scholarly journals Comparing the differentiation potential of Brachyury+ mesodermal cells generated from 3-D and 2-D culture systems

2017 ◽  
Author(s):  
Jing Zhou ◽  
Antonius Plagge ◽  
Patricia Murray
Keyword(s):  
Ex Vivo ◽  
Expression Profiles ◽  
Embryonic Stem ◽  
Gene Expression Profiles ◽  
Embryoid Bodies ◽  
Pcr Analysis ◽  
Lateral Plate ◽  
Differentiation Potential ◽  
Culture Systems

AbstractMesodermal populations can be generated in vitro from mouse embryonic stem cells (mESCs) using three-dimensional (3-D) aggregates called embryoid bodies or two-dimensional (2-D) monolayer culture systems. Here, we investigated whether Brachyury-expressing mesodermal cells generated using 3-D or 2-D culture systems are equivalent, or instead, have different properties. Using a Brachyury-GFP/E2-Crimson reporter mESC line, we isolated Brachyury-GFP+ mesoderm cells using flow-activated cell sorting and compared their gene expression profiles and ex vivo differentiation patterns. Quantitative RT-PCR analysis showed significant up-regulation of Cdx2, Foxf1 and Hoxb1 in the Brachyury-GFP+ cells isolated from the 3-D system compared with those isolated from the 2-D system. Furthermore, using an ex vivo mouse kidney rudiment assay, we found that irrespective of their source, Brachyury-GFP+ cells failed to integrate into developing nephrons, which are derived from the intermediate mesoderm. However, Brachyury-GFP+ cells isolated under 3-D conditions appeared to differentiate into endothelial-like cells within the kidney rudiments, whereas the Brachyury-GFP+ isolated from the 2-D conditions only did so to a limited degree. The high expression of Foxf1 in the 3-D Brachyury-GFP+ cells combined with their tendency to differentiate into endothelial-like cells suggests these mesodermal cells may represent lateral plate mesoderm.

scholarly journals Hematopoietic-Specific Genes Are Not Induced During In Vitro Differentiation of scl-Null Embryonic Stem Cells

Blood ◽  
1997 ◽  
Vol 90 (4) ◽  
pp. 1435-1447 ◽  
Author(s):  
Andrew G. Elefanty ◽  
Lorraine Robb ◽  
Raquella Birner ◽  
C. Glenn Begley
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Expression Profiles ◽  
Embryonic Stem ◽  
Gene Expression Profiles ◽  
Embryoid Bodies ◽  
Globin Genes ◽  
Wild Type ◽  
Temporal Expression Pattern

Abstract The helix-loop-helix transcription factor, scl, plays an essential role in hematopoietic development. Embryos in which the gene has been disrupted fail to develop yolk sac erythropoiesis, and scl-null embryonic stem cells do not contribute to hematopoiesis in chimeric mice. To analyze the molecular consequences of scl deficiency, we compared the gene expression profiles of control (wild-type and scl-heterozygous) and scl-null embryonic stem cells differentiated in vitro for up to 12 days. In control and scl-null embryoid bodies the temporal expression pattern of genes associated with the formation of ventral mesoderm, such as Brachyury, bone morphogenetic protein-4, and flk-1, was identical. Similarly, GATA-2, CD34, and c-kit, which are coexpressed in endothelial and hematopoietic lineages, were expressed normally in scl-null embryonic stem cell lines. However, hematopoietic-restricted genes, including the transcription factors GATA-1, EKLF, and PU.1 as well as globin genes and myeloperoxidase, were only expressed in wild-type and scl-heterozygous embryonic stem cells. Indirect immunofluorescence was used to confirm the observations that GATA-1 and globins were only present in control embryoid bodies but that CD34 was found on both control and scl-null embryoid bodies. These data extend the previous gene ablation studies and support a model whereby scl is absolutely required for commitment of a putative hemangioblast to the hematopoietic lineage but that it is dispensable for endothelial differentiation.

scholarly journals Hematopoietic-Specific Genes Are Not Induced During In Vitro Differentiation of scl-Null Embryonic Stem Cells

Blood ◽  
1997 ◽  
Vol 90 (4) ◽  
pp. 1435-1447 ◽  
Author(s):  
Andrew G. Elefanty ◽  
Lorraine Robb ◽  
Raquella Birner ◽  
C. Glenn Begley
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Expression Profiles ◽  
Embryonic Stem ◽  
Gene Expression Profiles ◽  
Embryoid Bodies ◽  
Globin Genes ◽  
Wild Type ◽  
Temporal Expression Pattern

