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1.
Embryonic stem cells (ES) can self-replicate and differentiate into all cell types including insulin-producing, beta-like
cells and could, therefore, be used to treat diabetes mellitus. To date, results of stem cell differentiation into beta cells
have been debated, largely due to difficulties in defining the identity of a beta cell. We have recently differentiated non-human
primate (rhesus) embryonic stem (rES) cell lines into insulin producing, beta-like cells with the beta cell growth factor,
Exendin-4 and using C-peptide as a phenotype marker. Cell development was characterized at each stage by gene and protein
expression. Insulin, NKX6.1 and glucagon mRNA were expressed in stage 4 cells but not in early undifferentiated cells. We
concluded that rES cells could be differentiated ex vivo to insulin producing cells. These differentiated rES cells could be used to develop a non-human primate model for evaluating
cell therapy to treat diabetes. To facilitate the identification of beta-like cells and to track the cells post-transplantation,
we have developed a marker gene construct: fusing the human insulin promoter (HIP) to the green fluorescent protein (GFP)
gene. This construct was transfected into stage 3 rES derived cells and subsequent GFP expression was identified in C-peptide
positive cells, thereby substantiating endogenous insulin production by rES derived cells. Using this GFP detection system,
we will enrich our population of insulin producing rES derived cells and track these cells post-transplantation in the non-human
primate model. 相似文献
2.
Kuniko Akama Tomoe Horikoshi Takashi Nakayama Masahiro Otsu Noriaki Imaizumi Megumi Nakamura Tosifusa Toda Michiko Inuma Hisashi Hirano Yasushi Kondo Yutaka Suzuki Nobuo Inoue 《Biochimica et Biophysica Acta - Proteins and Proteomics》2013,1834(2):601-610
Understanding astrocytogenesis is valuable for the treatment of nervous system disorders, as astrocytes provide structural, metabolic and defense support to neurons, and regulate neurons actively. However, there is limited information about the molecular events associated with the differentiation from primate ES cells to astrocytes. We therefore investigated the differentially expressed proteins in early astrocytogenesis, from cynomolgus monkey ES cells (CMK6 cell line) into astrocyte progenitor (AstP) cells via the formation of primitive neural stem spheres (Day 4), mature neural stem spheres (NSS), and neural stem (NS) cells in vitro, using two-dimensional gel electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS-MS). We identified 66 differentially expressed proteins involved in these five differentiation stages. Together with the results of Western blotting, RT-PCR, and a search of metabolic pathways related to the identified proteins, these results indicated that collapsin response mediator protein 2 (CRMP2), its phosphorylated forms, and cellular retinoic acid binding protein 1 (CRABP1) were upregulated from ES cells to Day 4 and NSS cells, to which differentiation stages apoptosis-associated proteins such as caspases were possibly related; Phosphorylated CRMP2s were further upregulated but CRABP1 was downregulated from NSS cells to NS cells, during which differentiation stage considerable axon guidance proteins for development of growth cones, axon attraction, and repulsion were possibly readied; Nonphosphorylated CRMP2 was downregulated but CRABP1 was re-upregulated from NS cells to AstP cells, in which differentiation stage reorganization of actin cytoskeleton linked to focal adhesion was possibly accompanied. These results provide insight into the molecular basis of early astrocytogenesis in monkey. 相似文献
3.
