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Late-onset Alzheimer’s disease (AD) has become the paradigm of a non-mendelian complex neurodegenerative disease, for which a major genetic determinant is known, the APOE locus. A rare APOE variant named Christchurch (APOEch) yielding a missense mutation from Arginine to Serine at amino acid 136, has been suggested to exert a protective effect in an individual carrying the most penetrant form of Familial AD (Paisa mutation in PSEN1 gene, E280A). We describe here a new set of induced pluripotent stem cell (iPSC) lines, where the Christchurch mutation (Ch) has been introduced by gene editing into the APOE locus of three isogenic iPSC lines carrying the more common APOE variants (APOE 2/2, APOE 3/3, and an APOE 4/4) in homozygosity. Brain cells derived from these iPSC lines will enable a better understanding of APOE biology in general and facilitate the study of how the Christchurch variant affects the function of each APOE genotype. This set of iPSC lines are globally available via the European Bank of iPSCs, EBiSC.org.

Abstract Huntington’s disease (HD) is caused by a CAG trinucleotide repeat expansion in the first exon of the huntingtin (HTT) gene coding for the huntingtin (HTT) protein. The misfolding and consequential aggregation of CAG-expanded mutant HTT (mHTT) underpin HD pathology. Our interest in the life cycle of HTT led us to consider the development of high-affinity small-molecule binders of HTT oligomerized/amyloid-containing species that could serve as either cellular and in vivo imaging tools or potential therapeutic agents. We recently reported the development of PET tracers CHDI-180 and CHDI-626 as suitable for imaging mHTT aggregates, and here we present an in-depth pharmacological investigation of their binding characteristics. We have implemented an array of in vitro and ex vivo radiometric binding assays using recombinant HTT, brain homogenate-derived HTT aggregates, and brain sections from mouse HD models and humans post-mortem to investigate binding affinities and selectivity against other pathological proteins from indications such as Alzheimer’s disease and spinocerebellar ataxia 1. Radioligand binding assays and autoradiography studies using brain homogenates and tissue sections from HD mouse models showed that CHDI-180 and CHDI-626 specifically bind mHTT aggregates that accumulate with age and disease progression. Finally, we characterized CHDI-180 and CHDI-626 regarding their off-target selectivity and binding affinity to beta amyloid plaques in brain sections and homogenates from Alzheimer’s disease patients.

Neurogenin 2 encodes a neural-specific transcription factor (NGN2) able to drive neuronal fate on somatic and stem cells. NGN2 is expressed in neural progenitors within the developing central and peripheral nervous systems. Overexpression of NGN2 in human induced pluripotent stem cells (hiPSCs) or human embryonic stem cells has been shown to efficiently trigger conversion to neurons. Here we describe two gene-edited hiPSC lines harbouring a doxycycline (DOX)-inducible cassette in the AAVS1 locus driving expression of NGN2 (BIONi010-C-13) or NGN2-T2A-GFP (BIONi010-C-15). By introducing NGN2-expressing cassette, we reduce variability associated with conventional over-expression methods such as viral transduction, making these lines amenable for scale-up production and screening processes. DOX-treated hiPSCs convert to neural phenotype within one week and display the expression of structural neuronal markers such as Beta-III tubulin and tau. We performed functional characterization of NGN2-neurons co-cultured with hiPSC-derived astrocytes in a “fully-humanized” set up. Passive properties of NGN2-neurons were indistinguishable from mouse primary cells while displaying variable activity in extracellular recordings performed in multi-electrode arrays (MEAs). We demonstrate that hiPSC-derived astrocytes and neurons can be co-cultured and display functional properties comparable to the gold standard used in electrophysiology. Both lines are globally available via EBiSC repository at https://cells.ebisc.org/.

