Introduction:
Oligodendrocytes (OLs) generate myelin essential for CNS function, yet the physical and biochemical cues regulating myelination remain incompletely defined. Existing in vitro models often fail to reproduce the softness, geometry, and extracellular matrix (ECM) environment of native axons, limiting mechanistic insight and contributing to poor translational predictability of pro-myelinating compounds. To address this, we developed a fully hydrogel-based axon-mimetic micropillar system with independently tuneable stiffness, diameter, and spacing that supports formation of multilayered compact myelin by rodent and human OLs.
Aims/Objectives:
We aimed to (1) determine how biophysical and biochemical features (axon-like stiffness, geometry, and ECM coatings) influence OL differentiation and myelination, (2) validate the platform using ultrastructural and transcriptomic analyses, and (3) assess whether substrate stiffness modulates pharmacological responses to known myelination-promoting drugs.
Methods:
Micropillar arrays (diameters 3–10 µm, spacing 5–15 µm) were fabricated from polyacrylamide hydrogels of defined stiffness (0.5, 5, 20, 50 kPa). Primary rat O4⁺ OLs or human foetal / hPSC-derived OLs were cultured for 7–14 days. Myelination was quantified by MBP immunostaining using a standardised wrapping score (0–3). Myelin ultrastructure was assessed by TEM. Transcriptomic profiling of OLs on flat vs micropillar gels was performed via RNA-seq (n = 3 biological replicates/condition). For compound testing, OLs were treated for 5 days with benztropine, clemastine, GSK239512, simvastatin, or wiskostatin (n = 3 replicates/condition). Statistical analyses used one-way ANOVA with Tukey post-hoc testing.
Results:
Rodent OLs robustly differentiated and formed compact multilayered myelin around micropillars, with 51.9 ± 15.1% of pillars showing multilayer wraps and an average sheath thickness of 45.9 ± 20.7 nm (n = 3). Myelin thickness correlated strongly with lamellae number (R² = 0.94). Geometry critically regulated OL behaviour: 10 µm pillars exhibited significantly higher full-wrapping (score 3) than 3 or 5 µm pillars (p < 0.001), recapitulating diameter-dependent myelination. Stiffness modulated myelination in a diameter-dependent manner: for 10 µm pillars, wrapping increased progressively from 0.5 kPa to 50 kPa (p < 0.01). ECM cues also influenced outcomes, with laminin increasing the proportion of fully wrapped pillars compared to PDL (p < 0.05). RNA-seq revealed >800 differentially expressed genes on micropillars versus flat controls, including upregulation of key myelin-related genes (Mog, Mag, Mbp), ECM remodelling pathways, and Vegfa. Drug testing demonstrated stiffness-dependent effects: benztropine and clemastine increased full wrapping (p < 0.01), but effects were attenuated on softer 5 kPa pillars. Human foetal and hPSC-derived OLs also myelinated micropillars, with human cells producing multilayered myelin by day 14.
Conclusion:
This tuneable hydrogel micropillar platform captures key biomechanical determinants of CNS myelination and improves the physiological relevance of in vitro assays. Its ability to reveal stiffness-dependent drug responses suggests it could reduce false-positive compound identification and advance translational discovery for demyelinating diseases.
Ethical Standards:
All animal procedures complied with UK Animals (Scientific Procedures) Act (1986) and institutional ethical guidelines. Human stem cell–derived and foetal cells were used in accordance with approved ethical protocols.