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BACKGROUND: The histone variant H2A.Z has been implicated in nucleosome exchange,transcriptional activation and Polycomb repression. However,the relationships among these seemingly disparate functions remain obscure.backslashnbackslashnRESULTS: We mapped H2A.Z genome-wide in mammalian ES cells and neural progenitors. H2A.Z is deposited promiscuously at promoters and enhancers,and correlates strongly with H3K4 methylation. Accordingly,H2A.Z is present at poised promoters with bivalent chromatin and at active promoters with H3K4 methylation,but is absent from stably repressed promoters that are specifically enriched for H3K27 trimethylation. We also characterized post-translational modification states of H2A.Z,including a novel species dually-modified by ubiquitination and acetylation that is enriched at bivalent chromatin.backslashnbackslashnCONCLUSIONS: Our findings associate H2A.Z with functionally distinct genomic elements,and suggest that post-translational modifications may reconcile its contrasting locations and roles. View Publication

FUS/TLS (fused in sarcoma/translocated in liposarcoma) and TDP-43 are integrally involved in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. We found that FUS/TLS binds to RNAs from textgreater5,500 genes in mouse and human brain,primarily through a GUGGU-binding motif. We identified a sawtooth-like binding pattern,consistent with co-transcriptional deposition of FUS/TLS. Depletion of FUS/TLS from the adult nervous system altered the levels or splicing of textgreater950 mRNAs,most of which are distinct from RNAs dependent on TDP-43. Abundance of only 45 RNAs was reduced after depletion of either TDP-43 or FUS/TLS from mouse brain,but among these were mRNAs that were transcribed from genes with exceptionally long introns and that encode proteins that are essential for neuronal integrity. Expression levels of a subset of these were lowered after TDP-43 or FUS/TLS depletion in stem cell-derived human neurons and in View Publication

Immature primary and stem cell-derived cardiomyocytes provide useful models for fundamental studies of heart development and cardiac disease,and offer potentialbackslashrbackslashnfor patient specific drug testing and differentiation protocols aimed at cardiac grafts. To assess their potential for augmenting heart function,and to gain insight into cardiac growth and disease,tissue engineers must quantify the contractile forces of these single cells. Currently,axial contractile forces of isolated adult heart cells can only be measuredbackslashrbackslashnby two-point methods such as carbon fiber techniques,which cannot be applied to neonatal and stem cell-derived heart cells because they are more difficult to handle and lack a persistent shape. Here we present a novel axial technique for measuring the contractile forces of isolated immature cardiomyocytes. We overcome cell manipulation and patterning challenges by using a thermoresponsive sacrificialbackslashrbackslashnsupport layer in conjunction with arrays of widely separated elastomeric microposts. Our approach has the potential to be high-throughput,is functionally analogous to current gold-standard axial force assays for adult heart cells,and prescribes elongated cell shapes without protein patterning. Finally,we calibrate these force posts withbackslashrbackslashnpiezoresistive cantilevers to dramatically reduce measurement error typical for soft polymer-based force assays. We report quantitative measurements of peak contractile forces up to 146 nN with post stiffness standard error (26 nN) far betterbackslashrbackslashnthan that based on geometry and stiffness estimates alone. The addition of sacrificial layers to future 2D and 3D cell culturebackslashrbackslashnplatforms will enable improved cell placement and the complex suspension of cells across 3D constructs. View Publication

Forced expression of selected transcription factors can transform somatic cells into embryonic stem cell (ESC)-like cells,termed induced pluripotent stem cells (iPSCs). There is no consensus regarding the preferred tissue from which to harvest donor cells for reprogramming into iPSCs,and some donor cell types may be more prone than others to accumulation of epigenetic imprints and somatic cell mutations. Here,we present a simple,reproducible,noninvasive method for generating human iPSCs from renal tubular cells present in urine. This procedure eliminates many problems associated with other protocols,and the resulting iPSCs display an excellent ability to differentiate. These data suggest that urine may be a preferred source for generating iPSCs. View Publication

