftment had a cardioprotective effect after myocardial injury,as assessed by magnetic resonance imaging. Mechanistically,costimulation-adhesion therapy is associated with systemic and intragraft upregulation of T-cell immunoglobulin and mucin domain 3 (TIM3) and a reduced proinflammatory cytokine profile.backslashnbackslashnCONCLUSIONS: Costimulation-adhesion therapy is a superior alternative to current clinical immunosuppressive strategies for preventing the post-transplant rejection of hESC derivatives. By extending the window for cellular engraftment,costimulation-adhesion therapy enhances functional preservation following ischemic injury. This regimen may function through a TIM3-dependent mechanism. View Publication