Proteomic profiling of the pectoralis muscle of Thai indigenous chickens during growth period was analyzed using two-dimensional gel electrophoresis (2-DE) and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF/MS). A total of 259, 161, 120 and 107 protein spots were found to be expressed in the chicken pectoralis muscles at 0, 3, 6 and 18 weeks of age, respectively. From these expressed proteins, five distinct protein spots were significantly associated with chicken age. These protein spots were characterized and showed homology with phosphoglycerate mutase 1 (PGAM1), apolipoprotein A1 (APOA1), triosephosphate isomerase 1 (TPI1), heat shock protein 25 kDa (HSP25) and fatty acid binding protein 3 (FABP3). These five protein spots were categorized as follows: (i) the expression levels of PGAM1 and TPI1 proteins were positively correlated with chicken aging (p<0.05), (ii) the expression levels of APOA1 and FABP3 proteins were negatively correlated with chicken aging (p<0.05) and (iii) the expression levels of the HSP25 protein were up- and down-regulated during growth period. Moreover, the mRNA expression levels of the FABP3 and HSP25 genes were significantly decreased in muscle during the growth period (p<0.05), whereas no significant changes of the PGAM1, TPI1 and APOA1 gene expression from the chicken muscle was observed. The identified proteins were classified as metabolic and stress proteins. This demonstrates a difference in energy metabolism and stress proteins between age groups and shows that proteomics is a useful tool to uncover the molecular basis of physiological differences in muscle during the growth period. 相似文献
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The ubiquitin-proteasome system is a major proteolytic system for nonlysosomal degradation of cellular proteins. Here, we investigated the response of mouse embryonic stem (ES) cells under proteotoxic stress. Proteasome inhibitors induced expression of heat shock protein 70 (HSP70) in a concentration- and time-dependent manner, and also induced apoptosis of ES cells. Importantly, more apoptotic cells were observed in ES cells compared with other somatic cells. To understand this phenomenon, we further investigated the expression of HSP70 and pluripotent cell markers. HSP70 expression was more significantly increased in somatic cells than in ES cells, and expression levels of pluripotent cell markers such as Oct4 and Nanog were decreased in ES cells. These results suggest that higher sensitivity of ES cells to proteotoxic stress may be related with lower capacity of HSP70 expression and decreased pluripotent cell marker expression, which is essential for the survival of ES cells. 相似文献
6.
Akama K Tatsuno R Otsu M Horikoshi T Nakayama T Nakamura M Toda T Inoue N 《Biochimica et biophysica acta》2008,1784(5):773-782
Embryonic stem (ES) cells are pluripotent stem cells and give rise to a variety of differentiated cell types including neurons. To study a molecular basis for differentiation from ES cells to neural cells, we searched for proteins involved in mouse neurogenesis from ES cells to neural stem (NS) cells and neurons by two-dimensional gel electrophoresis (2-DE) and peptide mass fingerprinting, using highly homogeneous cells differentiated from ES cells in vitro. We newly identified seven proteins with increased expression and one protein with decreased expression from ES cells to NS cells, and eight proteins with decreased expression from NS cells to neurons. Western blot analysis confirmed that a tumor-specific transplantation antigen, HS90B, decreased, and an extracellular matrix and membrane glycoprotein (such as laminin)-binding protein, galectin 1 (LEG1), increased in NS cells, and LEG1 and a cell adhesion receptor, laminin receptor (RSSA), decreased in neurons. The results of RT-PCR showed that mRNA of LEG1 was also up-regulated in NS cells and down-regulated in neurons, implying an important role of LEG1 in regulating the differentiation. The differentially expressed proteins identified here provide insight into the molecular basis of neurogenesis from ES cells to NS cells and neurons. 相似文献
7.
Doxorubicin (DOX), an effective chemotherapeutic drug used in the treatment of various cancers, is limited in its clinical applications due to cardiotoxicity. Recent studies suggest that transplanted adult stem cells inhibit DOX-induced cardiotoxicity. However, the effects of transplanted embryonic stem (ES) and induced pluripotent stem (iPS) cells are completely unknown in DOX-induced left ventricular dysfunction following myocardial infarction (MI). In brief, C57BL/6 mice were divided into five groups: Sham, DOX-MI, DOX-MI+cell culture (CC) media, DOX-MI+ES cells, and DOX-MI+iPS cells. Mice were injected with cumulative dose of 12 mg/kg of DOX and 2 weeks later, MI was induced by coronary artery ligation. Following ligation, 5×104 ES or iPS cells were delivered into the peri-infarct region. At day 14 post-MI, echocardiography was performed, mice were sacrificed, and hearts were harvested for further analyses. Our data reveal apoptosis was significantly inhibited in ES and iPS cell transplanted hearts compared with respective controls (DOX-MI+ES: 0.48±0.06% and DOX-MI+iPS: 0.33±0.05% vs. DOX-MI: 1.04±0.07% and DOX-MI+CC: 0.96±0.21%; p<0.05). Furthermore, a significant increase in levels of Notch-1 (p<0.05), Hes1 (p<0.05), and pAkt (p<0.05) were observed whereas a decrease in the levels of PTEN (p<0.05), a negative regulator of Akt, was evident following stem cell transplantation. Moreover, hearts transplanted with stem cells demonstrated decreased vascular and interstitial fibrosis (p<0.05) as well as MMP-9 expression (p<0.01) compared with controls. Additionally, heart function was significantly improved (p<0.05) in both cell-transplanted groups. In conclusion, our data show that transplantation of ES and iPS cells blunt DOX-induced adverse cardiac remodeling, which is associated with improved cardiac function, and these effects are mediated by the Notch pathway. 相似文献
8.
