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Culture Variabilities of Human iPSC-Derived Cerebral Organoids Are a Major Issue for the Modelling of Phenotypes Observed in Alzheimer’s Disease

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Abstract

Apolipoprotein E (APOE) is the most important susceptibility gene for late onset of Alzheimer’s disease (AD), with the presence of APOE-ε4 associated with increased risk of developing AD. Here, we reprogrammed human fibroblasts from individuals with different APOE-ε genotypes into induced pluripotent stem cells (iPSCs), and generated isogenic lines with different APOE profiles. Following characterisation of the newly established iPSC lines, we used an unguided/unpatterning differentiation method to generate six-month-old cerebral organoids from all iPSC lines to assess the suitability of this in vitro system to measure APOE, β amyloid, and Tau phosphorylation levels. We identified variabilities in the organoids’ cell composition between cell lines, and between batches of differentiation for each cell line. We observed more homogenous cerebral organoids, and similar levels of APOE, β amyloid, and Tau when using the CRISPR-edited APOE isogenic lines, with the exception of one site of Tau phosphorylation which was higher in the APOE-ε4/ε4 organoids. These data describe that pathological hallmarks of AD are observed in cerebral organoids, and that their variation is mainly independent of the APOE-ε status of the cells, but associated with the high variability of cerebral organoid differentiation. It demonstrates that the cell-line-to-cell-line and batch-to-batch variabilities need to be considered when using cerebral organoids.

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Data Availability

Cell lines are available upon request to the corresponding authors.

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Funding

We thank Sophie Chevalier (CERA), Vikrant Singh (University of Tasmania) and Vanta Jameson (Melbourne Cytometry Platform, Melbourne Brain Centre Node) for technical assistance. This work was supported by grants from the Yulgilbar Alzheimer’s Research Program, the DHB Foundation, the Brain Foundation, Dementia Australia, a National Health and Medical Research Council (NHMRC) Practitioner Fellowship (AWH), an NHMRC Senior Research Fellowship (AP, 1154389), the University of Melbourne and Operational Infrastructure Support from the Victorian Government.

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Contributions

D.H.: concept and design, experimental work, interpretation of data, financial support, writing of manuscript, final approval of the manuscript; L.A.R., M.D., L.G., H.H.L.: experimental work, interpretation of data, final approval of the manuscript; A.L.C.: provision of samples, final approval of the manuscript; A.W.H.: provision of biopsies, interpretation of data, final approval of the manuscript; A.P.: concept and design, interpretation of data, financial support, writing of manuscript, final approval of the manuscript.

Corresponding authors

Correspondence to Damián Hernández or Alice Pébay.

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Ethical Approval

All experimental work performed in this study was approved by the Human Research Ethics committees of the Royal Victorian Eye and Ear Hospital (11/1031H), University of Melbourne (1545394), University of Tasmania (H0014124) with the requirements of the National Health & Medical Research Council of Australia (NHMRC) and conformed with the Declaration of Helsinki [46].

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This article belongs to the Topical Collection: Special issue on Neurogenesis and Neurodegeneration: Basic Research and Clinic Applications

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Supplementary Information

Figure S1

Schematic representation of CRISPR/Cas9 gene editing sequencing of APOE4 to APOE3 genotype. A single SNP rs429358 located in the fourth exon of the APOE gene differentiates the APOE4 to the APOE3 by affecting the amino acid in the position 130 of the resulting protein (Arg-130 and Cys-130 respectively). The codon (CGC) for Arg-130 (R130) in APOE4 gene was genetically modified to codon (TGC) for Cyst-130 (C130) present in APOE3 gene with CRISPR/Cas9 and the addition of a single strand (ss) DNA donor (to enhance homology directed repair) (PNG 595 kb)

High resolution image (TIFF 807 kb)

Figure S2

Characterisation of TOB0121. (A) Generation of iPSC lines. Representative images of TOB0121 showing expression of the pluripotency markers TRA-1-60 and OCT4, with DAPI counterstained and merged. Negative IgG controls are also displayed. (B) Representative germ layer immunostaining of TOB0121. TOB0121 demonstrates pluripotency by positive staining for markers of each embryonic germ layer; endoderm (AFP), mesoderm (SMA) and ectoderm (NESTIN). (C) APOE genotyping. Sanger sequencing of rs429358 and rs7412 in TOB0121 confirms APOE4/4 status. (D) Copy Number Variation Analysis of TOB0121 in their original fibroblasts and iPSCs (p8). Each panel shows the B allele frequency (BAF) and the log R ratio (LRR). BAF at values others then 0, 0.5 or 1 indicate an abnormal copy number. Similarly, the LRR represents a logged ratio of “observed probe intensity to expected intensity”. A deviation from zero corresponds to a change in copy number (PNG 2973 kb)

High resolution image (TIFF 1219 kb)

Figure S3

Characterisation of MBE2968. (A) Generation of iPSC lines. Representative images of TOB2968 clone (c) 2 showing expression of the pluripotency markers TRA-1-60 and OCT4, with DAPI counterstained and merged. (B) Representative germ layer immunostaining of MBE2968. MBE2968 c2 demonstrates pluripotency by positive staining for markers of each embryonic germ layer; endoderm (AFP), mesoderm (SMA) and ectoderm (NESTIN). (C) APOE genotyping. Sanger sequencing of rs429358 and rs7412 in MBE2968 c1 and c2 confirms APOE3/3 status. Note that c1 genotype was already published in [23] (D) Copy Number Variation Analysis of MBE2968 in their original fibroblasts and iPSCs (p8). Each panel shows the B allele frequency (BAF) and the log R ratio (LRR). BAF at values others then 0, 0.5 or 1 indicate an abnormal copy number. Similarly, the LRR represents a logged ratio of “observed probe intensity to expected intensity”. A deviation from zero corresponds to a change in copy number (PNG 2525 kb)

High resolution image (TIFF 3152 kb)

Figure S4

Representative immunostaining of 6-month-old cerebral organoids. Tile (A, B) and single (A’, B′) images of immunostaining of sectioned TOB0002 cerebral organoids for MAP2 with PAX6 (green and red respectively, A, A’), and β-Tubulin III with GFAP (green and red respectively, B, B′), counterstained with DAPI (blue) and merged (A’, B′). Images are representative of organoids obtained with all cell lines. Scale bars: 200 μm (PNG 1977 kb)

High resolution image (TIFF 9936 kb)

Figure S5

Western blots, uncropped membranes. Uncropped membranes for PAX6, GFAP, β-tubulin and β-actin negative isotype controls. Note that membranes were stripped and reprobed one time in the following order: PAX6 followed β-actin; β-tubulin followed by β-actin; GFAP followed by β-actin; rabbit IgG isotype control followed by mouse IgG isotype control. Relates to Fig. 3A (PNG 1245 kb)

High resolution image (TIFF 2790 kb)

Figure S6

Western blots, uncropped membranes. Uncropped membranes for PAX6, GFAP, β-tubulin and β-actin. Note that membranes were stripped and reprobed one time in the following order: PAX6 followed by β-actin; β-tubulin followed by β-actin; GFAP followed by β-actin. Relates to Fig. 3b, e (PNG 807 kb)

High resolution image (TIFF 1867 kb)

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Hernández, D., Rooney, L.A., Daniszewski, M. et al. Culture Variabilities of Human iPSC-Derived Cerebral Organoids Are a Major Issue for the Modelling of Phenotypes Observed in Alzheimer’s Disease. Stem Cell Rev and Rep 18, 718–731 (2022). https://doi.org/10.1007/s12015-021-10147-5

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