技术资料

Developing an efficient culture system for controlled human pluripotent stem cell (hPSC) differentiation into selected lineages is a major challenge in realizing stem cell-based clinical applications. Here,we report the use of chitin-alginate 3D microfibrous scaffolds,previously developed for hPSC propagation,to support efficient neuronal differentiation and maturation under chemically defined culture conditions. When treated with neural induction medium containing Noggin/retinoic acid,the encapsulated cells expressed much higher levels of neural progenitor markers SOX1 and PAX6 than those in other treatment conditions. Immunocytochemisty analysis confirmed that the majority of the differentiated cells were nestin-positive cells. Subsequently transferring the scaffolds to neuronal differentiation medium efficiently directed these encapsulated neural progenitors into mature neurons,as detected by RT-PCR and positive immunostaining for neuron markers βIII tubulin and MAP2. Furthermore,flow cytometry confirmed that textgreater90% βIII tubulin-positive neurons was achieved for three independent iPSC and hESC lines,a differentiation efficiency much higher than previously reported. Implantation of these terminally differentiated neurons into SCID mice yielded successful neural grafts comprising MAP2 positive neurons,without tumorigenesis,suggesting a potential safe cell source for regenerative medicine. These results bring us one step closer toward realizing large-scale production of stem cell derivatives for clinical and translational applications. View Publication

AIM: The generation and characterization of a human embryonic stem cell (hESC) line stably expressing red fluorescent mCherry protein.backslashnbackslashnMETHODS: Lentiviral transduction of a ubiquitously-expressed human EF-1α promoter driven mCherry transgene was performed in MEL2 hESC. Red fluore-scence was assessed by immunofluorescence and flow cytometry. Pluripotency of stably transduced hESC was determined by immunofluorescent pluripotency marker expression,flow cytometry,teratoma assays and embryoid body-based differentiation followed by reverse transcriptase-polymerase chain reaction. Quantification of cell motility and survival was performed with time lapse microscopy.backslashnbackslashnRESULTS: Constitutively fluorescently-labeled hESCs are useful tools for facile in vitro and in vivo tracking of survival,motility and cell spreading on various surfaces before and after differentiation. Here we describe the generation and characterization of a hESC line (MEL2) stably expressing red fluorescent protein,mCherry. This line was generated by random integration of a fluorescent protein-expressing cassette,driven by the ubiquitously-expressed human EF-1α promoter. Stably transfected MEL2-mCherry hESC were shown to express pluripotency markers in the nucleus (POU5F1/OCT4,NANOG and SOX2) and on the cell surface (SSEA4,TRA1-60 and TG30/CD9) and were shown to maintain a normal karyotype in long-term (for at least 35 passages) culture. MEL2-mCherry hESC further readily differentiated into representative cell types of the three germ layers in embryoid body and teratoma based assays and,importantly,maintained robust mCherry expression throughout differentiation. The cell line was next adapted to single-cell passaging,rendering it compatible with numerous bioengineering applications such as measurement of cell motility and cell spreading on various protein modified surfaces,quantification of cell attachment to nanoparticles and rapid estimation of cell survival.backslashnbackslashnCONCLUSION: The MEL2-mCherry hESC line conforms to the criteria of bona fide pluripotent stem cells and maintains red fluorescence throughout differentiation,making it a useful tool for bioengineering and in vivo tracking experiments. View Publication

Electronic cigarettes (EC) and refill fluids are distributed with little information on their pre- and postnatal health effects. This study compares the cytotoxicity of EC refill fluids using embryonic and adult cells and examines the chemical characteristics of refill fluids using HPLC. Refill solutions were tested on human embryonic stem cells (hESC),mouse neural stem cells (mNSC),and human pulmonary fibroblasts (hPF) using the MTT assay,and NOAELs and IC50s were determined from dose-response curves. Spectral analysis was performed when products of the same flavor had different MTT outcomes. hESC and mNSC were generally more sensitive to refill solutions than hPF. All products from one company were cytotoxic to hESC and mNSC,but non-cytotoxic to hPF. Cytotoxicity was not due to nicotine,but was correlated with the number and concentration of chemicals used to flavor fluids. Additional studies are needed to fully assess the prenatal effect of refill fluids. ?? 2012 Elsevier Inc. View Publication