The helix-loop-helix transcription factor, scl, plays an essential role in hematopoietic development. Embryos in which the gene has been disrupted fail to develop yolk sac erythropoiesis, and scl-null embryonic stem cells do not contribute to hematopoiesis in chimeric mice. To analyze the molecular consequences of scl deficiency, we compared the gene expression profiles of control (wild-type and scl-heterozygous) and scl-null embryonic stem cells differentiated in vitro for up to 12 days. In control and scl-null embryoid bodies the temporal expression pattern of genes associated with the formation of ventral mesoderm, such as Brachyury, bone morphogenetic protein-4, and flk-1, was identical. Similarly, GATA-2, CD34, and c-kit, which are coexpressed in endothelial and hematopoietic lineages, were expressed normally in scl-null embryonic stem cell lines. However, hematopoietic-restricted genes, including the transcription factors GATA-1, EKLF, and PU.1 as well as globin genes and myeloperoxidase, were only expressed in wild-type and scl-heterozygous embryonic stem cells. Indirect immunofluorescence was used to confirm the observations that GATA-1 and globins were only present in control embryoid bodies but that CD34 was found on both control and scl-null embryoid bodies. These data extend the previous gene ablation studies and support a model whereby scl is absolutely required for commitment of a putative hemangioblast to the hematopoietic lineage but that it is dispensable for endothelial differentiation.

scholarly journals Discovery of novel mechanosensitive genes in vivo using mouse carotid artery endothelium exposed to disturbed flow

Blood ◽  
2010 ◽  
Vol 116 (15) ◽  
pp. e66-e73 ◽  
Author(s):  
Chih-Wen Ni ◽  
Haiwei Qiu ◽  
Amir Rezvan ◽  
Kihwan Kwon ◽  
Douglas Nam ◽  
...  
Keyword(s):  
Carotid Artery ◽  
Ex Vivo ◽  
Expression Profiles ◽  
Quantitative Polymerase Chain Reaction ◽  
Gene Expression Profiles ◽  
Disturbed Flow ◽  
A Genome ◽  
Pattern Comparison

Abstract Recently, we showed that disturbed flow caused by a partial ligation of mouse carotid artery rapidly induces atherosclerosis. Here, we identified mechanosensitive genes in vivo through a genome-wide microarray study using mouse endothelial RNAs isolated from the flow-disturbed left and the undisturbed right common carotid artery. We found 62 and 523 genes that changed significantly by 12 hours and 48 hours after ligation, respectively. The results were validated by quantitative polymerase chain reaction for 44 of 46 tested genes. This array study discovered numerous novel mechanosensitive genes, including Lmo4, klk10, and dhh, while confirming well-known ones, such as Klf2, eNOS, and BMP4. Four genes were further validated for protein, including LMO4, which showed higher expression in mouse aortic arch and in human coronary endothelium in an asymmetric pattern. Comparison of in vivo, ex vivo, and in vitro endothelial gene expression profiles indicates that numerous in vivo mechanosensitive genes appear to be lost or dysregulated during culture. Gene ontology analyses show that disturbed flow regulates genes involved in cell proliferation and morphology by 12 hours, followed by inflammatory and immune responses by 48 hours. Determining the functional importance of these novel mechanosensitive genes may provide important insights into understanding vascular biology and atherosclerosis.

Abstract 15751: A New Role of Sox6 in Renin Regulation

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Jose Gomez ◽  
Eric Sum ◽  
Anna Keyte ◽  
Conrad Hodgkinson ◽  
Mary Hutson ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Cell Fate ◽  
Kidney Development ◽  
Ex Vivo ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Renin Angiotensin System ◽  
Adult Kidney ◽  
Fold Reduction

Introduction: The renin-angiotensin system (RAS) is an important component of blood pressure regulation in mammals. Renin catalyzes the rate limiting step of RAS, is produced and stored by Juxtaglomerular (JG) cells in the kidney. However, the transcriptional mechanisms that govern the specification of renin expressing cells under normal or pathophysiological conditions remain poorly understood. During blood pressure changes the number of adult renal cells expressing renin increase through a process termed JG recruitment. We found that this process involves differentiation mesenchymal stromal-like cells (MSC) to renin expressing cells. Our aim in this study was to determine new regulators of renin cell fate during kidney development and JG recruitment. Methods: Gene expression profiles of MSC and JG cells were performed with Affymetrix Mouse 430 2.0 array. In vitro assays were performed in adult renal MSCs isolated from C57BL6 Ren1c YFP mice. Renin expression in vitro was induced by treatment with IBMX and Forskolin. MSC were transduced with lentivirus carrying vectors for Sox6, Sox6 shRNA or controls. Ex vivo analysis was performed in embryonic kidneys (14.5 dpc) isolated and transduced with Sox6 or scrambled shRNA, kidneys were then cultured for 4 days and the expression of Sox6 and Renin analyzed by IHC. Results: Data showed that the transcription factor Sox6 is expressed in renin producing cells in the developing kidney (n=4) and in the adult kidney after stimulation that promotes JG recruitment (n=3). Overexpression of Sox6 (n=3, P<0.05) enhanced differentiation of renal MSCs to renin producing cells in vitro , and Sox6 knockdown reduced differentiation of renal MSC to renin producing cells in vitro (6-fold, n=4, P<0.01). Furthermore, knockdown of Sox6 in an ex vivo model of kidney development resulted in a 5-fold reduction in renin expressing cells (n=4, P<0.05). Conclusion: These results support a novel role for Sox6 in the development of renin expressing cells. This may have implications for renal development and physiology, opening new possibilities of addressing questions regarding both developmental and physiological regulation of renin.

