194 EPIGENETIC REMODELING OF ADULT SOMATIC CELLS

2014 ◽  
Vol 26 (1) ◽  
pp. 211 ◽  
Author(s):  
G. Pennarossa ◽  
S. Maffei ◽  
F. Gandolfi ◽  
T. A. L. Brevini
Keyword(s):  
Granulosa Cells ◽  
Muscle Development ◽  
Morphological Changes ◽  
Primary Cultures ◽  
Cell Lineage ◽  
Cell Types ◽  
Plastic State ◽  
Specific Marker ◽  
Germ Layer

Mammalian differentiation is obtained through epigenetic regulations that shape the genome, which is identical in all cells, to distinct phenotypes and tissue specific identities. The differentiated state of mature cells in an adult organism is therefore acquired through epigenetic restrictions that lead to a gradual loss of differentiative potency. In agreement with this, recent experiments demonstrate that terminally differentiated cells can be induced to de-differentiate in vitro and increase their plasticity in response to epigenetic modifiers that are capable of reverting cells from their lineage commitment to a more plastic state. Here we describe experiments where we prepared porcine skin fibroblasts and granulosa primary cultures and exposed them to an inhibitor of DNA methylation, the 5-aza-cytidine (5-aza-CR), to increase cell plasticity. Taking advantage of the obtained increased permissivity window, we investigated the ability of 5-aza-CR treated cells to respond to specific differentiation conditions and be re-addressed to a different cell lineage either within the same germ layer or to a different germ layer. Cells were evaluated for their morphological changes and assessed using RT-PCR and immunocytochemical studies during the treatment. Following the exposure to 5-aza-CR the phenotype of both cell types changed. Treated cells displayed an oval or round shape, and appeared smaller with larger nuclei and granular and vacuolated cytoplasm. This was accompanied by an active expression of the main pluripotency-related genes OCT4, NANOG, SOX2, and REX1, originally undetectable in untreated fibroblasts and granulosa cells. 5-aza-CR treated granulosa cells cultured with recombinant human vascular endothelial growth factor to induce myogenic specification (different lineage within the same germ layer) suppressed the expression of granulosa specific marker (Cytokeratin) as well as of the pluripotency genes, and expressed MYOD, MYF5, and MYOG (earliest myogenic markers that are involved in the coordination of skeletal muscle development or myogenesis). In order to trans-differente 5-aza-CR treated fibroblasts to cells of a different germ layer, they were exposed to activin A to promote endoderm commitment. Cells down-regulated Vimentin (fibroblast marker) as well as pluripotent gene expression and transcribed Nestin (transiently involved in multi-lineage progenitor cell differentiation), SOX17, FOXA2 (induction of definitive endoderm), and HNF4A, HNF1 (primitive gut tube specific genes). Altogether these results suggest that it is possible to obtain a direct inter-lineage conversion by removing epigenetic restriction, using demethylating agents such as 5-aza-CR, and avoiding a stable pluripotent state. This novel approach may represent a promising tool for regenerative medicine because it does not involve the use of any transgenic modifications, retroviral transfection, or both. Supported by Network Lombardo iPS (NetLiPS) Project ID 30190629.

scholarly journals Dedifferentiated follicular granulosa cells derived from pig ovary can transdifferentiate into osteoblasts

2012 ◽  
Vol 447 (2) ◽  
pp. 239-248 ◽  
Author(s):  
Yoshinao Oki ◽  
Hiromasa Ono ◽  
Takeharu Motohashi ◽  
Nobuki Sugiura ◽  
Hiroyuki Nobusue ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Cell Biology ◽  
Cell Lineage ◽  
Marker Genes ◽  
Specific Cell ◽  
Specific Marker ◽  
Matrix Mineralization ◽  
Differentiated Cells

Transdifferentiation is the conversion of cells from one differentiated cell type into another. How functionally differentiated cells already committed to a specific cell lineage can transdifferentiate into other cell types is a key question in cell biology and regenerative medicine. In the present study we show that porcine ovarian follicular GCs (granulosa cells) can transdifferentiate into osteoblasts in vitro and in vivo. Pure GCs isolated and cultured in Dulbecco's modified Eagle's medium supplemented with 20% FBS (fetal bovine serum) proliferated and dedifferentiated into fibroblast-like cells. We referred to these cells as DFOG (dedifferentiated follicular granulosa) cells. Microarray analysis showed that DFOG cells lost expression of GC-specific marker genes, but gained the expression of osteogenic marker genes during dedifferentiation. After osteogenic induction, DFOG cells underwent terminal osteoblast differentiation and matrix mineralization in vitro. Furthermore, when DFOG cells were transplanted subcutaneously into SCID mice, these cells formed ectopic osteoid tissue. These results indicate that DFOG cells derived from GCs can differentiate into osteoblasts in vitro and in vivo. We suggest that GCs provide a useful model for studying the mechanisms of transdifferentiation into other cell lineages in functionally differentiated cells.

