Figure 1.
Scheme for the applied differentiation protocol.
In order to initiate human AFS cell differentiation to a Schwann cell phenotype AFS cells were first treated in serum free α-MEM with 1 mM β-mercaptoethanol (Diff. I) for 24 hours. Afterwards cells were incubated in α-MEM supplemented with 10% fetal bovine serum and 35 ng/ml retinoic acid (Diff. II) for 72 hours. Subsequently, cells were cultured in α-MEM containing 10% fetal bovine serum supplemented with 20 ng/mL epidermal growth factor, 20 ng/mL basic fibroblast growth factor, 5 mM forskolin, 5 ng/mL platelet-derived growth factor-AA and 200 ng/mL recombinant human heregulin-beta1 (Diff. III) until day 15 of differentiation. Media was changed every 3 days, indicated by arrows. Pharmacologic (pharm.) treatment, consisting of rapamycin or statin, was applied together with Diff. III media.
Figure 2.
Human monoclonal amniotic fluid stem cells can be differentiated into a early Schwann cell phenotype.
(A) AFS cells are small cells with omnidirectional protruding filopodia and upon differentiation to Schwann-like cells, at day 15 of treatment, cells exhibited an increase in cellular volume and an elongated cell morphology. Scale bar represents 50 µm. (B) Immunofluorescence staining of AFS cells differentiated for 15 days (dAFS) compared to undifferentiated AFS cells (AFS) and MCM1 neural crest-derived cells (control), for the Schwann cell markers NGFR, GFAP and S100b (labeled in red, nuclei labeled in green). Purity of cells is indicated as percent positive cells versus total amount of cells ± S.D. Scale bar represents 10 µm. (C) Quantitative RT-PCR of cDNA derived from AFS cells and from AFS cells subjected to Schwann cell differentiation after different time points was performed. Results are shown as fold change expression of respective genes compared to undifferentiated AFS cells. The results are expressed as means ± SEM of three independent experiments. P<0.05 for * vs undifferentiated AFS cells.
Figure 3.
mTOR signaling is active in differentiated AFS cells and important for the differentiation process.
(A) AKT phosphorylation at Ser 473 and ribosomal protein S6 phosphorylation at Ser 240/244 were quantified at the indicated time points during differentiation with and without rapamycin treatment. (B) NGFR, a marker for early differentiated AFS cells (labeled in green), was co-stained with phosphorylated S6 at Ser 240/244 protein (labeled in red) with and without rapamycin treatment (nuclei labeled in blue). Scale bar represents 10 µm. (C) Accumulation of free cholesterol was monitored by filipin III staining. Scale bar represents 10 µm.
Figure 4.
Rapamycin treatment down-regulates Schwann cell marker expression in differentiated human AFS cells and in sciatic nerves from juvenile mice.
(A) Quantitative RT-PCR of cDNA derived from AFS cells differentiated towards Schwann cells for 15 days with and without rapamycin was performed to assess Schwann cell marker expression. Results are shown as fold change of respective gene expression from rapamycin-treated cells compared to control treated cells. (B) Sciatic nerves were isolated from everolimus- or control-treated mice and cDNA generated thereof was assessed for Schwann cell marker expression. Results are shown as fold change of respective gene expression from everolimus-treated mice compared to control-treated mice. The results are expressed as means ± SEM of three independent experiments. P<0.05 for * vs control treated cells or animals. (C) Sciatic nerves from untreated or treated mice were subjected to Luxol fast blue staining and immunohistochemical staining for S100b and S6 phosphorylation was performed (stained in red, nuclei in blue). Panel in upper right shows control treated sciatic nerve tissue stained for active S6 protein (red) and nuclei (blue), insert shows control antibody staining. Scale bar represents 20 µm.
Figure 5.
Rapamycin decreases Schwann cell markers, whereas statin induces Schwann cell markers.
(A) During the last 72 hrs of differentiation, AFS cells were treated with 5 µM and 10 µM statin. After 15 days cDNA was generated and used for quantitative PCR of respective genes. The results are expressed as means ± SEM of three independent experiments. P<0,05 for * vs control treated cells. (B) AFS cells were differentiated for 15 days and since day 5 continuously treated either with 25 nM rapamycin or 1 µM of statin. Fixed cells were stained with indicated antibodies (labeled in red, nuclei in green). Scale bar represents 10 µm. (C) Western blotting of cells differentiated for 15 days and since day 5 continuously treated either with 25 nM rapamycin or 1 µM of statin. GFAP was detected at about 50 kDa, LDLR at 160 kDa and HMGCR as a double band at 90 kDa.
Figure 6.
Rapamycin resistant S6K1 induces S100b, but not LDLR or HMGCR expression.
(A) AFS cells were differentiated without or (B) in the presence of rapamycin and at day 15 cells were transfected with an HA-fused S6K1 rapamycin-resistant mutant (HA-S6K1-RR). After 72 hours in differentiation media containing rapamycin, cells were fixed and stained with anti-HA antibody (shown in green) combined with antibodies detecting S100b, LDLR, HMGCR and phosphorylated S6 (shown in red). Scale bar represents 25 µm.
Figure 7.
Model of mTORC1 involvement in Schwann cell differentiation.
Rapamycin blocks mTORC1 and results in the down regulation of Schwann cell markers (e.g.: S100b) and in the down regulation of lipogenic genes (e.g.: LDLR, HMGCR). Our data indicates that S6K1 regulates the expression of S100b, but not of LDLR and HMGCR.