共查询到20条相似文献,搜索用时 15 毫秒
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Lee YJ Nah HY Hong SH Lee JW Jeon I Pak JH Huh JR Kim SH Chae HD Kang BM Kim CG Kim CH 《The International journal of developmental biology》2008,52(1):43-53
Human embryonic stem cells (hESCs) are pluripotent, whereby they can proliferate endlessly and differentiate into many different cell types. At the molecular level, little is known of the mechanisms underlying their capability for self-renewal and differentiation. In the present study, we established two new hESC lines (AMC-hES1 and AMC-hES2) and demonstrated the existence of a regulator that may be a key molecule in hESC dynamics. Spa-1 is a principal Ras-proximate 1 (Rap1) GTPase-activating protein in hematopoietic progenitor cells that regulates Rap1-related signal transduction and is expressed restrictively in human adult tissues (bone marrow, thymus, and spleen). To investigate its functions in hESCs, we examined spa-1 expression profiles during hESC differentiation and used RNA interference (RNAi) to downregulate spa-1 in these cells. Our results show that Spa-1 is expressed in undifferentiated hESCs and is downregulated during hESC differentiation. In addition, the process of passing from the mode of self-renewal to that of differentiation in hESCs was regulated by spa-1 via Rap1/Raf/mitogen-activated protein kinase kinase/extracellular signal-related kinase signaling. An RNAi expression vector against spa-1 (pSUPER.retro.puro) was transfected into hESCs, which were seen to differentiate into three germ layers in spite of being in the undifferentiated condition. Based on our findings, therefore, it appears that spa-1 may be involved in hESC dynamics, and our results provide fundamental information regarding the self-renewal and differentiation of hESCs. 相似文献
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During human pregnancy, the production of 17-beta-estradiol (E2) rises steadily to eighty fold at term, and placenta has been
found to specifically bind estrogens. We have recently demonstrated the expression of estrogen receptor alpha (ER-alpha) protein
in human placenta and its localization in villous cytotrophoblast (CT), vascular pericytes, and amniotic fibroblasts. In vitro,
E2 stimulated development of large syncytiotrophoblast (ST) aggregates. In the present study we utilized ER-beta affinity
purified polyclonal (N19:sc6820) and ER-alpha monoclonal (clone h-151) antibodies. Western blot analysis revealed a single
~52 kDa ER-beta band in chorionic villi (CV) protein extracts. In CV, strong cytoplasmic ER-beta immunoreactivity was confined
to ST. Dual color immunohistochemistry revealed asymmetric segregation of ER-alpha in dividing villous CT cells. Prior to
separation, the cell nuclei more distant from ST exhibited high ER-alpha, while cell nuclei associated with ST showed diminution
of ER-alpha and appearance of ER-beta. In trophoblast cultures, development of ST aggregates was associated with diminution
of ER-alpha and appearance of ER-beta immunoreactivity. ER-beta was also detected in endothelial cells, amniotic epithelial
cells and fibroblasts, extravillous trophoblast (nuclear and cytoplasmic) and decidual cells (cytoplasmic only). In addition,
CFK-E12 (E12) and CWK-F12 (F12) monoclonal antibodies, which recognize ~64 kDa ER-beta with hormone binding domain, showed
nuclear-specific reactivity with villous ST, extravillous trophoblast, and amniotic epithelium and fibroblasts. Western blot
analysis indicated abundant expression of a ~64 kDa ER-beta variant in trophoblast cultures, significantly higher when compared
to the chorionic villi and freshly isolated trophoblast cell protein extracts. This is the first report on ER-beta expression
in human placenta and cultured trophoblast. Our data indicate that during trophoblast differentiation, the ER-alpha is associated
with a less, and ER-beta with the more differentiated state. Enhanced expression of ~64 kDa ER-beta variant in trophoblast
cultures suggests a unique role of ER-beta hormone binding domain in the regulation of trophoblast differentiation. Our data
also indicate that asymmetric segregation of ER-alpha may play a role in asymmetric division of estrogen-dependent cells. 相似文献
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There is scarce information about the factors associated with estrogen receptors (ER) at menopause. In 113 volunteers pre- and post-menopausal healthy women, grouped as with and without obesity, estrogen receptors-alpha and -beta, and progesterone receptor (PR) were measured by immunohistochemistry in skin punch biopsies obtained from the external gluteal area. In pre-menopausal women, biopsies and a blood sample were performed between days 7 and 14 of the cycle. Serum hormone levels were measured by immunoradiometric assay or radioimmunoassay. After menopause, ER and PR amounts decreased significantly. At pre-menopause, obese women had lower PR levels than non obese (P<.006). In the post-menopausal group, obese women showed higher ER-alpha (P<.03) and ER-beta (P<.02) levels than the non obese group. In the analysis of