共查询到20条相似文献,搜索用时 15 毫秒
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Saswati Banerjee 《Experimental cell research》2010,316(2):172-412
Understanding endothelial cell (EC) differentiation is a step forward in tissue engineering, controlling angiogenesis, and endothelial dysfunction. We hypothesized that epigenetic activation of EC lineage specification genes is an important mediator of embryonic stem cell (ESC) differentiation into EC. Mouse ESC was differentiated by removing leukemia inhibitory factor (LIF) from the maintenance media in the presence or absence of the specific DNA methyltransferase (DNMT) inhibitor 5′-aza-2′-deoxycytidine (aza-dC). Expression of EC specification and marker genes was monitored by quantitative PCR, western, immunocytochemistry, and flow cytometry. Functionality of differentiated EC was assessed by angiogenesis assay. The methylation status in the proximal promoter CpGs of the mediators of EC differentiation VEGF-A, BMP4, and EPAS-1 as well as of the mature EC marker VE-cadherin was determined by bisulfite sequencing. ESC differentiation resulted in repression of OCT4 expression in both the absence and presence of aza-dC treatment. However, significant increase in angiogenesis and expression of the mediators of EC differentiation and EC-specific genes was only observed in aza-dC-treated cells. The DNMT inhibition-mediated increase in EC specification and marker gene expression was not associated with demethylation of these genes. These studies suggest that DNMT inhibition is an efficient inducer of EC differentiation from ESC. 相似文献
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Makoolati Z Movahedin M Forouzandeh-Moghadam M 《In vitro cellular & developmental biology. Animal》2011,47(5-6):391-398
Presence of specific growth factors and feeder layers are thought to be important for in vitro embryonic stem cell (ESCs) differentiation. In this study, the effect of bone morphogenetic protein 4 (BMP4) and mouse embryonic fibroblasts (MEFs) co-culture system on germ cell differentiation from mouse ESCs was evaluated. One-day-old embryoid body was cultured for 4?d in simple culture systems or on top of the MEFs, both in the presence or absence of BMP4. Data showed significant higher viability percent and proliferation rate in simple culture media compared to co-culture systems. Analysis of gene expression indicated that the germ cell-specific genes (VASA and Stra8) were expressed in a significant higher ratio in BMP4-treated cells in simple culture system. Also, the results of immunocytochemistry in simple culture systems showed that the mean percentage of immunostaining cells of VASA, the primordial germ cell (PGC) marker, was increased significantly in BMP4-treated cells compared with BMP4-free group. Meanwhile, CDH1, the late premiotic germ cell marker, showed no significant difference between these two groups. The results suggest that BMP4 is an efficient inducer in PGC derivation from mouse ESC. However, the employment of MEFs as feeder has no apparent effect on PGC derivation. 相似文献
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Nishikawa M Yanagawa N Kojima N Yuri S Hauser PV Jo OD Yanagawa N 《Biochemical and biophysical research communications》2012,417(2):897-902
The in vitro derivation of renal lineage progenitor cells is essential for renal cell therapy and regeneration. Despite extensive studies in the past, a protocol for renal lineage induction from embryonic stem cells remains unestablished. In this study, we aimed to induce renal lineages from mouse embryonic stem cells (mESC) by following in vivo developmental stages, i.e., the induction of mesoderm (Stage I), intermediate mesoderm (Stage II) and renal lineages (Stage III). For stage I induction, in accordance with known signaling pathways involved in mesoderm development in vivo, i.e., Nodal, bone morphogenic proteins (BMPs) and Wnt, we found that the sequential addition of three factors, i.e., Activin-A (A), a surrogate for Nodal signaling, during days 0-2, A plus BMP-4 (4) during days 2-4, and A4 plus lithium (L), a surrogate for Wnt signaling, during days 4-6, was most effective to induce the mesodermal marker, Brachyury. For stage II induction, the addition of retinoic acid (R) in the continuous presence of A4L during days 6-8 was most effective to induce nephrogenic intermediate mesodermal markers, such as Pax2 and Lim1. Under this condition, more than 30% of cells were stained positive for Pax2, and there was a concomitant decrease in the expression of non-mesodermal markers. For stage III induction, in resemblance to the reciprocal induction between ureteric bud (UB) and metanephric mesenchyme (MM) during kidney development, we found that the exposure to conditioned media derived from UB and MM cells was effective in inducing MM and UB markers, respectively. We also observed the emergence and gradual increase of cell populations expressing progenitor cell marker CD24 from Stage I to Stage III. These CD24(+) cells correlated with higher levels of expression of Brachyury at stage I, Pax2 and Lim1 at stage II and MM markers, such as WT1 and Cadherin 11, after exposure to UB-conditioned media at stage III. In conclusion, our results show that stepwise induction by tracing in vivo developmental stages was effective to generate renal lineage progenitor cells from mESC, and CD24 may serve as a useful surface marker for renal lineage cells at stage II and MM cells at stage III. 相似文献
