技术资料

Characterization of pluripotent stem cells is required for the registration of stem cell lines and allows for an impartial and objective comparison of the results obtained when generating multiple lines. It is therefore crucial to establish specific,fast and reliable protocols to detect the hallmarks of pluripotency. Such protocols should include immunocytochemistry (takes 2 d),identification of the three germ layers in in vitro-derived embryoid bodies by immunocytochemistry (immunodetection takes 3 d) and detection of differentiation markers in in vivo-generated teratomas by immunohistochemistry (differentiation marker detection takes 4 d). Standardization of the immunodetection protocols used ensures minimum variations owing to the source,the animal species,the endogenous fluorescence or the inability to collect large amounts of cells,thereby yielding results as fast as possible without loss of quality. This protocol provides a description of all the immunodetection procedures necessary to characterize mouse and human stem cell lines in different circumstances. View Publication

IMPORTANCE Although leukocyte telomere length is associated with mortality and many chronic diseases thought to be manifestations of age-related functional decline,it is not known whether it relates to acute disease in younger healthy populations. OBJECTIVE To determine whether shorter telomeres in leukocytes,especially CD8CD28- T cells,are associated with decreased resistance to upper respiratory infection and clinical illness in young to midlife adults. DESIGN,SETTING,AND PARTICIPANTS Between 2008 and 2011,telomere length was assessed in peripheral blood mononuclear cells (PBMCs) and T-cell subsets (CD4,CD8CD28+,CD8CD28-) from 152 healthy 18- to 55-year-old residents of Pittsburgh,Pennsylvania. Participants were subsequently quarantined (single rooms),administered nasal drops containing a common cold virus (rhinovirus 39),and monitored for 5 days for development of infection and clinical illness. MAIN OUTCOME MEASURES Infection (virus shedding or 4-fold increase in virus-specific antibody titer) and clinical illness (verified infection plus objective signs of illness). RESULTS Rates of infections and clinical illness were 69{\%} (n = 105) and 22{\%} (n = 33),respectively. Shorter telomeres were associated with greater odds of infection,independent of prechallenge virus-specific antibody,demographics,contraceptive use,season,and body mass index (PBMC: odds ratio [OR] per 1-SD decrease in telomere length,1.71 [95{\%} CI,1.08-2.72]; n = 128 [shortest tertile 77{\%} infected; middle,66{\%}; longest,57{\%}]; CD4: OR,1.76 [95{\%} CI,1.15-2.70]; n = 146 [shortest tertile 80{\%} infected; middle,71{\%}; longest,54{\%}]; CD8CD28+: OR,1.93 [95{\%} CI,1.21-3.09],n = 132 [shortest tertile 84{\%} infected; middle,64{\%}; longest,58{\%}]; CD8CD28-: OR,2.02 [95{\%} CI,1.29-3.16]; n = 144 [shortest tertile 77{\%} infected; middle,75{\%}; longest,50{\%}]). CD8CD28- was the only cell population in which shorter telomeres were associated with greater risk of clinical illness (OR,1.69 [95{\%} CI,1.01-2.84]; n = 144 [shortest tertile,26{\%}; middle,22{\%}; longest,13{\%}]). The association between CD8CD28- telomere length and infection increased with age (CD8CD28- telomere length × age interaction,b = 0.09 [95{\%} CI,0.02-0.16],P = .01,n = 144). CONCLUSION AND RELEVANCE In this preliminary study among a cohort of healthy 18- to 55-year-olds,shorter CD8CD28- T-cell telomere length was associated with increased risk for experimentally induced acute upper respiratory infection and clinical illness. View Publication

Haematopoietic stem cell (HSC) gene therapy has demonstrated potential to treat many diseases. However,current state of the art requires sophisticated ex vivo gene transfer in a dedicated Good Manufacturing Practices facility,limiting availability. An automated process would improve the availability and standardized manufacture of HSC gene therapy. Here,we develop a novel program for semi-automated cell isolation and culture equipment to permit complete benchtop generation of gene-modified CD34+ blood cell products for transplantation. These cell products meet current manufacturing quality standards for both mobilized leukapheresis and bone marrow,and reconstitute human haematopoiesis in immunocompromised mice. Importantly,nonhuman primate autologous gene-modified CD34+ cell products are capable of stable,polyclonal multilineage reconstitution with follow-up of more than 1 year. These data demonstrate proof of concept for point-of-care delivery of HSC gene therapy. Given the many target diseases for gene therapy,there is enormous potential for this approach to treat patients on a global scale. View Publication