factors associated with the amount of steroid receptors for the total group, log[ER-alpha], log[ER-beta], and log[PR] were associated with age (P<.002, <.005, and <.004, respectively). The log[ER-alpha] was also associated with log[FSH] (P<.0008); meanwhile, the log[PR] showed a marginal correlation with log[FSH]. In pre-menopausal women no factor associated with any of the three receptors was found. In post-menopausal women log[ER-alpha] was associated with log[estrone] and log[DHEAS] (P<.003 and <.02, respectively). log[PR] was associated with BMI (P<.002), years since menopause (P<.05), and log[DHEAS] (P<.003). We concluded that ER and PR diminish sharply at post-menopause. At this stage the amount of receptors depends on several factors such as BMI, years since menopause, and androgen precursors. 相似文献
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Vittoria Rago Laura Siciliano Saveria Aquila Amalia Carpino 《Reproductive biology and endocrinology : RB&E》2006,4(1):36-6
Background
A key role of estrogens in human sperm biology has been recently suggested by aromatase and estrogen receptor detection in human testicular germ cells and ejaculated spermatozoa. However, the involvement of these hormones in the sperm maturation process is still not defined. The aim of this work was to investigate the expression of estrogen receptors, ER-alpha and ER-beta, in human ejaculated immature spermatozoa with excess residual cytoplasm. 相似文献9.
目的:比较通过慢病毒方法获得的人诱导多能性干细胞(iPSCs)与人胚胎干细胞(hESCs)分化过程中全能性基因Oct4、Nanog的表达变化。方法:收集分化不同时间点的拟胚体(EBs),检测三胚层分化基因以及全能性基因Oct4/Nanog的表达,并通过畸胎瘤组织切片的荧光染色分析Oct4的表达。结果:iPSCs获得的EB中内外三胚层分化基因表达的出现明显晚于hESCs来源的EB。不同于hESCs,iPSCs悬浮培养获得的EBs在体外培养18天未见内源性Oct4、Nanog基因表达的下调。未分化的iPSCs注射严重联合免疫缺陷(SCID)小鼠培养10周后获得的畸胎瘤中仍存在Oct4阳性的细胞,但iPS-#2中明显少于iPS-#5。结论:通过慢病毒方法获得的iPSCs虽然具有向三胚层分化的能力,但在分化过程中仍维持较高水平的全能性基因Oct4、Nanog的表达。 相似文献
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Prion protein (PrPC), is a glycoprotein that is expressed on the cell surface. The current study examines the role of PrPC in early human embryogenesis using human embryonic stem cells (hESCs) and tetracycline‐regulated lentiviral vectors that up‐regulate or suppresses PrPC expression. Here, we show that expression of PrPC in pluripotent hESCs cultured under self‐renewal conditions induced cell differentiation toward lineages of three germ layers. Silencing of PrPC in hESCs undergoing spontaneous differentiation altered the dynamics of the cell cycle and changed the balance between the lineages of the three germ layers, where differentiation toward ectodermal lineages was suppressed. Moreover, over‐expression of PrPC in hESCs undergoing spontaneous differentiation inhibited differentiation toward lineages of all three germ layers and helped to preserve high proliferation activity. These results illustrate that PrPC is involved in key activities that dictate the status of hESCs including regulation of cell cycle dynamics, controlling the switch between self‐renewal and differentiation, and determining the fate of hESCs differentiation. This study suggests that PrPC is at the crossroads of several signaling pathways that regulate the switch between preservation of or departure from the self‐renewal state, control cell proliferation activity, and define stem cell fate. 相似文献
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The protein ER-alpha has been exhaustively characterized in estrogen-sensitive tissues and cell lines. However, little is known regarding the expression and cellular distribution of the newly identified ER-beta protein. We first quantified the specific estradiol binding site content in the estrogen-responsive cell lines MCF-7 (mammary) and SHM (myometrial). In the two cell types, these sites were associated to the expression of both ER-alpha and -beta isoforms. Native ER-beta was visualized to reside inside the nucleus by means of conventional indirect immunofluorescence. The cells expressed ER-beta as a tight approximately 50 kDa triplet when resolved by sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and blotted using antibodies mapping different domains of the cloned ER-beta version. When the cells were subjected to homogenization and differential centrifugation, a substantial proportion of ER-beta immunolabeling was localized at membrane subfractions. ER-beta expression and partitioning was confirmed by Ligand blotting assays using estrogen derivatives coupled to different macromolecular tags. However, ER-alpha was expressed as the major estrogen binding protein in both cell lines. Similar localization experiments were performed on HeLa cells (cervix). Though usually considered ER-negative, this cell line displayed basal significant estrogen binding capacity and co-expression of both ER isoforms. Taken as a whole, the results indicate that ER-beta could be expressed as functional estrogen binding proteins among a dominant population of ER-alpha sites in the cell lines under study. 相似文献
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Pluripotent human embryonic stem cell (hESC) lines are a promising model system in developmental and tissue regeneration research.
