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

1 Scientific RepoRts | 6:21563 | DOI: 10.1038/srep21563 www.nature.com/scientificreports Modulation of oligodendrocyte differentiation and maturation by combined biochemical and mechanical cues Tânia Lourenço1,2, Joana Paes de Faria3,4, Christian A. Bippes5, João Maia6, José A. Lopes-da-Silva7, João B. Relvas3,4 & Mário Grãos1,2 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 OPCor OL-like morphology in response to braincompliant 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. Oligodendrocytes (OLs) are the myelin-forming cells of the central nervous system (CNS), wrapping axons and providing insulation to accelerate the transmission of action potentials1. The process of myelination occurs mostly during embryonic development and in early post-natal stages and is strictly regulated by several molecular elements, such as growth factors and hormones. While basic Fibroblast Growth Factor (bFGF) and Platelet Derived Growth Factor (PDGF) contribute to the proliferation of OL progenitors — OPCs2, the thyroid hormones [Triiodo-L-thyronine (T3) and Thyroxin (T4)] control the specification and differentiation of oligodendrocytes, also playing a role during the myelination of axons3–7. The loss of OLs and consequently their myelin sheaths causes anomalous nerve transmission and neuronal cell death, as it is the case in the course of demyelinating diseases such as multiple sclerosis8. In demyelinating diseases, the remyelination process may be incomplete for reasons yet unclear9–11. Possible reasons are the exhaustion of OPCs or the presence of inhibitory or absence of stimulatory factors at lesioned areas which prevent the differentiation of existing progenitors9,12. Another hypothesis is the presence of a disturbed extracellular milieu, since a particular balance between extracellular adhesion and matrix rigidity seems to be required for successful myelination and remyelination to occur13. The extracellular matrix (ECM) is the acellular component of organs and tissues. It is composed essentially by water, proteins and polysaccharides, providing not only physical support to cells, but also biochemical and mechanical signals necessary for tissue morphogenesis, differentiation and homeostasis (reviewed in Frantz, C. 1Biocant, Technology Transfer Association, Cantanhede, Portugal. 2Centre for Neuroscience and Cell Biology (CNC), University of Coimbra, Coimbra, Portugal. 3IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal. 4i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal. 5Nanosurf AG, Liestal, Switzerland. 6Chemical Engineering Department, Faculty of Science and Technology, University of Coimbra, Coimbra, Portugal. 7QOPNA, Chemistry Department, University of Aveiro, Aveiro, Portugal. Correspondence and requests for materials should be addressed to M.G. (email: [email protected]) received: 31 March 2015 Accepted: 25 January 2016 Published: 16 February 2016 OPEN www.nature.com/scientificreports/ 2 Scientific RepoRts | 6:21563 | DOI: 10.1038/srep21563 et al.)14. The biochemical composition of the extracellular matrix of the brain plays a key role during oligodendrogenesis. While the ECM proteins fibronectin (FN) or vitronectin (VN) are involved in the proliferation and maintenance of oligodendrocyte progenitors, laminin α 2 (also known as merosin — MN) promotes their differentiation into mature OLs15–20. Furthermore, the extracellular matrix also comprises mechanical support. It is known that the stiffness of the extracellular milieu can modulate the fate of distinct cell types. To illustrate this idea, mesenchymal stem cells cultured on substrates compliant with the rigidity of the brain, muscle or bone were shown to display a neurogenic, myogenic or osteogenic phenotype21. Additionally, neural stem cells (NSCs) were shown to become specified into neuronal or glial lineages depending on substrate stiffness. Very soft platforms (100–500 Pa) promoted neuronal differentiation, while slightly stiffer ones (1,000–10,000 Pa) favoured the appearance of glial (astrocytic) cells22. Similarly, substrate stiffness was shown to modulate survival and proliferation of OPCs and oligodendrocyte morphology23,24. In the present work, we sought to develop a hydrogel-based platform functionalised with extracellular matrix proteins. Combining mechanical and biochemical properties typical of the brain’s ECM allowed studying the combined effect of such factors during oligodendrocyte differentiation and maturation (unlike existing studies focusing on the effect of each component separately16,18,23,24). The results presented here indicate that the combination of mechanical and biochemical properties of the ECM present in vivo play a crucial role during oligodendroglial differentiation, suggesting that such factors should be taken into account when studying the biology of oligodendrocytes and in putative future clinical applications using oligodendrocyte progenitors. Results Characterization of mechanical properties of polyacrylamide hydrogels. Polyacrylamide polymers are widely used in a cell biology context due to their capacity of modelling different degrees