技术资料

Data suggest that clinical applications of human induced pluripotent stem cells (hiPSCs) will be realized. Nonetheless,clinical applications will require hiPSCs that are free of exogenous DNA and that can be manufactured through Good Manufacturing Practice (GMP). Optimally,derivation of hiPSCs should be rapid and efficient in order to minimize manipulations,reduce potential for accumulation of mutations and minimize financial costs. Previous studies reported the use of modified synthetic mRNAs to reprogram fibroblasts to a pluripotent state. Here,we provide an optimized,fully chemically defined and feeder-free protocol for the derivation of hiPSCs using synthetic mRNAs. The protocol results in derivation of fully reprogrammed hiPSC lines from adult dermal fibroblasts in less than two weeks. The hiPSC lines were successfully tested for their identity,purity,stability and safety at a GMP facility and cryopreserved. To our knowledge,as a proof of principle,these are the first integration-free iPSCs lines that were reproducibly generated through synthetic mRNA reprogramming that could be putatively used for clinical purposes. View Publication

Integrating viruses represent robust tools for cellular reprogramming; however,the presence of viral transgenes in induced pluripotent stem cells (iPSCs) is deleterious because it holds the risk of insertional mutagenesis leading to malignant transformation. Here,we combine the robustness of lentiviral reprogramming with the efficacy of Cre recombinase protein transduction to derive iPSCs devoid of transgenes. By genome-wide analysis and targeted differentiation towards the cardiomyocyte lineage,we show that transgene-free iPSCs are superior to iPSCs before Cre transduction. Our study provides a simple,rapid and robust protocol for the generation of clinical-grade iPSCs suitable for disease modeling,tissue engineering and cell replacement therapies. View Publication

GANT61 is a small-molecule inhibitor of glioma-associated oncogene 1 (GLI1)- and GLI2-mediated transcription at the nuclear level that exerts its effect by preventing DNA binding. It has been demonstrated to induce cell death against Ewing's sarcoma family tumor (ESFT) cell lines in a dose-dependent manner. The most sensitive cell line was SK-N-LO,which expresses the EWS-FLI1 fusion gene. SK-N-LO cells treated with GANT61 showed cellular and nuclear morphological changes,including cell shrinkage,chromatin condensation and nuclear fragmentation,in a concentration-dependent manner,as visualized by Hoechst 33342 staining. Furthermore,annexin V-propidium iodide (PI) double-staining revealed a significant increase in the number of late apoptotic cells. GANT61 induced a significant decrease in the proportion of cells in the S phase. Significant decrease of the protein levels of GLI2,survivin,cyclin A and claspin,and significant increase of p21 expression was also observed in the cells treated with GANT61. Moreover,poly (ADP-ribose) polymerase (PARP) cleavage was observed,but no cleavage of caspase-3 or -7,or any change in the expressions of Bcl-2 or p53 were observed. These findings suggest that GANT61 induces cell death of SK-N-LO cells in a caspase-independent manner,by inhibiting DNA replication in the S phase. View Publication

As a critical tumor suppressor,p53 is inactivated in human cancer cells by somatic gene mutation or disruption of pathways required for its activation. Therefore,it is critical to elucidate the mechanism underlying p53 activation after genotoxic and cellular stresses. Accumulating evidence has indicated the importance of posttranslational modifications such as acetylation in regulating p53 stability and activity. However,the physiological roles of the eight identified acetylation events in regulating p53 responses remain to be fully understood. By employing homologous recombination,we introduced various combinations of missense mutations (lysine to arginine) into eight acetylation sites of the endogenous p53 gene in human embryonic stem cells (hESCs). By determining the p53 responses to DNA damage in the p53 knock-in mutant hESCs and their derivatives,we demonstrate physiological importance of the acetylation events within the core domain (K120 and K164) and at the C-terminus (K370/372/373/381/382/386) in regulating human p53 responses to DNA damage. View Publication

The development of human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) facilitates in vitro studies of human disease mechanisms,speeds up the process of drug screening,and raises the feasibility of using cell replacement therapy in clinics. However,the study of genotype-phenotype relationships in ESCs or iPSCs is hampered by the low efficiency of site-specific gene editing. Transcription activator-like effector nucleases (TALENs) spurred interest due to the ease of assembly,high efficiency and faithful gene targeting. In this study,we optimized the TALEN design to maximize its genomic cutting efficiency. We showed that using optimized TALENs in conjunction with single-strand oligodeoxynucleotide (ssODN) allowed efficient gene editing in human cells. Gene mutations and gene deletions for up to 7.8 kb can be accomplished at high efficiencies. We established human tumor cell lines and H9 ESC lines with homozygous deletion of the microRNA-21 (miR-21) gene and miR-9-2 gene. These cell lines provide a robust platform to dissect the roles these genes play during cell differentiation and tumorigenesis. We also observed that the endogenous homologous chromosome can serve as a donor template for gene editing. Overall,our studies demonstrate the versatility of using ssODN and TALEN to establish genetically modified cells for research and therapeutic application. View Publication

