Int J Stem Cells.  2023 Aug;16(3):293-303. 10.15283/ijsc22168.

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

• Herrmann A
• Meyer AK
• Braunschweig L
• Wagenfuehr L
• Markert F
• Kolitsch D
• Vukicevic V
• Hartmann C
• Siebert M
• Ehrhart-Bornstein M
• Hermann A
• Storch A

• KMID: 2545229
• DOI: http://doi.org/10.15283/ijsc22168

Abstract

Background and Objectives
The physiological oxygen tension in fetal brains (∼3%, physioxia) is beneficial for the maintenance of neural stem cells (NSCs). Sensitivity to oxygen varies between NSCs from different fetal brain regions, with midbrain NSCs showing selective susceptibility. Data on Hif-1α/Notch regulatory interactions as well as our observations that Hif-1α and oxygen affect midbrain NSCs survival and proliferation prompted our investigations on involvement of Notch signalling in physioxia-dependent midbrain NSCs performance.
Methods and Results
Here we found that physioxia (3% O₂ ) compared to normoxia (21% O 2 ) increased proliferation, maintained stemness by suppression of spontaneous differentiation and supported cell cycle progression. Microarray and qRT-PCR analyses identified significant changes of Notch related genes in midbrain NSCs after long-term (13 days), but not after short-term physioxia (48 hours). Consistently, inhibition of Notch signalling with DAPT increased, but its stimulation with Dll4 decreased spontaneous differentiation into neurons solely under normoxic but not under physioxic conditions.
Conclusions
Notch signalling does not influence the fate decision of midbrain NSCs cultured in vitro in physioxia, where other factors like Hif-1α might be involved. Our findings on how physioxia effects in midbrain NSCs are transduced by alternative signalling might, at least in part, explain their selective susceptibility to oxygen.

Keyword

Hypoxia; Physioxia; Hif-1α; Notch; Cell cycle; Stem cell maintenance; Neural stem cells

Figure

• 

  Fig. 1 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% O2. Scale bar is 25 μm. (B) Representative images of mNSCs stained for Tuj1, Map2, GFAP, Nestin and Hoechst. A total of 105 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% O2 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% O2. Plots show the relative distribution of cells grown under 3% or 21% O2 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% O2 (unpaired two-sided t-test with Bonferroni correction).

• 

  Fig. 2 The Notch pathway is active in mNSCs. (A) Representative images of mNSCs stained for NICD and Hoechst. A total of 105 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% O2. (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 (Hiflox/lox) of mNSCs Hif-1α CKO grown in 3% or 21% O2. 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 Hiflox/lox (D; unpaired two-sided t-test with Bonferroni correction).

• 

  Fig. 3 Notch inhibits spontaneous differentiation under normoxic conditions but has no influence on the cell cycle. (A) Relative mRNA levels of Notch target genes Hes1, 3, 5, Hey1 and Hif-1α target gene Vegf in respect to Hmbs, normalised to the respective control condition of mNSCs treated with DAPT or Dll4 and grown in 3% or 21% O2. Red dotted line indicates threshold for relevant changed genes. (B) Representative bright-field images of mNSCs cultured with (+) or without (−) DAPT. mNSCs were cultured 13 days in 3% or 21% of oxygen and treated with DAPT and their respective solvent as a control (DMSO). Scale bar is 50 μm. (C) Cell counts of mNSCs grown in 3% (blue) or 21% O2 (red). Cells were cultured for 13 days with (+) or without (−) DAPT or their respective solvent (DMSO) as control. Cells were counted as Hoechst+ cells per mm2. (D) Survival of mNSCs under physioxia (blue) and normoxia (red) cultured for 13 days. The number of PI+ cells normalised to the total mNSC number is displayed. (E) Quantitative analysis displaying the cellular distribution in the different cell cycle phases. The plot shows the relative distribution of mNSCs grown under physioxia or normoxia and treated with DAPT, Dll4 and their respective controls across the different cell cycle phases G0-G1, S or G2-M in percent. (F) Representative images of mNSCs stained for Tuj1, GFAP and Hoechst. A total of 105 cells were seeded on a coverslip and grown for three days under hypoxic or normoxic conditions in absence or presence of DAPT, soluble or membrane bound Dll4. Scale bar is 50 μm. (G) Quantitative analysis of the relative amount of Tuj1+ or GFAP+ cells normalised to the total number of cells (Hoechst+) and compared to the control condition. *p<0.05, **p<0.01, ***p<0.001 in respect to control condition using unpaired two-sided t-test (A, G); Two-way ANOVA with Bonferroni-adjusted post-hoc t-test **p<0.01, ***p<0.001 in respect to cells grown in 21% O2 (C∼E); ++p<0.01 +++p<0.001 in respect to cells grown without DAPT (C∼E).

Reference

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