Combination Therapy With Fingolimod and Neural Stem Cells Promotes Functional Myelination in vivo Through a Non-immunomodulatory Mechanism

Abstract

Myelination, which occurs predominantly postnatally and continues throughout life, is important for proper neurologic function of the mammalian central nervous system (CNS). We have previously demonstrated that the combination therapy of fingolimod (FTY720) and transplanted neural stem cells (NSCs) had a significantly enhanced therapeutic effect on the chronic stage of experimental autoimmune encephalomyelitis, an animal model of CNS autoimmunity, compared to using either one of them alone. However, reduced disease severity may be secondary to the immunomodulatory effects of FTY720 and NSCs, while whether this therapy directly affects myelinogenesis remains unknown. To investigate this important question, we used three myelination models under minimal or non-inflammatory microenvironments. Our results showed that FTY720 drives NSCs to differentiate into oligodendrocytes and promotes myelination in an ex vivo brain slice culture model, and in the developing CNS of healthy postnatal mice in vivo. Elevated levels of neurotrophic factors, e.g., brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor, were observed in the CNS of the treated infant mice. Further, FTY720 and NSCs efficiently prolonged the survival and improved sensorimotor function of shiverer mice. Together, these data demonstrate a direct effect of FTY720, beyond its known immunomodulatory capacity, in NSC differentiation and myelin development as a novel mechanism underlying its therapeutic effect in demyelinating diseases.

Keywords: combination therapy; fingolimod; myelination; neural stem cells; oligodendrocytes.

Figure 1

Co-treatment with FTY720 and NSCs promoted myelination in the brain slice culture model. (A) Schematic diagram illustrating experimental design followed throughout this study. Slices obtained from newborn (P0-3) C57/Bl6 mouse pups were grown for 3 days, and NSCs (2 μl, ~5 × 10⁴ cells /slice) and/or FTY720 (1 nM) were added for the subsequent 14 days, after which the slices were fixed and immunostained. (B) Representative confocal images displaying GFP + cells (green; NSCs) and MBP (red) immunoreactivity after 14 days of treatment. Scale bar = 20 μm. (C) MBP intensity was measured using Image-Pro. (D) GFP + MBP + intensity was measured using Image-Pro. Representative images are shown, and quantitative data refer to mean ± SD (n = 6 slices/group). ^**p < 0.01, ^***p < 0.001. One-way ANOVA with Tukey's multiple comparisons test and unpaired Student's t-test. One representative of 3 independent experiments is shown.

Figure 2

Impact of FTY720 plus NSC treatment on neural cell lineages in brain slice culture model. Brain slices obtained from newborn (P0-3) C57/Bl6 mouse pups were grown for 3 days in culture, and NSCs (2 μl, ~ 5 × 10⁴ cells /slice) and/or FTY720 (1 nM) were added for the subsequent 7 days; the slices were then fixed and immunostained. (A) Quantitative analysis of the number of transplanted NSCs (GFP+) in brain slices. (B) Immunofluorescence images of brain slices. Cells co-labeled with GFP and neural specific markers (red) were identified as differentiated cells derived from NSCs (yellow); cells positive only for neural-specific markers (red) were endogenous cells. CC1⁺: oligodendrocytes, NeuN+: neurons, GFAP+, astrocytes. Scale bar = 20 μm. (C) High-magnification confocal images show that GFP (green) was highly colocalized with the oligodendrocyte marker MBP (red). Scale bar = 10 μm. (D) Quantification of total CC1⁺ cell numbers. (E) Quantitative analysis of differentiation of transplanted NSCs in the CNS as shown in (B). Symbols represent mean ± SD; n = 10 random areas per group. ^***p < 0.001. One-way ANOVA with Tukey's multiple comparisons test and unpaired Student's t-test. One representative of 3 independent experiments is shown.

