Chapter 5 166 ◀ Figure 4: Axonal segment number increased in presence of ALD microglia-like compared to control microglia-like cell co-cultures (A) Immunofluorescent image of neuronal co-cultures showing nuclei (DAPI, blue), microglia (Iba1, green), and reactive microglia marker CD68 (magenta). (B) Quantification of CD68+ reactive microglia-like cells in ALD and control co-cultures. High-content analysis of nuclear debris (C), mature NeuN+ neurons (D), axonal (NF+) segments (E), and dendritic (MAP2+) segments (F) revealed significant differences in axonal segments between ALD and control co-cultures (p = 0.032, Fisher’s method). (G, H) Detailed post-hoc analysis of axonal and dendritic morphology parameters. (I) Synaptic density (Synaptophysin+ puncta per dendritic area) comparison between ALD and control microglia-like cell co-cultures. Larger circles denote control (gray) and ALD (green) group means and smaller symbols indicating line observations per line on different time points (circles: 8 weeks post plating of neurons (WPP), triangles: 10 WPP, squares: 12 WPP). Statistical analysis was performed on line means across time points. (J) Comparison of mean active channels over time (7–12 WPP) in ALD (green) and control (gray) microglia-like cell co-cultures. Scale bar = 50 µm. *P < 0.05 4H microglia-like cells suitable for co-culture In addition, we generated hPSC-derived microglia from five 4H patient lines (three with POLR3A and two with POLR3B mutations) and three control lines and co-cultured them on one neuron background that consisted of a mixture of 4 control lines in equal parts. Visualization of Iba1 positive cells shows that 4H microglia also remain present during the entire length of the co-culture. Similarly, to ALD microglia-like cells, there are no indications for increased intrinsic stress in 4H microglia-like cells as determined by their numbers and roundness (Fisher’s method, p = 0.570, Supplementary Fig. 7A-7B), even though one of the 4H lines had few microglia-like cells in the co-cultures, their morphology was similar to the other lines (Supplementary Fig. 7A-F, Supplementary Table VII). We conclude that hPSCderived microglia of 4H are suitable for co-culture and do not show signs of intrinsic impairment due to 4H. 4H microglia-like cells shape the neuronal circuit in a similar way as control microglia Next, we wanted to investigate if the modulations made by 4H microglia-like cells are similar to the changes made by control cells. Again, we investigated nuclear debris, neuron numbers and the axonal and dendritic protrusions. We did not identify differences in cultures with control and 4H microglia-like cells (Fisher’s method, p = 0.836). Since there were no changes found in co-culture morphology parameters we did not do any further post-hoc testing. Also the neuronal activity, measured by the amount of active channels, did not show any difference between co-cultures with control and 4H microglia-like cells (Fig. 5E, T-test, p = 0.532).
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