Sandy S. C. Hung Raymond C. B. Wong Alexei A. Sharov Yuhki Nakatake Hong Yu Minoru S. H. Ko 《DNA research》2013,20(4):391-402
Mouse embryonic stem (ES) cells are prototypical stem cells that remain undifferentiated in culture for long periods, yet maintain the ability to differentiate into essentially all cell types. Previously, we have reported that ES cells oscillate between two distinct states, which can be distinguished by the transient expression of Zscan4 genes originally identified for its specific expression in mouse two-cell stage embryos. Here, we report that the nascent protein synthesis is globally repressed in the Zscan4-positive state of ES cells, which is mediated by the transient expression of newly identified eukaryotic translation initiation factor 1A (Eif1a)-like genes. Eif1a-like genes, clustered on Chromosome 12, show the high sequence similarity to the Eifa1 and consist of 10 genes (Eif1al1–Eif1al10) and 9 pseudogenes (Eif1al-ps1–Eif1al-ps9). The analysis of the expressed sequence tag database showed that Eif1a-like genes are expressed mostly in the two-cell stage mouse embryos. Microarray analyses and quantitative real-time polymerase chain reaction analyses show that Eif1a-like genes are expressed specifically in the Zscan4-positive state of ES cells. These results indicate a novel mechanism to repress protein synthesis by Eif1a-like genes and a unique mode of protein synthesis regulation in ES cells, which undergo a transient and reversible repression of global protein synthesis in the Zscan4-positive state. 相似文献
9.
Jung‐Il Chae Janghwan Kim Sun‐Mi Woo Hyo‐Won Han Young Keun Cho Keon‐Bong Oh Ki‐Hoan Nam Yong‐Kook Kang 《Proteomics》2009,9(5):1128-1141
The ability to generate neural lineages from human embryonic stem cells (hESCs) in a controlled manner would further investigation of human neurogenesis and development of potential cell therapeutic applications to treat neurological diseases; however, generating such neural stem cells (NSCs) remains a challenge. In an attempt to characterize the cellular mechanisms involved in hESC differentiation into neuroprogenitor cells, we performed 2‐DE using protein extracts from hESC‐derived embryoid bodies (EBs) and neuroectodermal spheres (NESs) bearing neuroprogenitors. Of 47 differentially expressed protein spots, 28 nonredundant spots were shown to be upregulated in the NESs; these protein spots included neurogenesis‐related proteins (TAF1, SEPT2, NPH3, and CRABP), as expected. Interestingly, 6 of these 28 protein spots were cytoskeleton‐associated proteins (CSAP) such as Fascin‐1, Cofilin‐1, and Stathmin‐1. Western‐blot analyses confirmed the increased levels of these proteins in the NESs. Furthermore, immunostaining analysis showed that both Fascin‐1 and Stathmin‐1 were preferentially expressed in the inner rims of neural rosettes, which are characteristic features of neuroprogenitors in culture. We also confirmed prominent expression of Fascin‐1 in (sub‐)ventricular zone in E15.5 mouse fetal brain. Our results suggest that, in addition to the induction of those genes involved in neural development, hESC differentiation into the NES is associated with a marked reorganization of the cellular cytoskeleton. 相似文献
10.
