Fig 2: GDNF secretion enhanced the neural-like cell differentiation capacity of hAMSC in mouse models of PD. (A, D, G, and J) Representative immunofluorescence staining pictures of GDNF, Nestin, GFAP, and Tuj-1 expression and neural-like cell differentiation in PD/hAMSC-vector and PD/hAMSC-GDNF groups, scale bar, 50 μm. (B and C) Immunofluorescence staining assay indicated that the hAMSCs could secrete GDNF steadily in the PD/hAMSC-GDNF group but not in the PD/hAMSC-vector group. (E & F, H & I, and K & L) The PD/hAMSC-GDNF group showed a higher number of GDNF-, Nestin-, GFAP-, and Tuj-1-positive cells compared to the PD/hAMSC-vector group. Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001. GDNF: glial cell line-derived neurotrophic factor; GFAP: glial fibrillary acidic protein; hAMSCs: human primary adipose-derived mesenchymal stem cells; SEM: standard error of the mean.

Fig 3: Exogenous GDNF promotes the proliferation of hAMSCs and induces hAMSC differentiation in vitro. (A) MTT assays showed that hAMSCs pretreated with GDNF exhibited a greater proliferation capacity when compared with the control group. (B and C) Similar results were confirmed by Ki67 immunofluorescence staining assay, scale bar, 50 μm. (D) Representative immunofluorescence staining pictures of vimentin, Nestin, GFAP, and Tuj-1 in the different groups, scale bar, 50 μm. (E–H) The hAMSCs which were exposed to GDNF displayed a significantly higher percentage of Nestin, GFAP, and Tuj-1 expressions compared to control groups. (I) Western blot assays were used to detect GFRa1 expression in the hAMSCs. Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001. GDNF: glial cell line-derived neurotrophic factor; GFAP: glial fibrillary acidic protein; hAMSCs: human primary adipose-derived mesenchymal stem cells; MTT: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; SEM: standard error of the mean.

Fig 4: hAMSC-GDNF alleviates the motor behavior of 6-OHDA-lesioned mice. (A) Schematic of the behavior tests performed in PD mice after 6-OHDA lesioning. (B) Representative images showing the number of TH-positive immunostaining cells in the SNc of 6-OHDA-lesioned mice. (C and D) The number of TH-positive cells in the PD/hAMSC-GDNF group was significantly higher than that in the PD/saline, PD/GDNF, and PD/ hAMSC-vector groups in 6-OHDA-lesioned mice. (E) The average number of rotations per minute was significantly lower in the PD/hAMSC-GDNF group than in the PD/saline, PD/GDNF, and PD/hAMSC-Vector groups. (F) Time to fall displayed a significant difference in the PD/hAMSC-GDNF group compared to other PD groups. The PD/hAMSC-GDNF group exhibited a significantly longer time to fall than the PD/saline, PD/GDNF, and PD/hAMSC-Vector groups. Scale bar, 200 μm. Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.001. 6-OHDA: 6-hydroxydopamine; APO: apomorphine-induced rotation; DAPI: 4′,6-diamidino-2-phenylindole; GDNF: glial cell line-derived neurotrophic factor; hAMSCs: human primary adipose-derived mesenchymal stem cells; PD: Parkinson’s disease; SEM: standard error of the mean; SNc: substantia nigra; TH: tyrosine hydroxylase.

Fig 5: Effects of erythropoietin on total, small, and large RGCs under TFW-induced toxicity. (A) Morphology of TFW-treated RGCs. The cells were cultured in a medium lacking BDNF, CNTF, and bFGF. After culturing, RGCs were immunostained with anti-Thy-1 antibody (red), anti-NF-L antibody (green), and DAPI nuclear staining (blue). Survival rate of (B) total, (C) small, and (D) large RGCs. The cells were cultured in a standard medium, TFW medium, TFW medium+GDNF (1–100 ng/mL), or TFW medium+EPO (1–100 ng/mL). The number of RGCs was counted and normalized versus the control group. Data are presented as mean ± standard error of the mean (n = 3–12). #P<0.05, ##P<0.01, and ###P<0.001 versus the control group according to Student’s t-test. *P<0.05, **P<0.01 and ***P<0.001 versus the TFW group according to 1-way ANOVA, followed by Dunnett’s test.