of stiffness, which may be achieved by obtaining different crosslinking degrees by simply varying the percentage of the acrylamide (AC) and/or bis-acrylamide (BAC) monomers. The mechanical properties of six formulations of polyacrylamide hydrogels (PAHs) were measured using a rheometer, by performing 0.1–10 Hz frequency sweeps (Fig.1A). The shear storage modulus (G′) of all hydrogel formulations was essentially independent of the oscillatory frequency across the tested range, meaning that we are dealing with true elastic gels where any macromolecular rearrangements are very limited (Fig.1A). The Young’s modulus (E) of each PAH formulation tested was calculated from the G′ values at 1 Hz as described in the “Materials and Methods” section. Increased percentage of AC and/or BAC correlated with higher stiffness of the hydrogels produced (Fig.1B), with a range between 9720 ± 1352 Pa (gel number 1) and 362 ± 65 Pa (gel number 6), as shown in Table1 and Fig.1B. These formulations are therefore compliant with the range of stiffness described for central nervous system (CNS) tissue between 100 and 10,000 Pa25. The hydrogels were also analysed by performing AFM measurements (Fig. S1). It could be confirmed that substrate stiffness was directly proportional to the final AC/BAC concentration used in each formulation. The 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). Gel number % AC/% BAC Swelling ratio Mean ± SD E – Young’s modulus (Pa) Mean ± SD 1 12.5%/0.37% 1.06 ± 0.024 9720 ± 1352 2 10%/0.3% 1.05 ± 0.034 6629 ± 2691 3 5%/0.2% 1.06 ± 0.041 2442 ± 858 4 4%/0.2% 1.09 ± 0.036 2032 ± 738 5 3%/0.2% 1.14 ± 0.048 952 ± 320 6 3%/0.05% 1.40 ± 0.140 362 ± 65 Table 1. Formulation (in percentage of acrylamide — AC — and bis-acrylamide — BAC), swelling ratio and Young’s modulus measured by rheometry of distinct polyacrylamide hydrogels (numbers 1–6). www.nature.com/scientificreports/ 3 Scientific RepoRts | 6:21563 | DOI: 10.1038/srep21563 absolute Young’s modulus values obtained by AFM (Fig. S1) were higher than the ones obtained by rheometry (Table1 and Fig.1B), although still within the kPa range. Similar differences between the nanoscale elastic modulus (AFM-nanoindentation) and the macroscopic shear modulus (assessed by rheometry) have been previously observed for a reconstituted basement membrane-like complex26. These discrepancies may be partially explained by different strain modes (shear or compressive, in case of rheometry or AFM, respectively) and distinct measuring time scales. Moreover, it is well known that the moduli of soft substrates (like the ones analysed here) obtained by AFM-based measurements are dependent on the conditions used. In particular, the usually unknown exact tip geometry and size as well as viscous effects of the samples that depend on the indentation velocity may lead to discrepancies27–29. It is known that some formulations of PAHs may present high swelling ratio (SR) upon immersion in aqueous media, which could lead to different mechanical properties of the hydrogel during culture22 and surface creasing when SR is higher than 1.5, resulting from heterogeneous swelling due to the attachment of the substrate to a rigid support (like a glass coverslip)30. All PAHs produced in this study presented low SR values (lower than 1.5) — Table1 and Fig.1C —, hence stable in an aqueous environment. Namely, the formulations used for biological experiments (gels #1, #2 and #3) presented swelling ratio values very close to 1 [1.06 ± 0.024 (SD), 1.05 ± 0.034 (SD) and 1.06 ± 0.041 (SD), respectively]. Progenitoror mature-like morphology of CG-4 cells induced by fibronectin or laminin-2 was enhanced by compliant hydrogels. Fibronectin is known to favour the maintenance of the progenitor state of oligodendrocytes18, while laminin-2 (also known as merosin — MN) promotes their differentiation16. We aimed to test whether these extracellular matrix (ECM) proteins could influence the morphology of oligodendrocytes (OLs) when coupled with substrates presenting distinct degrees of stiffness. For that, CG-4 cells (a rat CNS glial precursor cell line)31 were cultured in proliferation medium (to rule out the direct influence of soluble differentiation factors) for 48 h on ~6.5 kPa brain-compliant substrates (gel number 2 — Table1) functionalised with fibronectin (FN) or poly-D-lysine/merosin (PDLMN) and, as control, on stiff (~1 GPa) glass coverslips coated with the same proteins. Cells were then stained with agglutinin (Fig.2A,C) to assess the morphological complexity27 (i.e., to assess the presence of elaborate process branching) of the cells. Morphological complexity of oligodendrocytes is proportional to their differentiation state, and was quantified by fractal dimension (D) analysis, as described by others32,33. Hence, lower D values are typical of cells with progenitor phenotype presenting low process branching (Fig.2C, left panel), while higher D values reflect a higher degree of morphological complexity 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 μ m. 