Targeted genome editing with engineered nucleases has transformed the ability to introduce precise sequence modifications at almost any site within the genome. A major obstacle to probing the efficiency and consequences of genome editing is that no existing method enables the frequency of different editing events to be simultaneously measured across a cell population at any endogenous genomic locus.We have developed a method for quantifying individual genome-editing outcomes at any site of interest with single-molecule real-time (SMRT) DNA sequencing. We show that this approach can be applied at various loci using multiple engineered nuclease platforms,including transcription-activator-like effector nucleases (TALENs),RNA-guided endonucleases (CRISPR/Cas9),and zinc finger nucleases (ZFNs),and in different cell lines to identify conditions and strategies in which the desired engineering outcome has occurred. This approach offers a technique for studying double-strand break repair,facilitates the evaluation of gene-editing technologies,and permits sensitive quantification of editing outcomes in almost every experimental system used. ?? 2014 The Authors. View Publication

The prevalence of hearing loss after damage to the mammalian cochlea has been thought to be due to a lack of spontaneous regeneration of hair cells,the primary receptor cells for sound. Here,we show that supporting cells,which surround hair cells in the normal cochlear epithelium,differentiate into new hair cells in the neonatal mouse following ototoxic damage. Using lineage tracing,we show that new hair cells,predominantly outer hair cells,arise from Lgr5-expressing inner pillar and third Deiters cells and that new hair cell generation is increased by pharmacological inhibition of Notch. These data suggest that the neonatal mammalian cochlea has some capacity for hair cell regeneration following damage alone and that Lgr5-positive cells act as hair cell progenitors in the cochlea. View Publication

Cancer stem cells (CSCs) are a promising target for treating cancer,yet how CSC plasticity is maintained in vivo is unclear and is difficult to study in vitro. Here we establish a sustainable primary culture of Oct3/4(+)/Nanog(+) lung CSCs fed with CD90(+) cancer-associated fibroblasts (CAFs) to further advance our knowledge of preserving stem cells in the tumour microenvironment. Using transcriptomics we identify the paracrine network by which CAFs enrich CSCs through de-differentiation and reacquisition of stem cell-like properties. Specifically,we find that IGF1R signalling activation in cancer cells in the presence of CAFs expressing IGF-II can induce Nanog expression and promote stemness. Moreover,this paracrine signalling predicts overall and relapse-free survival in stage I non-small cell lung cancer (NSCLC) patients. IGF-II/IGF1R signalling blockade inhibits Nanog expression and attenuates cancer stem cell features. Our data demonstrate that CAFs constitute a supporting niche for cancer stemness,and targeting this paracrine signalling may present a new therapeutic strategy for NSCLC. View Publication

TDP-43 is found in cytoplasmic inclusions in 95% of amyotrophic lateral sclerosis (ALS) and 60% of frontotemporal lobar degeneration (FTLD). Approximately 4% of familial ALS is caused by mutations in TDP-43. The majority of these mutations are found in the glycine-rich domain,including the variant M337V,which is one of the most common mutations in TDP-43. In order to investigate the use of allele-specific RNA interference (RNAi) as a potential therapeutic tool,we designed and screened a set of siRNAs that specifically target TDP-43(M337V) mutation. Two siRNA specifically silenced the M337V mutation in HEK293T cells transfected with GFP-TDP-43(wt) or GFP-TDP-43(M337V) or TDP-43 C-terminal fragments counterparts. C-terminal TDP-43 transfected cells show an increase of cytosolic inclusions,which are decreased after allele-specific siRNA in M337V cells. We then investigated the effects of one of these allele-specific siRNAs in induced pluripotent stem cells (iPSCs) derived from an ALS patient carrying the M337V mutation. These lines showed a two-fold increase in cytosolic TDP-43 compared to the control. Following transfection with the allele-specific siRNA,cytosolic TDP-43 was reduced by 30% compared to cells transfected with a scrambled siRNA. We conclude that RNA interference can be used to selectively target the TDP-43(M337V) allele in mammalian and patient cells,thus demonstrating the potential for using RNA interference as a therapeutic tool for ALS. View Publication

