Academic literature on the topic 'Mice embryonic fibroblast'

Analysis of embryonic fibroblasts from GFP reporter mice indicates that the fibroblast cell type harbors a large collection of developmentally and phenotypically heterogeneous subtypes. Some of these cells exhibit multipotency, whereas others do not. Multiparameter flow cytometry analysis shows that a large number of distinct populations of fibroblast-like cells can be found in cultures initiated from different embryonic organs, and cells sorted according to their surface phenotype typically retain their characteristics on continued propagation in culture. Similarly, surface phenotypes of individual cloned fibroblast-like cells exhibit significant variation. The fibroblast cell class appears to contain a very large number of denumerable subtypes.

ABSTRACT Human fibroblast activation protein (FAP), a member of the serine prolyl oligopeptidase family, is a type II cell surface glycoprotein selectively expressed by fibroblastic cells in areas of active tissue remodeling, such as the embryonic mesenchyme, areas of wound healing, the gravid uterus, and the reactive stroma of epithelial cancers. Homologues of FAP have been identified in the mouse and Xenopus laevis. FAP is a dual-specificity enzyme that acts as a dipeptidyl peptidase and collagenase in vitro. To explore the role of FAP in vivo, Fap −/− mice were generated by homologous recombination. RNase protection analysis and reverse transcription-PCR confirmed the absence of full-length Faptranscripts in mouse embryonic tissues. No FAP protein was detected inFap −/− animals by immunohistochemistry, and no FAP-specific dipeptidyl peptidase activity was found. We report thatFap −/− mice are fertile, show no overt developmental defects, and have no general change in cancer susceptibility.

The highly homologous β (βcyto) and γ (γcyto) cytoplasmic actins are hypothesized to carry out both redundant and unique essential functions, but studies using targeted gene knockout and siRNA-mediated transcript knockdown to examine βcyto- and γcyto-isoform–­specific functions in various cell types have yielded conflicting data. Here we quantitatively characterized actin transcript and protein levels, as well as cellular phenotypes, in both gene- and transcript-targeted primary mouse embryonic fibroblasts. We found that the smooth muscle αsm-actin isoform was the dominantly expressed actin isoform in WT primary fibroblasts and was also the most dramatically up-regulated in primary βcyto- or β/γcyto-actin double-knockout fibroblasts. Gene targeting of βcyto-actin, but not γcyto-actin, led to greatly decreased cell proliferation, decreased levels of cellular ATP, and increased serum response factor signaling in primary fibroblasts, whereas immortalization induced by SV40 large T antigen supported fibroblast proliferation in the absence of βcyto-actin. Consistent with in vivo gene-targeting studies in mice, both gene- and transcript-targeting approaches demonstrate that the loss of βcyto-actin protein is more disruptive to primary fibroblast function than is the loss of γcyto-actin.

Abstract We have previously reported that the population of mesenteric lymph node cells from normal BALB/c mice infected 14 days with the rodent nematode Nippostrongylus brasiliensis (Nb-MLN) contains a nongranulated mast cell-committed progenitor (MCCP) which does not require IL-3 for proliferation and differentiation if either a fibroblast monolayer or soluble factors produced by monolayers of 3T3 fibroblasts or embryonic skin are present in the culture. When Nb-MLN were cloned in a methylcellulose culture system using fibroblast conditioned medium as the only source of growth factors, numerous colonies of pure mast cells developed. We wished to determine whether the mast cell deficiency of W/Wv or S1/S1d mice could be explained by the failure of these mice to make either the MCCP or the factor to support proliferation and differentiation of the MCCP. We found that Nb-MLN from W/Wv mice were only able to produce mast cell colonies in response to a source of IL-3 such as conditioned medium from pokeweed mitogen-stimulated spleen cells (CM), and cultures given fibroblast conditioned medium as the only source of growth factors did not produce mast cell colonies. In contrast, Nb-MLN from mast cell deficient S1/S1d mice developed many mast cell colonies in methylcellulose cultures supplemented with either fibroblast conditioned medium or conditioned medium from PWM-stimulated spleen cells. These data suggest that S1/S1d mice but not W/Wv mice produce the mast cell progenitor that responds to fibroblast conditioned medium. To determine if mast cell deficient mice make the fibroblast derived factors that support development of the MCCP, monolayers were prepared from skin connective tissues of S1/S1d and W/Wv mice and Nb-MLN from normal BALB/c mice were cloned in the presence of conditioned medium from these monolayers. Fibroblast conditioned medium from monolayers prepared from W/Wv but not S1/S1d mice supported development of numerous mast cell colonies. Taken together, these data demonstrate that W/Wv mice are incapable of producing normal MCCP whereas S1/S1d fibroblasts fail to produce the appropriate factor to support the MCCP. In accordance with these data, a candidate for the gene product of each of these mutant alleles is discussed.