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Naoto Ninomiya Tatsuo Michiue Makoto Asashima Akira Kurisaki 《In vitro cellular & developmental biology. Animal》2013,49(3):230-237
Somatic stem/progenitor cells are known to be present in most adult tissues. However, those in the lung have limited abilities for tissue regeneration after serious damage as a result of chronic disease. Therefore, regenerative medicine using exogenous stem cells has been suggested for the treatment of progressive lung diseases such as chronic obstructive pulmonary disease and pulmonary fibrosis. Embryonic stem (ES) cells and induced pluripotent stem cells, with their potent differentiation abilities, are promising sources for the generation of various tissue cells. In this study, we investigated the effects of various differentiation-inducing growth factors on the differentiation of lung cells from ES cells in vitro. Several factors, including activin, nodal, and noggin, significantly promoted the induction of Nkx2.1-positive lung progenitor cells when cells were cultured as embryoid bodies. Bone morphogenetic protein (BMP) 4 signaling controls the lineage commitment of lung cells along the proximal–distal axis. BMP4 promotes the induction of distal cell lineages of alveolar bud, such as Clara cells and mucus-producing goblet cells. These results suggest that several developmentally essential factors, including nodal/activin and BMP signaling, are important in the control of the differentiation of lung epithelial cells from mouse ES cells in vitro. 相似文献
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Santiago Nahuel Villegas Maurice Canham Joshua M. Brickman 《Journal of cellular biochemistry》2010,110(1):10-20
The fibroblast growth factor (FGF) signalling pathway is one of the most ubiquitous in biology. It has diverse roles in development, differentiation and cancer. Embryonic stem (ES) cells are in vitro cell lines capable of differentiating into all the lineages of the conceptus. As such they have the capacity to differentiate into derivatives of all three germ layers and to some extent the extra‐embryonic lineages as well. Given the prominent role of FGF signalling in early embryonic development, we explore the role of this pathway in early ES cell differentiation towards the major lineages of the embryo. As early embryonic differentiation is intricately choreographed at the level of morphogenetic movement, adherent ES cell culture affords a unique opportunity to study the basic steps in early lineage specification in the absence of ever shifting complex in vivo microenvironments. Thus recent experiments in ES cell differentiation are able to pinpoint specific FGF dependent lineage transitions that are difficult to resolve in vivo. Here we review the role of FGF signalling in early development alongside the ES cell data and suggest that FGF dependent signalling via phospho‐Erk activation maybe a major mediator of transitions in lineage specification. J. Cell. Biochem. 110: 10–20, 2010. © 2010 Wiley‐Liss, Inc. 相似文献
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Zahra Shokati Eshkiki Mohammad Hossein Ghahremani Parisa Shabani Sattar Gorgani Firuzjaee Asie Sadeghi Hossein Ghanbarian Reza Meshkani 《Molecular and cellular biochemistry》2017,425(1-2):95-102
Protein tyrosine phosphatase 1B (PTP1B) has been shown to regulate multiple cellular events such as differentiation, cell growth, and proliferation; however, the role of PTP1B in differentiation of embryonic stem (ES) cells into cardiomyocytes remains unexplored. In the present study, we investigated the effects of PTP1B inhibition on differentiation of ES cells into cardiomyocytes. PTP1B mRNA and protein levels were increased during the differentiation of ES cells into cardiomyocytes. Accordingly, a stable ES cell line expressing PTP1B shRNA was established. In vitro, the number and size of spontaneously beating embryoid bodies were significantly decreased in PTP1B-knockdown cells, compared with the control cells. Decreased expression of cardiac-specific markers Nkx2-5, MHC-α, cTnT, and CX43, as assessed by real-time PCR analysis, was further confirmed by immunocytochemistry of the markers. The results also showed that PTP1B inhibition induced apoptosis in both differentiated and undifferentiated ES cells, as presented by increasing the level of cleaved caspase-3, cytochrome C, and cleaved PARP. Further analyses revealed that PTP1B inhibition did not change proliferation and pluripotency of undifferentiated ES cells. Taken together, the data presented here suggest that PTP1B is essential for proper differentiation of ES cells into cardiomyocytes. 相似文献
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Tadao Atsumi Mariko Nagayoshi Hiroko Anzai Yoji Ikawa 《Development, growth & differentiation》1995,37(2):167-172
Embryonic stem (ES) cells effectively differentiated into primitive erythroid/mesodermal cells when grown in the absence of both a feeder layer and leukemia inhibitory factor (LIF). The formation of a three-dimensional structure, exogenous mesoderm induction factors and exogenous hematopoietic growth factors were not essential for their differentiation. Primitive erythroid cells were first detected on day 5 in the differentiation-permissive cultures. Differentiation into other mesodermal cells was always preceded by that into primitive erythroid cells. Precursor cells of erythroid cells but of other hematoid cells were also detected in this system. This model system is useful for studying the early steps of mesoderm formation in mouse embryogenesis. 相似文献