Abstract 313: Twist Contributes to Cardiomyocyte Renewal and is Required for Maintenance of Adult Cardiac Function

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Yi-Li Min ◽  
Svetlana Bezprozvannaya ◽  
Drazen Šošic ◽  
Young-Jae Nam ◽  
Hesham Sadek ◽  
...  
Keyword(s):  
Cardiac Function ◽  
Cardiac Fibroblasts ◽  
Cell Lineage ◽  
Cardiac Regeneration ◽  
Cell Types ◽  
Bhlh Transcription Factor ◽  
Adult Heart ◽  
Twist 1 ◽  
Essential Contribution

Cardiomyocyte renewal occurs very slowly in adult mammals, and little is known of the genetic basis of cardiac regeneration. Twist is a highly conserved bHLH transcription factor responsible for Drosophila mesoderm formation during embryogenesis. Recent studies have shown that Twist protein is essential for muscle regeneration in adult Drosophila, but the potential role of Twist in the mammalian heart has not been explored. There are two Twist genes in vertebrates, Twist-1 and -2. We show that Twist-1 and -2 are expressed in epicardium and interstitial cells but not in differentiated cardiomyocytes in mice. To understand the potential function of Twist-dependent lineages in the adult heart, we generated inducible Twist2CreERT2; ROSA26-tdTomato reporter mice. By treating these mice with tamoxifen at 8 weeks of age, we observed progressive labeling of various cell types, such as epithelial cells, cardiac fibroblasts, and cardiomyocytes in the heart. We isolated Tomato-positive nonmyocytes from these mice and found that these cells can differentiate into cardiomyocytes and other cell types in vitro. Furthermore, cardiac-specific deletion of both Twist1 and Twist2 resulted in an age-dependent lethal cardiomyopathy. These findings reveal an essential contribution of Twist to long-term maintenance of cardiac function and support the concept of slow, lifelong renewal of cardiomyocytes from a Twist-dependent cell lineage in the adult heart.

scholarly journals Lymphoid Tumors ofXenopus laeviswith Different Capacities for Growth in Larvae and Adults

1994 ◽  
Vol 3 (4) ◽  
pp. 297-307 ◽  
Author(s):  
Jacques Robert ◽  
Chantal Guiet ◽  
Louis Du Pasquier
Keyword(s):  
Cell Surface ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Mhc Class I ◽  
Mhc Class Ii ◽  
Cell Lineage ◽  
Cell Types ◽  
Class Ii ◽  
Class I ◽  
Specific Marker

Three new lymphoid tumors offering an assortment of variants in terms of MHC class I expressions, MHC class II expression, and Ig gene transcription have been discovered in the amphibianXenopus. One was developed in an individual of the isogenic LG15 clone (LG15/0), one in a frog of the LG15/40 clone (derived from a small egg recombinant of LG15), and one (ff-2) in a maleffsib of the individual in which MAR1, the first lymphoid tumor in Xenopus was found 2 years ago. These tumors developed primarily as thymus outgrowths and were transplantable in histocompatible tadpoles but not in nonhistocompatible hosts. Whereas LG15/0 and LG15/40 tumor cells also grow in adult LG15 frogs, theff-2 tumor, like the MAR1 cell line, is rejected by adultffanimals. Using flow cytometry with fluorescence-labeled antibodies and immunoprecipitation analysis, we could demonstrate that, like MAR1, these three new tumors express on their cell surface lymphopoietic markers recognized by mAbs FIF6 and RC47, as well as T-cell lineage markers recognized by mAbs AM22 (CD8-1ike) and X21.2, but not by immunologobulin (Ig) nor MHC class II molecules. Another lymphocyte-specific marker AM15 is expressed by 15/0 and 15/40 but notff-2 tumor cells. Theff-2 tumor cell expresses MHC class molecule in association withβ2-microglobulin on the surface, 15/40 cells contain cytoplasmic Iαchain that is barely detected at the cell surface by fluocytometry, and 15/0 cells do not synthesize class Iαchain at all. The three new tumors all produce large amounts of IgM mRNA of two different sizes but no Ig protein on the membrane nor in the cytoplasm. All tumor cell types synthesize large amount of Myc mRNA and MHC class I-like transcripts considered to be non classical.

