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ABSTRACT: Previously we reported that feeders formed from human placental fibroblasts (hPFs) support derivation and long-term self-renewal of human embryonic stem cells (hESCs) under serum-free conditions. Here, we show, using antibody array and ELISA platforms, that hPFs secrete ∼6-fold higher amounts of the CXC-type chemokine, GROα, than IMR 90, a human lung fibroblast line, which does not support hESC growth. Furthermore, immunocytochemistry and immunoblot approaches revealed that hESCs express CXCR, a GROα receptor. We used this information to develop defined culture medium for feeder-free propagation of hESCs in an undifferentiated state. Cells passaged as small aggregates and maintained in the GROα-containing medium had a normal karyotype, expressed pluripotency markers, and exhibited apical-basal polarity, i.e., had the defining features of pluripotent hESCs. They also differentiated into the three primary (embryonic) germ layers and formed teratomas in immunocompromised mice. hESCs cultured as single cells in the GROα-containing medium also had a normal karyotype, but they downregulated markers of pluripotency, lost apical-basal polarity, and expressed markers that are indicative of the early stages of neuronal differentiation-βIII tubulin, vimentin, radial glial protein, and nestin. These data support our hypothesis that establishing and maintaining cell polarity is essential for the long-term propagation of hESCs in an undifferentiated state and that disruption of cell-cell contacts can trigger adoption of a neuronal fate.
Differentiation 03/2011; 81(4):222-32. · 2.81 Impact Factor
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ABSTRACT: We have derived hESC from biopsied blastomeres of cleavage stage embryos under virtually the same conditions we used for the derivation of hESC lines from inner cell mass of blastocyst stage embryos. Blastomere-derived hESC lines exhibited all the standard characteristics of hESC including undifferentiated proliferation, genomic stability, expression of pluripotency markers and the ability to differentiate into the cells of all three germ layers both in vitro and in vivo. To examine whether hESC lines derived from two developmental stages of the embryo differ in gene expression, we have subjected three blastomere-derived hESC lines and two ICM-derived hESC lines grown under identical culture conditions to transcriptome analysis using gene expression arrays. Unlike previously reported comparisons of hESC lines which demonstrated, apart from core hESC-associated pluripotency signature, significant variations in gene expression profiles of different lines, our data show that hESC lines derived and grown under well-controlled defined culture conditions adopt nearly identical gene expression profiles. Moreover, blastomere-derived and ICM-derived hESC exhibited very similar transcriptional profiles independent of the developmental stage of the embryo from which they originated. Furthermore, this profile was evident in very early passages of the cells and did not appear to be affected by extensive passaging. These results suggest that during derivation process cells which give rise to hESC acquire virtually identical stable phenotype and are not affected by the developmental stage of the starting cell population.
PLoS ONE 01/2011; 6(10):e26570. · 4.09 Impact Factor
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ABSTRACT: The success rate of human embryonic stem cell (hESC) derivation depends on both culture conditions and embryo quality and is routinely determined by morphological criteria. However, high incidence of chromosomal abnormality even in high-grade cleavage embryos from in vitro fertilization (IVF) patients suggests that the morphological grade of supernumerary embryos obtained from IVF clinics may not be a good prediction factor for successful hESC derivation. We show here that from one donor under identical derivation conditions 12 karyotypically abnormal post-bioptic embryos did not yield hESC lines, whereas two out of four normal embryos did. This suggests that the capacity of embryos to give rise to hESC line is likely to be influenced by their genetic status.
