Towards a 3D spheroid system for modelling leukodystrophies 113 4 RESULTS OL spheroids develop white matter-like cell types capable of myelination To investigate whether OL spheroids effectively model white matter-like cell development, we generated 3D spheroids from induced pluripotent stem cells (iPSCs) derived from five healthy donor (HD) lines and eight leukodystrophy (LD) lines of which three globoid leukodystrophy (GLD), two 4H leukodystrophy and three Canavan disease lines (CD). Spheroids were cultured for 150 days using a previously published protocol (Marton et al., 2019), at a centralized facility to ensure consistency across experimental conditions (Fig. 1A). Growth curve analysis of spheroid diameter revealed steady growth during the first 50 days of culture, followed by a plateau phase (Fig. 1B). While overall growth dynamics were comparable between HD and LD spheroids, subtle differences in the growth plateau phase were observed among specific lines. Immunofluorescence (IF) staining confirmed the presence of key white matter cell types, including neurons (neurofilament-positive), astrocytes (GFAP-positive), and oligodendrocytes (MBP-positive) in both HD and LD spheroids (Fig. 1C, 1E, 1I). Interestingly, NF+ area decreases between day 100 and day 150 (Fig. 1C, P = 0.0002). While marker for inhibitory neurons (GABA) increased (Fig. 1D, P = 0.0488). We further analysed oligodendrocyte lineage progression by quantifying Olig2+ and MBP+ area. Olig2+ area was consistent across all lines and differentiation stages, with no significant differences observed between HD and LD lines (Fig. 1F). Myelinating oligodendrocytes, marked by MBP positive area, showed a significant difference by time (Fig. 1G, P = 0.0085). Specifically, an increase in positive area from day 50 to day 100 and from day 50 to day 150 in both HD and LD spheroids (Fig. 1G, P = 0.0160 and P = 0.0127). Electron microscopy further confirmed the presence of compacted myelin structures surrounding neuronal axons (Fig. 1H), demonstrating that OL spheroids are capable of generating functional myelinating cells. Lastly, area of astrocytic marker expression (GFAP) showed significant difference on time (P < 0.0001), disease status (P = 0.0014) and the interaction of time and disease status (P = 0.0012). Post-hoc tests showed significant increase in GFAP positive area for both control (P = 0.0256) and LDs (P < 0.0001) between day 100 and 150 as well as significant higher GFAP expression in LDs compared to control at day 150 (Fig. 1I, P < 0.0001). Overall, these findings demonstrate that OL spheroids can model key aspects of WM cell development and myelination.
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