Abstract T lymphocyte clones were established from mice with acute and chronic graft-vs-host disease (GVHD) [C57B6/6 (B6) mice transplanted i.v. with LP/J spleen cells] and immune mice (LP/J mice immunized intraperitoneally with B6 spleen cells). To determine the role of leukokines in the increased collagen production that characterizes chronic GVHD, the supernatants of in vitro stimulated clones were assayed for their effect on 1) B6 embryonic fibroblast proliferation, 2) total fibroblast collagen secretion, and 3) collagen secretion per fibroblast. Four of nine chronic GVHD (CG) clones stimulated both total collagen secretion and collagen secretion per fibroblast, but none stimulated fibroblast proliferation. Six of 10 immune (I) clones stimulated fibroblast proliferation, and none stimulated collagen secretion per fibroblast. Acute GVHD (G) clones were heterogeneous; 2/10 G clones stimulated fibroblast proliferation, 5/10 stimulated total collagen secretion, and 4/10 stimulated collagen secretion per fibroblast. I and CG clones may act synergistically in vivo. The presence of CG-like clones in mice with acute GVHD suggest that the immunologic events that culminate in chronic GVHD are initiated early after transplantation.

ABSTRACT Cited2 (CBP/p300 interacting transactivator with ED-rich tail 2) is required for embryonic development, coactivation of transcription factor AP-2, and inhibition of hypoxia-inducible factor 1 transactivation. Cited2 is induced by multiple growth factors and cytokines and oncogenically transforms cells. Here, we show that the proliferation of Cited2 −/− mouse embryonic fibroblasts ceases prematurely. This is associated with a reduction in growth fraction, senescent cellular morphology, and increased expression of the cell proliferation inhibitors p16INK4a, p19ARF, and p15INK4b. Deletion of INK4a/ARF (encoding p16INK4a and p19ARF) completely rescued the defective proliferation of Cited2−/− fibroblasts. However, the deletion of INK4a/ARF did not rescue the embryonic malformations observed in Cited2 −/− mice, indicating that INK4a/ARF-independent pathways are likely to be involved here. We found that Cited2 −/− fibroblasts had reduced expression of the polycomb-group genes Bmi1 and Mel18, which function as INK4a/ARF and Hox repressors. Complementation with CITED2-expressing retrovirus enhanced proliferation, induced Bmi1/Mel18 expression, and decreased INK4a/ARF expression. Bmi1- and Mel18-expressing retroviruses enhanced the proliferation of Cited2 −/− fibroblasts, indicating that they function downstream of Cited2. Our results provide genetic evidence that Cited2 controls the expression of INK4a/ARF and fibroblast proliferation, at least in part via the polycomb-group genes Bmi1 and Mel18.

