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Effects of physiological hypoxia (physioxia) on proliferation, spontaneous differentiation and cell cycle phase distribution of fetal mNSCs. (A) Representative bright-field images of E13.5 fetal mNSCs cultured for 6 or 13 days in 3% or 21% O 2 . Scale bar is 25 μm. (B) Representative images of mNSCs stained for Tuj1, Map2, GFAP, Nestin and Hoechst. A total of 10 5 cells were seeded on a coverslip and grown for three days under physioxic or normoxic conditions. Scale bar is 50 μm. (C) Comparison of a marker selection of midbrain NSC development (from left to right side) showed additional reduction of late neuronal markers such as Lmo3 or Sox6 . (D) Quantitative analysis of immunoreactivity of mNSCs grown in 3% or 21% O 2 for Tuj1, Map2, GFAP and Nestin normalised to the total number of cells (Hoechst + ) in percent. (E) Quantitative analysis of the fraction of mNSCs in the different cell cycle phases of mNSCs grown in 3 and 21% O 2 . Plots show the relative distribution of cells grown under 3% or 21% O 2 across the different cell cycle phases G0-G1, S or G2-M in percent. (F, G) Comparison of cell cycle markers (F) and senescence/quiescence markers (G) of mNSCs by <t>microarray</t> analysis. Heat-maps represent the fold change of the relative mRNA expression levels of mNSCs cultured under physioxia for 48 h or 13 d in comparison to those cultured under normoxia. Colour bar displays the colour contrast level of the heat-map with red and green indicating high and low expression levels for (C, F, G). *p<0.05, **p<0.01, ***p<0.001 in respect to 21% O 2 (unpaired two-sided t-test with Bonferroni correction).
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Effects of physiological hypoxia (physioxia) on proliferation, spontaneous differentiation and cell cycle phase distribution of fetal mNSCs. (A) Representative bright-field images of E13.5 fetal mNSCs cultured for 6 or 13 days in 3% or 21% O 2 . Scale bar is 25 μm. (B) Representative images of mNSCs stained for Tuj1, Map2, GFAP, Nestin and Hoechst. A total of 10 5 cells were seeded on a coverslip and grown for three days under physioxic or normoxic conditions. Scale bar is 50 μm. (C) Comparison of a marker selection of midbrain NSC development (from left to right side) showed additional reduction of late neuronal markers such as Lmo3 or Sox6 . (D) Quantitative analysis of immunoreactivity of mNSCs grown in 3% or 21% O 2 for Tuj1, Map2, GFAP and Nestin normalised to the total number of cells (Hoechst + ) in percent. (E) Quantitative analysis of the fraction of mNSCs in the different cell cycle phases of mNSCs grown in 3 and 21% O 2 . Plots show the relative distribution of cells grown under 3% or 21% O 2 across the different cell cycle phases G0-G1, S or G2-M in percent. (F, G) Comparison of cell cycle markers (F) and senescence/quiescence markers (G) of mNSCs by <t>microarray</t> analysis. Heat-maps represent the fold change of the relative mRNA expression levels of mNSCs cultured under physioxia for 48 h or 13 d in comparison to those cultured under normoxia. Colour bar displays the colour contrast level of the heat-map with red and green indicating high and low expression levels for (C, F, G). *p<0.05, **p<0.01, ***p<0.001 in respect to 21% O 2 (unpaired two-sided t-test with Bonferroni correction).
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Effects of physiological hypoxia (physioxia) on proliferation, spontaneous differentiation and cell cycle phase distribution of fetal mNSCs. (A) Representative bright-field images of E13.5 fetal mNSCs cultured for 6 or 13 days in 3% or 21% O 2 . Scale bar is 25 μm. (B) Representative images of mNSCs stained for Tuj1, Map2, GFAP, Nestin and Hoechst. A total of 10 5 cells were seeded on a coverslip and grown for three days under physioxic or normoxic conditions. Scale bar is 50 μm. (C) Comparison of a marker selection of midbrain NSC development (from left to right side) showed additional reduction of late neuronal markers such as Lmo3 or Sox6 . (D) Quantitative analysis of immunoreactivity of mNSCs grown in 3% or 21% O 2 for Tuj1, Map2, GFAP and Nestin normalised to the total number of cells (Hoechst + ) in percent. (E) Quantitative analysis of the fraction of mNSCs in the different cell cycle phases of mNSCs grown in 3 and 21% O 2 . Plots show the relative distribution of cells grown under 3% or 21% O 2 across the different cell cycle phases G0-G1, S or G2-M in percent. (F, G) Comparison of cell cycle markers (F) and senescence/quiescence markers (G) of mNSCs by <t>microarray</t> analysis. Heat-maps represent the fold change of the relative mRNA expression levels of mNSCs cultured under physioxia for 48 h or 13 d in comparison to those cultured under normoxia. Colour bar displays the colour contrast level of the heat-map with red and green indicating high and low expression levels for (C, F, G). *p<0.05, **p<0.01, ***p<0.001 in respect to 21% O 2 (unpaired two-sided t-test with Bonferroni correction).
