Modulation of oligodendrocyte differentiation and maturation by combined biochemical and mechanical cues

Abstract

Extracellular matrix (ECM) proteins play a key role during oligodendrogenesis. While fibronectin (FN) is involved in the maintenance and proliferation of oligodendrocyte progenitor cells (OPCs), merosin (MN) promotes differentiation into oligodendrocytes (OLs). Mechanical properties of the ECM also seem to affect OL differentiation, hence this study aimed to clarify the impact of combined biophysical and biochemical elements during oligodendrocyte differentiation and maturation using synthetic elastic polymeric ECM-like substrates. CG-4 cells presented OPC- or OL-like morphology in response to brain-compliant substrates functionalised with FN or MN, respectively. The expression of the differentiation and maturation markers myelin basic protein--MBP--and proteolipid protein--PLP--(respectively) by primary rat oligodendrocytes was enhanced in presence of MN, but only on brain-compliant conditions, considering the distribution (MBP) or amount (PLP) of the protein. It was also observed that maturation of OLs was attained earlier (by assessing PLP expression) by cells differentiated on MN-functionalised brain-compliant substrates than on standard culture conditions. Moreover, the combination of MN and substrate compliance enhanced the maturation and morphological complexity of OLs. Considering the distinct degrees of stiffness tested ranging within those of the central nervous system, our results indicate that 6.5 kPa is the most suitable rigidity for oligodendrocyte differentiation.

Figure 1. Properties of polyacrylamide hydrogels.

(A) Representative rheological measurements of the shear storage modulus G’ (by rheometry) of six distinct formulations of polyacrylamide hydrogels (PAHs) across a frequency sweep (0.1–10 Hz) at a constant strain (2 millistrain) and 37 °C. Mean ± SD of the Young’s modulus (B) or swelling ratio (C) of at least three independent batches of six distinct formulations of PAHs (1–6).

Figure 2. Cell morphology assessment of CG-4 cells by fractal dimension analysis.

(A) Representative fluorescence microscopy images of oligodendroglial CG-4 cells plated on glass coverslips or 6.5 kPa polyacrylamide hydrogels (PAHs) functionalised with fibronectin (FN) or poly-D-lysine and merosin (PDLMN) and maintained in proliferation medium for 2 days. Scale bar corresponds to 50 μm. The D values (fractal dimension) are shown in (B,C). Values in (B) represent at least n = 13 cells analysed from three independent experiments and in (C) are depicted representative images of cells analysed in (B). Statistical analysis was performed by t-test using the software GraphPad Prism 6. Statistical comparisons were represented using connectors (n.s.: non-significant, ***p < 0.001).

Figure 3. Differentiation of primary rat oligodendrocyte progenitor cells (OPCs) using distinct substrates and conditions.

(A) Representative immunofluorescence microscopy images of primary rat oligodendrocytes stained for MBP (in red, and nuclei were counterstained with DAPI, in blue), cultured for 2 days in proliferation medium (2d PM), or for 3 or 5 days in differentiation medium (3d DM or 5d DM). Cells were maintained on TCPs or 6.5 kPa PAHs coated/functionalised with PDL or PDLMN, as indicated. Scale bars correspond to 50 μm. (B) The percentage of MBP-positive cells was quantified for each experimental condition (3 or 5 days in DM) and platform. Data represent mean ± SEM of at least five independent experiments. Statistical analysis was performed by two-way ANOVA followed by Bonferroni post-test. (C) Quantification of CTCF of MBP signal of primary rat oligodendrocytes cultured with DM for 3 or 5 days on TCPs or 6.5 kPa PAHs coated/functionalised with PDL or PDLMN, as indicated. Data represent mean ± SEM of at least 5 independent experiments. Statistical analysis was performed by t-test. (D,F) Measurement of MBP signal area of primary rat oligodendrocytes cultured with differentiation medium for 3 days (D) or 5 days (F) on TCPs or PAHs coated/functionalised with PDL or PDLMN. Data represent mean ± SEM of at least 5 independent experiments. Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparison test. (E,G) Mean fluorescence intensity (MFI) of MBP signal of oligodendrocytes cultured on TCPs or PAHs for 3 days (E) or for 5 days (G). Data represent mean ± SEM of at least 5 independent experiments. Statistical analysis was performed by t-test. All statistical analysis was performed using the software GraphPad Prism 6 and the statistical significant differences were represented using the connectors (n.s non-significant, *p < 0.05, **p < 0.01, ***p < 0.001).

Figure 4. Maturation of primary rat OPCs using distinct substrates and conditions.

