技术资料

The use of human pluripotent cell progeny for cardiac disease modeling,drug testing and therapeutics requires the ability to efficiently induce pluripotent cells into the cardiomyogenic lineage. Although direct activation of the Activin-A and/or Bmp pathways with growth factors yields context-dependent success,recent studies have shown that induction of Wnt signaling using low molecular weight molecules such as CHIR,which in turn induces the Activin-A and Bmp pathways,is widely effective. To further enhance the reproducibility of CHIR-induced cardiomyogenesis,and to ultimately promote myocyte maturation,we are using exogenous growth factors to optimize cardiomyogenic signaling downstream of CHIR induction. As indicated by RNA-seq,induction with CHIR during Day 1 (Days 0-1) was followed by immediate expression of Nodal ligands and receptors,followed later by Bmp ligands and receptors. Co-induction with CHIR and high levels of the Nodal mimetic Activin-A (50-100 ng/ml) during Day 0-1 efficiently induced definitive endoderm,whereas CHIR supplemented with Activin-A at low levels (10 ng/ml) consistently improved cardiomyogenic efficiency,even when CHIR alone was ineffective. Moreover,co-induction using CHIR and low levels of Activin-A apparently increased the rate of cardiomyogenesis,as indicated by the initial appearance of rhythmically beating cells by Day 6 instead of Day 8. By contrast,co-induction with CHIR plus low levels (3-10 ng/ml) of Bmp4 during Day 0-1 consistently and strongly inhibited cardiomyogenesis. These findings,which demonstrate that cardiomyogenic efficacy is improved by optimizing levels of CHIR-induced growth factors when applied in accord with their sequence of endogenous expression,are consistent with the idea that Nodal (Activin-A) levels toggle the entry of cells into the endodermal or mesodermal lineages,while Bmp levels regulate subsequent allocation into mesodermal cell types. View Publication

Endogenous molecular and cellular mediators modulate tissue repair and regeneration. We have recently described antibody mediated osseous regeneration (AMOR) as a novel strategy for bioengineering bone in rat calvarial defect. This entails application of anti-BMP-2 antibodies capable of in vivo capturing of endogenous osteogenic BMPs (BMP-2,BMP-4,and BMP-7). The present study sought to investigate the feasibility of AMOR in other animal models. To that end,we examined the efficacy of a panel of anti-BMP-2 monoclonal antibodies (mAbs) and a polyclonal Ab immobilized on absorbable collagen sponge (ACS) to mediate bone regeneration within rabbit calvarial critical size defects. After 6 weeks,de novo bone formation was demonstrated by micro-CT imaging,histology,and histomorphometric analysis. Only certain anti-BMP-2 mAb clones mediated significant in vivo bone regeneration,suggesting that the epitopes with which anti-BMP-2 mAbs react are critical to AMOR. Increased localization of BMP-2 protein and expression of osteocalcin were observed within defects,suggesting accumulation of endogenous BMP-2 and/or increased de novo expression of BMP-2 protein within sites undergoing bone repair by AMOR. Considering the ultimate objective of translation of this therapeutic strategy in humans,preclinical studies will be necessary to demonstrate the feasibility of AMOR in progressively larger animal models. View Publication

Human induced pluripotent stem cells (hiPSCs) provide a platform for studying human disease in vitro,increase our understanding of human embryonic development,and provide clinically relevant cell types for transplantation,drug testing,and toxicology studies. Since their discovery,numerous advances have been made in order to eliminate issues such as vector integration into the host genome,low reprogramming efficiency,incomplete reprogramming and acquisition of genomic instabilities. One of the ways to achieve integration-free reprogramming is by using RNA-based Sendai virus. Here we describe a method to generate hiPSCs with Sendai virus in both feeder-free and feeder-dependent culture systems. Additionally,we illustrate methods by which to validate pluripotency of the resulting stem cell population. View Publication

The role of intermediate methylation states in DNA is unclear. Here,to comprehensively identify regions of intermediate methylation and their quantitative relationship with gene activity,we apply integrative and comparative epigenomics to 25 human primary cell and tissue samples. We report 18,452 intermediate methylation regions located near 36% of genes and enriched at enhancers,exons and DNase I hypersensitivity sites. Intermediate methylation regions average 57% methylation,are predominantly allele-independent and are conserved across individuals and between mouse and human,suggesting a conserved function. These regions have an intermediate level of active chromatin marks and their associated genes have intermediate transcriptional activity. Exonic intermediate methylation correlates with exon inclusion at a level between that of fully methylated and unmethylated exons,highlighting gene context-dependent functions. We conclude that intermediate DNA methylation is a conserved signature of gene regulation and exon usage. View Publication