scholarly journals Isolation, Characterization and Differentiation Potential of Chicken Spermatogonial Stem Cell Derived Embryoid Bodies

2016 ◽  
Vol 16 (1) ◽  
pp. 115-128 ◽  
Author(s):  
Thanh Luan Nguyen ◽  
Jae Gyu Yoo ◽  
Neelesh Sharma ◽  
Sung Woo Kim ◽  
Yong Jun Kang ◽  
...  
Keyword(s):  
Developmental Biology ◽  
Regenerative Medicine ◽  
Connexin 43 ◽  
Es Cells ◽  
Periodic Acid ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Significant Finding ◽  
Differentiation Potential

Abstract Human, murine and monkey spermatogonial stem cells (SSCs) have the capability to undergo self-renewal and differentiation into different body cell types in vitro, which are expected to serve as a powerful tool and resource for the developmental biology and regenerative medicine. We have successfully isolated and characterized the chicken SSCs from 3-day-old chicken testicular cells. The pluripotency was using Periodic Acid-Schiff (PAS ) staining or alkaline phosphatase staining, and antibodies to stage-specific embryonic antigens. In suspension culture conditions SSCs formed embryoid bodies (EBs) like embryonic stem (ES) cells. Subsequently EB differentiated into osteoblasts, adipocytes and most importantly into cardiomyocytes under induced differentiation conditions. The differentiation potential of EBs into cardiomyocyte-like cells was confirmed by using antibodies against sarcomeric α-actinin, cardiac troponin T and connexin 43. Cardiomyocytes-like cells were also confirmed by RT-PCR analysis for several cardiac cell genes like GATA-4, Nkx2-5, α-MHC, and ANF. We have successfully established an in vitro differentiation system for chicken SSCs into different body cells such as osteoblasts, adipocytes and cardiomyocytes. The most significant finding of this study is the differentiation potential of chicken SSCs into cardiomyocytes. Our findings may have implication in developmental biology and regenerative medicine by using chicken as the most potential animal model.

scholarly journals Transcriptome profiling of peanut (Arachis hypogaea) gynophores in gravitropic response

2013 ◽  
Vol 40 (12) ◽  
pp. 1249 ◽  
Author(s):  
Hai-fen Li ◽  
Xiao-Ping Chen ◽  
Fang-he Zhu ◽  
Hai-Yan Liu ◽  
Yan-Bin Hong ◽  
...  
Keyword(s):  
Differentially Expressed Genes ◽  
Arachis Hypogaea ◽  
Molecular Mechanisms ◽  
Carbon Fixation ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Pentose Phosphate ◽  
Differentially Expressed ◽  
Pcr Analysis

Peanut (Arachis hypogaea L.) produces flowers aerially, but the fruit develops underground. This process is mediated by the gynophore, which always grows vertically downwards. The genetic basis underlying gravitropic bending of gynophores is not well understood. To identify genes related to gynophore gravitropism, gene expression profiles of gynophores cultured in vitro with tip pointing upward (gravitropic stimulation sample) and downward (control) at both 6 and 12 h were compared through a high-density peanut microarray. After gravitropic stimulation, there were 174 differentially expressed genes, including 91 upregulated and 83 downregulated genes at 6 h, and 491 differentially expressed genes including 129 upregulated and 362 downregulated genes at 12 h. The differentially expressed genes identified were assigned to 24 functional categories. Twenty pathways including carbon fixation, aminoacyl-tRNA biosynthesis, pentose phosphate pathway, starch and sucrose metabolism were identified. The quantitative real-time PCR analysis was performed for validation of microarray results. Our study paves the way to better understand the molecular mechanisms underlying the peanut gynophore gravitropism.

scholarly journals Comparison of gene expression profiles in erythroid-like cells derived from mouse embryonic stem cells differentiated in simple and co-culture systems

2008 ◽  
Vol 83 (2) ◽  
pp. 109-115 ◽  
Author(s):  
Mandana B. Boroujeni ◽  
Mojdeh Salehnia ◽  
Mojtaba R. Valojerdi ◽  
Seyed J. Mowla ◽  
Mehdi Forouzandeh ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Expression Profiles ◽  
Embryonic Stem ◽  
Gene Expression Profiles ◽  
Mouse Embryonic Stem Cells ◽  
Culture Systems

scholarly journals Effect ofEx VivoCulture Conditions on Immunosuppression by Human Mesenchymal Stem Cells