Embryonic haemopoietic stem cells can differentiate from mouse blastocysts grown in vitro. Mouse blastocysts were cultured for 3 or 4 days and the resultant cells were injected intravenously into lethally X-irradiated or genetically anaemic recipient mice. Blastocysts grown in vitro did not maintain normal embryonic morphology. The presence of donor haemoglobin and donor lymphocytic glucose phosphate isomerase in grafted recipients, demonstrates the presence of embryonic haemopoietic stem cells. Recipients of embryonic haemopoietic stem cells, obtained from growth in vitro, were haematologically stable with no evidence of neoplasia. Pluripotent embryonic cells, maintained on fibroblast feeder layers, were unable to colonize X-irradiated or genetically anaemic mice. Recipients of pluripotent cells died at the same time as saline-injected controls.

Mutant Gy male mice, which have previously been described as having disruption of the phosphate-regulating Phex gene and a spermine synthase gene [Meyer, Henley, Meyer, Morgan, McDonald, Mills and Price (1998) Genomics, 48, 289–295; Lorenz, Francis, Gempel, Böddrich, Josten, Schmahl and Schmidt (1998) Hum. Mol. Genet. 7, 541–547], as well as mutant Hyp male mice, which have disruption of the Phex gene only, were examined along with their respective normal male littermates. Biochemical analyses of extracts of brains, hearts and livers of 5-week-old mice showed that Gy males lacked any significant spermine synthase activity as well as spermine content. Organs of Gy males had a higher spermidine content. This was caused not only by the lack of conversion of spermidine into spermine, but also because of compensatory increases in the activities of other polyamine biosynthetic enzymes. Gy males were half the body weight of their normal male littermates at weaning age. Hyp males, however, were no different in size when compared with their controls. High mortality of Gy males occurs by weaning age and this mortality was shown to be largely post-natal. Embryonic fibroblasts were isolated from Gy males and their normal male littermates and were similarly shown to lack any significant spermine synthase activity as well as spermine content. The lack of spermine, however, had no significant effect on the growth of immortalized fibroblasts or of primary fibroblast cultures. Similarly, there was no difference in the time of senescence of primary fibroblast cultures from Gy males compared with cultures derived from normal male littermates. However, the lack of spermine did increase the sensitivity of immortalized fibroblasts to killing by the chloroethylating agent 1,3-bis(2-chloroethyl)-N-nitrosourea. Therefore both the Gy male mice and derived embryonic fibroblasts provide valuable models to study the importance of spermine and spermine synthase, without the use of inhibitors which may have additional side effects.

The molecular characterization of a cardiac determination gene has been an elusive goal for the past several years. Prior to cloning of the skeletal muscle determination factor MyoD, the presence of a dominantly acting skeletal muscle determination factor had been inferred from the observation that the skeletal muscle phenotype was dominant in skeletal muscle-fibroblast heterokaryons (H. M. Blau, G. K. Pavlath, E. C. Hardeman, C.-P. Chiu, L. Siberstein, S. G. Webster, S. C. Miller, and D. Webster, Science 230:758-766, 1985). In these experiments, we have examined cardiac-fibroblast heterokaryons to investigate the existence of a dominantly acting cardiac determination factor. We have employed a novel experimental approach using primary embryonic fibroblasts from transgenic mice as a means of assaying for the activation of a cardiac promoter-luciferase reporter transgene within fibroblast nuclei. This approach provides a potential means of genetic selection for a dominantly acting positive factor and can be generalized to other systems. We have examined the expression of three markers of the cardiac lineage: a myofibrillar protein promoter (MLC2), a secreted protein (ANF), and a transcription factor (MEF2). MEF2 is specific to both cardiac and skeletal muscle cells. Our results indicate that in a majority of heterokaryons with an equal ratio of cardiac to fibroblast nuclei, none of these cardiac markers are expressed, indicating that the cardiac phenotype is not dominant over the embryonic fibroblast phenotype. The distinction from previous results with skeletal muscle is emphasized by our results with MEF2, which is dominantly expressed in skeletal muscle-fibroblast but not cardiac-fibroblast heterokaryons, supporting its divergent regulation in the two cell types.

