Chapter 3 86 Supplementary Fig. 2H). The variation between the individual GLD lines was large, potentially pointing to line-specific changes. For example, the GLD KO line exhibited a consistent high percentage of anterograde transport. And the 4H hvs450 line initially displayed high rates of mitochondria moving in both directions, while at DIV7 there was more unidirectional movement. The distance and velocity of mitochondria transport was not significant different between control and leukodystrophies, though both parameters were lowest in the GLD KO line across both timepoints (Fig. 2I–J, Supplementary Fig. 2I), but these trends did not reach statistical significance. To determine if psychosine accumulation was more pronounced in the absence of astrocytes, we measured psychosine levels in the neuron mono-culture model. Contrary to our expectations, psychosine levels were not significantly elevated in the GLD lines under mono-culture conditions (Fig. 2J). Again, there are also no indication of other disturbances in glycosphingolipid metabolism (Supplementary Fig. 1D-F). In summary, these results display potential differences in mitochondrial motility, specifically the direction of movement, as a distinguishing feature between 4H and control neurons in mono-culture settings. While these models show potential for investigating neuron intrinsic defects and detection of leukodystrophy-related phenotypes, minimal psychosine accumulation in GLD cultures and variability across individual lines warrant refinement of experimental designs to draw solid conclusions. ▶ Figure 2: hiPSC-derived neuron mono-cultures as a more suitable model for leukodystrophy related neuronal defects. A) Schematic representation of mono-culture set-up. Quantification of B) Neurite length, C) Axon length, D) EB3 comet density and E) EB3 Comet Velocity and F) Mitochondrial (Mito.) motility. Line graphs connect the mean values for each cell line across timepoints while bar graphs summarize the mean for control, 4H and GLD with dots representing individual cell line means. Mitochondrial G) Direction, H) Distance, and I) Speed are displayed individually for mitochondria travelling retrograde, anterograde or both directions. Related bar graphs can be found in the supplement. J) Psychosine concentrations (pmol/gram) in iPSC and neuron mono-cultures at DIV7 from control (ctrl) or GLD cells. DIV = Days in vitro.
RkJQdWJsaXNoZXIy MTk4NDMw