PMC

In vitro abnormality analysis of the induced NSPCs. Karyotype abnormalities were seen in the 1231A3 NSPCs and CNV abnormalities were seen in the 1231A3 and 1201C1 NSPCs. a Cellular doubling times were calculated by measuring ATP consumption for each of the NSPC lines. b The cell cycle for each NSPC cell line. Although a higher percentage of 1231A3-NSPCs were in S phase, there was no significant difference between the cell lines. c Results of karyotype analysis by G band-staining are shown. Only the 1231A3 NR-NSPCs had a karyotype abnormality. See also Additional file : Figure S2, and Additional file : Tables S3, Additional file : Table S4, Additional file : Table S5 and Additional file : Table S6

Representative images of DNT, UDNT, and BLT. a, b Representative images of the DNT with zone formation around the central canal resembling cysts, which were observed in the 1231A3 NR-NSPC transplanted spinal cords 6 months after transplantation. a The red arrows indicate the cyst, blue arrows indicate the VZ, and black arrows indicate the IZ. Upper panel: H&E, STEM121, hNestin, and hGFAP. (Scale = 500 μm.) Lower panel: STEM121, hNestin, and hGFAP. (Captured in boxed area 1 in the first panel. Scale = 100 μm.) (b) STEM121+ scattered neural cells (SNCs) observed in the MZ that formed in the rostral end of boxed area 2 in Fig. 5A are indicated by white arrows. There were many more migrating neural cells than is indicated. (Scale = 50 μm.) (c) Representative histologic features of the UDNT, as observed in a 1201C1 EB-NSPCs transplanted spinal cord at the 12th week after transplantation. Upper panel: STEM121 and H&E. (Scale = 500 μm.) Lower panel: H&E, hNestin, hGFAP, hKi67, Alcian Blue, and HNA. (Captured in the boxed area in the upper panel. Scale = 50 μm.) (d) Representative histologic features of the BLT as observed in a 1210B2 NR-NSPC transplanted spinal cord at the 12th week after transplantation. The low power field views of the STEM121 staining is the same image in Fig. . Upper panel: STEM121 and H&E. (Scale = 500 μm.) Lower panel: H&E, hNestin, hGFAP, and hKi67. (Captured in the boxed area in the upper panel. Scale = 50 μm.)

Schematic neural induction diagrams and characterization of the NSPCs generated from human PBMC-derived iPSCs. a Schematics of the NSPC induction protocols used in this study. (Scale = 200 μm for the images of neurospheres.) (b, c, d) Representative data taken by 1210B2-NSPCs for characterization analysis of the NSPCs. b Cell surface markers of the induced NSPCs. c The quantitative RT-PCR analysis results are depicted by ΔCt values. Quantitative RT − PCR analysis confirmed the decrease in the iPSC markers, and an increase in NSPC markers following the differentiation of iPSCs into NSPCs. (n = 2 for each iPSC-NSPC lines) d Representative immunocytochemistry data collected to confirm the differentiation potential of NSPCs into neuronal and glial lineages. (Scale = 100 μm.) e The correlation coefficients of the expression profiles among the NSPCs. The microarray profiles were similar between all the NSPCs regardless of the induction protocols or iPSCs. (n = 2 for each iPSC-NSPC lines) See also Additional file : Figure S1 and Additional file : Table S1

Histology revealed proliferative characteristics of the 1231A3-NSPCs both in intact brains and injured spinal cords. a Schematic of the in vivo transplantation protocol that was used. b Representative tissue sections of the spinal cord (upper row, 12 weeks after transplant) and brain (lower row, 26 weeks after transplant) after the transplantation of each cell line. Immunohistochemistry results for STEM121 and DAB, which were positive in the cytoplasm of the transplanted human cells. (Scale = 500 μm.) (c, d) The mean graft volume percentages in the injured spinal cord (c) and brain (d) sections are shown. Although the volumes were generally smaller in the brains compared with the injured spinal cords, the 1231A3 EB-NSPCs showed significantly greater proliferation levels in both the spinal cord and brain, and the 1231A3 NR-NSPCs also formed large tissues. Values are means ± SEM. *P < 0.05 (c: n = 4, 4, 4, 6, 3, 3. d n = 2, 3, 6, 6, 6, 6)

NSPC histology schematic in injured spinal cord and brain, according to the maturational pattern. A, B The transplant fate schematics in an injured spinal cord (A) and an intact brain (B). The transplanted cells reached the edge of a cyst that resembled the central canal in the injured spinal cords (a), the ventricle of brain (a’), the inner pia mater (b, b’), or vessels in the brain striatum (c’), which recapitulated fetal central nervous system development consisting of DNT. Medullary DNT arose from the edge of the central canal where transplanted cells transformed into neuroepithelium and neuroblasts (closed diamond), which gave rise to three cellular zones with different maturation patterns (ventricular zone (VZ)-, intermediate zone (IZ)-, and marginal zone (MZ)-like structures), as observed in fetal brain development. The migrating neural cells attached to the inner pia mater become flattened in shape (subpial neural cells, closed square), which had an equivalent immunophenotype to a VZ component of the DNT. UDNT are composed of cells that show a VZ equivalent immunophenotype, which are thought to be formed as the consequence of a maturational delay in the DNT formation process (broken arrows). c The migrating transplanted cells that did not reach the above neurogenesis initiation sites may differentiate into mature neural cells (scattered neural cells: SNC, indicated by a star) or fail to differentiate and may form a UDNT (broken arrow). SNCs might also be derived from DNT. d, d’ The transplanted cells that failed to engraft into the parenchyma exhibit arrested or incomplete neural differentiation (BLT, UDNT) or mesenchymal differentiation (MES)