The molecular characterization of a cardiac determination gene has been an elusive goal for the past several years. Prior to cloning of the skeletal muscle determination factor MyoD, the presence of a dominantly acting skeletal muscle determination factor had been inferred from the observation that the skeletal muscle phenotype was dominant in skeletal muscle-fibroblast heterokaryons (H. M. Blau, G. K. Pavlath, E. C. Hardeman, C.-P. Chiu, L. Siberstein, S. G. Webster, S. C. Miller, and D. Webster, Science 230:758-766, 1985). In these experiments, we have examined cardiac-fibroblast heterokaryons to investigate the existence of a dominantly acting cardiac determination factor. We have employed a novel experimental approach using primary embryonic fibroblasts from transgenic mice as a means of assaying for the activation of a cardiac promoter-luciferase reporter transgene within fibroblast nuclei. This approach provides a potential means of genetic selection for a dominantly acting positive factor and can be generalized to other systems. We have examined the expression of three markers of the cardiac lineage: a myofibrillar protein promoter (MLC2), a secreted protein (ANF), and a transcription factor (MEF2). MEF2 is specific to both cardiac and skeletal muscle cells. Our results indicate that in a majority of heterokaryons with an equal ratio of cardiac to fibroblast nuclei, none of these cardiac markers are expressed, indicating that the cardiac phenotype is not dominant over the embryonic fibroblast phenotype. The distinction from previous results with skeletal muscle is emphasized by our results with MEF2, which is dominantly expressed in skeletal muscle-fibroblast but not cardiac-fibroblast heterokaryons, supporting its divergent regulation in the two cell types.

Une exposition prénatale aux radiations ionisantes est associée au développement de pathologies neurodéveloppementales liées à l’induction de dommages à l’ADN dans les cellules souches et progéniteurs neuraux (CSPN). Ainsi, la stabilité génétique des CSPN est cruciale pour le développement et l’homéostasie du cerveau. Cependant, des altérations génomiques au niveau des CSPN au cours du développement pourraient promouvoir la diversité neuronale. XLF est un composant de la voie de réparation d’ADN par NHEJ (pour Non-Homologous End-Joining). Nous avons montré une augmentation de l’instabilité des CSPN dans le cerveau embryonnaire des souris Xlf-/- qui pourrait perturber la neurogenèse au cours du développement, et ainsi être responsable d’altérations comportementales identifiées chez ces souris à l’âge adulte. A l’aide d’approches cytogénétiques, nous avons comparés la stabilité chromosomique des CSPN et des fibroblastes embryonnaires murins (MEF) exposés à un stress génotoxique aigue (irradiation γ) ou chronique (incorporation de thymidine tritiée dans l’ADN). Nos résultats démontrent que les CSPN maintiennent leur intégrité génétique de façon plus efficace que les MEF. En effet, les CSPN semblent avoir de meilleures capacités de réparation des dommages à l’ADN que les MEF, ce qui leur permet de développer une réponse adaptative à un stress génotoxique chronique. Cette réponse adaptative implique XLF et agit conjointement avec les points de contrôle du cycle cellulaire et l'apoptose pour préserver la stabilité du génome et éliminer les cellules endommagées. L’ensemble de nos résultats apporte la démonstration d’une réponse robuste aux dommages de l'ADN dans les CSPN et souligne l'importance de XLF lors du développement du cerveau
Prenatal exposure to ionizing radiation has been associated with many neurodevelopmental disorders due to the DNA damage induced in neural stem and progenitors cells (NSPC). Thus, genetic stability of NSPC is crucial for brain development and homeostasis. Nevertheless, genomic alterations occurring during development in NSPC may have a potential impact on the physiological neuronal diversity. XLF is a component of the NHEJ (Non-Homologous End-Joining) repair pathway. Here, we show that NSPC from Xlf-/- embryos exhibit increased chromosome instability, leading to premature neurogenesis and consequently neurobehavioral disorders. Using cytogenetic approaches, we compared the chromosome stability of mouse embryonic NSPC and fibroblasts (MEF) exposed to acute (γ-irradiation) or chronic (incorporation of tritiated thymidine into DNA) genotoxic stress. Our results demonstrate the higher capacity of NSPC as compared to MEF to maintain their genomic integrity. We evidenced that NSPC have more efficient DNA repair activity than MEF, allowing them to develop an adaptive response to chronic genotoxic stress. This adaptive response involves XLF and acts together with apoptosis and cell cycle checkpoints to preserve the stability of the genome and to eliminate damaged cells. Altogether, our results provide new insights into the robust DNA damage response in NSPC and highlight the importance of Xlf during brain development

