技术资料

GABAergic interneurons regulate cortical neural networks by providing inhibitory inputs,and their malfunction,resulting in failure to intricately regulate neural circuit balance,is implicated in brain diseases such as Schizophrenia,Autism,and Epilepsy. During early development,GABAergic interneuron progenitors arise from the ventral telencephalic area such as medial ganglionic eminence (MGE) and caudal ganglionic eminence (CGE) by the actions of secreted signaling molecules from nearby organizers,and migrate to their target sites where they form local synaptic connections. In this study,using combinatorial and temporal modulation of developmentally relevant dorsoventral and rostrocaudal signaling pathways (SHH,Wnt,and FGF8),we efficiently generated MGE cells from multiple human pluripotent stem cells. Most importantly,modulation of FGF8/FGF19 signaling efficiently directed MGE versus CGE differentiation. Human MGE cells spontaneously differentiated into Lhx6-expressing GABAergic interneurons and showed migratory properties. These human MGE-derived neurons generated GABA,fired action potentials,and displayed robust GABAergic postsynaptic activity. Transplantation into rodent brains results in well-contained neural grafts enriched with GABAergic interneurons that migrate in the host and mature to express somatostatin or parvalbumin. Thus,we propose that signaling modulation recapitulating normal developmental patterns efficiently generate human GABAergic interneurons. This strategy represents a novel tool in regenerative medicine,developmental studies,disease modeling,bioassay,and drug screening. View Publication

Mesenchymal stem cells (MSCs) are being tested in a wide range of human diseases; however,loss of potency and inconsistent quality severely limit their use. To overcome these issues,we have utilized a developmental precursor called the hemangioblast as an intermediate cell type in the derivation of a highly potent and replenishable population of MSCs from human embryonic stem cells (hESCs). This method circumvents the need for labor-intensive hand-picking,scraping,and sorting that other hESC-MSC derivation methods require. Moreover,unlike previous reports on hESC-MSCs,we have systematically evaluated their immunomodulatory properties and in vivo potency. As expected,they dynamically secrete a range of bioactive factors,display enzymatic activity,and suppress T-cell proliferation that is induced by either allogeneic cells or mitogenic stimuli. However,they also display unique immunophenotypic properties,as well as a smaller size and textgreater30,000-fold proliferative capacity than bone marrow-derived MSCs. In addition,this is the first report which demonstrates that hESC-MSCs can inhibit CD83 up-regulation and IL-12p70 secretion from dendritic cells and enhance regulatory T-cell populations induced by interleukin 2 (IL-2). This is also the first report which shows that hESC-MSCs have therapeutic efficacy in two different autoimmune disorder models,including a marked increase in survival of lupus-prone mice and a reduction of symptoms in an autoimmune model of uveitis. Our data suggest that this novel and therapeutically active population of MSCs could overcome many of the obstacles that plague the use of MSCs in regenerative medicine and serve as a scalable alternative to current MSC sources. View Publication

The purpose of this study was to examine the in vitro and in vivo osteogenic potential of human induced pluripotent stem cells (hiPSCs) against that of human bone marrow mesenchymal stem cells (hBMMSCs). Embryoid bodies (EBs),which were formed from undifferentiated hiPSCs,were dissociated into single cells and underwent osteogenic differentiation using the same medium as hBMMSCs for 14 days. Osteoinduced hiPSCs were implanted on the critical-size calvarial defects and long bone segmental defects in rats. The healing of defects was evaluated after 8 weeks and 12 weeks of implantation,respectively. Osteoinduced hiPSCs showed relatively lower and delayed in vitro expressions of the osteogenic marker COL1A1 and bone sialoprotein,as well as a weaker osteogenic differentiation through alkaline phosphatase staining and mineralization through Alizarin red staining compared with hBMMSCs. Calvarial defects treated with osteoinduced hiPSCs had comparable quality of new bone formation,including full restoration of bone width and robust formation of trabeculae,to those treated with hBMMSCs. Both osteoinduced hiPSCs and hBMMSCs persisted in regenerated bone after 8 weeks of implantation. In critical-size long bone segmental defects,osteoinduced hiPSC treatment also led to healing of segmental defects comparable to osteoinduced hBMMSC treatment after 12 weeks. In conclusion,despite delayed in vitro osteogenesis,hiPSCs have an in vivo osteogenic potential as good as hBMMSCs. 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

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

Antiserum to epithelial membrane antigen and three monoclonal antibodies (MAb) to milk-fat globule membranes immunocytochemically stain only epithelial cells,whereas a fourth reacts also with myoepithelial cells in inter- and intralobular ducts of human breast. Staining with peanut lectin shows a gradual increase for epithelial cells,from little or no staining in ducts through variable staining in ductules to intense staining in secretory alveoli. Antisera and MAb to vimentin,smooth-muscle actin,MAb to the common acute lymphoblastic leukemia antigen and to a glycoprotein of 135 KD stain myoepithelial cells in main ducts,but this staining is reduced in inter- and intralobular ducts and ductules. MAb to epithelial-specific keratin 18 stain a minor population of ductal epithelial cells,the major population of epithelial cells in interlobular (ILD) and extralobular terminal ducts (ETD),and epithelial cells in a minority of ductules. In lactating glands most epithelial cells in ductules are stained,but the alveolar and myoepithelial cells are unstained. Keratin MAb PKK2 and LP34 strongly stain myoepithelial cells,but only a minor population of epithelial cells in main ducts. However,these MAb stain principally the epithelial cells in ILD,ETD,and a minority of ductules. In lactating glands most epithelial cells are stained in ductules,but the myoepithelial and not the alveolar cells are stained intensely in secretory lobules. It is suggested that the unusual staining pattern of cells found principally in the ILD,ETD,and some ductules may represent regions of growth and/or subpopulation(s) of cells intermediate between epithelial and myoepithelial cells. View Publication

