NSC2 are generated from mature astrocytes

Although the mAGES population is very homogeneous with more than 99% of the cells expressing the astrocyte marker GFAP (Kleiderman et al. in press), we cannot be sure, if NSC2 really arise from individual mAGES, or if a minor subpopulation of non-differen-tiated GFAP-negative cells produce NSC2. So there exist two hypotheses for the genera-tion of NSC2 (Fig. 19A): First, some cells within the mAGES populagenera-tion did not differ-entiate into mature astrocytes, and stay in an intermediate state. They start to proliferate in response to FGF2, and overgrow the mAGES, which reply with cell death. And second, mAGES re-enter the cell cycle, and convert into neural stem cells. Data obtained on the de-differentiation of 1 month-old mAGES decreases the probability of hypothesis 1. Ev-idence on the homogeneity of the mAGES population, and the absence of immature pre-cursor or quiescent stem cells (Ki67^-, p27⁺, CD133^-), which has been investigated before, further suggest hypothesis 2.

When a minor subpopulation of cells produces NSC2, which overgrow mAGES, the loss of GFAP expression during de-differentiation would assume extensive cell death of mAGES. Therefore, LDH release was measured during the first 8 days of exposure to FGF2, where GFAP expression was already reduced. During FGF2 exposure, no increas-ing LDH release could be observed, indicatincreas-ing that there does not occur measureable cell death (Supplemental Fig. 21A). Thus, the loss of GFAP expression is not due to cell death of GFAP-expressing cells, but due to a downregulation of the astrocyte marker, which is in contrast to hypothesis 1.

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Fig. 18. Gene expression of NSC-selectivity genes

Heatmap of gene expression values for NSC-selectivity marker genes previously identified (Gotz et al., 2015). The normalized (z-scores) expression data are plotted for four NSC, 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 2 to -6. The full gene names and the corresponding absolute expression values are listed in Supplemental Fig. 20.

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Fig. 19. Evidence for NSC2 generation from differentiated astrocytes

(A) Two working hypotheses on the origin of NSC2 have been formulated for further testing. Hypothesis 1: putative nestin-positive stem cells (red) within the mAGES population (<1%) are stimulated by FGF2 to proliferate (green nuclei), while GFAP-positive, non-proliferating mAGES (blue) die. Hypothesis 2: FGF2 induces cell cycle re-entry of a large subpopulation of mAGES (green nuclei); these convert to GFAP+/nes-tin+-positive cells, and later to nestin+/GFAP- neural stem cells. (B) Experimental design to investigate the phenotype of transition cells: mAGES were exposed to 20 ng/ml FGF2 or medium without additional fac-tors (no FGF2, ctrl) for up to 7 days, and EdU was added to the medium for the last 48 h before cells were immunostained. (C) Cells were treated and analyzed as in (B). EdU/GFAP-double-positive, and EdU sin-gle- positive cells were counted by high throughput imaging. (D) The experiment described in (C) was performed with Aquaporin 4 (Aqp4)-staining instead of GFAP. (E) The mAGES were exposed to FGF for 10 days, and all of the original mAGES that divided at least once during this period were identified. To calculate the percentage of dividing cells, either live-cell continuous time-lapse imaging (tracking 84 DsRed-transfected cells (details in Supplemental Fig. 21,22)), or measurement of the label dilution of car-boxyfluorescein-stained (CFSE) mAGES (Supplemental Fig. 21) was used. All data presented are means ± SEM of two/three biological replicates.

To more directly prove hypothesis 2, the proliferation of mAGES has been investigated in more detail. If single mAGES give rise to NSC2, GFAP- and/or Aqp4-positive astro-cytes should re-enter the cell cycle and incorporate EdU into DNA. Therefore, mAGES were exposed to FGF2 for up to 8 days and incubated for the last 48 h respectively with EdU (Fig. 19B). Afterwards, cells were fixed, and stained for EdU incorporation and GFAP, AQP4, or nestin expression. Up to day 7, more than 90% of the proliferating (EdU-positive) cells co-expressed GFAP (Fig. 19C). After day 7, the amount of GFAP

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and EdU double-positive cells decreased, consistent with the downregulation of GFAP after prolonged FGF2 exposure (data not shown). Moreover, most of the EdU-positive cells co-expressed the mature astrocyte marker AQP4 (Fig. 19D). Interestingly, all of the EdU-positive cells were also positive for nestin, indicating that proliferation of cells is accompanied by a simultaneous upregulation of the neural stem cells marker (data not shown). Thus, proliferating cells expressed the astrocyte marker GFAP and/or AQP4, indicating that mature astrocytes re-entered the cell cycle in response to FGF2.

