Combinatory Multifactor Treatment Effects on Primary Nanofiber Oligodendrocyte Cultures

Abstract

Multiple sclerosis (MS) is a chronic inflammatory demyelinating and neurodegenerative disease of the central nervous system. Neurological deficits are attributed to inflammatory demyelination, which compromises axonal function and survival. These are mitigated in experimental models by rapid and often complete remyelination of affected axons, but in MS this endogenous repair mechanism frequently fails, leaving axons increasingly vulnerable to the detrimental effects of inflammatory and metabolic stress. Understanding the molecular basis of remyelination and remyelination failure is essential to develop improved therapies for this devastating disease. However, recent studies suggest that this is not due to a single dominant mechanism, but rather represents the biological outcome of multiple changes in the lesion microenvironment that combine to disrupt oligodendrocyte differentiation. This identifies a pressing need to develop technical platforms to investigate combinatory and/or synergistic effects of factors differentially expressed in MS lesions on oligodendrocyte proliferation and differentiation. Here we describe protocols using primary oligodendrocyte cultures from Bl6 mice on 384-well nanofiber plates to model changes affecting oligodendrogenesis and differentiation in the complex signaling environment associated with multiple sclerosis lesions. Using platelet-derived growth factor (PDGF-AA), fibroblast growth factor 2 (FGF2), bone morphogenetic protein 2 (BMP2) and bone morphogenetic protein 4 (BMP4) as representative targets, we demonstrate that we can assess their combinatory effects across a wide range of concentrations in a single experiment. This in vitro model is ideal for assessing the combinatory effects of changes in availability of multiple factors, thus more closely modelling the situation in vivo and furthering high-throughput screening possibilities.

Keywords: multiple sclerosis; myelin; nanofibers; oligodendrocyte; remyelination; screening.

Figure 1

Experimental set up and cell culture characterization. (a) Illustration of the experimental timeline showing the sacrifice of the mice, two-week growth of the oligospheres, and plating on the nanofibers. (b) Schematic drawing of the experimental set-up of the 384-nanofiber plates. (c–l) Representative images of the different stainings. (c) The cells proliferated in clusters growing along the nanofibers. (d) Most of the nuclei (stained with DAPI) colocalized with the oligodendrocyte lineage marker OLIG2 (arrows, well treated with 20 ng/mL platelet-derived growth factor subunit A dimer (PDGF–AA) and fibroblast growth factor 2 (FGF2). (e) Note, some nuclei showed a dense and sometimes fragmented morphology and were not positive for oligodendrocyte lineage factor 2 (OLIG2) (arrowheads, (d and e)). This was more pronounced in the wells with less PDGF–AA and FGF2 and more bone morphogenetic protein 2 (BMP2) (well treated with 0.625 ng/mL PDGF–AA and FGF2). (f) Dense cell clusters were not eligible for accurate counting of the nuclei. (g, h, and k) PDGFRa and MBP were mainly expressed within the clusters, while (i and j) the larger MOG positive cells were usually situated in proximity but not within the clusters. (l) Possible contamination with glial fibrillary acidic protein (GFAP) positive astrocytes was very rare, with most wells showing no staining for GFAP.

Figure 2

Oligodendrocyte progenitor cells (OPC) cell density after 14 days of multifactor treatment. (a) Heatmap showing the percent of area positive for DAPI, as a measure for OPC cell density, dependent on the platelet-derived growth factor subunit A dimer (PDGF–AA) and fibroblast growth factor 2 (FGF2) treatment. (b) Representative microscopy images showing the DAPI signal upon different PDGF–AA and FGF2 treatments. (c) Heatmap showing the percent of area positive for DAPI dependent on the PDGF–AA/FGF2 and bone morphogenetic protein 2 (BMP2) treatment. (d) Representative microscopy images of PDGF–AA and FGF2 concentrated at 20 ng/mL and with BMP2 levels of 0–100 ng/mL. (e) Heatmap showing the percent of area positive for DAPI dependent on the PDGF–AA/FGF2 and BMP4 treatment. (f) Representative microscopy images of PDGF–AA and FGF2 concentrated at 20 ng/mL and with BMP4 levels of 0–100 ng/mL.

Figure 3

Early oligodendrocyte progenitor cells (OPC) differentiation after 14 days of multifactor treatment. (a) Heatmap showing the percent of area positive for platelet-derived growth factor alpha (PDGFRa), as a measure for early OPC differentiation, dependent on the platelet-derived growth factor subunit A dimer (PDGF–AA) and fibroblast growth factor 2 (FGF2) treatment. (b) Representative microscopy images showing the PDGFRa expression upon different PDGF–AA and FGF2 treatments. (c) Heatmap showing the PDGFRa expression in dependency of the PDGF–AA/FGF2 and bone morphogenetic protein 2 (BMP2) treatment. (d) Representative microscopy images of PDGF–AA and FGF2 concentrated at 20 ng/mL and with BMP2 levels of 0–100 ng/mL. (e) Heatmap showing the PDGFRa expression in dependency of the PDGF–AA/FGF2 and BMP4 treatment. (f) Representative microscopy images of PDGF and FGF2 concentrated at 20 ng/mL and with BMP4 levels of 0–100 ng/mL.

Figure 4

Late oligodendrocyte progenitor cells (OPC) differentiation after 14 days of multifactor treatment. (a) Heatmap showing the influence of platelet-derived growth factor subunit A dimer (PDGF–AA) and fibroblast growth factor 2 (FGF2) on late differentiation measured as percent of area positive for myelin basic protein (MBP). (b). Representative microscopy images showing the MBP expression. (c) Heatmap showing the influence of bone morphogenetic protein 2 (BMP2) on MBP expression. (d) Representative microscopy images of PDGF–AA and FGF2 concentrated at 20 ng/mL and with BMP2 levels of 0–100 ng/mL. (e) Heatmap showing the influence of BMP4 on MBP expression. (f) Representative microscopy images of PDGF–AA and FGF2 concentrated at 20 ng/mL and with BMP4 levels of 0–100 ng/mL.