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• W2607140359 abstract "The role of striatin interacting protein 2 (Strip2) in differentiation of embryonic stem cells (ESCs) is still under debate. Strip2-silenced murine (KD) ESCs were differentiated for 4, 8, 12, and 16 days. We show that Strip2 is distributed in the perinucleus or nuclei of wild-type (WT) undifferentiated ESCs, but is localized in high-density nuclear bodies in differentiated cells. CellNet analysis of microarray gene expression data for the KD and scrambled control (SCR) embryoid bodies (EBs), as well as immunostainings of key pluripotent factors, demonstrated that differentiation of KD ESCs is repressed. This occurs even in 16-day-old EBs, which possessed a high tumorigenic potential. Correlated with very high expression levels of epigenetic regulator genes, Hat1 and Dnmt3, enzymatic activities of the histone acetyltransferase type B (Hat1) and DNA (cytosine-5)-methyltransferase 3 beta (Dnmt3b) were higher in differentiated 16-day-old KD EBs than in SCR or WT EBs. The expression levels of let-7, 290, and 302 microRNA families were opposed in KD ESCs, while KD EBs had levels comparable to WT and SCR ESCs during differentiation. Strip2 is critical for the regular differentiation of ESCs. Moreover, Strip2 deficient ESCs showed a dysregulation of epigenetic regulators and microRNAs regulating pluripotency. The role of striatin interacting protein 2 (Strip2) in differentiation of embryonic stem cells (ESCs) is still under debate. Strip2-silenced murine (KD) ESCs were differentiated for 4, 8, 12, and 16 days. We show that Strip2 is distributed in the perinucleus or nuclei of wild-type (WT) undifferentiated ESCs, but is localized in high-density nuclear bodies in differentiated cells. CellNet analysis of microarray gene expression data for the KD and scrambled control (SCR) embryoid bodies (EBs), as well as immunostainings of key pluripotent factors, demonstrated that differentiation of KD ESCs is repressed. This occurs even in 16-day-old EBs, which possessed a high tumorigenic potential. Correlated with very high expression levels of epigenetic regulator genes, Hat1 and Dnmt3, enzymatic activities of the histone acetyltransferase type B (Hat1) and DNA (cytosine-5)-methyltransferase 3 beta (Dnmt3b) were higher in differentiated 16-day-old KD EBs than in SCR or WT EBs. The expression levels of let-7, 290, and 302 microRNA families were opposed in KD ESCs, while KD EBs had levels comparable to WT and SCR ESCs during differentiation. Strip2 is critical for the regular differentiation of ESCs. Moreover, Strip2 deficient ESCs showed a dysregulation of epigenetic regulators and microRNAs regulating pluripotency. Recently, increasing attention has been directed toward identifying and understanding the function and intracellular signaling pathways of the striatin-interacting phosphatase and kinase (STRIPAK) complex in regulating biological processes of multiple organisms.1Bai S.W. Herrera-Abreu M.T. Rohn J.L. Racine V. Tajadura V. Suryavanshi N. Bechtel S. Wiemann S. Baum B. Ridley A.J. Identification and characterization of a set of conserved and new regulators of cytoskeletal organization, cell morphology and migration.BMC Biol. 2011; 9: 54Crossref PubMed Scopus (116) Google Scholar, 2Goudreault M. D’Ambrosio L.M. Kean M.J. Mullin M.J. Larsen B.G. Sanchez A. Chaudhry S. Chen G.I. Sicheri F. Nesvizhskii A.I. et al.A PP2A phosphatase high density interaction network identifies a novel striatin-interacting phosphatase and kinase complex linked to the cerebral cavernous malformation 3 (CCM3) protein.Mol. Cell. Proteomics. 2009; 8: 157-171Crossref PubMed Scopus (271) Google Scholar, 3Hwang J. Pallas D.C. STRIPAK complexes: structure, biological function, and involvement in human diseases.Int. J. Biochem. Cell Biol. 2014; 47: 118-148Crossref PubMed Scopus (155) Google Scholar There is growing evidence that striatin interacting protein 2 (Strip2; also known as Fam40b) is a member of the STRIPAK complex, which may be involved in the regulation of cell growth, proliferation, cell migration and adhesion, neural and vascular development, and ultimately may regulate cardiac function.1Bai S.W. Herrera-Abreu M.T. Rohn J.L. Racine V. Tajadura V. Suryavanshi N. Bechtel S. Wiemann S. Baum B. Ridley A.J. Identification and characterization of a set of conserved and new regulators of cytoskeletal organization, cell morphology and migration.BMC Biol. 2011; 9: 54Crossref PubMed Scopus (116) Google Scholar, 2Goudreault M. D’Ambrosio L.M. Kean M.J. Mullin M.J. Larsen B.G. Sanchez A. Chaudhry S. Chen G.I. Sicheri F. Nesvizhskii A.I. et al.A PP2A phosphatase high density interaction network identifies a novel striatin-interacting phosphatase and kinase complex linked to the cerebral cavernous malformation 3 (CCM3) protein.Mol. Cell. Proteomics. 