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Effects of physiological hypoxia (physioxia) on proliferation, spontaneous differentiation and cell cycle phase distribution of fetal mNSCs. (A) Representative bright-field images of E13.5 fetal mNSCs cultured for 6 or 13 days in 3% or 21% O 2 . Scale bar is 25 μm. (B) Representative images of mNSCs stained for Tuj1, Map2, GFAP, Nestin and Hoechst. A total of 10 5 cells were seeded on a coverslip and grown for three days under physioxic or normoxic conditions. Scale bar is 50 μm. (C) Comparison of a marker selection of midbrain NSC development (from left to right side) showed additional reduction of late neuronal markers such as Lmo3 or Sox6 . (D) Quantitative analysis of immunoreactivity of mNSCs grown in 3% or 21% O 2 for Tuj1, Map2, GFAP and Nestin normalised to the total number of cells (Hoechst + ) in percent. (E) Quantitative analysis of the fraction of mNSCs in the different cell cycle phases of mNSCs grown in 3 and 21% O 2 . Plots show the relative distribution of cells grown under 3% or 21% O 2 across the different cell cycle phases G0-G1, S or G2-M in percent. (F, G) Comparison of cell cycle markers (F) and senescence/quiescence markers (G) of mNSCs by <t>microarray</t> analysis. Heat-maps represent the fold change of the relative mRNA expression levels of mNSCs cultured under physioxia for 48 h or 13 d in comparison to those cultured under normoxia. Colour bar displays the colour contrast level of the heat-map with red and green indicating high and low expression levels for (C, F, G). *p<0.05, **p<0.01, ***p<0.001 in respect to 21% O 2 (unpaired two-sided t-test with Bonferroni correction).
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Effects of physiological hypoxia (physioxia) on proliferation, spontaneous differentiation and cell cycle phase distribution of fetal mNSCs. (A) Representative bright-field images of E13.5 fetal mNSCs cultured for 6 or 13 days in 3% or 21% O 2 . Scale bar is 25 μm. (B) Representative images of mNSCs stained for Tuj1, Map2, GFAP, Nestin and Hoechst. A total of 10 5 cells were seeded on a coverslip and grown for three days under physioxic or normoxic conditions. Scale bar is 50 μm. (C) Comparison of a marker selection of midbrain NSC development (from left to right side) showed additional reduction of late neuronal markers such as Lmo3 or Sox6 . (D) Quantitative analysis of immunoreactivity of mNSCs grown in 3% or 21% O 2 for Tuj1, Map2, GFAP and Nestin normalised to the total number of cells (Hoechst + ) in percent. (E) Quantitative analysis of the fraction of mNSCs in the different cell cycle phases of mNSCs grown in 3 and 21% O 2 . Plots show the relative distribution of cells grown under 3% or 21% O 2 across the different cell cycle phases G0-G1, S or G2-M in percent. (F, G) Comparison of cell cycle markers (F) and senescence/quiescence markers (G) of mNSCs by <t>microarray</t> analysis. Heat-maps represent the fold change of the relative mRNA expression levels of mNSCs cultured under physioxia for 48 h or 13 d in comparison to those cultured under normoxia. Colour bar displays the colour contrast level of the heat-map with red and green indicating high and low expression levels for (C, F, G). *p<0.05, **p<0.01, ***p<0.001 in respect to 21% O 2 (unpaired two-sided t-test with Bonferroni correction).