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). www.nature.com/scientificreports/ 4 Scientific RepoRts | 6:21563 | DOI: 10.1038/srep21563 (with elaborate process branching) typical of more differentiated cells27 (Fig.2C, right panel). CG-4 cells showed a tendency to display a lower D value in presence of FN and higher in presence of MN (as expected) when cultured on both substrates (Fig.2A,B). However, cells cultured on 6.5 kPa PAHs functionalised with merosin (PDLMN) presented a D value that was significantly higher (p < 0.0001) than that of cells cultured on the same substrate but functionalised with FN, whereas no statistically significant differences (p = 0.1023) were observed for cells cultured on stiff coverslips coated with the same proteins (Fig.2B). These results suggest that the effects of merosin and fibronectin to induce higher or lower morphological complexity (respectively) of oligodendroglial cells was enhanced by the mechanical properties of brain-compliant 6.5 kPa substrates when compared to stiff (~1 GPa) glass coverslips. Substrate compliance and ECM protein composition modulate the differentiation and maturation of OPCs into OLs. It was demonstrated that ECM stiffness has an impact during the differentiation of oligodendrocyte progenitor cells23,24, although the effect of the combination of ECM proteins and stiffness has not yet been reported. In the current study, the influence of substrate stiffness associated with the presence or absence of laminin-2 on the differentiation of OPCs was tested using full differentiation conditions (i.e., OPCs cultured for 3 to 5 days in presence of oligodendrocyte differentiation culture medium, as described in the ‘Materials and Methods’ section). To monitor the outcome of OPC differentiation in these distinct conditions, the expression of differentiation and maturation markers, cellular area and morphology were assessed by fluorescence microscopy. Because CG-4 cells presented low differentiation efficiency (reflected by a low percentage of MBP-positive cells — data not shown), further studies were performed using primary rat OPCs. OPCs were differentiated (using differentiation medium — DM) on 6.5 kPa PAHs and stiff tissue culture polystyrene plates (TCPs) functionalised or coated (respectively) with PDL or PDLMN for the indicated periods of time (Figs3A and 4A) and the expression of MBP (oligodendrocyte differentiation marker) and PLP (OL maturation marker) were assessed by immunocytochemistry. Undifferentiated OPCs kept on proliferation medium (PM) were used as a negative control (Figs3A and 4A). The percentage of MBPand PLP-positive cells was similar across substrates (Figs3B and 4B), but increased along time of differentiation, as expected, although the only statistically significant differences between day 5 and day 3 were observed when cells were cultured on compliant 6.5 kPa substrates and not on stiff TCPs surfaces. In order to quantify the differentiation state of oligodendrocytes between experimental conditions, the corrected total fluorescence (CTCF)34,35 per cell (from fluorescence microscopy images) using antibodies that recognize the differentiation and maturation markers MBP (Fig.3C) and PLP (Fig.4C), respectively, was calculated. CTCF of MBP and PLP reflects the differentiation state of oligodendrocytes35 by taking into account (i) the staining area of the differentiation/maturation marker36, correlating with the morphological differentiation state, and (ii) the mean fluorescence intensity (MFI) value37, that mirrors the expression level of the protein labelled per cell38. After 5 days in differentiation conditions, the CTCF of MBP was statistically higher (p = 0.0003) in primary rat oligodendrocytes cultured on 6.5 kPa substrates functionalised with PDLMN comparing with those on PDL alone (Fig.3C). In contrast, the CTCF for MBP of OLs cultured on TCPs in presence of the same proteins was not statistically different (p = 0.7407) — Fig.3C. This indicates that the combined effect of the presence of merosin and substrate compliance (6.5 kPa) was determinant for the full differentiation of oligodendrocytes. Western-blot analysis data was in line with these results, revealing a tendency for a higher increase of MBP expression induced by laminin-2 when OPCs were differentiated on compliant substrates (6.5 kPa) compared with those maintained on stiff TCPs (Fig. S2). Since the CTCF of MBP reflects differentiation of cells based on the combination of morphological aspects and expression level of the protein (a well-established differentiation marker), we sought to decouple the two parameters — signal area and MFI — in order to get further insight into the contribution of substrate rigidity and the presence or absence of merosin on each differentiation component. After 5 days of differentiation, MBP area was significantly higher for cells cultured on PAHs functionalised with PDLMN than on TCPs (either in presence or absence of MN), which indicates that oligodendrocytes cultured on compliant substrates in presence of merosin acquired a more mature morphological state when compared with cells differentiated on standard culture conditions (Fig.3F). Moreover, the presence of MN also seemed to favour the expression of MBP, as observed by a statistically significant increase of