Functional and phenotypic resemblance between NSC2 and NSC

cell marker genes were measured by qPCR, and compared with expression levels in mAGES and NSC (Fig. 17A). All astrocyte markers were strongly downregulated, and expressed in a similar range in NSC and NSC2. Glt-1 and S100B were even lower ex-pressed in NSC2 compared with NSC. Gfap was not exex-pressed at all in NSC as well as NSC2. By contrast, neural stem cell markers such as Nestin and Olig2 were upregulated in NSC2 compared with mAGES. Thus, qPCR revealed a clear resemblance between NSC2 with NSC, demonstrating high expression of neural stem cell markers, and the absence or at least strong downregulation of astrocyte marker genes.

Furthermore, expression of over 34,000 genes (covered by 45,000 probesets) was meas-ured by microarray profiling. For each cell type (mESC, NSC, mAGES, NSC2), mRNA was prepared from four independent differentiations, and principal component analysis of global gene expression demonstrated close similarity of NSC2 and NSC, which clus-tered together (Fig. 17B). The replicates of NSC2 revealed one outlier, while the other three replicates strongly overlapped with each other and the replicates of NSC. This out-lier has not been used for further analysis.

NSC2 microarray expression data was referred to NSC, to identify differentially ex-pressed genes (DEG) between both populations. However, not a single DEG could be identified, which was significantly up- or downregulated in NSC2 compared with NSC.

Thus, microarray profiling confirmed a very strong resemblance between NSC2 and NSC.

The genes, which have been identified before as suitable marker genes to distinguish as-trocytes from neural stem cells (Fig. 21) (Kleiderman et al. in press), were used here to analyze NSC2 expression. A heatmap of the normalized expression data confirmed the resemblance between NSC2 and NSC (Fig. 17C). While NSC marker genes were highly expressed, astrocyte marker genes were consistently downregulated in NSC2. The cell type-specific genes, which we identified before, focused more on astrocyte-specific marker genes. For a characterization of NSC2, however, neural stem cell-specific marker genes would be more suitable. Only recently, marker genes specific for neural stem cells, as in contrast to mature astrocytes, have been identified (Gotz et al. 2015). These genes,

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derived from in vivo data, were used to characterize NSC2 compared with NSC and mAGES. A heatmap of normalized expression data for NSC-selectivity genes demon-strated high expression of more than 70% of the genes in NSC2 and NSC, while they were less abundant in mAGES (Fig. 18). The remaining 30% revealed high variety in mAGES samples and were not suitable to distinguish NSC and mAGES. Thus, microar-ray profiling clearly demonstrated a high correlation of NSC2 and NSC. By the upregu-lation of neural stem cell and the downreguupregu-lation of astrocyte marker genes, gene expres-sion analysis confirmed a complete converexpres-sion of astrocytic mAGES to the neural stem cell-like NSC2.

For a functional comparison of NSC2 with NSC and mAGES, those features have been tested, which were clearly different between mAGES and NSC (Fig. 21) (Kleiderman et al., in press). The proliferation and neurogenic capacity of NSC2, as specific features for neural stem cells, have already been tested. Regarding astrocyte-specific functions, the inflammatory capacity of NSC2 has been assessed. NSC2 as well as NSC, mAGES, and mAGES2 were exposed to the inflammatory cytokine mix (CCM) for 30 min, and NFkB translocation was measured (Fig. 17D). NSC2 and NSC did not activate NFkB in contrast to mAGES and mAGES2, in which NFkB was translocated into the nucleus in more than 90% of the cells. Moreover, mAGES2 revealed the same metabolic pattern as mAGES.

Both cell types released citrate (Fig. 17E), and exhibited similar glucose consumption and lactate release rates (Fig. 17F). In contrast, NSC and NSC2 did not release citrate and showed higher metabolic rates compared with mAGES or mAGES2. Thus, NSC2 are not only functional neural stem cells, but they also lose astrocyte-specific functions, confirm-ing a complete conversion from an astrocytic to a neural stem cell identity.

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Fig. 17. Comparison of NSC and NSC2 for gene expression, metabolism, and inflammatory compe-tence

(A) Astrocyte-specific mRNA (Gfap (glial fibrillary acidic protein), Aqp4 (aquaporin), Glt-1 (Slc1A2, glu-tamate transporter), inwardly-rectifying potassium channel (Kir4.1), Aldh1L1 (aldehyde dehydrogenase) and S100b (S100beta)), or neural stem cell-specific mRNA (nestin, oligodendrocyte lineage transcription factor (Olig2), and brain lipid binding protein (Blbp)) were quantified by qPCR and normalized to Gapdh.

Data are means ± SEM. ***, p <.0001; **, p <.001; *, p <.01; ns, not significant (relative to NSC2). (B) Whole transcriptome data were obtained by microarray analysis for murine embryonic stem cells (mESC), NSC, mAGES, and NSC2 of 4 independent differentiations. Data are presented in a 2D-principal compo-nent analysis plot. The 95% confidence interval for gene expression of each cell type is indicated by shaded elypses. (C) Heatmap of gene expression values for astrocyte (blue) and NSC (red) marker genes previously identified (Kleiderman et al., in press). The normalized (z-scores) expression data are plotted for four NSC,

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four mAGES, and three NSC2 samples for each of the marker genes. Blue colors represent low, red colors high expression, with z-scores ranging from 1.4 to -1.4. The full gene names and the corresponding absolute expression values are listed in Supplemental Fig. 19. (D) All cell types were exposed to a cytokine mix (=10 ng/ml TNFα, 10 ng/ml IL1β, and 20 ng/ml IFNγ) for 30 min, and immunostained for the transcription factor NF-κB (NFkB) p65 subunit. NFkB translocation was measured by high throughput imaging. Data are means ± SEM. (E) Citrate concentrations were measured in the supernatant of all cell populations after 3/24 h, and citrate release (normalized to total protein content) was calculated. Data are means ± SEM. ***, p <.0001. (F) Glucose and lactate concentrations were measured in the supernatant after 3/24 h. Uptake/re-lease rates were calculated, and normalized to total protein content. Data are means ± SEM of two biological replicates. *, p <.01.