Summary: Polymorphism in the microglial receptor CD33 gene has been linked to late-onset Alzheimer disease (AD), and reduced expression of the CD33 sialic acid-binding domain confers protection. Thus, CD33 inhibition might be an effective therapy against disease progression. Progress toward discovery of selective CD33 inhibitors has been hampered by the absence of an atomic resolution structure. We report here the crystal structures of CD33 alone and bound to a subtype-selective sialic acid mimetic called P22 and use them to identify key binding residues by site-directed mutagenesis and binding assays to reveal the molecular basis for its selectivity toward sialylated glycoproteins and glycolipids. We show that P22, when presented on microparticles, increases uptake of the toxic AD peptide, amyloid-β (Aβ), into microglial cells. Thus, the sialic acid-binding site on CD33 is a promising pharmacophore for developing therapeutics that promote clearance of the Aβ peptide that is thought to cause AD. : Molecular Structure; Neuroscience; Molecular Neuroscience; Components of the Immune System; Protein Structure Aspects Subject Areas: Molecular Structure, Neuroscience, Molecular Neuroscience, Components of the Immune System, Protein Structure Aspects

APOE genotype is the strongest genetic risk factor for Alzheimer’s Disease (AD). The low degree of homology between mouse and human APOE is a concerning issue in preclinical models currently used to study the role of this gene in AD pathophysiology. A key objective of ADAPTED (Alzheimer's Disease Apolipoprotein Pathology for Treatment Elucidation and Development) project was to generate in vitro models that better recapitulate human APOE biology. We describe a new set of induced pluripotent stem cells (iPSC) lines carrying common APOE variants (Ɛ2, Ɛ3, and Ɛ3/Ɛ4) and a knock-out isogenic to the parental APOE Ɛ4/Ɛ4 line (UKBi011-A).

Neurog2 is the gene encoding the neuronal transcription factor NGN2, which can convert stem cells into functional neurons in a fast and efficient way. Here we report the generation of two iPS cell lines, where DOX inducible constructs of neurog2 either without or with T2A-eGFP were inserted into the safe-site locus AAVS1. These iPS cell lines, BIONi010-C-13 and BIONi010-C-15, respectively, stay pluripotent without DOX but differentiate to (GFP positive) neurons when DOX is added without the need of differentiation factors.

The European Bank for induced Pluripotent Stem Cells (EBiSC), a non-profit repository for storage, banking, Quality Control (QC) and subsequent distribution of research-grade human induced Pluripotent Stem Cell (iPSC) lines, has centralised iPSC lines generated internationally across >35 disease areas and made them available to users via the EBiSC Catalogue, for research use (cells.ebisc.org/). Comprehensive datasets are accessible prior to purchase detailing the disease background of the original tissue sample, background of iPSC reprogramming and cell line characterisation data. EBiSC also performs robust QC screening to ensure supply of reliable, well-characterised iPSC lines, compliant with ISO9001:2015 principles. Whole Genome Sequencing data for specific iPSC lines can be downloaded from the European Genome Archive, subject to application to the EBiSC Data Access Committee. The EBiSC Access and Use Agreement, required to be completed prior to shipping, can be downloaded from the website along with specific Cell Line Information Packs; together these documents clarify how EBiSC lines can be used for research and detail any specific Third Party Obligations and/or restrictions for use which may apply. A protocol for how to culture and monitor iPSC lines including implementation of routine cell line screening is also available. A second project phase will continue collecting iPSC lines generated internationally, provide iPSC derived differentiated products using improved automation strategies for upscaling and develop the current services provided by EBiSC, including iPSC reprogramming, gene-editing and characterisation.

Human pluripotent stem cells (hPSC) represent a unique opportunity to study fundamental biological processes in a human- and cell-specific setting. Its translational potential and the impact on human health makes this technology revolutionary. The possibility to generate stem cells from almost any somatic cell, and their capacity to be differentiated in virtually all cells of the body has been demonstrated extensively during the last decade of research. Target-centric as well as phenotypic screenings using differentiated cells have become a reality, while the use of these cells for “disease modelling” is still challenging due to the paucity of relevant and reproducible phenotypes. The combination of hPSCs with gene editing technologies aiming to e.g. reduce immunogenic response has enabled promising clinical trials that will eventually demonstrate their therapeutic potential in tissue regeneration and cancer treatment. Maximizing the therapeutic applications of hPSCs requires systematic data comparison, consensus between scientists and health care professionals, as well as a close collaboration between research labs, clinics, and regulators. The goal of this review is to provide a comprehensive outlook of the current use of hPSCs in drug development and regenerative medicine for the treatment of central nervous system (CNS) disorders. In the first part, we analyse how hPSCs are currently used in drug development and discuss their use in challenging paradigms such as neurodegeneration. In the second part we review the status of hPSCs in regenerative medicine. Finally, key challenges and pitfalls of the technology will be discussed, and strategies proposed to improve hPSC research and to benefit patients across different therapeutic areas.