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Epidermal growth factor inhibits morphogenesis and cell differentiation in cultured mouse embryonic teeth 总被引:3,自引:0,他引:3
Although local epithelial-mesenchymal tissue interactions which are presumably mediated by extracellular matrix molecules are important regulators of tooth morphogenesis and differentiation, our studies have indicated that these developmental processes also depend on circulating molecules. The iron-carrying serum protein transferrin is necessary for the early morphogenesis of mouse tooth in organ culture (A-M. Partanen, I. Thesleff, and P. Ekblom, 1984, Differentiation 27, 59-66). In the present study we have examined the effects of other growth factors on mouse tooth germs grown in a chemically defined medium containing transferrin. Fibroblast growth factor and platelet derived growth factor had no detectable effects but epidermal growth factor (EGF) inhibited dramatically the morphogenesis of teeth, and prevented odontoblast and ameloblast cell differentiation. EGF stimulated cell proliferation in the explants measured as [3H]thymidine incorporation in DNA. However, when the distribution of dividing cells was visualized in autoradiographs, it was observed that cell proliferation was stimulated in the dental epithelium but was inhibited in the dental mesenchyme. The inhibition of cell proliferation in the dental mesenchyme apparently caused the inhibition of morphogenesis. We do not know whether the dental epithelium or mesenchyme was the primary target for the action of EGF in the inhibition of morphogenesis. It is, however, apparent that the response of the dental mesenchymal cells to EGF (inhibition of proliferation) is regulated by their local environment, since EGF enhanced proliferation when these cells were disaggregated and cultured as monolayers. This indicates that the organ culture system where the various embryonic cell lineages are maintained in their original environment corresponds better to the in vivo situation when the roles of exogenous growth factors during development are examined. 相似文献
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Epigenetic gene control is involved in mechanisms of development. Little is known about the cooperation of nuclear and chromatin events in programmed differentiation from mouse embryonic stem cells (ESC). To address this, Oct3/4-positive ESC and differentiated progenies, Sox1-positive neural precursor cells (NPC) and post-mitotic neurons (PMN), were isolated using a stage-selected culture system. We first investigated global nuclear organization at the each stage. Chromocenter preexists in ESC, disperses in NPC and becomes integrated into large heterochromatic foci in PMN, while the formation of PML bodies markedly decreases in neural differentiation. We next focused on the gene-dense MHC-Oct3/4 region. Oct3/4 gene is expressed preferentially adjacent to PML bodies in ESC and are repressed in the absence of chromocenter association in NPC and PMN. Histone deacetylation in NPC, demethylation of lysine 4 of histone H3 (H3K4), tri-methylation of H3K27, and CpG methylation in PMN are targeted for the Oct3/4 promoter within the region. Interestingly, di-methyl H3K4 mark is present in Oct3/4 promoter in NPC as well as ESC. These findings provide insights into the molecular basis of global nuclear reorganization and euchromatic gene silencing in differentiation through the spatiotemporal order of epigenetic controls. 相似文献
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Perinatal dexamethasone (Dx) alters the immune system leading to increased infections and developmental abnormalities. Dendritic cells (DCs) derived from cord-blood monocytes are especially Dx sensitive and we sought to determine the effects of Dx on cord-blood CD34+-DCs. Distinct stages of cord-blood CD34+-DC development were delineated: pre-DC, immature, and mature DCs. Dx added during development of pre-DCs did not suppress precursor number, or translocate the glucocorticoid receptor (GcR) from the cytoplasm to the nucleus. However, Dx added during pre-DCs differentiation into immature DCs, prompted GcR translocation to the nucleus, enhanced DC apoptosis, suppressed differentiation to CD1a+ cells, inhibited expression of CD86, reduced subsequent CD83 expression, maintained DC endocytic activity, suppressed IL-6 secretion, enhanced IL-10 secretion, and reduced DC-mediated T cell stimulation. Dx added during the maturation stage caused less dramatic effects. Thus, Dx stalled maturation, selectively induced apoptosis of developing DCs and the sensitivity peaked during pre-DCs differentiation into immature DCs. 相似文献
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目的 应用旋转生物反应器(RCCS)和微载体培养体系尝试建立一种实现批量培养干细胞的新方法.方法 应用RCCS和微载体培养体系对小鼠胚胎干细胞(mESCs)进行体外培养扩增,定期收集细胞样品,镜下观察mESCs在RCCS生长的形态特征,并定量绘制细胞生长曲线,利用MATLAB软件计算细胞生长参数并对照平面培养体系,利用H&E染色、免疫荧光及RT-PCR技术对RCCS内培养的mESCs的细胞形态,未分化标志蛋白(SSEA-1)和标志基因(oct-4)的表达进行定性或半定量分析.结果 mESCs可在RCCS内以贴附于微载体表面的形式实现三维生长,其生长增殖状态良好,且伴随培养时间的延长,SSEA-1蛋白及oct-4 基因的表达水平逐渐降低.这表明RCCS内培养扩增的mESCs逐渐走向分化,该分化进程同步于平面对照培养体系.结论 RCCS可以为mESCs的体外规模化扩增培养提供良好的培养体系. 相似文献
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