rES (rhesus monkey embryonic stem) cells have similar characteristics to human ES (embryonic stem) cells, and might be useful as a substitute model for preclinical research. Before their clinical application, it is critical to understand the roles of factors that control the differentiation of ES cells into hepatocytes. Here, we analysed the effect of collagen gels on rES cells differentiation into hepatocytes by stepwise protocols. About 80% of DE (definitive endoderm) cells were generated from rES cells after being treated with activin A. The DE cells were then plated on to collagen gels or type I collagen-coated wells with growth factors to induce hepatocyte differentiation. In type I collagen systems, characteristics of immature hepatocytes were observed, including the expression of immature hepatic genes and the generation of 15±3% AFP (alpha fetoprotein)/CK (cytokeratin)18 double-positive cells. In collagen gel culture, differentiated cells exhibited typical hepatocyte morphology and expressed adult liver-specific genes. The mRNA expression of AFP (immature hepatic gene) was detected at day 11 but decreased at day 18. In contrast, mRNA expression of albumin (mature hepatic gene) was detected at day 11 and increased at day 18. Compared with type I collagen systems, significantly higher AFP/CK18 double-positive cells (68±7%) were produced in collagen gel culture. Furthermore, some differentiated cells acquired the hepatocytic function of glycogen storage. However, only immature hepatic genes were observed in collagen gel systems if growth factors were absent. Thus, collagen gels combined with hepatocyte-inducing growth factors efficiently promoted differentiation of hepatocytes from rES. 相似文献
11.
Lin SL Chang DC Chang-Lin S Lin CH Wu DT Chen DT Ying SY 《RNA (New York, N.Y.)》2008,14(10):2115-2124
Renewal of stem cells differs from cancer cell growth in self-controlled cell division. The mir-302 microRNA (miRNA) family (mir-302s) is expressed most abundantly in slow-growing human embryonic stem (ES) cells, and quickly decreases after cell differentiation and proliferation. Therefore, mir-302s was investigated as one of the key factors essential for maintenance of ES cell renewal and pluripotency in this study. The Pol-II-based intronic miRNA expression system was used to transgenically transfect the mir-302s into several human cancer cell lines. The mir-302 – transfected cells, namely, miRNA-induced pluripotent stem (mirPS) cells, not only expressed many key ES cell markers, such as Oct3/4, SSEA-3, SSEA-4 ,Sox2, and Nanog, but also had a highly demethylated genome similar to a reprogrammed zygotic genome. Microarray analyses further revealed that genome-wide gene expression patterns between the mirPS and human ES H1 and H9 cells shared over 86% similarity. Using molecular guidance in vitro, these mirPS cells could differentiate into distinct tissue cell types, such as neuron-, chondrocyte-, fibroblast-, and spermatogonia-like primordial cells. Based on these findings, we conclude that mir-302s not only function to reprogram cancer cells into an ES-like pluripotent state but also to maintain this state under a feeder-free cultural condition, which may offer a great opportunity for therapeutic intervention. 相似文献
12.
Odile Costa Pascale Schneider Laurent Coquet Philippe Chan Dominique Penther Elisabeth Legrand Thierry Jouenne Marc Vasse Jean-Pierre Vannier 《Clinical proteomics》2014,11(1):31
Background
Until now, the major prognostic factors for pediatric acute lymphoblastic leukemia (ALL), age, white blood cell count and chromosomal alterations are initially taken into account for the risk stratification of patients. In the light of protein marker studies to classify subtypes of Acute Myeloblastic Leukemia efficiently, we have compared the lymphoblastes proteome in Childhood ALL in accordance with the presence of t(12;21), indicator of good prognosis, usually.Methods