scholarly journals Corticotrophin-releasing hormone inhibits insulin-like growth factor-I release from primary cultures of rat granulosa cells

2002 ◽  
Vol 174 (3) ◽  
pp. 493-498 ◽  
Author(s):  
AE Calogero ◽  
A Barreca ◽  
N Burrello ◽  
I Palermo ◽  
G Giordano ◽  
...  
Keyword(s):  
Growth Factor ◽  
Granulosa Cells ◽  
Primary Cultures ◽  
Cell Types ◽  
Suppressive Effect ◽  
Insulin Like Growth Factor ◽  
Sprague Dawley ◽  
Releasing Hormone ◽  
Igf I ◽  
Igfbp 3

Corticotrophin-releasing hormone (CRH), a neuropeptide which modulates gonadal function during stress, is expressed by several cell types of the rat ovary and is able to suppress oestrogen release from rat granulosa cells. The mechanism of this effect is, however, not known. Since insulin-like growth factor (IGF)-I is produced by rat granulosa cells and exerts a synergistic role with FSH on granulosa cell steroidogenesis, we hypothesised that CRH may suppress oestrogen release from granulosa cells by inhibiting IGF-I release and/or stimulating the release of its binding protein (IGFBP-3). To test this hypothesis, granulosa cells were obtained from immature female Sprague-Dawley rats primed with diethylstilboestrol, and hormone concentrations were measured in the conditioned medium by radioimmunoassay. CRH suppressed oestrogen and IGF-I release stimulated by FSH used at a concentration of 1 IU/l, whereas it did not have any statistically significant effect on oestrogen and IGF-I release in basal conditions or in response to 5 IU/l FSH. The suppressive effects of CRH on oestrogen and IGF-I release were antagonised by a selective CRH receptor antagonist. CRH had no effects on IGFBP-3 release. CRH did not have any effect on oestrogen release stimulated by increasing concentrations of IGF-I and its suppressive effect on FSH-stimulated oestrogen release was overcome by the addition of low doses of exogenous IGF-I. In conclusion, CRH suppressed the release of oestrogen and IGF-I, but not of IGFBP-3. Thus, the inhibitory effects of CRH on oestrogen release could be mediated, partly, by a suppression of the autocrine/paracrine action of IGF-I.

scholarly journals Expanded equine cumulus–oocyte complexes exhibit higher meiotic competence and lower glucose consumption than compact cumulus–oocyte complexes

2018 ◽  
Vol 30 (2) ◽  
pp. 297 ◽  
Author(s):  
L. González-Fernández ◽  
M. J. Sánchez-Calabuig ◽  
M. G. Alves ◽  
P. F. Oliveira ◽  
S. Macedo ◽  
...  
Keyword(s):  
Glucose Metabolism ◽  
Granulosa Cells ◽  
Glucose Consumption ◽  
Cell Types ◽  
Solute Carrier Family ◽  
Necrosis Factor Alpha ◽  
Maturation Rate ◽  
Solute Carrier ◽  
Meiotic Competence

Equine cumulus–oocyte complexes (COCs) are classified as compact (cCOC) or expanded (eCOC) and vary in their meiotic competence. This difference could be related to divergent glucose metabolism. To test this hypothesis in the present study, eCOCs, cCOCs and expanded or compact mural granulosa cells (EC and CC respectively) were matured in vitro for 30 h, at which time maturation rate, glucose metabolism and the expression of genes involved in glucose transport, glycolysis, apoptosis and meiotic competence were determined. There were significant differences between eCOCs and cCOCs in maturation rate (50% vs 21.7% (n = 192 and 46) respectively; P < 0.001), as well as mean (± s.e.m.) glucose consumption (1.8 ± 0.5 vs 27.9 ± 5.9 nmol per COC respectively) and pyruvate (0.09 ± 0.01 vs 2.4 ± 0.8 nmol per COC respectively) and lactate (4.7 ± 1.3 vs 64.1 ± 20.6 nmol per COC respectively; P < 0.05 for all) production. Glucose consumption in EC and CC did not differ significantly. Expression of hyaluronan-binding protein (tumour necrosis factor alpha induced protein 6; TNFAIP6) was increased in eCOCs and EC, and solute carrier family 2 member 1 (SLC2A1) expression was increased in eCOCs, but there were no differences in the expression of glycolysis-related enzymes and solute carrier family 2 member 3 (SLC2A3) between the COC or mural granulosa cell types. The findings of the present study demonstrate that metabolic and genomic differences exist between eCOCs and cCOCs and mural granulosa cells in the horse.

