Accelerated and enhanced effect of CCR5-transduced bone marrow neural stem cells on autoimmune encephalomyelitis

Abstract

The suppressive effect of neural stem cells (NSCs) on experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), has been reported. However, the migration of NSCs to inflammatory sites was relatively slow as was the onset of rather limited clinical benefit. Lack of, or low expression of particular chemokine receptors on NSCs could be an important factor underlying the slow migration of NSCs. To enhance the therapeutic effect of NSCs, in the present study we transduced bone marrow (BM)-derived NSCs with CCR5, a receptor for CCL3, CCL4, and CCL5, chemokines that are abundantly produced in CNS-inflamed foci of MS/EAE. After i.v. injection, CCR5-NSCs rapidly reached EAE foci in larger numbers, and more effectively suppressed CNS inflammatory infiltration, myelin damage, and clinical EAE than GFP-NSCs used as controls. CCR5-NSC-treated mice also exhibited augmented remyelination and neuron/oligodendrocyte repopulation compared to PBS- or GFP-NSC-treated mice. We inferred that the critical mechanism underlying enhanced effect of CCR5-transduced NSCs on EAE is the early migration of chemokine receptor-transduced NSCs into the inflamed foci. Such migration at an earlier stage of inflammation enables NSCs to exert more effective immunomodulation, to reduce the extent of early myelin/neuron damage by creating a less hostile environment for remyelinating cells, and possibly to participate in the remyelination/neural repopulation process. These features of BM-derived transduced NSCs, combined with their easy availability (the subject's own BM) and autologous properties, may lay the groundwork for an innovative approach to rapid and highly effective MS therapy.

Figure 1. Chemotaxis assay for CCR5-transduced NSCs

(A)NSCs at the 5^th passage were transduced with CCR5. After 5 days, cells were plated into the upper well of a 48-well chemotaxis chamber, and different concentrations of CCL5 were added into the lower well for 5 hrs. Cells that migrated into the lower well were stained with Diff-Quik dye (blue) and counted as described in “Methods”. (B) Quantitative analysis of cell migration. Data represent mean values ± SE of three wells in Graph A. P values refer to comparisons between CCR5-transduced NSCs with GFP-transducedNSCs. *, p<0.05; **, p<0.01. One representative experiments of three is shown.

Fig. 2. Enhanced suppression of adoptive transfer EAE by CCR5-overexpressing NSCs

CCR5-transduced NSCs were injected i.v. to EAE mice, 1.5 x10⁶ cells/mouse, at the peak of disease (day 22 post T-cell transfer). Mice receiving the same number of GFP-NSCs or PBS only served as controls. (A) Clinical scores were checked daily by two researchers blindly according to a 0–5 scale (n = 8 in each group). (B) H&E staining to detect CNS inflammation. EAE mice were sacrificed 2 weeks p.t., and spinal cords were harvested for H&E staining. A decrease in inflammatory infiltrates in the white matter of spinal cord and reduced mean score of inflammation (C) in H&E staining were found in mice treated with CCR5-NSCs as compared with control NSC-treated and sham-EAE (PBS-treated) mice; Scale bar: 50 µm in B. *p<0.05, **p<0.01, comparison between sham-EAE and other groups. # p<0.05, comparison between GFP-NSC-injected and CCR5-NSC-injected mice. (n = 8 each group).

Figure 3. Effect of CCR5-transduced NSCs on actively induced EAE and peripheral immune system

(A) CCR5-transduced NSCs were injected i.v. to EAE mice, 1.5 x10⁶ cells / mouse, at disease peak of active EAE (day 22 p.i.). Mice receiving the same number of GFP-NSCs or PBS only served as controls. Clinical scores were checked daily by two researchers blindly according to a 0–5 scale. *p<0.05, **p<0.01, comparison between sham-EAE and other groups. # p<0.05, comparison between GFP-NSC-injected and CCR5-NSC-injected mice. (n = 6–8 each group). (B-C) H&E staining to determine CNS inflammation. EAE mice were sacrificed 2 weeks p.t., and spinal cords were harvested for H&E staining as described in Fig. 2B and C (n = 6–8 each group). (D-E) Splenocytes from each group of EAE mice described in Fig. 2B were harvested two weeks p.t., cultured at 1.5×10⁶/ml and stimulated with MOG_35–55 (10 µg/ml) or Con A (5 µg/ml) for 3 days. (D) Proliferative responses were determined by ³H-thymidine incorporation and (E) cytokine production in culture supernatants analyzed by ELISA. *, p<0.05, comparisons between sham-EAE and other groups (n = 6–8 each group).

