技术资料

Recent evidence suggests human embryonic stem cell (hESC) and induced pluripotent stem (iPS) cell lines have differences in their epigenetic marks and transcriptomes,yet the impact of these differences on subsequent terminally differentiated cells is less well understood. Comparison of purified,homogeneous populations of somatic cells derived from multiple independent human iPS and ES lines will be required to address this critical question. Here,we report a differentiation protocol based on embryonic development that consistently yields large numbers of endothelial cells (ECs) derived from multiple hESCs or iPS cells. Mesoderm differentiation of embryoid bodies was maximized,and defined growth factors were used to generate KDR+ EC progenitors. Magnetic purification of a KDR+ progenitor subpopulation resulted in an expanding,homogeneous pool of ECs that expressed EC markers and had functional properties of ECs. Comparison of the transcriptomes revealed limited gene expression variability between multiple lines of human iPS-derived ECs or between lines of ES- and iPS-derived ECs. These results demonstrate a method to generate large numbers of pure human EC progenitors and differentiated ECs from pluripotent stem cells and suggest individual lineages derived from human iPS cells may have significantly less variance than their pluripotent founders. STEM Cells2013;31:92–103 View Publication

The Hippo pathway is crucial in organ size control,and its dysregulation contributes to tumorigenesis. However,upstream signals that regulate the mammalian Hippo pathway have remained elusive. Here,we report that the Hippo pathway is regulated by G-protein-coupled receptor (GPCR) signaling. Serum-borne lysophosphatidic acid (LPA) and sphingosine 1-phosphophate (S1P) act through G12/13-coupled receptors to inhibit the Hippo pathway kinases Lats1/2,thereby activating YAP and TAZ transcription coactivators,which are oncoproteins repressed by Lats1/2. YAP and TAZ are involved in LPA-induced gene expression,cell migration,and proliferation. In contrast,stimulation of Gs-coupled receptors by glucagon or epinephrine activates Lats1/2 kinase activity,thereby inhibiting YAP function. Thus,GPCR signaling can either activate or inhibit the Hippo-YAP pathway depending on the coupled G protein. Our study identifies extracellular diffusible signals that modulate the Hippo pathway and also establishes the Hippo-YAP pathway as a critical signaling branch downstream of GPCR. View Publication

Cell Research 23,122 (2013). doi:10.1038/cr.2012.119 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

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

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

Trisomy 21 is associated with hematopoietic abnormalities in the fetal liver,a preleukemic condition termed transient myeloproliferative disorder,and increased incidence of acute megakaryoblastic leukemia. Human trisomy 21 pluripotent cells of various origins,human embryonic stem (hES),and induced pluripotent stem (iPS) cells,were differentiated in vitro as a model to recapitulate the effects of trisomy on hematopoiesis. To mitigate clonal variation,we isolated disomic and trisomic subclones from the same parental iPS line,thereby generating subclones isogenic except for chromosome 21. Under differentiation conditions favoring development of fetal liver-like,γ-globin expressing,definitive hematopoiesis,we found that trisomic cells of hES,iPS,or isogenic origins exhibited a two- to fivefold increase in a population of CD43(+)(Leukosialin)/CD235(+)(Glycophorin A) hematopoietic cells,accompanied by increased multilineage colony-forming potential in colony-forming assays. These findings establish an intrinsic disturbance of multilineage myeloid hematopoiesis in trisomy 21 at the fetal liver stage. View Publication

AIM: Commercial regenerative medicine will require large quantities of clinical-specification human cells. The cost and quality of manufacture is notoriously difficult to control due to highly complex processes with poorly defined tolerances. As a step to overcome this,we aimed to demonstrate the use of 'quality-by-design' tools to define the operating space for economic passage of a scalable human embryonic stem cell production method with minimal cell loss. MATERIALS & METHODS: Design of experiments response surface methodology was applied to generate empirical models to predict optimal operating conditions for a unit of manufacture of a previously developed automatable and scalable human embryonic stem cell production method. RESULTS & CONCLUSION: Two models were defined to predict cell yield and cell recovery rate postpassage,in terms of the predictor variables of media volume,cell seeding density,media exchange and length of passage. Predicted operating conditions for maximized productivity were successfully validated. Such 'quality-by-design' type approaches to process design and optimization will be essential to reduce the risk of product failure and patient harm,and to build regulatory confidence in cell therapy manufacturing processes. View Publication

