技术资料

OBJECTIVE The differentiation program for human thyroid follicular cells (TFCs) relies on the interplay between sequence-specific transcription factors and transcriptional co-regulators. Transcriptional co-activator with PDZ-binding motif (TAZ) is a co-activator that regulates several transcription factors,including PAX8 and NKX2-1,which play a central role in thyroid-specific gene transcription. TAZ and PAX8/NKX2-1 are co-expressed in the nuclei of thyroid cells,and TAZ interacts directly with both PAX8 and NKX2-1,leading to their enhanced transcriptional activity on the thyroglobulin (TG) promoter and additional genes. METHODS The use of a small molecule,ethacridine,recently identified as a TAZ activator,in the differentiation of thyroid cells from human embryonic stem (hES) cells was studied. First,endodermal cells were derived from hES cells using Activin A,followed by induction of differentiation into thyroid cells directed by ethacridine and thyrotropin (TSH). RESULTS The expression of TAZ was increased in the Activin A-derived endodermal cells by ethacridine in a dose-dependent manner and followed by increases in PAX8 and NKX2-1 when assessed by both quantitative polymerase chain reaction and immunostaining. Following further differentiation with the combination of ethacridine and TSH,the thyroid-specific genes TG,TPO,TSHR,and NIS were all induced in the differentiated hES cells. When these cells were cultured with extracellular matrix-coated dishes,thyroid follicle formation and abundant TG protein expression were observed. Furthermore,such hES cell-derived thyroid follicles showed a marked TSH-induced and dose-dependent increase in radioiodine uptake and protein-bound iodine accumulation. CONCLUSION These data show that fully functional human thyroid cells can be derived from hES cells using ethacridine,a TAZ activator,which induces thyroid-specific gene expression and promotes thyroid cell differentiation from the hES cells. These studies again demonstrate the importance of transcriptional regulation in thyroid cell development. This approach also yields functional human thyrocytes,without any gene transfection or complex culture conditions,by directly manipulating the transcriptional machinery without interfering with intermediate signaling events. View Publication

Embryoid bodies (EBs) can be generated by culturing human pluripotent stem cells in ultra-low attachment culture vessels,under conditions that are adverse to pluripotency and proliferation. EBs generated in suspension cultures are capable of differentiating into cells of the ectoderm,mesoderm,and endoderm. In this chapter,we describe techniques for generation of EBs from human pluripotent stem cells. Once formed,the EBs can then be dissociated using specific enzymes to acquire a single cell population that has the potential to differentiate into cells of all three germ layers. This population can then be cultured in specialized conditions to obtain progenitor cells of specific lineages. Pure populations of progenitor cells generated on a large scale basis can be used for research,drug discovery/development,and cellular transplantation therapy. View Publication

Genetic causes of many familial arrhythmia syndromes remain elusive. In this study,whole-exome sequencing (WES) was carried out on patients from three different families that presented with life-threatening arrhythmias and high risk of sudden cardiac death (SCD). Two French Canadian probands carried identical homozygous rare variant in TECRL gene (p.Arg196Gln),which encodes the trans-2,3-enoyl-CoA reductase-like protein. Both patients had cardiac arrest,stress-induced atrial and ventricular tachycardia,and QT prolongation on adrenergic stimulation. A third patient from a consanguineous Sudanese family diagnosed with catecholaminergic polymorphic ventricular tachycardia (CPVT) had a homozygous splice site mutation (c.331+1GtextgreaterA) in TECRL Analysis of intracellular calcium ([Ca(2+)]i) dynamics in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) generated from this individual (TECRLHom-hiPSCs),his heterozygous but clinically asymptomatic father (TECRLHet-hiPSCs),and a healthy individual (CTRL-hiPSCs) from the same Sudanese family,revealed smaller [Ca(2+)]i transient amplitudes as well as elevated diastolic [Ca(2+)]i in TECRLHom-hiPSC-CMs compared with CTRL-hiPSC-CMs. The [Ca(2+)]i transient also rose markedly slower and contained lower sarcoplasmic reticulum (SR) calcium stores,evidenced by the decreased magnitude of caffeine-induced [Ca(2+)]i transients. In addition,the decay phase of the [Ca(2+)]i transient was slower in TECRLHom-hiPSC-CMs due to decreased SERCA and NCX activities. Furthermore,TECRLHom-hiPSC-CMs showed prolonged action potentials (APs) compared with CTRL-hiPSC-CMs. TECRL knockdown in control human embryonic stem cell-derived CMs (hESC-CMs) also resulted in significantly longer APs. Moreover,stimulation by noradrenaline (NA) significantly increased the propensity for triggered activity based on delayed afterdepolarizations (DADs) in TECRLHom-hiPSC-CMs and treatment with flecainide,a class Ic antiarrhythmic drug,significantly reduced the triggered activity in these cells. In summary,we report that mutations in TECRL are associated with inherited arrhythmias characterized by clinical features of both LQTS and CPVT Patient-specific hiPSC-CMs recapitulated salient features of the clinical phenotype and provide a platform for drug screening evidenced by initial identification of flecainide as a potential therapeutic. These findings have implications for diagnosis and treatment of inherited cardiac arrhythmias. View Publication