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Effects of physiological hypoxia (physioxia) on proliferation, spontaneous differentiation and cell cycle phase distribution of fetal mNSCs. (A) Representative bright-field images of E13.5 fetal mNSCs cultured for 6 or 13 days in 3% or 21% O 2 . Scale bar is 25 μm. (B) Representative images of mNSCs stained for Tuj1, Map2, GFAP, Nestin and Hoechst. A total of 10 5 cells were seeded on a coverslip and grown for three days under physioxic or normoxic conditions. Scale bar is 50 μm. (C) Comparison of a marker selection of midbrain NSC development (from left to right side) showed additional reduction of late neuronal markers such as Lmo3 or Sox6 . (D) Quantitative analysis of immunoreactivity of mNSCs grown in 3% or 21% O 2 for Tuj1, Map2, GFAP and Nestin normalised to the total number of cells (Hoechst + ) in percent. (E) Quantitative analysis of the fraction of mNSCs in the different cell cycle phases of mNSCs grown in 3 and 21% O 2 . Plots show the relative distribution of cells grown under 3% or 21% O 2 across the different cell cycle phases G0-G1, S or G2-M in percent. (F, G) Comparison of cell cycle markers (F) and senescence/quiescence markers (G) of mNSCs by <t>microarray</t> analysis. Heat-maps represent the fold change of the relative mRNA expression levels of mNSCs cultured under physioxia for 48 h or 13 d in comparison to those cultured under normoxia. Colour bar displays the colour contrast level of the heat-map with red and green indicating high and low expression levels for (C, F, G). *p<0.05, **p<0.01, ***p<0.001 in respect to 21% O 2 (unpaired two-sided t-test with Bonferroni correction).
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Effects of physiological hypoxia (physioxia) on proliferation, spontaneous differentiation and cell cycle phase distribution of fetal mNSCs. (A) Representative bright-field images of E13.5 fetal mNSCs cultured for 6 or 13 days in 3% or 21% O 2 . Scale bar is 25 μm. (B) Representative images of mNSCs stained for Tuj1, Map2, GFAP, Nestin and Hoechst. A total of 10 5 cells were seeded on a coverslip and grown for three days under physioxic or normoxic conditions. Scale bar is 50 μm. (C) Comparison of a marker selection of midbrain NSC development (from left to right side) showed additional reduction of late neuronal markers such as Lmo3 or Sox6 . (D) Quantitative analysis of immunoreactivity of mNSCs grown in 3% or 21% O 2 for Tuj1, Map2, GFAP and Nestin normalised to the total number of cells (Hoechst + ) in percent. (E) Quantitative analysis of the fraction of mNSCs in the different cell cycle phases of mNSCs grown in 3 and 21% O 2 . Plots show the relative distribution of cells grown under 3% or 21% O 2 across the different cell cycle phases G0-G1, S or G2-M in percent. (F, G) Comparison of cell cycle markers (F) and senescence/quiescence markers (G) of mNSCs by <t>microarray</t> analysis. Heat-maps represent the fold change of the relative mRNA expression levels of mNSCs cultured under physioxia for 48 h or 13 d in comparison to those cultured under normoxia. Colour bar displays the colour contrast level of the heat-map with red and green indicating high and low expression levels for (C, F, G). *p<0.05, **p<0.01, ***p<0.001 in respect to 21% O 2 (unpaired two-sided t-test with Bonferroni correction).
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Effects of physiological hypoxia (physioxia) on proliferation, spontaneous differentiation and cell cycle phase distribution of fetal mNSCs. (A) Representative bright-field images of E13.5 fetal mNSCs cultured for 6 or 13 days in 3% or 21% O 2 . Scale bar is 25 μm. (B) Representative images of mNSCs stained for Tuj1, Map2, GFAP, Nestin and Hoechst. A total of 10 5 cells were seeded on a coverslip and grown for three days under physioxic or normoxic conditions. Scale bar is 50 μm. (C) Comparison of a marker selection of midbrain NSC development (from left to right side) showed additional reduction of late neuronal markers such as Lmo3 or Sox6 . (D) Quantitative analysis of immunoreactivity of mNSCs grown in 3% or 21% O 2 for Tuj1, Map2, GFAP and Nestin normalised to the total number of cells (Hoechst + ) in percent. (E) Quantitative analysis of the fraction of mNSCs in the different cell cycle phases of mNSCs grown in 3 and 21% O 2 . Plots show the relative distribution of cells grown under 3% or 21% O 2 across the different cell cycle phases G0-G1, S or G2-M in percent. (F, G) Comparison of cell cycle markers (F) and senescence/quiescence markers (G) of mNSCs by microarray analysis. Heat-maps represent the fold change of the relative mRNA expression levels of mNSCs cultured under physioxia for 48 h or 13 d in comparison to those cultured under normoxia. Colour bar displays the colour contrast level of the heat-map with red and green indicating high and low expression levels for (C, F, G). *p<0.05, **p<0.01, ***p<0.001 in respect to 21% O 2 (unpaired two-sided t-test with Bonferroni correction).