Functional endothelial-like cells (EC) have been successfully derived from different cell sources and potentially used for treatment of cardiovascular diseases; however,their relative therapeutic efficacy remains unclear. We differentiated functional EC from human bone marrow mononuclear cells (BM-EC),human embryonic stem cells (hESC-EC) and human induced pluripotent stem cells (hiPSC-EC),and compared their in-vitro tube formation,migration and cytokine expression profiles,and in-vivo capacity to attenuate hind-limb ischemia in mice. Successful differentiation of BM-EC was only achieved in 1/6 patient with severe coronary artery disease. Nevertheless,BM-EC,hESC-EC and hiPSC-EC exhibited typical cobblestone morphology,had the ability of uptaking DiI-labeled acetylated low-density-lipoprotein,and binding of Ulex europaeus lectin. In-vitro functional assay demonstrated that hiPSC-EC and hESC-EC had similar capacity for tube formation and migration as human umbilical cord endothelial cells (HUVEC) and BM-EC (Ptextgreater0.05). While increased expression of major angiogenic factors including epidermal growth factor,hepatocyte growth factor,vascular endothelial growth factor,placental growth factor and stromal derived factor-1 were observed in all EC cultures during hypoxia compared with normoxia (Ptextless0.05),the magnitudes of cytokine up-regulation upon hypoxic were more dramatic in hiPSC-EC and hESC-EC (Ptextless0.05). Compared with medium,transplanting BM-EC (n = 6),HUVEC (n = 6),hESC-EC (n = 8) or hiPSC-EC (n = 8) significantly attenuated severe hind-limb ischemia in mice via enhancement of neovascularization. In conclusion,functional EC can be generated from hECS and hiPSC with similar therapeutic efficacy for attenuation of severe hind-limb ischemia. Differentiation of functional BM-EC was more difficult to achieve in patients with cardiovascular diseases,and hESC-EC or iPSC-EC are readily available as off-the-shelf" format for the treatment of tissue ischemia." View Publication

Faithful execution of developmental gene expression programs occurs at multiple levels and involves many different components such as transcription factors,histone-modification enzymes,and mRNA processing proteins. Recent evidence suggests that nucleoporins,well known components that control nucleo-cytoplasmic trafficking,have wide-ranging functions in developmental gene regulation that potentially extend beyond their role in nuclear transport. Whether the unexpected role of nuclear pore proteins in transcription regulation,which initially has been described in fungi and flies,also applies to human cells is unknown. Here we show at a genome-wide level that the nuclear pore protein NUP98 associates with developmentally regulated genes active during human embryonic stem cell differentiation. Overexpression of a dominant negative fragment of NUP98 levels decreases expression levels of NUP98-bound genes. In addition,we identify two modes of developmental gene regulation by NUP98 that are differentiated by the spatial localization of NUP98 target genes. Genes in the initial stage of developmental induction can associate with NUP98 that is embedded in the nuclear pores at the nuclear periphery. Alternatively,genes that are highly induced can interact with NUP98 in the nuclear interior,away from the nuclear pores. This work demonstrates for the first time that NUP98 dynamically associates with the human genome during differentiation,revealing a role of a nuclear pore protein in regulating developmental gene expression programs. View Publication

BACKGROUND: Fragile X syndrome (FXS) is a common form of inherited intellectual disability caused by an expansion of CGG repeats located in the 5' untranslated region (UTR) of the FMR1 gene,which leads to hypermethylation and silencing of this locus. Although a dramatic increase in DNA methylation of the FMR1 full mutation allele is well documented,the extent to which these changes affect DNA methylation throughout the rest of the genome has gone unexplored. METHODS: Here we examined genome-wide methylation in both peripheral blood (N = 62) and induced pluripotent stem cells (iPSCs; N = 10) from FXS individuals and controls. RESULTS: We not only found the expected significant DNA methylation differences in the FMR1 promoter and 5' UTR,we also saw that these changes inverse in the FMR1 gene body. Importantly,we found no other differentially methylated loci throughout the remainder of the genome,indicating the aberrant methylation of FMR1 in FXS is locus-specific. CONCLUSIONS: This study provides a comprehensive methylation profile of FXS and helps refine our understanding of the mechanisms behind FMR1 silencing. View Publication

The unique ability of Sox2 to cooperate with Oct4 at selective binding sites in the genome is critical for reprogramming somatic cells into induced pluripotent stem cells (iPSCs). We have recently demonstrated that Sox17 can be converted into a reprogramming factor by alteration of a single amino acid (Sox17EK) within its DNA binding HMG domain. Here we expanded this study by introducing analogous mutations to 10 other Sox proteins and interrogated the role of N-and C-termini on the reprogramming efficiency. We found that point-mutated Sox7 and Sox17 can convert human and mouse fibroblasts into iPSCs,but Sox4,Sox5,Sox6,Sox8,Sox9,Sox11,Sox12,Sox13,and Sox18 cannot. Next we studied regions outside the HMG domain and found that the C-terminal transactivation domain of Sox17 and Sox7 enhances the potency of Sox2 in iPSC assays and confers weak reprogramming potential to the otherwise inactive Sox4EK and Sox18EK proteins. These results suggest that the glutamate (E) to lysine (K) mutation in the HMG domain is necessary but insufficient to swap the function of Sox factors. Moreover,the HMG domain alone fused to the VP16 transactivation domain is able to induce reprogramming,albeit at low efficiency. By molecular dissection of the C-terminus of Sox17,we found that the β-catenin interaction region contributes to the enhanced reprogramming efficiency of Sox17EK. To mechanistically understand the enhanced reprogramming potential of Sox17EK,we analyzed ChIP-sequencing and expression data and identified a subset of candidate genes specifically regulated by Sox17EK and not by Sox2. View Publication