Stem cells and development 07/2009; 19(1):39-46. · 4.15 Impact Factor
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ABSTRACT: Reproductive toxicity encompasses harmful effects of various agents on all aspects and stages of the reproductive cycle, including infertility and the induction of adverse effects in the embryo/fetus. In developing a model for reproductive toxicity screening, it is important to define the stage of the human reproductive cycle that this specific model is going to recreate in vitro and to identify molecular targets that are critical for this stage of development. In this review, we focus our discussion on modeling pre-implantation embryotoxicity. The rationale for this is that despite advances on both clinical and biological levels, many unresolved infertility cases may be due to our lack of knowledge regarding environmental influences on this short, but critical stage of development. Data from in vitro fertilization practice suggest that the early-dividing embryo is very sensitive to numerous factors present in its microenvironment. In vivo, as the embryo travels down the oviduct, physical or chemical insults can directly damage the embryo and/or prevent implantation, and cause infertility. Multiple lines of evidence point to the differences between mouse and human pre-implantation development and between mouse and human embryonic stem cells (hESCs). In light of these data we present the case that hESCs and their derivatives are better suited as in vitro models for human pre-implantation development than their mouse counterparts. We then describe some of the most promising hESC-based systems that are used today to model certain aspects of development in the human pre-implantation embryo and that have the potential to be used for embryo toxicity screening tests in the near future. Described systems model two major events during differentiation of the human pre-implantation embryo: differentiation of the trophectoderm and segregation of the inner cell mass into epiblast and hypoblast. The first event is replicated in vitro by triggering either direct or indirect (through embryoid body stage) differentiation into trophectoderm. The second event can be modeled using the recently described system of high-throughput generation of embryoid bodies that recapitulate segregation of inner cell mass. We conclude by discussing the potential of these existing models in toxicology studies and the possibilities for their improvement in the future.
Regenerative Medicine 06/2009; 4(3):449-59. · 3.72 Impact Factor
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ABSTRACT: In a continuous effort to improve the generation of therapeutic grade human embryonic stem cell (hESC) lines, we focused on preserving developmental capacity of the embryos, minimizing the exposure to xenomaterials, increasing derivation efficacy, and reducing the complexity of the derivation procedure. In this study, we describe an improved method for efficient derivation of hESC lines from blastomeres of biopsied embryos. Our protocol substituted feeder cells of mouse origin with human foreskin fibroblasts (HFFs), limited serum exposure of cells to formation of the initial outgrowth, and increased derivation efficacy from 12.5% (one hESC line out of 13 biopsies) to 50% (3 out of 6 biopsies) by using early population doubling (PD) HFFs. In addition, it eliminated a need for embryo-blastomere coculture, thus reducing the complexity of the culture and enabling continued development of the biopsied embryo under optimal conditions. All derived lines maintained normal karyotype and expressed totipotent phenotype including the ability to differentiate into trophectoderm and all three germ layers.
Stem cells and development 03/2009; 18(9):1343-50. · 4.15 Impact Factor
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ABSTRACT: It is often difficult to determine molecular mechanisms leading to early embryonic lethality of genetically modified mice due to lack of cells for further analyses. The authors describe here establishment of mouse embryonic fibroblast (MEF) cell lines from gastrulation stage embryos. In this example, using a combination of in vivo and in vitro techniques, the authors successfully generated MEF cell lines that lack both fibronectin (FN) and focal adhesion kinase (FAK).
Cell Communication & Adhesion 12/2008; 15(4):379-83. · 1.18 Impact Factor
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ABSTRACT: Ubiquitously expressed focal adhesion kinase (FAK), a critical component in transducing signals from sites of cell contacts with extracellular matrix, was named after its typical localization in focal adhesions. A nuclear localization of FAK has been also reported and its scaffolding role in nucleus and requirement for p53 ubiquitination were only recently described. Whereas FAK nuclear localization signal (NLS) was found in F2 lobe of FERM domain, nuclear export signal (NES) sequences have not been yet determined. Here we demonstrate that FAK has two NES sequences, NES1 in F1 lobe of FERM domain and NES2 in kinase domain. Although, both NES1 and NES2 are evolutionary conserved, and present as well in FAK-related protein kinase Pyk2, only NES2 demonstrates full biological nuclear export activity.
FEBS Letters 08/2008; 582(16):2402-6. · 3.54 Impact Factor
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ABSTRACT: The first human embryonic stem cell lines (hESCs) were derived using mouse embryonic fibroblasts as feeder cells. In attempts to replace mouse embryonic fibroblasts with feeders of human origin, irradiated human placental fibroblasts were successfully used as feeder cells for the derivation and propagation of hESCs. Here we describe a protocol for the isolation and expansion of fibroblasts from placental villous stroma. We include a description of placental architecture to provide the background for a stepwise tissue digestion that leads to the isolation of villous stroma. Villous stroma from the first trimester tissue is different from term placenta and contains mesenchymal, fibroblast-like cells, only a few blood vessels, and a network of matrix fibers. The fibroblasts isolated from a single placenta of 6- to 8-weeks gestation proliferate rapidly and retain the ability to support hESC growth between passage doubling (PD) 8 and PD 12.