Mesenchymal stem cells (MSCs) possess tumor-tropic properties and consequently have been used to deliver therapeutic agents for cancer treatment. Their potential in cancer therapy highlights the need for a consistent and renewable source for the production of uniform human MSCs suitable for clinical applications. In this study,we seek to investigate whether human embryonic stem cells can be used as a cell source to fulfill this goal. We generated MSC-like cells from two human embryonic stem cell lines,HuES9 and H1,and observed that MSC-like cells derived from human embryonic stem cells were able to migrate into human glioma intracranial xenografts after being injected into the cerebral hemisphere contralateral to the tumor inoculation site. We engineered these cells with baculoviral and lentiviral vectors,respectively,for transient and stable expression of the herpes simplex virus thymidine kinase gene. In tumor-bearing mice the engineered MSC-like cells were capable of inhibiting tumor growth and prolonging survival in the presence of ganciclovir after they were injected either directly into the xenografts or into the opposite hemisphere. Our findings suggest that human embryonic stem cell-derived MSCs may be a viable and attractive alternative for large-scale derivation of targeting vehicles for cancer therapy. View Publication

Male germline stem cells (mGSCs) are stem cells present in male testis responsible for spermatogenesis during their whole life. Studies have shown that mGSCs can be derived in vitro and resemble embryonic stem cells (ESCs) properties both in the mouse and humans. However,little is know about these cells in domestic animals. Here we report the first successful establishment of goat GSCs derived from 2-5-month fetal testis,and developmental potential assay of these cells both in vitro and in vivo. These cells express pluripotent markers such as Oct4,Sox2,C-myc,and Tert when cultured as human ESCs conditions. Embryoid bodies (EBs) formed by goat mGSCs were induced with 2 × 10(-6) M retinoic acid (RA). Immunofluorescence analysis showed that some cells inside of the EBs were positive for meiosis marker-SCP3,STRA8,and germ cell marker-VASA,and haploid marker-FE-J1,PRM1,indicating their germ cell lineage differentiation. Some cells become elongated sperm-like cells after induction. Approximately 34.88% (30/86) embryos showed cleavage and four embryos were cultured on murine fibroblast feeder and formed small embryonic stem like colonies. However,most stalled at four-cell stage after intracytoplasmic sperm injection (ICSI) of these cells. Transplantation of DAPI labeled mGSCs into the seminiferous tubules of busulfan-treated mice,and showed that mGSCs can colonize,self-renew,and differentiate into germ cells. Thus,we have established a goat GSC cell line and these cells could be differentiated into sperm-like cells in vivo and sperms in vitro,providing a promising platform for generation of transgenic goat for production of specific humanized proteins. View Publication

We re-examine the individual components for human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) culture and formulate a cell culture system in which all protein reagents for liquid media,attachment surfaces and splitting are chemically defined. A major improvement is the lack of a serum albumin component,as variations in either animal- or human-sourced albumin batches have previously plagued human ESC and iPSC culture with inconsistencies. Using this new medium (E8) and vitronectin-coated surfaces,we demonstrate improved derivation efficiencies of vector-free human iPSCs with an episomal approach. This simplified E8 medium should facilitate both the research use and clinical applications of human ESCs and iPSCs and their derivatives,and should be applicable to other reprogramming methods. View Publication

