技术资料

Sorting (or isolation) and manipulation of rare cells with high recovery rate and purity are of critical importance to a wide range of physiological applications. In the current paper,we report on a generic single cell manipulation tool that integrates optical tweezers and microfluidic chip technologies for handling small cell population sorting with high accuracy. The laminar flow nature of microfluidics enables the targeted cells to be focused on a desired area for cell isolation. To recognize the target cells,we develop an image processing methodology with a recognition capability of multiple features,e.g.,cell size and fluorescence label. The target cells can be moved precisely by optical tweezers to the desired destination in a noninvasive manner. The unique advantages of this sorter are its high recovery rate and purity in small cell population sorting. The design is based on dynamic fluid and dynamic light pattern,in which single as well as multiple laser traps are employed for cell transportation,and a recognition capability of multiple cell features. Experiments of sorting yeast cells and human embryonic stem cells are performed to demonstrate the effectiveness of the proposed cell sorting approach. View Publication

Activin/Nodal signaling via SMAD2/3 maintains human embryonic stem cell (hESC) pluripotency by direct transcriptional regulation of NANOG or,alternatively,induces mesoderm and definitive endoderm (DE) formation. In search of an explanation for these contrasting effects,we focused on SNON (SKIL),a potent SMAD2/3 corepressor that is expressed in hESCs but rapidly down-regulated upon differentiation. We show that SNON predominantly associates with SMAD2 at the promoters of primitive streak (PS) and early DE marker genes. Knockdown of SNON results in premature activation of PS and DE genes and loss of hESC morphology. In contrast,enforced SNON expression inhibits DE formation and diverts hESCs toward an extraembryonic fate. Thus,our findings provide novel mechanistic insight into how a single signaling pathway both regulates pluripotency and directs lineage commitment. View Publication

BACKGROUND Neuritic degeneration is an important early pathological step in neurodegeneration. AIM The purpose of this study was to explore the mechanisms connecting neuritic degeneration to the functional and morphological remodeling of endoplasmic reticulum (ER) and mitochondria. METHODS Here,we set up neuritic degeneration models by neurite cutting-induced neural degeneration in human-induced pluripotent stem cell-derived neurons. RESULTS We found that neuritic ER becomes fragmented and forms complexes with mitochondria,which induces IP3R-dependent mitochondrial Ca(2+) elevation and dysfunction during neuritic degeneration. Furthermore,mitochondrial membrane potential is required for ER fragmentation and mitochondrial Ca(2+) elevation during neuritic degeneration. Mechanically,tightening of the ER-mitochondria associations by expression of a short synthetic linker" and ER Ca(2+) releasing together could promote mitochondrial Ca(2+) elevation� View Publication

Metabolomics analysis was performed on the supernatant of human embryonic stem (hES) cell cultures exposed to a blinded subset of 11 chemicals selected from the chemical library of EPA's ToxCast™ chemical screening and prioritization research project. Metabolites from hES cultures were evaluated for known and novel signatures that may be indicative of developmental toxicity. Significant fold changes in endogenous metabolites were detected for 83 putatively annotated mass features in response to the subset of ToxCast chemicals. The annotations were mapped to specific human metabolic pathways. This revealed strong effects on pathways for nicotinate and nicotinamide metabolism,pantothenate and CoA biosynthesis,glutathione metabolism,and arginine and proline metabolism pathways. Predictivity for adverse outcomes in mammalian prenatal developmental toxicity studies used ToxRefDB and other sources of information,including Stemina Biomarker Discovery's predictive DevTox® model trained on 23 pharmaceutical agents of known developmental toxicity and differing potency. The model initially predicted developmental toxicity from the blinded ToxCast compounds in concordance with animal data with 73% accuracy. Retraining the model with data from the unblinded test compounds at one concentration level increased the predictive accuracy for the remaining concentrations to 83%. These preliminary results on a 11-chemical subset of the ToxCast chemical library indicate that metabolomics analysis of the hES secretome provides information valuable for predictive modeling and mechanistic understanding of mammalian developmental toxicity. View Publication

Human induced pluripotent stem cells (iPSCs) have been generated with varied efficiencies from multiple tissues. Yet,acquiring donor cells is,in most instances,an invasive procedure that requires laborious isolation. Here we present a detailed protocol for generating human iPSCs from exfoliated renal epithelial cells present in urine. This method is advantageous in many circumstances,as the isolation of urinary cells is simple (30 ml of urine are sufficient),cost-effective and universal (can be applied to any age,gender and race). Moreover,the entire procedure is reasonably quick--around 2 weeks for the urinary cell culture and 3-4 weeks for the reprogramming--and the yield of iPSC colonies is generally high--up to 4% using retroviral delivery of exogenous factors. Urinary iPSCs (UiPSCs) also show excellent differentiation potential,and thus represent a good choice for producing pluripotent cells from normal individuals or patients with genetic diseases,including those affecting the kidney. View Publication

