Journal: bioRxiv

Article Title: Engineered micropillars to unveil oligodendrocyte responses to physical cues

doi: 10.1101/2025.03.16.643578

Figure Lengend Snippet: (A) Representative confocal microscopy images of OLs on micropillars at day 5 (D5) of differentiation (DIFF) expressing myelin basic protein (MBP, orange) and Olig2 (magenta) (nuclei counterstained with Hoechst, blue). I and II represent bottom and top z-planes of OLs on micropillars, respectively (MBP, white). OLs extend long myelin sheaths and wrap around the micropillars as depicted by the myelin rings observed in II and depicted by the arrows. Scale bar indicates 50 μm. (B) Scanning electron microscopy (SEM) of an OL wrapping around a micropillar at D5 DIFF. OLs wrap micropillars axially but also longitudinally. Scale bar indicates 5 μm. (C) mRNA levels of OL specific genes on micropillars and flat PDMS surfaces at D5 DIFF. Levels were normalized to the Oaz1 housekeeping gene, and results indicate mean values from 3 independent experiments (8 mixed diameter micropillar-/flat-structures pooled together). At this differentiation stage, OLs expressed their common markers, highlighting the feasibility of the PDMS structures in promoting OL lineage commitment and differentiation patterns. (D) Stimulated emission depletion microscopy (STED) representative images of OLs wrapping the micropillars and expressing MBP (white). Some micropillars displayed thick myelin sheaths with more than one visible concentric layer (noted by the arrows). Scale bar indicates 2 μm. (E) Representative fluorescent images of OLs loaded with the calcium indicator Fluo-4 at D5 DIFF. The image pseudo colours indicate differences in pixel intensities. Scale bar indicates 100 μm. (F) Representative calcium signals overtime of an active (blue) and inactive (orange) cell at D5 DIFF. Calcium events were scored as increases of ΔF/F 0 equal to or more than 40% of the baseline.

Article Snippet: Image acquisition was carried on a Leica Stellaris 8 STED Falcon microscope (single point scanning confocal equipped with a fully motorized inverted Leica DMI8 microscope, a white light laser (WLL), Falcon and STED modalities) from Leica Microsystems.

Techniques: Confocal Microscopy, Expressing, Electron Microscopy, Microscopy

Journal: bioRxiv

Article Title: Engineered micropillars to unveil oligodendrocyte responses to physical cues

doi: 10.1101/2025.03.16.643578

Figure Lengend Snippet: (A) Diagram of the sequential steps for image acquisition and analysis. OLs were cultured on PLL/lam211 coated micropillars or flat surfaces iand fixed at D1, 5 or 7 DIFF. Confocal images were acquired for myelin basic protein (MBP, orange) and Olig2 (magenta) markers, while nuclei were counterstained with Hoechst (blue). (B) Representative images of OLs cultured on flat, 1 μm and 5 μm micropillar platforms. Scale bar indicates 25 μm. Zoomed in images show regions of wrapped micropillars. (C) mRNA levels of OLs cultured on different diameter and stiffness micropillars at D1 DIFF. Lineage commitment (Sox10, Nkx2.2, and Olig2) as well as differentiation (CNP, MBP, PLP and MAG) markers were analyzed. Results show mean values of levels normalized to Oaz1 housekeeping gene and to respective flat surfaces (2 -ΔΔCt , n>3 independent experiments, 8 micropillar platforms pooled together). Statistical analysis performed using a two-way ANOVA. (D) and (E) Nkx2.2 and Mag mRNA levels of OLs cultured on different diameter and stiffness micropillars at D5 DIFF. Results show mean values of levels normalized to Oaz1 housekeeping gene and to respective flat surfaces (2 -ΔΔCt , n>3 independent experiments, 8 micropillar platforms pooled together). Statistical analysis performed using a two-way ANOVA. (F) OL branching ability at D1, 5 and 7 DIFF. Quantification was performed based on the “Collar Occupancy method” . Results represent mean ± standard deviation (SD), n > 5 micropillar platforms from 4 independent experiments. Two-way ANOVA. (G) Percentage of wrapped micropillars by OLs in different diameter and stiffness micropillars. Results show mean ± SD, n>6 micropillar platforms analyzed from 4 different independent experiments. Two-way ANOVA. (H) Myelin thickness of OLs on micropillars at D5 (i) and D7 DIFF (ii). Measurements were performed from images acquired by STED microscopy. Six measurements per z-plane from a total of four planes per micropillar were made. At least 10 micropillars per condition were acquired. Real values of the diameter of the micropillars were estimated based on the length of the mid-plane acquired. Results show median values. Statistical analysis was performed using Two-way ANOVA.

Article Snippet: Image acquisition was carried on a Leica Stellaris 8 STED Falcon microscope (single point scanning confocal equipped with a fully motorized inverted Leica DMI8 microscope, a white light laser (WLL), Falcon and STED modalities) from Leica Microsystems.

Techniques: Cell Culture, Standard Deviation, Microscopy