Fig. 2

Neurons and glia are highly functional in hiNS mimicking early brain physiology. A Immunofluorescence of MAP2, GFAP and IBA1 in a mature neurosphere at day in vitro (DIV) 60. B Redox imaging of hiNS(−) following transduction with AAV.php.eb.hSyn-roGFP1. A redox ratio is quantified by dividing green emission acquired at 405 nm by green emission acquired at 488 nm. As controls, DTT (5 mM) was used to fully reduce neurospheres and diamide (2 mM) to fully oxidize the tissue. Resting redox states of hiNS(−) (N = 6 hiNS) were only slightly higher than that of fully reduced DTT -treated hiNS (N = 5 hiNS) indicating healthy neurons throughout the 3D tissue. Full oxidation by diamide (N = 4 hiNS) indicates maximum roGFP ratios. Statistical testing performed using one-way ANOVA followed by Holm-Sidak post-hoc test. C Functional intracellular neuronal calcium (AAV.php.eb.-hSyn-GCaMP6f or AAV2/9.-hSyn-jRGECO1) and astrocytic calcium (AAV2/9-gfaABC1D-GCaMP8 s) imaging shows highly synchronized and simultaneous calcium activity in both neurons and astrocytes in hiNS(−). D Elevated extracellular glutamate (detected by hSyn-iGluSnFr expression) coincides with calcium wave activity in hiNS (detected by hSyn-jRECO1 expression). E Spontaneous neuronal calcium activity traverses through hiNS in a wave-like manner. F Pharmacological block of calcium waves by TTX (1 µM) (N = 6 hiNS), AP5 (100 µM)/CNQX (20 µM) (N = 6 hiNS) and GABA (300 µM) (N = 6 hiNS). Application of picrotoxin (100 µM) (N = 4 hiNS) resulted in a significant increase in calcium wave frequencies. Statistical testing performed using paired student t-tests for each drug application vs its baseline. G: Infiltration of hiMG in hiNS(+) results in a significant reduction in calcium wave frequencies with longer wave durations (hiNS(−): N = 10; hiNS(+): N = 12). Statistical testing performed using unpaired student t-tests. H Long-term confocal imaging of transduced hiNS(+) (N = 4) with AAV.php.eb-hSyn-mCherry (red) and AAV.php.eb-gfaABC1D-EGFP (green) and tomato lectin DyLight 649 stained hiMG. hiMG infiltration into the tissue was quantified within the first 24 h post-transfer (see yellow arrows and areas). I hiMG within hiNS display surveillance behaviour with rapidly moving processes and a more stationary soma. Tissue damage induced by a laser lesion leads to rapid process outgrowth of microglia towards the lesion. J Electrophysiological properties of hiMG 7–10 days post infiltration (N = 9 cells)