Protein expression in pre-B2 lymphoblastic cells, collected from residual bone marrow cells after diagnostic procedures, was analyzed using two dimensional gel electrophoresis protocol. Protein spots whose average normalized volumes were statistically different in the two patients groups (n = 13; student t test p < 0.01), were excised. Tryptic peptides were then analyzed using a nano-LC1200 system coupled to a 6340 Ion Trap mass spectrometer equipped with a HPLC-chip cube interface. The tandem mass spectrometry peak lists extracted using the DataAnalysis program, were compared with the protein database Mascot Daemon.Results
We focused on twelve spots corresponding to sixteen identified candidate proteins among the 26 found differentially expressed (p ≤ 0.05) regarding the presence of t(12;21). Among over expressed proteins, two proteins were implicated in cellular growth arrest (i.e. calponine 2, p ≤ 0.001 and phosphatidylinositol transfer protein beta, p ≤ 0.001) in accordance with good prognosis, while two other proteins favored cell cycle proliferation (i.e. methionine adenosyl transferase 2β, p ≤ 0.005 and heterogeneous nuclear ribonucleo-proteins A2 p ≤ 0.01) and could therefore be good marker candidates of aggressiveness. Level of expression of proteasome subunit beta type-2 (p ≤ 0.01) and protein casein kinase 2α (p ≤ 0.01) which both favored apoptosis, deubiquitinating enzyme OTUB1 (p ≤ 0.05) and MLL septin-like fusion protein MSF-B, septin 9 i4 (p ≤ 0.01) were in accord with a good prognosis related to t(12;21) lymphoblasts.Conclusion
By drawing up the protein map of leukemic cells, these new data identified marker candidates of leukemic aggressiveness and new t(12;21) patients subgroups. These preliminary results will be in the near future confirmed by using a larger sample of pre-B2 childhood ALLs from national lymphoblastic cell collections. 相似文献13.
Ruo-Chiau Wang Chien-Yu Huang Tai-Long Pan Wei-Yu Chen Chun-Te Ho Tsan-Zon Liu Yu-Jia Chang 《PloS one》2015,10(10)
To search for reliable biomarkers and drug targets for management of hepatocellular carcinoma (HCC), we performed a global proteomic analysis of a pair of HCC cell lines with distinct differentiation statuses using 2-DE coupled with MALDI-TOF MS. In total, 106 and 55 proteins were successfully identified from the total cell lysate and the cytosolic, nuclear and membrane fractions in well-differentiated (HepG2) and poorly differentiated (SK-Hep–1) HCC clonal variants, respectively. Among these proteins, nine spots corresponding to proteins differentially expressed between HCC cell types were selected and confirmed by immunofluorescence staining and western blotting. Notably, Annexin 1 (ANX1), ANX–2, vimentin and stress-associated proteins, such as GRP78, HSP75, HSC–70, protein disulfide isomerase (PDI), and heat shock protein–27 (HSP27), were exclusively up-regulated in SK-Hep–1 cells. Elevated levels of ANX–4 and antioxidant/metabolic enzymes, such as MnSOD, peroxiredoxin, NADP-dependent isocitrate dehydrogenase, α-enolase and UDP-glucose dehydrogenase, were observed in HepG2 cells. We functionally demonstrated that ANX1 and HSP27 were abundantly overexpressed only in highly invasive types of HCC cells, such as Mahlavu and SK-Hep–1. Knockdown of ANX1 or HSP27 in HCC cells resulted in a severe reduction in cell migration. The in-vitro observations of ANX1 and HSP27 expressions in HCC sample was demonstrated by immunohistochemical stains performed on HCC tissue microarrays. Poorly differentiated HCC tended to have stronger ANX1 and HSP27 expressions than well-differentiated or moderately differentiated HCC. Collectively, our findings suggest that ANX1 and HSP27 are two novel biomarkers for predicting invasive HCC phenotypes and could serve as potential treatment targets. 相似文献
14.
