Anti-Muscarinic Adjunct Therapy Accelerates Functional Human Oligodendrocyte Repair

Transcriptional profile of CD140a/O4-sorted cells reveals distinct stages of human oligodendrocyte lineage differentiation. a, Principle components were calculated for each gene expression profile and plotted on a 3D scatter plot. The RNAs for each sorted fraction clustered together, indicating similar expression profiles within each sorted fraction and significant differences between samples. b, Between-sample distances were calculated using maximum distance (uniform norm) between samples. Samples with closely related gene expression patterns and low intersample distances are colored blue according to the color bar. Samples within each sorted fraction from distinct fetal donors were more closely related than between sorted fractions obtained from the same donor brain. c, Several known cell-type-specific marker genes were significantly regulated between each glial fraction and double-negative cells. Gene expression shown indicates expression relative to median of all samples. d, Probes significantly regulated in each cell fraction relative to CD140a⁻O4⁻ cells. The overlapping genes between each comparison are colored according to their relative abundance. This shows that there is extensive overlap between both CD140⁺ and O4⁺ fractions. Only six probes were shared between CD140a⁺O4⁻ and CD140a⁻O4⁺ fractions. e, Real-time RT-PCR confirmed very high enrichment of OPC-expressed PDGFRA and MBP mRNA immediately after CD140a/O4 multicolor FACS (mean ± SEM, n = 5 fetal samples). *p < 0.05, **p < 0.01, ***p < 0.001, Dunnett's posttest, versus CD140a⁻O4⁻ cells (1-way ANOVA).

mAchR activity regulates oligodendrocyte commitment by hOPCs. CD140a/PDGFαR⁺ hOPCs were treated with 0–40 μm OxoM for 4 d and stained for O4 (red) and DAPI (blue) (a, b). The proportion of O4⁺ oligodendrocytes was determined at each dose (mean ± SEM, n = 3 seperate brain preparations, 19–22 week gestational age) (c). d–e, CD140a⁺ hOPCs were cocultured with human neuronal reaggregate cultures for 14 d in vitro (DIV). The fate of human OPCs was tracked by prior infection with mCherry-expressing lentivirus at −2 DIV. Oligodendrocyte differentiation was assessed at 14 DIV by colocalization of mCherry-expressing cells (red) with O4 (green) (d, null control). e, Daily treatment of matched cultures with 50 nm darifenacin, an M₃R antagonist, induced oligodendrocyte differentiation. f, Proportion of mCherry⁺ OPCs expressing O4 was determined (mean ± SEM, n = 3 separate brain preparations). *p < 0.05, t test. Scale bar, 50 μm.

Systemic treatment with an M3R antagonist promotes precocious oligodendrocyte differentiation in vivo. Neonatal mice received daily subcutaneous injections of either saline or 10 mg/kg solifenacin, a blood–brain barrier-permeable M₃R antagonist, from 4 d postnatally until being killedon day 9 (n = 4). a, b, Oligodendrocyte differentiation and myelination was assessed by immunoflourescence staining for MBP in saline- (a) or antagonist (b)-injected animals. The percentage area of myelinated corpus callosum was quantified in each animal and shown in c (mean ± SEM). M₃R antagonist treatment induced a significant increase in myelinated area (n = 4 animals per group). d, Solifenacin increased the amount of MBP protein normalized to tubulin (n = 3 mice per group). CC1⁺OLIG2⁺ oligodendrocyte cells were counted in corpus callosum and compared with total OLIG2⁺ oligodendrocyte lineage cells (e). There was a significant increase in the proportion of differentiating oligodendrocytes in solifenacin-treated animals. f, Overall density of CC1⁺ cells was also increased in corpus callosum. *p < 0.05, t test. Scale bars: 500 μm (a, b), 50 μm (c).

Solifenacin promotes human oligodendrocyte differentiation after xenograft into shiverer/rag2 mice. Newborn homozygous shiverer/rag2 mice were transplanted with human CD140a/PDGFαR⁺ OPCs. Four days later, animals were injected daily with either saline or solifenacin (10 mg/kg) for 8 weeks (n = 4 animals per group). Human cells were identified using human nuclear antigen staining (hNA, red) and myelinating oligodendrocytes using MBP (green). Compared with saline controls (a), solifenacin treatment resulted in a substantial increase in the number of and area myelinated by MBP⁺ human oligodendrocytes in the corpus callosum at 8 weeks (b, c). d, e, Confocal images showing individual myelinated fibers in corpus callosum of saline-treated (d) and solifenacin-treated (e) animals. f, Human CC1⁺hNA⁺ oligodendrocytes were counted in xenografted corpus callosum and compared with total hNA⁺ human cells (n = 4 animals). *p < 0.05, t test. Scale bars: 100 μm (a–c), 10 μm (d, e).