技术资料

The application of genome-wide expression analysis to a large-scale,multicentered program in critically ill patients poses a number of theoretical and technical challenges. We describe here an analytical and organizational approach to a systematic evaluation of the variance associated with genome-wide expression analysis specifically tailored to study human disease. We analyzed sources of variance in genome-wide expression analyses performed with commercial oligonucleotide arrays. In addition,variance in gene expression in human blood leukocytes caused by repeated sampling in the same subject,among different healthy subjects,among different leukocyte subpopulations,and the effect of traumatic injury,were also explored. We report that analytical variance caused by sample processing was acceptably small. Blood leukocyte gene expression in the same individual over a 24-h period was remarkably constant. In contrast,genome-wide expression varied significantly among different subjects and leukocyte subpopulations. Expectedly,traumatic injury induced dramatic changes in apparent gene expression that were greater in magnitude than the analytical noise and interindividual variance. We demonstrate that the development of a nation-wide program for gene expression analysis with careful attention to analytical details can reduce the variance in the clinical setting to a level where patterns of gene expression are informative among different healthy human subjects,and can be studied with confidence in human disease. View Publication

Megakaryocytopoiesis gives rise to platelets by proliferation and differentiation of lineage-specific progenitors,identified in vitro as Colony Forming Unit-Megakaryocytes (CFU-Mk). The aim of this study was to refine and optimize the in vitro Standard Operating Procedure (SOP) of the CFU-Mk assay for detecting drug-induced thrombocytopenia and to prevalidate a model for predicting the acute exposure levels that cause maximum tolerated decreases in the platelets count,based on the correlation with the maximal plasma concentrations (C max) in vivo. The assay was linear under the SOP conditions,and the in vitro endpoints (percentage of colonies growing) were reproducible within and across laboratories. The protocol performance phase was carried out testing 10 drugs (selected on the base of their recognised or potential in vivo haematotoxicity,according to the literature). Results showed that a relationship can be established between the maximal concentration in plasma (C max) and the in vitro concentrations that inhibited the 10-50-90 percent of colonies growth (ICs). When C max is lower than IC10,it is possible to predict that the chemicals have no direct toxicity effect on CFU-Mk and could not induce thrombocytopenia due to bone marrow damage. When the C max is higher than IC90 and/or IC50,thrombocytopenia can occur due to direct toxicity of chemicals on CFU-Mk progenitors. View Publication

Tissue engineering provides a new paradigm for periodontal tissue regeneration in which proper stem cells and effective cellular factors are very important. The objective of this study was,for the first time,to investigate the capabilities and advantages of periodontal tissue regeneration using induced pluripotent stem (iPS) cells and enamel matrix derivatives (EMD). In this study the effect of EMD gel on iPS cells in vitro was first determined,and then tissue engineering technique was performed to repair periodontal defects in three groups: silk scaffold only; silk scaffold + EMD; and silk scaffold + EMD + iPS cells. EMD greatly enhanced the mRNA expression of Runx2 but inhibited the mRNA expression of OC and mineralization nodule formation in vitro. Transplantation of iPS cells showed higher expression levels of OC,Osx,and Runx2 genes,both 12 and 24 days postsurgery. At 24 days postsurgery in the iPS cell group,histological analysis showed much more new alveolar bone and cementum formation with regenerated periodontal ligament between them. The results showed the commitment role that EMD contributes in mesenchymal progenitors to early cells in the osteogenic lineage. iPS cells combined with EMD provide a valuable tool for periodontal tissue engineering,by promoting the formation of new cementum,alveolar bone,and normal periodontal ligament. View Publication

Advancing cardiac tissue engineering requires innovative fabrication techniques,including 3D bioprinting and tissue maturation,to enable the generation of new muscle for repairing or replacing damaged heart tissue. Recent advances in tissue engineering have highlighted the need for rapid,high-resolution bioprinting methods that preserve cell viability and maintain structural fidelity. Traditional collagen-based bioinks gel slowly,limiting their use in bioprinting. Here,we implement TRACE (tunable rapid assembly of collagenous elements),a macromolecular crowding-driven bioprinting technique that enables the immediate gelation of collagen bioinks infused with cells. This overcomes the need for extended incubation,allowing for direct bioprinting of engineered cardiac tissues with high fidelity. Unlike methods that rely on high-concentration acidic collagen or fibrin for gelation,TRACE achieves rapid bioink stabilization without altering the biochemical composition. This ensures greater versatility in bioink selection while maintaining functional tissue outcomes. Additionally,agarose slurry provides stable structural support,preventing tissue collapse while allowing nutrient diffusion. This approach better preserves complex tissue geometries during culture than gelatin-based support baths or polydimethylsiloxane (PDMS) molds. Our results demonstrate that TRACE enables the bioprinting of structurally stable cardiac tissues with high resolution. By supporting the fabrication of biomimetic tissues,TRACE represents a promising advancement in bioprinting cardiac models and other engineered tissues. View Publication

