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Neural cells derived from induced pluripotent stem cells (iPSCs) have the potential for autologous cell therapies in treating patients with severe neurological disorders or injury. However,further study of efficacy and safety are needed in large animal preclinical models that have similar neural anatomy and physiology to humans such as the pig. The pig model for pluripotent stem cell therapy has been made possible for the first time with the development of pig iPSCs (piPSCs) capable of in vitro and in vivo differentiation into tissues of all three germ layers. Still,the question remains if piPSCs are capable of undergoing robust neural differentiation using a system similar to those being used with human iPSCs. In this study,we generated a new line of piPSCs from fibroblast cells that expressed pluripotency markers and were capable of embryoid body differentiation into all three germ layers. piPSCs demonstrated robust neural differentiation forming βIII-TUB/MAP2+ neurons,GFAP+ astrocytes,and O4+ oligodendrocytes and demonstrated strong upregulation of neural cell genes representative of all three major neural lineages of the central nervous system. In the presence of motor neuron signaling factors,piPSC-derived neurons showed expression of transcription factors associated with motor neuron differentiation (HB9 and ISLET1). Our findings demonstrate that SSEA4 expression is required for piPSCs to differentiate into neurons,astrocytes,and oligodendrocytes and furthermore develop specific neuronal subtypes. This indicates that the pigs can fill the need for a powerful model to study autologous neural iPSC therapies in a system similar to humans. View Publication

Human pluripotent stem cells (hPSCs) are a promising cell source for tissue engineering and regenerative medicine,especially in the field of neurobiology. Neural differentiation protocols have been developed to differentiate hPSCs into specific neural cells,but these predominantly rely on biochemical cues. Recently,differentiation protocols have incorporated topographical cues to increase the total neuronal yield. However,the means by which these topographical cues improve neuronal yield remains unknown. In this study,we explored the effect of topography on the neural differentiation of hPSC by quantitatively studying the changes in marker expression at a transcript and protein level. We found that 2 ??m gratings increase the rate of neural differentiation,and that an additional culture period of 2 ??m gratings in the absence of neurotrophic signals can improve the neural differentiation of hPSCs. We envisage that this work can be incorporated into future differentiation protocols to decrease the differentiation period as well as the biochemical signals added,thus generating hPSC-derived neural cells in a more cost effective and efficient manner. ?? 2012 Elsevier Ltd. View Publication

MicroRNAs (miRNAs) have emerged as critical regulators of gene expression through translational inhibition and RNA decay and have been implicated in the regulation of cellular differentiation,proliferation,angiogenesis,and apoptosis. In this study,we analyzed global miRNA and mRNA microarrays to predict novel miRNA-mRNA interactions in human embryonic stem cells and induced pluripotent stem cells (iPSCs). In particular,we demonstrate a regulatory feedback loop between the miR-302 cluster and two transcription factors,NR2F2 and OCT4. Our data show high expression of miR-302 and OCT4 in pluripotent cells,while NR2F2 is expressed exclusively in differentiated cells. Target analysis predicts that NR2F2 is a direct target of miR-302,which we experimentally confirm by reporter luciferase assays and real-time polymerase chain reaction. We also demonstrate that NR2F2 directly inhibits the activity of the OCT4 promoter and thus diminishes the positive feedback loop between OCT4 and miR-302. Importantly,higher reprogramming efficiencies were obtained when we reprogrammed human adipose-derived stem cells into iPSCs using four factors (KLF4,C-MYC,OCT4,and SOX2) plus miR-302 (this reprogramming cocktail is hereafter referred to as KMOS3") when compared to using four factors ("KMOS"). Furthermore� View Publication

Aim: Reprogramming of somatic cells utilizing viral free methods provide a remarkable method to generate human induced pluripotent stem cells (hiPSCs) for regenerative medicine. In this study,we evaluate developmental ontogeny of cardiomyocytes following induced differentiation of hiPSCs. Main Methods: Fibroblasts were reprogrammed with episomal vectors to generate hiPSC and were subsequently differentiated to cardiomyocytes. Ontogenic development of cardiomyocytes was studied by real-time PCR. Key findings: Human iPSCs derived from episomal based vectors maintain classical pluripotency markers,generate teratomas and spontaneously differentiate into three germ layers in vitro. Cardiomyogenic induction of these hiPSCs efficiently generated cardiomyocytes. Ontogenic gene expression studies demonstrated that differentiation of cardiomyocytes was initiated by increased expression of mesodermal markers,followed by early cardiac committed markers,structural and ion channel genes. Furthermore,our correlation analysis of gene expression studies with human heart demonstrated that pivotal structural genes like cardiac troponin,actinin,myosin light chain maintained a high correlation with ion channel genes indicating coordinated activation of cardiac transcriptional machinery. Finally,microelectrode recordings show that these cardiomyocytes could respond aptly to pharmacologically active drugs. Cardiomyocytes showed a chronotropic response to isoproterenol,reduced Na+ influx with quinidine,prolongation of beating rate corrected field potential duration (cFPD) with E-4031 and reduced beating frequency and shortened cFPD with verapamil. Significance: Our study shows that viral free hiPSCs efficiently differentiate into cardiomyocytes with cardiac-specific molecular,structural,and functional properties that recapitulate developmental ontogeny of cardiogenesis. These results,coupled with the potential to generate patient-specific hiPSC lines hold great promise for the development of in vitro platform for drug pharmacogenomics; disease modeling and regenerative medicine. textcopyright 2012 Elsevier Inc. All rights reserved. View Publication

