技术资料

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

Embryonic stem (ES) and induced-pluripotent stem (iPS) cells can be grown indefinitely under appropriate conditions whilst retaining the ability to differentiate to cells representative of the three primary germ layers. Such cells have the potential to revolutionize medicine by offering treatment options for a wide range of diseases and disorders as well as providing a model system for elucidating mechanisms involved in development and disease. In recent years,evidence for the function of E-cadherin in regulating pluripotent and self-renewal signaling pathways in ES and iPS cells has emerged. In this review,we discuss the function of E-cadherin and its interacting partners in the context of development and disease. We then describe relevant literature highlighting the function of E-cadherin in establishing and maintaining pluripotent and self-renewal properties of ES and iPS cells. In addition,we present experimental data demonstrating that exposure of human ES cells to the E-cadherin neutralizing antibody SHE78.7 allows culture of these cells in the absence of FGF2-supplemented medium. View Publication

Transcription factors NANOG,OCT4,and SOX2 regulate self-renewal and pluripotency in human embryonic stem (hES) cells; however,their expression profiles during early differentiation of hES cells are unclear. In this study,we used multiparameter flow cytometric assay to detect all three transcription factors (NANOG,OCT4,and SOX2) simultaneously at single cell level and monitored the changes in their expression during early differentiation towards endodermal lineage (induced by sodium butyrate). We observed at least four distinct populations of hES cells,characterized by specific expression patterns of NANOG,OCT4,and SOX2 and differentiation markers. Our results show that a single cell can express both differentiation and pluripotency markers at the same time,indicating a gradual mode of developmental transition in these cells. Notably,distinct regulation of SOX2 during early differentiation events was detected,highlighting the potential importance of this transcription factor for self-renewal of hES cells during differentiation. View Publication

Post-translational modification of histones is fundamental to the regulation of basic nuclear processes and subsequent cellular events,including differentiation. In this study,we analyzed acetylated forms of histones H2A,H2B,and H4 during induced differentiation in mouse (mESCs) and human (hESCs) embryonic stem cells and during induced enterocytic differentiation of colon cancer cells in vitro. Endoderm-like differentiation of mESCs induced by retinoic acid and enterocytic differentiation induced by histone deacetylase inhibitor sodium butyrate were accompanied by increased mono-,di-,and tri-acetylation of histone H2B and a pronounced increase in di- and tri-acetylation of histone H4. In enterocytes,mono-acetylation of histone H2A also increased and tetra-acetylation of histone H4 appeared only after induction of this differentiation pathway. During differentiation of hESCs,we observed increased mono-acetylation and decreased tri-acetylation of H2B. Mono-,di-,and tri-acetylation of H4 were reduced,manifested by a significant increase in nonacetylated H4 histones. Levels of acetylated histones increased during induced differentiation in mESCs and during histone deacetylase (HDAC) inhibitor-induced enterocytic differentiation,whereas differentiation of human ESCs was associated with reduced acetylation of histones H2B and H4. 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

PURPOSE Demonstrate a novel manufacturing method to generate extracellular matrix scaffolds from cardiac fibroblasts (CF-ECM) as a therapeutic mesenchymal stem cell-transfer device. MATERIALS AND METHODS Rat CF were cultured at high-density (˜1.6×10(5)/cm(2)) for 10-14 days. Cell sheets were removed from the culture dish by incubation with EDTA and decellularized with water and peracetic acid. CF-ECM was characterized by mass spectrometry,immunofluorescence and scanning electron microscopy. CF-ECM seeded with human embryonic stem cell derived mesenchymal stromal cells (hEMSCs) were transferred into a mouse myocardial infarction model. 48 hours later,mouse hearts were excised and examined for CF-ECM scaffold retention and cell transfer. RESULTS CF-ECM scaffolds are composed of fibronectin (82%),collagens type I (13%),type III (3.4%),type V (0.2%),type II (0.1%) elastin (1.3%) and 18 non-structural bioactive molecules. Scaffolds remained intact on the mouse heart for 48 hours without the use of sutures or glue. Identified hEMSCs were distributed from the epicardium to the endocardium. CONCLUSIONS High density cardiac fibroblast culture can be used to generate CF-ECM scaffolds. CF-ECM scaffolds seeded with hEMSCs can be maintained on the heart without suture or glue. hEMSC are successfully delivered throughout the myocardium. View Publication