Genesis of life begins with the fusion of female and male haploid gametes through a process of fertilization leading to the formation of a diploid cell, the zygote. This undergoes successive cleavage divisions forming 2-, 4- and 8- cell embryos and their individual cells (blastomeres) are totipotent. As development proceeds, there is a gradual restriction in their totipotency, resulting in the generation of two distinct cell lineages i.e., the differentiated trophectoderm (TE) cells and the undifferentiated, inner cell mass (ICM) during blastocyst morphogenesis (Rossant and Tam 2009). During the course of development, the ICM cells can give rise to all cell types of an organism and can also provide embryonic stem (ES)-cells when cultured in vitro (Evan and Kaufman 1981). ES-cells are pluripotent cells, having the ability to self-renew indefinitely and differentiate into all the three primary germ layers (ectoderm, mesoderm and endoderm) derived-cell types. ES-cells are an excellent developmental model system to understand basic mechanisms of self-renewal, cell differentiation and function of various genes in vitro and in vivo (Capecchi 2001). Importantly, their cell derivatives could potentially be used for experimental cell-based therapy for a number of diseases. Although, human ES-cell lines have been successfully derived and differentiated to various cell types (Thomson et al., 1998; Odorico et al., 2001), their cell-therapeutic potential is far from being tested, in view of the lack of our understanding of lineage-specific differentiation, homing and structural-functional integration of differentiated cell types in the host environment. To understand these mechanisms, it is desirable to have fluorescently-marked ES-cells and their differentiated cell-types, which could facilitate experimental cell transplantation studies. In this regard, our laboratory has earlier generated enhanced green fluorescent protein (EGFP)-expressing FVB/N transgenic ‘green’ mouse, under the control of ubiquitous chicken -actin promoter (Devgan et al., 2003). This transgenic mouse has been an excellent source of intrinsically green fluorescent cell types. We have been attempting to derive ES-cell line from this transgenic mouse. Because the derivation of ES-cell line is genetic strain-dependent, with some strains being relatively permissible for ES-cell derivation while others are quite resistant (non permissive), it has been extremely difficult to derive ES-cell line from the FVB/N mouse strain. There is a need to evolve experimental strategies to derive ES-cell line from FVB/N mouse, a strain extensively used for transgenesis. Thus, the aims of the study described in the thesis are to: (1) develop an experimental system to derive EGFP-expressing fluorescently-marked ES-cell line from a non-permissive FVB/N mouse strain; (2) characterize the established ES-cell line; (3) achieve differentiation of various cell types from EGFP-expressing ES-cell line and (4) understand role of FGF signaling in cardiac differentiation from the established ES-cell line. In order to have an appropriate and relevant literature background, the 1st chapter in this thesis describes a comprehensive up-to-date review of literature, pertaining to the early mammalian development and differentiation of blastocyst, followed by origin and properties of ES-cells. Various ES-cell derivation strategies from genetically permissive and non-permissive mouse strains are described and also the ES-cell differentiation potential to various progenitors and differentiated cell types. Subsequently, details on molecular basis of cardiac differentiation and the therapeutic potential of ES-cell derived differentiated cell types to treat disease(s) are described. This chapter is followed by three data chapters (II-IV). Chapter-II describes the issues related to non-permissiveness of FVB/N strain for ES-cell derivation and strategies to overcome this hurdle. This is