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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

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

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

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

The mitochondrial contribution to the maintenance of human embryonic stem cell (hESC) pluripotency and culture homeostasis remains poorly understood. Here,we sought to determine whether hESC adaptation to different feeder-free culture conditions is linked to a mitochondrial adaptation. The expression of ESC pluripotency factors and parameters of mitochondrial contribution including mitochondrial membrane potential,mtDNA content,and the expression of master mitochondrial genes implicated in replication,transcription,and biogenesis were determined in 8 hESC lines maintained in 2 distinct human feeders-conditioned media (CM): human foreskin fibroblast-CM (HFF-CM) and mesenchymal stem cell-CM (MSC-CM). We show a robust parallel trend between the expression of ESC pluripotency factors and the mitochondrial contribution depending on the culture conditions employed to maintain the hESCs,with those in MSC-CM consistently displaying increased levels of pluripotency markers associated to an enhanced mitochondrial contribution. The differences in the mitochondrial status between hESCs maintained in MSC-CM versus HFF-CM respond to coordinated changes in mitochondrial gene expression and biogenesis. Importantly,the culture conditions determine the mitochondrial distribution within the stage-specific embryonic antigen 3 positive (SSEA3(+)) and negative (SSEA3(-)) isolated cell subsets. hESC colonies in MSC-CM display an intrinsic" high mitochondrial status which may suffice to support undifferentiated growth� View Publication

As known from model organisms,such as frog,fish,mouse and chicken,the anterior-posterior patterning of the definitive endoderm (DE) into distinct domains is controlled by a variety of signaling interactions between the DE and its surrounding mesoderm. This includes Wnt/FGFs and BMPs in the posterior half and all-trans-retinoic acid,TGF-$$-ligands,Wnt- and BMP-inhibitors in the anterior half of the DE sheet. However,it is currently unclear how these embryonic tissue interactions can be translated into a defined differentiation protocol for human embryonic stem cells. Activin A has been proposed to direct DE into a SOX2-positive foregut-like cell type. Due to the pleiotropic nature of SOX2 in pluripotency and developing cells of the foregut we purified DE-cells by magnetic cell sorting and tested the effects of anteriorizing and posteriorizing factors on pure endoderm. We show in contrast to previous studies that the generation of the foregut marked by SOX2/FOXA2 double-positive cells does not depend on activin A/TGF-$$-signaling but is mediated by the inhibition of Wnt- and BMP-signaling. Retinoic acid can posteriorize and at the same time dorsalize the foregut towards a PDX1-positive pancreatic duodenal cell type whereas active Wnt/beta-catenin signaling synergistically with FGF-2,BMP-4 and RA induces the formation of CDX2-positive posterior endoderm. Thus,these results provide new insights into the mechanisms behind cell specification of human DE derived from pluripotent stem cells. This article is protected by copyright. All rights reserved. View Publication

The role of autophagy,a lysosomal degradation pathway which prevents cellular damage,in the maintenance of adult mouse hematopoietic stem cells (HSCs) remains unknown. Although normal HSCs sustain life-long hematopoiesis,malignant transformation of HSCs leads to leukemia. Therefore,mechanisms protecting HSCs from cellular damage are essential to prevent hematopoietic malignancies. In this study,we crippled autophagy in HSCs by conditionally deleting the essential autophagy gene Atg7 in the hematopoietic system. This resulted in the loss of normal HSC functions,a severe myeloproliferation,and death of the mice within weeks. The hematopoietic stem and progenitor cell compartment displayed an accumulation of mitochondria and reactive oxygen species,as well as increased proliferation and DNA damage. HSCs within the Lin(-)Sca-1(+)c-Kit(+) (LSK) compartment were significantly reduced. Although the overall LSK compartment was expanded,Atg7-deficient LSK cells failed to reconstitute the hematopoietic system of lethally irradiated mice. Consistent with loss of HSC functions,the production of both lymphoid and myeloid progenitors was impaired in the absence of Atg7. Collectively,these data show that Atg7 is an essential regulator of adult HSC maintenance. View Publication

Neurogenin3 (NEUROG3) is a basic helix-loop-helix transcription factor required for development of the endocrine pancreas in mice. In contrast,humans with NEUROG3 mutations are born with endocrine pancreas function,calling into question whether NEUROG3 is required for human endocrine pancreas development. To test this directly,we generated human embryonic stem cell (hESC) lines where both alleles of NEUROG3 were disrupted using CRISPR/Cas9-mediated gene targeting. NEUROG3(-/-) hESC lines efficiently formed pancreatic progenitors but lacked detectible NEUROG3 protein and did not form endocrine cells in vitro. Moreover,NEUROG3(-/-) hESC lines were unable to form mature pancreatic endocrine cells after engraftment of PDX1(+)/NKX6.1(+) pancreatic progenitors into mice. In contrast,a 75-90% knockdown of NEUROG3 caused a reduction,but not a loss,of pancreatic endocrine cell development. We conclude that NEUROG3 is essential for endocrine pancreas development in humans and that as little as 10% NEUROG3 is sufficient for formation of pancreatic endocrine cells. View Publication