共查询到20条相似文献，搜索用时 0 毫秒
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The regulation of embryonic stem cell differentiation by leukaemia inhibitory factor (LIF) 总被引：8，自引：0，他引：8
LIF (leukaemia inhibitory factor) is commonly used to maintain mouse embryonic stem cells in an undifferentiated state. These cells spontaneously differentiate when allowed to aggregate in the absence of LIF, forming embryoid bodies in which early embryonic cell lineages develop. Using embryoid bodies cultured in the presence and absence of LIF, we show that although LIF inhibited the development of visceral and parietal endodermal cells, it did not affect the differentiation of the primitive endodermal cell precursors of these extraembryonic cell lineages. Furthermore, deposition of the basement membrane produced by the primitive endodermal cells, which separates them from the remaining cells of the embryoid body, still occurred. The differentiation of primitive ectodermal cells and their progeny was inhibited by LIF, as evidenced by the lack of expression of FGF-5, muscle, and neuronal markers. However, cavitation of the embryoid body and maintenance of the cells in contact with the primitive endodermal basement membrane as an epiblast epithelium still occurred normally in the presence of LIF. These results indicate that cavitation and formation of the epiblast epithelium are regulated by mechanisms distinct from those controlling the differentiation of epiblast cell lineages. Furthermore, although epithelium formation and cavitation do not require the differentiation of visceral endodermal cells, the results are consistent with the hypothesis that the primitive endodermal basement membrane is sufficient to induce the epithelialization of undifferentiated embryonic stem cells necessary for cavitation. 相似文献
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Nagaoka M Ise H Harada I Koshimizu U Maruyama A Akaike T 《Journal of cellular biochemistry》2008,103(1):296-310
Rearrangement of cell-cell adhesion is a critical event in embryonic development and tissue formation. We investigated the regulatory function of E-cadherin, a key adhesion protein, in the developmental process by using E-cadherin/IgG Fc fusion protein as an adhesion matrix in cell culture. F9 embryonal carcinoma cells usually form colonies when cultured on gelatin or fibronectin matrices. However, F9 cells cultured on the E-cadherin/IgG Fc fusion protein matrix formed a scattered distribution, with a different cytoskeletal organization and E-cadherin-rich protrusions that were regulated by Rac1 activity. The same scattering activity was observed in P19 embryonal carcinoma cells. In contrast, three types of differentiated cells, NMuMG mammary gland cells, MDCK kidney epithelial cells, and mouse primary isolated hepatocytes, did not show the scattering activity observed in F9 and P19 cells. These results suggest that migratory behavior on an E-cadherin-immobilized surface is only observed in embryonic cells, and that the regulatory mechanisms underlying E-cadherin-mediated cell adhesion vary with the state of differentiation. 相似文献
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Disabled-2 is essential for endodermal cell positioning and structure formation during mouse embryogenesis 总被引：4，自引：0，他引：4
Yang DH Smith ER Roland IH Sheng Z He J Martin WD Hamilton TC Lambeth JD Xu XX 《Developmental biology》2002,251(1):27-44
The signal transduction adapter protein Disabled-2 (Dab2) is one of the two mammalian orthologs of the Drosophila Disabled. The brain-specific Disabled-1 (Dab1) functions in positional organization of brain cells during development. Dab2 is widely distributed and is highly expressed in many epithelial cell types. The dab2 gene was interrupted by in-frame insertion of beta-galactosidase (LacZ) in embryonic stem cells and transgenic mice were produced. Dab2 expression was first observed in the primitive endoderm at E4.5, immediately following implantation. The homozygous Dab2-deficient mutant is embryonic lethal (earlier than E6.5) due to defective cell positioning and structure formation of the visceral endoderm. In E5.5 dab2 (-/-) conceptus, visceral