followed by detailed results pertaining to generation of homozygous EGFP-expressing transgenic mice and development of a two-pronged ES-cell derivation approach to successfully establish a permanent ES-cell line (named ‘GS-2’ ES-cell line) from the EGFP-transgenic ‘green’ mouse. This chapter also provides results pertaining to detailed characterization of the ‘GS-2’ ES-cell line which includes colony morphology, expansion efficiency, alkaline phosphatase staining, expression analysis of pluripotent markers by RT-PCR and immunostaining approaches and karyotyping. Following this, the outcome of results and significance in the context of reported information are discussed in detail. Having successfully derived the ‘GS-2’ ES-cell line, it is necessary to thoroughly assess the differentiation competence of the ‘GS-2’ ES-cell line. Therefore, the Chapter-III describes detailed assessment of the in vitro and in vivo differentiation potential of the ‘GS-2’ ES-cell line. For in vitro differentiation, results pertaining to ES-cell derived embryoid body (EB) formation and their differentiation to ectodermal, mesodermal and endodermal cell types, expressing nestin, BMP-4 and α-fetoprotein, respectively, are described. Besides, the robustness of adaptability of ‘GS-2’ ES-cells to various culture conditions for their maintenance and differentiation are described. Also shown in the chapter is the relatively greater propensity of this cell line to cardiac differentiation. For in vivo differentiation, the ‘GS-2’ ES-cell derived teratoma formation in nude mice and its detailed histological analysis showing three germ layer cell types and their derivatives are described. Last part of the data described in this chapter, pertains to generation of chimeric blastocysts by aggregation method. Because the ‘GS-2’ ES-cell line exhibited a robust differentiation potential, including an efficient cardiomyocyte differentiation, it is of interest to enhance the efficiency of cardiomyocyte differentiation by exogenous addition of one of the key growth factors i.e., FGF8b since this has been implicated to be critical for cardiogenesis in non-mammalian verterbrate species. Therefore, Chapter-IV is focused on assessing the ability of ‘GS-2’ ES-cell line for its cardiomyocyte differentiation property with particular emphasis on the FGF-induced cardiac differentiation. Results pertaining to the expressions of various FGF ligands and their receptors during differentiation of ES-cells are described. Besides, increases in the cardiac efficiency, following FGF8b treatment and the associated up-regulation of cardiac-specific markers such as GATA-4, ISL-1 and α-MHC are shown. At the end of data chapters, separate sections are devoted for ‘Summary and Conclusion’ and for ‘Bibliography’.

The use of transgenic mice is an incredibly powerful tool in understanding the underlying etiology of disease. To understand the usefulness of specific transgenic mice, the systems of interest should be characterized. We have created TGFβ2-deficient mouse fetuses that develop widespread aortic and coronary artery aneurysms [1]. Several studies have pointed to a strong connection between elevated TGFβ signaling and aortic aneurysm [2]. In situ hybridization has shown that Tgfb2 and Tgfb3 are major ligands expressed in the aortic medial wall. Further reduction of TGFβ signaling by combining TGFβ2- and TGFβ3-deficient mice exacerbated cardiovascular aneurysms in TGFβ2/TGFβ3-doubly deficient embryos. In vitro cell culture experiments demonstrated an impaired ability of TGFβ2-deficient mouse embryonic fibroblasts to reorganize collagen. Previous data indicate reduced levels of TGFβ2 leading to a higher susceptibility to aortic aneurysm. We present here the macroscopic biomechanical characterization of the aorta of a transgenic mouse line showing this susceptibility and compare it to wild-type mice. We also present results comparing the microstructure between mouse lines.