the MFI of MBP at day 5, but in this case, independent of the substrate used [p = 0.0016 (PAHs) and p = 0.0017 (TCPs)] (Fig.3G). In summary, data indicate that at day 5 of differentiation the combination of substrate compliance and merosin contributed to the morphological maturation of MBP + oligodendrocytes (Fig.3F), whereas the presence of MN alone contributed mostly to increased expression of MBP (Fig.3G). Surprisingly, after only 3 days of differentiation, the CTCF of MBP in rat oligodendrocytes cultured on 6.5 kPa PAHs functionalised with PDLMN was lower than that of OLs cultured on 6.5 kPa PAHs with PDL (Fig.3B). The lower CTCF value found in the PDLMN condition may be explained by the marked decrease in cellular MBP area (Fig.3D), but no changes on the intensity of the signal were observed (Fig.3E). Concomitantly, the number of adherent cells per field after 3 days of differentiation was also significantly higher on 6.5 kPa PAHs functionalised with PDLMN than with PDL alone (p = 0.0351) (Fig. S3A), which could explain the observed decrease in cellular area due to higher cellular density and consequently lower CTCF of MBP. Concerning the MFI of MBP, it was slightly higher in presence of MN comparing with PDL alone after 3 days (Fig.3E), showing the same trend as observed at day 5 of differentiation (Fig.3G), although this increase was only statistically significant for the latter time point. In summary, data indicate that the differentiation period of 5 days was the most appropriate time point for the differentiation of primary rat OPCs in vitro and that the combined presence of MN and compliant substrates www.nature.com/scientificreports/ 5 Scientific RepoRts | 6:21563 | DOI: 10.1038/srep21563 enhanced the differentiation of the cells when compared with cells cultured on PDL alone, in contrast to what was observed on TCPs, where no significant differences were found between PDLMN versus PDL alone (Fig.3C). Assessment of the maturation of OPCs into OLs. The maturation of oligodendrocytes cultured on the distinct platforms was assessed by analysing the expression of PLP (an oligodendrocyte maturation marker), using a similar approach as described above for the differentiation marker MBP. OPCs cultured for 3 days in 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 μ m. (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). www.nature.com/scientificreports/ 6 Scientific RepoRts | 6:21563 | DOI: 10.1038/srep21563 differentiation conditions on 6.5 kPa PAHs in presence of MN displayed a higher CTCF value for PLP than cells cultured on PDL alone (p = 0.0051). This CTCF value was similarly high at day 5 of differentiation, but at this time point independent of the presence of MN (Fig.4C, right panel). On the other hand, no effects of MN were observed on the maturation of OPCs when cultured on TCPs (Fig.4C, left panel), suggesting that the presence of laminin-2 accelerated the maturation of oligodendrocytes only when cultured on compliant substrates (~6.5 kPa). Data also suggest that substrate compliance allows cells to eventually achieve a high level of maturation regardless of the presence of MN — as observed for cells after 5 days of differentiation cultured on 6.5 kPa PAHs (Fig.4C, 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 μ m. (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). www.nature.com/scientificreports/ 7 Scientific RepoRts | 6:21563 | DOI: 10.1038/srep21563 right panel). In fact the combined effect of laminin-2 and substrate compliance seems to be reflected instead by an early maturation of the cells, as observed by the increase of CTCF of PLP already at day 3 of differentiation on PAHs functionalised with PDLMN when compared to PDL alone (Fig.4C, right panel). After 3 days of differentiation, no significant differences were found regarding the area of PLP expression by PLP-positive oligodendrocytes cultured in the distinct conditions tested (Fig.4D). However, cells differentiated for 5 days on 6.5 kPa PAHs displayed higher PLP area than those cultured on stiff TCPs (Fig.4F), reinforcing the idea that substrate compliance favours OL maturation. Regarding the influence of MN on the expression level of proteolipid protein, the MFI values obtained were higher in the presence than in the absence of MN after 3 days of differentiation on PAHs (p = 0.0165) (Fig.4E, right panel), but not on TCPs (p = 0.7927) (Fig.4E, left panel). No significant differences were found for cells on either substrate at a later stage (day 5) of differentiation (Fig.4G). These results indicate a higher expression of PLP in response to laminin-2 only in cells cultured on compliant substrates at an earlier time point (3 days), but not on stiff TCPs. Taken together, these results indicate that compliant substrates (6.5 kPa) promote the maturation of OPCs (mainly at the morphological level — Fig.4F), and that the combined presence of MN and soft substrate leads to earlier expression of PLP (at day 3 of differentiation — Fig.4E), suggesting earlier maturation of the cells (Fig.4C). Compliant substrates favour the maturation of OLs, as evidenced by the formation of membranousand myelin sheet-containing