Serial in vivo imaging of transplanted allogeneic neural stem cell survival in a mouse model of amyotrophic lateral sclerosis

Abstract

Neural stem cells (NSCs) are being investigated as a possible treatment for amyotrophic lateral sclerosis (ALS) through intraspinal transplantation, but no longitudinal imaging studies exist that describe the survival of engrafted cells over time. Allogeneic firefly luciferase-expressing murine NSCs (Luc⁺-NSCs) were transplanted bilaterally (100,000 cells/2μl) into the cervical spinal cord (C5) parenchyma of pre-symptomatic (63day-old) SOD1^G93A ALS mice (n=14) and wild-type age-matched littermates (n=14). Six control SOD1^G93A ALS mice were injected with saline. Mice were immunosuppressed using a combination of tacrolimus+sirolimus (1mg/kg each, i.p.) daily. Compared to saline-injected SOD1^G93A ALS control mice, a transient improvement (p<0.05) in motor performance (rotarod test) was observed after NSC transplantation only at the early disease stage (weeks 2 and 3 post-transplantation). Compared to day one post-transplantation, there was a significant decline in bioluminescent imaging (BLI) signal in SOD1^G93A ALS mice at the time of disease onset (71.7±17.9% at 4weeks post-transplantation, p<0.05), with a complete loss of BLI signal at endpoint (120day-old mice). In contrast, BLI signal intensity was observed in wild-type littermates throughout the entire study period, with only a 41.4±8.7% decline at the endpoint. In SOD1^G93A ALS mice, poor cell survival was accompanied by accumulation of mature macrophages and the presence of astrogliosis and microgliosis. We conclude that the disease progression adversely affects the survival of engrafted murine Luc⁺-NSCs in SOD1^G93A ALS mice as a result of the hostile ALS spinal cord microenvironment, further emphasizing the challenges that face successful cell therapy of ALS.

Keywords: Amyotrophic lateral sclerosis; Bioluminescence imaging; Cell survival; Neural stem cell; Transplantation.

Figure 1

(A) In vitro BLI of plated Luc⁺-NSCs GRPs at several cell densities. (B) Linear correlation between the number of luciferase-expressing cells and BLI signal (R² =0.99).

Figure 2

Co-registration of CT (gray scale) and BLI (color scale) to confirm the correct placement of cells at the targeted injection site (C5 spinal cord region). Images were obtained at day 1 post-transplantation.

Figure 3

Rotarod test behavioral analysis shows a transient improvement in the motor performance of ALS mice transplanted with Luc⁺-NSC (n=6) compared to ALS mice injected with saline (n=6, *p<0.05).

Figure 4

(A) Representative BL images of Luc⁺-NSCs engrafted SOD1^G39A mice at the pre-symptomatic, onset, and post-symptomatic (end) stage of the disease. Wild-type mice were engrafted at the same time points. (B) Percentage loss of BLI signal compared to day 1 post-transplantation (set as 100%) (n=10, *=p<0.05).

Figure 5

(A) Astrogliosis in the spinal cord of SOD1^G39A mice. Representative micrographs of anti-GFAP (red) and anti-luciferase (green) staining demonstrate activated astrocytes at the site of transplantation in ALS spinal cord at the post-symptomatic stage of the disease (8 weeks after transplantation). Wild-type littermates exhibit less expression of GFAP around the injection site of luciferase-positive cells at the same time point. Scale bar =100 μm. (b) Microgliosis in the spinal cord of SOD1^G39A mice. Representative micrographs of anti-Iba1 (red) and anti-luciferase (green) staining demonstrate activated microglia at the site of transplantation in the ALS spinal cord at the post-symptomatic stage of the disease (8 weeks after transplantation). Wild-type littermates exhibit less expression of Iba1 around the injection site of luciferase-positive cells at the same time point. Scale bar =100 μm.

Figure 6

Luc⁺-NSCs 2 weeks do not express differentiation markers at 2 weeks post-transplantation in SOD1^G39A mice spinal cord. Shown are stainings for anti-NeuN (red), anti-GFAP (red), anti-Iba1 (red), anti-Olig2 (red), and anti-luciferase (green). Scale bar = 100 μm, inset scale bar = 50 μm.

Figure 7

Accumulation of mature macrophages in the spinal cord of SOD1^G39A mice. Shown are stainings for anti-F4/80 (red) and anti-NeuN (green) at 8 weeks post-transplantation demonstrate accumulation of mature macrophages at the site of transplantation in the ALS spinal cord. Wild-type littermates demonstrate far fewer F4/80⁺ cells. Scale bar=50 μm.