We recently reported that chronic lymphocytic leukemia (CLL) subgroups with distinct clonotypic BCRs present discrete patterns of TLR expression,function,and/or tolerance. In this study,to explore whether specific types of BCR/TLR collaboration exist in CLL,we studied the effect of single versus concomitant BCR and/or TLR stimulation on CLL cells from mutated (M-CLL) and unmutated CLL (U-CLL) cases. We stimulated negatively isolated CLL cells by using anti-IgM,imiquimod,and CpG oligodeoxynucleotide for BCR,TLR7,and TLR9,respectively,alone or in combination for different time points. After in vitro culture in the absence of stimulation,differences in p-ERK were identified at any time point,with higher p-ERK levels in U-CLL versus M-CLL. Pronounced p-ERK induction was seen by single stimulation in U-CLL,whereas BCR/TLR synergism was required in View Publication

We have previously reported the genetic correction of Huntington's disease (HD) patient-derived induced pluripotent stem cells using traditional homologous recombination (HR) approaches. To extend this work,we have adopted a CRISPR-based genome editing approach to improve the efficiency of recombination in order to generate allelic isogenic HD models in human cells. Incorporation of a rapid antibody-based screening approach to measure recombination provides a powerful method to determine relative efficiency of genome editing for modeling polyglutamine diseases or understanding factors that modulate CRISPR/Cas9 HR. View Publication

INTRODUCTION: We previously demonstrated that the lifespan of primary human keratinocytes could be extended indefinitely by culture in the presence of the Rho kinase (ROCK) inhibitor Y-27632. This technique has proven to be very useful in diverse areas of basic and clinical research. METHODS: In this follow-up study we determine whether the continual presence of Y-27632 is required for sustained proliferation. We also test whether different ROCK inhibitors can be used for this technique and whether it can also promote indefinite proliferation of animal keratinocytes. We measure keratinocyte gene expression,proliferation,behaviour and lifespan in the presence and absence of Y-27632. RESULTS: We demonstrate that the extension of lifespan observed by culture of keratinocytes in the presence of fibroblast feeders and a ROCK inhibitor is reversible and that cells senesce gradually when the inhibitor is removed from the medium. Conversely,keratinocytes that are close to the end of their replicative life span can be revived by ROCK inhibition. We demonstrate that different inhibitors of ROCK can also efficiently extend the lifespan of human keratinocytes and that ROCK inhibition extends the lifespan of animal keratinocytes derived from mouse and bovine epithelia. Gene expression analysis of human epidermal keratinocytes cells grown in the presence of Y-27632 demonstrates that ROCK inhibition primarily inhibits keratinocyte differentiation. Live-imaging of keratinocytes cultured with ROCK inhibitors show that the effect of ROCK inhibition on cellular proliferation is immediate and ROCK inhibited cells proliferate rapidly without differentiation or stratification. CONCLUSIONS: ROCK inhibition rapidly and conditionally induces indefinite proliferation of keratinocytes. This method has far-reaching applications for basic research,as well as for regenerative and personalized medicine. View Publication

Spleen tyrosine kinase (Syk) is an attractive drug target in autoimmune,inflammatory,and oncology disease indications. The most advanced Syk inhibitor,R406,1 (or its prodrug form fostamatinib,2),has shown efficacy in multiple therapeutic indications,but its clinical progress has been hampered by dose-limiting adverse effects that have been attributed,at least in part,to the off-target activities of 1. It is expected that a more selective Syk inhibitor would provide a greater therapeutic window. Herein we report the discovery and optimization of a novel series of imidazo[1,2-a]pyrazine Syk inhibitors. This work culminated in the identification of GS-9973,68,a highly selective and orally efficacious Syk inhibitor which is currently undergoing clinical evaluation for autoimmune and oncology indications. View Publication

Vascular endothelial growth factor (VEGF) was purified from media conditioned by bovine pituitary folliculostellate cells (FC). VEGF is a heparin-binding growth factor specific for vascular endothelial cells that is able to induce angiogenesis in vivo. Complementary DNA clones for bovine and human VEGF were isolated from cDNA libraries prepared from FC and HL60 leukemia cells,respectively. These cDNAs encode hydrophilic proteins with sequences related to those of the A and B chains of platelet-derived growth factor. DNA sequencing suggests the existence of several molecular species of VEGF. VEGFs are secreted proteins,in contrast to other endothelial cell mitogens such as acidic or basic fibroblast growth factors and platelet-derived endothelial cell growth factor. Human 293 cells transfected with an expression vector containing a bovine or human VEGF cDNA insert secrete an endothelial cell mitogen that behaves like native VEGF. View Publication

Several transcription factors (TFs) have been implicated in neuroectoderm (NE) development,and recently,the TF PAX6 was shown to be critical for human NE specification. However,microRNA networks regulating human NE development have been poorly documented. We hypothesized that microRNAs activated by PAX6 should promote NE development. Using a genomics approach,we identified PAX6 binding sites and active enhancers genome-wide in an in vitro model of human NE development that was based on neural differentiation of human embryonic stem cells (hESC). PAX6 binding to active enhancers was found in the proximity of several microRNAs,including hsa-miR-135b. MiR-135b was activated during NE development,and ectopic expression of miR-135b in hESC promoted differentiation toward NE. MiR-135b promotes neural conversion by targeting components of the TGF-β and BMP signaling pathways,thereby inhibiting differentiation into alternate developmental lineages. Our results demonstrate a novel TF-miRNA module that is activated during human neuroectoderm development and promotes the irreversible fate specification of human pluripotent cells toward the neural lineage. View Publication