Fig. 4

Spinally grafted clonal-derived NSCs show a long-term engraftment, no tumor formation, and time-dependent expression of human-specific markers characteristic of immature and mature neurons and glial cells. a Single suspension of NSCs was injected bilaterally into central gray matter of lumbar spinal cord segments in immunodeficient or G93A ALS rat using glass capillary. b Grafted cells were identified by expression of human-specific markers such as hNSE (green; white arrows). c, d H&E staining of lumbar spinal cord section at 6 months after NSCs grafting show well engrafted cells (red dotted area) with no detectable tumor formation. e Quantitative analysis of neuronal and glial differentiation at 3 weeks, 8 weeks, and 6 months after spinal NSC grafting in immunodeficient rats. Data are expressed as percent of double-stained hNUMA/DCX, hNUMA/hNSE, hNUMA/NeuN, hNUMA/GFAP, and hNUMA/Vim relative to hNUMA+ cells. Data are presented as mean ± SD. f, g At 3 weeks after grafting, a marker characteristic for proliferating immature glial precursors (Vimentin) and early post-mitotic neurons (DCX) are seen in grafted hNUMA+ cells. Extensive axo-dendritic sprouting of DCX+ positive processes surrounding the host interneurons and α-motoneurons can be seen (g). h, i At 6–8 weeks after NSCs transplantation, a more advanced cell migration and neuronal maturation were seen. Numerous double hNUMA/DCX+ neurons residing outside of the graft core were identified in the gray matter (h). Similarly, extensive axonal sprouting of HO14+ human axons was seen in the host gray matter (i). j, k At 6 months after NSCs grafting the appearance of mature neuronal and glial markers was identified throughout the graft. A high intensity of human-specific NSE was seen in grafted areas with several hNSE+ neurons identified outside of the graft (j, white arrow). Staining with human-specific GFAP antibody showed a high density of GFAP+ network with numerous hGFAP+ processes found in the ventral gray matter between α-motoneurons of the host (k). l, m Analysis of grafted NSCs at 56 days after grafting in G93A ALS rat lumbar spinal cord. A high density of double hNUMA/DCX-stained grafts was seen throughout the grafted segments (l). Staining with hGFAP showed only relatively few hGFAP+ astrocytes and these were preferentially found at the borders of individual hNUMA+ grafts (m) (scale bars: b, c 500 μm; f, g 100 μm; h, i 300 μm; j 300 μm; k 100 μm; l 300 μm; m 200 μm)