Werner syndrome (WS) patients exhibit premature aging predominantly in mesenchyme-derived tissues,but not in neural lineages,a consequence of telomere dysfunction and accelerated senescence. The cause of this lineage-specific aging remains unknown. Here,we document that reprogramming of WS fibroblasts to pluripotency elongated telomere length and prevented telomere dysfunction. To obtain mechanistic insight into the origin of tissue-specific aging,we differentiated iPSCs to mesenchymal stem cells (MSCs) and neural stem/progenitor cells (NPCs). We observed recurrence of premature senescence associated with accelerated telomere attrition and defective synthesis of the lagging strand telomeres in MSCs,but not in NPCs. We postulate this aging" discrepancy is regulated by telomerase. Expression of hTERT or p53 knockdown ameliorated the accelerated aging phenotypein MSC� View Publication

High telomerase activity is a characteristic of human embryonic stem cells (hESCs),however,the regulation and maintenance of correct telomere length in hESCs is unclear. In this study we investigated telomere elongation in hESCs in vitro and found that telomeres lengthened from their derivation in blastocysts through early expansion,but stabilized at later passages. We report that the core unit of telomerase,hTERT,was highly expressed in hESCs in blastocysts and throughout long-term culture; furthermore,this was regulated in a Wnt-β-catenin-signaling-dependent manner. Our observations that the alternative lengthening of telomeres (ALT) pathway was suppressed in hESCs and that hTERT knockdown partially inhibited telomere elongation,demonstrated that high telomerase activity was required for telomere elongation. We observed that chromatin modification through trimethylation of H3K9 and H4K20 at telomeric regions decreased during early culture. This was concurrent with telomere elongation,suggesting that epigenetic regulation of telomeric chromatin may influence telomerase function. By measuring telomere length in 96 hESC lines,we were able to establish that telomere length remained relatively stable at 12.02±1.01 kb during later passages (15-95). In contrast,telomere length varied in hESCs with genomic instability and hESC-derived teratomas. In summary,we propose that correct,stable telomere length may serve as a potential biomarker for genetically stable hESCs. View Publication

Patients with dyskeratosis congenita (DC),a disorder of telomere maintenance,suffer degeneration of multiple tissues. Patient-specific induced pluripotent stem (iPS) cells represent invaluable in vitro models for human degenerative disorders like DC. A cardinal feature of iPS cells is acquisition of indefinite self-renewal capacity,which is accompanied by induction of the telomerase reverse transcriptase gene (TERT). We investigated whether defects in telomerase function would limit derivation and maintenance of iPS cells from patients with DC. Here we show that reprogrammed DC cells overcome a critical limitation in telomerase RNA component (TERC) levels to restore telomere maintenance and self-renewal. We discovered that TERC upregulation is a feature of the pluripotent state,that several telomerase components are targeted by pluripotency-associated transcription factors,and that in autosomal dominant DC,transcriptional silencing accompanies a 3' deletion at the TERC locus. Our results demonstrate that reprogramming restores telomere elongation in DC cells despite genetic lesions affecting telomerase,and show that strategies to increase TERC expression may be therapeutically beneficial in DC patients. View Publication

