技术资料

Elevated numbers of primitive Philadelphia chromosome-positive (Ph+) progenitors,including long-term culture-initiating cells (LTC-IC) as well as colony-forming cells (CFC),have been previously described in the blood of patients with chronic myeloid leukemia (CML) in chronic phase with high white blood cell counts. In the present study,which focused primarily on an analysis of circulating progenitors present in such patients at diagnosis,we discovered the frequent and occasionally exclusive presence of circulating normal (Ph-) LTC-IC,often at levels above those seen for LTC-IC in the blood of normal individuals. The presence of detectable numbers of circulating Ph- LTC-IC was independent of the fact that the same peripheral blood samples also contained elevated numbers of predominantly or exclusively Ph+ CFC. Interestingly,both the Ph+ and Ph- LTC-IC in these samples were CD34+CD71- and variably CD38- and Thy-1+,as previously documented for LTC-IC in normal marrow. Thus,neither CD38 nor Thy-1 expression was useful for discriminating between Ph+ and Ph- LTC-IC in mixed populations. Nevertheless,an association of these phenotypes with LTC-IC function did allow highly enriched (textgreater 5% pure) suspensions of either Ph+ or Ph- LTC-IC to be obtained from selected samples of CML blood in which the initial LTC-IC population was either predominantly Ph+ or Ph-,respectively. These findings suggest that the mechanisms causing mobilization of leukemic stem cells in untreated CML patients may affect their normal counterparts. They also indicate a possible new source of autologous cells for the support of intensive therapy of CML patients. Finally,they provide a method for obtaining the most highly purified populations of Ph+ LTC-IC described to date. This method should be useful for further analyses of the molecular activities of these very primitive neoplastic cells. View Publication

Small Kinetochore-Associated Protein (SKAP)/Kinastrin is a multifunctional protein with proposed roles in mitosis,apoptosis and cell migration. Exact mechanisms underlying its activities in these cellular processes are not completely understood. SKAP is predicted to have different isoforms,however,previous studies did not differentiate between them. Since distinct molecular architectures of protein isoforms often influence their localization and functions,this study aimed to examine the expression profile and functional differences between SKAP isoforms in human and mouse. Analyses of various human tissues and cells of different origin by RT-PCR,and by Western blotting and immunocytochemistry applying newly generated anti-SKAP monoclonal antibodies revealed that human SKAP exists in two protein isoforms: ubiquitously expressed SKAP16 and testis/sperm-specific SKAP1. In mouse,SKAP1 expression is detectable in testis at 4 weeks postnatally,when the first wave of spermatogenesis in mice is complete and the elongated spermatids are present in the testes. Furthermore,we identified Pontin as a new SKAP1 interaction partner. SKAP1 and Pontin co-localized in the flagellar region of human sperm suggesting a functional relevance for SKAP1-Pontin interaction in sperm motility. Since most previous studies on SKAP were performed with the testis-specific isoform SKAP1,our findings provide a new basis for future studies on the role of SKAP in both human somatic cells and male germ cells,including studies on male fertility. View Publication

The physical culture parameters have important influences on the proliferation and differentiation fate of hematopoietic stem cells. Recently,we have demonstrated that CD34+ cord blood (CB) cells undergo accelerated and increased megakaryocyte (Mk) differentiation when incubated under mild hyperthermic conditions (i.e.,39 degrees C). In this study,we investigated in detail the impacts of mild hyperthermia on Mk differentiation and maturation,and explored potential mechanisms responsible for these phenomena. Our results demonstrate that the qualitative and quantitative effects on Mk differentiation at 39 degrees C appear rapidly within 7 days,and that early transient culture at 39 degrees C led to even greater Mk yields (ptextless0.03). Surprisingly,cell viability was only found to be significantly reduced in the early stages of culture,suggesting that CB cells are able with time to acclimatize themselves to 39 degrees C. Although mild hyperthermia accelerated differentiation and maturation of CB-derived Mks,it failed to promote their polyploidization further but rather led to a small reduction in the proportion of polyploid Mks (p=0.01). Conversely,gene arrays analysis demonstrated that Mks derived at 39 degrees C have a normal gene expression program consistent with an advanced maturation state. Finally,two independent mechanisms that could account for the accelerated Mk differentiation were investigated. Our results suggest that the accelerated and increased Mk differentiation induced by mild hyperthermia is not mediated by cell-secreted factors but could perhaps be mediated by the increased expression of Mk transcription factors. View Publication

