Characterization of isolated MSCs. (A) FACS analysis of rat MSCs. Numbers in panels represent mean fluorescent intensity of the cells expressing each marker. (B and C) Phase-contrast microscopy of rat (B) and human (C) MSCs. (D–K) Immunocytochemistry of CD29 (D), CD90 (E), and CD34 (F) of human MSCs and PDGF receptor β (PDGFR) (G), and smooth muscle actin (sm-actin) (H), CD31 (I), CD45 (J), and neurofilamen-M (K) of rat MSCs. Bars = 50 ∝m. (L–P) Adipogenic (L and M), chondrogenic (N), and osteogenic (O) induction from human (L–N) and rat (O) MSCs. Micrographs in (M) show oil red staining of lipid droplet in adipocytes in (N) immunocytochemistry of collagen type II of chondrogenic induction and in (O) alkaline phosphatase in osteocytes. (L) Phase-contrast image of adipocytes. Bars = 50 ∝m. (P) Alkaline phosphatase (ALP) activity of rat and human MSCs before and after osteogenic induction. **P < 0.01.

Analysis of TF-MSCs (5 days after trophic factor induction). (A–C) Phase contrast of TF-MSCs from rats (A and B) and humans (C). Bars, A = 200 ∝m, B and C = 50 ∝m. (D–F and H–J) Immunocytochemical analysis of neuronal and glial markers in rat (F, H–J) and human (D and E) TF-MSCs. MAP-2ab (D), neurofilament-M (E), and β-tubulin isotype 3 (F) were detected. None of the cells were reactive to GFAP (H), GalC (I), and O4 (J). (G) The Brd-U labeling of rat TF-MSCs. MAP-2ab–positive cells (green) did not incorporate Brd-U (red), whereas negative cells were occasionally incorporated with Brd-U. Bars in D–J = 100 ∝m. (K) Western blot analysis of MAP-2ab and GFAP rat samples. Brain, positive control; TF-MSC. β-tubulin (tub) as a loading control. (L–Q) Patch clamp. K⁺ current increased with trophic factor induction up to approximately 1,600 pA and 4,000 pA in rat (L) and human (M) TF-MSCs, respectively. (N) Phase contrast of human TF-MSCs recorded in (M). (O) Voltage-gated inward current recorded in rat BDNF + NGF–treated TF-MSCs. A series of Na current to show the process of block by TTX. Capacity current was blanked. (P) Action potentials from rat BDNF + NGF–treated TF-MSCs; subthreshold, threshold, and suprathreshold current injections were made. (Q) Immunocytochemistry of sodium channel (green). Bar = 30 –m. (R) Relative promoter activities of NeuroD and GFAP in rat MSCs, N-MSCs, and TF-MSCs.

Induction of TH-positive cells. (A) The percentage of each transmitter in rat TF-MSCs and TH in rat G-MSCs. NPY, neuropeptide Y; CGRP, calcitonin gene-related peptide; VACht, vesicular acetylcholine transporter; SP, substance P; Glu, glutamate; Ser, serotonin transporter; Vip, vasoactive intestinal peptide; GABA, γ-aminobutyric acid. (B–D) TH expression (green) in rat (B) and human G-MSCs (C and D). Bars in B and C = 100 ∝m; D = 30 ∝m. (E) RT-PCR of each factor in rat cells. Upregulation is recognized in G-MSCs compared with TF-MSCs. (F) Western blot of TH. Adrenal medulla (Ad M), as a positive control. β-tubulin (tub) as a loading control. (G) Analysis of apomorphine-induced rotations after transplantation of rat cells. Sham operation group (diamonds), MSC group (triangles), TF-MSC group (circles), and G-MSC group (squares) (*0.01 < P < 0.05; **P < 0.01, statistical difference to sham group). (H) Apomorphine-induced rotation in sham-operated rats (triangles) and after the transplantation of human MSCs (hMSCs; squares) and human G-MSCs (hG-MSCs; circles). (I and J) Adjusting step test (I) and paw-reaching test (J) after transplantation of rat G-MSCs. Adjusting step test: the ratios of the number of steps of the lesioned side to the contralateral intact side paw. Paw-reaching test: the ratios of the number of pellets eaten by the lesioned side paw to the contralateral intact side paw. Control group received no grafting after 6-OHDA administration.

Phenotype of N-MSCs. (A) Immuno-cytochemistry of GLAST, 3-PGDH, and nestin in rat MSCs and N-MSCs. Bar = 50 ∝m. (B) Promoter activity of 3-PGDH in rat MSCs and N-MSCs (NICD transfection followed by 11 days of G418 selection). Both full-length and truncated (M1965) forms of 3-PGDH showed nine- to tenfold increases in promoter activity in N-MSCs. **P < 0.01.

Transcription factors during induction. (A) RT-PCR of rat MSCs, N-MSCs, 24 hours and 5 days after trophic factor induction in TF-MSCs, and positive control. (B) Western blot of Notch extracellular domain (NECD) and NICD in rat MSCs, N-MSCs, TF-MSCs (5 days after trophic factor induction), and positive control from brain. β-tubulin (tub) as a loading control. (C and D) Generation of MAP-2ab⁺ cells (green in D) by JAK/STAT inhibitor WHI-P131 administration and subsequent trophic factor induction in rat MSCs. (E and F) Rat MSCs cotransfected with active form of STAT3 (anti-FLAG; green) and NICD, followed by trophic factor induction. Only a small number of cells were positive for MAP-2ab (red). (G and H) Hes1/5 transfection to rat MSCs. Bars in C–H = 80 ∝m.

Histological analysis of striatum 10 weeks after transplantation in G-MSC group. (A) GFP-labeled rat transplanted cells were found in the gray regions in the schematic diagrams. (B) Box B in panel A is subjected to HE staining. Implantation zone is indicated by a dotted line. Bar = 100 ∝m. (C) Immunostaining of anti-GFP (green) and –TH (red) of box C in panel B from an adjacent section of panel B. TH-positive processes are extending to the outside of the implantation zone (arrows). Bar = 30 ∝m. (D–G) Immunostaining for TH (D), DAT (E), GFAP (F), and O4 (G) (all red color-coded) taken from the area of boxes D–G in panel A. Bar = 50 ∝m.