技术资料

The advance in stem cell research relies largely on the efficiency and biocompatibility of technologies used to manipulate stem cells. In our previous study,we had designed an amphipathic peptide RV24 that can deliver proteins into cancer cell lines efficiently without significant side effects. Encouraged by this observation,we moved forward to test whether RV24 could be used to deliver proteins into human embryonic stem cells and human induced pluripotent stem cells. RV24 successfully mediated protein delivery into these pluripotent stem cells,as well as their derivatives including neural stem cells and dendritic cells. Based on NMR studies and particle surface charge measurements,we proposed that hydrophobic domain of RV24 interacts with ??-sheet structures of the proteins,followed by formation of peptide cage" to facilitate delivery across cellular membrane. These findings suggest the feasibility of using amphipathic peptide to deliver functional proteins intracellularly for stem cell research. ?? 2012 Elsevier Inc." View Publication

Human embryonic stem (hES) cells activate a rapid apoptotic response after DNA damage but the underlying mechanisms are unknown. A critical mediator of apoptosis is Bax,which is reported to become active and translocate to the mitochondria only after apoptotic stimuli. Here we show that undifferentiated hES cells constitutively maintain Bax in its active conformation. Surprisingly,active Bax was maintained at the Golgi rather than at the mitochondria,thus allowing hES cells to effectively minimize the risks associated with having preactivated Bax. After DNA damage,active Bax rapidly translocated to the mitochondria by a p53-dependent mechanism. Interestingly,upon differentiation,Bax was no longer active,and cells were not acutely sensitive to DNA damage. Thus,maintenance of Bax in its active form is a unique mechanism that can prime hES cells for rapid death,likely to prevent the propagation of mutations during the early critical stages of embryonic development. View Publication

We describe a method using atomic force microscopy (AFM) to quantify the mechanobiological properties of pluripotent,stem cell-derived cardiomyocytes,including contraction force,rate,duration,and cellular elasticity. We measured beats from cardiomyocytes derived from induced pluripotent stem cells of healthy subjects and those with dilated cardiomyopathy,and from embryonic stem cell lines. We found that our AFM method could quantitate beat forces of single cells and clusters of cardiomyocytes. We demonstrate the dose-responsive,inotropic effect of norepinephrine and beta-adrenergic blockade of metoprolol. Cardiomyocytes derived from subjects with dilated cardiomyopathy showed decreased force and decreased cellular elasticity compared to controls. This AFM-based method can serve as a screening tool for the development of cardiac-active pharmacological agents,or as a platform for studying cardiomyocyte biology. View Publication

Embryonic stem cell (ESC) markers are molecules specifically expressed in ES cells. Understanding of the functions of these markers is critical for characterization and elucidation for the mechanism of ESC pluripotent maintenance and self-renewal,therefore helping to accelerate the clinical application of ES cells. Unfortunately,different cell types can share single or sometimes multiple markers; thus the main obstacle in the clinical application of ESC is to purify ES cells from other types of cells,especially tumor cells. Currently,the marker-based flow cytometry (FCM) technique and magnetic cell sorting (MACS) are the most effective cell isolating methods,and a detailed maker list will help to initially identify,as well as isolate ESCs using these methods. In the current review,we discuss a wide range of cell surface and generic molecular markers that are indicative of the undifferentiated ESCs. Other types of molecules,such as lectins and peptides,which bind to ESC via affinity and specificity,are also summarized. In addition,we review several markers that overlap with tumor stem cells (TSCs),which suggest that uncertainty still exists regarding the benefits of using these markers alone or in various combinations when identifying and isolating cells. View Publication

Human pluripotent stem cell (hPSC-) derived cardiomyocytes have potential applications in drug discovery,toxicity testing,developmental studies,and regenerative medicine. Before these cells can be reliably utilized,characterization of their functionality is required to establish their similarity to native cardiomyocytes. We tracked fluorescent beads embedded in 4.4-99.7 kPa polyacrylamide hydrogels beneath contracting neonatal rat cardiomyocytes and cardiomyocytes generated from hPSCs via growth-factor-induced directed differentiation to measure contractile output in response to changes in substrate mechanics. Contraction stress was determined using traction force microscopy,and morphology was characterized by immunocytochemistry for α-actinin and subsequent image analysis. We found that contraction stress of all types of cardiomyocytes increased with substrate stiffness. This effect was not linked to beating rate or morphology. We demonstrated that hPSC-derived cardiomyocyte contractility responded appropriately to isoprenaline and remained stable in culture over a period of 2 months. This study demonstrates that hPSC-derived cardiomyocytes have appropriate functional responses to substrate stiffness and to a pharmaceutical agent,which motivates their use in further applications such as drug evaluation and cardiac therapies. View Publication