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmia syndrome characterized by adrenaline induced ventricular tachycardia. The primary genetic aetiologies underlying CPVT are either autosomal dominant or autosomal recessive inheritance,resulting from heterozygous mutations in cardiac ryanodine receptor (RYR2) and homozygous mutations in cardiac calsequestrin-2 (CASQ2),respectively. Recently,a large family with autosomal dominant CPVT due to a heterozygous mutation in CASQ2,p.Lys180Arg,was reported. This resource is the first induced pluripotent stem cell line generated from a patient with autosomal dominant CPVT due to a heterozygous mutation in CASQ2. Induced pluripotent stem cells were generated from the whole blood of a 40-year-old woman with severe CPVT who is heterozygous for the p.Lys180Arg CASQ2 mutation. Induced pluripotent stem cell (iPSC) characterization confirmed expression of pluripotency makers,trilineage differentiation potential,and the absence of exogenous pluripotency vector expression. View Publication

Although transcriptional and posttranscriptional events are detected in RNA-Seq data from second-generation sequencing,full-length mRNA isoforms are not captured. On the other hand,third-generation sequencing,which yields much longer reads,has current limitations of lower raw accuracy and throughput. Here,we combine second-generation sequencing and third-generation sequencing with a custom-designed method for isoform identification and quantification to generate a high-confidence isoform dataset for human embryonic stem cells (hESCs). We report 8,084 RefSeq-annotated isoforms detected as full-length and an additional 5,459 isoforms predicted through statistical inference. Over one-third of these are novel isoforms,including 273 RNAs from gene loci that have not previously been identified. Further characterization of the novel loci indicates that a subset is expressed in pluripotent cells but not in diverse fetal and adult tissues; moreover,their reduced expression perturbs the network of pluripotency-associated genes. Results suggest that gene identification,even in well-characterized human cell lines and tissues,is likely far from complete. View Publication

Due to viral envelope glycoprotein D binding to cellular membrane HVEM receptor,HSV-1 can infect certain dendritic cells,which becomes an event in the viral strategy to interfere with the host's immune system. We previously generated the HSV-1 mutant strain M6,which produced an attenuated phenotype in mice and rhesus monkeys. The attenuated M6 strain was used to investigate how HSV-1 infection of dendritic cells interferes with both innate and adaptive immunity. Our study showed that dendritic cells membrane HVEM receptors could mediate infection of the wild-type strain and attenuated M6 strain and that dendritic cells infected by both viruses in local tissues of animals exhibited changes in transcriptional profiles associated with innate immune and inflammatory responses. The infection of pDCs and cDCs by the two strains promoted cell differentiation to the CD103+ phenotype,but varied transcriptional profiles were observed,implying a strategy that the HSV-1 wild-type strain interferes with antiviral immunity,probably due to viral modification of the immunological phenotype of dendritic cells during processing and presentation of antigen to T cells,leading to a series of deviations in immune responses,ultimately generating the deficient immune phenotype observed in infected individuals in the clinical. View Publication

