技术资料

Fibrodysplasia ossificans progressiva (FOP) is a rare and intractable disease characterized by extraskeletal bone formation through endochondral ossification. Patients with FOP harbor point mutations in ACVR1,a type I receptor for BMPs. Although mutated ACVR1 (FOP-ACVR1) has been shown to render hyperactivity in BMP signaling,we and others have uncovered a mechanism by which FOP-ACVR1 mistransduces BMP signaling in response to Activin-A,a molecule that normally transduces TGF-β signaling. Although Activin-A evokes enhanced chondrogenesis in vitro and heterotopic ossification (HO) in vivo,the underlying mechanisms have yet to be revealed. To this end,we developed a high-throughput screening (HTS) system using FOP patient-derived induced pluripotent stem cells (FOP-iPSCs) to identify pivotal pathways in enhanced chondrogenesis that are initiated by Activin-A. In a screen of 6,809 small-molecule compounds,we identified mTOR signaling as a critical pathway for the aberrant chondrogenesis of mesenchymal stromal cells derived from FOP-iPSCs (FOP-iMSCs). Two different HO mouse models,an FOP model mouse expressing FOP-ACVR1 and an FOP-iPSC-based HO model mouse,revealed critical roles for mTOR signaling in vivo. Moreover,we identified ENPP2,an enzyme that generates lysophosphatidic acid,as a linker of FOP-ACVR1 and mTOR signaling in chondrogenesis. These results uncovered the crucial role of the Activin-A/FOP-ACVR1/ENPP2/mTOR axis in FOP pathogenesis. View Publication

This report describes the establishment directly from normal preimplantation mouse embryos of a cell line that forms teratocarcinomas when injected into mice. The pluripotency of these embryonic stem cells was demonstrated conclusively by the observation that subclonal cultures,derived from isolated single cells,can differentiate into a wide variety of cell types. Such embryonic stem cells were isolated from inner cell masses of late blastocysts cultured in medium conditioned by an established teratocarcinoma stem cell line. This suggests that such conditioned medium might contain a growth factor that stimulates the proliferation or inhibits the differentiation of normal pluripotent embryonic cells,or both. This method of obtaining embryonic stem cells makes feasible the isolation of pluripotent cells lines from various types of noninbred embryo,including those carrying mutant genes. The availability of such cell lines should made possible new approaches to the study of early mammalian development. View Publication

Under appropriate conditions in culture,embryonic stem cells will differentiate and form embryoid bodies that have been shown to contain cells of the hematopoietic,endothelial,muscle and neuronal lineages. Many aspects of the lineage-specific differentiation programs observed within the embryoid bodies reflect those found in the embryo,indicating that this model system provides access to early cell populations that develop in a normal fashion. Recent studies involving the differentiation of genetically altered embryonic stem cells highlight the potential of this in vitro differentiation system for defining the function of genes in early development. View Publication

The available techniques for directed gene manipulation in the mouse are unprecedented in any multicellular organism and make the mouse an invaluable tool for unraveling all aspects of mammalian biology. To realize fully the potential of these genetic tools requires that phenotypic analysis be efficient,rapid,and complete. Genetic chimeras and mosaics,in which mutant cells are mixed with wild-type cells,can be used to augment standard analysis of intact mutant animals and alleviate the time required and the expense involved in generating and maintaining multiple strains of mutant mice. View Publication

Evi1 (ecotropic viral integration site 1) is essential for proliferation of hematopoietic stem cells and implicated in the development of myeloid disorders. Particularly,high Evi1 expression defines one of the largest clusters in acute myeloid leukemia and is significantly associated with extremely poor prognosis. However,mechanistic basis of Evi1-mediated leukemogenesis has not been fully elucidated. Here,we show that Evi1 directly represses phosphatase and tensin homologue deleted on chromosome 10 (PTEN) transcription in the murine bone marrow,which leads to activation of AKT/mammalian target of rapamycin (mTOR) signaling. In a murine bone marrow transplantation model,Evi1 leukemia showed modestly increased sensitivity to an mTOR inhibitor rapamycin. Furthermore,we found that Evi1 binds to several polycomb group proteins and recruits polycomb repressive complexes for PTEN down-regulation,which shows a novel epigenetic mechanism of AKT/mTOR activation in leukemia. Expression analyses and ChIPassays with human samples indicate that our findings in mice models are recapitulated in human leukemic cells. Dependence of Evi1-expressing leukemic cells on AKT/mTOR signaling provides the first example of targeted therapeutic modalities that suppress the leukemogenic activity of Evi1. The PTEN/AKT/mTOR signaling pathway and the Evi1-polycomb interaction can be promising therapeutic targets for leukemia with activated Evi1. View Publication