scholarly journals The Human c-Fes Tyrosine Kinase Binds Tubulin and Microtubules through Separate Domains and Promotes Microtubule Assembly

2004 ◽  
Vol 24 (21) ◽  
pp. 9351-9358 ◽  
Author(s):  
Charles E. Laurent ◽  
Frank J. Delfino ◽  
Haiyun Y. Cheng ◽  
Thomas E. Smithgall
Keyword(s):  
Tyrosine Kinase ◽  
Protein Tyrosine Kinase ◽  
Morphological Changes ◽  
Neuronal Cells ◽  
Coiled Coil ◽  
Cell Types ◽  
Sh2 Domain ◽  
Microtubule Assembly

ABSTRACT The c-Fes protein-tyrosine kinase (Fes) has been implicated in the differentiation of vascular endothelial, myeloid hematopoietic, and neuronal cells, promoting substantial morphological changes in these cell types. The mechanism by which Fes promotes morphological aspects of cellular differentiation is unknown. Using COS-7 cells as a model system, we observed that Fes strongly colocalizes with microtubules in vivo when activated via coiled-coil mutation or by coexpression with an active Src family kinase. In contrast, wild-type Fes showed a diffuse cytoplasmic localization in this system, which correlated with undetectable kinase activity. Coimmunoprecipitation and immunofluorescence microscopy showed that the N-terminal Fes/CIP4 homology (FCH) domain is involved in Fes interaction with soluble unpolymerized tubulin. However, the FCH domain was not required for colocalization with polymerized microtubules in vivo. In contrast, a functional SH2 domain was essential for microtubule localization of Fes, consistent with the strong tyrosine phosphorylation of purified tubulin by Fes in vitro. Using a microtubule nucleation assay, we observed that purified c-Fes also catalyzed extensive tubulin polymerization in vitro. Taken together, these results identify c-Fes as a regulator of the tubulin cytoskeleton that may contribute to Fes-induced morphological changes in myeloid hematopoietic and neuronal cells.

scholarly journals miR-375 mediates CRH signaling pathway in inhibiting E2 synthesis in porcine ovary

Reproduction ◽  
2017 ◽  
Vol 153 (1) ◽  
pp. 63-73 ◽  
Author(s):  
Chulin Yu ◽  
Meiling Li ◽  
Yue Wang ◽  
Ying Liu ◽  
Chengzhi Yan ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Granulosa Cells ◽  
Reproductive System ◽  
Follicular Development ◽  
Cell Types ◽  
Signaling System ◽  
Key Factor ◽  
The Relationship

The corticotropin-releasing hormone (CRH) signaling system is involved in numbers of stress-related physiological and pathological responses, including its inhibiting effects on estradiol (E2) synthesis and follicular development in the ovary. In addition, there are reports that microRNAs (miRNAs) can control the function of animal reproductive system. The aim of present study was to investigate the functions of miR-375 and the relationship between miR-375 and CRH signaling molecules in the porcine ovary. First, our common PCR results show that miR-375 and the CRH receptor 1 (CRHR1) are expressed in porcine ovary, whereas CRH receptor 2 (CRHR2) is not detected. We further have located the cell types of miR-375 and CRHR1 by in situ hybridization (ISH), and the results show that miR-375 is located only in the granulosa cells, whereas CRHR1 is positive in all of granulosa cells and oocytes, inferring that miR-375 and CRHR1 are co-localized in granulosa cells. Second, we show that overexpression of miR-375 in cultured granulosa cells suppresses the E2 production, whereas miR-375 knockdown demonstrates the opposite result. Besides, our in vitro results demonstrate that miR-375 mediates the signaling pathway of CRH inhibiting E2 synthesis. Finally, our data show that the action of miR-375 is accomplished by directly binding to the 3′UTR of specificity protein1 (SP1) mRNA to decrease the SP1 protein level. Thus, we conclude that miR-375 is a key factor in regulating E2 synthesis by mediating the CRH signaling pathway.

scholarly journals The homeobox gene, TGIF1, is required for chicken ovarian cortical development and generation of the juxtacortical medulla

Development ◽  
2021 ◽  
Author(s):  
Martin Andres Estermann ◽  
Claire Elizabeth Hirst ◽  
Andrew Thomas Major ◽  
Craig Allen Smith
Keyword(s):  
Granulosa Cells ◽  
Interstitial Cell ◽  
Cell Lineage ◽  
Homeobox Gene ◽  
Cell Types ◽  
Surface Epithelium ◽  
Cell Populations ◽  
Supporting Cells ◽  
Homeobox Transcription Factor ◽  
Steroidogenic Cells

