技术资料

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

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

We show here that the process of megakaryocytic differentiation requires the presence of the recently discovered protein tescalcin. Tescalcin is dramatically upregulated during the differentiation and maturation of primary megakaryocytes or upon PMA-induced differentiation of K562 cells. This upregulation requires sustained signaling through the ERK pathway. Overexpression of tescalcin in K562 cells initiates events of spontaneous megakaryocytic differentiation,such as expression of specific cell surface antigens,inhibition of cell proliferation,and polyploidization. Conversely,knockdown of this protein in primary CD34+ hematopoietic progenitors and cell lines by RNA interference suppresses megakaryocytic differentiation. In cells lacking tescalcin,the expression of Fli-1,Ets-1,and Ets-2 transcription factors,but not GATA-1 or MafB,is blocked. Thus,tescalcin is essential for the coupling of ERK cascade activation with the expression of Ets family genes in megakaryocytic differentiation. View Publication

Human urine cells (HUCs) can be reprogrammed into neural progenitor cells (NPCs) or induced pluripotent stem cells (iPSCs) with defined factors and a small molecule cocktail,but the underlying fate choice remains unresolved. Here,through sequential removal of individual compound from small molecule cocktail,we showed that A8301,a TGF$$ signaling inhibitor,is sufficient to switch the cell fate from iPSCs into NPCs in OSKM-mediated HUCs reprogramming. However,TGF$$ exposure at early stage inhibits HUCs reprogramming by promoting EMT. Base on these data,we developed an optimized approach for generation of NPCs or iPSCs from HUCs with significantly improved efficiency by regulating TGF$$ activity at different reprogramming stages. This approach provides a simplified and improved way for HUCs reprogramming,thus would be valuable for banking human iPSCs or NPCs from people with different genetic background. View Publication

During pregnancy the uterine decidua is populated by large numbers of natural killer (NK) cells with a phenotype CD56(superbright)CD16(-)CD9(+)KIR(+) distinct from both subsets of peripheral blood NK cells. Culture of highly purified CD16(+)CD9(-) peripheral blood NK cells in medium containing TGFbeta1 resulted in a transition to CD16(-)CD9(+) NK cells resembling decidual NK cells. Decidual stromal cells,when isolated and cultured in vitro,were found to produce TGFbeta1. Incubation of peripheral blood NK cells with conditioned medium from decidual stromal cells mirrored the effects of TGFbeta1. Similar changes may occur upon NK cell entry into the decidua or other tissues expressing substantial TGFbeta. In addition,Lin(-)CD34(+)CD45(+) hematopoietic stem/progenitor cells could be isolated from decidual tissue. These progenitors also produced NK cells when cultured in conditioned medium from decidual stromal cells supplemented with IL-15 and stem cell factor. View Publication

THAP1 encodes a transcription factor but its regulation is largely elusive. TOR1A was shown to be repressed by THAP1 in vitro. Notably,mutations in both of these genes lead to dystonia (DYT6 or DYT1). Surprisingly,expressional changes of TOR1A in THAP1 mutation carriers have not been detected indicating additional levels of regulation. Here,we investigated whether THAP1 is able to autoregulate its own expression. Using in-silico prediction,luciferase reporter gene assays,and (quantitative) chromatin immunoprecipitation (ChIP),we defined the THAP1 minimal promoter to a 480. bp-fragment and demonstrated specific binding of THAP1 to this region which resulted in repression of the THAP1 promoter. This autoregulation was disturbed by different DYT6-causing mutations. Two mutants (Ser6Phe,Arg13His) were shown to be less stable than wildtype THAP1 adding to the effect of reduced binding to the THAP1 promoter. Overexpressed THAP1 is preferably degraded through the proteasome. Notably,endogenous THAP1 expression was significantly reduced in cells overexpressing wildtype THAP1 as demonstrated by quantitative PCR. In contrast,higher THAP1 levels were detected in induced pluripotent stem cell (iPS)-derived neurons from THAP1 mutation carriers. Thus,we identified a feedback-loop in the regulation of THAP1 expression and demonstrated that mutant THAP1 leads to higher THAP1 expression levels. This compensatory autoregulation may contribute to the mean age at onset in the late teen years or even reduced penetrance in some THAP1 mutation carriers. View Publication

Phenotypic markers associated with human hematopoietic stem cells (HSCs) were developed and validated using uncultured cells. Because phenotype and function can be dissociated during culture,better markers to prospectively track and isolate HSCs in ex vivo cultures could be instrumental in advancing HSC-based therapies. Using an expansion system previously shown to increase hematopoietic progenitors and SCID-repopulating cells (SRCs),we demonstrated that the rhodamine-low phenotype was lost,whereas AC133 expression was retained throughout culture. Furthermore,the AC133(+)CD38(-) subpopulation was significantly enriched in long-term culture-initiating cells (LTC-IC) and SRCs after culture. Preculture and postculture analysis of total nucleated cell and LTC-IC number,and limiting dilution analysis in NOD/SCID mice,showed a 43-fold expansion of the AC133(+)CD38(-) subpopulation that corresponded to a 7.3-fold and 4.4-fold expansion of LTC-ICs and SRCs in this subpopulation,respectively. Thus,AC133(+)CD38(-) is an improved marker that tracks and enriches for LTC-IC and SRC in ex vivo cultures. View Publication

BACKGROUND The Activin A and bone morphogenetic protein (BMP) pathways are critical regulators of the immune system and of bone formation. Inappropriate activation of these pathways,as in conditions of congenital heterotopic ossification,are thought to activate an osteogenic program in endothelial cells. However,if and how this occurs in human endothelial cells remains unclear. METHODS We used a new directed differentiation protocol to create human induced pluripotent stem cell (hiPSC)-derived endothelial cells (iECs) from patients with fibrodysplasia ossificans progressiva (FOP),a congenital disease of heterotopic ossification caused by an activating R206H mutation in the Activin A type I receptor (ACVR1). This strategy allowed the direct assay of the cell-autonomous effects of ACVR1 R206H in the endogenous locus without the use of transgenic expression. These cells were challenged with BMP or Activin A ligand,and tested for their ability to activate osteogenesis,extracellular matrix production,and differential downstream signaling in the BMP/Activin A pathways. RESULTS We found that FOP iECs could form in conditions with low or absent BMP4. These conditions are not normally permissive in control cells. FOP iECs cultured in mineralization media showed increased alkaline phosphatase staining,suggesting formation of immature osteoblasts,but failed to show mature osteoblastic features. However,FOP iECs expressed more fibroblastic genes and Collagen 1/2 compared to control iECs,suggesting a mechanism for the tissue fibrosis seen in early heterotopic lesions. Finally,FOP iECs showed increased SMAD1/5/8 signaling upon BMP4 stimulation. Contrary to FOP hiPSCs,FOP iECs did not show a significant increase in SMAD1/5/8 phosphorylation upon Activin A stimulation,suggesting that the ACVR1 R206H mutation has a cell type-specific effect. In addition,we found that the expression of ACVR1 and type II receptors were different in hiPSCs and iECs,which could explain the cell type-specific SMAD signaling. CONCLUSIONS Our results suggest that the ACVR1 R206H mutation may not directly increase the formation of mature chondrogenic or osteogenic cells by FOP iECs. Our results also show that BMP can induce endothelial cell dysfunction,increase expression of fibrogenic matrix proteins,and cause differential downstream signaling of the ACVR1 R206H mutation. This iPSC model provides new insight into how human endothelial cells may contribute to the pathogenesis of heterotopic ossification. View Publication