Adult Fibroblast Cells Research Articles

Embryonic stem cell as nuclear donor could promote in vitro development of the heterogeneous reconstructed embryo

The nucleus of a somatic cell could be dedifferentiated and reprogrammed in an enucleated heterogeneous oocyte. Some reconstructed oocytes could develop into blastocysts in vitro, and a few could develop into term normally after transferred into foster mothers, but most of cloning embryos fail to develop to term. In order to evaluate the efficacy of embryonic stem cell as nucleus donor in interspecific animal cloning, we reconstructed enucleated rabbit oocytes with nuclei from mouse ES cells, and analyzed the developmental ability of reconstructed embryos in vitro. Two kinds of fibroblast cells were used as donor control, one derived from ear skin of an adult Kunming albino mouse, and the other derived from a mouse fetus. Three types of cells were transferred into perivitelline space under zona pellucida of rabbit oocytes respectively. The reconstructed oocytes were fused and activated by electric pulses, and cultured in vitro. The developmental rate of reconstructed oocytes derived from embryonic stem cells was 16.1%, which was significantly higher than that of both the adult mouse fibroblast cells (0%–3.1%, P< 0.05) and fetus mouse fibroblast cells (2.1%–3.7%, P<0.05). Chromosome analysis confirmed that blastocyst cells were derived from ES donor cell. These observations show that reprogramming is easier in interspecific embryos reconstructed with ES cells than that reconstructed with somatic cells, and that ES cells have the higher ability to direct the reconstructed embryos development normally than fibroblast cells.

Differential patterns of histone methylation and acetylation distinguish active and repressed alleles at X-linked genes

In female mammals, one of the two X chromosomes is inactivated to compensate for the difference in dosage of X-linked genes between males and females. X inactivation involves sequential alterations to the chromatin that ultimately lead to the transcriptional repression of genes on the X chromosome. Here, histone methylation and acetylation along X-linked genes are investigated by chromatin immunoprecipitation (ChIP) of adult fibroblast cell lines. At Pgk1 and Hprt, chromatin on the active X chromosome reveals H3 lysine 4 methylation and acetylation of histones H3 and H4. These modifications are absent on the repressed allele, which is marked by H3 lysine 9 methylation. On the expressed allele of Xist (on the inactive X chromosome), we found that H3 acetylation is confined to the promoter, whereas H3 lysine 4 methylation and H4 acetylation are present along the entire gene. On the repressed Xist allele, in contrast, the promoter and gene exhibit H3 lysine 9 methylation. At only 1.5 kb upstream of the Xist gene, chromatin on the inactive X chromosome has strongly reduced levels of H4 acetylation and is marked by both H3 lysine 9 and H3 lysine 4 methylation. These data demonstrate that patterns of histone methylation and acetylation are distinct along and upstream of Xist and suggest that the inactive X chromatin configuration occurs at a region close to the 5′ end of the gene.

Development of cloned embryos from adult rabbit fibroblasts: effect of activation treatment and donor cell preparation.

This research was to study the in vitro and in vivo development of cloned embryos derived from adult rabbit fibroblasts following various activation protocols. Effects of serum starvation and passage number of donor cells on the efficiency of cloning were also examined. In experiment I, oocytes were activated either by electric pulses or by electric pulses followed by culture with 6-dimethylaminopurin (DMAP). For experiment II, the best activation protocol from experiment I was employed for cloning using adult rabbit fibroblasts that were cultured for 0-15 passages. In experiment III, the effect of serum starvation of the donor cells on cloning was examined. Finally, in experiment IV, embryo transfers were conducted. These experiments showed that combined electrical pulse and DMAP treatment resulted in superior parthenogenetic blastocyst development (up to 29%), and that activation of the cytoplast before versus after fusion was not different in supporting the in vitro development of nuclear transferred embryos (16%-18% blastocysts). Adult fibroblasts derived from nonpassaged cells were less capable of developing into blastocysts than passaged cells (6% vs. 17%). Serum starvation of donor cells improved cleavage (up to 71%) but did not improve blastocyst development (13%), and no progeny was obtained, irrespective of the treatment. Cell-cycle analysis of adult rabbit fibroblast cells showed that passage 6 and 12 cells were more likely to be in G(0)/G(1) than passage 0 cells, which agrees with the improved embryo development in the passaged-cell groups.

High developmental rates of vitrified bovine oocytes following parthenogenetic activation, in vitro fertilization, and somatic cell nuclear transfer.

