技术资料

A human cell-based in vitro model that can accurately predict drug penetration into the brain as well as metrics to assess these in vitro models are valuable for the development of new therapeutics. Here,human induced pluripotent stem cells (hPSCs) are differentiated into a polarized monolayer that express blood-brain barrier (BBB)-specific proteins and have transendothelial electrical resistance (TEER) values greater than 2500 Ωtextperiodcenteredcm(2). By assessing the permeabilities of several known drugs,a benchmarking system to evaluate brain permeability of drugs was established. Furthermore,relationships between TEER and permeability to both small and large molecules were established,demonstrating that different minimum TEER thresholds must be achieved to study the brain transport of these two classes of drugs. This work demonstrates that this hPSC-derived BBB model exhibits an in vivo-like phenotype,and the benchmarks established here are useful for assessing functionality of other in vitro BBB models. View Publication

Fibroblasts from a male patient with compound heterozygous variants in the tyrosine hydroxylase gene (TH; OMIM: 191290; c.[385-CtextgreaterT]; [692-GtextgreaterC]/p.[R129*]; [R231P]),the rate-limiting enzyme for dopamine synthesis,were reprogrammed to iPSCs using episomal reprogramming delivering the reprogramming factors Oct3/4,Sox2,L-Myc,Lin28,Klf4 and p53 shRNA Okita et al. (2011). Pluripotency of TH-1 iPSC was verified by immunohistochemistry and RT-PCR analysis. Cells exhibited a normal karyotype and differentiated spontaneously into the 3 germ layers in vitro. TH-1 iPSC represents the first model system to study the pathomechanism of this rare metabolic disease and provides a useful tool for drug testing. View Publication

Spinocerebellar ataxia type3 (SCA3) is an autosomal dominant neurodegenerative disorder. Human embryonic stem cell line chHES-472 was derived from abnormal embryo donated by SCA3 patient after preimplantation genetic diagnosis (PGD) treatment. This cell line had a normal karyotype and retained the disease-causing mutant in ATXN3 gene. Characteristic tests proved that the embryonic stem cell line presented typical markers of pluripotency and had the capability to form the three germlayers in vivo. View Publication

In vitro differentiation of human pluripotent stem cells (hPSCs) recapitulates early aspects of human embryogenesis,but the underlying processes are poorly understood and controlled. Here we show that modulating the bulk cell density (BCD: cell number per culture volume) deterministically alters anteroposterior patterning of primitive streak (PS)-like priming. The BCD in conjunction with the chemical WNT pathway activator CHIR99021 results in distinct paracrine microenvironments codifying hPSCs towards definitive endoderm,precardiac or presomitic mesoderm within the first 24 h of differentiation,respectively. Global gene expression and secretome analysis reveals that TGFß superfamily members,antagonist of Nodal signalling LEFTY1 and CER1,are paracrine determinants restricting PS progression. These data result in a tangible model disclosing how hPSC-released factors deflect CHIR99021-induced lineage commitment over time. By demonstrating a decisive,functional role of the BCD,we show its utility as a method to control lineage-specific differentiation. Furthermore,these findings have profound consequences for inter-experimental comparability,reproducibility,bioprocess optimization and scale-up. View Publication

Previous studies in the mouse indicated that ARID3A plays a critical role in the first cell fate decision required for generation of trophectoderm (TE). Here,we demonstrate that ARID3A is widely expressed during mouse and human placentation and essential for early embryonic viability. ARID3A localizes to trophoblast giant cells and other trophoblast-derived cell subtypes in the junctional and labyrinth zones of the placenta. Conventional Arid3a knockout embryos suffer restricted intrauterine growth with severe defects in placental structural organization. Arid3a null placentas show aberrant expression of subtype-specific markers as well as significant alteration in cytokines,chemokines and inflammatory response-related genes,including previously established markers of human placentation disorders. BMP4-mediated induction of trophoblast stem (TS)-like cells from human induced pluripotent stem cells results in ARID3A up-regulation and cytoplasmic to nuclear translocation. Overexpression of ARID3A in BMP4-mediated TS-like cells up-regulates TE markers,whereas pluripotency markers are down-regulated. Our results reveal an essential,conserved function for ARID3A in mammalian placental development through regulation of both intrinsic and extrinsic developmental programs. View Publication