Since the derivation of the first human embryonic stem cell (hESC) lines by Thomson and coworkers in 1998,more than 1,200 different hESC lines have been established worldwide. Nevertheless,there is still a recognized interest in the establishment of new lines of hESC,particularly from HLA types and ethnic groups currently underrepresented among the available lines. The methodology of hESC derivation has evolved significantly since 1998,when human LIF (hLIF) was used for maintenance of pluripotency. However,there are a number of different strategies for the several steps involved in establishing a new line of hESC. Here we make a survey of the most relevant parameters used between 1998 and 2010 for the derivation of the 375 hESC lines deposited in two international stem cell registries,and able to form teratomas in immunocompromised mice. Although we identify some trends in the methodology for establishing hESC lines,our data reveal a much greater heterogeneity of strategies than what is used for derivation of murine ESC lines,indicating that optimum conditions have not been consolidated yet,and thus,hESC establishment is still an evolving field of research. View Publication

Stem cells hold great promise for therapies aimed at regenerating damaged tissue,drug screening and studying in vitro models of human disease. However,many challenges remain before these applications can become a reality. One such challenge is developing chemically defined and scalable culture conditions for derivation and expansion of clinically viable human pluripotent stem cells,as well as controlling their differentiation with high specificity. Interaction of stem cells with their extracellular microenvironment plays an important role in determining their differentiation commitment and functions. Regenerative medicine approaches integrating cell-matrix and cell-cell interactions,and soluble factors could lead to development of robust microenvironments to control various cellular responses. Indeed,several of these recent developments have provided significant insight into the design of microenvironments that can elicit the targeted cellular response. In this article,we will focus on some of these developments with an emphasis on matrix-mediated expansion of human pluripotent stem cells while maintaining their pluripotency. We will also discuss the role of matrix-based cues and cell-cell interactions in the form of soluble signals in directing stem cell differentiation into musculoskeletal lineages. View Publication

We have developed a synthetic polymer interface for the long-term self-renewal of human embryonic stem cells (hESCs) in defined media. We successfully cultured hESCs on hydrogel interfaces of aminopropylmethacrylamide (APMAAm) for over 20 passages in chemically-defined mTeSR™1 media and demonstrated pluripotency of multiple hESC lines with immunostaining and quantitative RT-PCR studies. Results for hESC proliferation and pluripotency markers were both qualitatively and quantitatively similar to cells cultured on Matrigel™-coated substrates. Mechanistically,it was resolved that bovine serum albumin (BSA) in the mTeSR™1 media was critical for cell adhesion on APMAAm hydrogel interfaces. This study uniquely identified a robust long-term culture surface for the self-renewal of hESCs without the use of biologic coatings (e.g.,peptides,proteins,or Matrigel™) in completely chemically-defined media that employed practical culturing techniques amenable to clinical-scale cell expansion. View Publication

Rett syndrome (RTT) is a progressive neurologic disorder representing one of the most common causes of mental retardation in females. To date mutations in three genes have been associated with this condition. Classic RTT is caused by mutations in the MECP2 gene,whereas variants can be due to mutations in either MECP2 or FOXG1 or CDKL5. Mutations in CDKL5 have been identified both in females with the early onset seizure variant of RTT and in males with X-linked epileptic encephalopathy. CDKL5 is a kinase protein highly expressed in neurons,but its exact function inside the cell is unknown. To address this issue we established a human cellular model for CDKL5-related disease using the recently developed technology of induced pluripotent stem cells (iPSCs). iPSCs can be expanded indefinitely and differentiated in vitro into many different cell types,including neurons. These features make them the ideal tool to study disease mechanisms directly on the primarily affected neuronal cells. We derived iPSCs from fibroblasts of one female with p.Q347X and one male with p.T288I mutation,affected by early onset seizure variant and X-linked epileptic encephalopathy,respectively. We demonstrated that female CDKL5-mutated iPSCs maintain X-chromosome inactivation and clones express either the mutant CDKL5 allele or the wild-type allele that serve as an ideal experimental control. Array CGH indicates normal isogenic molecular karyotypes without detection of de novo CNVs in the CDKL5-mutated iPSCs. Furthermore,the iPS cells can be differentiated into neurons and are thus suitable to model disease pathogenesis in vitro. View Publication

