Neural Cell Adhesion Molecule L1-Transfected Embryonic Stem Cells Promote Functional Recovery after Excitotoxic Lesion of the Mouse Striatum

L1 expression increases neuronal differentiation and decreases proliferation in vitro. A, Generation of neurons from L1 ⁻ and L1 ⁺ ES cells was determined at day 7 of stage 5 by immunostaining for β-tubulinIII (β-tub; red). GFP ⁺ cells, green; β-tubulinIII/GFP ⁺ cells, yellow. The proportion of β-tubulinIII ⁺ cells of all GFP ⁺ cells was greater in L1 ⁺ cells than in L1 ⁻ cells. Scale bar, 20 μm. EGFP, Enhanced GFP. B, Percentages of β-tubulinIII ⁺ cells of all GFP ⁺ cells are shown at day 7 of stage 5 (mean ± SEM). Student's t test was performed for statistical analysis (**p < 0.01). C, At day 14 of stage 5, mature neuronal proteins NF-200 (NF), GAD, and TH and the neural stem cell protein nestin (all in red) were determined by immunocytochemistry in L1 ⁻ and L1 ⁺ cells. The proportion of NF ⁺, GAD ⁺, and TH ⁺ cells was greater in the L1 ⁺ than in the L1 ⁻ cells, whereas the proportion of nestin ⁺ cells was higher in L1 ⁻ cells. Scale bar, 10 μm. D, Percentages of NF ⁺, GAD ⁺, TH ⁺, and nestin ⁺ cells at day 14 of stage 5 (mean ± SEM). *p < 0.05, **p < 0.01 (Student's t test). E, BrdU incorporation was determined at day 7 of stage 4 and at days 3, 7, and 14 of stage 5 by immunostaining for BrdU (red) after a pulse labeling of 8 h with 10 mm BrdU. Scale bar, 10 μm. F, Percentages of BrdU ⁺ cells of all cells are shown at day 7 of stage 4 and days 3, 7, and 14 of stage 5 (mean ± SEM). Student's t test was performed for statistical analysis (*p < 0.05; **p < 0.01). 3d, Day 3; 7d, day 7; 14d, day 14.

L1 ⁺ cells grafted into the quinolinic acid-lesioned striatum show increased neuronal differentiation. A, Laser-scanning microscopy illustrating immunohistochemical analysis of grafts of early stage 5 cells derived from L1 ⁻ and L1 ⁺ ES cells with the neuronal marker NF and the astrocytic marker GFAP (all in red) 4 weeks after transplantation of GFP ⁺ (green) cells. Donor-derived marker ⁺ cells in merged images appear yellow. NF staining of an L1 ⁺ graft with Z-series is displayed at top right. Note the increased expression of NF and the decreased expression of GFAP in L1 ⁺ grafts compared with L1 ⁻ grafts. Scale bar, 10 μm. B, Percentages of cell type-specific marker ⁺ cells in L1 ⁺ (n = 6) or L1 ⁻ (n = 6) grafts 4 weeks after transplantation (mean ± SEM). *p < 0.05 (Student's t test).

Analysis of proliferation in grafted cells by Ki-67 immunolabeling and of neurogenesis in the SVZ by BrdU incorporation and expression of DCX 1 and 4 weeks after transplantation. A, The fraction of Ki-67 ⁺ cells of all GFP ⁺ transplanted cells is given 1 and 4 weeks after transplantation of L1 ⁺ (n = 4) and L1 ⁻ (n = 4) cells. No difference was observed. B, Mice transplanted with L1 ⁺ (n = 4) or L1 ⁻ (n = 4) cells into the lesioned striatum were labeled with BrdU on days 3–7 after transplantation and killed 7 d and 4 weeks after grafting. The ratio of BrdU ⁺ cells in the SVZ ipsilateral and contralateral to the lesioned hemisphere is shown 1 and 4 weeks after transplantation. No difference was observed between the L1 ⁺ and L1 ⁻ groups. C, Immunohistochemical staining for DCX (red) in the SVZ ipsilateral to the lesion in sham-injected animals 4 weeks after grafting. D, Immunostaining for DCX in the SVZ was assessed 4 weeks after grafting. The ratio of DCX ⁺ cells in the SVZ ipsilateral and contralateral to the lesioned and transplanted hemisphere is given for mice transplanted with L1 ⁺ (n = 4) or L1 ⁻ (n = 4) cells into the lesioned striatum. No difference between L1 ⁺ and L1 ⁻ groups was observed. Error bars indicate SEM. ipsi, Ipsilateral; contra, contralateral.

The number of GABAergic neurons is increased in L1 ⁺ grafts. A, Laser-scanning microscopy illustrating immunohistochemical analysis of grafts of early stage 5 cells derived from L1 ⁻ and L1 ⁺ ES cells with the neuronal markers GAD (red) 8 weeks after transplantation of GFP ⁺ (green) cells. Donor-derived marker ⁺ cells in merged images appear yellow. Scale bar, 10 μm. B, Percentages of GAD ⁺ cells of GFP ⁺ cells in L1 ⁺ (n = 16) or L1 ⁻ (n = 16) grafts 1–8 weeks after transplantation. ANOVA followed by Student's t test was performed for statistical analysis (*p < 0.05). C, Total number of GAD ⁺ cells per graft 1–8 weeks after transplantation. ANOVA followed by Student's t test was performed for statistical analysis (*p < 0.05). Note the increased number of GAD ⁺ cells in L1 ⁺ grafts 1–8 weeks after transplantation compared with L1 ⁻ grafts and the number of GAD ⁺ cells in both groups decreasing 8 weeks after transplantation and reflecting a decrease in total cell number. D, Total numbers of graft-derived GFP ⁺ cells per graft 1–8 weeks after transplantation. Error bars indicate mean ± SEM.