endoderm-like cells are present in the deformed primitive egg cylinder; however, the visceral endoderm cells are not organized, the cells of the epiblast have not expanded, and the proamniotic cavity fails to form. Disorganization of the visceral endodermal layer is evident, as cells with positive visceral endoderm markers are scattered throughout the dab2 (-/-) conceptus. Only degenerated remains were observed at E6.5 for dab2 (-/-) embryos, and by E7.5, the defective embryos were completely reabsorbed. In blastocyst in vitro culture, initially cells with characteristics of endoderm, trophectoderm, and inner cell mass were observed in the outgrowth of the hatched dab2 (-/-) blastocysts. However, the dab2 (-/-) endodermal cells are much more dispersed and disorganized than those from wild-type blastocysts, the inner cell mass fails to expand, and the outgrowth degenerates by day 7. Thus, Dab2 is required for visceral endodermal cell organization during early mouse development. The absence of an organized visceral endoderm in Dab2-deficient conceptus leads to the growth failure of the inner cell mass. We suggest that Dab2 functions in a signal pathway to regulate endodermal cell organization using endocytosis of ligands from the blastocoel cavity as a positioning cue. 相似文献
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Shinjirou Kawazoe Nobuhito Ikeda Kengo Miki Masayuki Shibuya Kumi Morikawa Seiji Nakano Mitsuo Oshimura Ichiro Hisatome Yasuaki Shirayoshi 《Development, growth & differentiation》2009,51(2):81-93
Embryonic carcinoma (EC) cells, which are malignant stem cells of teratocarcinoma, have numerous morphological and biochemical properties in common with pluripotent stem cells such as embryonic stem (ES) cells. However, three EC cell lines (F9, P19 and PCC3) show different developmental potential and self‐renewal capacity from those of ES cells. All three EC cell lines maintain self‐renewal capacity in serum containing medium without Leukemia Inhibitory factor (LIF) or feeder layer, and show limited differentiation capacity into restricted lineage and cell types. To reveal the underlying mechanism of these characteristics, we took the approach of characterizing extrinsic factors derived from EC cells on the self‐renewal capacity and pluripotency of mouse ES cells. Here we demonstrate that EC cell lines F9 and P19 produce factor(s) maintaining the undifferentiated state of mouse ES cells via an unidentified signal pathway, while P19 and PCC3 cells produce self‐renewal factors of ES cells other than LIF that were able to activate the STAT3 signal; however, inhibition of STAT3 activation with Janus kinase inhibitor shows only partial impairment on the maintenance of the undifferentiated state of ES cells. Thus, these factors present in EC cells‐derived conditioned medium may be responsible for the self‐renewal capacity of EC and ES cells independently of LIF signaling. 相似文献
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《Cell Stem Cell》2019,24(6):983-994.e7
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Moore RN Dasgupta A Rajaei N Yarmush ML Toner M Larue L Moghe PV 《Biotechnology and bioengineering》2008,101(6):1332-1343
We examined the effects of co-cultivated hepatocytes on the hepatospecific differentiation of murine embryonic stem (ES) cells. Utilizing an established mouse ES cell line expressing high or low levels of E-cadherin, that we have previously shown to be responsive to hepatotrophic growth factor stimulation (Dasgupta et al., 2005. Biotechnol Bioeng 92(3):257-266), we compared co-cultures of cadherin-expressing ES (CE-ES) cells with cultured rat hepatocytes, allowing for either paracrine interactions (indirect co-cultures) or both juxtacrine and paracrine interactions (direct co-cultures, random and patterned). Hepatospecific differentiation of ES cells was evaluated in terms of hepatic-like cuboidal morphology, heightened gene expression of late maturation marker, glucose-6-phosphatase in relation to early marker, alpha-fetoprotein (AFP), and the intracellular localization of albumin. Hepatocytes co-cultured with growth factor primed CE-ES cells