Magnesium (Mg) is a promising conductive metallic biomaterial due to its desirable mechanical properties for load bearing and biodegradability in human body. Controlling the rapid degradation of Mg in physiological environment continues to be the key challenge toward clinical translation. In this study,we investigated the effects of conductive poly(3,4-ethylenedioxythiophene) (PEDOT) coating on the degradation behavior of Mg substrates and their cytocompatibility. Human embryonic stem cells (hESCs) were used as the in vitro model system to study cellular responses to Mg degradation because they are sensitive and can potentially differentiate into many cell types of interest (e.g.,neurons) for regenerative medicine. The PEDOT was deposited on Mg substrates using electrochemical deposition. The greater number of cyclic voltammetry (CV) cycles yielded thicker PEDOT coatings on Mg substrates. Specifically,the coatings produced by 2,5,and 10 CV cycles (denoted as 2×-PEDOT-Mg,5×-PEDOT-Mg,and 10×-PEDOT-Mg) had an average thickness of 31,63,and 78 µm,respectively. Compared with non-coated Mg samples,all PEDOT coated Mg samples showed slower degradation rates,as indicated by Tafel test results and Mg ion concentrations in the post-culture media. The 5×-PEDOT-Mg showed the best coating adhesion and slowest Mg degradation among the tested samples. Moreover,hESCs survived for the longest period when cultured with the 5×-PEDOT-Mg samples compared with the non-coated Mg and 2×-PEDOT-Mg. Overall,the results of this study showed promise in using PEDOT coating on biodegradable Mg-based implants for potential neural recording,stimulation and tissue engineering applications,thus encouraging further research. View Publication

It has been suggested that the isolation of scalable populations of limbal stem cells may lead to radical changes in ocular therapy. In particular,the derivation and transplantation of corneal stem cells from these populations may result in therapies providing clinical normality of the diseased or damaged cornea. Although feasible in theory,the lack of donor material in sufficient quantity and quality currently limits such a strategy. A potential scalable source of corneal cells could be derived from pluripotent stem cells (PSCs). We developed an in vitro and serum-free corneal differentiation model which displays significant promise. Our stepwise differentiation model was designed with reference to development and gave rise to cells which displayed similarities to epithelial progenitor cells which can be specified to cells displaying a corneal epithelial phenotype. We believe our approach is novel,provides a robust model of human development and in the future,may facilitate the generation of corneal epithelial cells that are suitable for clinical use. Additionally,we demonstrate that following continued cell culture,stem cell-derived corneal epithelial cells undergo transdifferentiation and exhibit squamous metaplasia and therefore,also offer an in vitro model of disease. View Publication

Vascular smooth muscle cells (SMCs) arise from diverse developmental origins. Regional distribution of vascular diseases may,in part,be attributed to this inherent heterogeneity in SMC lineage. Therefore,systems for generating human SMC subtypes of distinct embryonic origins would represent useful platforms for studying the influence of SMC lineage on the spatial specificity of vascular disease. Here we describe how human pluripotent stem cells can be differentiated into distinct populations of SMC subtypes under chemically defined conditions. The initial stage (days 0-5 or 0-7) begins with the induction of three intermediate lineages: neuroectoderm,lateral plate mesoderm and paraxial mesoderm. Subsequently,these precursor lineages are differentiated into contractile SMCs (days 5-19+). At key stages,the emergence of lineage-specific markers confirms recapitulation of embryonic developmental pathways and generation of functionally distinct SMC subtypes. The ability to derive an unlimited supply of human SMCs will accelerate applications in regenerative medicine and disease modeling. 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