Since EdU incorporation is only a measure of DNA synthesis, but not necessarily of cell division, CFSE label dilution has been used to calculate the percentage of dividing cells during FGF2 exposure. Therefore, adherent cultures of mAGES were incubated with 10 µM CFSE for 15 min, and treated with 20 ng/ml FGF2 for up to 10 days. As control, CFSE-labeled astrocytes were maintained in medium without growth factors (BMP4 or FGF2). CFSE intensity was measured in live cells with a high throughput imaging device.

A histrogram of total CFSE intensity in 20,000 cells showed that CFSE-label decreased in FGF2-exposed cells, demonstrating cell proliferation or rather the decrease of label during cell division (Supplemental Fig. 21B). To define the classes of non-dividing and dividing cells, a threshold has been set, which comprises 95% of the cells in control cul-tures. On day 10, 28% of the FGF2-exposed mAGES revealed a CFSE-label intensity above this threshold, and were non-dividing cells. However, the amount of non-dividing cells was referred to total cell count measured on day 10, where most of the cells already divided and at least doubled the amount of cells compared with the starting population.

To normalize the data with regard to the starting population (day 0), cells were grouped into cells that have divided once, twice, or three times. For this categorization, the thresh-old was consecutively divided by 2, since CFSE label is bisected with every cell division.

Then, the amount of cells, which divided once, was divided by 2, the amount of cells, which divided twice, was divided by 4, and so forth (Supplemental Fig. 21B). With this approach, the frequency of cells, which divided up to three times, could be calculated and the percentage of cells in the starting population, which were dividing or non-dividing, could be determined. Within 10 days of FGF2 exposure, 50% of the mAGES re-entered the cell cycle (Fig. 19E).

To confirm the CFSE-labeling data, and to directly follow mAGES division, imaging of live cells during FGF2 exposure has been performed. The mAGES were transfected with DsRed to track single cells (even in high density cultures). Then, mAGES were exposed

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to FGF2 for up to 10 days, and time-lapse microscopy was performed taking pictures of DsRed-labeled cells every 5 h (Supplemental Fig. 22B,D). Single cells have been tracked using Timm’s Tracking Tool, which has been developed to track freshly isolated neural stem cells in vitro. With this approach, cell division could be directly recorded, and the percentage of dividing cells as well as further grouping in one, two, three, or more cell divisions could be calculated. Lineage trees were produced for 84 cells, of which 42 (50%) divided during 10 days of FGF2 exposure (Supplemental Fig. 21C), consistent with the 50% dividing cells measured with CFSE-labeling. Cell division of mAGES was heterogeneous, with most of the cells dividing once (19%) or three or more times (19%) (Supplemental Fig. 21C). Most of the cells (60%) achieved their first cell division be-tween day 6 and 8 of FGF2 exposure (Supplemental Fig. 21D), which is consistent with EdU incorporation that starts to increase at day 5.

Time-lapse microscopy of mAGES (low density cultures), with phase contrast pictures taken every 15 min, also revealed that around 50% of the astrocytes (flat cells with a star-shaped morphology) divided (Supplemental Fig. 22A,C). After two to three divisions, cells changed their appearance and adopted a morphology similar to that of NSC. No cell division at all was observed in control cultures maintained in medium containing no fac-tors or 10 ng/ml BMP4 (data not shown). Thus, CFSE-label dilution, time-lapse micros-copy, as well as EdU incorporation of GFAP- and/or Aqp4-positive cells, confirmed hy-pothesis 2 with 50% of mAGES re-entering the cell cycle, self-renewing, and converting into neural stem-like NSC2.