2009; 8: 157-171Crossref PubMed Scopus (271) Google Scholar, 3Hwang J. Pallas D.C. STRIPAK complexes: structure, biological function, and involvement in human diseases.Int. J. Biochem. Cell Biol. 2014; 47: 118-148Crossref PubMed Scopus (155) Google Scholar Striatin, a core of the STRIPAK complex, is highly expressed in the central and peripheral nervous systems, in heart muscle, testes, and lymphocytes.3Hwang J. Pallas D.C. STRIPAK complexes: structure, biological function, and involvement in human diseases.Int. J. Biochem. Cell Biol. 2014; 47: 118-148Crossref PubMed Scopus (155) Google Scholar Thus, Striatin and Strip2 may play important roles in several biological processes, such as differentiation, cell growth, development differentiation, and cancer.4Shi Z. Jiao S. Zhou Z. STRIPAK complexes in cell signaling and cancer.Oncogene. 2016; 35: 4549-4557Crossref PubMed Scopus (53) Google Scholar More recently, it has been shown that Strip2 plays an important role in cancer development and metastasis.5Madsen C.D. Hooper S. Tozluoglu M. Bruckbauer A. Fletcher G. Erler J.T. Bates P.A. Thompson B. Sahai E. STRIPAK components determine mode of cancer cell migration and metastasis.Nat. Cell Biol. 2015; 17: 68-80Crossref PubMed Scopus (117) Google Scholar Previously, we provided evidence that Strip2 is required for proper differentiation of embryonic stem cells (ESCs). In this context, we have shown that short hairpin RNAs (shRNAs) mediated silencing of Fam40b expression in ESCs led to perturbed differentiation to different somatic cell types at 12-day embryoid bodies (EBs), including a complete abrogation of cardiomyogenesis. Interestingly, pluripotency factors, such as Nanog, Oct4, and Sox2, as well as epigenetic factors, such as histone acetyltransferase type B (Hat1) and DNA (cytosine-5)-methyltransferase 3b (Dnmt3b), were highly upregulated in Strip2-silenced 12-day EBs (KD EBs), compared with scrambled control (SCR) EBs. We identified the gene product of Strip2 in undifferentiated ESCs, as a nuclear and perinuclear protein, with a molecular weight of 96 kDa.6Wagh V. Doss M.X. Sabour D. Niemann R. Meganathan K. Jagtap S. Gaspar J.A. Ardestani M.A. Papadopoulos S. Gajewski M. et al.Fam40b is required for lineage commitment of murine embryonic stem cells.Cell Death Dis. 2014; 5: e1320Crossref PubMed Scopus (18) Google Scholar To prove that Strip2 is essential for differentiation of ESCs toward defined somatic cells, and to determine its underlying mechanisms, we extended our study by performing detailed parallel transcriptome and microRNA (miRNA) expression studies following differentiation of KD, SCR, and wild-type (WT) ESCs for periods of 4, 8, 12, and 16 days. The cell types differentiated from KD, SCR, and WT ESCs have been classified, using the online CellNet bioinformatics platform (http://cellnet.hms.harvard.edu/run/). In addition, the activities of the epigenetic regulators Hat1 and Dnmt3b have been investigated during differentiation of the KD and SCR ESCs. Here, we show that Strip2 is located in differentiated cells, in dense nuclear bodies, demonstrating that Strip2 is a key nuclear factor, acting during the very early stages to differentiate all three germ layers. Moreover, Strip2 regulates the activity of the nuclear epigenetic regulators Hat1 and Dnmt3b and the expression of a set of miRNAs required for switching the pluripotency state of the ESCs to a differentiation state. Using a Strip2 (Fam40b) shRNA expression vector targeting mouse Strip2 and a scrambled shRNA control vector with a non-active scrambled sequence cassette, we previously generated a constitutive Strip2 KD ESC line. In this cell line, Strip2 