The putative behavioral effects of the enantiomers of BAY K 8644 and the behavioral responses to (+/-)-BAY K 8644 following chronic injection were assessed on motor function in mice. The interaction of the enantiomers of BAY K 8644 with mouse brain dihydropyridine binding sites was also evaluated. The calcium channel activating enantiomer (-)-S-BAY K 8644 impaired rotarod and motor activity with an ED50 value of 0.5 mg/kg. The calcium channel blocker enantiomer (+)-R-BAY K 8644 neither affected rotarod nor motor activity. (+)-R-BAY K 8644,and the structurally related dihydropyridine calcium channel blockers nifedipine and (-)-202-791 inhibited the impairment of rotarod activity by (-)-S-BAY K 8644 in a dose-dependent manner. (+/-)-BAY K 8644 produced convulsions in mice with a CD50 of 5 mg/kg. Chronic injection of (+/-)-BAY K 8644 (8 mg/kg IP once each day for four days) resulted in a significant tolerance to,and increase in recovery from,the motor deficits produced by (+/-)-BAY K 8644. Furthermore,chronic treatment with (+/-)-BAY K 8644 increased the onset time,but did not reduce the number of mice having convulsions to (+/-)-BAY K 8644. Chronic injection of nifedipine did not affect the motor deficit and convulsive activity of (+/-)-BAY K 8644. The behavioral effects of (+/-)-BAY K 8644 were observed at significant brain levels of drug. [3H]Nitrendipine binding to mouse brain dihydropyridine binding sites was unchanged in mice chronically injected with either (+/-)-BAY K 8644 or nifedipine.(ABSTRACT TRUNCATED AT 250 WORDS) View Publication

A major concern in Pluripotent Stem Cell (PSC)-derived cell replacement therapy is the risk of teratoma formation from contaminating undifferentiated cells. Removal of undifferentiated cells from differentiated cultures is an essential step before PSC-based cell therapies can be safely deployed in a clinical setting. We report a group of novel small molecules that are cytotoxic to PSCs. Our data indicates that these molecules are specific and potent in their activity allowing rapid eradication of undifferentiated cells. Experiments utilizing mixed PSC and primary human neuronal and cardiomyocyte cultures demonstrate that up to a 6-fold enrichment for specialized cells can be obtained without adversely affecting cell viability and function. Several structural variants were synthesized to identify key functional groups and to improve specificity and efficacy. Comparative microarray analysis and ensuing RNA knockdown studies revealed involvement of the PERK/ATF4/DDIT3 ER stress pathway. Surprisingly,cell death following ER stress induction was associated with a concomitant decrease in endogenous ROS levels in PSCs. Undifferentiated cells treated with these molecules preceding transplantation fail to form teratomas in SCID mice. Furthermore,these molecules remain non-toxic and non-teratogenic to zebrafish embryos suggesting that they may be safely used in vivo. View Publication

Obesity increases the risk of developing multiple myeloma (MM). Adiponectin is a cytokine produced by adipocytes,but paradoxically decreased in obesity,that has been implicated in MM progression. Herein,we evaluated how prolonged exposure to adiponectin affected the survival of MM cells as well as putative signaling mechanisms. Adiponectin activates protein kinase A (PKA),which leads to decreased AKT activity and increased AMP-activated protein kinase (AMPK) activation. AMPK,in turn,induces cell cycle arrest and apoptosis. Adiponectin-induced apoptosis may be mediated,at least in part,by the PKA/AMPK-dependent decline in the expression of the enzyme acetyl-CoA-carboxylase (ACC),which is essential to lipogenesis. Supplementation with palmitic acid,the preliminary end product of fatty acid synthesis,rescues MM cells from adiponectin-induced apoptosis. Furthermore,5-(tetradecyloxy)-2-furancarboxylic acid (TOFA),an ACC inhibitor,exhibited potent antiproliferative effects on MM cells that could also be inhibited by fatty acid supplementation. Thus,adiponectin's ability to reduce survival of MM cells appears to be mediated through its ability to suppress lipogenesis. Our findings suggest that PKA/AMPK pathway activators,or inhibitors of ACC,may be useful adjuvants to treat MM. Moreover,the antimyeloma effect of adiponectin supports the concept that hypoadiponectinemia,as occurs in obesity,promotes MM tumor progression. View Publication