Lifelong blood cell production is governed through the poorly understood integration of cell-intrinsic and -extrinsic control of hematopoietic stem cell (HSC) quiescence and activation. MicroRNAs (miRNAs) coordinately regulate multiple targets within signaling networks,making them attractive candidate HSC regulators. We report that miR-126,a miRNA expressed in HSC and early progenitors,plays a pivotal role in restraining cell-cycle progression of HSC in vitro and in vivo. miR-126 knockdown by using lentiviral sponges increased HSC proliferation without inducing exhaustion,resulting in expansion of mouse and human long-term repopulating HSC. Conversely,enforced miR-126 expression impaired cell-cycle entry,leading to progressively reduced hematopoietic contribution. In HSC/early progenitors,miR-126 regulates multiple targets within the PI3K/AKT/GSK3β pathway,attenuating signal transduction in response to extrinsic signals. These data establish that miR-126 sets a threshold for HSC activation and thus governs HSC pool size,demonstrating the importance of miRNA in the control of HSC function. View Publication

Articular cartilage,which is mainly composed of collagen II,enables smooth skeletal movement. Degeneration of collagen II can be caused by various events,such as injury,but degeneration especially increases over the course of normal aging. Unfortunately,the body does not fully repair itself from this type of degeneration,resulting in impaired movement. Microfracture,an articular cartilage repair surgical technique,has been commonly used in the clinic to induce the repair of tissue at damage sites. Mesenchymal stem cells (MSC) have also been used as cell therapy to repair degenerated cartilage. However,the therapeutic outcomes of all these techniques vary in different patients depending on their age,health,lesion size and the extent of damage to the cartilage. The repairing tissues either form fibrocartilage or go into a hypertrophic stage,both of which do not reproduce the equivalent functionality of endogenous hyaline cartilage. One of the reasons for this is inefficient chondrogenesis by endogenous and exogenous MSC. Drugs that promote chondrogenesis could be used to induce self-repair of damaged cartilage as a non-invasive approach alone,or combined with other techniques to greatly assist the therapeutic outcomes. The recent development of human induced pluripotent stem cell (iPSCs),which are able to self-renew and differentiate into multiple cell types,provides a potentially valuable cell resource for drug screening in a more relevant" cell type. Here we report a screening platform using human iPSCs in a multi-well plate format to identify compounds that could promote chondrogenesis." View Publication

Differences in global levels of histone acetylation occur in normal and cancer cells,although the reason why cells regulate these levels has been unclear. Here we demonstrate a role for histone acetylation in regulating intracellular pH (pH(i)). As pH(i) decreases,histones are globally deacetylated by histone deacetylases (HDACs),and the released acetate anions are coexported with protons out of the cell by monocarboxylate transporters (MCTs),preventing further reductions in pH(i). Conversely,global histone acetylation increases as pH(i) rises,such as when resting cells are induced to proliferate. Inhibition of HDACs or MCTs decreases acetate export and lowers pH(i),particularly compromising pH(i) maintenance in acidic environments. Global deacetylation at low pH is reflected at a genomic level by decreased abundance and extensive redistribution of acetylation throughout the genome. Thus,acetylation of chromatin functions as a rheostat to regulate pH(i) with important implications for mechanism of action and therapeutic use of HDAC inhibitors. View Publication

PURPOSE This study aimed to develop a feasible and efficient method for generating embryonic stem cell (ESC)-like induced pluripotent stem (iPS) cells from human Tenon's capsule fibroblasts (HTFs) through the expression of a defined set of transcription factors,which will have significant application value for ophthalmic personalized regenerative medicine. METHODS HTFs were harvested from fresh samples,and reprogramming was induced by the exogenous expression of the four classic transcription factors,OCT-3/4,SOX-2,KLF-4,and C-MYC. The HTF-derived iPS (TiPS) cells were analyzed with phase contrast microscopy,real-time PCR,immunofluorescence,FACS analysis,alkaline phosphatase activity analysis,and a teratoma formation assay. Human ESC colonies were used as the positive control. RESULTS The resulting HTF-derived iPS cell colonies were indistinguishable from human ESC colonies regarding morphology,gene expression levels,pluripotent gene expression,alkaline phosphatase activity,and the ability to generate all three embryonic germ layers. CONCLUSIONS This study presents a simple,efficient,practical procedure for generating patient-tailored iPS cells from HTFs. These cells will serve as a valuable and preferred candidate donor cell population for ophthalmological regenerative medicine. View Publication