was constitutively knocked down (referred to as KD ESCs) in mRNA and protein levels, as well as in SCR ESCs.6Wagh V. Doss M.X. Sabour D. Niemann R. Meganathan K. Jagtap S. Gaspar J.A. Ardestani M.A. Papadopoulos S. Gajewski M. et al.Fam40b is required for lineage commitment of murine embryonic stem cells.Cell Death Dis. 2014; 5: e1320Crossref PubMed Scopus (18) Google Scholar Here, we also use control WT ESCs for our comparative investigations. Strip2 detection by immunostaining in undifferentiated and differentiated KD and SCR ESCs is shown in Figure 1A. Strip2 was sporadically distributed within the nuclei of the undifferentiated ESCs. No significant expression could be observed in the nuclei of the KD ESCs. Figure 1B shows that Strip2 was intensely detected in several areas of the EBs, consisting of several somatic cell types. No significant expression could be detected in the 16-day-old KD EBs (Figure 1B). Detection of Strip2 in differentiated single cells, generated after trypsinization of 16-day-old EBs and further culturing for 24 hr, is shown in Figure 1C. Results suggest that Strip2 is located in nuclear bodies of the 16-day-old differentiated cells, but was absent in the 16-day-old KD EBs. In our previous study, KD, SCR, and WT ESCs were differentiated for 12 days (12-day-old EBs), using the hanging drop protocol, and their transcriptomes were characterized.6Wagh V. Doss M.X. Sabour D. Niemann R. Meganathan K. Jagtap S. Gaspar J.A. Ardestani M.A. Papadopoulos S. Gajewski M. et al.Fam40b is required for lineage commitment of murine embryonic stem cells.Cell Death Dis. 2014; 5: e1320Crossref PubMed Scopus (18) Google Scholar Transcriptome characterization of the undifferentiated KD ESCs and the 12-day-old EBs indicated that Strip2 was essential for the differentiation of ESCs into somatic cells present in 12-day-old EBs via mechanisms controlling pluripotency. However, our findings did not allow us to determine whether Strip2 was required for the development of germ layers from ESCs or for the development of somatic cells from germ layer cells. To answer this question, we extended our transcriptome studies by performing a similar study, including early (4-day), late (8-day and 12-day), and very late (16-day) differentiation stages of the KD, SCR, and WT ESCs and carrying out a detailed transcriptome analysis of these stages. In parallel, a miRNA expression analysis was performed, with the same total RNA. In general, 12,156 as well as 12,440 differentially expressed genes (at least 2-fold) have been identified in WT as well as in SCR 4-day (d4), 8-day (d8), 12-day (d12), and 16-day (d16) EBs in comparison to WT ESC (d0) as well as SCR ESCs (d0), respectively. In contrast, only 4,075 genes were found to be differentially expressed in KD d4, d8, d12, and d16 EBs in comparison to the KD ESCs (d0). Significance of the variance in expression levels in undifferentiated (KD, SCR, and WT ESCs) and in their respective differentiated 4-day, 8-day, 12-day, and 16-day-old EBs were evaluated using a principal component analysis (PCA) (Figure 2A). PC 1 shows the highest variance in transcriptome variability among these biological samples, followed by variation in PC2. As shown in Figure 2A, there are relatively large differences in the transcriptomes of all three cell populations. Briefly, PCA indicated that the transcriptomes of the undifferentiated KD, SCR, and WT ESCs were very similar, while they were significantly different from the transcriptomes of the differentiated SCR and WT (4-, 8-, 12-, and 16-day-old EBs). As expected, the transcriptomes of the differentiated SCR and WT (4-, 8-, 12-, and 16-day EBs) were very similar. Among them, the transcriptomes of the 4-day-old SCR and WT EBs (clustered together) differ significantly in their PC1 and PC2 weightings, compared with the transcriptomes of 8-, 12-, and 16-day-old EBs. These later stages show only small differences in PC2. Notably, the transcriptomes of the KD 4-, 8-, 12-, and 16-day-old EBs were similar to the transcriptomes of undifferentiated KD, SCR, and WT ESCs, having only small differences in PC2. There were 12 transcriptome clusters of different cell populations that were identified using the K-means clustering algorithm. These represent