Host proteins are essential for HIV entry and replication and can be important nonviral therapeutic targets. Large-scale RNA interference (RNAi)-based screens have identified nearly a thousand candidate host factors,but there is little agreement among studies and few factors have been validated. Here we demonstrate that a genome-wide CRISPR-based screen identifies host factors in a physiologically relevant cell system. We identify five factors,including the HIV co-receptors CD4 and CCR5,that are required for HIV infection yet are dispensable for cellular proliferation and viability. Tyrosylprotein sulfotransferase 2 (TPST2) and solute carrier family 35 member B2 (SLC35B2) function in a common pathway to sulfate CCR5 on extracellular tyrosine residues,facilitating CCR5 recognition by the HIV envelope. Activated leukocyte cell adhesion molecule (ALCAM) mediates cell aggregation,which is required for cell-to-cell HIV transmission. We validated these pathways in primary human CD4(+) T cells through Cas9-mediated knockout and antibody blockade. Our findings indicate that HIV infection and replication rely on a limited set of host-dispensable genes and suggest that these pathways can be studied for therapeutic intervention. View Publication

Cellular therapy using stem cells in bone regeneration has gained increasing interest. Various studies suggest the clinical utility of osteoprogenitors-like mesenchymal stem cells in bone regeneration. However,limited availability of mesenchymal stem cells and conflicting evidence on their therapeutic efficacy limit their clinical application. Human embryonic stem cells (hESCs) are potentially an unlimited source of healthy and functional osteoprogenitors (OPs) that could be utilized for bone regenerative applications. However,limited ability to track hESC-derived progenies in vivo greatly hinders translational studies. Hence,in this study,we aimed to establish hESC-derived OPs (hESC-OPs) expressing green fluorescent protein (GFP) and to investigate their osteogenic differentiation potential in vitro. We fluorescently labelled H9-hESCs using a plasmid vector encoding GFP. The GFP-expressing hESCs were differentiated into hESC-OPs. The hESC-OPs(GFP+) stably expressed high levels of GFP,CD73,CD90,and CD105. They possessed osteogenic differentiation potential in vitro as demonstrated by increased expression of COL1A1,RUNX2,OSTERIX,and OPG transcripts and mineralized nodules positive for Alizarin Red and immunocytochemical expression of osteocalcin,alkaline phosphatase,and collagen-I. In conclusion,we have demonstrated that fluorescently labelled hESC-OPs can maintain their GFP expression for the long term and their potential for osteogenic differentiation in vitro. In future,these fluorescently labelled hESC-OPs could be used for noninvasive assessment of bone regeneration,safety,and therapeutic efficacy. View Publication

BACKGROUND Human rhinoviruses (HRVs) are a major trigger of asthma exacerbations,with the bronchial epithelium being the major site of HRV infection and replication. Mast cells (MCs) play a key role in asthma where their numbers are increased in the bronchial epithelium with increasing disease severity. OBJECTIVE In view of the emerging role of MCs in innate immunity and increased localization to the asthmatic bronchial epithelium,we investigated whether HRV infection of MCs generated innate immune responses which were protective against infection. METHODS The LAD2 MC line or primary human cord blood-derived MCs (CBMCs) were infected with HRV or UV-irradiated HRV at increasing multiplicities of infection (MOI) without or with IFN-β or IFN-λ. After 24 h,innate immune responses were assessed by RT-qPCR and IFN protein release by ELISA. Viral replication was determined by RT-qPCR and virion release by TCID50 assay. RESULTS HRV infection of LAD2 MCs induced expression of IFN-β,IFN-λ and IFN-stimulated genes. However,LAD2 MCs were permissive for HRV replication and release of infectious HRV particles. Similar findings were observed with CBMCs. Neutralization of the type I IFN receptor had minimal effects on viral shedding,suggesting that endogenous type I IFN signalling offered limited protection against HRV. However,augmentation of these responses by exogenous IFN-β,but not IFN-λ,protected MCs against HRV infection. CONCLUSION AND CLINICAL RELEVANCE MCs are permissive for the replication and release of HRV,which is prevented by exogenous IFN-β treatment. Taken together,these findings suggest a novel mechanism whereby MCs may contribute to HRV-induced asthma exacerbations. View Publication

Mouse embryonic fibroblasts (MEFs) and human foreskin fibroblasts (HFFs) are used for the culture of human embryonic stem cells (hESCs). MEFs and HFFs differed in their capacity to support the proliferation and pluripotency of hESCs and could affect cardiac differentiation potential of hESCs. The aim of this study was to evaluate the effect of MEFs and HFFs feeders on dopaminergic differentiation of hESCs lines. To minimize the impact of culture condition variation,two hESCs lines were cultured on mixed feeder cells (MFCs,MEFs: HFFs = 1:1) and HFFs feeder,respectively,and then were differentiated into dopaminergic (DA) neurons under the identical protocol. Dopaminergic differentiation was evaluated by immunocytochemistry,quantitative fluorescent real-time PCR,transmission and scanning electron microscopy,and patch clamp. Our results demonstrated that these hESCs-derived neurons were genuine and functional DA neurons. However,compared to hESCs line on MFCs feeder,hESCs line on HFFs feeder had a higher proportion of tyrosine hydroxylase (TH) positive cells and expressed higher levels of FOXA2,PITX3,NURR1,and TH genes. In addition,the values of threshold intensity and threshold membrane potential of DA neurons from hESCs line on HFFs feeder were lower than those of DA neurons from hESCs line on the MFCs feeder. In conclusion,HFFs feeder not only facilitated the differentiation of hESCs cells into dopaminergic neurons,but also induced hESCs-derived DA neurons to express higher electrophysiological excitability. Therefore,feeder cells could affect not only dopaminergic differentiation potential of different hESCs lines,but also electrophysiological properties of hESCs-derived DA neurons. View Publication

