Intragraft gene expression profile associated with the induction of tolerance-0

R, transduced bone marrow cells and bone marrow cells from GalT BMT mice at day 14 post-BMT express the galactosyltransferase gene as identified by real-time PCR. Relative cDNA expression levels were normalized with respect to GAPDH gene expression as an internal control. The samples were run in triplicate and the experiment was repeated twice. Standard deviations are shown for each experiment.<p><b>Copyright information:</b></p><p>Taken from "Intragraft gene expression profile associated with the induction of tolerance"</p><p>http://www.biomedcentral.com/1471-2172/9/5</p><p>BMC Immunology 2008;9():5-5.</p><p>Published online 11 Feb 2008</p><p>PMCID:PMC2275216.</p><p></p

Intragraft gene expression profile associated with the induction of tolerance-5

R, transduced bone marrow cells and bone marrow cells from GalT BMT mice at day 14 post-BMT express the galactosyltransferase gene as identified by real-time PCR. Relative cDNA expression levels were normalized with respect to GAPDH gene expression as an internal control. The samples were run in triplicate and the experiment was repeated twice. Standard deviations are shown for each experiment.<p><b>Copyright information:</b></p><p>Taken from "Intragraft gene expression profile associated with the induction of tolerance"</p><p>http://www.biomedcentral.com/1471-2172/9/5</p><p>BMC Immunology 2008;9():5-5.</p><p>Published online 11 Feb 2008</p><p>PMCID:PMC2275216.</p><p></p

Intragraft gene expression profile associated with the induction of tolerance-2

Ts (GalT BMT) compared to syngeneic control grafts. Nine functional subsets were identified using Gene Ontology Biological Process categories.<p><b>Copyright information:</b></p><p>Taken from "Intragraft gene expression profile associated with the induction of tolerance"</p><p>http://www.biomedcentral.com/1471-2172/9/5</p><p>BMC Immunology 2008;9():5-5.</p><p>Published online 11 Feb 2008</p><p>PMCID:PMC2275216.</p><p></p

Intragraft gene expression profile associated with the induction of tolerance-4

Tolerance group (GalT BMT) compared to syngeneic controls were analyzed by real-time PCR to determine whether the data obtained by microarray analysis could be validated using an alternative technique. Relative cDNA expression levels were normalized with respect to GAPDH gene expression as internal control. Results are shown as the logarithmic value of mean fold-change of gene expression.<p><b>Copyright information:</b></p><p>Taken from "Intragraft gene expression profile associated with the induction of tolerance"</p><p>http://www.biomedcentral.com/1471-2172/9/5</p><p>BMC Immunology 2008;9():5-5.</p><p>Published online 11 Feb 2008</p><p>PMCID:PMC2275216.</p><p></p

Simulated Microgravity Exerts an Age-Dependent Effect on the Differentiation of Cardiovascular Progenitors Isolated from the Human Heart

<div><p>Microgravity has a profound effect on cardiovascular function, however, little is known about the impact of microgravity on progenitors that reside within the heart. We investigated the effect of simulated microgravity exposure on progenitors isolated from the neonatal and adult human heart by quantifying changes in functional parameters, gene expression and protein levels after 6-7 days of 2D clinorotation. Utilization of neonatal and adult cardiovascular progenitors in ground-based studies has provided novel insight into how microgravity may affect cells differently depending on age.</p><p>Simulated microgravity exposure did not impact AKT or ERK phosphorylation levels and did not influence cell migration, but elevated transcripts for paracrine factors were identified in neonatal and adult cardiovascular progenitors. Age-dependent responses surfaced when comparing the impact of microgravity on differentiation. Endothelial cell tube formation was unchanged or increased in progenitors from adults whereas neonatal cardiovascular progenitors showed a decline in tube formation (p<0.05). Von Willebrand Factor, an endothelial differentiation marker, and MLC2v and Troponin T, markers for cardiomyogenic differentiation, were elevated in expression in adult progenitors after simulated microgravity. DNA repair genes and telomerase reverse transcriptase which are highly expressed in early stem cells were increased in expression in neonatal but not adult cardiac progenitors after growth under simulated microgravity conditions. Neonatal cardiac progenitors demonstrated higher levels of MESP1, OCT4, and brachyury, markers for early stem cells. MicroRNA profiling was used to further investigate the impact of simulated microgravity on cardiovascular progenitors. Fifteen microRNAs were significantly altered in expression, including microRNAs-99a and 100 (which play a critical role in cell dedifferentiation). These microRNAs were unchanged in adult cardiac progenitors.</p><p>The effect of exposure to simulated microgravity in cardiovascular progenitors is age-dependent. Adult cardiac progenitors showed elevated expression of markers for endothelial and cardiomyogenic differentiation whereas neonatal progenitors acquired characteristics of dedifferentiating cells.</p></div

Cardiac progenitor cell differentiation after 6–7 days of simulated microgravity.