Journal: International Journal of Stem Cells

Article Title: Notch Is Not Involved in Physioxia-Mediated Stem Cell Maintenance in Midbrain Neural Stem Cells

doi: 10.15283/ijsc22168

Figure Lengend Snippet: Effects of physiological hypoxia (physioxia) on proliferation, spontaneous differentiation and cell cycle phase distribution of fetal mNSCs. (A) Representative bright-field images of E13.5 fetal mNSCs cultured for 6 or 13 days in 3% or 21% O 2 . Scale bar is 25 μm. (B) Representative images of mNSCs stained for Tuj1, Map2, GFAP, Nestin and Hoechst. A total of 10 5 cells were seeded on a coverslip and grown for three days under physioxic or normoxic conditions. Scale bar is 50 μm. (C) Comparison of a marker selection of midbrain NSC development (from left to right side) showed additional reduction of late neuronal markers such as Lmo3 or Sox6 . (D) Quantitative analysis of immunoreactivity of mNSCs grown in 3% or 21% O 2 for Tuj1, Map2, GFAP and Nestin normalised to the total number of cells (Hoechst + ) in percent. (E) Quantitative analysis of the fraction of mNSCs in the different cell cycle phases of mNSCs grown in 3 and 21% O 2 . Plots show the relative distribution of cells grown under 3% or 21% O 2 across the different cell cycle phases G0-G1, S or G2-M in percent. (F, G) Comparison of cell cycle markers (F) and senescence/quiescence markers (G) of mNSCs by microarray analysis. Heat-maps represent the fold change of the relative mRNA expression levels of mNSCs cultured under physioxia for 48 h or 13 d in comparison to those cultured under normoxia. Colour bar displays the colour contrast level of the heat-map with red and green indicating high and low expression levels for (C, F, G). *p<0.05, **p<0.01, ***p<0.001 in respect to 21% O 2 (unpaired two-sided t-test with Bonferroni correction).

Article Snippet: Microarray chips were then immediately scanned using an Agilent microarray confocal laser scanner.

Techniques: Cell Culture, Staining, Comparison, Marker, Selection, Microarray, Expressing

The Notch pathway is active in mNSCs. (A) Representative images of mNSCs stained for NICD and Hoechst. A total of 10 5 cells were seeded on a coverslip and cultured for three days under hypoxic or normoxic conditions. Scale bar is 50 μm. (B) Comparison of mRNA expression of mNSCs by microarray analysis. Heat-map represents the log of the fold change of the relative mRNA expression levels of mNSCs cultured under physioxia for 48 h or 13 d in comparison to those cultured under normoxia. Colour bar displays the colour contrast level of the heat-map with red and green indicating high and low expression levels. (C) Relative mRNA levels of Hes1, 3, 5, Id1, Hey1 and Hif-1α target genes Vegf, and Pgk1 in respect to Hmbs levels as housekeeping gene of mNSCs grown in 3% or 21% O 2 . (D) Relative mRNA levels of Hes1, 3, 5, Hey1, and Notch 1 and Hif-1α target genes Vegf, and Pgk1 in respect to Hmbs, normalised to the respective control condition (Hif lox/lox ) of mNSCs Hif-1α CKO grown in 3% or 21% O 2 . Red dotted line indicates threshold for relevant changed genes. *p<0.05, **p<0.01 and ***p<0.001 in respect to normoxia (B, C) or control condition Hif lox/lox (D; unpaired two-sided t-test with Bonferroni correction).

Journal: International Journal of Stem Cells

Article Title: Notch Is Not Involved in Physioxia-Mediated Stem Cell Maintenance in Midbrain Neural Stem Cells

doi: 10.15283/ijsc22168

Figure Lengend Snippet: The Notch pathway is active in mNSCs. (A) Representative images of mNSCs stained for NICD and Hoechst. A total of 10 5 cells were seeded on a coverslip and cultured for three days under hypoxic or normoxic conditions. Scale bar is 50 μm. (B) Comparison of mRNA expression of mNSCs by microarray analysis. Heat-map represents the log of the fold change of the relative mRNA expression levels of mNSCs cultured under physioxia for 48 h or 13 d in comparison to those cultured under normoxia. Colour bar displays the colour contrast level of the heat-map with red and green indicating high and low expression levels. (C) Relative mRNA levels of Hes1, 3, 5, Id1, Hey1 and Hif-1α target genes Vegf, and Pgk1 in respect to Hmbs levels as housekeeping gene of mNSCs grown in 3% or 21% O 2 . (D) Relative mRNA levels of Hes1, 3, 5, Hey1, and Notch 1 and Hif-1α target genes Vegf, and Pgk1 in respect to Hmbs, normalised to the respective control condition (Hif lox/lox ) of mNSCs Hif-1α CKO grown in 3% or 21% O 2 . Red dotted line indicates threshold for relevant changed genes. *p<0.05, **p<0.01 and ***p<0.001 in respect to normoxia (B, C) or control condition Hif lox/lox (D; unpaired two-sided t-test with Bonferroni correction).

Article Snippet: Microarray chips were then immediately scanned using an Agilent microarray confocal laser scanner.

Techniques: Staining, Cell Culture, Comparison, Expressing, Microarray