markedly enhanced ES cell differentiation toward the hepatic lineage, an effect that was reversed through E-cadherin blockage and inhibited in control ES cells with reduced cadherin expression. Comparison of single ES cell cultures versus co-cultures show that direct contact co-cultures of hepatocytes and CE-ES cells maximally promoted ES cell commitment towards hepatodifferentiation, suggesting cooperative effects of cadherin-based juxtacrine and paracrine interactions. In contrast, E-cadherin deficient mouse ES (CD-ES) cells co-cultured with hepatocytes failed to show increased G6P expression, confirming the role of E-cadherin expression. To establish whether albumin expression in CE-ES cells was spatially regulated by co-cultured hepatocytes, we co-cultivated CE-ES cells around micropatterned, pre-differentiated rat hepatocytes. Albumin localization was enhanced \"globally\" within CE-ES cell colonies and was inhibited through E-cadherin antibody blockage in all but an interfacial band of ES cells. Thus, stem cell based cadherin presentation may be an effective tool to induce hepatotrophic differentiation by leveraging both distal/paracrine and contact/juxtacrine interactions with primary cells of the liver. 相似文献
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Farnaz Mohajertehran Amirhossein Sahebkar Reza Zare Nooshin Mohtasham 《Journal of cellular physiology》2018,233(11):8499-8507
Oral squamous cell carcinoma (OSCC) is the most common type of head and neck cancer. Epithelial dysplasia is often initiated in the cells and cell nuclei adjacent to the epithelial cell membrane. Reduced cell–cell adhesions enable cancer cells to detach from the tumor and disseminate to other organs. The mutations in epithelial dysplasia markers such as E‐cadherin and epithelial cell adhesion molecules (CD326) can lead to proliferation, growth and survival of the tumor cells and persistence of numerous malignancies that play a key role in epithelial dysplasia of OSCC. Accordingly, these genes can be considered prognostic markers or potential therapeutic targets for the tailored management of patients with OSCC. The gene expression profile of OSCC stem cells indicates a differential pattern that facilitates establishing a cell signature. Owing to the highly tumorigenic behavior of cancer stem cells and the role of these cells in tumor differentiation, treatment resistance, relapse, and metastasis, we reviewed the role of stem cell markers in epithelial dysplasia and OSCC. 相似文献
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为了探索5-氨基咪唑-4-氨甲酰核糖核苷（AICAR）抑制小鼠F9 细胞（F9 embryonal carcinoma cells）增殖的作用机制,本文构建了Foxc1的慢病毒真核表达载体,通过实时定量PCR、免疫荧光染色、双荧光素酶报告基因检测系统以及细胞增殖检测试验,探索AICAR抑制小鼠F9细胞的增殖作用机制. 结果发现,AICAR可以在RNA和蛋白水平促进Foxc1的基因表达,并可以作用于核转录因子κB通路. 另外在培养液中添加AICAR或过表达Foxc1都能抑制F9细胞的增殖. 信号通路报告载体检测发现Foxc1可以激活核转录因子κB通路以及细胞周期相关的通路. 总之,本研究证明,AICAR 通过激活Foxc1通路及其下游多条信号通路来抑制F9细胞增殖. 相似文献
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Hao H Chen C Rao XM Gomez-Gutierrez JG Zhou HS McMasters KM 《Journal of cellular and molecular medicine》2012,16(3):605-615
E2F-1-deleted mutant, 'truncated E2F' (E2Ftr, E2F-1[1-375]), lacking the carboxy-terminal transactivation domain, was shown to be more potent at inducing cancer cell apoptosis than wild-type E2F-1 (wtE2F-1; full-length E2F-1). Mechanisms by which wtE2F-1 and E2Ftr induce apoptosis, however, are not fully elucidated. Our study demonstrates molecular effects of pro-apoptotic BH3-only Bcl-2 family member Harakiri (Hrk) in wtE2F-1- and E2Ftr-induced melanoma cell apoptosis. We found that Hrk mRNA and Harakiri (HRK) protein expression was highly up-regulated in melanoma cells in response to wtE2F-1 and E2Ftr overexpression. HRK up-regulation did not require the E2F-1 transactivation domain. In addition, Hrk gene up-regulation and HRK protein expression did not require p53 in cancer cells. Hrk knockdown by Hrk siRNA was associated with significantly reduced wtE2F-1- and E2Ftr-induced apoptosis. We also found that an upstream factor, 'downstream regulatory element antagonist modulator' (DREAM), may be involved in HRK-mediated apoptosis in response to wtE2F-1 and E2Ftr overexpression. DREAM