Oxidative stress contributes to tissue injury and cell death during the development of various diseases. The present study aims at investigating whether oxidative stress triggered by the exposure to hydrogen peroxide (H2O2) can induce apoptosis of induced pluripotent stem cells (iPS cells) in a mechanism mediated by insulin-like growth factor (IGF-1) and microRNA-1 (miR-1). iPS cells treated with H2O2 showed increases in miR-1 expression,mitochondria dysfunction,cytochrome-c release and apoptosis,Addition of IGF-1 into the iPS cell cultures reduced the H2O2 cytotoxicity. Prediction algorithms showed that 3'-untranslated regions of IGF-1 gene as a target of miR-1. Moreover,miR-1 mimic,but not miR-1 mimic negative control,diminished the protective effect of IGF-1 on H2O2-induced mitochondrial dysfunction,cytochrome-c release and apoptosis in iPS cells. In conclusion,IGF-1 inhibits H2O2-induced mitochondrial dysfunction,cytochrome-c release and apoptosis. IGF-1's effect is,at least partially,regulated by miR-1 in iPS cells. ?? 2012 Elsevier Inc. View Publication

Embryonic stem cells can replicate continuously in the absence of senescence and,therefore,are immortal in culture. Although genome stability is essential for the survival of stem cells,proteome stability may have an equally important role in stem-cell identity and function. Furthermore,with the asymmetric divisions invoked by stem cells,the passage of damaged proteins to daughter cells could potentially destroy the resulting lineage of cells. Therefore,a firm understanding of how stem cells maintain their proteome is of central importance. Here we show that human embryonic stem cells (hESCs) exhibit high proteasome activity that is correlated with increased levels of the 19S proteasome subunit PSMD11 (known as RPN-6 in Caenorhabditis elegans) and a corresponding increased assembly of the 26S/30S proteasome. Ectopic expression of PSMD11 is sufficient to increase proteasome assembly and activity. FOXO4,an insulin/insulin-like growth factor-I (IGF-I) responsive transcription factor associated with long lifespan in invertebrates,regulates proteasome activity by modulating the expression of PSMD11 in hESCs. Proteasome inhibition in hESCs affects the expression of pluripotency markers and the levels of specific markers of the distinct germ layers. Our results suggest a new regulation of proteostasis in hESCs that links longevity and stress resistance in invertebrates to hESC function and identity. View Publication

RATIONALE: Molecular imaging has proven to be a vital tool in the characterization of stem cell behavior in vivo. However,the integration of reporter genes has typically relied on random integration,a method that is associated with unwanted insertional mutagenesis and positional effects on transgene expression.backslashnbackslashnOBJECTIVE: To address this barrier,we used genome editing with zinc finger nuclease (ZFN) technology to integrate reporter genes into a safe harbor gene locus (PPP1R12C,also known as AAVS1) in the genome of human embryonic stem cells and human induced pluripotent stem cells for molecular imaging.backslashnbackslashnMETHODS AND RESULTS: We used ZFN technology to integrate a construct containing monomeric red fluorescent protein,firefly luciferase,and herpes simplex virus thymidine kinase reporter genes driven by a constitutive ubiquitin promoter into a safe harbor locus for fluorescence imaging,bioluminescence imaging,and positron emission tomography imaging,respectively. High efficiency of ZFN-mediated targeted integration was achieved in both human embryonic stem cells and induced pluripotent stem cells. ZFN-edited cells maintained both pluripotency and long-term reporter gene expression. Functionally,we successfully tracked the survival of ZFN-edited human embryonic stem cells and their differentiated cardiomyocytes and endothelial cells in murine models,demonstrating the use of ZFN-edited cells for preclinical studies in regenerative medicine.backslashnbackslashnCONCLUSION: Our study demonstrates a novel application of ZFN technology to the targeted genetic engineering of human pluripotent stem cells and their progeny for molecular imaging in vitro and in vivo. View Publication

