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. 2022 Sep 15;9:1006497. doi: 10.3389/fmolb.2022.1006497

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Copyright © 2022 Scholz, Lewis, Saulich, Endres, Boehmerle and Huehnchen.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Characterization of mature brain organoids. (A–H) Immunohistochemistry analyses of important cell type markers in iPSC derived human brain organoids show the presence of neuronal progenitor cells (NPCs) by expression of Sox2 (A) and Nestin (B), mature neuronal cells by NeuN (C) and MAP2 (D), astrocytes by Sox9 (E) and GFAP (F) at day 64, as well as oligodendrocytes by Olig2 (G) and MBP (H) at day 85 of the differentiation protocol. (I) Western blot analyses of cell lysates from d10 (NPCs), d36 (early organoids) and d58 (late organoids), demonstrate a decrease of NPC markers (left panel), and an increase of mature neuron as well as astrocyte markers (middle panel and right panel, respectively) during the time of the differentiation protocol. (J,K) Stimulation of day 86 organoids with 100 µM glutamate after 20 s of baseline measurement and analyzation of three regions of interest (ROI) in live calcium imaging (J) demonstrated a depth dependent response, expressed as change in fluorescent signal (ΔF/F) (K). Scale bars represent 50 µm.