expression levels increased following wtE2F-1 and E2Ftr overexpression. Western blotting detected increased DREAM primarily in dimeric form. The homodimerization of DREAM resulting from wtE2F-1 and E2Ftr overexpression may contribute to the decreased binding activity of DREAM to the 3'-untranslated region of the Hrk gene as shown by electromobility shift assay. Results showed wtE2F-1- and E2Ftr-induced apoptosis is partially mediated by HRK. HRK function is regulated in response to DREAM. Our findings contribute to understanding the mechanisms that regulate wtE2F-1- and E2Ftr-induced apoptosis and provide insights into the further evaluation of how E2Ftr-induced apoptosis may be used for therapeutic gain. 相似文献
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Teratocarcinoma stem cell F9 expressed a potent fucosyltransferase activity acting on asialofetuin. A majority of the product was susceptible to alpha-L-fucosidase I from almond emulsin, indicating that the linkage formed was mainly Fuc alpha 1 leads to 3GlcNAc. The specific activity of the transferase decreased when the stem cells were induced to differentiate into parietal endoderm cells by retinoic acid and dibutyryl cyclic AMP. Furthermore, PYS-2 cell, a parietal endoderm cell line virtually lacked the transferase. The change in the fucosyltransferase activity could be correlated with cell surface changes occurring during differentiation. 相似文献
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Previous studies have shown that there is a strict requirement for fibroblast growth factor-4 (FGF-4) during mammalian embryogenesis, and that FGF-4 expression in embryonic stem (ES) cells and embryonal carcinoma (EC) cells are controlled by a powerful downstream distal enhancer. More recently, mouse ES cells were shown to express significantly more FGF-4 mRNA than human ES cells. In the work reported here, we demonstrate that mouse EC cells also express far more FGF-4 mRNA than human EC cells. Using a panel of FGF-4 promoter/reporter gene constructs, we demonstrate that the enhancer of the mouse FGF-4 gene is approximately tenfold more active than its human counterpart. Moreover, we demonstrate that the critical difference between the mouse and the human FGF-4 enhancer is a 4 bp difference in the sequence of an essential GT box. Importantly, we demonstrate that changing 4 bp in the human enhancer to match the sequence of the mouse GT box elevates the activity of the human FGF-4 enhancer to the same level as that of the mouse enhancer. We extended these studies by examining the roles of Sp1 and Sp3 in FGF-4 expression. Although we demonstrate that Sp3, but not Sp1, can activate the FGF-4 promoter when artificially tethered to the FGF-4 enhancer, we show that Sp3 is not essential for expression of FGF-4 mRNA in mouse ES cells. Finally, our studies with human EC cells suggest that the factor responsible for mediating the effect of the mouse GT box is unlikely to be Sp1 or Sp3, and this factor is either not expressed in human EC cells or it is not sufficiently active in these cells. 相似文献
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Sin Y. Choi Hae J. Kee Thomas Kurz Finn K. Hansen Yuhee Ryu Gwi R. Kim Ming Q. Lin Li Jin Zhe H. Piao Myung H. Jeong 《Journal of cellular and molecular medicine》2016,20(12):2289-2298
Epithelial‐mesenchymal transition (EMT) and renal fibrosis are closely involved in chronic kidney disease. Inhibition of histone deacetylase (HDAC) has an anti‐fibrotic effect in various diseases. However, the pathophysiological role of isoform‐specific HDACs or class‐selective HDACs in renal fibrosis remains unknown. Here, we investigated EMT markers and extracellular matrix (ECM) proteins in a human proximal tubular cell line (HK‐2) by using HDAC inhibitors or by knockdown of class I HDACs (HDAC1, 2, 3 and 8). Trichostatin A (TSA), MS275, PCI34051 and LMK235 inhibited ECM proteins such as collagen type I or fibronectin in transforming growth factor β1 (TGF‐β1)‐induced HK2 cells. However, restoration of TGF‐β1‐induced E‐cadherin down‐regulation was only seen in HK‐2 cells treated with TSA or MS275, but not with PCI34051, whereas TGF‐β1‐induced N‐cadherin expression was not affected by the inhibitors. ECM protein and EMT marker levels were prevented or restored by small interfering RNA transfection against HDAC8, but not against other class I HDACs (HDAC1, 2 and 3). E‐cadherin regulation is mediated by HDAC8 expression, but not by HDAC8 enzyme activity. Thus, class I HDACs (HDAC1, 2, 3 and 8) play a major role in regulating ECM and EMT, whereas class IIa HDACs (HDAC4 and 5) are less effective. 相似文献