The ability of human embryonic stem cells (hESCs) to proliferate indefinitely is attributed to its high telomerase activity and associated long telomere. However,factors regulating telomere length in hESCs remain largely uncharacterized. The aims of this study were,to identify factors which modulate telomere length of hESCs,and to determine if the telomere length of hESCs influences cellular senescence of its differentiated progeny cells. Telomerase reverse transcriptase (TERT) gene expression,telomerase activity and telomere length of hESCs cultured in different culture systems were compared. Genetically identical hESCs of different telomere lengths were differentiated into fibroblasts simultaneously,and the population doubling and cellular senescence levels were determined. We found that telomere lengths were significantly different in different culture systems and Fibroblast growth factor-2 (FGF-2) upregulated TERT expression,telomerase activity and telomere length via Wnt/β-catenin signaling pathway in hESCs in a significant manner. We also provide evidence that fibroblast differentiated from hESCs with longer telomere exhibited significant more population doublings and longer life span than those derived from hESCs with shorter telomeres. Thus,FGF-2 levels in hESCs culture systems can be manipulated to generate cells with longer telomere which would be advantageous in the applications of hESCs in regenerative medicine. View Publication

The differentiation of patient-derived induced pluripotent stem cells (iPSCs) to committed fates such as neurons,muscle and liver is a powerful approach for understanding key parameters of human development and disease. Whether undifferentiated iPSCs themselves can be used to probe disease mechanisms is uncertain. Dyskeratosis congenita is characterized by defective maintenance of blood,pulmonary tissue and epidermal tissues and is caused by mutations in genes controlling telomere homeostasis. Short telomeres,a hallmark of dyskeratosis congenita,impair tissue stem cell function in mouse models,indicating that a tissue stem cell defect may underlie the pathophysiology of dyskeratosis congenita. Here we show that even in the undifferentiated state,iPSCs from dyskeratosis congenita patients harbour the precise biochemical defects characteristic of each form of the disease and that the magnitude of the telomere maintenance defect in iPSCs correlates with clinical severity. In iPSCs from patients with heterozygous mutations in TERT,the telomerase reverse transcriptase,a 50% reduction in telomerase levels blunts the natural telomere elongation that accompanies reprogramming. In contrast,mutation of dyskerin (DKC1) in X-linked dyskeratosis congenita severely impairs telomerase activity by blocking telomerase assembly and disrupts telomere elongation during reprogramming. In iPSCs from a form of dyskeratosis congenita caused by mutations in TCAB1 (also known as WRAP53),telomerase catalytic activity is unperturbed,yet the ability of telomerase to lengthen telomeres is abrogated,because telomerase mislocalizes from Cajal bodies to nucleoli within the iPSCs. Extended culture of DKC1-mutant iPSCs leads to progressive telomere shortening and eventual loss of self-renewal,indicating that a similar process occurs in tissue stem cells in dyskeratosis congenita patients. These findings in iPSCs from dyskeratosis congenita patients reveal that undifferentiated iPSCs accurately recapitulate features of a human stem cell disease and may serve as a cell-culture-based system for the development of targeted therapeutics. View Publication