Cockayne syndrome (CS) is a human premature aging disorder associated with neurological and developmental abnormalities,caused by mutations mainly in the CS group B gene (ERCC6). At the molecular level,CS is characterized by a deficiency in the transcription-couple DNA repair pathway. To understand the role of this molecular pathway in a pluripotent cell and the impact of CSB mutation during human cellular development,we generated induced pluripotent stem cells (iPSCs) from CSB skin fibroblasts (CSB-iPSC). Here,we showed that the lack of functional CSB does not represent a barrier to genetic reprogramming. However,iPSCs derived from CSB patient's fibroblasts exhibited elevated cell death rate and higher reactive oxygen species (ROS) production. Moreover,these cellular phenotypes were accompanied by an up-regulation of TXNIP and TP53 transcriptional expression. Our findings suggest that CSB modulates cell viability in pluripotent stem cells,regulating the expression of TP53 and TXNIP and ROS production. View Publication

PURPOSE: The presence of multipotent human limbal stromal cells resembling mesenchymal stromal cells (MSC) provides new insights to the characteristic of these cells and its therapeutic potential. However,little is known about the expression of stage-specific embryonic antigen 4 (SSEA-4) and the embryonic stem cell (ESC)-like properties of these cells. We studied the expression of SSEA-4 surface protein and the various ESC and MSC markers in the ex vivo cultured limbal stromal cells. The phenotypes and multipotent differentiation potential of these cells were also evaluated.backslashnbackslashnMETHODS: Limbal stromal cells were derived from corneoscleral rims. The SSEA-4(+) and SSEA-4(-) limbal stromal cells were sorted by fluorescence-activated cells sorting (FACS). Isolated cells were expanded and reanalyzed for their expression of SSEA-4. Expression of MSC and ESC markers on these cells were also analyzed by FACS. In addition,expression of limbal epithelial and corneal stromal proteins such as ATP-binding cassette sub-family G member 2 (ABCG2),tumour protein p63 (p63),paired box 6 (Pax6),cytokeratin 3 (AE5),cytokeratin 10,and keratocan sulfate were evaluated either by immunofluorecence staining or reverse transcription polymerase chain reaction. Appropriate induction medium was used to differentiate these cells into adipocytes,osteocytes,and chondrocytes.backslashnbackslashnRESULTS: Expanded limbal stromal cells expressed the majority of mesenchymal markers. These cells were negative for ABCG2,p63,Pax6,AE-5,and keratocan sulfate. After passaged,a subpopulation of these cells showed low expression of SSEA-4 but were negative for other important ESC surface markers such as Tra-1-60,Tra-1-81,and transcription factors like octamer-binding transcription factor 4 (Oct4),SRY(sex determining region Y)-box 2 (Sox2),and Nanog. Early passaged cells when induced were able to differentiate into adipocytes,osteocytes and chondrocytes.backslashnbackslashnCONCLUSIONS: The expanded limbal stromal cells showed features of multipotent MSC. Our study confirmed the expression of SSEA-4 by a subpopulation of cultured limbal stromal cells. However,despite the expression of SSEA-4,these cells did not express any other markers of ESC. Therefore,we conclude that the cells did not show properties of ESC. View Publication

The blood-brain barrier (BBB) is crucial to the health of the brain and is often compromised in neurological disease. Moreover,because of its barrier properties,this endothelial interface restricts uptake of neurotherapeutics. Thus,a renewable source of human BBB endothelium could spur brain research and pharmaceutical development. Here we show that endothelial cells derived from human pluripotent stem cells (hPSCs) acquire BBB properties when co-differentiated with neural cells that provide relevant cues,including those involved in Wnt/β-catenin signaling. The resulting endothelial cells have many BBB attributes,including well-organized tight junctions,appropriate expression of nutrient transporters and polarized efflux transporter activity. Notably,they respond to astrocytes,acquiring substantial barrier properties as measured by transendothelial electrical resistance (1,450 ± 140 Ω cm2),and they possess molecular permeability that correlates well with in vivo rodent blood-brain transfer coefficients. View Publication