Differentiation of hESCs towards the three germ layers and finally tissue specific cell types is often performed through the
formation of embryoid bodies (EBs) in suspension or hanging droplet culture systems. However, these systems are inefficient
regarding embryoid body (EB) formation, structural support to the EB and long term differentiation capacity. The present study
investigates if agarose, as a semi solid matrix, can facilitate EB formation and support differentiation of hESC lines. The
results showed that agarose culture is able to enhance EB formation efficiency with 10% and increase EB growth by 300%. The
agarose culture system was able to maintain expression of the three germ layers over 8 weeks of culture. All of the four hESC
lines tested developed EBs in the agarose system although with a histological heterogeneity between cell lines as well as
within cell lines. In conclusion, a 3-D agarose culture of spherical hESC colonies improves EB formation and growth in a cost
effective, stable and non-laborious technique. 相似文献
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Differential expression of mRNAs for PACAP and its receptors during neural differentiation of embryonic stem cells 总被引:1,自引:0,他引:1
Hirose M Hashimoto H Shintani N Nakanishi M Arakawa N Iga J Niwa H Miyazaki J Baba A 《Regulatory peptides》2005,126(1-2):109-113
The expressions of mRNAs for pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal peptide (VIP), and their receptors (PAC1, VPAC1 and VPAC2) were examined in the five steps of the in vitro neuronal culture model of embryonic stem (ES) cell differentiation. mRNAs for PACAP, VIP, PAC1 receptor, and VPAC2 receptor were moderately expressed in neural stem cell-enriched cultures, while VPAC1 receptor mRNA was most prominently expressed in embryoid bodies (EBs). The expression of PAC1 receptor mRNA was further upregulated after terminal differentiation into neurons. In contrast, the expressions of PAC1 receptor and PACAP mRNAs were markedly decreased after glial differentiation. These results suggest that this in vitro neuronal culture system will be a useful model for future studies on the functional role of the PACAPergic system during different stages of neuronal development. 相似文献
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目的:比较通过慢病毒方法获得的人诱导多能性干细胞(iPSCs)与人胚胎干细胞(hESCs)分化过程中全能性基因Oct4、Nanog的表达变化。方法:收集分化不同时间点的拟胚体(EBs),检测三胚层分化基因以及全能性基因Oct4/Nanog的表达,并通过畸胎瘤组织切片的荧光染色分析Oct4的表达。结果:iPSCs获得的EB中内外三胚层分化基因表达的出现明显晚于hESCs来源的EB。不同于hESCs,iPSCs悬浮培养获得的EBs在体外培养18天未见内源性Oct4、Nanog基因表达的下调。未分化的iPSCs注射严重联合免疫缺陷(SCID)小鼠培养10周后获得的畸胎瘤中仍存在Oct4阳性的细胞,但iPS-#2中明显少于iPS-#5。结论:通过慢病毒方法获得的iPSCs虽然具有向三胚层分化的能力,但在分化过程中仍维持较高水平的全能性基因Oct4、Nanog的表达。 相似文献
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Maria Sundberg Linda Jansson Johanna Ketolainen Harri Pihlajamäki Riitta Suuronen Heli Skottman José Inzunza Outi Hovatta Susanna Narkilahti 《Stem cell research》2009,2(2):113-124
Human embryonic stem cells (hESCs) are pluripotent cells that can differentiate into neural cell lineages. These neural populations are usually heterogeneous and can contain undifferentiated pluripotent cells that are capable of producing teratomas in cell grafts. The characterization of surface protein profiles of hESCs and their neural derivatives is important to determine the specific markers that can be used to exclude undifferentiated cells from neural populations. In this study, we analyzed the cluster of differentiation (CD) marker expression profiles of seven undifferentiated hESC lines using flow-cytometric analysis and compared their profiles to those of neural derivatives. Stem cell and progenitor marker CD133 and epithelial adhesion molecule marker CD326 were more highly expressed in undifferentiated hESCs, whereas neural marker CD56 (NCAM) and neural precursor marker (chemokine receptor) CD184 were more highly expressed in hESC-derived neural cells. CD326 expression levels were consistently higher in all nondifferentiated hESC lines than in neural cell derivatives. In addition, CD326-positive hESCs produced teratomas in SCID mouse testes, whereas CD362-negative neural populations did not. Thus, CD326 may be useful as a novel marker of undifferentiated hESCs to exclude undifferentiated hESCs from differentiated neural cell populations prior to transplantation. 相似文献