Successful cryopreservation of mammalian oocytes would provide a steady source of materials for nuclear transfer and in vitro embryo production. Our goal was to develop an effective vitrification protocol to cryopreserve bovine oocytes for research and practice of parthenogenetic activation, in vitro fertilization, and nuclear transfer. Bovine oocytes matured in vitro were placed in 4% ethylene glycol (EG) in TCM 199 plus 20% fetal bovine serum (FBS) at 39 degrees C for 12-15 min, and then transferred to a vitrification solution (35% EG, 5% polyvinyl-pyrrolidone, 0.4 M trehalose in TCM 199 and 20% FBS). Oocytes were vitrified in microdrops on a precooled (-150 degrees C) metal surface (solid-surface vitrification). The vitrified microdrops were stored in liquid nitrogen and were either immediately thawed or were thawed after storage for 2-3 wk. Surviving oocytes were subjected to 1) parthenogenetic activation, 2) in vitro fertilization, or 3) nuclear transfer with cultured adult fibroblast cells. Treated oocytes were cultured in KSOM containing BSA or FBS for 9 to 10 days. Embryo development rates were recorded daily and morphologically high-quality blastocysts were cryopreserved for nuclear transfer-derived embryos at Day 7 or Day 8 of culture. Immediate survival of vitrified/thawed oocytes varied between 77% and 86%. Cleavage and blastocyst development rates of vitrified oocytes following in vitro fertilization or activation were lower than those of the controls. For nuclear transfer, however, vitrified oocytes supported embryonic development as equally well as fresh oocytes.

Increased expression of transforming growth factor-beta1, acidic fibroblast growth factor, and basic fibroblast growth factor in fetal versus adult fibroblast cell lines.

Fetal wound healing occurs without scar tissue formation. Differences in growth factor expression between fetal and adult fibroblasts have been explored. Recently we used RNA expression studies to demonstrate that transforming growth factor (TGF)-beta1, acidic fibroblast growth factor (alpha-FGF), and basic fibroblast growth factor (beta-FGF) could be detected in both fetal and adult fibroblast cell lines. In addition, adult fibroblasts showed greater relative expression of these growth factors than fetal fibroblasts. The aim of this study was to identify the level of protein expression in fetal and adult fibroblasts. Fetal and adult fibroblasts were grown in culture using standard and serum-free media. After protein extraction, Western blot studies were performed to detect the presence and amount of TGFbeta-1, alpha-FGF, and beta-FGF growth factor proteins. beta-Tubulin was used as a control. TGFbeta-1, alpha-FGF, and beta-FGF proteins were detected in fetal and adult fibroblasts grown in standard and serum-free media. The fetal fibroblasts showed higher levels of all three growth factor proteins compared with the adult fibroblasts. Western blot studies suggest higher levels of TGFbeta-1, alpha-FGF, and beta-FGF expression in fetal fibroblasts. It is clear that significant differences exist in the expression/production of these growth factors and it seems likely that further study of these differences will help elucidate the unique healing capabilities of fetal fibroblasts.

Six cloned calves produced from adult fibroblast cells after long-term culture

Cloning whole animals with somatic cells as parents offers the possibility of targeted genetic manipulations in vitro such as "gene knock-out" by homologous recombination. However, such manipulation requires prolonged culture of nuclear donor cells. Previous successes in cloning have been limited to the use of cells collected either fresh or after short-term culture. Therefore, demonstration of genetic totipotency of cells after prolonged culture is pivotal to combining site-specific genetic manipulations and cloning. Here we report birth of six clones of an aged (17-year-old) Japanese Black Beef bull using ear skin fibroblast cells as nuclear donor cells after up to 3 months of in vitro culture (10-15 passages). We observed higher developmental rates for embryos derived from later passages (10 and 15) as compared with those embryos from an early passage (passage 5). The four surviving clones are now 10-12 months of age and appear normal, similar to their naturally reproduced peers. These data show that fibroblasts of aged animals remain competent for cloning, and prolonged culture does not affect the cloning competence of adult somatic donor cells.

