Fig 1.
Scheme of the different models for neuronal differentiation assessment.
(A) Neuronal differentiation of NT2 cells was performed throughout 3 weeks, with medium exchanges three times a week in alternated days. After 24 days of differentiation (d24), the obtained mixed (undifferentiated and neuronal) cell population was treated with anti-mitotic compounds for neuronal enrichment during 10 days (d34). B) SH-SY5Y cells were induced to differentiate during 7 days (d7), subjected to a differentiation medium exchange at day 4. After 7 days of differentiation (d7), a mixed population of undifferentiated cells and post-mitotic neurons was obtained. In order to obtain an enriched neuronal population, cultures are treated with anti-mitotic compounds for 5 days (d12). C) After 11 days of in vitro culture, the differentiation medium is added to the OHSC (11 DIV). The medium is exchanged twice a week during 2 weeks. Then, fully mature and differentiated slice cultures were obtained (26DIV).
Fig 2.
CORM-A1 increases final yield of enriched neurons.
(A) Neuronal yield is calculated based on nuclei count per volume of NT2 derived post-mitotic neurons (neuron/mL) after 24 days of differentiation and 10 days of neuronal enrichment; (B) Nuclei count per volume of SH-SY5Y derived post-mitotic neurons (neuron/mL) after 7 days of differentiation and 5 days of neuronal enrichment (C) Characterization of NT2 derived post-mitotic neurons by immunocytochemistry (green staining: Tuj1; blue staining: DAPI; magnification 200x); (D) Characterization of SH-SY5Y derived post-mitotic neurons by immunocytochemistry (green staining: Tuj1; blue staining: DAPI; magnification 100x); (E,F) Characterization of neuronal functionality by neurotransmitter quantification (glutamate and GABA) in mixed cell populations of NT2 and SH-SY5Y cells after 24 and 7 days of differentiation, respectively. Glutamate and GABA quantification is normalized by total protein amount.
Fig 3.
CO increases total mixed cellular population during differentiation process: precursor cells, early stage neurons and mature neurons.
Characterization of mixed cell population was assessed following neuronal differentiation and before neuronal enrichment. (A) Nuclei count of NT2 mixed population after 24 days of differentiation; (B) Nuclei count of SH-SY5Y mixed population after 7 days of differentiation. mRNA expression of specific neuronal differentiation markers (Nestin for neuronal precursors, Tuj1 for early-differentiated neurons and MAP2 for mature neurons) was quantified for (C) NT2 mixed population and for (D) SH-SY5Y mixed population.
Fig 4.
CORM-A1 does not increase the expression of retinoic acid receptors in mixed cell population (after neuronal differentiation procedure).
(A) mRNA expression of cellular and nuclear retinoic acid receptors were measured in NT2 mixed population after 24 days of differentiation; (B) Quantification of mRNA of cellular and nuclear retinoic acid receptors in SH-SY5Y mixed population after 7 days of differentiation.
Fig 5.
CORM-A1 promotes cell proliferation.
(A) Ki67 mRNA expression was assessed in NT2 mixed cell population at two distinct time points, day 17 and day 24, during neuronal differentiation process; (B) quantification of ki67 mRNA expression in SH-SY5Y mixed population after 7 days of differentiation.
Fig 6.
CORM-A1 prevents cell death in mixed cell population (following neuronal differentiation procedure).
(A) NT2 PI incorporation evaluation after 24 days of differentiation; (B) SH-SY5Y PI incorporation evaluation after 7 days of differentiation; (C) NT2 mixed population’s Bcl-2 expression at two distinct time points, day 17 and day 24, during neuronal differentiation process; (D) SH-SY5Y mixed population’s Bcl-2 expression after 7 days of differentiation; (E) NT2 mixed population’s Bax expression after 24 days of differentiation; (F) SH-SY5Y mixed population’s Bax expression after 7 days of differentiation; (G) cleaved caspase-3 protein quantification by western blot analysis of cell extracts obtained after 24 days of NT2 neuronal differentiation; (H) cleaved caspase-3 protein quantification by western blot analysis of cell extracts obtained after 7 days of SH-SY5Y neuronal differentiation.
Fig 7.
Role of reactive oxygen species (ROS) in CORM-A1 modulation of neuronal differentiation.
(A) Quantification of intracellular ROS in NT2 mixed cell populations after 24 days of neuronal differentiation (values normalized by total cell count); (B) Intracellular ROS quantification in SH-SY5Y mixed population 1h after treatment with RA 10μM ± CORM-A1 25μM and after 7 days of differentiation (values normalized by total cell count); (C) Phase contrast images of SH-SY5Y cells after 7 days of differentiation under different conditions (including N-acetylcysteine treatment); (D) Quantification of cell number and protein levels of SH-SY5Y cells after 7 days of differentiation under different conditions.
Fig 8.
Validation of CORM-A1 role in neuronal differentiation in ex-vivo model of OHSC.
(A)OHSC immunohistochemistry at 26 DIV (green staining: Tuj1; blue staining: DAPI; magnification 50x); (B) phase contrast images during neuronal differentiation process (magnification 40x); (C) OHSC immunohistochemistry at 14 DIV (red staining: ki67; blue staining: Hoechst33342; magnification 50x); (D) Proliferative cells (ki67 positive cells) per slice. Ratio calculated taking in account the area positively stained for ki67 and Hoechst33342; (E) OHSC PI uptake (red staining) at 26 DIV (magnification 50x); (F) PI uptake per slice—count of the positively marked cells in each slice; (G) OHSC Active caspase-3 and Bcl-2 expression at two distinct time points, 18 DIV and 26 DIV, during neuronal differentiation process (red staining: active caspase-3; blue staining: Hoescht33342; green staining: Bcl2) (magnification 40x); Area per slice. Ratio calculated taking in account the area positively stained for active caspase-3 and Bcl2 at two distinct time points, 18 DIV and 26 DIV, during neuronal differentiation process; (H) Immunohistochemistry quantification of active caspase-3 positive cells in HOSC after 26DIV, treated with N-acetyl-cysteine and CORM-A1 during neuronal differentiation process.