技术资料

Recent genome-wide association studies (GWASs) have identified a locus on chromosome 1p13 strongly associated with both plasma low-density lipoprotein cholesterol (LDL-C) and myocardial infarction (MI) in humans. Here we show through a series of studies in human cohorts and human-derived hepatocytes that a common noncoding polymorphism at the 1p13 locus,rs12740374,creates a C/EBP (CCAAT/enhancer binding protein) transcription factor binding site and alters the hepatic expression of the SORT1 gene. With small interfering RNA (siRNA) knockdown and viral overexpression in mouse liver,we demonstrate that Sort1 alters plasma LDL-C and very low-density lipoprotein (VLDL) particle levels by modulating hepatic VLDL secretion. Thus,we provide functional evidence for a novel regulatory pathway for lipoprotein metabolism and suggest that modulation of this pathway may alter risk for MI in humans. We also demonstrate that common noncoding DNA variants identified by GWASs can directly contribute to clinical phenotypes. View Publication

Thirdhand cigarette smoke (THS) was recently recognized as an environmental health hazard; however,little is known about it effects on cells. Mitochondria are sensitive monitors of cell health and report on environmentally-induced stress. We tested the effects of low levels of THS extracted from terry cloth on mitochondrial morphology and function using stem cells with well-defined mitochondria. Concentrations of THS that did not kill cells caused stress-induced mitochondrial hyperfusion (SIMH),which was characterized by changes in mitochondrial morphology indicative of fusion,increased mitochondrial membrane potential (MMP),increased ATP levels,increased superoxide production,and increased oxidation of mitochondrial proteins. SIMH was accompanied by a decrease in Fis1 expression,a gene responsible for mitochondrial fission,and a decrease in apoptosis-related genes,including Aifm2,Bbc3 and Bid There was also down regulation of Ucp2,Ucp4 and Ucp5,genes that decrease MMP thereby reducing oxidative phosphorylation,while promoting glycolysis. These effects,which collectively accompany SIMH,are a pro-survival mechanism to rescue damaged mitochondria and protect cells from apoptosis. Prolonged exposure to THS caused a reduction in MMP and decreased cell proliferation,which likely leads to apoptosis. View Publication

Rapid technological advances have made the acquisition of large numbers of spectra not only feasible,but also routine. As a result,a significant research effort is focused on semi-automated and fully automated spectral processing techniques. However,the need to provide initial estimates of the number of peaks,their band shapes,and the initial parameters of these bands presents an obstacle to the full automation of peak fitting and its incorporation into fully automated spectral-preprocessing workflows. Moreover,the sensitivity of peak-fit routines to initial parameter settings and the resultant variations in solution quality further impede user-free operation. We have developed a technique to perform fully automated peak fitting on fully automated preconditioned spectra-specifically,baseline-corrected and smoothed spectra that are free of cosmic-ray-induced spikes. Briefly,the tallest peak in a spectrum is located and a Gaussian peak-fit is performed. The fitted peak is then subtracted from the spectrum,and the procedure is repeated until the entire spectrum has been processed. In second and third passes,all the peaks in the spectrum are fitted concurrently,but are fitted to a Pearson Type VII model using the parameters for the model established in the prior pass. The technique is applied to a synthetic spectrum with several peaks,some of which have substantial overlap,to test the ability of the method to recover the correct number of peaks,their true shape,and their appropriate parameters. Finally the method is tested on measured Raman spectra collected from human embryonic stem cells and samples of red blood cells. View Publication

Photoreceptor degeneration due to retinitis pigmentosa (RP) is a primary cause of inherited retinal blindness. Photoreceptor cell-replacement may hold the potential for repair in a completely degenerate retina by reinstating light sensitive cells to form connections that relay information to downstream retinal layers. This study assessed the therapeutic potential of photoreceptor progenitors derived from human embryonic and induced pluripotent stem cells (ESCs and iPSCs) using a protocol that is suitable for future clinical trials. ESCs and iPSCs were cultured in four specific stages under defined conditions,resulting in generation of a near-homogeneous population of photoreceptor-like progenitors. Following transplantation into mice with end-stage retinal degeneration,these cells differentiated into photoreceptors and formed a cell layer connected with host retinal neurons. Visual function was partially restored in treated animals,as evidenced by two visual behavioral tests. Furthermore,the magnitude of functional improvement was positively correlated with the number of engrafted cells. Similar efficacy was observed using either ESCs or iPSCs as source material. These data validate the potential of human pluripotent stem cells for photoreceptor replacement therapies aimed at photoreceptor regeneration in retinal disease. View Publication

