Chapter 5 164 Since microglia health is suspected to be influenced by the ALD mutations, we first confirmed the sustained presence of microglia-like cells of ALD throughout the experiment using immunofluorescence (Fig. 4A). After establishing the presence, we determined if there are indications for increased intrinsic stress in ALD microglia. We defined increased stress by decreased numbers and changes in morphology, specifically increased roundness. We did not find evidence for changed numbers or roundness in ALD microglia-like cells compared to control microglia-like cells (Supplementary Fig. 6A-B, p = 0.909, Fisher’s method). Also, other morphological parameters were similar between ALD and control microglia-like cells (Supplementary Fig. 6C-F, Supplementary Table VI). Additional investigation about the reactive state of the cells using CD68+, showed no difference in reactivity between ALD and control microglia-like cells (Fig. 4A-B, T-test, p = 0.616). Together, we show that hPSC-derived microglia of ALD remain in co-culture and there is no evidence for increased death or reactivity caused by ALD. Axons are differentially affected by ALD microglia-like cells Now that we established that ALD microglia-like cells can be used in the co-culture model. We wanted to investigate the effect of the ALD-microglia like cells on the neurons. We hypothesized that ALD microglia-like cells might be hampered in their function compared to control microglia-like cells. To address this, we again investigated nuclear debris, neuron count and dendrite and axonal protrusions. The combination of these four parameters was indeed significantly different between co-cultures with control and ALD microglia-like cells (Fisher’s method p = 0.032). Additional post-hoc testing shows that this alteration is caused by increased axonal segments (p = 0.012). To elaborate on the change in axonal segments, we did additional post-hoc analysis we only find a trend of reduced axon segment length in cultures with ALD microglia (Fig. 4G, uncorrected p = 0.053). Since previous experiments showed a large effect of microglia-like cells on dendrites and synapses, we investigated if ALD microglia-like cells affect synapse density differential to control cells, we did not find evidence for this (Fig. 4H and 4I). Lastly, we previously showed that microglia-like cells increase neuronal activity. Here, we determined that ALD microglia-like cells can alter neuronal activity in the same way as control microglia-like cells (Fig. 4J, p = 0.897). Combined, ALD microglia-like cells are largely affecting the neurons similar to control microglia-like cells. However, neuron axonal morphology measures are changed in presence of ALD microglia-like cells, suggesting a possible impairment in ALD microglia which requires further research.
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