During early embryogenesis in amniotic vertebrates, the gonads differentiate into either ovaries or testes. The first cell lineage to differentiate gives rise to the supporting cells; Sertoli cells in males and pre-granulosa cells in females. These key cell types direct the differentiation of the other cell types in the gonad, including steroidogenic cells. The gonadal surface epithelium and the interstitial cell populations are less well studied, and little is known about their sexual differentiation programs. Here, we show the requirement of the homeobox transcription factor gene TGIF1 for ovarian development in the chicken embryo. TGIF1 is expressed in the two principal ovarian somatic cell populations, the cortex and the pre-granulosa cells of the medulla. TGIF1 expression is associated with an ovarian phenotype in estrogen-mediated sex reversal experiments. Targeted mis-expression and gene knockdown indicate that TGIF1 is required, but not sufficient, for proper ovarian cortex formation. In addition, TGIF1 is identified as the first known regulator of juxtacortical medulla development. These findings provide new insights into chicken ovarian differentiation and development, specifically cortical and juxtacortical medulla formation.

Surface Morphological Changes Of Hela Cells Exposed To Tamoxifen And Taxol

1999 ◽  
Vol 5 (S2) ◽  
pp. 1270-1271
Author(s):  
S.K. Majumdar ◽  
J. Valdellon
Keyword(s):  
Morphological Changes ◽  
Cell Types ◽  
Chemotherapeutic Agents ◽  
Taxus Brevifolia ◽  
Antimitotic Agent ◽  
The Pacific ◽  
Cervical Cancer Cells ◽  
Mammary Gland Cells ◽  
Human Cervical Cancer Cells

Tamoxifen and taxol are two promising chemotherapeutic agents currently used in cancer treatment, but each has its own unique mechanism of action. Tamoxifen, known to inhibit the activity of estrogen on malignant mammary gland cells, was recently approved by the FDA for the treatment of breast cancer. Taxol, extracted from the Pacific Yew tree, Taxus brevifolia, is an antimitotic agent that binds specifically to the β-subunit, promoting the assembly and stabilization of tubulin polymers. Studies have shown that tamoxifen and taxol induce cytostatic and cytotoxic effects leading to the death of several cancer cell types in vitro.In this research, the surface ultrastructural effects of both tamoxifen and taxol were investigated in human cervical cancer cells (HeLa).Exponentially growing cells were treated with 5, 10, 15, and 20 μg/ml of tamoxifen and 1 μg/ml of taxol (LC50) for 24, 48, 72, and 96 hours. For scanning electron microscopy (SEM), the cells were grown on coverslips, fixed in 2% glutaraldehyde followed by 1% osmium tetroxide.

scholarly journals Drug-Induced Naïve iPS Cells Exhibit Better Performance than Primed iPS Cells with Respect to the Ability to Differentiate into Pancreatic β-Cell Lineage

2020 ◽  
Vol 9 (9) ◽  
pp. 2838
Author(s):  
Yuki Kiyokawa ◽  
Masahiro Sato ◽  
Hirofumi Noguchi ◽  
Emi Inada ◽  
Yoko Iwase ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Cell Lineage ◽  
Ips Cells ◽  
Embryoid Bodies ◽  
Specific Marker ◽  
Β Cells ◽  
Β Cell ◽  
Pancreatic Β Cells

Pluripotent stem cells are classified as naïve and primed cells, based on their in vitro growth characteristics and potential to differentiate into various types of cells. Human-induced pluripotent stem cells (iPSCs, also known as epiblast stem cells [EpiSCs]) have limited capacity to differentiate and are slightly more differentiated than naïve stem cells (NSCs). Although there are several in vitro protocols that allow iPSCs to differentiate into pancreatic lineage, data concerning generation of β-cells from these iPSCs are limited. Based on the pluripotentiality of NSCs, it was hypothesized that NSCs can differentiate into pancreatic β-cells when placed under an appropriate differentiation induction condition. We examined whether NSCs can be efficiently induced to form potentially pancreatic β cells after being subjected to an in vitro protocol. Several colonies resembling in vitro-produced β-cell foci, with β-cell-specific marker expression, were observed when NSC-derived embryoid bodies (EBs) were induced to differentiate into β-cell lineage. Conversely, EpiSC-derived EBs failed to form such foci in vitro. Intrapancreatic grafting of the in vitro-formed β-cell foci into nude mice (BALB/c-nu/nu) generated a cell mass containing insulin-producing cells (IPCs), without noticeable tumorigenesis. These NSCs can be used as a promising resource for curing type 1 diabetes.

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