Long QT syndrome (LQTS) is a genetic disease characterized by a prolonged QT interval in an electrocardiogram (ECG),leading to higher risk of sudden cardiac death. Among the 12 identified genes causal to heritable LQTS,∼90% of affected individuals harbor mutations in either KCNQ1 or human ether-a-go-go related genes (hERG),which encode two repolarizing potassium currents known as I(Ks) and I(Kr). The ability to quantitatively assess contributions of different current components is therefore important for investigating disease phenotypes and testing effectiveness of pharmacological modulation. Here we report a quantitative analysis by simulating cardiac action potentials of cultured human cardiomyocytes to match the experimental waveforms of both healthy control and LQT syndrome type 1 (LQT1) action potentials. The quantitative evaluation suggests that elevation of I(Kr) by reducing voltage sensitivity of inactivation,not via slowing of deactivation,could more effectively restore normal QT duration if I(Ks) is reduced. Using a unique specific chemical activator for I(Kr) that has a primary effect of causing a right shift of V(1/2) for inactivation,we then examined the duration changes of autonomous action potentials from differentiated human cardiomyocytes. Indeed,this activator causes dose-dependent shortening of the action potential durations and is able to normalize action potentials of cells of patients with LQT1. In contrast,an I(Kr) chemical activator of primary effects in slowing channel deactivation was not effective in modulating action potential durations. Our studies provide both the theoretical basis and experimental support for compensatory normalization of action potential duration by a pharmacological agent. 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

The expression and function of several multidrug transporters (including ABCB1 and ABCG2) have been studied in human cancer cells and in mouse and human adult stem cells. However,the expression of ABCG2 in human embryonic stem cells (hESCs) remains unclear. Limited and contradictory results in the literature from two research groups have raised questions regarding its expression and function. In this study,we used quantitative real-time PCR,Northern blots,whole genome RNA sequencing,Western blots,and immunofluorescence microscopy to study ABCG2 expression in hESCs. We found that full-length ABCG2 mRNA transcripts are expressed in undifferentiated hESC lines. However,ABCG2 protein was undetectable even under embryoid body differentiation or cytotoxic drug induction. Moreover,surface ABCG2 protein was coexpressed with the differentiation marker stage-specific embryonic antigen-1 of hESCs,following constant BMP-4 signaling at days 4 and 6. This expression was tightly correlated with the downregulation of two microRNAs (miRNAs) (i.e.,hsa-miR-519c and hsa-miR-520h). Transfection of miRNA mimics and inhibitors of these two miRNAs confirmed their direct involvement in the regulation ABCG2 translation. Our findings clarify the controversy regarding the expression of the ABCG2 gene and also provide new insights into translational control of the expression of membrane transporter mRNAs by miRNAs in hESCs. View Publication

Haematopoietic stem cells (HSCs) regenerate blood cells throughout the lifespan of an organism. With age,the functional quality of HSCs declines,partly owing to the accumulation of damaged DNA. However,the factors that damage DNA and the protective mechanisms that operate in these cells are poorly understood. We have recently shown that the Fanconi anaemia DNA-repair pathway counteracts the genotoxic effects of reactive aldehydes. Mice with combined inactivation of aldehyde catabolism (through Aldh2 knockout) and the Fanconi anaemia DNA-repair pathway (Fancd2 knockout) display developmental defects,a predisposition to leukaemia,and are susceptible to the toxic effects of ethanol-an exogenous source of acetaldehyde. Here we report that aged Aldh2(-/-) Fancd2(-/-) mutant mice that do not develop leukaemia spontaneously develop aplastic anaemia,with the concomitant accumulation of damaged DNA within the haematopoietic stem and progenitor cell (HSPC) pool. Unexpectedly,we find that only HSPCs,and not more mature blood precursors,require Aldh2 for protection against acetaldehyde toxicity. Additionally,the aldehyde-oxidizing activity of HSPCs,as measured by Aldefluor stain,is due to Aldh2 and correlates with this protection. Finally,there is more than a 600-fold reduction in the HSC pool of mice deficient in both Fanconi anaemia pathway-mediated DNA repair and acetaldehyde detoxification. Therefore,the emergence of bone marrow failure in Fanconi anaemia is probably due to aldehyde-mediated genotoxicity restricted to the HSPC pool. These findings identify a new link between endogenous reactive metabolites and DNA damage in HSCs,and define the protective mechanisms that counteract this threat. View Publication