Current protocols in stem cell biology 07/2008; Chapter 1:Unit 1C.6.
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Dusko Ilic
Regenerative Medicine 04/2008; 3(2):137-43. · 3.72 Impact Factor
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Young Chung,
Irina Klimanskaya,
Sandy Becker,
Tong Li,
Marc Maserati,
Shi-Jiang Lu,
Tamara Zdravkovic, Dusko Ilic,
Olga Genbacev,
Susan Fisher,
Ana Krtolica,
Robert Lanza
Cell stem cell 03/2008; 2(2):113-7. · 23.56 Impact Factor
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Yangmi Lim,
Ssang-Taek Lim,
Alok Tomar,
Margaret Gardel,
Joie A Bernard-Trifilo,
Xiao Lei Chen,
Sean A Uryu,
Rafaela Canete-Soler,
Jinbin Zhai,
Hong Lin,
William W Schlaepfer,
Perihan Nalbant,
Gary Bokoch, Dusko Ilic,
Clare Waterman-Storer,
David D Schlaepfer
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ABSTRACT: Integrin binding to matrix proteins such as fibronectin (FN) leads to formation of focal adhesion (FA) cellular contact sites that regulate migration. RhoA GTPases facilitate FA formation, yet FA-associated RhoA-specific guanine nucleotide exchange factors (GEFs) remain unknown. Here, we show that proline-rich kinase-2 (Pyk2) levels increase upon loss of focal adhesion kinase (FAK) in mouse embryonic fibroblasts (MEFs). Additionally, we demonstrate that Pyk2 facilitates deregulated RhoA activation, elevated FA formation, and enhanced cell proliferation by promoting p190RhoGEF expression. In normal MEFs, p190RhoGEF knockdown inhibits FN-associated RhoA activation, FA formation, and cell migration. Knockdown of p190RhoGEF-related GEFH1 does not affect FA formation in FAK(-/-) or normal MEFs. p190RhoGEF overexpression enhances RhoA activation and FA formation in MEFs dependent on FAK binding and associated with p190RhoGEF FA recruitment and tyrosine phosphorylation. These studies elucidate a compensatory function for Pyk2 upon FAK loss and identify the FAK-p190RhoGEF complex as an important integrin-proximal regulator of FA formation during FN-stimulated cell motility.
The Journal of Cell Biology 02/2008; 180(1):187-203. · 10.26 Impact Factor
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ABSTRACT: FAK is known as an integrin- and growth factor-associated tyrosine kinase promoting cell motility. Here we show that, during mouse development, FAK inactivation results in p53- and p21-dependent mesodermal cell growth arrest. Reconstitution of primary FAK-/-p21-/- fibroblasts revealed that FAK, in a kinase-independent manner, facilitates p53 turnover via enhanced Mdm2-dependent p53 ubiquitination. p53 inactivation by FAK required FAK FERM F1 lobe binding to p53, FERM F2 lobe-mediated nuclear localization, and FERM F3 lobe for connections to Mdm2 and proteasomal degradation. Staurosporine or loss of cell adhesion enhanced FERM-dependent FAK nuclear accumulation. In primary human cells, FAK knockdown raised p53-p21 levels and slowed cell proliferation but did not cause apoptosis. Notably, FAK knockdown plus cisplatin triggered p53-dependent cell apoptosis, which was rescued by either full-length FAK or FAK FERM re-expression. These studies define a scaffolding role for nuclear FAK in facilitating cell survival through enhanced p53 degradation under conditions of cellular stress.
