FGF2 signaling is required for cell cycle reactivation

For further investigation of the signaling pathways, which are involved in the de-differ-entiation of mAGES, a western blot of phosphorylated proteins downstream of FGF2 was performed. FGF2 signaling can be mediated by different MAPK pathways including p38, JNK, ERK (p42/44), as well as the PI3K/Akt pathway. To minimize phosphorylation of downstream proteins by other medium components such as BMP4, mAGES were starved in N2B27-medium without any additional factors for 24 h. Then, medium without N2 and B27 supplement was added for 2 h. In this condition, cells were exposed to 20 ng/ml FGF2 for 20 min before cells were lysed. An increase in phosphorylated ERK (pERK) as well as Akt (pAkt) could be observed in response to FGF2, while total protein (un-phos-phorylated) was not affected (Fig. 15A). Levels of phosphorylated p38 or JNK did not change after stimulation with FGF2, although both pathways revealed basic activity (Sup-plemental Fig. 16A). As control, cells were pre-incubated for 30 min with 10 µM SU5402, an inhibitor of the FGFR tyrosine kinase, before FGF2 was added. SU5402 clearly inhib-ited the phosphorylation of both ERK and Akt. While Akt phosphorylation was com-pletely inhibited by SU5402, ERK levels were decreased by 75% compared with the stim-ulation with FGF2 alone (Fig. 15B).

As a consequence of ERK and Akt phosphorylation inhibition, the de-differentiation of mAGES in the presence of SU5402 was disturbed. SU5402 did not affect the viability of mAGES, maintained in BMP4-containing medium, as measured by resazurin reduction (Supplemental Fig. 16B). However, it clearly inhibited the proliferation of mAGES ex-posed to FGF2 for 8 days at concentrations above 2.5 µM (Fig. 15C). The inhibition of proliferation could be further confirmed by a strong decrease of EdU incorporation (Fig.

15D). Besides proliferation, SU5402 also inhibited the upregulation of nestin in mAGES exposed to FGF2 by 50% (Fig. 15D), clearly demonstrating that mAGES fail to de-dif-ferentiate in the presence of SU5402.

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Fig. 14. Transformation of mature mAGES to neural stem cells by FGF2

(A) The mAGES were exposed to 20 ng/ml FGF2 for 1-9 days. EdU was added to the medium at 48 h before cell fixation, and its incorporation was visualized by immunocytochemistry. EdU-positive nuclei were counted by high throughput imaging. (B) Cells were treated as in (A). The glial fibrillary acidic protein (GFAP) and nestin were visualized by immunocytochemistry, and the number of positive cells was counted by high throughput imaging. (C) As shown in the schematic, neural stem cells (NSC) were differentiated to fully mature mAGES with BMP4 for 30 days (normally for 5 days, as in (A) and (B)), before cells were exposed to 20 ng/ml FGF2 or medium containing no factors for 8 days; EdU was added for the last 48 h.

GFAP (green) and nestin (red) were visualized by immunocytochemistry. Nuclei were stained with H-33342 (blue). The right part of the panel visualizes EdU incorporation. FGF2 treatment induced a conver-sion of fully mature mAGES into neural stem-like cells (NSC2). (D) Embryonic stem cell-derived NSC (pool), three single cells clones of these NSC (# 1-3), and induced pluripotent stem cell (iPS)-derived NSC were differentiated to mAGES (3 days 20 ng/ml BMP4). Then, mAGES were exposed to 20 ng/ml FGF2 for 8 days, and incubated with EdU for the last 48 h. GFAP/nestin protein and EdU incorporation were visualized by immunocytochemistry, and positive cells were counted. n.d., not detectable. All data are means ± SEM.