transcripts that were significantly deregulated among the different cell types (at least a 2-fold change) (Figure 2B). These clusters comprise genes (Table S1) that were similarly regulated during the differentiation of the KD, SCR, and WT ESCs. Figure 2C shows representative gene expression levels of the genes in these clusters (Table S1) during differentiation of the WT and KD ESCs. As shown in Figure S1A, the expression levels of the genes in differentiated SCR ESCs were very similar to the expression levels in the differentiated SCR ESCs. To categorize the biological significance of the differentially expressed genes, the Database for Annotation, Visualization and Integrated Discovery (DAVID) was used (https://david.ncifcrf.gov/). DAVID assigns a biological process, molecular function, and cellular component, based on a statistical enrichment score. The main developmental/differentiation associated Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for biological processes identified in the annotation enrichment analysis for all 12 clusters are shown in Table 1. The complete GO and KEGG pathways for all clusters are given in Table S2. Our detailed cluster analysis using DAVID indicated that clusters 3, 11, and 12 are of particular interest (Table 1). In cluster 3, 521 probe sets (PSs) (421 genes) have been identified. Interestingly, among them, the GO:0019827 stem cell maintenance (p < 2.55 × 10−8) and GO:0045596 negative regulation of cell differentiation (p < 1.37 × 10−4) were highly enriched. The GO:0019827 stem cell maintenance category includes several pluripotency-associated genes (Nanog, Rif1, Esrrb, Pou5f1, Nodal, Sox2, Klf4, Fgf4, and Tcl1). Figure S2 shows that the expression level of these genes is maximal in an undifferentiated state, while during differentiation of WT and SCR, a rapid downregulation of the pluripotent genes began on day 4, continuing until day 16 (e.g., as seen in genes Pou5f1, Nodal, and Sox2). In striking contrast, the level of the pluripotent genes remained high during differentiation of the KD ESCs, after up to 16 days of differentiation (Figure S2). In cluster 12, 173 PSs (157 genes) have been identified. Among them, the GO:0001704 formation of primary germ layers (ectoderm, mesoderm, and endoderm) was highly enriched (p < 4.15 × 10−11) (Table 1). This GO set includes Bmp4, Hand1, Lhx1, Eomes, Lef1, Smad1, Bmp7, Mesp1, Mixl1, and Bmpr1a. As expected, the GO:0001707 mesoderm formation (p < 2.00 × 10−8; Bmp4, Hand1, Eomes, Lef1, Smad1, Bmp7, Mesp1, and Bmpr1a), the GO:0007492 endoderm development (p < 1.06 × 10−4; Lhx1, Eomes, Cfc1, Mixl1, and Bmpr1a), and the GO:0007398 ectoderm development (p < 0.0010) were also highly enriched (Table 1). In Figure 2C, like Mesp1, genes in cluster 12 show maximal expression levels only on day 4 in differentiated WT and SCR ESCs, but have low levels in undifferentiated cells (d0), as well as 8-, 12-, and 16-day-old EBs. Interestingly, the expression levels of these genes in KD ESCs were very low at all stages of differentiation, even in 4-day-old EBs (Figure 2).Figure 2Transcriptome Analysis of Undifferentiated and Differentiated Strip2 KD, Control SCR, and WT ESCsShow full captionDifferentiation of these cells was performed for 4-, 8-, 12-, and 16-day periods. (A) PCA of genome-wide gene expression. Each sphere represents an individual sample from a color-coded triplicate sample set. (B) Visualization of K-means clustering of 10,548 differentially expressed probe sets (with at least a 2-fold change in expression), using a Euclidean distance measurement and k = 12 group clusters. The replicates are displayed on the vertical axis, with genes on the horizontal axis. Log2 transformed signal intensities are depicted, using a color gradient. The heatmap indicates high expression levels in red, intermediate expression in dark gray, and low expression levels in green. (C) Representative diagrams showing the gene expression pattern of various clusters of genes during differentiation of KD and WT ESCs (*p < 0.05 for the differentiated KD versus WT ESCs, mean ± SD, n = 3). (D) Validation of the microarray data by the real-time qPCR analysis of six genes (*p < 0.05 for differentiated KD ESCs versus SCR ESCs, mean ± SD n = 3).