技术资料

Telomere length maintenance ensures self-renewal of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs); however,the mechanisms governing telomere length homeostasis in these cell types are unclear. Here,we report that telomere length is determined by the balance between telomere elongation,which is mediated by telomerase,and telomere trimming,which is controlled by XRCC3 and Nbs1,homologous recombination proteins that generate single-stranded C-rich telomeric DNA and double-stranded telomeric circular DNA (T-circles),respectively. We found that reprogramming of differentiated cells induces T-circle and single-stranded C-rich telomeric DNA accumulation,indicating the activation of telomere trimming pathways that compensate telomerase-dependent telomere elongation in hiPSCs. Excessive telomere elongation compromises telomere stability and promotes the formation of partially single-stranded telomeric DNA circles (C-circles) in hESCs,suggesting heightened sensitivity of stem cells to replication stress at overly long telomeres. Thus,tight control of telomere length homeostasis is essential to maintain telomere stability in hESCs. View Publication

The realization of personalized medicine through human induced pluripotent stem cell (iPSC) technology can be advanced by transcriptomics,epigenomics,and bioinformatics that inform on genetic pathways directing tissue development and function. When possible,population diversity should be included in new studies as resources become available. Previously we derived replicate iPSC lines of African American,Hispanic-Latino and Asian self-designated ethnically diverse (ED) origins with normal karyotype,verified teratoma formation,pluripotency biomarkers,and tri-lineage in vitro commitment. Here we perform bioinformatics of RNA-Seq and ChIP-seq pluripotency data sets for two replicate Asian and Hispanic-Latino ED-iPSC lines that reveal differences in generation of contractile cardiomyocytes but similar and robust differentiation to multiple neural,pancreatic,and smooth muscle cell types. We identify shared and distinct genes and contributing pathways in the replicate ED-iPSC lines to enhance our ability to understand how reprogramming to iPSC impacts genes and pathways contributing to cardiomyocyte contractility potential. View Publication

PURPOSE Nonexudative (dry) age-related macular degeneration (AMD),a leading cause of blindness in the elderly,is associated with the loss of retinal pigmented epithelium (RPE) cells and the development of geographic atrophy,which are areas devoid of RPE cells and photoreceptors. One possible treatment option would be to stimulate RPE attachment and proliferation to replace dying/dysfunctional RPE and bring about wound repair. Clinical trials are underway testing injections of RPE cells derived from pluripotent stem cells to determine their safety and efficacy in treating AMD. However,the factors regulating RPE responses to AMD-associated lesions are not well understood. Here,we use cell culture to investigate the role of RhoA coiled coil kinases (ROCKs) in human embryonic stem cell-derived RPE (hESC-RPE) attachment,proliferation,and wound closure. METHODS H9 hESC were spontaneously differentiated into RPE cells. hESC-RPE cells were treated with a pan ROCK1/2 or a ROCK2 only inhibitor; attachment,and proliferation and cell size within an in vitro scratch assay were examined. RESULTS Pharmacological inhibition of ROCKs promoted hESC-RPE attachment and proliferation,and increased the rate of closure of in vitro wounds. ROCK inhibition decreased phosphorylation of cofilin and myosin light chain,suggesting that regulation of the cytoskeleton underlies the mechanism of action of ROCK inhibition. CONCLUSIONS ROCK inhibition promotes attachment,proliferation,and wound closure in H9 hESC-RPE cells. ROCK isoforms may have different roles in wound healing. TRANSLATIONAL RELEVANCE Modulation of the ROCK-cytoskeletal axis has potential in stimulating wound repair in transplanted RPE cells and attachment in cellular therapies. View Publication