Development of cloned embryos from adult rabbit fibroblast cells

Le phénomène d'empreinte parentale émergeant chez les mammifères est à l'origine d'une barrière qui interdit le développement normal des conceptions parthénogénétiques et androgéniques que sont respectivement les tératomes bénins de l'ovaire et les môles hydatiformes. L'évolution hautement pathologique et diamétralement opposée de ces conceptus s'inscrit dans une dualité des rôles respectifs des chromosomes maternels et paternels dans le développement de l'embryon et des annexes. Ces pathologies provenant généralement d'un dysfonctionnement accidentel du système gamétogenèse–fécondation sont plus souvent sporadiques, mais il existe des facteurs favorisants, des cas familiaux, suggérant l'implication de facteurs génétiques qui ne sont pas totalement élucidés.Les conceptions andro- et parthénogénétiques humaines et plus particulièrement les môles peuvent dégénérer en cancer. Le déséquilibre des gènes soumis à empreinte, événement initiateur du développement anarchique du conceptus, l'expression anarchique de nombreux autres gènes par effet cascade et l'acquisition et/ou la perte secondaire d'oncogènes ou d'anti-oncogènes, sont autant d'éléments pouvant intervenir sur cette dérive cancéreuse. Du fait du caractère très particulier de ces tumeurs (matériel chromosomique paternel pour les choriocarcinomes), les facteurs immunologiques, voire environnementaux doivent également être pris en compte. Un nombre croissant de travaux est publié sur ce sujet qui intéresse de nombreuses disciplines de la médecine et de la génétique. Des avancées importantes ont été faites. Par exemple, le rôle de certains gènes (parmi eux : HOX, Tim P3, E-cad et P16) et la découverte d'anomalies chromosomiques récurrentes (7q21 + et 8p21-) dans les processus qui conduisent de la môle au choriocarcinome. Mais l'ensemble de ces phénomènes est complexe et hétérogène et si le modèle des andro- et parthénogénotes est très attrayant pour de nombreuses études, beaucoup de questions restent en suspens.Genomic imprinting is a process that appeared in mammals. This phenomenon blocks the normal development of parthenogenic and androgenic conceptuses, that is to say benign ovarian teratomas and hydatidiform moles respectively. Pathological modifications of these conceptuses depend on whether the chromosomes come from the mother or father.These pathologies are associated with an accidental anomaly during gametogenesis and/or fertilizing. These reproductive anomalies are sporadic and some familial cases may exist suggesting a genetic control of such diseases. The human andro- and parthenogenetic conceptuses, but more frequently the moles, may be invasive (choriocarcinoma). An imbalance of the imprinting genes may initiate the deregulation of other genes, including oncogenes and anti-oncogenes, which can explain the cancerous modification. Immunological and environmental factors must be also considered (presence of the only paternal chromosomes in the choriocarcinoma).Numerous works on this subject are published and some recent important discoveries underline the roles of genes HOX, Tim P3, E-cad and p-16, and the recurrent chromosome anomalies 7q21 + and 8p21- in the mole to choriocarcinoma processing.Although these phenomena are complex and heterogeneous, the andro- and parthenogenote conceptuses are particularly interesting models with which to understand developmental disorders and cancerous progression.

The use of activated and aged-cooled oocytes as host ooplasts in bovine nuclear transfer

This study reconstructed heterogeneous embryos using camel skin fibroblast cells as donor karyoplasts and ovine oocytes as recipient cytoplasts for investigating the developmental potential of the reconstructed embryos. Serum-starved adult camel skin fibroblast cells were used as donor somatic cells. Ovine oocytes matured in vitro were employed as recipient cytoplasts. The fusion of fibroblast cells into recipient cytoplasm was induced by electrofusion. The fused oocytes were activated by 5 mM/ml inomycin with 2 mM/ml 6-dimethylaminopurine (6-DMAP). The activated reconstructed embryos were co-cultured with ovine cumulus cells in synthetic oviduct fluid supplemented with amino acid (SOFaa) and 10% fetal calf serum (FCS) for 168 h. A total of 300 enucleated ovine oocytes were available for xenonuclear embryo reconstruction. The results showed that 71% of the nuclear transfer couplets were successfully fused, 55% of the fused oocytes cleaved within 48 h after activation, 82% of the cleaved oocytes developed to 2–16-cell embryo stages and 18% of the cleaved nuclear transfer zygotes developed to the morula stage. This study demonstrated that the xenonuclear transfer camel embryos can undergo the first embryonic division and subsequent development to morula stage in vitro.