(A) Representative immunofluorescence microscopy images of primary rat oligodendrocytes stained for PLP (in green, and nuclei were counterstained with DAPI, in blue), cultured for 2 days in proliferation medium (2d PM), or for 3 or 5 days in differentiation medium (3d DM or 5d DM, respectively). Cells were maintained on TCPs or 6.5 kPa PAHs coated/functionalised with PDL or PDLMN, as indicated. Scale bars correspond to 50 μm. (B) The percentage of PLP-positive cells was quantified for each experimental condition (3 or 5 days in DM) and platform. Data represent the mean ± SEM of at least three independent experiments. Statistical analysis was performed by two-way ANOVA followed by Bonferroni post-test. (C) Quantification of CTCF for PLP signal of primary rat oligodendrocytes cultured with DM for 3 or 5 days on TCPs or 6.5 kPa PAHs coated/functionalised with PDL or PDLMN. Data represent mean ± SEM of at least 3 independent experiments. (D,F) Measurement of the PLP signal area of primary rat oligodendrocytes cultured with DM for 3 days (D) or 5 days (F) on TCPs or PAHs coated/functionalised with PDL or PDLMN. Data represent mean ± SEM of at least 3 independent experiments. Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparison test. (E,G) Mean fluorescence intensity (MFI) of PLP signal of oligodendrocytes cultured on TCPs or PAHs for 3 days (E) or for 5 days (G). Data represent mean ± SEM of CTCF of at least 3 independent experiments. Statistical analysis was performed by t-test. All statistical analysis was performed using the software GraphPad Prism 6 and the statistical significant differences were represented using the connectors (n.s.: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001).

Figure 5. Morphological characterization of oligodendrocytes differentiated using distinct substrates and conditions.

(A) Representative fluorescence microscopy images of primary rat oligodendrocytes cultured for 5 days in differentiation medium using distinct combinations of substrates (TCPs or 6.5 kPa PAHs) coated/functionalised with PDL or PDLMN, as indicated. Immunostaining was performed using an anti-MBP antibody (red) and DAPI for nuclear counterstaining (blue). Scale bars correspond to 50 μm. (C) Quantification of the percentage of cells bearing a non-membranous, membranous, or myelin sheet distribution of MBP, as depicted in the representative images in (B) — left, centre and right panels, respectively. The graph represents data from six independent experiments. Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparison test using the software GraphPad Prism 6. Statistically significant differences within cellular categories between substrates were represented (*, ^#p < 0.05, ^##p < 0.01, where *represent comparisons relative to TCPs PDL and ^#comparisons relative to TCPs PDLMN).

Figure 6. Modulation of oligodendrocyte differentiation and MBP levels by substrate stiffness within a narrow, brain-compliant range.

(A) Representative fluorescence microscopy images of primary rat oligodendrocytes cultured for 5 days in differentiation medium using PAHs with distinct degrees of stiffness (2.5, 6.5 or 10 kPa) functionalised with PDL or PDLMN, as indicated, using an anti-MBP antibody (red) and DAPI for nuclear counterstaining (blue). Scale bars correspond to 50 μm. (B) Percentage of MBP-positive cells on the distinct substrates and (C) quantification of CTCF for MBP of oligodendrocytes cultured with differentiation medium for 5 days on 2.5, 6.5 and 10 kPa PAHs functionalised with PDL or PDLMN (as indicated). (D) Measurement of MBP signal area and (E) mean fluorescence intensity (MFI) of MBP signal (six fields per experiment from three independent experiments were analysed). Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparison test or Kruskal-Wallis test [in (B)] using the software GraphPad Prism 6. Statistically significant differences between substrate stiffness were represented using connectors (*p < 0.05, **p < 0.01).

Figure 7. Modulation of oligodendrocyte maturation and PLP levels by substrate stiffness within a narrow, brain-compliant range.

(A) Representative fluorescence microscopy images of primary rat oligodendrocytes cultured for 5 days in differentiation medium using PAHs with distinct degrees of stiffness (2.5, 6.5 or 10 kPa) functionalised with PDL +/− MN, as indicated, using an anti-PLP antibody (green) and DAPI for nuclear counterstaining (blue). Scale bars correspond to 50 μm. (B) Percentage of the PLP-positive cells on distinct substrates and (C) quantification of CTCF for PLP of primary oligodendrocytes cultured with differentiation medium for 5 days on 2.5, 6.5 and 10 kPa PAHs functionalised with PDL or PDLMN (as indicated). (D) Measurement of the PLP signal area and (E) mean fluorescence intensity (MFI) of the PLP signal (six fields from three independent experiments were analysed). Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparison or Kuskal-Wallis test [in (B)] using the software GraphPad Prism 6. Statistically significant differences between substrate stiffness were represented using connectors (*p < 0.05, **p < 0.01).

Figure 8. Effect of substrate stiffness, merosin and actomyosin contractility inhibition on morphological features of oligodendrocytes during early-stage differentiation.

(A) Representative fluorescence microscopy images of OPCs cultured for 24 h in differentiation medium on 6.5 kPa PAHs functionalised with PDLMN in absence (left) or presence (right) of 15 μM of non-muscle myosin-II (NMM-II) inhibitor blebbistatin. Cells were immunostained for the oligodendrocyte lineage marker Olig2 (nuclear staining, in red) and for alpha-tubulin to stain the processes (green) and nuclei were counterstained with DAPI (blue). Scale bar corresponds to 50 μm. The area of oligodendrocyte processes of Olig2-positive cells — e.g.: cells highlighted by pink arrowheads in (A) — maintained on 6.5 kPa PAHs (B) or on TCPs (C) were normalised to the conditions PAHs PDL (B) or TCPs PDL (C). Data in graphs represent n = 30 cells obtained from three independent experiments. Statistical analysis was performed by t-test using the software GraphPad Prism 6. Statistical significance between conditions under comparison was represented using connectors (n.s.: non-significant, *p < 0.05).