Dual transplantation of human neural stem cells into cervical and lumbar cord ameliorates motor neuron disease in SOD1 transgenic rats

Abstract

Stem cells provide novel sources of cell therapies for motor neuron disease that have recently entered clinical trials. In the present study, we transplanted human neural stem cells (NSCs) into the ventral horn of both the lumbar (L4-L5) and cervical (C4-C5) protuberance of SOD1 G93A rats, in an effort to test the feasibility and general efficacy of a dual grafting paradigm addressing several muscle groups in the front limbs, hind limbs and the respiratory apparatus. Transplantation was done prior to the onset of motor neuron disease. Compared with animals that had received dead NSC grafts (serving as controls), rats with live NSCs grafted at the two spinal levels lived 17 days longer. Disease onset in dually grafted animals was delayed by 10 days compared to control animals. Disease duration in NSC-grafted animals was longer by 7 days compared to controls. Our results support the potential of NSC grafts at multiple levels of spinal cord as future cellular therapy for motor neuron disease.

Figure 1

Survival, migration, neuronal differentiation (A, B) and motor circuit integration (C, D) of human NSC grafts in rat cervical cord parenchyma. A. Grafted NSCs survive in large numbers and migrated in the gray and white matter of the cervical cord as shown by HNu immunoreactivity (red). Inoculation site is indicated with arrows. Migration of NSCs towards ventral horn is marked with arrowheads. Some grafted cells are located in the white matter (extreme left). Surviving host motor neurons are shown as large TUJ1 (+) nerve cells. B. This confocal image illustrates the neuronal differentiation of grafted NSCs (arrows) on the basis of dual staining with the graft-selective marker HNu (red) and the generic neuronal marker TUJ1 (green) on X, Y and Z different sectioning planes. C–D. NSC-derived neurons innervate the cell bodies and proximal dendrites of cervical motor neurons of SOD1 G93A rats. Immunoreactivity for human synaptophysin (SYN, red) serves as a selective marker for graft-derived terminals. Postsynaptic host structures are labeled with the motor neuron phenotypic marker choline acetyltransferase (ChAT, green). In (C), a large number of human synaptophysin (+) boutons (SYN, red; arrows) contacts host ChAT motor neurons (green). B is a confocal image taken from the framed area of (C) to confirm the apposition of human synaptophysin boutons to the cell body and dendrites of the motor neuron at the center of (C). Arrows in (D) depict boutons that are positionally validated with x and y resectioning. Scale bars: A, 100 μm; B, 20 μm; C, 50 μm, D, 10 μm

Figure 2

Effects of human NSC transplantation on disease onset (A), life span/survival (B–C) and muscle weakness (D–E) in G93A SOD1 rats. A. Disease onset was 10 days later in the live-cell group (129.5 ± 3.234 days, n=11) compared to the dead-cell group (119.2 ± 3.709 days, N=11) (P=0.0481). B. End-point analysis shows that the live-cell group has significantly longer life span compared to dead-cell group (158.9 ± 6.256 days and 141.3 ± 5.494 days, respectively, n=11) (P=0.0469). C. This Kaplan-Meier plot shows a significant separation between live (L) and dead cell group (D) survival throughout the course of the study (p=0.0402), suggesting animals with live NSCs have better survival. D–E. BBB (D) and inclined plane (E) scores show a significant separation in these two measures of muscle weakness between live-cell (L) and dead-cell (D) groups (p=0.0183 and 0.045, respectively), suggesting muscle weakness progress is significantly slowed down in animals with live NSCs compared with control animals. Bar= Mean ±SEM, * P<0.05.