Nted with SPC-01 cells. Staining for GAP43 demonstrates more-intensive sprouting in the injured spinal cord tissue of rats transplanted with SPC-01 cells (A) compared with control animals (B). (C) Higher-magnification view of the transplant area, marked by the white line in (A). (D) Quantitative analysis of the number of GAP43-positive fibers in the spinal cord tissue of control and transplanted animals.positive for Nkx6.1 (Figure 6A), whereas qPCR analysis did not reveal changes in the expression of this marker compared with the levels seen in SPC-01 cells before transplantation (Figure 6B). However, a highly significant (P PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25751659 this population consists of approximately 25 of the total number of transplanted cells.Seventeen weeks (4 months) after grafting, transplanted SPC-01 cells were less positive for GFAP (Figure 7A) and nestin (Figure 7B) than 8 weeks after grafting, and the expression was confined to individual fibers. However, at this time, the grafted cells were not positive for Olig2, as was the case 8 weeks after transplantation, and were positive for CNPase (Additional file 4: Figure S2F), which suggests their differentiation toward an oligodendroglial phenotype. Co-staining for NF200 (Figure 7C), Tau (Figure 7D), CGRP (Figure 7E), and NG2 (Figure 7F) together with markers of human cells (MTCO2 or HuNu), revealed that newly forming host tissue elements are intensively incorporating into the graft and communicating with grafted cells, because the staining for endogenous tissue elements and the staining for human markers did not colocalize. In the host nervous tissue adjacent to the transplants, endogenous cells positive for Olig2 (Figure 7G) and MOSP (Figure 7J) were found.Amemori et al. Stem Cell Research Therapy 2013, 4:68 http://stemcellres.com/content/4/3/Page 11 ofFigure 6 Differentiation of transplanted SPC-01 cells toward a motor neuronal phenotype. The expression of motor neuron-specific markers in SPC-01 cells at 8 and 17 weeks after transplantation. Eight weeks after transplantation, 25.31 ?2.15 of SPC-01 cells were positive for Nkx6.1 (A, A1, A2). At the same time, according to the results of qPCR analysis, the expression of the Nkx6.1 gene in SPC-01 cells did Lenalidomide not change, compared with the level of expression seen in the cells before transplantation. However, the expression of the more advanced motor neuron genes Islet2 and HB9 in transplanted cells significantly increased (**P < 0.005) compared with that observed in SPC-01 cells before transplantation (B). Seventeen weeks after transplantation, SPC-01 cells were positive for Islet2 (C; C1, C2, C3: higher magnification of C) and ChAT (D and D2: higher magnification of D; D1: staining for ChAT in combination with GFP and DAPI).Discussion In the present study, we used clone 01 of human fetal neural stem cells (SPC-01) derived from fetal spinal.