技术资料

Autologous feeder cells have been developed by various methods to minimize the presence of xenogenic entities in human embryonic stem cell (hESC) cultures. However,there was no systematic comparison of supportive effects of the feeder cells on hESC growth,nor comparison to the supportive effects of various feeder-free culture systems and standard mouse feeder cells. In this study,we aimed to compare the supportive abilities of autologous feeders derived either directly from H9 hESCs (H9 dF) or from outgrowth of embryoid body predifferentiated in suspension from H9 hESCs (H9 ebF). Mouse feeder system and matrigel-mTeSR1 feeder-free system were used as controls. H9 ebF was found to secrete more basic fibroblast growth factor in the conditioned medium than H9 dF did. The undifferentiated state of H9 hESCs was sustained more stably on H9 ebF than on H9 dF,and the differentiation potential of H9 hESCs on H9 ebF was higher than on H9 dF. We concluded that H9 ebF was an optimal autologous feeder to maintain the long-term undifferentiated state of hESCs in our current culture system. This study helps to standardize the autologous culture of hESCs. It also suggests a more definite direction for future development of xeno-free culture system for hESCs. View Publication

Since mouse embryonic stem (ES) cells was first derived in 1981,the ability of this unprecedented cell type to self-renew and differentiate without limit has revolutionized the discovery tools that are used to study gene functions and development. Furthermore,they have inspired others to hunt for similar cells from other species. The derivation of human ES cells in 1998 has accelerated these discoveries and has also widely provoked public interest,due to both the scientific significance of these cells for human tissue regeneration and the ethical disputes over the use of donated early human embryos. However,this is no longer a barrier,with the recent discovery of methods that can convert differentiated somatic cells into ES-like cells or induced pluripotent stem (iPS) cells,by using defined reprogramming factors. This review attempts to summarize the progresses in the derivation of ES cells (as well as other embryo-derived pluripotent cells) and iPS cells from various species. We will focus on the molecular and biological features of the cells,as well as the different determinants identified thus far to sustain their pluripotency. 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

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

The recent technique of transducing key transcription factors into unipotent cells (fibroblasts) to generate pluripotent stem cells (induced pluripotent stem cells [iPSCs]) has significantly changed the stem cell field. These cells have great promise for many clinical applications,including that of regenerative medicine. Our findings show that iPSCs can be derived from human adipose-derived stromal cells (hASCs),a notable advancement in the clinical applicability of these cells. To investigate differences between two iPS cell lines (fibroblast-iPSC and hASC-iPSC),and also the gold standard human embryonic stem cell,we looked at cell stiffness as a possible indicator of cell differentiation-potential differences. We used atomic force microscopy as a tool to determine stem cell stiffness,and hence differences in material properties between cells. Human fibroblast and hASC stiffness was also ascertained for comparison. Interestingly,cells exhibited a noticeable difference in stiffness. From least to most stiff,the order of cell stiffness was as follows: hASC-iPSC,human embryonic stem cell,fibroblast-iPSC,fibroblasts,and,lastly,as the stiffest cell,hASC. In comparing hASC-iPSCs to their origin cell,the hASC,the reprogrammed cell is significantly less stiff,indicating that greater differentiation potentials may correlate with a lower cellular modulus. The stiffness differences are not dependent on cell culture density; hence,material differences between cells cannot be attributed solely to cell-cell constraints. The change in mechanical properties of the cells in response to reprogramming offers insight into how the cell interacts with its environment and might lend clues to how to efficiently reprogram cell populations as well as how to maintain their pluripotent state. View Publication

The introduction of double stranded RNA (dsRNA) into the cytoplasm of mammalian cells usually leads to a potent antiviral response resulting in the rapid induction of interferon beta (IFNβ). This response can be mediated by a number of dsRNA sensors,including TLR3,MDA5,RIG-I and PKR. We show here that pluripotent human cells (human embryonic stem (hES) cells and induced pluripotent (iPS) cells) do not induce interferon in response to cytoplasmic dsRNA,and we have used a variety of approaches to learn the underlying basis for this phenomenon. Two major cytoplasmic dsRNA sensors,TLR3 and MDA5,are not expressed in hES cells and iPS cells. PKR is expressed in hES cells,but is not activated by transfected dsRNA. In addition,RIG-I is expressed,but fails to respond to dsRNA because its signaling adapter,MITA/STING,is not expressed. Finally,the interferon-inducible RNAse L and oligoadenylate synthetase enzymes are also expressed at very low levels. Upon differentiation of hES cells into trophoblasts,cells acquire the ability to respond to dsRNA and this correlates with a significant induction of expression of TLR3 and its adaptor protein TICAM-1/TRIF. Taken together,our results reveal that the lack of an interferon response may be a general characteristic of pluripotency and that this results from the systematic downregulation of a number of genes involved in cytoplasmic dsRNA signaling. View Publication

