参考文献

Unintended exposure to teratogenic compounds can lead to various birth defects; however current animal-based testing is limited by time,cost and high inter-species variability. Here,we developed a human-relevant in vitro model,which recapitulated two cellular events characteristic of embryogenesis,to identify potentially teratogenic compounds. We spatially directed mesoendoderm differentiation,epithelial-mesenchymal transition and the ensuing cell migration in micropatterned human pluripotent stem cell (hPSC) colonies to collectively form an annular mesoendoderm pattern. Teratogens could disrupt the two cellular processes to alter the morphology of the mesoendoderm pattern. Image processing and statistical algorithms were developed to quantify and classify the compounds' teratogenic potential. We not only could measure dose-dependent effects but also correctly classify species-specific drug (Thalidomide) and false negative drug (D-penicillamine) in the conventional mouse embryonic stem cell test. This model offers a scalable screening platform to mitigate the risks of teratogen exposures in human. View Publication

Previous studies have demonstrated that normal mouse mammary tissue contains a rare subset of mammary stem cells. We now describe a method for detecting an analogous subpopulation in normal human mammary tissue. Dissociated cells are suspended with fibroblasts in collagen gels,which are then implanted under the kidney capsule of hormone-treated immunodeficient mice. After 2-8 weeks,the gels contain bilayered mammary epithelial structures,including luminal and myoepithelial cells,their in vitro clonogenic progenitors and cells that produce similar structures in secondary transplants. The regenerated clonogenic progenitors provide an objective indicator of input mammary stem cell activity and allow the frequency and phenotype of these human mammary stem cells to be determined by limiting-dilution analysis. This new assay procedure sets the stage for investigations of mechanisms regulating normal human mammary stem cells (and possibly stem cells in other tissues) and their relationship to human cancer stem cell populations. View Publication

Introduction: Human embryonic stem cells (hESC) provide an invaluable model for assessing the effects of environmental chemicals and drugs on human prenatal development. However,hESC are difficult to adapt to 96-well plate screening assays,because they survive best when plated as colonies,which are difficult to count and plate accurately. The purpose of this study is to present an experimental method and analysis procedure to accomplish reliable screening of toxicants using hESC. Methods: We present a method developed to rapidly and easily determine the number of cells in small colonies of hESC spectrophotometerically and then accurately dispense equivalent numbers of cells in 96-well plates. The MTT assay was used to evaluate plating accuracy,and the method was tested using known toxicants. Results: The quality of the plate set-up and analysis procedure was evaluated with NIH plate validation and assessment software. All statistical parameters measured by the software were acceptable,and no drift or edge effects were observed. The 96-well plate MTT assay with hESC was tested by performing a dose-response screen of commercial products,which contain a variety of chemicals. The screen was done using single wells/dose,and the reliability of this method was demonstrated in a subsequent screen of the same products repeated three times. The single and triple screens were in good agreement,and NOAELs and IC50s could be determined from the single screen. The effects of vapor from volatile chemicals were studied,and methods to monitor and avoid vapor effects were incorporated into the assay. Discussion: Our method overcomes the difficulty of using hESC for reliable quantitative 96-well plate assays. It enables rapid dose-response screening using equipment that is commonly available in laboratories that culture hESC. This method could have a broad application in studies of environmental chemicals and drugs using hESC as models of prenatal development. ?? 2012 Elsevier Inc. View Publication

Embryos allocate cells to the three germ layers in a spatially ordered sequence. Human embryonic stem cells (hESCs) can generate the three germ layers in culture; however,differentiation is typically heterogeneous and spatially disordered. We show that geometric confinement is sufficient to trigger self-organized patterning in hESCs. In response to BMP4,colonies reproducibly differentiated to an outer trophectoderm-like ring,an inner ectodermal circle and a ring of mesendoderm expressing primitive-streak markers in between. Fates were defined relative to the boundary with a fixed length scale: small colonies corresponded to the outer layers of larger ones. Inhibitory signals limited the range of BMP4 signaling to the colony edge and induced a gradient of Activin-Nodal signaling that patterned mesendodermal fates. These results demonstrate that the intrinsic tendency of stem cells to make patterns can be harnessed by controlling colony geometries and provide a quantitative assay for studying paracrine signaling in early development. View Publication

Modeling tissues and organs using conventional 2D cell cultures is problematic as the cells rapidly lose their in vivo phenotype. In microfluidic bioreactors the cells reside in microstructures that are continuously perfused with cell culture medium to provide a dynamic environment mimicking the cells natural habitat. These micro scale bioreactors are sometimes referred to as organs-on-chips and are developed in order to improve and extend cell culture experiments. Here,we describe the two manufacturing techniques photolithography and soft lithography that are used in order to easily produce microfluidic bioreactors. The use of these bioreactors is exemplified by a toxicity assessment on 3D clustered human pluripotent stem cells (hPSC)-derived cardiomyocytes by beating frequency imaging. View Publication