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 1Specific GO and KEGG Pathways of the Cluster Specific Genes Indicated in Figure 2GO and KEGGGene No.p ValueCluster 1GO:0006665 sphingolipid metabolic process60.007178GO:0006643 membrane lipid metabolic process60.008196mmu04144:Endocytosis110.008332mmu00603:Glycosphingolipid biosynthesis30.032475Cluster 2GO:0007167 enzyme linked receptor protein signaling pathway207.38 × 10−5GO:0048514 blood vessel morphogenesis155.55 × 10−4mmu04360:Axon guidance90.009831mmu04310:Wnt signaling pathway90.020142Cluster 3GO:0006520 cellular amino acid metabolic process286.93 × 10−9GO:0019827 stem cell maintenance96.51 × 10−8GO:0045596 negative regulation of cell differentiation150.005mmu00330:Arginine and proline metabolism109.33 × 10−5Cluster 4GO:0006281 DNA repair231.23 × 10−4GO:0019318 hexose metabolic process192.06 × 10−4mmu00030:Pentose phosphate pathway92.68 × 10−6mmu00480:Glutathione metabolism111.56 × 10−5Cluster 5GO:0002526 acute inflammatory response225.67 × 10−16GO:0009888 tissue development403.31 × 10−7mmu04610:Complement and coagulation cascades229.89 × 10−15mmu00980:Metabolism of xenobiotics by cytochrome P450131.13 × 10−6Cluster 6GO:0009888 tissue development846.31 × 10−13GO:0009887 organ morphogenesis736.56 × 10−10mmu04512:ECM-receptor interaction201.42 × 10−7mmu05414:Dilated cardiomyopathy211.65 × 10−7Cluster 7GO:0042981 regulation of apoptosis241.40 × 10−4GO:0043067 regulation of programmed cell death241.68 × 10−4mmu04142:Lysosome103.92 × 10−4mmu03320:PPAR signaling pathway70.003697Cluster 8GO:0043229 intracellular organelle1638.06 × 10−8GO:0005634 nucleus984.50 × 10−6mmu04810:Regulation of actin cytoskeleton100.003562664mmu04520:Adherens junction60.004405387Cluster 9GO:0030500 regulation of bone mineralization57.73 × 10−5GO:0004867 serine-type endopeptidase inhibitor activity94.52 × 10−4mmu04142:Lysosome114.68 × 10−5Cluster 10GO:0009887 organ morphogenesis698.67 × 10−12GO:0007507 heart development363.63 × 10−10mmu04360:Axon guidance243.32 × 10−9mmu04340:Hedgehog signaling pathway142.19 × 10−7Cluster 11GO:0005730 nucleolus841.72 × 10−49GO:0034470 ncRNA processing424.05 × 10−24GO:0030529 ribonucleoprotein complex571.06 × 10−15GO:0040029 regulation of gene expression, epigenetic90.003GO:0022618 ribonucleoprotein complex assembly50.033GO:0031080 Nup107-160 complex75.70 × 10−8GO:0005643 nuclear pore131.04 × 10−6GO:0051028 mRNA transport131.90 × 10−6Cluster 12GO:0001704 formation of primary germ layer104.15 × 10−11GO:0001707 mesoderm formation82.00 × 10−8GO:0007492 endoderm development51.06 × 10−4GO:0007398 ectoderm development70.001 Open table in a new tab Differentiation of these cells was performed for 4-, 8-, 12-, and 16-day periods. (A) PCA of genome-wide gene expression. Each sphere represents an individual sample from a color-coded triplicate sample set. (B) Visualization of K-means clustering of 10,548 differentially expressed probe sets (with at least a 2-fold change in expression), using a Euclidean distance measurement and k = 12 group clusters. The replicates are displayed on the vertical axis, with genes on the horizontal axis. Log2 transformed signal intensities are depicted, using a color gradient. The heatmap indicates high expression levels in red, intermediate expression in dark gray, and low expression levels in green. (C) Representative diagrams showing the gene expression pattern of various clusters of genes during differentiation of KD and WT ESCs (*p < 0.05 for the differentiated KD versus WT ESCs, mean ± SD, n = 3). (D) Validation of the microarray data by the real-time qPCR analysis of six genes (*p < 0.05 for differentiated KD ESCs versus SCR ESCs, mean ± SD n = 3). In cluster 11, 842 PSs (676 genes) have been identified. Among them, genes encoding for nucleus proteins (GO:0005730 nucleolus; p < 1.72 × 10−49), non-coding (nc)RNAs processing genes (GO:0034470 ncRNA processing; p < 4.05 × 10−24), ribonucleoproteins (GO:0030529 ribonucleoprotein complex; p < 1.06 × 10−15), and RNA polymerases (KEGG, mmu03020:RNA polymerase; p < 2.07 × 10−4) were highly enriched. Genes participating in the regulation of gene expression via epigenetic mechanisms (GO:0040029 regulation of gene expression, epigenetic; p < 0.0037; Tarbp2, Dnmt3a, Hat1, Lin28a, Dnmt3b, Sirt1, Mphosph8, Brca1, and