A Matter of Choice: Inhibition of c-Rel Shifts Neuronal to Oligodendroglial Fate in Human Stem Cells

The molecular mechanisms underlying fate decisions of human neural stem cells (hNSCs) between neurogenesis and gliogenesis are critical during neuronal development and neurodegenerative diseases. Despite its crucial role in the murine nervous system, the potential role of the transcription factor NF-κB in the neuronal development of hNSCs is poorly understood. Here, we analyzed NF-κB subunit distribution during glutamatergic differentiation of hNSCs originating from neural crest-derived stem cells. We observed several peaks of specific NF-κB subunits. The most prominent nuclear peak was shown by c-REL subunit during a period of 2–5 days after differentiation onset. Furthermore, c-REL inhibition with pentoxifylline (PTXF) resulted in a complete shift towards oligodendroglial fate, as demonstrated by the presence of OLIG2+/O4+-oligodendrocytes, which showed PDGFRα, NG2 and MBP at the transcript level. In addition c-REL impairment further produced a significant decrease in neuronal survival. Transplantation of PTXF-treated predifferentiated hNSCs into an ex vivo oxidative-stress-mediated demyelination model of mouse organotypic cerebellar slices further led to integration in the white matter and differentiation into MBP+ oligodendrocytes, validating their functionality and therapeutic potential. In summary, we present a human cellular model of neuronal differentiation exhibiting a novel essential function of NF-κB-c-REL in fate choice between neurogenesis and oligodendrogenesis which will potentially be relevant for multiple sclerosis and schizophrenia.

Figure S2. Immunocytochemical analysis of p65 (RELA). A-F) NCSC-derived NSCs labeled against RELA after 0, 1, 2, 5, 9.5 and 10 days of glutamatergic differentiation. Each panel shows RELA protein on the left-side and DNA co-localization with DAPI staining on the right-side.
Intensity scale indicates white as highest intensity level and black as lowest intensity level. G) Quantification of immunocytochemical analyses showing nuclear mean integrated density of p65 (RELA) during early differentiation (mean ± SEM, n=3). Normality of the data was refuted using Shapiro-Wilk normality test. Non-parametric Kruskal-Wallis (***p≤0.001) and Bonferroni corrected post-test (**p<0.01) revealed a significant nuclear translocation of NF-κB-p65 at day 0.
H) Fluorescence intensity profiles measured at three different time points (0, 1, 2 days of differentiation), for different cells following transects as shown, to reveal the difference between the nuclear and cytoplasmic fluorescence. NCSC: neural crest-derived stem cells, NSCs: neural stem cells, SEM: Standard error of the mean.

Figure S3
Figure S3. Immunocytochemical analysis of RELB. A-F) NCSC-derived NSCs labeled against RELB after 0, 1, 2, 5, 9.5 and 10 days of glutamatergic differentiation. Each panel shows RELB protein on the left-side and DNA co-localization with DAPI staining on the right-side. Intensity scale indicates white as highest and black as lowest intensity levels. G) Quantification of immunocytochemical analyses show nuclear mean integrated density of RELB during early differentiation (mean ± SEM, n=3). Normality of the data was refuted using Shapiro-Wilk normality test. Non-parametric Kruskal-Wallis (***p≤0.001) and Bonferroni corrected post-test (***p<0.001) revealed a significant peak in nuclear translocation of NF-B-RELB at day 0. H) Fluorescence intensity profiles measured at three different time points (0, 1 and 2 days of differentiation), for different cells following transects as shown, to elucidate the difference between the nuclear and cytoplasmic fluorescence. NCSC: neural crest-derived stem cells, NSCs: neural stem cells, SEM: Standard error of the mean.            Table S1. Primers sequences for quantitative polymerase chain reaction.

Supplemental materials and methods section
Extended detailed methods

Immunocytochemistry
Differentiated NCSC-derived NSCs were fixed in phosphate-buffered 4% paraformaldehyde (pH 7.4) for 15 minutes at room temperature (RT) followed by 3 wash steps in phosphate-buffered saline (1xPBS). Cells were permeabilized with 0.02% Triton X-100 and blocked using 5% of appropriate serum or 3% bovine serum albumin for 30 minutes at RT, followed by incubation with primary antibodies for 1 hour at RT.