cells. Since it was not advisable to compare the CTCF and MFI of MBP and PLP directly between cells differentiated on distinct substrates because the materials (TCPs and PAHs) had potentially different optical properties, putatively interfering with the absolute values of MFI (panels C, E and G of Figs3 and 4), a semi-quantitative approach was used in order to compare directly the maturation state of cells kept on the different platforms. For that end, MBP-positive oligodendrocytes were classified into three categories: (i) non-membranous, (ii) membranous and (iii) with myelin sheets (Fig.5A,B). Myelin membrane sheets have been described to form only in the later stages of oligodendrocyte differentiation39, thus the formation of such myelin structures is restricted to highly mature OLs. The morphological classification of the MBP-positive cells showed that fully matured OLs displaying myelin sheets were essentially inexistent on TCPs after 5 days of differentiation, but were present on 6.5 kPa substrates (Fig.5C). Moreover, there was also a significant increase in the percentage of membranous oligodendrocytes at the expense of non-membranous cells on PAHs when compared with TCPs (Fig.5). These results were observed both in the presence and in the absence of merosin, in line with the PLP and MBP signal area results (Figs3F and 4F) showing an overall higher morphological maturation state of the cells on PAHs than on TCPs. Taken together, these results show that morphological maturation of OLs is highly dependent on the stiffness of the substrate, but less dependent on the presence of MN, which seems to play a more important role in terms of expression of differentiation (Fig.3G) and maturation markers, which in case of PLP is also dependent on the combined presence of a compliant substrate (Fig.4E). Fine-tuning of the extracellular matrix stiffness as a modulator of oligodendrocyte differentiation. It was proposed that alterations of ECM stiffness could lead to anomalous myelination in vivo13. In order to evaluate more deeply the influence of substrate’s stiffness on the differentiation of OPCs in vitro, distinct cell-culture substrates with degrees of stiffness within the range of rigidity attributed to the brain — between 0.1 and 10 kPa25 — were tested. Namely, PAHs with Young’s moduli of 2.5, 6.5 and 10 kPa (gels number 3, 2 and 1, respectively — Table1 and Fig.1) functionalised with PDLMN were tested. After 5 days of differentiation, the percentage of MBPand PLP-positive cells was similar between substrates (Figs6A,B and 7A,B and S3B). However, differences were observed in terms of CTCF and MFI of MBP in cells cultured on the distinct substrates. The CTCF value was higher on 6.5 kPa PAHs than on 2.5 or 10 kPa, and the difference was statistically significant between 6.5 and 10 kPa PAHs (p = 0.0006) (Fig.6C). The area of MBP signal was similar between conditions (Fig.6D), nevertheless the MFI of MBP was statistically higher on 6.5 kPa PAHs than on the other substrates tested (Fig.6E), indicating an increased level of MBP expression in OLs cultured on 6.5 kPa hydrogels. These results reinforced the idea that substrate rigidity influences the expression level of MBP in addition to the morphological maturation of the cells as already described in Figs3F and 5. Regarding PLP staining, the CTCF, area and MFI were also calculated. The CTCF for PLP was higher on 6.5 kPa PAHs than on other degrees of stiffness, being statistically significant between 6.5 and 10 kPa PAHs (p = 0.0011) (Fig.7C), following the same trend as observed for MBP. The expression level of PLP seems to be similar between substrates, since the MFI did not change within the three conditions tested (Fig.7E). However, the area of expression of PLP per cell was statistically higher on 6.5 kPa PAHs, supporting the idea that this particular stiffness promotes the differentiation and maturation of OPCs more than 2.5 or 10 kPa. Overall, these results indicate that 6.5 kPa is the most appropriated stiffness within the tested range, suggesting that this could be close to the optimal rigidity to promote the differentiation and maturation of OPCs. Inhibition of non-muscle myosin-II promotes the elongation of processes of early-stage differentiation OPCs only when cultured on compliant substrates. The presence of the inhibitor of non-muscle myosin-II (NM-II) blebbistatin is known to induce relaxation of the cellular actin network and was already described to favour oligodendrocyte membrane extension during differentiation24,40. In such studies, cells were treated with blebbistatin throughout a period of 3 days in differentiating culture conditions, coincident with the expression of MBP, hence its effect was assessed only after differentiation being essentially achieved. In contrast, we aimed to understand the effect of actomyosin inhibition on early-stage differentiating oligodendrocytes. For that, primary rat OPCs were cultured in differentiation conditions on compliant substrates (6.5 kPa PAHs) or www.nature.com/scientificreports/ 8 Scientific RepoRts | 6:21563 | DOI: 10.1038/srep21563 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 μ m. (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). www.nature.com/scientificreports/ 9 Scientific RepoRts | 6:21563 | DOI: 10.1038/srep21563 TCPs functionalised or coated (respectively) with PDL or PDLMN for 24 h, in presence or absence of blebbistatin. Cells were stained using antibodies against Olig2 and alpha-tubulin (to identify the oligodendrocyte-lineage specific cells and to stain the cellular processes, respectively) and the full area of the processes of oligodendrocytes was measured as a read-out of the process extension (Fig.8A), as previously described by others41. In this experiment, only Olig2-positive cells were considered oligodendrocytes — Fig.8A, pink arrowheads —, being the Olig2-negative cells considered as contaminating glia — Fig.8A, white arrowheads. It could be observed that blebbistatin promoted the extension of processes when OPCs were cultured on 6.5 kPa PAHs, notably only when comparing cells cultured on substrates functionalised with merosin, but not with PDL (Fig.8B). Conversely, the effect of blebbistatin on OPCs cultured on TCPs was not statistically significant, when comparing the presence or absence of blebbistatin between PDLor PDLMN-coated surfaces (Fig.8C). It should be noted that for data represented in Fig.8B,C the effect of MN in enhancing the extension of the processes of oligodendrocytes in absence of blebbistatin was also observed when comparing PDLversus PDLMN-functionalised PAHs (p = 0.0003, two-tailed t-test), but not between PDLand PDLMN-coated TCPs (p = 0.068, two-tailed t-test). These results indicate that the combined effect of blebbistatin, compliant substrates and presence of merosin promotes the early (24 h) differentiation of OPCs. Discussion Oligodendrocyte progenitor cells (primary rat OPCs and CG-4 cells) responded to mechanical and biochemical cues provided by brain-compliant substrates functionalised with ECM proteins, changing morphological complexity and expression of differentiation and maturation markers (MBP and PLP, respectively). Non-differentiated CG-4 cells exhibited distinct morphological features when cultured in proliferation medium (under non-differentiating conditions) in presence of FN or MN, exhibiting a bipolar morphology characteristic of oligodendrocyte progenitors or a branched morphology typical of differentiated cells, respectively. This effect was only statistically significant when cells were cultured on brain-compliant substrates — 6.5 kPa, as assessed by rheometry (Fig.1 and Table1) —, but not when kept on stiff TCPs (Fig.2B). These results indicate that oligodendrocyte progenitor cells become particularly responsive when cultured on compliant substrates to the well-established effect of fibronectin of maintaining the progenitor state of oligodendrocytes and the effect of laminin-2/merosin (MN) of inducing the differentiation of the cells15–20, even in presence of non-differentiating medium (as reported here). Such morphological changes may be related with changes in the contractility of actomyosin of OLs in response to soft substrates, in agreement with what has been described in the literature24,41,42, 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 with7 PDL or PDLMN, as indicated, using an anti-MBP antibody (red) and DAPI for nuclear counterstaining (blue). Scale bars correspond to 50 μ m. (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). www.nature.com/scientificreports/ 16 Scientific RepoRts | 6:21563 | DOI: 10.1038/srep21563 8. Lourenco, A. S., Baldeiras, I., Graos, M. & Duarte, C. B. Proteomics-based technologies in the discovery of biomarkers for multiple sclerosis in the cerebrospinal fluid. Curr Mol Med 11, 326–349, doi: 10.2174/156652411795677981 (2011). 9. Câmara, J. & ffrench-Constant, C. Lessons from oligodendrocyte biology on promoting repair in multiple sclerosis. Journal of Neurology 254, I15–I22, doi: 10.07/s00415-007-1004-y (2007). 10. Franklin, R. J. & Ffrench-Constant, C. Remyelination in the CNS: from biology to therapy. Nat Rev Neurosci 9, 839–855, doi: 10.38/ nrn2480 (2008). 11. McTigue, D. M. & Tripathi, R. B. The life, death, and replacement of oligodendrocytes in the adult CNS. J Neurochem 107, 1–19, doi: 10.1111/j.1471-4159.2008.05570.x (2008). 12. Baumann, N. & Pham-Dinh, D. Biology of oligodendrocyte and myelin in the mammalian central nervous system. Physiol Rev 81, 871–927 (2001). 13. Bauer, N. G. & ffrench-Constant, C. Physical forces in myelination and repair: a question of balance? J Biol 8, 78, doi: 10.1186/ jbiol169 (2009). 14. Frantz, C., Stewart, K. M. & Weaver, V. M. The extracellular matrix at a glance. J Cell Sci 123, 4195–4200, doi: 10.1242/jcs.023820 (2010). 15. Baron, W., Decker, L., Colognato, H. & ffrench-Constant, C. Regulation of integrin growth factor interactions in oligodendrocytes by lipid raft microdomains. Curr Biol 13, 151–155, doi: 10.16/S0960-9822(02)01437-9 (2003). 16. Buttery, P. C. & ffrench-Constant, C. Laminin-2/integrin interactions enhance myelin membrane formation by oligodendrocytes. Mol Cell Neurosci 14, 199–212, doi: 10.06/mcne.1999.0781 (1999). 