As a critical tumor suppressor,p53 is inactivated in human cancer cells by somatic gene mutation or disruption of pathways required for its activation. Therefore,it is critical to elucidate the mechanism underlying p53 activation after genotoxic and cellular stresses. Accumulating evidence has indicated the importance of posttranslational modifications such as acetylation in regulating p53 stability and activity. However,the physiological roles of the eight identified acetylation events in regulating p53 responses remain to be fully understood. By employing homologous recombination,we introduced various combinations of missense mutations (lysine to arginine) into eight acetylation sites of the endogenous p53 gene in human embryonic stem cells (hESCs). By determining the p53 responses to DNA damage in the p53 knock-in mutant hESCs and their derivatives,we demonstrate physiological importance of the acetylation events within the core domain (K120 and K164) and at the C-terminus (K370/372/373/381/382/386) in regulating human p53 responses to DNA damage. View Publication

The CCCTC-binding factor CTCF is the only known vertebrate insulator protein and has been shown to regulate important developmental processes such as imprinting,X-chromosome inactivation and genomic architecture. In this study,we examined the role of CTCF in human embryonic stem cell (hESC) biology. We demonstrate that CTCF associates with several important pluripotency genes,including NANOG,SOX2,cMYC and LIN28 and is critical for hESC proliferation. CTCF depletion impacts expression of pluripotency genes and accelerates loss of pluripotency upon BMP4 induced differentiation,but does not result in spontaneous differentiation. We find that CTCF associates with the distal ends and internal sites of the co-regulated 160 kb NANOG-DPPA3-GDF3 locus. Each of these sites can function as a CTCF-dependent enhancer-blocking insulator in heterologous assays. In hESCs,CTCF exists in multisubunit protein complexes and can be poly(ADP)ribosylated. Known CTCF cofactors,such as Cohesin,differentially co-localize in the vicinity of specific CTCF binding sites within the NANOG locus. Importantly,the association of some cofactors and protein PARlation selectively changes upon differentiation although CTCF binding remains constant. Understanding how unique cofactors may impart specialized functions to CTCF at specific genomic locations will further illuminate its role in stem cell biology. View Publication

BACKGROUND: Differentiation of human embryonic stem cells into endothelial cells (hESC-ECs) has the potential to provide an unlimited source of cells for novel transplantation therapies of ischemic diseases by supporting angiogenesis and vasculogenesis. However,the endothelial differentiation efficiency of the conventional embryoid body (EB) method is low while the 2-dimensional method of co-culturing with mouse embryonic fibroblasts (MEFs) require animal product,both of which can limit the future clinical application of hESC-ECs. Moreover,to fully understand the beneficial effects of stem cell therapy,investigators must be able to track the functional biology and physiology of transplanted cells in living subjects over time. METHODOLOGY: In this study,we developed an extracellular matrix (ECM) culture system for increasing endothelial differentiation and free from contaminating animal cells. We investigated the transcriptional changes that occur during endothelial differentiation of hESCs using whole genome microarray,and compared to human umbilical vein endothelial cells (HUVECs). We also showed functional vascular formation by hESC-ECs in a mouse dorsal window model. Moreover,our study is the first so far to transplant hESC-ECs in a myocardial infarction model and monitor cell fate using molecular imaging methods. CONCLUSION: Taken together,we report a more efficient method for derivation of hESC-ECs that express appropriate patterns of endothelial genes,form functional vessels in vivo,and improve cardiac function. These studies suggest that hESC-ECs may provide a novel therapy for ischemic heart disease in the future. View Publication

Realizing the potential that human embryonic stem cells (hESCs) hold,both for the advancement of biomedical science and the development of new treatments for many human disorders,will be greatly facilitated by the introduction of standardized methods for assessing and altering the biological properties of these cells. The 7-day in vitro alkaline phosphatase colony-forming cell (AP(+)-CFC) assay currently offers the most sensitive and specific method to quantify the frequency of undifferentiated cells present in a culture. In this regard,it is superior to any phenotypic assessment protocol. The AP(+)-CFC assay,thus,provides a valuable tool for monitoring the quality of hESC cultures,and also for evaluating quantitative changes in pluripotent cell numbers following manipulations that may affect the self-renewal and differentiation properties of the treated cells. Two other methods routinely used to evaluate hESC pluripotency involve either culturing the cells under conditions that promote the formation of nonadherent differentiating cell aggregates (termed embryoid bodies),or transplanting the cells into immunodeficient mice to obtain teratomas containing differentiated cells representative of endoderm,mesoderm,and ectoderm lineages. View Publication