Long non-coding RNAs (lncRNAs) are a numerous class of newly discovered genes in the human genome,which have been proposed to be key regulators of biological processes,including stem cell pluripotency and neurogenesis. However,at present very little functional characterization of lncRNAs in human differentiation has been carried out. In the present study,we address this using human embryonic stem cells (hESCs) as a paradigm for pluripotency and neuronal differentiation. With a newly developed method,hESCs were robustly and efficiently differentiated into neurons,and we profiled the expression of thousands of lncRNAs using a custom-designed microarray. Some hESC-specific lncRNAs involved in pluripotency maintenance were identified,and shown to physically interact with SOX2,and PRC2 complex component,SUZ12. Using a similar approach,we identified lncRNAs required for neurogenesis. Knockdown studies indicated that loss of any of these lncRNAs blocked neurogenesis,and immunoprecipitation studies revealed physical association with REST and SUZ12. This study indicates that lncRNAs are important regulators of pluripotency and neurogenesis,and represents important evidence for an indispensable role of lncRNAs in human brain development. View Publication

We present a method to perform absolute quantification of glycogen in human embryonic stem cells (hESCs) in situ based on the use of Raman microspectroscopy. The proposed quantification method was validated by comparison to a commonly used commercial glycogen assay kit. With Raman microspectroscopy,we could obtain the glycogen content of hESCs faster and apparently more accurately than with the kit. In addition,glycogen distributions across a colony could be obtained. Raman spectroscopy can provide reliable estimates of the in situ glycogen content in hESCs,and this approach should also be extensible to their other biochemical constituents as well as to other cell types. View Publication

BACKGROUND: Human pluripotent stem cells have the ability to generate all cell types present in the adult organism,therefore harboring great potential for the in vitro study of differentiation and for the development of cell-based therapies. Nonetheless their use may prove challenging as incomplete differentiation of these cells might lead to tumoregenicity. Interestingly,many cancer types have been reported to display metabolic modifications with features that might be similar to stem cells. Understanding the metabolic properties of human pluripotent stem cells when compared to their differentiated counterparts can thus be of crucial importance. Furthermore recent data has stressed distinct features of different human pluripotent cells lines,namely when comparing embryo-derived human embryonic stem cells (hESCs) and induced pluripotent stem cells (IPSCs) reprogrammed from somatic cells.backslashnbackslashnMETHODOLOGY/PRINCIPAL FINDINGS: We compared the energy metabolism of hESCs,IPSCs,and their somatic counterparts. Focusing on mitochondria,we tracked organelle localization and morphology. Furthermore we performed gene expression analysis of several pathways related to the glucose metabolism,including glycolysis,the pentose phosphate pathway and the tricarboxylic acid (TCA) cycle. In addition we determined oxygen consumption rates (OCR) using a metabolic extracellular flux analyzer,as well as total intracellular ATP levels by high performance liquid chromatography (HPLC). Finally we explored the expression of key proteins involved in the regulation of glucose metabolism.backslashnbackslashnCONCLUSIONS/FINDINGS: Our results demonstrate that,although the metabolic signature of IPSCs is not identical to that of hESCs,nonetheless they cluster with hESCs rather than with their somatic counterparts. ATP levels,lactate production and OCR revealed that human pluripotent cells rely mostly on glycolysis to meet their energy demands. Furthermore,our work points to some of the strategies which human pluripotent stem cells may use to maintain high glycolytic rates,such as high levels of hexokinase II and inactive pyruvate dehydrogenase (PDH). View Publication