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To investigate if both Akt and ERK phosphorylation are necessary for the de-differentia-tion of mAGES, inhibitors of downstream proteins specific for one of the respective path-ways were tested. Ly294002, an inhibitor of the PI3K, which is responsible for Akt phos-phorylation, did not show any effect at all, when mAGES were exposed to FGF2 in the presence of up to 10 µM inhibitor for 8 days (data not shown). However, U0126, an in-hibitor of MEK1/2, which phosphorylates ERK, inhibited the proliferation of mAGES in response to FGF2 at concentrations above 10 µM, as measured by resazurin reduction (Supplemental Fig. 17A). Viability of mAGES cultured with BMP4 was not affected by U0126 up to 50 µM (Supplemental Fig. 17D). Moreover, 10 µM U0126 completely in-hibited DNA synthesis in mAGES exposed to FGF2 as measured by EdU incorporation (Supplemental Fig. 17B). Interestingly, nestin expression or rather nestin upregulation in response to FGF2 was not affected, indicating that the proliferation of cells and the change in phenotype might be regulated by different pathways (Supplemental Fig. 17B). As a control for specificity of the inhibitor U0126, phosphorylation of ERK and Akt was meas-ured after FGF2 stimulation in the presence or absence of 10 µM U0126. While ERK phosphorylation was completely blocked by U0126, Akt phosphoralytion was not af-fected (Supplemental Fig. 17C).

FGF2 alone has been shown to be sufficient for the de-differentiation of mAGES ( Fig.

13C). In combination with EGF, the de-differentiation was slightly enhanced, although EGF alone did not show any effect regarding the proliferation or the phenotype of cells within 8 days of exposure. However, since many studies described an involvement of EGF in the de-differentiation of astrocytes, we wanted to disclose, if endogenously pro-duced EGF might be involved. Therefore, we exposed mAGES to gefitinib, an antibody binding to the EGF receptor, and inhibiting its receptor tyrosine kinase. Exposure of mAGES in BMP4-containing medium to the inhibitor revealed that gefitinib was toxic to the cells at ≥ 5 µM, as measured by resazurin reduction, indicating that endogenously produced EGF is necessary for the maintenance of mAGES (Supplemental Fig. 18A). To determine the effective concentration of gefitinib, phosphorylation of proteins down-stream of EGF receptor were tested. Interestingly, EGF signaling also involved a phos-phorylation of Akt and ERK, which was completely blocked by gefitinib at 1 µM and already strongly inhibited at 0.1 µM (Fig. 15E). Gefitinib was not toxic to mAGES at this concentration (Supplemental Fig. 18A).

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Fig. 15. Need for FGF2 signaling, but not EGF, for cell cycle reactivation

(A) The mAGES were pre-incubated for 30 min with 10 µM SU5402 (FGF receptor inhibitor), and exposed to 20 ng/ml FGF2 for 20 min. Cell lysates were analyzed by Western blot for Akt and ERK, an their phos-phorylated forms (pAkt, pERK). (B) Densitometric quantification of the ratio between phosphos-phorylated and un-phosphorylated proteins from three experiments as in (A). ***, p <.0001; **, p <.001. (C) The mAGES were exposed for 8 days either to 0 (ctrl) or 20 ng/ml FGF2 plus increasing concentrations of SU5402.

Proliferation was measured by quantification of additional resazurin reduction. (D) The mAGES were treated as in (C), and incubated with EdU for the last 48 h. Nestin protein and EdU incorporation were visualized by immunocytochemistry. Positive cells were counted by high throughput imaging. (E) The mAGES were pre-incubated for 30 min with gefitinib (Gef, EGF receptor inhibitor), and exposed to 20 ng/ml EGF or 20 ng/ml FGF2 for 20 min, before Akt, ERK, and their phosphorylated forms were deter-mined by Western blot analysis. (F) The mAGES were exposed for 8 days either to 0 (ctrl) or 20 ng/ml FGF2 plus increasing concentrations of gefitinib. EdU was added for the last 48 h. EdU incorporation and nestin protein were visualized by immunocytochemistry. Positive cells were counted by high throughput imaging. All data presented are means ± SEM.

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Since EGF and FGF2 shared the same signaling pathways, we tested whether gefitinib only affects EGF and not FGF2 signaling. Therefore, cells were stimulated with FGF2 for 20 min in combination with gefitinib. Gefitinib, in contrast to SU5402, did not affect phosphorylation of Akt and ERK induced by FGF2 (Fig. 15E). A phosphorylation of JNK or p38 could not be observed (data not shown). To test if EGF receptor activation is nec-essary for the de-differentiation, mAGES were exposed to FGF2 in combination with gefitinib up to its effective concentration of 1 µM. Gefitinib did not significantly affect EdU incorporation or nestin expression (Fig. 15F), and a decrease in proliferation in mAGES exposed to FGF2 could not be observed below cytotoxic concentrations (Sup-plemental Fig. 18B,C). Thus, EGF is neither sufficient nor necessary for the de-differen-tiation of mAGES.