BACKGROUND: Human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) are a promising source of cells for regenerating myocardium. However,several issues,especially the large-scale preparation of hiPS-CMs and elimination of undifferentiated iPS cells,must be resolved before hiPS cells can be used clinically. The cell-sheet technique is one of the useful methods for transplanting large numbers of cells. We hypothesized that hiPS-CM-sheet transplantation would be feasible,safe,and therapeutically effective for the treatment of ischemic cardiomyopathy.backslashnbackslashnMETHODS AND RESULTS: Human iPS cells were established by infecting human dermal fibroblasts with a retrovirus carrying Oct3/4,Sox2,Klf4,and c-Myc. Cardiomyogenic differentiation was induced by WNT signaling molecules,yielding hiPS-CMs that were almost 90% positive for α-actinin,Nkx2.5,and cardiac troponin T. hiPS-CM sheets were created using thermoresponsive dishes and transplanted over the myocardial infarcts in a porcine model of ischemic cardiomyopathy induced by ameroid constriction of the left anterior descending coronary artery (n=6 for the iPS group receiving sheet transplantation and the sham-operated group; both groups received tacrolimus daily). Transplantation significantly improved cardiac performance and attenuated left ventricular remodeling. hiPS-CMs were detectable 8 weeks after transplantation,but very few survived long term. No teratoma formation was observed in animals that received hiPS-CM sheets.backslashnbackslashnCONCLUSIONS: The culture system used yields a large number of highly pure hiPS-CMs,and hiPS-CM sheets could improve cardiac function after ischemic cardiomyopathy. This newly developed culture system and the hiPS-CM sheets may provide a basis for the clinical use of hiPS cells in cardiac regeneration therapy. View Publication

The inactivation of p53 functions enhances the efficiency and decreases the latency of producing induced pluripotent stem cells (iPSC) in culture. The formation of iPSCs in culture starts with a rapid set of cell divisions followed by an epigenetic reprogramming of the DNA and chromatin. The mechanisms by which the p53 protein inhibits the formation of iPSCs are largely unknown. Using a temperature sensitive mutant of the p53 (Trp53) gene,we examined the impact of the temporal expression of wild type p53 in preventing stem cell induction from somatic cells. We also explored how different p53 mutant alleles affect the reprogramming process. We found that little or no p53 activity favors the entire process of somatic cell reprogramming. Reactivation of p53 at any time point during the reprogramming process not only interrupted the formation of iPSCs,but also induced newly formed stem cells to differentiate. Among p53-regulated genes,p21 (Cdkn1a),but not Puma (Bbc3) played a partial role in iPSCs formation probably by slowing cell division. Activation of p53 functions in iPSCs induced senescence and differentiation in stem cell populations. High rate of birth defects and increases in DNA methylation at the IGF2-H19 loci in female offspring of p53 knockout mice suggested that the absence of p53 may give rise to epigenetic instability in a stochastic fashion. Consistently,selected p53 missense mutations showed differential effects on the stem cell reprogramming efficiency in a c-Myc dependent manner. The absence of p53 activity and functions also contributed to an enhanced efficiency of iPSC production from cancer cells. The production of iPSCs in culture from normal and cancer cells,although different from each other in several ways,both responded to the inhibition of reprogramming by the p53 protein. View Publication

LIN28 is a conserved RNA-binding protein implicated in pluripotency,reprogramming,and oncogenesis. It was previously shown to act primarily by blocking let-7 microRNA (miRNA) biogenesis,but here we elucidate distinct roles of LIN28 regulation via its direct messenger RNA (mRNA) targets. Through crosslinking and immunoprecipitation coupled with high-throughput sequencing (CLIP-seq) in human embryonic stem cells and somatic cells expressing exogenous LIN28,we have defined discrete LIN28-binding sites in a quarter of human transcripts. These sites revealed that LIN28 binds to GGAGA sequences enriched within loop structures in mRNAs,reminiscent of its interaction with let-7 miRNA precursors. Among LIN28 mRNA targets,we found evidence for LIN28 autoregulation and also direct but differing effects on the protein abundance of splicing regulators in somatic and pluripotent stem cells. Splicing-sensitive microarrays demonstrated that exogenous LIN28 expression causes widespread downstream alternative splicing changes. These findings identify important regulatory functions of LIN28 via direct mRNA interactions. ?? 2012 Elsevier Inc. View Publication