Molecular Cell 02/2008; 29(1):9-22. · 14.18 Impact Factor
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Dusko Ilic,
Man Mao-Qiang,
Debra Crumrine,
Gregory Dolganov,
Nicholas Larocque,
Pu Xu,
Marianne Demerjian,
Barbara E Brown,
Ssang-Taek Lim,
Valeria Ossovskaya,
David D Schlaepfer,
Susan J Fisher,
Kenneth R Feingold,
Peter M Elias,
Theodora M Mauro
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ABSTRACT: Ubiquitously expressed focal adhesion kinase (FAK), linked to multiple intracellular signaling pathways, has previously been shown to control cell motility, invasion, proliferation, and survival. Using mice with a keratinocyte-restricted deletion of fak (FAK(K5 KO)), we report here a novel role for FAK: maintenance of adult epidermal permeability barrier homeostasis. Abundant lacunae of unprocessed lipids in stratum corneum (SC) of FAK(K5 KO) mice and delayed barrier recovery pointed to malfunction of pH-dependent enzymes active in extracellular space of SC. Measuring the SC pH gradient showed significantly more neutral pH values in FAK(K5 KO) mice, suggesting the importance of FAK for acidification. Moreover, normal functions were restored when FAK(K5 KO) mice were exposed to a surface pH typical of mouse SC (pH = 5.5). Baseline levels and response to barrier disruption of secretory phospholipase A2 isoforms, enzymes that mediate generation of free fatty acids in epidermis, appeared similar in both FAK(K5 KO) and control littermates. We found that the critical SC acidification regulator Na(+)/H(+) exchanger 1 failed to localize to the plasma membrane in FAK-deficient keratinocytes both in vivo and in vitro. Thus, for plasma membrane localization in terminally differentiated keratinocytes, Na(+)/H(+) exchanger 1 requires an intact actin cytoskeleton, which is impaired in FAK-deficient cells.
American Journal Of Pathology 07/2007; 170(6):2055-67. · 4.89 Impact Factor
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ABSTRACT: This unit describes protocols for culturing human embryos and deriving human embryonic stem cells from the intact blastocyst. Description of the culturing begins with methods for obtaining human blastocysts using pronuclear or cleavage stage embryos left over after in vitro fertilization. Then there is a description of methods that can be used to derive human embryonic stem cell lines from the blastocyst without trophectoderm removal. Also included is a discussion of the critical steps and parameters such as zona pellucida removal, embryo quality assessment, feeder selection, when and how to transfer early embryonic outgrowths. In addition, there are protocols for embryo thawing, seeding of feeder cells, gelatin coating of plates, cleavage and blastocyst stage embryo grading, preparation and storage of reagents and solutions. Finally, there is a discussion of alternative derivation approaches as well as the timeline for the procedures.
Current protocols in stem cell biology 07/2007; Chapter 1:Unit 1A.2.
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ABSTRACT: Integrins are a family of heterodimeric alpha/beta transmembrane cell adhesion receptors that play important roles in the regulation of cell migration, proliferation, and survival. Integrins do not possess intrinsic catalytic activity, and signaling events are mediated by their lateral association with other cell surface receptors or clustering of their cytoplasmic domains with signaling proteins. Rapid activation of protein-tyrosine kinases is one of the first signaling events associated with integrin binding to the extracellular matrix protein fibronectin. The intracellular focal adhesion kinase (FAK) is recruited to sites of integrin clustering, and this unit describes the methods with which to analyze FAK phosphorylation, activity, and localization within fibroblasts. Additional methods on how to grow primary FAK+/+ and FAK-/- fibroblasts and measure integrin-stimulated cell motility are described as well as methods for evaluating the activity of the FAK-related kinase, Pyk2, which is expressed in FAK-/- cells.
Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.] 05/2006; Chapter 14:Unit 14.7.
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03/2006: pages 14.7.1 - 14.7.35; , ISBN: 9780471143031
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Dusko Ilic
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ABSTRACT: Among the major obstacles impeding successful derivation and continuous culture of human embryonic stem cells (hESC) for therapeutic purposes, are the presence of feeder cells and feeder-conditioned media of animal origin. The risk of contamination with xenopathogens makes hESC cultured in this way unsafe for future use in regenerative medicine. A holy grail for investigators in the field will be to establish and maintain new hESC lines in completely feeder-free and serum-free defined conditions. Recently, propagation of hESC has become possible, using mammalian- or human-derived extracellular matrix (ECM) and conditioned medium from feeder cells. In addition, providing a three-dimensional ECM environment can even support the derivation of new hESC. In this review, we examine recent advances in the use and development of substrates suitable for the derivation and maintenance of hESC, and our current understanding of the effects of a three-dimensional ECM milieu on cellular behavior.
