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Cerebral organoids are a promising model to study human brain function and disease, though the high inter-organoid variability of the mini-brains is still challenging. To overcome this limitation, we introduce the method of labeled mixed organoids generated from two different human induced pluripotent stem cell (hiPSC) lines, which enables the identification of cells from different origin within a single organoid. The method combines a gene editing workflow and subsequent organoid differentiation and offers a unique tool to study gene function in a complex human 3D tissue-like model. Using a CRISPR/Cas9 gene editing approach, different fluorescent proteins were fused to β-actin or lamin B1 in hiPSCs and subsequently used as a marker to identify each cell line. Mixtures of differently edited cells were seeded to induce embryoid body formation and cerebral organoid differentiation. As a consequence, the development of the 3D tissue was detectable by live confocal fluorescence microscopy and immunofluorescence staining in fixed samples. Analysis of mixed organoids allowed the identification and examination of specifically labeled cells in the organoid that belong to each of the two hiPSC donor lines. We demonstrate that a direct comparison of the individual cells is possible by having the edited and the control (or the two differentially labeled) cells within the same organoid, and thus the mixed organoids overcome the inter-organoid inhomogeneity limitations. The approach aims to pave the way for the reliable analysis of human genetic disorders by the use of organoids and to fundamentally understand the molecular mechanisms underlying pathological conditions.

BackgroundThe amyloid precursor protein (APP) is a type I transmembrane protein with unknown physiological function. Amyloidogenic processing of APP causes the generation of different cleavage products amongst b-amyloid and the APP C-terminal (APP-CT) fragment.METHODS1D, cerebral organoid 3D cell culture and human hippocampal post-mortem tissue were used to study APP-CT signalling in combination with immunofluorescence, -precipitation, confocal and STED imaging.RESULTSThe current study demonstrates that APP-CT signals to the nucleus causing the generation of dynamic aggregates consisting of FE65 and the tumour suppressors p53 and PML. PML aggregates fuse over time depending on the APP nuclear signalling and complexes of the APP-CT/p53/PML and FE65 are present in the aged human post-mortem brain but not in cerebral organoids. Precise quantification revealed that AD brains show a significant loss of these nuclear aggregates in areas with high plaque load. The PML aggregates are co-localized to the herpes simplex virus in the human brain.CONCLUSIONSOur data show that APP-CT signalling recruits the nuclear PML body machinery and is of central relevance for neurodegeneration in AD with a potential function in viral defence.