Diverse Ab effector functions mediated by the Fc domain have been commonly associated with reduced risk of infection in a growing number of nonhuman primate and human clinical studies. This study evaluated the anti-HIV Ab effector activities in polyclonal serum samples from HIV-infected donors,VAX004 vaccine recipients,and healthy HIV-negative subjects using a variety of primary and cell line-based assays,including Ab-dependent cellular cytotoxicity (ADCC),Ab-dependent cell-mediated viral inhibition,and Ab-dependent cellular phagocytosis. Additional assay characterization was performed with a panel of Fc-engineered variants of mAb b12. The goal of this study was to characterize different effector functions in the study samples and identify assays that might most comprehensively and dependably capture Fc-mediated Ab functions mediated by different effector cell types and against different viral targets. Deployment of such assays may facilitate assessment of functionally unique humoral responses and contribute to identification of correlates of protection with potential mechanistic significance in future HIV vaccine studies. Multivariate and correlative comparisons identified a set of Ab-dependent cell-mediated viral inhibition and phagocytosis assays that captured different Ab activities and were distinct from a group of ADCC assays that showed a more similar response profile across polyclonal serum samples. The activities of a panel of b12 monoclonal Fc variants further identified distinctions among the ADCC assays. These results reveal the natural diversity of Fc-mediated Ab effector responses among vaccine recipients in the VAX004 trial and in HIV-infected subjects,and they point to the potential importance of polyfunctional Ab responses. View Publication

We have compared the transcriptomic profiles of human induced pluripotent stem cells after their differentiation in hepatocytes like cells in plates and microfluidic biochips. The biochips provided a 3D and dynamic support during the cell differentiation when compared to the 2D static cultures in plates. The microarray have demonstrated the up regulation of important pathway related to liver development and maturation during the culture in biochips. Furthermore,the results of the transcriptomic profile,coupled with immunostaining,and RTqPCR analysis have shown typical biomarkers illustrating the presence of responders of biliary like cells,hepatocytes like cells,and endothelial like cells. However,the overall tissue still presented characteristic of immature and foetal patterns. Nevertheless,the biochip culture provided a specific micro-environment in which a complex multicellular differentiation toward liver could be oriented. View Publication

The advent of human induced pluripotent stem cell (hiPSC) technology has produced patient-specific hiPSC derived cardiomyocytes (hiPSC-CMs) that can be used as a platform to study cardiac diseases and to explore new therapies.The ability to genetically manipulate hiPSC-CMs not only is essential for identifying the structural and/or functional role of a protein but can also provide valuable information regarding therapeutic applications. In this chapter,we describe protocols for culture,maintenance,and cardiac differentiation of hiPSCs. Then,we provide a basic procedure to transduce hiPSC-CMs. View Publication

The cytogenetic analysis of plasma cell myeloma (PCM) allows stratification of patients so that prognosis may be determined and appropriate therapeutic options can be discussed. Owing to the patchy nature of the disease in the bone marrow (BM),the low proliferative activity of plasma cells and the cryptic nature of some PCM-associated cytogenetic changes,karyotypic analysis in this disease should be augmented with targeted interphase fluorescence in situ hybridization (FISH). Immunofluorescent revelation of cytoplasmic immunoglobulin light chains,together with interphase FISH (cIg-FISH),allows the identification of plasma cells within a sample so that they may be scored preferentially. This is particularly useful in situations where there are only a small percentage of plasma cells in a sample. Where an underlying myeloid disease is suspected the cIg-FISH-negative cells can be scored separately. Two methods are provided in this chapter: the technique for cIg-FISH in fresh PCM BM samples and a procedure for use in fixed cytogenetics preparations. View Publication

Human pluripotent stem cells (hPSCs) provide a valuable model for the study of human development and a means to generate a scalable source of cells for therapeutic applications. This protocol specifies cell fate efficiently into cardiac and endothelial lineages from hPSCs. The protocol takes 2 weeks to complete and requires experience in hPSC culture and differentiation techniques. Building on lessons taken from early development,this monolayer-directed differentiation protocol uses different concentrations of activin A and bone morphogenetic protein 4 (BMP4) to polarize cells into mesodermal subtypes that reflect mid-primitive-streak cardiogenic mesoderm and posterior-primitive-streak hemogenic mesoderm. This differentiation platform provides a basis for generating distinct cardiovascular progenitor populations that enable the derivation of cardiomyocytes and functionally distinct endothelial cell (EC) subtypes from cardiogenic versus hemogenic mesoderm with high efficiency without cell sorting. ECs derived from cardiogenic and hemogenic mesoderm can be matured into textgreater90% CD31(+)/VE-cadherin(+) definitive ECs. To test the functionality of ECs at different stages of differentiation,we provide methods for assaying the blood-forming potential and de novo lumen-forming activity of ECs. To our knowledge,this is the first protocol that provides a common platform for directed differentiation of cardiomyocytes and endothelial subtypes from hPSCs. This protocol yields endothelial differentiation efficiencies exceeding those of previously published protocols. Derivation of these cell types is a critical step toward understanding the basis of disease and generating cells with therapeutic potential. View Publication