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Denning C Allegrucci C Priddle H Barbadillo-Muñoz MD Anderson D Self T Smith NM Parkin CT Young LE 《The International journal of developmental biology》2006,50(1):27-37
Development of generic differentiation protocols that function in a range of independently-derived human embryonic stem cell (hESC) lines remains challenging due to considerable diversity in culture methods practiced between lines. Maintenance of BG01 and HUES-7 has routinely been on mouse embryonic fibroblast (MEF) feeder layers using manual- and trypsin-passaging, respectively. We adapted both lines to trypsin-passaging on feeders or on Matrigel in feeder-free conditions and assessed proliferation and cardiac differentiation. On feeders, undifferentiated proliferation of BG01 and HUES-7 was supported by all three media tested (BG-SK, HUES-C and HUES-nL), although incidence of karyotypic instability increased in both lines in BG-SK. On Matrigel, KSR-containing conditioned medium (CM) promoted undifferentiated cell proliferation, while differentiation occurred in CM containing Plasmanate or ES-screened Fetal Bovine Serum (FBS) and in unconditioned medium containing 100 ng/ml bFGF. Matrigel cultures were advantageous for transfection but detrimental to embryoid body (EB) formation. However, transfer of hESCs from Matrigel back to feeders and culturing to confluence was found to rescue EB formation. EBs formed efficiently when hESCs on feeders were treated with collagenase, harvested by scraping and then cultured in suspension in CM. Subsequent culture in FBS-containing medium produced spontaneously contracting EBs, for which the mean beat rate was 37.2 +/- 2.3 and 41.1 +/- 3.1 beats/min for BG01-EBs and HUES-7-EBs, respectively. Derived cardiomyocytes expressed cardiac genes and responded to pharmacological stimulation. Therefore the same culture and differentiation conditions functioned in two independently-derived hESC lines. Similar studies in other lines may facilitate development of universal protocols. 相似文献
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Harmston WR Taddayon P Kolman K Chandar N 《In vitro cellular & developmental biology. Animal》2005,41(8-9):264-271
Summary Our study focused on investigating the mechanism of action of estrogen in regulating p53 levels within osteoblasts. In the
studies reported here, we attempted to understand the role of estrogen receptors, ER-alpha and ER-beta, in the regulation
of p53 and osteoblast differentiation. We stably expressed ER-alpha and ER-beta in ROS 17/2.8 cells and isolated several single
cell clones. These clones were initially characterized for expression of the exogenous receptors, and representative clones
from each type were chosen for further analyses. Cell proliferation, alkaline phosphatase activity, and the viability of these
clones in culture were tested. The cells expressing exogenous ER-alpha exhibited more differentiated characteristics than
cells expressing ER-beta. Morphologically, ER-beta-overexpressing cells were more rounded than the ER-alpha-overexpressing
cells, which were more elongated and fibroblastic in appearance. The ER-beta-expressing cells had a higher survival and growth
rate when compared with ER-alpha cells. The ER-alpha clones were not as viable as ER-beta clones, and some of the ER-alpha
cell lines showed signs of senescence, with an increase in senescence-associated (SA) galactosidase activity. The basal levels
of p53 functional activity were higher in cells expressing ER-alpha as was protein expression of the p53-regulated gene p21.
The significance of these receptors to osteoblast differentiation and p53 regulation is discussed. 相似文献