Figure 3

Co-treatment with FTY720 and NSCs promoted myelination in the CNS of newborn mice in vivo. (A) Schematic diagram illustrating experimental design followed throughout this study. Mice were i.c.v. injected with NSCs (2 μl, ~2 × 10⁵ cells/mouse) at day 3 postnatal, then injected daily with FTY720 i.p. (0.3 mg/mouse) or with PBS from day 3 postnatal. Spinal cords were harvested at day 14 postnatal, and stained with antibodies to MBP for myelin and to NFH for axonal fibers. (B) Representative confocal images displaying NFH (green) and MBP (red) immunoreactivity. Scale bar = 10 μm. (C) Quantification of myelinated axons from the total number of axons in 5 mouse litters each group, performed within 5 random standard 500 μm² fields in the white matter of the ventral lumbar. (D) Representative electron micrographs of transverse lumbar sections of the ventral region. Scale bar = 1.0 μm. (E) Quantification of the mean thickness of myelin sheaths. (F) Mean g ratio (axon diameter divided by the entire myelinated fiber diameter) was determined using Image-Pro Plus software. Symbols represent mean ± SD. ^*p < 0.05, ^**p < 0.01. One-way ANOVA with Tukey's multiple comparisons test and unpaired Student's t-test.

Figure 4

The effect of co-treatment with FTY720 and NSCs on the number of mature oligodendrocytes in the spinal cord of newborn mice. Spinal cords of mice described in Figure 3 were harvested at day 14 postnatal, and lumbar sections were stained for newly matured oligodendrocyte marker CC1. (A) Representative confocal images displaying CC1⁺ (red) immunoreactivity at day 14 postnatal. Scale bar = 10 μm. (B) Quantification of total CC1⁺ cell numbers. (C) Expression of growth factor genes in brain tissues was determined using Custom RT2 Profiler™ PCR Array Mouse Growth Factors (Qiagen, Valencia, CA). Relative expression was calculated by –ΔΔCt values from triplicate of PCR (n = 3; mean + SEM, Student's t-test; normalized to PBS group). (D) The effect of postnatal FTY720 or/and NSC administration on the performance of newborn mice in accelerating rotating rod test was examined at day 21 postnatal. Symbols represent mean ± SD. ^*p < 0.05, ^**p < 0.01, ^***p < 0.01. One-way ANOVA with Tukey's multiple comparisons test and unpaired Student's t test.

Figure 5

FTY720 in combination with transplanted NSCs improved neurological function and diminished seizures of shiverer mice. (A) Transplanted NSCs and FTY720-NSC co-treatment prolong the survival of shiverer mice. Newborn double-homozygous shiverer (shi/shi) mice were implanted i.c.v. with dissociated single NSCs (2.0 × 10⁵ cells/mouse), and injected i.p. with fingolimod (at 0.3 mg/kg daily) (n = 12) from the day of cell injection. Shiverer mice that received NSCs (n = 14), or fingolimod (n = 12) alone served as single-treatment controls, and mice that received the same volume of phosphate-buffered saline (PBS) served as a sham-treated control (n = 12). All these shiverer mice were maintained in small group housing and monitored daily until death. The Kaplan-Meier survival graph, plotting the percentage of each group alive in days, is shown. (B) Quantification of length of episodes. (C) Quantification of intervals of episodes. Symbols represent mean ± SD. ^***p < 0.01. One-way ANOVA with Tukey's multiple comparisons test and unpaired Student's t test.

Figure 6

FTY720 in combination with transplanted NSCs efficiently myelinated the shiverer brain. In separate experiments, mice were treated as described in Figure 5, and brains were harvested at day 90 postnatal for immunostaining. (A) Confocal micrographs showing FluoroMyelin staining in the midline of the corpus callosum of shiverer mice treated with NSCs and/or FTY720. (B) Averaged values (mean ± SEM) of myelin signal density in the midline of the callosal body from treatment groups shown in (A). ^*p < 0.05, ^**p < 0.01. One-way ANOVA with Tukey's multiple comparisons test and unpaired Student's t-test. One representative of 3 independent experiments is shown.