Requirement of Transforming Growth Factor-β (TGF-β) Type II Receptor for TGF-β-induced Proliferation and Growth Inhibition

Growth regulation of fibroblasts is important for lung development and repair of lung injury. In this study, we investigated the role of transforming growth factor-beta (TGF-beta) type II receptor in the TGF-beta-dependent proliferative response of lung fibroblasts. TGF-beta stimulated the proliferation of adult lung fibroblasts at a low concentration (1 ng/ml), but inhibited the growth of fetal lung fibroblasts in a dose-dependent fashion (0.1-10 ng/ml). Cross-linking and Northern analysis demonstrated that the two lung fibroblast cell lines expressed the TGF-beta type I receptor (T beta RI) and type II receptor (T beta RII). We overexpressed in lung fibroblasts a truncated derivative of T beta RII that lacked the cytoplasmic serine/threonine kinase domain (T beta RII delta K). T beta RII delta K was a dominant-negative inhibitor of TGF-beta signal transduction blocking not only TGF-beta-induced mitogenic action upon adult lung fibroblasts but also TGF-beta-induced growth inhibition of fetal lung fibroblasts. The results indicate that the type II receptor is indispensable for mediating both the mitogenic and antiproliferative effects of TGF-beta upon lung fibroblasts.

Identification of functional angiotensin II receptors on rat cardiac fibroblasts.

Cardiac hypertrophy results in an increased deposition of the extracellular matrix (ECM) proteins fibronectin and collagen. Recent evidence indicates that angiotensin II (Ang II) might have an important role in the development of myocardial fibrosis accompanying cardiac hypertrophy. We sought to determine whether fibroblasts of cardiac origin (isolated from neonatal and adult animals) express receptors for Ang II and to examine the ability of this peptide to regulate fibronectin and collagen gene expression in a cultured adult cardiac fibroblast cell preparation. Binding of 125I-Ang II to both neonatal and adult cardiac fibroblasts in culture was specific, reversible, and saturable, with the receptor evenly distributed over the cell population. Competition binding experiments with receptor-specific antagonists indicate that Ang II receptors found on both fibroblast types were of the AT1 subtype. Analysis of mRNA levels for the AT1 receptor indicates that adult cardiac fibroblasts express higher levels of the message than neonatal fibroblasts or cardiac myocytes. Addition of 10(-9) mol/L Ang II to adult cardiac fibroblasts resulted in an induction of ECM proteins above control levels, as determined through Northern blots and total collagen assays. Results from this study indicate that neonatal and adult rat cardiac fibroblasts in culture express AT1 receptors for Ang II. Ang II stimulation of AT1 receptors results in an increased gene expression for ECM proteins. These data suggest that Ang II might have important regulatory roles over cardiac fibroblast function under normal and pathological conditions.

Insulin-like growth factors (IGFs) reduce IGF-binding protein-4 (IGFBP-4) concentration and stimulate IGFBP-3 independently of IGF receptors in human fibroblasts and epidermal cells.

Recent studies have provided a consensus that insulin-like growth factor-I (IGF-I) stimulates IGF-binding protein-3 (IGFBP-3) in vivo and in vitro. While it also appears well established that IGFBP-1 is inversely related to insulin concentrations, evidence regarding regulation of other IGFBP is inconclusive. Using immunoprecipitation and Western ligand blot, we have characterized the IGFBPs released into conditioned medium (CM) by cells from the adult human fibroblast cell line N3652 and the human epidermal squamous cell carcinoma line SCL-1. N3652 cells expressed IGFBP-3, IGFBP-2, a 24-kilodalton (kDa) IGFBP presumed to be IGFBP-4, and IGFBPs at 30 and 28 kDa. SCL-1 expressed IGFBP-3 and a putative IGFBP-4, with intermediate bands at 34 and 30 kDa. As determined by ligand blot of CM from confluent cells 72 h after the addition of peptides to serum-free medium, IGF-I and IGF-II potently stimulated IGFBP-3 in both cell lines, but otherwise IGFBP regulation in the two cells diverged. In N3652 cells, IGFBP-3 concentrations in CM increased to 700% and 800% of basal levels in the presence of IGF-I and IGF-II (at 100 ng/ml; n = 5 experiments), respectively. IGFBP-3 was not affected by insulin up to 10 micrograms/ml. In contrast, IGFBP-4 levels were diminished 54% and 73% by 100 ng/ml IGF-I and IGF-II, respectively, with no response to insulin. In SCL-1 cells, IGF-I and IGF-II were virtually identical in stimulating a mean 200% increase in IGFBP-3 (n = 5 experiments). Insulin was less potent, but caused a significant stimulation of IGFBP-3 levels. IGF-I, IGF-II, and insulin all stimulated an approximately 50% increase in IGFBP-4 concentrations. To test the hypothesis that IGF-induced alterations in IGFBP-3 and IGFBP-4 concentrations were regulated via the type 1 IGF receptor, we attempted to block IGFBP changes with type 1 IGF receptor antibody alpha IR-3 and to induce IGFBP changes with an IGF-II analog, [Leu27]IGF-II, with little affinity for the type 1 receptor. alpha IR-3 failed to block either the IGF-induced rise in IGFBP-3 in each cell line or the decline in IGFBP-4 in N3652 CM. [Leu27]IGF-II was as potent as IGF-II or IGF-I in inducing changes in IGFBP-3 and IGFBP-4 concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)