<p>A) Quantification of tube formation using Image pro plus software. Reduced tube formation was present in neonatal CPCs after simulated microgravity whereas 2 of 3 adult CPCs exhibited elevated tube formation after simulated microgravity. B) Representative images of tube formation in neonatal and adult CPC after 6–7 days of simulated microgravity. C) Representative flow cytometry histogram of increased von Willebrand Factor (vWF) expression in adult CPCs after simulated microgravity. Positive staining is represented by a colored histogram, secondary alone staining is represented as a dotted line. D) Quantification of von Willebrand factor protein expression in adult CPCs (n = 3, p = 0.047). Mean fluorescence shift above secondary antibody alone is shown above. E) Relative expression of genes associated with cardiac differentiation in adult CPCs after 6–7 days of simulated microgravity by RT-PCR. Myosin light chain 2 (MLC2V) an early indicator of cardiovascular differentiation was significantly elevated in adult CPCs (n = 3, run in triplicate P = 0.017).</p

MicroRNA expression changes in neonatal CPCs after 6–7 days of simulated microgravity.

<p>A) Heat map of 15 microRNAs that were significantly altered in neonatal CPCs after simulated microgravity. Two representative neonatal CPC clones are shown above. Red color identifies maximum expression, black color represents average expression, blue color identifies microRNAs with minimum expression. Sets of co-regulated microRNAs were grouped together by RT² Profiler PCR Array Data Analysis software (SABiosciences). B) MicroRNAs expressed at significantly different levels during simulated microgravity were analyzed using DIANA mirPATH software. Pathways significantly regulated by altered microRNAs were grouped according to KEGG pathway classifications, and the percentage of pathways in each category is displayed above. C) MicroRNA-100-5p and miR-99a-5p whose expression levels negatively correlate with stemness, were significantly downregulated after 6–7 days of simulated microgravity in neonatal CPCs (n = 3, p<0.05). D) Expression of microRNA-99a-5p and miR-100-5p were unchanged in adult CPCs after simulated microgravity (n = 3, run in triplicate).</p

Cell Function after exposure to 6–7 days of simulated microgravity.

<p>A) Simulated microgravity significantly increased the expression of growth factors in both neonatal and adult CPCs by RT-PCR (n = 3, run in triplicate * = p<0.05). After simulated microgravity, neonatal and adult CPCs were plated in transwell plates to assess migration. Neither B) neonatal or C) adult CPCs demonstrated a change in the number of migrated cells after clinorotation. Phosphorylation of D) ERK and E) AKT was not significantly altered after simulated microgravity as shown by flow cytometry. Mean fluorescence shift (MFS) above secondary antibody alone is shown.</p

DNA repair and cell number after 6–7 days of simulated microgravity.

<p>A) DNA repair transcripts including RAD50, E2F1, ATM, RAD23 were elevated after simulated microgravity in neonatal (n = 4, run in triplicate, * = p<0.05) but not adult CPCs (n = 3, run in triplicate). B) A significantly higher frequency of neonatal CPCs entered the S phase of the cell cycle when compared with adult CPCs in simulated microgravity (12.4% vs 4.9%, p = 0.03). G1 phase and G2 phase did not differ significantly between neonatal and adult CPCs (G1 phase—93.3% vs 87.1%, p = 0.62 and G2 phase—3.9% vs 7.8%, p = 0.12 respectively). C) The average number of cells after 6–7 days of clinorotation was significantly higher in neonatal CPCs (5.89 x 10⁴, n = 12) when compared with adult CPCs (1.95 x 10⁴, n = 11). Number of cells seeded is shown in grey. D) The expression level of human telomerase reverse transcriptase was significantly elevated in neonatal (n = 4, run in triplicate) but not adult CPCs after simulated microgravity. Fold change above matched control is shown.</p

Transcripts associated with stemness are elevated in neonatal CPCs after 6–7 days of simulated microgravity.

<p>The expression of genes associated with early progenitor populations including MESP1, Oct-4, and Brachyury were significantly elevated in neonatal CPCs after simulated microgravity (n = 4, run in triplicate, p<0.05).</p