Nocodazole is a known destabiliser of microtubule dynamics and arrests cell-cycle at the G2/M phase. In the context of the human embryonic stem cell (hESC) it is important to understand how this arrest influences the pluripotency of cells. Here we report for the first time the changes in the expression of transcription markers Nanog and Oct4 as well as SSEA-3 and SSEA-4 in human embryonic cells after their treatment with nocodazole. Multivariate permeabilised-cell flow cytometry was applied for characterising the expression of Nanog and Oct4 during different cell cycle phases. Among untreated hESC we detected Nanog-expressing cells,which also expressed Oct4,SSEA-3 and SSEA-4. We also found another population expressing SSEA-4,but without Nanog,Oct4 and SSEA-3 expression. Nocodazole treatment resulted in a decrease of cell population positive for all four markers Nanog,Oct4,SSEA-3,SSEA-4. Nocodazole-mediated cell-cycle arrest was accompanied by higher rate of apoptosis and upregulation of p53. Twenty-four hours after the release from nocodazole block,the cell cycle of hESC normalised,but no increase in the expression of transcription markers Nanog and Oct4 was detected. In addition,the presence of ROCK-2 inhibitor Y-27632 in the medium had no effect on increasing the expression of pluripotency markers Nanog and Oct4 or decreasing apoptosis or the level of p53. The expression of SSEA-3 and SSEA-4 increased in Nanog-positive cells after wash-out of nocodazole in the presence and in the absence of Y-27632. Our data show that in hESC nocodazole reversible blocks cell cycle,which is accompanied by irreversible loss of expression of pluripotency markers Nanog and Oct4. 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 embryonic stem cells (hESCs) offer tremendous potential for not only treating neurological disorders but also for their ability to serve as vital reagents to model and investigate human disease. To further our understanding of a key protein involved in Alzheimer disease pathogenesis,we stably overexpressed amyloid precursor protein (APP) in hESCs. Remarkably,we found that APP overexpression in hESCs caused a rapid and robust differentiation of pluripotent stem cells toward a neural fate. Despite maintenance in standard hESC media,up to 80% of cells expressed the neural stem cell marker nestin,and 65% exhibited the more mature neural marker β-3 tubulin within just 5 days of passaging. To elucidate the mechanism underlying the effects of APP on neural differentiation,we examined the proteolysis of APP and performed both gain of function and loss of function experiments. Taken together,our results demonstrate that the N-terminal secreted soluble forms of APP (in particular sAPPβ) robustly drive neural differentiation of hESCs. Our findings not only reveal a novel and intriguing role for APP in neural lineage commitment but also identify a straightforward and rapid approach to generate large numbers of neurons from human embryonic stem cells. These novel APP-hESC lines represent a valuable tool to investigate the potential role of APP in development and neurodegeneration and allow for insights into physiological functions of this protein. View Publication

The use of small molecules to 'chemically direct' differentiation represents a powerful approach to promote specification of embryonic stem cells (ESCs) towards particular functional cell types for use in regenerative medicine and pharmaceutical applications. Here,we demonstrate a novel route for chemically directed differentiation of human ESCs (hESCs) into definitive endoderm (DE) exploiting a selective small-molecule inhibitor of glycogen synthase kinase 3 (GSK-3). This GSK-3 inhibitor,termed 1m,when used as the only supplement to a chemically defined feeder-free culture system,effectively promoted differentiation of ESC lines towards primitive streak (PS),mesoderm and DE. This contrasts with the role of GSK-3 in murine ESCs,where GSK-3 inhibition promotes pluripotency. Interestingly,1m-mediated induction of differentiation involved transient NODAL expression and Nodal signalling. Prolonged treatment of hESCs with 1m resulted in the generation of a population of cells displaying hepatoblast characteristics,that is expressing α-fetoprotein and HNF4α. Furthermore,1m-induced DE had the capacity to mature and generate hepatocyte-like cells capable of producing albumin. These findings describe,for the first time,the utility of GSK-3 inhibition,in a chemically directed approach,to a method of DE generation that is robust,potentially scalable and applicable to different hESC lines. View Publication

Microfluidic systems create significant opportunities to establish highly controlled microenvironmental conditions for screening pluripotent stem cell fate. However,since cell fate is crucially dependent on this microenvironment,it remains unclear as to whether continual perfusion of culture medium supports pluripotent stem cell maintenance in feeder-free,chemically defined conditions,and further,whether optimum perfusion conditions exist for subsequent use of human embryonic stem cell (hESCs) in other microfludic systems. To investigate this,we designed microbioreactors based on resistive flow to screen hESCs under a linear range of flowrates. We report that at low rates (conditions where glucose transport is convection-limited with Péclet number textless1),cells are affected by apparent nutrient depletion and waste accumulation,evidenced by reduced cell expansion and altered morphology. At higher rates,cells are spontaneously washed out,and display morphological changes which may be indicative of early-stage differentiation. However,between these thresholds exists a narrow range of flowrates in which hESCs expand comparably to the equivalent static culture system,with regular morphology and maintenance of the pluripotency marker TG30 in textgreater95% of cells over 7 days. For MEL1 hESCs the optimum flowrate also coincided with the time-averaged medium exchange rate in static cultures,which may therefore provide a good first estimate of appropriate perfusion rates. Overall,we demonstrate hESCs can be maintained in microbioreactors under continual flow for up to 7 days,a critical outcome for the future development of microbioreactor-based screening systems and assays for hESC culture. View Publication