Various preclinical models have been developed to clarify the pathophysiology of prostate cancer (PCa). Traditional PCa cell lines from clinical metastatic lesions,as exemplified by DU-145,PC-3,and LNCaP cells,are useful tools to define mechanisms underlying tumorigenesis and drug resistance. Cell line-based experiments,however,have limitations for preclinical studies because those cells are basically adapted to 2-dimensional monolayer culture conditions,in which the majority of primary PCa cells cannot survive. Recent tissue engineering enables generation of PCa patient-derived xenografts (PDXs) from both primary and metastatic lesions. Compared with fresh PCa tissue transplantation in athymic mice,co-injection of PCa tissues with extracellular matrix in highly immunodeficient mice has remarkably improved the success rate of PDX generation. PDX models have advantages to appropriately recapitulate the molecular diversity,cellular heterogeneity,and histology of original patient tumors. In contrast to PDX models,patient-derived organoid and spheroid PCa models in 3-dimensional culture are more feasible tools for in vitro studies for retaining the characteristics of patient tumors. In this article,we review PCa preclinical model cell lines and their sublines,PDXs,and patient-derived organoid and spheroid models. These PCa models will be applied to the development of new strategies for cancer precision medicine. View Publication

AIM: Commercial regenerative medicine will require large quantities of clinical-specification human cells. The cost and quality of manufacture is notoriously difficult to control due to highly complex processes with poorly defined tolerances. As a step to overcome this,we aimed to demonstrate the use of 'quality-by-design' tools to define the operating space for economic passage of a scalable human embryonic stem cell production method with minimal cell loss. MATERIALS & METHODS: Design of experiments response surface methodology was applied to generate empirical models to predict optimal operating conditions for a unit of manufacture of a previously developed automatable and scalable human embryonic stem cell production method. RESULTS & CONCLUSION: Two models were defined to predict cell yield and cell recovery rate postpassage,in terms of the predictor variables of media volume,cell seeding density,media exchange and length of passage. Predicted operating conditions for maximized productivity were successfully validated. Such 'quality-by-design' type approaches to process design and optimization will be essential to reduce the risk of product failure and patient harm,and to build regulatory confidence in cell therapy manufacturing processes. View Publication

In a previous study of prevalidation,a standard operating procedure (SOP) for two independent in vitro tests (human and mouse) had been developed,to evaluate the potential hematotoxicity of xenobiotics from their direct and the adverse effects on granulocyte-macrophages (CFU-GM). A predictive model to calculate the human maximum tolerated dose (MTD) was set up,by adjusting a mouse-derived MTD for the differential interspecies sensitivity. In this paper,we describe an international blind trial designed to apply this model to the clinical neutropenia,by testing 20 drugs,including 14 antineoplastics (Cytosar-U,5-Fluorouracil,Myleran,Thioguanine,Fludarabine,Bleomycin,Methotrexate,Gemcitabine,Carmustine,Etoposide,Teniposide,Cytoxan,Taxol,Adriamycin); two antivirals (Retrovir,Zovirax,); three drugs for other therapeutic indications (Cyclosporin,Thorazine,Indocin); and one pesticide (Lindane). The results confirmed that the SOP developed generates reproducible IC90 values with both human and murine GM-CFU. For 10 drugs (Adriamycin,Bleomycin,Etoposide,Fludarabine,5-Fluorouracil,Myleran,Taxol,Teniposide,Thioguanine,and Thorazine),IC90 values were found within the range of the actual drug doses tested (defined as the actual IC90). For the other 10 drugs (Carmustine,Cyclosporin,Cytosar-U,Cytoxan,Gemcitabine,Indocin,Lindane,Methotrexate,Retrovir,and Zovirax) extrapolation on the regression curve out of the range of the actual doses tested was required to derive IC90 values (extrapolated IC90). The model correctly predicted the human MTD for 10 drugs out of 10 that had actual IC90 values" and 7 drugs out of 10 for those having only an extrapolated IC90. Two of the incorrect predictions (Gemcitabine and Zovirax) were within 6-fold of the correct MTD� View Publication