Mitogen-activated protein (MAP) kinase cascades represent one of the major signal systems used by eukaryotic cells to transduce extracellular signals into cellular responses. Four MAP kinase subgroups have been identified in humans: ERK,JNK (SAPK),ERK5 (BMK),and p38. Here we characterize a new MAP kinase,p38beta. p38beta is a 372-amino acid protein most closely related to p38. It contains a TGY dual phosphorylation site,which is required for its kinase activity. Like p38,p38beta is activated by proinflammatory cytokines and environmental stress. A comparison of events associated with the activation of p38beta and p38 revealed differences,most notably in the preferred activation of p38beta by MAP kinase kinase 6 (MKK6),whereas p38 was activated nearly equally by MKK3,MKK4,and MKK6. Moreover,in vitro and in vivo experiments showed a strong substrate preference by p38beta for activating transcription factor 2 (ATF2). Enhancement of ATF2-dependent gene expression by p38beta was approximately20-fold greater than that of p38 and other MAP kinases tested. The data reported here suggest that while closely related,p38beta and p38 may be regulated by differing mechanisms and may exert their actions on separate downstream targets. View Publication

Plasmacytoid dendritic cells (pDCs) are multifunctional immune cells with roles in both the innate and adaptive immune system. Their hallmark function is production of large amounts of type I interferons in response to viral infections,but they are also capable of producing a range of other cytokines,antigen presentation,and cytotoxicity. Their potential as an immunotherapy for cancer and infectious disease is being explored,but broad application of these cells is challenged by low frequency in the blood and low viability during ex vivo culturing. We have previously developed an effective in vitro differentiation protocol for producing pDCs from CD34+ hematopoietic stem and progenitor cells (HSPC-pDCs),which provides an attainable and large source of pDCs. HSPC-pDCs present pDC characteristics and functions,and like naturally occurring pDCs they exhibit large phenotypic and functional heterogeneity. Here,we characterize different cell subsets from in vitro pDC differentiation and identify a distinct population,which is the major producer of IFNα in response to TLR9 stimulation and display a transcriptomic profile similar to what is seen for pDCs circulating in the blood. We also investigate the possibility of rerouting subset specification during HSPCs-to-pDC differentiation by controlling gene expression of key master transcription factors (TFs). We identify TFs associated with the pDC differentiation trajectory that are essential for the development of TLR9-responsive HSPC-pDCs,and we also identify TFs that increase their frequency. In conclusion,we phenotypically and functionally characterize different cell subsets and modulate their relative frequencies by manipulating TF expression during pDC differentiation. These findings provide a deeper understanding of in vitro-differentiated pDC cultures that may spur further developments in their use as an immunomodulatory cell therapy. View Publication

BACKGROUND Massive liquid crystal droplets have been found during embryonic development in more than twenty different tissues and organs,including the liver,brain and kidney. Liquid crystal deposits have also been identified in multiple human pathologies,including vascular disease,liver dysfunction,age-related macular degeneration,and other chronic illnesses. Despite the involvement of liquid crystals in such a large number of human processes,this phenomenon is poorly understood and there are no in vitro systems to further examine the function of liquid crystals in biology. RESULTS We report the presence of tubular birefringent structures in embryoid bodies (EBs) differentiated in culture. These birefringent tubular structures initiate at the EB surface and penetrated the cortex at a variety of depths. Under crossed polarized light,these tubules are seen as a collection of birefringent Maltese crosses and tubules with birefringent walls and a non-birefringent lumen. The fluidity of these birefringent structures under pressure application led to elongation and widening,which was partially recoverable with pressure release. These birefringent structures also displayed heat triggered phase transition from liquid crystal to isotropic status that is partially recoverable with return to ambient temperature. These pressure and temperature triggered changes confirm the birefringent structures as liquid crystals. The first report of liquid crystal so early in development. CONCLUSION The structure of the liquid crystal tubule network we observed distributed throughout the differentiated embryoid bodies may function as a transportation network for nutrients and metabolic waste during EB growth,and act as a precursor to the vascular system. This observation not only reveals the involvement of liquid crystals earlier than previously known,but also provides a method for studying liquid crystals in vitro. View Publication

