技术资料

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

Intracellular delivery of macromolecules is a challenge in research and therapeutic applications. Existing vector-based and physical methods have limitations,including their reliance on exogenous materials or electrical fields,which can lead to toxicity or off-target effects. We describe a microfluidic approach to delivery in which cells are mechanically deformed as they pass through a constriction 30–80% smaller than the cell diameter. The resulting controlled application of compression and shear forces results in the formation of transient holes that enable the diffusion of material from the surrounding buffer into the cytosol. The method has demonstrated the ability to deliver a range of material,such as carbon nanotubes,proteins,and siRNA,to 11 cell types,including embryonic stem cells and immune cells. When used for the delivery of transcription factors,the microfluidic devices produced a 10-fold improvement in colony formation relative to electroporation and cell-penetrating peptides. Indeed,its ability to deliver structurally diverse materials and its applicability to difficult-to-transfect primary cells indicate that this method could potentially enable many research and clinical applications. View Publication

Many long noncoding RNA (lncRNA) species have been identified in mammalian cells,but the genomic origin and regulation of these molecules in individual cell types is poorly understood. We have generated catalogs of lncRNA species expressed in human and murine embryonic stem cells and mapped their genomic origin. A surprisingly large fraction of these transcripts (textgreater60%) originate from divergent transcription at promoters of active protein-coding genes. The divergently transcribed lncRNA/mRNA gene pairs exhibit coordinated changes in transcription when embryonic stem cells are differentiated into endoderm. Our results reveal that transcription of most lncRNA genes is coordinated with transcription of protein-coding genes. View Publication

The shortage of human organs and tissues for transplant has led to significant interest in xenotransplantation of pig tissues for human patients. However,transplantation of pig organs results in an acute immune rejection,leading to death of the organ within minutes. The $\$-1,3-galactosyltransferase (GALT) gene has been knocked out in pigs to reduce rejection,yet additional genes need to be modified to ultimately make pig tissue immunocompatible with humans. The development of pig induced pluripotent stem cells (piPSCs) from GALT knockout (GALT-KO) tissue would provide an excellent cell source for complex genetic manipulations (e.g.,gene targeting) that often require highly robust and proliferative cells. In this report,we generated GALT-KO piPSCs by the overexpression of POU5F1,SOX2,NANOG,LIN28,KLF-4,and C-MYC reprogramming genes. piPSCs showed classical stem cell morphology and characteristics,expressing integrated reprogramming genes in addition to the pluripotent markers AP,SSEA1,and SSEA4. GALT-KO piPSCs were highly proliferative and possessed doubling times and telomerase activity similar to human embryonic stem cells. These results demonstrated successful reprogramming of GALT-KO fibroblasts into GALT-KO piPSCs. GALT-KO piPSCs are potentially an excellent immortal cell source for the generation of pigs with complex genetic modifications for xenotransplantation,somatic cell nuclear transfer,or chimera formation. View Publication

Pluripotent stem cells hold great promise for regenerative medicine,due to their unlimited self-renewal potential and the ability to differentiate into all somatic cell types. Differences between the rodent disease models and the situation in humans can be narrowed down with non-human primate models. The common marmoset monkey (Callithrix jacchus) is an interesting model for biomedical research because these animals are easy to breed,get relatively old (≤ 13 years),are small in size,are relatively cost-effective and have a high genetic proximity to the human. In particular,diseases of the liver and pancreas are interesting for cell replacement therapies but the in vitro differentiation of ESCs into the definitive endoderm germ layer is still a demanding task. Membrane-permeable,chemically defined small molecules can possibly replace recombinant growth factors used in most directed differentiation protocols. However,the potent small molecules IDE-1 and IDE-2 were not able to induce definitive endoderm-like cells when ESCs from the common marmoset were treated with these compounds,whereas the recombinant growth factor Activin A could force the differentiation into this lineage. Our results indicate that ESCs from the common marmoset are less sensitive or even insensitive to these small molecules. Thus,differences between the species of human ESCs and ESCs of this non-human primate might be a useful model to further evaluate the exact mode of action of these compounds. View Publication