Linker histones are essential components of chromatin,but the distributions and functions of many during cellular differentiation are not well understood. Here,we show that H1.5 binds to genic and intergenic regions,forming blocks of enrichment,in differentiated human cells from all three embryonic germ layers but not in embryonic stem cells. In differentiated cells,H1.5,but not H1.3,binds preferentially to genes that encode membrane and membrane-related proteins. Strikingly,37% of H1.5 target genes belong to gene family clusters,groups of homologous genes that are located in proximity to each other on chromosomes. H1.5 binding is associated with gene repression and is required for SIRT1 binding,H3K9me2 enrichment,and chromatin compaction. Depletion of H1.5 results in loss of SIRT1 and H3K9me2,increased chromatin accessibility,deregulation of gene expression,and decreased cell growth. Our data reveal for the first time a specific and novel function for linker histone subtype H1.5 in maintenance of condensed chromatin at defined gene families in differentiated human cells. View Publication

Embryonic development can be partially recapitulated in vitro by differentiating human embryonic stem cells (hESCs). Thalidomide is a developmental toxicant in vivo and acts in a species-dependent manner. Besides its therapeutic value,thalidomide also serves as a prototypical model to study teratogenecity. Although many in vivo and in vitro platforms have demonstrated its toxicity,only a few test systems accurately reflect human physiology. We used global gene expression and proteomics profiling (two dimensional electrophoresis (2DE) coupled with Tandem Mass spectrometry) to demonstrate hESC differentiation and thalidomide embryotoxicity/teratogenecity with clinically relevant dose(s). Proteome analysis showed loss of POU5F1 regulatory proteins PKM2 and RBM14 and an over expression of proteins involved in neuronal development (such as PAK2,PAFAH1B2 and PAFAH1B3) after 14 days of differentiation. The genomic and proteomic expression pattern demonstrated differential expression of limb,heart and embryonic development related transcription factors and biological processes. Moreover,this study uncovered novel possible mechanisms,such as the inhibition of RANBP1,that participate in the nucleocytoplasmic trafficking of proteins and inhibition of glutathione transferases (GSTA1,GSTA2),that protect the cell from secondary oxidative stress. As a proof of principle,we demonstrated that a combination of transcriptomics and proteomics,along with consistent differentiation of hESCs,enabled the detection of canonical and novel teratogenic intracellular mechanisms of thalidomide. View Publication

Stochastic processes and imprinting,along with genetic factors,lead to monoallelic or allele-biased gene expression. Stochastic monoallelic expression fine-tunes information processing in immune cells and the olfactory system,and imprinting plays an important role in development. Recent studies suggest that both stochastic events and imprinting may be more widespread than previously considered. We are interested in allele-biased gene expression occurring in the brain because parent-of-origin effects suggestive of imprinting appear to play a role in the transmission of schizophrenia (SZ) and autism spectrum disorders (ASD) in some families. In addition,allele-biased expression could help explain monozygotic (MZ) twin discordance and reduced penetrance. The ability to study allele-biased expression in human neurons has been transformed with the advent of induced pluripotent stem cell (iPSC) technology and next generation sequencing. Using transcriptome sequencing (RNA-Seq) we identified 801 genes in differentiating neurons that were expressed in an allele-biased manner. These included a number of putative SZ and ASD candidates,such as A2BP1 (RBFOX1),ERBB4,NLGN4X,NRG1,NRG3,NRXN1,and NLGN1. Overall,there was a modest enrichment for SZ and ASD candidate genes among those that showed evidence for allele-biased expression (chi-square,p = 0.02). In addition to helping explain MZ twin discordance and reduced penetrance,the capacity to group many candidate genes affecting a variety of molecular and cellular pathways under a common regulatory process - allele-biased expression - could have therapeutic implications. View Publication