2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
Myoung Woo Lee ◽  
Dae Seong Kim ◽  
Somi Ryu ◽  
In Keun Jang ◽  
Hye Jin Kim ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ex Vivo ◽  
Expression Profiles ◽  
Human Mesenchymal Stem Cells ◽  
Gene Expression Profiles ◽  
Culture Conditions ◽  
Seeding Density ◽  
Initial Seeding ◽  
Density Treatment

A microarray analysis was performed to investigate whetherex vivoculture conditions affect the characteristics of MSCs. Gene expression profiles were mainly influenced by the level of cell confluence rather than initial seeding density. The analysis showed that 276 genes were upregulated and 230 genes downregulated in MSCs harvested at~90% versus~50% confluence (P<0.05,FC>2). The genes that were highly expressed in MSCs largely corresponded to chemotaxis, inflammation, and immune responses, indicating direct or indirect involvement in immunomodulatory functions. Specifically, PTGES and ULBP1 were up-regulated in MSCs harvested at high density. Treatment of MSCs withPTGESorULBP1siRNA reversed their inhibition of T-cell proliferationin vitro. The culture conditions such as cell confluence at harvest seem to be important for gene expression profile of MSCs; therefore, the results of this study may provide useful guidelines for the harvest of MSCs that can appropriately suppress the immune response.

scholarly journals A Comparison of Ex Vivo Expanded Human Regulatory T Cells Using Allogeneic Stimulated B Cells or Monocyte-Derived Dendritic Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Linda M. Lee ◽  
Hong Zhang ◽  
Karim Lee ◽  
Horace Liang ◽  
Alexander Merleev ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
B Cells ◽  
Regulatory T Cells ◽  
Ex Vivo ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Low Frequencies ◽  
Almost All

Alloreactive regulatory T cells (arTregs) are more potent than polyclonal Tregs at suppressing immune responses to transplant antigens. Human arTregs can be expanded with allogeneic CD40L-stimulated B cells (sBcs) or stimulated-matured monocyte-derived dendritic cells (sDCs). Here, we compared the expansion efficiency and properties of arTregs stimulated ex vivo using these two types of antigen-presenting cells. Compared to sBcs, sDCs stimulated Tregs to expand two times more in number. The superior expansion-inducing capacity of sDCs correlated with their higher expression of CD80, CD86, and T cell-attracting chemokines. sBc- and sDC-arTregs expressed comparable levels of FOXP3, HELIOS, CD25, CD27, and CD62L, demethylated FOXP3 enhancer and in vitro suppressive function. sBc- and sDCs-arTregs had similar gene expression profiles that were distinct from primary Tregs. sBc- and sDC-arTregs exhibited similar low frequencies of IFN-γ, IL-4, and IL-17A-producing cells, and the cytokine-producing arTregs expressed high levels of FOXP3. Almost all sBc- and sDC-arTregs expressed CXCR3, which may enable them traffic to inflammatory sites. Thus, sDCs-arTregs that expand more readily, are phenotypically similar to sBc-arTregs, supporting sDCs as a viable alternative for arTreg production for clinical evaluation.

scholarly journals Transcriptomic Analysis of Rat Macrophages

2021 ◽  
Vol 11 ◽  
Author(s):  
Clare Pridans ◽  
Katharine M. Irvine ◽  
Gemma M. Davis ◽  
Lucas Lefevre ◽  
Stephen J. Bush ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Profiles ◽  
Embryonic Stem ◽  
Gene Expression Profiles ◽  
Gene Signature ◽  
Colony Stimulating Factor ◽  
Blood Monocytes ◽  
Macrophage Colony ◽  
Stimulating Factor

The laboratory rat is widely used as a model for human diseases. Many of these diseases involve monocytes and tissue macrophages in different states of activation. Whilst methods for in vitro differentiation of mouse macrophages from embryonic stem cells (ESC) and bone marrow (BM) are well established, these are lacking for the rat. The gene expression profiles of rat macrophages have also not been characterised to the same extent as mouse. We have established the methodology for production of rat ESC-derived macrophages and compared their gene expression profiles to macrophages obtained from the lung and peritoneal cavity and those differentiated from BM and blood monocytes. We determined the gene signature of Kupffer cells in the liver using rats deficient in macrophage colony stimulating factor receptor (CSF1R). We also examined the response of BM-derived macrophages to lipopolysaccharide (LPS). The results indicate that many, but not all, tissue-specific adaptations observed in mice are conserved in the rat. Importantly, we show that unlike mice, rat macrophages express the CSF1R ligand, colony stimulating factor 1 (CSF1).

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