To develop a purification strategy for isolating the most primitive hematopoietic stem cells present in normal human marrow we have combined cell separation techniques with an assay for cells that initiate sustained hematopoiesis in vitro in the presence of irradiated human marrow adherent cells. These feeders" were established by subculturing 2- to 6-week-old primary long-term marrow culture adherent layers at a density of 3 x 10(4) irradiated cells per square centimeter. Test "long-term culture (LTC)-initiating cells" were plated on top of the feeders and the cocultures then maintained as standard long-term marrow cultures with half-media changes and removal of half of the nonadherent cells each week. The total number of myeloid View Publication

Human embryonic stem cells (hESCs) are used as platforms for disease study,drug screening and cell-based therapy. To facilitate these applications,it is frequently necessary to genetically manipulate the hESC genome. Gene editing with engineered nucleases enables site-specific genetic modification of the human genome through homology-directed repair (HDR). However,the frequency of HDR remains low in hESCs. We combined efficient expression of engineered nucleases and integration-defective lentiviral vector (IDLV) transduction for donor template delivery to mediate HDR in hESC line WA09. This strategy led to highly efficient HDR with more than 80% of the selected WA09 clones harboring the transgene inserted at the targeted genomic locus. However,certain portions of the HDR clones contained the concatemeric IDLV genomic structure at the target site,probably resulted from recombination of the IDLV genomic input before HDR with the target. We found that the integrase protein of IDLV mediated the highly efficient HDR through the recruitment of a cellular protein,LEDGF/p75. This study demonstrates that IDLV-mediated HDR is a powerful and broadly applicable technology to carry out site-specific gene modification in hESCs. View Publication

The host responds to virus infection by activating type I interferon (IFN) signaling leading to expression of IFN-stimulated genes (ISGs). Dysregulation of the IFN response results in inflammatory diseases and chronic infections. In this study,we demonstrate that IFN regulatory factor 2 (IRF2),an ISG and a negative regulator of IFN signaling,influences alphavirus neuroinvasion and pathogenesis. A Sindbis virus strain that in wild-type (WT) mice only causes disease when injected into the brain leads to lethal encephalitis in Irf2(-/-) mice after peripheral inoculation. Irf2(-/-) mice fail to control virus replication and recruit immune infiltrates into the brain. Reduced B cells and virus-specific IgG are observed in the Irf2(-/-) mouse brains despite the presence of peripheral neutralizing antibodies,suggesting a defect in B cell trafficking to the central nervous system (CNS). B cell-deficient μMT mice are significantly more susceptible to viral infection,yet WT B cells and serum are unable to rescue the Irf2(-/-) mice. Collectively,our data demonstrate that proper localization of B cells and local production of antibodies in the CNS are required for protection. The work advances our understanding of host mechanisms that affect viral neuroinvasion and their contribution to immunity against CNS infections. View Publication

TRAF1 is a signaling adaptor known for its role in tumor necrosis factor receptor-induced cell survival. Here we show that monocytes from healthy human subjects with a rheumatoid arthritis-associated single-nucleotide polymorphism (SNP) in the TRAF1 gene express less TRAF1 protein but greater amounts of inflammatory cytokines in response to lipopolysaccharide (LPS). The TRAF1 MATH domain binds directly to three components of the linear ubiquitination (LUBAC) complex,SHARPIN,HOIP and HOIL-1,to interfere with the recruitment and linear ubiquitination of NEMO. This results in decreased NF-κB activation and cytokine production,independently of tumor necrosis factor. Consistent with this,Traf1(-/-) mice show increased susceptibility to LPS-induced septic shock. These findings reveal an unexpected role for TRAF1 in negatively regulating Toll-like receptor signaling,providing a mechanistic explanation for the increased inflammation seen with a disease-associated TRAF1 SNP. View Publication