Embryonic stem cells (ES cells) can differentiate into endothelial cells and smooth muscle cells (SMCs), which participate in vascular angiogenesis. In this study, we differentiated mouse ES cells into Sca-1(+) cells, which have the potential to serve as vascular progenitor cells, and mapped their proteome by 2-DE using a pH 3-10 non-linear gradient and 12% SDS-polyacrylamide gels. A subset of 300 protein spots was analysed and mapped, with 241 protein spots being identified by their PMF using MALDI-TOF MS or by partial amino acid sequencing using MS/MS. Our protein map is the first of Sca-1(+) progenitor cells and will facilitate the identification of proteins differentially expressed during stem cell differentiation. The proteome of adult arterial SMCs is described in an accompanying paper (in this issue, DOI 10.1002/pmic.200402045). All data are made accessible on our website http://www.vascular-proteomics.com. 相似文献
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Ueda S Kawamata M Teratani T Shimizu T Tamai Y Ogawa H Hayashi K Tsuda H Ochiya T 《PloS one》2008,3(7):e2800
The rat is a reference animal model for physiological studies and for the analysis of multigenic human diseases such as hypertension, diabetes, neurological disorders, and cancer. The rats have long been used in extensive chemical carcinogenesis studies. Thus, the rat embryonic stem (rES) cell is an important resource for the study of disease models. Attempts to derive ES cells from various mammals, including the rat, have not succeeded. Here we have established two independent rES cells from Wister rat blastocysts that have undifferentiated characters such as Nanog and Oct3/4 genes expression and they have stage-specific embryonic antigen (SSEA) -1, -3, -4, and TRA-1-81 expression. The cells were successfully cultured in an undifferentiated state and can be possible over 18 passages with maintaining more than 40% of normal karyotype. Their pluripotent potential was confirmed by the differentiation into derivatives of the endoderm, mesoderm, and ectoderm. Most importantly, the rES cells are capable of producing chimera rats. Therefore, we established pluripotent rES cell lines that are widely used to produce genetically modified experimental rats for study of human diseases. 相似文献
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猕猴胚胎干细胞(rhesus monkey embryonic stem(rES))与人胚胎干细胞有相似的生物学特性,因此是理想的临床前研究的替代模型。Notch信号通路在胆管及胆管上皮细胞的形成中有重要的作用,然而,有关Notch信号通路在ES细胞的胆向分化中的作用了解甚少。该实验以rES为模型,对Notch信号通路对ES细胞的胆向分化过程中的作用进行了较为系统的研究。rES在细胞因子ActivinA诱导作用下产生约80%的限定性内胚层细胞。以Matrigel作为细胞外基质,在含BMP4和FGF1的无血清培养体系中继续诱导5~7d,rES细胞来源的限定性内胚层细胞分化产生约胆管样细胞。分化的细胞表达胆管细胞的特异性蛋白((CK7、CK18、CK19、CK20和OV-6)及基因(GSTPi、IB4和HNF1β)。在胆管样细胞的分化过程中检测到了Notch1和Notch2基因及下游信号分子hes1和hes5的表达。用Notch抑制剂L-685458处理分化过程中的细胞可导致Notch1和Notch2基因及下游信号分子hes1和hes5的表达下降,同时CK19阳性的胆管样细胞分化比率也从90%下降至约20%。这一... 相似文献
20.
The Pekin duck, bred from the mallard (Anas platyrhynchos) in china, is one of the most famous meat duck species in the world. However, it is more sensitive to heat stress than Muscovy duck, which is believed to have originated in South America. With temperature raising, mortality, laying performance, and meat quality of the Pekin duck are severely affected. This study aims to uncover the temperature-dependent proteins of two duck species using comparative proteomic approach. Duck was cultured under 39°C ± 0.5°C for 1 h, and then immediately returned to 20°C for a 3 h recovery period, the liver proteins were extracted and electrophoresed in two-dimensional mode. After analysis of gel images, 61 differentially expressed proteins were detected, 54 were clearly identified by MALDI TOF/TOF MS. Of the 54 differentially expressed protein spots identified, 7 were found in both species, whereas 47 were species specific (25 in Muscovy duck and 22 in Pekin duck). As is well known, chaperone proteins, such as heat shock protein (HSP) 70 and HSP10, were abundantly up-regulated in both species in response to heat stress. However, we also found that several proteins, such as α-enolase, and S-adenosylmethionine synthetase, showed different expression patterns in the 2 duck species. The enriched biological processes were grouped into 3 main categories according to gene ontology analysis: cell death and apoptosis (20.93%), amino acid metabolism (13.95%) and oxidation reduction (20.93%). The mRNA levels of several differentially expressed protein were investigated by real-time RT-PCR. To our knowledge, this study is the first to provide insights into the differential expression of proteins following heat stress in ducks and enables better understanding of possible heat stress response mechanisms in animals. 相似文献