Fig. 6: Functional characterization of hiPSC-astroglia.

A Top: Representative time projection image of a 2400 frame scan obtained by live-cell calcium imaging. Bottom: Representation of ROIs after segmentation, green ROIs are active cells. Scale bar equals 200 μm. B Representative time course of astrocytes response to addition of 2 mM CaCl₂ in the calcium-free buffer after 60 s. Black lines are non-active cells. Green lines are active cells. C Representative time course of astrocytes’ spontaneous CaCl₂ transients over a 20 min time period. D Averaged time courses of the Calcium response of each clone (left) and, CTRL and SCZ clones compared (right). E Percentage of astrocytes with a response to the addition of 2 mM CaCl₂ in the calcium-free buffer for each clone (left), and CTRL and SCZ clones compared (right). F Average amplitude of the response to the CaCl₂ stimulation of the active cells for each clone (left), and CTRL and SCZ clones compared (right). G Percentage of astrocytes with spontaneous CaCl₂ transients for each clone (left), and CTRL and SCZ clones compared (right). H Average amplitude of CaCl₂ transients for each clone (left), and CTRL and SCZ clones compared (right). I Glutamate uptake of resting iPSC astrocytes was assessed using a glutamate assay kit. Native AM cell medium (MC, ‘medium control’), and donor/clone-specific iPSC lines were used as controls (iPSC ctrl). Data of triplicate measurements of five independent donors per group are presented as mean ± SEM (in the case of MC, triplicate measurements were performed by sampling the same bottle of medium in which all cells were cultured). Asterisks represent significant differences relative to iPSC controls, and CTRL astrocytes versus SCZ astrocytes at p < 0.05. J General matrix metalloproteinase activity was measured at both baseline and following cell activation by 10 ng/ml IL-1β for 24 h. Samples from empty wells containing only AM medium were used as medium (negative) control. Fluorescence intensity was measured at E_x/E_m = 540/590 nm, and MMP activity is shown in relative fluorescence units (RFU). Data of triplicate measurements of five independent donors per group are presented as mean ± SEM. Asterisks represent significant differences at p-values <0.05.