Regenerative Medicine 02/2006; 1(1):95-101. · 3.72 Impact Factor
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ABSTRACT: The fibronectin binding integrins alpha5beta1 and alpha4beta1 generate signals pivotal for cell migration through distinct yet undefined mechanisms. For alpha5beta1, beta1-mediated activation of focal adhesion kinase (FAK) promotes c-Src recruitment to FAK and the formation of a FAK-Src signaling complex. Herein, we show that FAK expression is essential for alpha5beta1-stimulated cell motility and that exogenous expression of human alpha4 in FAK-null fibroblasts forms a functional alpha4beta1 receptor that promotes robust cell motility equal to the alpha5beta1 stimulation of wild-type and FAK-reconstituted fibroblasts. alpha4beta1-stimulated FAK-null cell spreading and motility were dependent on the integrity of the alpha4 cytoplasmic domain, independent of direct paxillin binding to alpha4, and were not affected by PRNK expression, a dominant-negative inhibitor of Pyk2. alpha4 cytoplasmic domain-initiated signaling led to a approximately 4-fold activation of c-Src which did not require paxillin binding to alpha4. Notably, alpha4-stimulated cell motility was inhibited by catalytically inactive receptor protein-tyrosine phosphatase alpha overexpression and blocked by the p50Csk phosphorylation of c-Src at Tyr-529. alpha4beta1-stimulated cell motility of triple-null Src(-/-), c-Yes(-/-), and Fyn(-/-) fibroblasts was dependent on c-Src reexpression that resulted in p130Cas tyrosine phosphorylation and Rac GTPase loading. As p130Cas phosphorylation and Rac activation are common downstream targets for alpha5beta1-stimulated FAK activation, our results support the existence of a novel alpha4 cytoplasmic domain connection leading to c-Src activation which functions as a FAK-independent linkage to a common motility-promoting signaling pathway.
Molecular and Cellular Biology 12/2005; 25(21):9700-12. · 5.53 Impact Factor
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ABSTRACT: Cell motility is stimulated by extracellular stimuli and initiated by intracellular signaling proteins that localize to sites of cell contact with the extracellular matrix termed focal contacts. Focal adhesion kinase (FAK) is an intracellular protein-tyrosine kinase (PTK) that acts to regulate the cycle of focal contact formation and disassembly required for efficient cell movement. FAK is activated by a variety of cell surface receptors and transmits signals to a range of targets. Thus, FAK acts as an integrator of cell motility-associated signaling events. We will review the stimulatory and regulatory mechanisms of FAK activation, the different signaling connections of FAK that are mediated by a growing number of FAK-interacting proteins, and the modulation of FAK function by tyrosine and serine phosphorylation. We will also summarize findings with regard to FAK function in vertebrate and invertebrate development as well as recent insights into the mechanistic role(s) of FAK in promoting cell migration. As increased FAK expression and tyrosine phosphorylation have been correlated with the progression to an invasive cell phenotype, there is growing interest in elucidating the important FAK-related signaling connections promoting invasive tumor cell movement. To this end, we will discuss the effects of FAK inhibition via the dominant-negative expression of the FAK C-terminal domain termed FAK-related non-kinase (FRNK) and how these studies have uncovered a distinct role for FAK in promoting cell invasion that may differ from its role in promoting cell motility.
Biochimica et Biophysica Acta 08/2004; 1692(2-3):77-102. · 4.66 Impact Factor
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Datsun A Hsia,
Satyajit K Mitra,
Christof R Hauck,
Daniel N Streblow,
Jay A Nelson, Dusko Ilic,
Shuang Huang,
Erguang Li,
Glen R Nemerow,
Jay Leng,
Kathryn S R Spencer,
David A Cheresh,
David D Schlaepfer
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ABSTRACT: Cell migration and invasion are fundamental components of tumor cell metastasis. Increased focal adhesion kinase (FAK) expression and tyrosine phosphorylation are connected with elevated tumorigenesis. Null mutation of FAK results in embryonic lethality, and FAK-/- fibroblasts exhibit cell migration defects in culture. Here we show that viral Src (v-Src) transformation of FAK-/- cells promotes integrin-stimulated motility equal to stable FAK reexpression. However, FAK-/- v-Src cells were not invasive, and FAK reexpression, Tyr-397 phosphorylation, and FAK kinase activity were required for the generation of an invasive cell phenotype. Cell invasion was linked to transient FAK accumulation at lamellipodia, formation of a FAK-Src-p130Cas-Dock180 signaling complex, elevated Rac and c-Jun NH2-terminal kinase activation, and increased matrix metalloproteinase expression and activity. Our studies support a dual role for FAK in promoting cell motility and invasion through the activation of distinct signaling pathways.
The Journal of Cell Biology 04/2003; 160(5):753-67. · 10.26 Impact Factor
