Fig 2: The effects of RNA interference of neuronal iconic proteins on podocyte morphology.Following RNA interference of nestin (A), synaptopodin (B), UCH-L1(C) and NSE (D) separately for 72 hours, Western blotting showed a decrease in the protein level of these indicators in treated podocyte compared with the control cells. Additionally, the statistical results show significant differences between the treated groups and the control group; the F-actin staining in treated podocytes showed significant morphological changes and F-actin misalignment compared with the control cells (A, B, C, D). The inhibition of NeuN (E) and S100 (F) mRNA resulted in few differences in the morphology and F-actin arrangement (E, F) compared with the control cells (compared with. Control *p<0.05, ***p<0.001). The data are presented from at least three individual experiments that were performed in duplicate. Fluorescence Phallotoxins ×400.

Fig 3: Positive ratios of NSCs-expressed neural markers in the different groups. The immunofluorescence staining was performed on the 5 groups simultaneously. The number of positive cells was measured (%), and expressed as χ±s. *P<0.05 and **P<0.01 vs. CON group; #P<0.05 vs. NC+N group; §P<0.05 vs. siNSCs+N group. NSC, neural stem cell; NSE, enolase 2; GFAP, glial fibrillary acidic protein; O4, oligodendrocyte cell surface antigen O4; CON, blank control group; NC, negative control plasmid-transfected cells; N, normal brain tissue; HIBD, hypoxic-ischemic brain damage tissue; siNSC, β-catenin small interfering RNA-transfected cell.

Fig 4: Expressions of NSE, O4 and GFAP in the experimental groups indicating NSC differentiation by fluorescence microscopy (×100). Immunofluorescence staining performed simultaneously on each group, red (CY3) represents the expression NSE or O4 in cytoplasm, green (FITC) represents the expression of GFAP in cytoplasm, blue (Hoechst 33258) represents the nucleus. NSC, neural stem cell; NSE, enolase 2; GFAP, glial fibrillary acidic protein; O4, oligodendrocyte cell surface antigen O4; CON, blank control group; NC, negative control plasmid-transfected cells; N, normal brain tissue; HIBD, hypoxic-ischemic brain damage tissue; siNSC, β-catenin small interfering RNA-transfected cells.

Fig 5: hMSC cell line from bone marrow has the ability to differentiate into spontaneously active neurons (A) Schematic representation of neuronal induction on hMSC cell line. (B) Plot indicates neuronal markers expression percentages of hMd-Neurons from hMSC cell line after neuronal induction during 12 days and almost %100 of neuronal induced cells express neuronal maturation proteins NeuN, Synaptophysin, NSE and PGP 9.5. Positively stained cells counted from 10 different area of staining and averages were calculated. Functionality of hMd-Neurons was evaluated upon labeling with Fluo-4 for real time Ca++ ion imaging without any outside stimulation chemically. (C) Immunofluorescence co-staining of hMSC cell lines in neuronal induction medium; NIM composed of NGF, BDNF, FGF-8, bFGF, EGF, dbcAMP, IBMX, B27 for 12 days reveals the presence of neuronal maturation proteins NeuN (a, e) and Synaptophysin (b, f) with DAPI nuclear staining (c, g). Merged images represent positive co-staining of NeuN and Synaptophysin for each individual cell (d, h). Dashed yellow squares magnified 3 folds (d’, h’). (D) Florescent images (a-f) demonstrates time dependent firing pattern of hMd-Neurons from hMSC cell line through imaging of Ca++ ion kinetics and arrows indicate firing of each hMd-Neuron separately (sec; seconds). (S1 Video) (E) Histogram indicates firing frequency and signal intensity of each individual hMd-Neuron while there is no signs of spontaneous activity from uninduced hMSCs. According to Ca++ influx/efflux through hMd-Neurons, (F) 78.5% of cells were recorded as spontaneously active with twice firing frequency in 4 minutes. Data are represented as mean ± S.E.M. Scale bars represent 50 μm.