Modulation of bFGF in lung fibroblasts by TGF-beta and PDGF

Growth factors produced by alveolar macrophages are thought to promote the fibroblast proliferation within interstitial spaces of fibrotic lungs. This study investigated the possibility that the macrophage-produced growth factors might modulate the expression of basic fibroblast growth factor (bFGF) by lung fibroblasts. To evaluate this question, bFGF gene expression and protein production were evaluated in normal adult human lung fibroblast cell lines. Under normal culture conditions, the fibroblasts expressed the bFGF gene as two major transcripts (7.1, 3.7 kb). The addition of fetal calf serum (FCS) to serum-starved fibroblasts caused a 5- to 10-fold increase in bFGF expression. Steady-state bFGF expression was increased 108% by platelet-derived growth factor (PDGF) and 602% by transforming growth factor-beta (TGF-beta). Insulin-like growth factor-1 had no significant effect on bFGF expression. Nuclear runoff studies demonstrated that both PDGF and TGF-beta increased the relative rates of bFGF transcription in the fibroblasts. Western blot analysis of lysates from fibroblasts treated with either PDGF or TGF-beta had no detectable increase in bFGF protein above unstimulated controls. However, the simultaneous addition of PDGF and TGF-beta, or FCS, produced a marked increase in bFGF. These experiments show that two growth factors present in the alveolar airspace compartment of fibrotic lungs can promote the expression of bFGF within lung fibroblasts.

Membrane cofactor protein of complement is present on human fibroblast, epithelial, and endothelial cells.

Membrane cofactor protein (MCP) of the complement system is a iC3/C3b binding molecule with cofactor activity that has been identified on all human peripheral blood cells except erythrocytes. Human mononuclear and platelet MCP is dimeric with molecular weights of 68,000 and 63,000 and is expressed in three phenotypic patterns. To further determine its tissue distribution, surface-labeled human fibroblast, epithelial, and endothelial cells and cell lines were assessed for the presence of MCP by iC3 affinity chromatography and by immunoprecipitation with a monospecific anti-MCP rabbit polyclonal antibody. All sources of adult and fetal fibroblast and epithelial cells and cell lines examined and umbilical vein endothelial cells expressed MCP. The molecular weight and phenotypic patterns of MCP were similar to those of peripheral blood cells. MCP was synthesized by fibroblast and epithelial cell lines. Solubilized extracts of these cell lines expressed factor I-dependent cofactor activity for the first cleavage of iC3/C3b which was abrogated by removal of MCP. Expression of MCP was modulated by SV40 transformation of two fetal fibroblast lines. There was a 5- to 10-fold increase in expression of MCP and a preferential expression of the lower species such that the phenotypic designation was changed. The wide tissue distribution and activity profile of MCP suggest that it is likely to play an important role in the regulation of the complement cascade.

Establishment of an adult rat fibroblast cell line for studies of collagenase regulation.

Several dermal fibroblast lines have been established from explants taken from adult rats. The cells have been cultured for 2 yr and possess stable and well-defined growth characteristics through subculture 18. The cells are readily stored in liquid nitrogen with good viability after thawing. Collagenase activity secreted into the culture medium of the cells at different periods of growth has been examined. There is an 88% drop in total enzyme activity present in the medium between 4 and 14 d of culture, when the cells were plated to reach confluence at Day 8 to 10. A more pronounced fall is noted at earlier times when the cells are plated at a higher density. The correlation between DNA content of the cell monolayer and enzyme activity was -0.895, indicating a possible relationship between the growth of cells and collagenase release.

Replicative potential of individual cell hybrids derived from young and old donor human skin fibroblasts.

Neonatal, adult, and aged donor human skin fibroblast cell cultures have been characterized for the population doubling potential and in vitro-in vivo relationship. Hybrid cells derived from individual whole-cell fusions of replicating GRC 387 (aged) and CSC 303 (neonatal) cells demonstrated that an intermediate mode of replication between that of the two parental cell lines occurred; therefore the longevity of the aged fibroblast cells was enhanced. The GRC 387 cells have an extended post-replicative phase-out period in comparison with the CSC 303 cells, and the experimental hybrids demonstrated a 20-25% increase of the period over that of the GRC 387 cells.