Whipworms are parasitic nematodes that live in the gut of more than 500 million people worldwide. Owing to the difficulty in obtaining parasite material,the mouse whipwormTrichuris murishas been extensively used as a model to study human whipworm infections. These nematodes secrete a multitude of compounds that interact with host tissues where they orchestrate a parasitic existence. Herein we provide the first comprehensive characterization of the excretory/secretory products ofT. muris. We identify 148 proteins secreted byT. murisand show for the first time that the mouse whipworm secretes exosome-like extracellular vesicles (EVs) that can interact with host cells. We use an Optiprep{\textregistered} gradient to purify the EVs,highlighting the suitability of this method for purifying EVs secreted by a parasitic nematode. We also characterize the proteomic and genomic content of the EVs,identifying {\textgreater}350 proteins,56 miRNAs (22 novel) and 475 full-length mRNA transcripts mapping toT. murisgene models. Many of the miRNAs putatively mapped to mouse genes are involved in regulation of inflammation,implying a role in parasite-driven immunomodulation. In addition,for the first time to our knowledge,colonic organoids have been used to demonstrate the internalization of parasite EVs by host cells. Understanding how parasites interact with their host is crucial to develop new control measures. This first characterization of the proteins and EVs secreted byT. murisprovides important information on whipworm-host communication and forms the basis for future studies. View Publication

SARS-CoV-2 Omicron variant has evolved into sublineages. Here,we compared the neutralization susceptibility and viral fitness of EG.5.1 and XBB.1.9.1. Serum neutralization antibody titer against EG.5.1 was 1.71-fold lower than that for XBB.1.9.1. However,there was no significant difference in virus replication between EG.5.1 and XBB.1.9.1 in human nasal organoids and TMPRSS2/ACE2 over-expressing A549 cells. No significant difference was observed in competitive fitness and cytokine/chemokine response between EG.5.1 and XBB.1.9.1. Both EG.5.1 and XBB.1.9.1 replicated more robustly in the nasal organoid from a younger adult than that from an older adult. Our findings suggest that enhanced immune escape contributes to the dominance of EG.5.1 over earlier sublineages. The combination of population serum susceptibility testing and viral fitness evaluation with nasal organoids may hold promise in risk assessment of upcoming variants. Utilization of serum specimens and nasal organoid derived from older adults provides a targeted risk assessment for this vulnerable population. Subject areas: Immunology,Immune response,Virology View Publication

Microenvironmental factors such as oxygen concentration mediate key effects on the biology of mesenchymal stromal cells (MSCs). Herein,we performed an in-depth characterization of the metabolic behavior of MSCs derived from the placenta,umbilical cord,and adipose tissue (termed hPMSCs,UC-MSCs,and AD-MSCs,respectively) at physiological (hypoxic; 5{\%} oxygen [O2]) and standardized (normoxic; 21{\%} O2) O2 concentrations using chemical isotope labeling liquid chromatography-mass spectrometry. 12C- and 13C-isotope dansylation (Dns) labeling was used to analyze the amine/phenol submetabolome,and 2574 peak pairs or metabolites were detected and quantified,from which 52 metabolites were positively identified using a library of 275 Dns-metabolite standards; 2189 metabolites were putatively identified. Next,we identified six metabolites using the Dns library,as well as 14 hypoxic biomarkers from the human metabolome database out of 96 altered metabolites. Ultimately,metabolic pathway analyses were performed to evaluate the associated pathways. Based on pathways identified using the Kyoto Encyclopedia of Genes and Genomes,we identified significant changes in the metabolic profiles of MSCs in response to different O2 concentrations. These results collectively suggest that O2 concentration has the strongest influence on hPMSCs metabolic characteristics,and that 5{\%} O2 promotes arginine and proline metabolism in hPMSCs and UC-MSCs but decreases gluconeogenesis (alanine-glucose) rates in hPMSCs and AD-MSCs. These changes indicate that MSCs derived from different sources exhibit distinct metabolic profiles. View Publication