A novel cardiomimetic biohybrid material,termed as the human ventricular cardiac anisotropic sheet (hvCAS) is reported. Well-characterized human pluripotent stem-cell-derived ventricular cardiomyocytes are strategically aligned to reproduce key electrophysiological features of native human ventricle,which,along with specific selection criteria,allows for a direct visualization of arrhythmic spiral re-entry and represents a revolutionary tool to assess preclinical drug-induced arrhythmogenicity. View Publication

Microbial growth in damp indoor environments has been correlated with risks to human health. This study was aimed to determine the cytotoxicity of 1-octen-3-ol (mushroom alcohol")� View Publication

Teratoma formation,the standard in vivo pluripotency assay,is also frequently used as a tumorigenicity assay. A common concern in therapeutic stem cell applications is the tumorigenicity potential of a small number of cell impurities in the final product. Estimation of this small number is hampered by the inaccurate methodology of the tumorigenicity assay. Hence,a protocol for tumorigenicity assay that can deliver a defined number of cells,without error introduced by leakage or migration of cells is needed. In this study,we tested our modified transplantation method that allows for transplant of small numbers of pluripotent stem cells (PSCs) under the kidney capsule with minimal cell leakage. A glass capillary with a finely shaped tip and an attached mouth pipette was used to inject PSCs into the rodent kidney capsule. H9 embryonic and induced PSCs were tagged with Fluc and green fluorescence protein reporter genes and divided in different cell doses for transplantation. Bioluminescence imaging (BLI) on the day of surgery showed that the cell signal was confined to the kidney and signal intensity correlated with increasing transplant cell numbers. The overall cell leakage rate was 17% and the rodent survival rate was 96%. Teratoma formation was observed in rodents transplanted with cell numbers between 1 × 10(5)-2 × 10(6). We conclude that this modified procedure for transplanting PSCs under the kidney capsule allows for transplantation of a defined number of PSCs with significant reduction of error associated with cell leakage from the transplant site. View Publication

Human induced pluripotent stem cells (hiPSCs) are being considered for use in understanding haematopoietic disorders and as a potential source of in vitro manufactured red cells. Here,we show that hiPSCs are able to recapitulate various stages of developmental erythropoiesis. We show that primitive erythroblasts arise first,express CD31(+) with CD235a(+),embryonic globins and red cell markers,but fail to express the hallmark red cell transcripts of adult erythropoiesis. When hiPSC-derived CD45(+) CD235a(-) haematopoietic progenitors are isolated on day 12 and further differentiated on OP9 stroma,they selectively express CD36(+) and CD235a(+),adult erythroid transcripts for transcription factors (e.g.,BCL11A,KLF1) and fetal/adult globins (HBG1/2,HBB). Importantly,hiPSC- and cord-derived CD36(+) CD235a(+) erythroblasts show a striking homology by transcriptome array profiling (only 306 transcripts with a 2Log fold change<1textperiodcentered5- or 2textperiodcentered8-fold). Phenotypic and transcriptome profiling of CD45(+) CD117(+) CD235a(+) pro-erythroblasts and terminally differentiated erythroblasts is also provided,including evidence of a HbF (fetal) to HbA (adult) haemoglobin switch and enucleation,that mirrors their definitive erythroblast cord-derived counterparts. These findings provide a molecular roadmap of developmental erythropoiesis from hiPSC sources at several critical stages,but also helps to inform on their use for clinical applications and modelling human haematopoietic disease. View Publication

Human pluripotent stem cells (hPSCs) exist in heterogeneous micro-environments with multiple subpopulations,convoluting fate-regulation analysis. We patterned hPSCs into engineered micro-environments and screened responses to 400 small-molecule kinase inhibitors,measuring yield and purity outputs of undifferentiated,neuroectoderm,mesendoderm,and extra-embryonic populations. Enrichment analysis revealed mammalian target of rapamycin (mTOR) inhibition as a strong inducer of mesendoderm. Dose responses of mTOR inhibitors such as rapamycin synergized with Bone Morphogenetic protein 4 (BMP4) and activin A to enhance the yield and purity of BRACHYURY-expressing cells. Mechanistically,small interfering RNA knockdown of RAPTOR,a component of mTOR complex 1,phenocopied the mesendoderm-enhancing effects of rapamycin. Functional analysis during mesoderm and endoderm differentiation revealed that mTOR inhibition increased the output of hemogenic endothelial cells 3-fold,with a concomitant enhancement of blood colony-forming cells. These data demonstrate the power of our multi-lineage screening approach and identify mTOR signaling as a node in hPSC differentiation to mesendoderm and its derivatives. View Publication