Human pluripotent stem cells (hPSCs) are a promising cell source for tissue engineering and regenerative medicine,especially in the field of neurobiology. Neural differentiation protocols have been developed to differentiate hPSCs into specific neural cells,but these predominantly rely on biochemical cues. Recently,differentiation protocols have incorporated topographical cues to increase the total neuronal yield. However,the means by which these topographical cues improve neuronal yield remains unknown. In this study,we explored the effect of topography on the neural differentiation of hPSC by quantitatively studying the changes in marker expression at a transcript and protein level. We found that 2 ??m gratings increase the rate of neural differentiation,and that an additional culture period of 2 ??m gratings in the absence of neurotrophic signals can improve the neural differentiation of hPSCs. We envisage that this work can be incorporated into future differentiation protocols to decrease the differentiation period as well as the biochemical signals added,thus generating hPSC-derived neural cells in a more cost effective and efficient manner. ?? 2012 Elsevier Ltd. View Publication

A significant clinical need exists to differentiate human pluripotent stem cells (hPSCs) into cardiomyocytes,enabling tissue modeling for in vitro discovery of new drugs or cell-based therapies for heart repair in vivo. Chemical and mechanical microenvironmental factors are known to impact the efficiency of stem cell differentiation,but cardiac differentiation protocols in hPSCs are typically performed on rigid tissue culture polystyrene (TCPS) surfaces,which do not present a physiological mechanical setting. To investigate the temporal effects of mechanics on cardiac differentiation,we cultured human embryonic stem cells (hESCs) and their derivatives on polyacrylamide hydrogel substrates with a physiologically relevant range of stiffnesses. In directed differentiation and embryoid body culture systems,differentiation of hESCs to cardiac troponin T-expressing (cTnT+) cardiomyocytes peaked on hydrogels of intermediate stiffness. Brachyury expression also peaked on intermediate stiffness hydrogels at day 1 of directed differentiation,suggesting that stiffness impacted the initial differentiation trajectory of hESCs to mesendoderm. To investigate the impact of substrate mechanics during cardiac specification of mesodermal progenitors,we initiated directed cardiomyocyte differentiation on TCPS and transferred cells to hydrogels at the Nkx2.5/Isl1+ cardiac progenitor cell stage. No differences in cardiomyocyte purity with stiffness were observed on day 15. These experiments indicate that differentiation of hESCs is sensitive to substrate mechanics at early stages of mesodermal induction,and proper application of substrate mechanics can increase the propensity of hESCs to differentiate to cardiomyocytes. textcopyright 2013 Acta Materialia Inc. View Publication

In culture conditions,human induced-pluripotent stem cells (hiPSC)-derived neurons form synaptic connections with other cells and establish neuronal networks,which are expected to be an in vitro model system for drug discovery screening and toxicity testing. While early studies demonstrated effects of co-culture of hiPSC-derived neurons with astroglial cells on survival and maturation of hiPSC-derived neurons,the population spiking patterns of such hiPSC-derived neurons have not been fully characterized. In this study,we analyzed temporal spiking patterns of hiPSC-derived neurons recorded by a multi-electrode array system. We discovered that specific sets of hiPSC-derived neurons co-cultured with astrocytes showed more frequent and highly coherent non-random synchronized spike trains and more dynamic changes in overall spike patterns over time. These temporally coordinated spiking patterns are physiological signs of organized circuits of hiPSC-derived neurons and suggest benefits of co-culture of hiPSC-derived neurons with astrocytes. View Publication

The recent identification of hemogenic endothelium (HE) in human pluripotent stem cell (hPSC) cultures presents opportunities to investigate signaling pathways that are essential for blood development from endothelium and provides an exploratory platform for de novo generation of hematopoietic stem cells (HSCs). However,the use of poorly defined human or animal components limits the utility of the current differentiation systems for studying specific growth factors required for HE induction and manufacturing clinical-grade therapeutic blood cells. Here,we identified chemically defined conditions required to produce HE from hPSCs growing in Essential 8 (E8) medium and showed that Tenascin C (TenC),an extracellular matrix protein associated with HSC niches,strongly promotes HE and definitive hematopoiesis in this system. hPSCs differentiated in chemically defined conditions undergo stages of development similar to those previously described in hPSCs cocultured on OP9 feeders,including the formation of VE-Cadherin(+)CD73(-)CD235a/CD43(-) HE and hematopoietic progenitors with myeloid and T lymphoid potential. View Publication