Hells) were also highly enriched. In Figure 2C, similar to expression levels of Hat1, the expression levels of cluster 11 genes was maximal in their undifferentiated state in all three ESCs lineages. During differentiation of WT and SCR ESCs, a rapid downregulation of genes occurred from their undifferentiated state through 4-day- to 16-day-old EBs. In contrast, the expression levels of genes remained high during differentiation of KD ESCs until day 16 of differentiation (Figure 2C). Genes in clusters 5, 6, 8, and 10 were downregulated at all stages of differentiation of KD ESCs, as well as in 16-day-old KD EBs comprising somatic cell type specific genes of all lineages, including cardiac cells, kidney, neurons, liver cells, and blood cells (Table S2). In contrast, expression of clusters 5 and 6 genes in WT and SCR ESCs began on day 12 and day 8 of differentiation, respectively. Their expression levels remained high even on day 16 of differentiation. Similarly, in both WT and SCR ESCs, expression levels of the genes in cluster 8 and 10 began on day 4, with maximal expression occurring on day 8 of differentiation, while still high expression levels were observed on day 12 and 16 of differentiation. Clusters 1 and 2 genes were downregulated in KD ESCs during differentiation, compared with SCR and WT ESCs. These genes belong mainly to metabolic and organ developmental processes, respectively. Cluster 4 genes showed upregulation in differentiated KD ESCs, compared with differentiated SCR and WT ESCs. These genes belong mainly to metabolic processes (Figure 2; Table S2). The expression of mainly apoptosis-related genes in Cluster 7 increased during differentiation of KD ESCs, compared with differentiated SCR and WT ESCs (Figure 2; Table S2). Validation of the microarray data has been performed by real-time qPCR analysis of six genes involved into the germ layer formation and their cell derivatives (Ncam1: ectoderm, neurogenesis; Brachyury T: mesoderm, cardiomyogenesis; Kdr: mesoderm, hematopoietic stem cells; Sox17: endoderm, endoderm derived organs such as gut; and Gata4 and Gata6: mesoderm and extraembryonic endoderm lineages, cardiomyogenesis, hepatogenesis) (Figure 2D). As shown, the microarray gene expression patterns of all six genes could be confirmed by the real-time qPCR method. Moreover, the expression levels in differentiated KD ESCs were significantly lower in comparison to the SCR ESCs at least at day 4 of differentiation. Overall, analysis suggests that Strip2 is essential for early differentiation of ESCs to germ layer cell types. To distinguish the specific cell types differentiated from KD, SCR, and WT ESCs within a 16-day period, we uploaded their gene expression data (.CEL files) to the CellNet bioinformatics platform, available at http://cellnet.hms.harvard.edu/run/. As described,7Cahan P. Li H. Morris S.A. Lummertz da Rocha E. Daley G.Q. Collins J.J. CellNet: network biology applied to stem cell engineering.Cell. 2014; 158: 903-915Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar such analysis allows prediction of the cell and tissue types, based on the cell and tissue specific gene expression levels derived from microarray gene expression data. The two main outputs of CellNet analysis are: (1) the cell versus tissue cell (C/T) classification score and (2) the C/T gene regulatory network (C/T GRNs) score. The values of the C/T classification score reflect the probability that a sample is indistinguishable from a given cell or tissue type based on its gene expression levels. These scores are typically represented as a heatmap, using a black > green > yellow color scale, representing values (0.0 > 0.5 > 1.0). A C/T value of 1 for a specific cell type has been derived from a set of training data for each cell type or tissue.7Cahan P. Li H. Morris S.A. Lummertz da Rocha E. Daley G.Q. Collins J.J. CellNet: network biology applied to stem cell engineering.Cell. 2014; 158: 903-915Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar Using this approach, 20 cell types can be distinguished, including those from ESCs; ovary, testis, neurons, glia, skin, heart, skeletal muscle, fibroblasts, white adipose, kidney, endothelial cells, hematopoietic stem and progenitor