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HSPs (heat shock proteins) have been recognized to maintain cellular homoeostasis during changes in microenvironment. The present study aimed to investigate the HSPs expression pattern in hierarchical neural differentiation stages from mouse embryonal carcinoma stem cells (P19) and its role in heat stressed exposed cells. For induction of HSPs, cells were heated at 42°C for 30 min and recovered at 37°C in different time points. For neural differentiation, EBs (embryoid bodies) were formed by plating P19 cells in bacterial dishes in the presence of 1 mM RA (retinoic acid) and 5% FBS (fetal bovine serum). Then, on the sixth day, EBs were trypsinized and plated in differentiation medium containing neurobasal medium, B27, N2 and 5% FBS and for an extra 4 days. The expression of HSPs and neural cell markers were evaluated by Western blot, flow cytometry and immunocytochemistry in different stages. Our results indicate that HSC (heat shock constant)70 and HSP60 expressions decreased following RA treatment, EB formation and in mature neural cells derived from heat-stressed single cells and not heat-treated EBs. While the level of HSP90 increased six times following maturation process, HSP25 was expressed constantly during neural differentiation; however, its level was enhanced with heat stress. Accordingly, heat shock 12 h before the initiation of differentiation did not affect the expression of neuroectodermal and neural markers, nestin and β-tubulin III, respectively. However, both markers increased when heat shock was induced after treatment and when EBs were formed. In conclusion, our results raise the possibility that HSPs could regulate cell differentiation and proliferation under both physiological and pathological conditions. 相似文献
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《中国生物化学与分子生物学报》2021,37(9):1188-1196
分化的体细胞可通过体外过表达多能性相关的转录因子重编程为诱导多能干细胞(induced pluripotent stem cells,iPSCs)。体细胞重编程的过程需要细胞内各个转录因子相互作用,协同调控细胞命运的转变。同源异形框D12(homeobox D12,Hoxd12)是调控脊椎动物胚胎发育的关键转录因子之一,对足趾发育、体轴形成和细胞内信号转导具有重要作用。但Hoxd12关于体细胞重编程和胚胎干细胞(embryonic stem cells,ESCs)方面的研究未见报道。本研究首先以7因子(Sall4-Esrrb-Jdp2-Glis1-Mkk6-Nanog-Kdm2b,7 factors)和Yamanka因子(Oct4-Klf4-Sox2,Yamanaka factors)诱导的重编程体系为模型,并结合RNA敲降(shRNA)和基因过表达技术,研究Hoxd12在体细胞重编程中的功能;其次利用CRISPR/Cas9基因编辑技术构建Hoxd12基因敲除的胚胎干细胞系,同时结合体外拟胚体形成(EB)和转录物组测序技术(RNA-seq),探索Hoxd12在ESCs多能性维持和退出中的功能。本研究得出以下结论:(1)敲降Hoxd12的表达抑制7因子诱导的重编程(**P<0.01),但对Yamanaka因子诱导的重编程无显著作用。(2)在7因子和Yamanaka因子诱导多能性的不同时期过表达Hoxd12对重编程无显著作用,且Hoxd12无法替代7因子当中的任何1个因子。(3)Hoxd12基因的敲除对ESCs多能性的维持无显著影响。(4)Hoxd12基因的敲除对ESCs在EB球形成中的谱系决定无显著影响。(5)Hoxd12基因的敲除抑制ESCs的增殖(Day_3t = 12.91, Day_4t = 4.87, Day_5t = 5.98)。本研究初步揭示了Hoxd12在体细胞重编程和ESCs增殖中具有重要作用,为进一步深入理解Hoxd12调控细胞命运转变提供新的参考。 相似文献
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《中国生物化学与分子生物学报》2021,37(9):1188-1196
分化的体细胞可通过体外过表达多能性相关的转录因子重编程为诱导多能干细胞(induced pluripotent stem cells,iPSCs)。体细胞重编程的过程需要细胞内各个转录因子相互作用,协同调控细胞命运的转变。同源异形框D12(homeobox D12,Hoxd12)是调控脊椎动物胚胎发育的关键转录因子之一,对足趾发育、体轴形成和细胞内信号转导具有重要作用。但Hoxd12关于体细胞重编程和胚胎干细胞(embryonic stem cells,ESCs)方面的研究未见报道。本研究首先以7因子(Sall4-Esrrb-Jdp2-Glis1-Mkk6-Nanog-Kdm2b,7 factors)和Yamanka因子(Oct4-Klf4-Sox2,Yamanaka factors)诱导的重编程体系为模型,并结合RNA敲降(shRNA)和基因过表达技术,研究Hoxd12在体细胞重编程中的功能;其次利用CRISPR/Cas9基因编辑技术构建Hoxd12基因敲除的胚胎干细胞系,同时结合体外拟胚体形成(EB)和转录物组测序技术(RNA-seq),探索Hoxd12在ESCs多能性维持和退出中的功能。本研究得出以下结论:(1)敲降Hoxd12的表达抑制7因子诱导的重编程(**P<0.01),但对Yamanaka因子诱导的重编程无显著作用。(2)在7因子和Yamanaka因子诱导多能性的不同时期过表达Hoxd12对重编程无显著作用,且Hoxd12无法替代7因子当中的任何1个因子。(3)Hoxd12基因的敲除对ESCs多能性的维持无显著影响。(4)Hoxd12基因的敲除对ESCs在EB球形成中的谱系决定无显著影响。(5)Hoxd12基因的敲除抑制ESCs的增殖(Day_3t = 12.91, Day_4t = 4.87, Day_5t = 5.98)。本研究初步揭示了Hoxd12在体细胞重编程和ESCs增殖中具有重要作用,为进一步深入理解Hoxd12调控细胞命运转变提供新的参考。 相似文献