17. Chun, S. J., Rasband, M. N., Sidman, R. L., Habib, A. A. & Vartanian, T. Integrin-linked kinase is required for laminin-2-induced oligodendrocyte cell spreading and CNS myelination. J Cell Biol 163, 397–408, doi: 10.83/jcb.200304154 (2003). 18. Colognato, H., Ramachandrappa, S., Olsen, I. M. & ffrench-Constant, C. Integrins direct Src family kinases to regulate distinct phases of oligodendrocyte development. J Cell Biol 167, 365–375, doi: 10.83/jcb.200404076 (2004). 19. Stoffels, J. M. et al. Fibronectin aggregation in multiple sclerosis lesions impairs remyelination. Brain 136, 116–131, doi: 10.93/brain/ aws313 (2013). 20. ffrench-Constant, C. & Colognato, H. Integrins: versatile integrators of extracellular signals. Trends Cell Biol 14, 678–686, doi: 10.16/j.tcb.2004.10.005 (2004). 21. Engler, A. J., Sen, S., Sweeney, H. L. & Discher, D. E. Matrix elasticity directs stem cell lineage specification. Cell 126, 677–689, doi: 10.16/j.cell.2006.06.044 (2006). 22. Saha, K. et al. Substrate modulus directs neural stem cell behavior. Biophys J 95, 4426–4438, doi: 10.1529/biophysj.108.132217 (2008). 23. Jagielska, A. et al. Mechanical environment modulates biological properties of oligodendrocyte progenitor cells. Stem Cells Dev 21, 2905–2914, doi: 10.89/scd.2012.0189 (2012). 24. Kippert, A., Fitzner, D., Helenius, J. & Simons, M. Actomyosin contractility controls cell surface area of oligodendrocytes. BMC Cell Biol 10, 71, doi: 10.1186/1471-2121-10-71 (2009). 25. Moore, S. W., Roca-Cusachs, P. & Sheetz, M. P. Stretchy proteins on stretchy substrates: the important elements of integrin-mediated rigidity sensing. Dev Cell 19, 194–206, doi: 10.16/j.devcel.2010.07.018 (2010). 26. Soofi, S. S., Last, J. A., Liliensiek, S. J., Nealey, P. F. & Murphy, C. J. The elastic modulus of Matrigel as determined by atomic force microscopy. J Struct Biol 167, 216–219, doi: 10.16/j.jsb.2009.05.005 (2009). 27. Trappmann, B. et al. Extracellular-matrix tethering regulates stem-cell fate. Nat Mater 11, 642–649, doi: 10.38/nmat3339 (2012). 28. Wen, J. H. et al. Interplay of matrix stiffness and protein tethering in stem cell differentiation. Nat Mater 13, 979–987, doi: 10.38/ nmat4051 (2014). 29. Gutierrez, E. & Groisman, A. Measurements of elastic moduli of silicone gel substrates with a microfluidic device. PLoS One 6, e25534, doi: 10.1371/journal.pone.0025534 (2011). 30. Saha, K. et al. Surface creasing instability of soft polyacrylamide cell culture substrates. Biophys J 99, L94–96, doi: 10.16/j. bpj.2010.09.045 (2010). 31. Louis, J. C., Magal, E., Muir, D., Manthorpe, M. & Varon, S. CG-4, a new bipotential glial cell line from rat brain, is capable of differentiating in vitro into either mature oligodendrocytes or type-2 astrocytes. J Neurosci Res 31, 193–204, doi: 10.02/jnr.490310125 (1992). 32. Behar, T. N. Analysis of fractal dimension of O2A glial cells differentiating in vitro. Methods 24, 331–339, doi: 10.06/meth.2001.1203 (2001). 33. Fernandez, E. & Jelinek, H. F. Use of fractal theory in neuroscience: methods, advantages, and potential problems. Methods 24, 309–321, doi: 10.06/meth.2001.1201 (2001). 34. Aulic, S. et al. Defined alpha-synuclein prion-like molecular assemblies spreading in cell culture. BMC Neurosci 15, 69, doi: 10.1186/1471-2202-15-69 (2014). 35. Dai, J., Bercury, K. K., Ahrendsen, J. T. & Macklin, W. B. Olig1 function is required for oligodendrocyte differentiation in the mouse brain. J Neurosci 35, 4386–4402, doi: 10.1523/JNEUROSCI.4962-14.2015 (2015). 36. Guardiola-Diaz, H. M., Ishii, A. & Bansal, R. Erk1/2 MAPK and mTOR signaling sequentially regulates progression through distinct stages of oligodendrocyte differentiation. Glia 60, 476–486, doi: 10.1002/glia.22281 (2012). 37. Fischer, R., Wajant, H., Kontermann, R., Pfizenmaier, K. & Maier, O. Astrocyte-specific activation of TNFR2 promotes oligodendrocyte maturation by secretion of leukemia inhibitory factor. Glia 62, 272–283, doi: 10.02/glia.22605 (2014). 38. Zhang, K. et al. A curve fitting method of quantifying green fluorescent protein expression level in Escherichia coli. J Microbiol Methods 80, 172–177, doi: 10.16/j.mimet.2009.12.003 (2010). 39. Osterhout, D. J., Wolven, A., Wolf, R. M., Resh, M. D. & Chao, M. V. Morphological differentiation of oligodendrocytes requires activation of Fyn tyrosine kinase. J Cell Biol 145, 1209–1218, doi: 10.83/jcb.145.6.1209 (1999). 40. Wang, H., Tewari, A., Einheber, S., Salzer, J. L. & Melendez-Vasquez, C. V. Myosin II has distinct functions in PNS and CNS myelin sheath formation. J Cell Biol 182, 1171–1184, doi: 10.83/jcb.200802091 (2008). 41. Wang, H. et al. Myosin II is a negative regulator of oligodendrocyte morphological differentiation. J Neurosci Res 90, 1547–1556, doi: 10.02/jnr.23036 (2012). 42. Bauer, N. G., Richter-Landsberg, C. & Ffrench-Constant, C. Role of the oligodendroglial cytoskeleton in differentiation and myelination. Glia 57, 1691–1705, doi: 10.02/glia.20885 (2009). 43. Kramer-Albers, E. M. & White, R. From axon-glial signalling to myelination: the integrating role of oligodendroglial Fyn kinase. Cell Mol Life Sci 68, 2003–2012, doi: 10.07/s00018-010-0616-z (2011). 44. Clark, K., Langeslag, M., Figdor, C. G. & van Leeuwen, F. N. Myosin II and mechanotransduction: a balancing act. Trends Cell Biol 17, 178–186, doi: 10.16/j.tcb.2007.02.002 (2007). 