Age-related macular degeneration (AMD) affects the retinal pigment epithelium (RPE),a cell monolayer essential for photoreceptor survival,and is the leading cause of vision loss in the elderly. There are no disease-altering therapies for dry AMD,which is characterized by accumulation of subretinal drusen deposits and complement-driven inflammation. We report the derivation of human-induced pluripotent stem cells (hiPSCs) from patients with diagnosed AMD,including two donors with the rare ARMS2/HTRA1 homozygous genotype. The hiPSC-derived RPE cells produce several AMD/drusen-related proteins,and those from the AMD donors show significantly increased complement and inflammatory factors,which are most exaggerated in the ARMS2/HTRA1 lines. Using a panel of AMD biomarkers and candidate drug screening,combined with transcriptome analysis,we discover that nicotinamide (NAM) ameliorated disease-related phenotypes by inhibiting drusen proteins and inflammatory and complement factors while upregulating nucleosome,ribosome,and chromatin-modifying genes. Thus,targeting NAM-regulated pathways is a promising avenue for developing therapeutics to combat AMD. View Publication

Genetic modification of cell lines and primary cells is an expensive and cumbersome approach,often involving the use of viral vectors. Electroporation using square-wave generating devices,like Lonza's Nucleofector,is a widely used option,but the costs associated with the acquisition of electroporation kits and the transient transgene expression might hamper the utility of this methodology. In the present work,we show that our in-house developed buffers,termed Chicabuffers,can be efficiently used to electroporate cell lines and primary cells from murine and human origin. Using the Nucleofector II device,we electroporated 14 different cell lines and also primary cells,like mesenchymal stem cells and cord blood CD34+,providing optimized protocols for each of them. Moreover,when combined with sleeping beauty-based transposon system,long-term transgene expression could be achieved in all types of cells tested. Transgene expression was stable and did not interfere with CD34+ differentiation to committed progenitors. We also show that these buffers can be used in CRISPR-mediated editing of PDCD1 gene locus in 293T and human peripheral blood mononuclear cells. The optimized protocols reported in this study provide a suitable and cost-effective platform for the genetic modification of cells,facilitating the widespread adoption of this technology. View Publication

Retinal degenerative diseases are among the leading causes of blindness worldwide,and cell replacement is considered as a promising therapeutic. However,the resources of seed cells are scarce. To further explore this type of therapy,we adopted a culture system that could harvest a substantial quantity of retinal progenitor cells (RPCs) from human embryonic stem cells (hESCs) within a relatively short period of time. Furthermore,we transplanted these RPCs into the subretinal spaces of Royal College of Surgeons (RCS) rats. We quantified the thickness of the treated rats' outer nuclear layers (ONLs) and explored the visual function via electroretinography (ERG). It was found that the differentiated cells expressed RPC markers and photoreceptor progenitor markers. The transplanted RPCs survived for at least 12 weeks,resulting in beneficial effects on the morphology of the host retina,and led to a significant improvement in the visual function of the treated animals. These therapeutic effects suggest that the hESCs-derived RPCs could delay degeneration of the retina and partially restore visual function. View Publication

Recently,deficiency in the cytosolic DNA sensor RNA Polymerase III was described in children with severe primary varicella-zoster virus (VZV) infection in the CNS and lungs. In the present study we examined adult patients with VZV CNS infection caused by viral reactivation. By whole exome sequencing we identified mutations in POL III genes in two of eight patients. These mutations were located in the coding regions of the subunits POLR3A and POLR3E. In functional assays,we found impaired expression of antiviral and inflammatory cytokines in response to the POL III agonist Poly(dA:dT) as well as increased viral replication in patient cells compared to controls. Altogether,this study provides significant extension on the current knowledge on susceptibility to VZV infection by demonstrating mutations in POL III genes associated with impaired immunological sensing of AT-rich DNA in adult patients with VZV CNS infection. View Publication

YAP,a key effector of Hippo pathway,is activated by its translocation from cytoplasm to nucleus to regulate gene expression and promote tumorigenesis. Although the mechanism by which YAP is suppressed in cytoplasm has been well-studied,how the activated YAP is sequestered in the nucleus remains unknown. Here,we demonstrate that YAP is a nucleocytoplasmic shuttling protein and its nuclear export is controlled by SET1A-mediated mono-methylation of YAP at K342,which disrupts the binding of YAP to CRM1. YAP mimetic methylation knockin mice are more susceptible to colorectal tumorigenesis. Clinically,YAP K342 methylation is reversely correlated with cancer survival. Collectively,our study identifies SET1A-mediated mono-methylation at K342 as an essential regulatory mechanism for regulating YAP activity and tumorigenesis. View Publication