Cultures of human embryonic stem cell typically rely on protein matrices or feeder cells to support attachment and growth,while mechanical,enzymatic or chemical cell dissociation methods are used for cellular passaging. However,these methods are ill defined,thus introducing variability into the system,and may damage cells. They also exert selective pressures favouring cell aneuploidy and loss of differentiation potential. Here we report the identification of a family of chemically defined thermoresponsive synthetic hydrogels based on 2-(diethylamino)ethyl acrylate,which support long-term human embryonic stem cell growth and pluripotency over a period of 2-6 months. The hydrogels permitted gentle,reagent-free cell passaging by virtue of transient modulation of the ambient temperature from 37 to 15 °C for 30 min. These chemically defined alternatives to currently used,undefined biological substrates represent a flexible and scalable approach for improving the definition,efficacy and safety of human embryonic stem cell culture systems for research,industrial and clinical applications. View Publication

UNLABELLED: Patient-specific induced pluripotent stem cells (iPSCs) represent a potential source for developing novel drug and cell therapies. Although increasing numbers of disease-specific iPSCs have been generated,there has been limited progress in iPSC-based drug screening/discovery for liver diseases,and the low gene-targeting efficiency in human iPSCs warrants further improvement. Using iPSC lines from patients with alpha-1 antitrypsin (AAT) deficiency,for which there is currently no drug or gene therapy available,we established a platform to discover new drug candidates and correct disease-causing mutation with a high efficiency. A high-throughput format screening assay,based on our hepatic differentiation protocol,was implemented to facilitate automated quantification of cellular AAT accumulation using a 96-well immunofluorescence reader. To expedite the eventual application of lead compounds to patients,we conducted drug screening utilizing our established library of clinical compounds (the Johns Hopkins Drug Library) with extensive safety profiles. Through a blind large-scale drug screening,five clinical drugs were identified to reduce AAT accumulation in diverse patient iPSC-derived hepatocyte-like cells. In addition,using the recently developed transcription activator-like effector nuclease technology,we achieved high gene-targeting efficiency in AAT-deficiency patient iPSCs with 25%-33% of the clones demonstrating simultaneous targeting at both diseased alleles. The hepatocyte-like cells derived from the gene-corrected iPSCs were functional without the mutant AAT accumulation. This highly efficient and cost-effective targeting technology will broadly benefit both basic and translational applications.backslashnbackslashnCONCLUSIONS: Our results demonstrated the feasibility of effective large-scale drug screening using an iPSC-based disease model and highly robust gene targeting in human iPSCs,both of which are critical for translating the iPSC technology into novel therapies for untreatable diseases. View Publication

Despite advances in detection and therapy,castration-resistant prostate cancer continues to be a major clinical problem. The aberrant activity of stem cell pathways,and their regulation by the Androgen Receptor (AR),has the potential to provide insight into novel mechanisms and pathways to prevent and treat advanced,castrate-resistant prostate cancers. To this end,we investigated the role of the embryonic stem cell regulator Sox2 [SRY (sex determining region Y)-box 2] in normal and malignant prostate epithelial cells. In the normal prostate,Sox2 is expressed in a portion of basal epithelial cells. Prostate tumors were either Sox2-positive or Sox2-negative,with the percentage of Sox2-positive tumors increasing with Gleason Score and metastases. In the castration-resistant prostate cancer cell line CWR-R1,endogenous expression of Sox2 was repressed by AR signaling,and AR chromatin-IP shows that AR binds the enhancer element within the Sox2 promoter. Likewise,in normal prostate epithelial cells and human embryonic stem cells,increased AR signaling also decreases Sox2 expression. Resistance to the anti-androgen MDV3100 results in a marked increase in Sox2 expression within three prostate cancer cell lines,and in the castration-sensitive LAPC-4 prostate cancer cell line ectopic expression of Sox2 was sufficient to promote castration-resistant tumor formation. Loss of Sox2 expression in the castration-resistant CWR-R1 prostate cancer cell line inhibited cell growth. Up-regulation of Sox2 was not associated with increased CD133 expression but was associated with increased FGF5 (Fibroblast Growth Factor 5) expression. These data propose a model of elevated Sox2 expression due to loss of AR-mediated repression during castration,and consequent castration-resistance via mechanisms not involving induction of canonical embryonic stem cell pathways. View Publication

Induced pluripotent stem cells (iPSCs) hold great clinical potential for regenerative medicine. Much work has been done to investigate the mechanisms of their generation,focusing on the cell nucleus. However,the roles of specific organelles and in particular mitochondria in the potential mechanisms of nuclear reprogramming remain unclear. In this study,we sought to determine the role of mitochondrial metabolism transition in nuclear reprogramming. We found that the mitochondrial cristae had remodeled in iPSCs. The efficiency of iPSC generation was significantly reduced by down-regulation of mitochondrial inner membrane protein (IMMT),which regulates the morphology of mitochondrial cristae. Moreover,cells with the oxidative phosphorylation (OXPHOS) advantage had higher reprogramming efficiency than normal cells and the glycolysis intermediate lactic acid enhanced the efficiency of iPSCs generation. Our results show that the remodeling of mitochondrial cristae couples with the generation of iPSCs,suggesting mitochondrial metabolism transition plays an important role in nuclear reprogramming. View Publication