45. Cretu, A., Castagnino, P. & Assoian, R. Studying the effects of matrix stiffness on cellular function using acrylamide-based hydrogels. J Vis Exp. e2089, doi: 10.3791/2089 (2010). 46. Hoffecker, I. T., Guo, W. H. & Wang, Y. L. Assessing the spatial resolution of cellular rigidity sensing using a micropatterned hydrogel-photoresist composite. Lab Chip 11, 3538–3544, doi: 10.39/c1lc20504h (2011). 47. Polio, S. R., Rothenberg, K. E., Stamenovic, D. & Smith, M. L. A micropatterning and image processing approach to simplify measurement of cellular traction forces. Acta Biomater 8, 82–88, doi: 10.16/j.actbio.2011.08.013 (2012). www.nature.com/scientificreports/ 17 Scientific RepoRts | 6:21563 | DOI: 10.1038/srep21563 48. Buxboim, A., Rajagopal, K., Brown, A. E. & Discher, D. E. How deeply cells feel: methods for thin gels. J Phys Condens Matter 22, 194116, doi: 10.88/0953-8984/22/19/194116 (2010). 49. Tse, J. R. & Engler, A. J. Preparation of hydrogel substrates with tunable mechanical properties. Curr Protoc Cell Biol Chapter 10, Unit 10 16, doi: 10.02/0471143030.cb1016s47 (2010). 50. Sader, J. E., Chon, J. W. M. & Mulvaney, P. Calibration of rectangular atomic force microscope cantilevers. Review of Scientific Instruments 70, 3967–3969, doi: 10.1063/1.1150021 (1999). 51. Sneddon, I. N. The relation between load and penetration in the axisymmetric boussinesq problem for a punch of arbitrary profile. Int J Eng Sci 3, 47–57 (1965). 52. Young, D. A., Choi, Y. S., Engler, A. J. & Christman, K. L. Stimulation of adipogenesis of adult adipose-derived stem cells using substrates that mimic the stiffness of adipose tissue. Biomaterials 34, 8581–8588, doi: 10.16/j.biomaterials.2013.07.103 (2013). 53. Domke, J. & Radmacher, M. Measuring the elastic properties of thin polymer films with the atomic force microscope. Langmuir 14, 3320–3325, doi: 10.1021/la9713006 (1998). 54. Engler, A. J., Rehfeldt, F., Sen, S. & Discher, D. E. Microtissue Elasticity: Measurements by Atomic Force Microscopy and Its Influence on Cell Differentiation. In Methods in Cell Biology. Vol. 83, 521–545 (Academic Press, 2007). 55. Liu, G. J. & Madsen, B. W. PACAP38 modulates activity of NMDA receptors in cultured chick cortical neurons. J Neurophysiol 78, 2231–2234 (1997). 56. Ji, S., Doucette, J. R. & Nazarali, A. J. Sirt2 is a novel in vivo downstream target of Nkx2.2 and enhances oligodendroglial cell differentiation. J Mol Cell Biol 3, 351–359, doi: 10.93/jmcb/mjr009 (2011). 57. McCarthy, K. D. & de Vellis, J. Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J Cell Biol 85, 890–902, doi: 10.83/jcb.85.3.890 (1980). 58. Milner, R. & ffrench-Constant, C. A developmental analysis of oligodendroglial integrins in primary cells: changes in alpha v-associated beta subunits during differentiation. Development 120, 3497–3506 (1994). 59. Bottenstein, J. E. & Sato, G. H. Growth of a rat neuroblastoma cell line in serum-free supplemented medium. Proceedings of the National Academy of Sciences 76, 514–517 (1979). 60. Leite, C. et al. Differentiation of human umbilical cord matrix mesenchymal stem cells into neural-like progenitor cells and maturation into an oligodendroglial-like lineage. PLoS One 9, e111059, doi: 10.1371/journal.pone.0111059 (2014). Acknowledgements Authors would like to thank Dr. Adil Nazarali (College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, Canada) for providing the CG-4 and B104 cell lines. This work was funded by the ERDF through Programa Operacional Factores de Competitividade - COMPETE and by national funds by FCT – Fundação para a Ciência e a Tecnologia (Portuguese Foundation for Science and Technology) through grants FCOMP01-0124-FEDER-021150 - PTDC/SAU-ENB/119292/2010 attributed to M.G., which included a research fellowship awarded to T.L., and COMPETE funding (Project “Stem cell based platforms for Regenerative and Therapeutic Medicine”, Centro-07-ST24-FEDER-002008). J.B.R acknowledges FCT for grant PTDC/SAUNMC/119937/2010 - FCOMP-01-0124-FEDER-021333 and FCT financially supported J.P.F. through fellowship SFRH/BPD/34834/2007. Thanks are also due to FCT/MEC for the financial support to the QOPNA research Unit (FCT UID/QUI/00062/2013), (also through national funds, co-financed by FEDER within the PT2020 Partnership Agreement). Author Contributions T.L. and C.A.B. designed and conducted experiments, and analysed data. J.P.F. and J.M. conducted experiments. J.B.R. designed experiments. J.A.L.S. and M.G. designed experiments and analysed data. T.L. and M.G. primarily wrote the paper with contribution of all authors. Additional Information Supplementary information accompanies this paper at http://www.nature.com/srep Competing financial interests: Christian A. Bippes is an employee of NanoSurf AG, a manufacturer of AFM equipment. The other authors declare no competing financial interests. How to cite this article: Lourenço, T. et al. Modulation of oligodendrocyte differentiation and maturation by combined biochemical and mechanical cues. Sci. Rep. 6, 21563; doi: 10.1038/srep21563 (2016). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/