Figure 4. Localization and migration of transplanted CCR5-NSCs in the CNS

Mice treated with NSCs i.v. at day 22 post T cell transfer were sacrificed 2, 4 and 6 weeks p.t.; brains and spinal cords were harvested for immunohistology. (A) Brain sections were immunostained with anti-CCR5 antibody and examined by confocal microscopy. Strong CCR5 expression (red) was observed in mice treated with CCR5-NSCs but not in GFP-NSCs, both of which were GFP⁺. Nuclei were stained with DAPI (blue). Scale bar: 10 µm. (B) The majority of transplanted CCR5-NSCs (green) had migrated to the parenchyma (examples indicated by arrow] by 4 weeks p.t., while most of the GFP-NSCs (green) were primarily confined to perivascular spaces (examples indicated by arrowhead). Blood vessels were stained with anti-vWF antibody (red). Nuclei were stained with DAPI (blue). (C) Quantitative analysis of GFP⁺ NSCs in spinal cord of EAE mice at 2, 4 and 6 weeks p.t. Symbols represent mean values and SD of 6–8 mice each group. * p<0.05, ** p<0.01, comparisons between CCR5-NSC- and GFP-NSC-treated groups. (D) GFP⁺ NSCs remained in inflammatory areas, where blood-borne CD45⁺ immune cells (red) that form the CNS inflammatory infiltrate persisted. Nuclei were stained with DAPI (blue). Scale bar: 10 µm in A, 50 µm in B, 25 µm in D. (E) Quantitative analysis of CD45⁺ T cells in spinal cord of EAE mice at 2, 4 and 6 weeks p.t. * p<0.05, ** p<0.01, comparisons between sham-EAE mice and mice treated with CCR5-NSCs or GFP-NSCs. # p<0.05, comparisons between CCR5-NSC- and GFP-NSC-treated groups.

Figure 5. Differentiation of transplanted NSCs in the CNS

Brains of EAE mice treated with GFP- and CCR5-NSCs were harvested at week 6 p.t., 7 µm cryosections were immunostained with anti-neural specific antibodies. a Cells co-labeled with GFP and neural specific markers (red) were identified as differentiated cells derived from transplanted NSCs (arrows); cells positive only for neural specific markers (red) were endogenous cells (dashed arrows). Co-localization of GFP⁺ and neural markers⁺ (red) in the CNS indicated that both transplanted GFP- and CCR5-NSCs had differentiated into β-tubulin⁺ neurons, GalC⁺ mature oligodendrocytes, GFAP⁺ astrocytes, and a small proportion of GFP⁺ cells remained undifferentiated (nestin⁺). A similar pattern was observed in both groups, suggesting a comparable differentiation potential in vivo of these two types of NSCs. Scale bar: 20 µm. b Absolute number and c percentage of differentiation of transplanted NSCs (GFP⁺) in the CNS at week 6 post NSC injection. (n = 8 each group). * p<0.05.

Figure 6. Transplanted CCR5-NSCs more efficiently promote remyelination

Spinal cords were harvested 6 weeks p.t. (A) Luxol fast blue (LFB) staining of spinal cord sections for detection of demyelination. Magnification × 50. (B) Electron microscopic analysis. The presence of thin myelin sheaths (remyelination; arrows) had been shown in the demyelinated lesions of spinal cords in both CCR5-NSC- and control-NSC-treated mice, while a large number of demyelinated axons (dashed arrows) were found in PBS-treated sham-EAE and EAE mice that were sacrificed at day 22 post disease induction, when NSC treatment was started in other groups (EAE before i.v.). Normal thick myelin sheaths (arrowheads) in naïve mice serve as control. Magnification × 500, insets × 2000.(C) Mean scores of demyelination. Symbols represent mean values and SD of 6–8 mice each group. (D) Quantification of percentage of myelinated axons among total axons as shown in electron micrographs. # p<0.05, # # p<0.01, comparisons between EAE before NSCs i.v. (day 22 p.i.) and other groups; ** p<0.01, comparisons between sham-EAE and other groups; ^@ p<0.05, comparison between GFP-NSCs i.v. and CCR5-aNSCs i.v. (E) Mean g ratio (axon diameter divided by entire myelinated fiber diameter) was determined using ImageJ software. * p<0.05, comparisons between naïve group and other groups, ^# p<0.05, comparison between GFP-NSC-i.v. and CCR5-NSC-i.v. Symbols represent mean values and

Figure 7. Schema: Mechanisms of action of CCR5-NSCs in EAE

Expressing CCR5 on BM-NSCs endows these cells with the capacity for rapid and guided migration to EAE foci after transplantation, thus reducing further demyelination and promoting more efficient remyelination at an earlier stage than control NSCs. Increased exogenous NSCs in demyelinated foci may also participate in the remyelination/neural re-population process. BBB: blood-brain barrier;NSCs: neural stem cells. Thick line/arrows: major pathways; thin line/arrows: secondary pathways.