Altering chromatin structure through histone posttranslational modifications has emerged as a key driver of transcriptional responses in cells. Modulation of these transcriptional responses by pharmacological inhibition of class I histone deacetylases (HDACs),a group of chromatin remodeling enzymes,has been successful in blocking the growth of some cancer cell types. These inhibitors also attenuate the pathogenesis of pathological cardiac remodeling by blunting and even reversing pathological hypertrophy. The mechanistic target of rapamycin (mTOR) is a critical sensor and regulator of cell growth that,as part of mTOR complex 1 (mTORC1),drives changes in protein synthesis and metabolism in both pathological and physiological hypertrophy. We demonstrated through pharmacological and genetic methods that inhibition of class I HDACs suppressed pathological cardiac hypertrophy through inhibition of mTOR activity. Mice genetically silenced for HDAC1 and HDAC2 had a reduced hypertrophic response to thoracic aortic constriction (TAC) and showed reduced mTOR activity. We determined that the abundance of tuberous sclerosis complex 2 (TSC2),an mTOR inhibitor,was increased through a transcriptional mechanism in cardiomyocytes when class I HDACs were inhibited. In neonatal rat cardiomyocytes,loss of TSC2 abolished HDAC-dependent inhibition of mTOR activity,and increased expression of TSC2 was sufficient to reduce hypertrophy in response to phenylephrine. These findings point to mTOR and TSC2-dependent control of mTOR as critical components of the mechanism by which HDAC inhibitors blunt pathological cardiac growth. These results also suggest a strategy to modulate mTOR activity and facilitate the translational exploitation of HDAC inhibitors in heart disease. View Publication

Human embryonic stem cells (hESCs) are thought to be a promising resource for cell therapy,while it has to face the major problem of graft immunological rejection. Major histocompatibility complex (MHC) class I expressed on the cell surface is the major cause of graft rejection. Transporter associated with antigen presentation 1 (TAP1) and TAP-associated glycoprotein (TAPBP) play important roles in regulating MHC class I expression. In this study,we generated TAP1- and TAPBP-deficient hESC lines,respectively,using transcription activator-like effector nucleases technique. These cells showed deficient expression of MHC class I on the cell surface and reduced immunogenicity compared with wild types,but maintained normal pluripotency,karyotypes,and differentiation ability. Thus,our findings are instrumental in developing a universal cell resource with both pluripotency and hypo-immunogenicity for transplantation therapy in the future. View Publication

Human embryonic stem cells (hESCs) provide a valuable window into the dissection of the molecular circuitry underlying the early formation of the human forebrain. However,dissection of signaling events in forebrain development using current protocols is complicated by non-neural contamination and fluctuation of extrinsic influences. Here,we show that SMAD7,a cell-intrinsic inhibitor of transforming growth factor-β (TGFβ) signaling,is sufficient to directly convert pluripotent hESCs to an anterior neural fate. Time course gene expression revealed downregulation of MAPK components,and combining MEK1/2 inhibition with SMAD7-mediated TGFβ inhibition promoted telencephalic conversion. Fibroblast growth factor-MEK and TGFβ-SMAD signaling maintain hESCs by promoting pluripotency genes and repressing neural genes. Our findings suggest that in the absence of these cues,pluripotent cells simply revert to a program of neural conversion. Hence,the primed" state of hESCs requires inhibition of the "default" state of neural fate acquisition. This has parallels in amphibians� View Publication

Pluripotent stem cell-derived cardiomyocytes are currently being investigated for in vitro human heart models and as potential therapeutics for heart failure. In this study,we have developed a differentiation protocol that minimizes the need for specific human embryonic stem cell (hESC) line optimization. We first reduced the heterogeneity that exists within the starting population of bulk cultured hESCs by using cells adapted to single-cell passaging in a 2-dimensional (2D) culture format. Compared with bulk cultures,single-cell cultures comprised larger fractions of TG30(hi)/OCT4(hi) cells,corresponding to an increased expression of pluripotency markers OCT4 and NANOG,and reduced expression of early lineage-specific markers. A 2D temporal differentiation protocol was then developed,aimed at reducing the inherent heterogeneity and variability of embryoid body-based protocols,with induction of primitive streak cells using bone morphogenetic protein 4 and activin A,followed by cardiogenesis via inhibition of Wnt signaling using the small molecules IWP-4 or IWR-1. IWP-4 treatment resulted in a large percentage of cells expressing low amounts of cardiac myosin heavy chain and expression of early cardiac progenitor markers ISL1 and NKX2-5,thus indicating the production of large numbers of immature cardiomyocytes (˜65,000/cm(2) or ˜1.5 per input hESC). This protocol was shown to be effective in HES3,H9,and,to a lesser,extent,MEL1 hESC lines. In addition,we observed that IWR-1 induced predominantly atrial myosin light chain (MLC2a) expression,whereas IWP-4 induced expression of both atrial (MLC2a) and ventricular (MLC2v) forms. The intrinsic flexibility and scalability of this 2D protocol mean that the output population of primitive cardiomyocytes will be particularly accessible and useful for the investigation of molecular mechanisms driving terminal cardiomyocyte differentiation,and potentially for the future treatment of heart failure. View Publication