Numerous studies have demonstrated that administration of antigen (Ag)-pulsed dendritic cells (DCs) is an effective strategy for enhancing immunity to tumors and infectious disease organisms. However,the generation and/or isolation of DCs can require substantial time and expense. Therefore,using inactivated F. tularensis (iFt) Ag as a model immunogen,we first sought to determine if DCs could be replaced with peripheral blood mononuclear cells (PBMCs) during the ex-vivo pulse phase and still provide protection against Ft infection. Follow up studies were then conducted using the S. pneumoniae (Sp) vaccine Prevnar {\textregistered}13 as the Ag in the pulse phase followed by immunization and Sp challenge. In both cases,we demonstrate that PBMCs can be used in place of DCs when pulsing with iFt and/or Prevnar {\textregistered}13 ex vivo and re-administering the Ag-pulsed PBMCs as a vaccine. In addition,utilization of the i.n. route for Ag-pulsed PBMC administration is superior to use of the i.v. route in the case of Sp immunization,as well as when compared to direct injection of Prevnar {\textregistered}13 vaccine i.m. or i.n. Furthermore,this PBMC-based vaccine strategy provides a more marked and enduring protective immune response and is also capable of serving as a multi-organism vaccine platform. The potential for this ex-vivo vaccine strategy to provide a simpler,less time consuming,and less expensive approach to DC-based vaccines and vaccination in general is also discussed. View Publication

X-linked chronic granulomatous disease (X-CGD) is an immune deficiency resulting from defective production of microbicidal reactive oxygen species (ROS) by phagocytes. Causative mutations occur throughout the CYBB gene,resulting in absent or defective gp91(phox) protein expression. To correct CYBB exon 5 mutations while retaining normal gene regulation,we utilized TALEN or Cas9 for exon 5 replacement in induced pluripotent stem cells (iPSCs) from patients,which restored gp91(phox) expression and ROS production in iPSC-derived granulocytes. Alternate approaches for correcting the majority of X-CGD mutations were assessed,involving TALEN- or Cas9-mediated insertion of CYBB minigenes at exon 1 or 2 of the CYBB locus. Targeted insertion of an exon 1-13 minigene into CYBB exon 1 resulted in no detectable gp91(phox) expression or ROS activity in iPSC-derived granulocytes. In contrast,targeted insertion of an exon 2-13 minigene into exon 2 restored both gp91(phox) and ROS activity. This demonstrates the efficacy of two correction strategies: seamless repair of specific CYBB mutations by exon replacement or targeted insertion of an exon 2-13 minigene to CYBB exon 2 while retaining exon/intron 1. Furthermore,it highlights a key issue for targeted insertion strategies for expression from an endogenous promoter: retention of intronic elements can be necessary for expression. View Publication

Insulin-like growth factor (IGF) is involved in regulating many processes during neural development,and IGF binding protein-4 (IGFBP4) functions as a modulator of IGF actions or in an IGF-independent manner (e.g.,via inhibiting Wnt/β-catenin signaling). In the present study,neural progenitor cells (NPCs) were isolated from the forebrain of newborn mice to investigate effects of IGFBP4 on the proliferation and differentiation of NPCs. The proliferation of NPCs was evaluated using Cell Counting Kit-8 (CCK-8) after treatment with or without IGFBP4 as well as blockers of IGF-IR and β-catenin. Phosphorylation levels of Akt,Erk1,2 and p38 were analyzed by Western blotting. The differentiation of NPCs was evaluated using immunofluorescence and Western blotting. It was shown that exogenous IGFBP4 significantly inhibited the proliferation of NPCs and it did not induce a more pronounced inhibition of cell proliferation after blockade of IGF-IR but it did after antagonism of β-catenin. Akt phosphorylation was significantly decreased and phosphorylation levels of Erk1,2 and p38 were not significantly changed in IGFBP4-treated NPCs. Excessive IGFBP4 significantly promoted NPCs to differentiate into astrocytes and neurons. These data suggested that exogenous IGFBP4 inhibits proliferation and promotes differentiation of neural progenitor cells mainly through IGF-IR signaling pathway. View Publication