BACKGROUND: Constitutive promoters that ensure sustained and high level gene expression are basic research tools that have a wide range of applications,including studies of human embryology and drug discovery in human embryonic stem cells (hESCs). Numerous cellular/viral promoters that ensure sustained gene expression in various cell types have been identified but systematic comparison of their activities in hESCs is still lacking. METHODOLOGY/PRINCIPAL FINDINGS: We have quantitatively compared promoter activities of five commonly used constitutive promoters,including the human β-actin promoter (ACTB),cytomegalovirus (CMV),elongation factor-1α,(EF1α),phosphoglycerate kinase (PGK) and ubiquitinC (UbC) in hESCs. Lentiviral gene transfer was used to ensure stable integration of promoter-eGFP constructs into the hESCs genome. Promoter activities were quantitatively compared in long term culture of undifferentiated hESCs and in their differentiated progenies. CONCLUSION/SIGNIFICANCE: The ACTB,EF1α and PGK promoters showed stable activities during long term culture of undifferentiated hESCs. The ACTB promoter was superior by maintaining expression in 75-80% of the cells after 50 days in culture. During embryoid body (EB) differentiation,promoter activities of all five promoters decreased. Although the EF1α promoter was downregulated in approximately 50% of the cells,it was the most stable promoter during differentiation. Gene expression analysis of differentiated eGFP+ and eGFP- cells indicate that promoter activities might be restricted to specific cell lineages,suggesting the need to carefully select optimal promoters for constitutive gene expression in differentiated hESCs. View Publication

Until recently,culturing human pluripotent stem cells was hampered by three prominent technical problems: a high degree of unwanted cellular stress when the cells are passaged,unacceptably low cloning efficiency and poor recovery of cryopreserved stocks. This review discusses recent developments that address these problems. A major focus of the review is the use of p160 Rho-associated coiled-coil kinase inhibitors for improving both the cloning efficiency and the recovery of cryopreserved human embryonic stem cells and human induced pluripotent stem cells. An underlying theme of this review is that the three problems have a common cause: separation of human pluripotent stem cells from one another increases cellular stress,which greatly decreases their viability unless special steps are taken. View Publication

Recent reports on the characteristics of naive human pluripotent stem cells (hPSCs) obtained using independent methods differ. Naive hPSCs have been mainly derived by conversion from primed hPSCs or by direct derivation from human embryos rather than by somatic cell reprogramming. To provide an unbiased molecular and functional reference,we derived genetically matched naive hPSCs by direct reprogramming of fibroblasts and by primed-to-naive conversion using different naive conditions (NHSM,RSeT,5iLAF and t2iLGöY). Our results show that hPSCs obtained in these different conditions display a spectrum of naive characteristics. Furthermore,our characterization identifies KLF4 as sufficient for conversion of primed hPSCs into naive t2iLGöY hPSCs,underscoring the role that reprogramming factors can play for the derivation of bona fide naive hPSCs. View Publication

INTRODUCTION The α-synuclein (SNCA) gene has been implicated in the etiology of Parkinson's disease (PD) and dementia with Lewy bodies (DLB). METHODS A computational analysis of SNCA 3' untranslated region to identify potential microRNA (miRNA) binding sites and quantitative real-time polymerase chain reaction (PCR) to determine their expression in isogenic induced pluripotent stem cell-derived dopaminergic and cholinergic neurons as a model of PD and DLB,respectively,were performed. In addition,we performed a deep sequencing analysis of the SNCA 3' untranslated region of autopsy-confirmed cases of PD,DLB,and normal controls,followed by genetic association analysis of the identified variants. RESULTS We identified four miRNA binding sites and observed a neuronal-type-specific expression profile for each miRNA in the different isogenic induced pluripotent stem cell-derived dopaminergic and cholinergic neurons. Furthermore,we found that the short structural variant rs777296100-polyT was moderately associated with DLB but not with PD. DISCUSSION We suggest that the regulation of SNCA expression through miRNAs is neuronal-type-specific and possibly plays a part in the phenotypic heterogeneity of synucleinopathies. Furthermore,genetic variability in the SNCA gene may contribute to synucleinopathies in a pathology-specific manner. View Publication

Mitochondrial disease is associated with a wide variety of clinical presentations,even among patients carrying heteroplasmic mitochondrial DNA (mtDNA) mutations,probably because of variations in mutant mtDNA proportions at the tissue and organ levels. Although several case reports and clinical trials have assessed the effectiveness of various types of drugs and supplements for the treatment of mitochondrial diseases,there are currently no cures for these conditions. In this study,we demonstrated for the first time that low dose resveratrol (RSV) ameliorated mitochondrial respiratory dysfunction in patient-derived fibroblasts carrying homoplasmic mtDNA mutations. Furthermore,low dose RSV also facilitated efficient cellular reprogramming of the patient-derived fibroblasts into induced pluripotent stem cells,partly due to improved cellular viability. Our results highlight the potential of RSV as a new therapeutic drug candidate for the treatment of mitochondrial diseases. View Publication

Vinculin is a highly conserved actin-binding protein that is localized in integrin-mediated focal adhesion complexes. Although critical roles have been proposed for integrins in hematopoietic stem cell (HSC) function,little is known about the involvement of intracellular focal adhesion proteins in HSC functions. This study showed that the ability of c-Kit(+)Sca1(+)Lin(-) HSCs to support reconstitution of hematopoiesis after competitive transplantation was severely impaired by lentiviral transduction with short hairpin RNA sequences for vinculin. The potential of these HSCs to differentiate into granulocytic and monocytic lineages,to migrate toward stromal cell-derived factor 1α,and to home to the bone marrow in vivo were not inhibited by the loss of vinculin. However,the capacities to form long term culture-initiating cells and cobblestone-like areas were abolished in vinculin-silenced c-Kit(+)Sca1(+)Lin(-) HSCs. In contrast,adhesion to the extracellular matrix was inhibited by silencing of talin-1,but not of vinculin. Whole body in vivo luminescence analyses to detect transduced HSCs confirmed the role of vinculin in long term HSC reconstitution. Our results suggest that vinculin is an indispensable factor determining HSC repopulation capacity,independent of integrin functions. View Publication