cells (hspc), B cells, T cells, macrophages, lung, liver, pancreas, small intestine, and colon. The C/T GRNs score for each different cell type is based on GRNs, specific to each cell type. Thus, a given GRN is associated with a particular cell or tissue type. The values in the bar blots in Figure 3B represent the extent of similarity between undifferentiated and differentiated ESCs for C/T GRN for these 20 different cell types. As indicated in Figure 3, the undifferentiated SCR ESCs and the 4-day-old EBs could be clearly distinguished from the 8-, 12-, and 16-day-old EBs. The C/T score was relatively high for undifferentiated SCR ESCs (close to 1), as well as for early differentiation stages in 4-day-old SCR EBs (scores close to 0.5), but very low for the 8-, 12-, and 16-day-old EBs (Figure 3A). Based on their GRN score status, we could distinguish ESCs, heart, fibroblasts, hspc, macrophages, lung, and liver cells (Figure 3B). As indicated in Figure 3B, maximal GRN scores for skin, hspc, macrophage, fibroblast, heart, and liver were overrepresented in 16-day, 8-day, 12-day, 12-day, 12-day, and 16-day-old EBs, respectively. All three independent experiments showed similar tendencies (Figure 3). Similar results were obtained for WT ESCs (Figure S3). In contrast, the KD ESCs, as well as their 4-, 8-, 12-, and the 16-day-old KD EBs had high scores (close to 1), typical for undifferentiated ESCs (Figure 4). No other somatic cell types could be distinguished by its C/T score.Figure 4CellNet Analysis of Differentiated KD ESCsShow full captionGene expression .CEL files from the undifferentiated Strip2-silenced KD ESCs, as well as 4-, 8-, 12-, and 16-day EBs were analyzed using online CellNet bioinformatics tools (http://cellnet.hms.harvard.edu/run/). (A) C/T classification score. (B) C/T gene regulatory network (GRN) status scores.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Gene expression .CEL files from the undifferentiated Strip2-silenced KD ESCs, as well as 4-, 8-, 12-, and 16-day EBs were analyzed using online CellNet bioinformatics tools (http://cellnet.hms.harvard.edu/run/). (A) C/T classification score. (B) C/T gene regulatory network (GRN) status scores. These findings again suggest perturbation of the differentiation processes of Strip2 deficient ESC toward somatic cells. Next, we determined the global expression profile for miRNAs during the differentiation of WT, SCR, and KD ESCs, using Affymetrix microRNA 3.0 arrays, as described previously.8Meganathan K. Jagtap S. Srinivasan S.P. Wagh V. Hescheler J. Hengstler J. Leist M. Sachinidis A. Neuronal developmental gene and miRNA signatures induced by histone deacetylase inhibitors in human embryonic stem cells.Cell Death Dis. 2015; 6: e1756Crossref PubMed Scopus (25) Google Scholar Figure 5 shows a PCA plot of these expression levels. Significance of the variances of these expression levels was evaluated using PCA. PC1 showed the highest variance in miRNA transcriptomes of these samples, followed by PC2. The miRNA transcriptomes of 4-day-old SCR and WT EBs were very similar and showed clear differences in PC1 and PC2, compared with other differentiation stages. The transcriptomes of the 8-, 12-, and 16-day-old WT, as well as SCR EBs were similar, characterized by small differences in PC2. The transcriptomes of the 4-, 8-, 12-, and 16-day-old KD EBs were similar to undifferentiated KD, SCR, and WT ESCs in PC1, with minor differences in PC2. Clusters containing miRNAs that were similarly regulated during differentiation of the KD, SCR, and WT ESCs are shown in Figure 5B (a representative time course of expression levels of different miRNAs clusters in differentiated WT and KD ESCs is shown in Figure 5C). As shown in Figure S1B, the expression levels of the miRNAs in differentiated SCR ESCs were very similar to the expression levels in the differentiated SCR ESCs. Of particular interest" @default.
• W2607140359 created "2017-04-28" @default.
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• W2607140359 date "2017-06-01" @default.
• W2607140359 modified "2023-10-16" @default.
• W2607140359 title "STRIP2 Is Indispensable for the Onset of Embryonic Stem Cell Differentiation" @default.
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