Immunotherapy treatments for cancer are becoming increasingly successful,however to further improve our understanding of the T-cell recognition involved in effective responses and to encourage moves towards the development of personalised treatments for leukaemia immunotherapy,precise antigenic targets in individual patients have been identified. Cellular arrays using peptide-MHC (pMHC) tetramers allow the simultaneous detection of different antigen specific T-cell populations naturally circulating in patients and normal donors. We have developed the pMHC array to detect CD8+ T-cell populations in leukaemia patients that recognise epitopes within viral antigens (cytomegalovirus (CMV) and influenza (Flu)) and leukaemia antigens (including Per Arnt Sim domain 1 (PASD1),MelanA,Wilms' Tumour (WT1) and tyrosinase). We show that the pMHC array is at least as sensitive as flow cytometry and has the potential to rapidly identify more than 40 specific T-cell populations in a small sample of T-cells (0.8-1.4 x 10(6)). Fourteen of the twenty-six acute myeloid leukaemia (AML) patients analysed had T cells that recognised tumour antigen epitopes,and eight of these recognised PASD1 epitopes. Other tumour epitopes recognised were MelanA (n = 3),tyrosinase (n = 3) and WT1(126-134) (n = 1). One of the seven acute lymphocytic leukaemia (ALL) patients analysed had T cells that recognised the MUC1(950-958) epitope. In the future the pMHC array may be used provide point of care T-cell analyses,predict patient response to conventional therapy and direct personalised immunotherapy for patients. View Publication

ROCK kinases,which play central roles in the organization of the actin cytoskeleton,are tantalizing targets for the treatment of human diseases. Deletion of ROCK I in mice revealed a role in the pathophysiological responses to high blood pressure,and validated ROCK inhibition for the treatment of specific types of cardiovascular disease. To date,the only ROCK inhibitor employed clinically in humans is fasudil,which has been used safely in Japan since 1995 for the treatment of cerebral vasospasm. Clinical trials,mostly focusing on the cardiovascular system,have uncovered beneficial effects of fasudil for additional indications. Intriguing recent findings also suggest significant potential for ROCK inhibitors in the production and implantation of stem cells for disease therapies. View Publication

The polarization and migration of neural progenitor cells (NPCs) are critical for embryonic brain development and neurogenesis after brain injury. Although stromal-derived factor-1α (SDF-1α,CXCL12) and its receptor CXCR4 are well-known to mediate the migration of NPCs in the developing brain,the dynamic cellular processes and structure-related molecular events remain elusive. Transwell and microfluidic-based assays are classical assays to effectively study cellular migration. However,both of them have limitations in the analysis of a single cell. In this study,we modified the stripe assay and extended its applications in the study of NPC polarization and intracellular molecular events associated with CXCL12-mediated migration. In response to localized CXCL12,NPCs formed lamellipodia in the stripe assay. Furthermore,CXCR4 and Rac1 quickly re-distributed to the area of lamellipodia,indicating their roles in NPC polarization upon CXCL12 stimulation. Although the chemokine stripes in the assay provided concentration gradients that can be best used to study cellular polarization and migration through immunocytochemistry,they can also generate live imaging data with comparable quality. In conclusion,stripe assay is a visual,dynamic and economical tool to study cellular mobility and its related molecule mechanisms. View Publication

SummaryThe BAF chromatin remodeler regulates lineage commitment including cranial neural crest cell (CNCC) specification. Variants in BAF subunits cause Coffin-Siris syndrome (CSS),a congenital disorder characterized by coarse craniofacial features and intellectual disability. Approximately 50% of individuals with CSS harbor variants in one of the mutually exclusive BAF subunits,ARID1A/ARID1B. While Arid1a deletion in mouse neural crest causes severe craniofacial phenotypes,little is known about the role of ARID1A in CNCC specification. Using CSS-patient-derived ARID1A+/? induced pluripotent stem cells to model CNCC specification,we discovered that ARID1A-haploinsufficiency impairs epithelial-to-mesenchymal transition (EMT),a process necessary for CNCC delamination and migration from the neural tube. Furthermore,wild-type ARID1A-BAF regulates enhancers associated with EMT genes. ARID1A-BAF binding at these enhancers is impaired in heterozygotes while binding at promoters is unaffected. At the sequence level,these EMT enhancers contain binding motifs for ZIC2,and ZIC2 binding at these sites is ARID1A-dependent. When excluded from EMT enhancers,ZIC2 relocates to neuronal enhancers,triggering aberrant neuronal gene activation. In mice,deletion of Zic2 impairs NCC delamination,while ZIC2 overexpression in chick embryos at post-migratory neural crest stages elicits ectopic delamination from the neural tube. These findings reveal an essential ARID1A-ZIC2 axis essential for EMT and CNCC delamination. Graphical abstract ARID1A modulates chromatin accessibility at enhancers of genes required for epithelial-to-mesenchymal transition,a process essential for cranial neural crest cell (CNCC) specification. Haploinsufficiency of ARID1A attenuates ZIC2 binding at these enhancers,resulting in impaired CNCC formation with an aberrant neuronal trajectory. This study reveals an ARID1A-ZIC2 axis essential for CNCC specification. 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