UNLABELLED In the classical form of $\$1-antitrypsin deficiency (ATD),aberrant intracellular accumulation of misfolded mutant $\$1-antitrypsin Z (ATZ) in hepatocytes causes hepatic damage by a gain-of-function,proteotoxic" mechanism. Whereas some ATD patients develop severe liver disease (SLD) that necessitates liver transplantation� View Publication

The ketone bodies β-hydroxybutyrate (BHB) and acetoacetate (AcAc) support mammalian survival during states of energy deficit by serving as alternative sources of ATP. BHB levels are elevated by starvation,caloric restriction,high-intensity exercise,or the low-carbohydrate ketogenic diet. Prolonged fasting reduces inflammation; however,the impact that ketones and other alternative metabolic fuels produced during energy deficits have on the innate immune response is unknown. We report that BHB,but neither AcAc nor the structurally related short-chain fatty acids butyrate and acetate,suppresses activation of the NLRP3 inflammasome in response to urate crystals,ATP and lipotoxic fatty acids. BHB did not inhibit caspase-1 activation in response to pathogens that activate the NLR family,CARD domain containing 4 (NLRC4) or absent in melanoma 2 (AIM2) inflammasome and did not affect non-canonical caspase-11,inflammasome activation. Mechanistically,BHB inhibits the NLRP3 inflammasome by preventing K(+) efflux and reducing ASC oligomerization and speck formation. The inhibitory effects of BHB on NLRP3 are not dependent on chirality or starvation-regulated mechanisms like AMP-activated protein kinase (AMPK),reactive oxygen species (ROS),autophagy or glycolytic inhibition. BHB blocks the NLRP3 inflammasome without undergoing oxidation in the TCA cycle,and independently of uncoupling protein-2 (UCP2),sirtuin-2 (SIRT2),the G protein-coupled receptor GPR109A or hydrocaboxylic acid receptor 2 (HCAR2). BHB reduces NLRP3 inflammasome-mediated interleukin (IL)-1β and IL-18 production in human monocytes. In vivo,BHB or a ketogenic diet attenuates caspase-1 activation and IL-1β secretion in mouse models of NLRP3-mediated diseases such as Muckle-Wells syndrome,familial cold autoinflammatory syndrome and urate crystal-induced peritonitis. Our findings suggest that the anti-inflammatory effects of caloric restriction or ketogenic diets may be linked to BHB-mediated inhibition of the NLRP3 inflammasome. View Publication

The glial cell line-derived neurotrophic factor (GDNF) has an important role in neuronal survival through binding to the GFRα1 (GDNF family receptor alpha-1) receptor and activation of the receptor tyrosine kinase Ret. Transient brain ischemia alters the expression of the GDNF signaling machinery but whether the GDNF receptor proteins are also affected,and the functional consequences,have not been investigated. We found that excitotoxic stimulation of cultured hippocampal neurons leads to a calpain-dependent downregulation of the long isoform of Ret (Ret51),but no changes were observed for Ret9 or GFRα1 under the same conditions. Cleavage of Ret51 by calpains was selectively mediated by activation of the extrasynaptic pool of N-methyl-d-aspartate receptors and leads to the formation of a stable cleavage product. Calpain-mediated cleavage of Ret51 was also observed in hippocampal neurons subjected to transient oxygen and glucose deprivation (OGD),a model of global brain ischemia,as well as in the ischemic region in the cerebral cortex of mice exposed to transient middle cerebral artery occlusion. Although the reduction of Ret51 protein levels decreased the total GDNF-induced receptor activity (as determined by assessing total phospho-Ret51 protein levels) and their downstream signaling activity,the remaining receptors still showed an increase in phosphorylation after incubation of hippocampal neurons with GDNF. Furthermore,GDNF protected hippocampal neurons when present before,during or after OGD,and the effects under the latter conditions were more significant in neurons transfected with human Ret51. These results indicate that the loss of Ret51 in brain ischemia partially impairs the neuroprotective effects of GDNF. View Publication