Neutrophils are short-lived cells that play important roles in both health and disease. Neutrophils and monocytes originate from the granulocyte monocyte progenitor (GMP) in bone marrow; however,unipotent neutrophil progenitors are not well defined. Here,we use cytometry by time of flight (CyTOF) and single-cell RNA sequencing (scRNA-seq) methodologies to identify a committed unipotent early-stage neutrophil progenitor (NeP) in adult mouse bone marrow. Importantly,we found a similar unipotent NeP (hNeP) in human bone marrow. Both NeP and hNeP generate only neutrophils. NeP and hNeP both significantly increase tumor growth when transferred into murine cancer models,including a humanized mouse model. hNeP are present in the blood of treatment-naive melanoma patients but not of healthy subjects. hNeP can be readily identified by flow cytometry and could be used as a biomarker for early cancer discovery. Understanding the biology of hNeP should allow the development of new therapeutic targets for neutrophil-related diseases,including cancer. View Publication

Neurologic melioidosis occurs in both human and animals; however,the mechanism by which the pathogen Burkholderia pseudomallei invades the central nervous system (CNS) remains unclear. B. pseudomallei-loaded Ly6C cells have been suggested as a putative portal; however,during melioidosis,lipopolysaccharide (LPS) can drive disruption of the blood-brain barrier (BBB). This study aims to test whether the Trojan horse-like mechanism occurs during endotoxemia. The expression levels of cerebral cytokines,chemokines and cell adhesion molecules; the activation of astrocytes,microglia and endothelial cells; and the increased vascular permeability and brain-infiltrating leukocytes were evaluated using B. pseudomallei,B. thailandensis,B. cenocepacia and B. multivorans LPS-induced brains. Accordingly,different degrees of BBB damage in those brains with endotoxemia were established. The B. multivorans LPS-induced brain exhibited the highest levels of disruptive BBB according to the above mediators/indicators. Into these distinct groups of endotoxemic mice,B. pseudomallei-loaded Ly6C cells or free B. pseudomallei were adoptively transferred at equal bacterial concentrations (103 CFU). The bacterial load and number of cases of meningeal neutrophil infiltration in the brains of animals treated with B. pseudomallei-loaded Ly6C cells were higher than those in brains induced by free B. pseudomallei in any of the endotoxemic groups. In particular,these results were reproducible in B. multivorans LPS-induced brains. We suggest that B. pseudomallei-loaded cells can act as a Trojan horse and are more effective than free B. pseudomallei in invading the CNS under septic or endotoxemic conditions even when there is a high degree of BBB disruption. View Publication

Cytokine responses from monocytes and macrophages exposed to bacteria are of particular importance in innate immunity. Focusing on the impact of the immunoregulatory cytokine interleukin (IL)-27 on control of innate immune system responses,we examined human immune responses to bacterial products and bacterial infection by E. coli and S. typhimurium. Since the effect of IL-27 treatment in human myeloid cells infected with bacteria is understudied,we treated human monocytes and macrophages with IL-27 and either LPS,flagellin,or bacteria,to investigate the effect on inflammatory signaling and cytokine responses. We determined that simultaneous stimulation with IL-27 and LPS derived from E. coli or S. typhimurium resulted in enhanced IL-12p40,TNF-$\alpha$,and IL-6 expression compared to that by LPS alone. To elucidate if IL-27 manipulated the cellular response to infection with bacteria,we infected IL-27 treated human macrophages with S. typhimurium. While IL-27 did not affect susceptibility to S. typhimurium infection or S. typhimurium-induced cell death,IL-27 significantly enhanced proinflammatory cytokine production in infected cells. Taken together,we highlight a role for IL-27 in modulating innate immune responses to bacterial infection. View Publication

As the sole target of broadly neutralizing antibodies (bnAbs) to HIV,the envelope glycoprotein (Env) trimer is the focus of vaccination strategies designed to elicit protective bnAbs in humans. Because HIV Env is densely glycosylated with 75-90 N-glycans per trimer,most bnAbs use or accommodate them in their binding epitope,making the glycosylation of recombinant Env a key aspect of HIV vaccine design. Upon analysis of three HIV strains,we here find that site-specific glycosylation of Env from infectious virus closely matches Envs from corresponding recombinant membrane-bound trimers. However,viral Envs differ significantly from recombinant soluble,cleaved (SOSIP) Env trimers,strongly impacting antigenicity. These results provide a benchmark for virus Env glycosylation needed for the design of soluble Env trimers as part of an overall HIV vaccine strategy. View Publication