BACKGROUND Definitive endoderm (DE) is one of the three germ layers which during in vivo vertebrate development gives rise to a variety of organs including liver,lungs,thyroid and pancreas; consequently efficient in vitro initiation of stem cell differentiation to DE cells is a prerequisite for successful cellular specification to subsequent DE-derived cell types [1,2]. In this study we present a novel approach to rapidly and efficiently down regulate pluripotency genes during initiation of differentiation to DE cells by addition of dimethyl sulfoxide (DMSO) to Activin A-based culture medium and report its effects on the downstream differentiation to hepatocyte-like cells. MATERIALS AND METHODS Human embryonic stem cells (hESC) were differentiated to DE using standard methods in medium supplemented with 100ng/ml of Activin A and compared to cultures where DE specification was additionally enhanced with different concentrations of DMSO. DE cells were subsequently primed to generate hepatic-like cells to investigate whether the addition of DMSO during formation of DE improved subsequent expression of hepatic markers. A combination of flow cytometry,real-time quantitative reverse PCR and immunofluorescence was applied throughout the differentiation process to monitor expression of pluripotency (POUF5/OCT4 & NANOG),definitive endoderm (SOX17,CXCR4 & GATA4) and hepatic (AFP & ALB) genes to generate differentiation stage-specific signatures. RESULTS Addition of DMSO to the Activin A-based medium during DE specification resulted in rapid down regulation of the pluripotency genes OCT4 and NANOG,accompanied by an increase expression of the DE genes SOX17,CXCR4 and GATA4. Importantly,the expression level of ALB in DMSO-treated cells was also higher than in cells which were differentiated to the DE stage via standard Activin A treatment. View Publication

The intestinal mucosa undergoes a continual process of proliferation,differentiation and apoptosis,which is regulated by multiple signaling pathways. Notch signaling is critical for the control of intestinal stem cell maintenance and differentiation. However,the precise mechanisms involved in the regulation of differentiation are not fully understood. Previously,we have shown that tuberous sclerosis 2 (TSC2) positively regulates the expression of the goblet cell differentiation marker,MUC2,in intestinal cells. Using transgenic mice constitutively expressing a dominant negative TSC2 allele,we observed that TSC2 inactivation increased mTORC1 and Notch activities,and altered differentiation throughout the intestinal epithelium,with a marked decrease in the goblet and Paneth cell lineages. Conversely,treatment of mice with either Notch inhibitor dibenzazepine (DBZ) or mTORC1 inhibitor rapamycin significantly attenuated the reduction of goblet and Paneth cells. Accordingly,knockdown of TSC2 activated,whereas knockdown of mTOR or treatment with rapamycin decreased,the activity of Notch signaling in the intestinal cell line LS174T. Importantly,our findings demonstrate that TSC2/mTORC1 signaling contributes to the maintenance of intestinal epithelium homeostasis by regulating Notch activity. View Publication

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes,smooth muscle cells (SMC),and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs,differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result,numerous strategies have been developed to derive CPCs from ESCs. In this protocol,differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs,ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus,CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs. View Publication

Neurogenin3 (NEUROG3) is a basic helix-loop-helix transcription factor required for development of the endocrine pancreas in mice. In contrast,humans with NEUROG3 mutations are born with endocrine pancreas function,calling into question whether NEUROG3 is required for human endocrine pancreas development. To test this directly,we generated human embryonic stem cell (hESC) lines where both alleles of NEUROG3 were disrupted using CRISPR/Cas9-mediated gene targeting. NEUROG3(-/-) hESC lines efficiently formed pancreatic progenitors but lacked detectible NEUROG3 protein and did not form endocrine cells in vitro. Moreover,NEUROG3(-/-) hESC lines were unable to form mature pancreatic endocrine cells after engraftment of PDX1(+)/NKX6.1(+) pancreatic progenitors into mice. In contrast,a 75-90% knockdown of NEUROG3 caused a reduction,but not a loss,of pancreatic endocrine cell development. We conclude that NEUROG3 is essential for endocrine pancreas development in humans and that as little as 10% NEUROG3 is sufficient for formation of pancreatic endocrine cells. View Publication

Factor induced reprogramming of fibroblasts is an orchestrated but inefficient process. At the epigenetic level,it results in drastic chromatin changes to erase the existing somatic memory" and to establish the pluripotent state. Accordingly� View Publication