Summary: Tauopathies such as frontotemporal dementia (FTD) remain incurable to date, partially due to the lack of translational in vitro disease models. The MAPT gene, encoding the microtubule-associated protein tau, has been shown to play an important role in FTD pathogenesis. Therefore, we used zinc finger nucleases to introduce two MAPT mutations into healthy donor induced pluripotent stem cells (iPSCs). The IVS10+16 mutation increases the expression of 4R tau, while the P301S mutation is pro-aggregant. Whole-transcriptome analysis of MAPT IVS10+16 neurons reveals neuronal subtype differences, reduced neural progenitor proliferation potential, and aberrant WNT/SHH signaling. Notably, these neurodevelopmental phenotypes could be recapitulated in neurons from patients carrying the MAPT IVS10+16 mutation. Moreover, the additional pro-aggregant P301S mutation revealed additional phenotypes, such as an increased calcium burst frequency, reduced lysosomal acidity, tau oligomerization, and neurodegeneration. This series of iPSCs could serve as a platform to unravel a potential link between pathogenic 4R tau and FTD. : In this article, Verheyen and colleagues introduced 2 MAPT mutations (IVS10+16/P301S) into healthy donor iPSCs. Characterization of MAPT IVS10+16 neurons revealed neurodevelopmental neuron subtype differences, reduced neural progenitor proliferation, and aberrant WNT/SHH signaling, while the additional pro-aggregant P301S mutation revealed phenotypes more related to neurodegeneration. These iPSCs could help to unravel a potential link between pathogenic 4R tau and FTD. Keywords: FTDP-17, MAPT, iPSC, ZFN, disease modelling

A fast track “Hot Start” process was implemented to launch the European Bank for Induced Pluripotent Stem Cells (EBiSC) to provide early release of a range of established control and disease linked human induced pluripotent stem cell (hiPSC) lines. Established practice amongst consortium members was surveyed to arrive at harmonised and publically accessible Standard Operations Procedures (SOPs) for tissue procurement, bio-sample tracking, iPSC expansion, cryopreservation, qualification and distribution to the research community. These were implemented to create a quality managed foundational collection of lines and associated data made available for distribution. Here we report on the successful outcome of this experience and work flow for banking and facilitating access to an otherwise disparate European resource, with lessons to benefit the international research community. eTOC: The report focuses on the EBiSC experience of rapidly establishing an operational capacity to procure, bank and distribute a foundational collection of established hiPSC lines. It validates the feasibility and defines the challenges of harnessing and integrating the capability and productivity of centres across Europe using commonly available resources currently in the field.

Alzheimer's disease (AD) is the most frequent neurodegenerative disease amongst the elderly. The SNPs rs429358 and rs7412 in the APOE gene are the most common risk factor for sporadic AD, and there are three different alleles commonly referred to as APOE-ε2, APOE-ε3 and APOE-ε4. Induced pluripotent stem cells (iPSCs) hold great promise to model AD as such cells can be differentiated in vitro to the required cell type. Here we report the use of CRISPR/Cas9 technology employed on iPSCs from a healthy individual with an APOE-ε3/ε4 genotype to obtain isogenic APOE-ε2/ε2, APOE-ε3/ε3, APOE-ε4/ε4 lines as well as an APOE-knock-out line.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin gene. Major pathological hallmarks of HD include inclusions of mutant huntingtin (mHTT) protein, loss of neurons predominantly in the caudate nucleus, and atrophy of multiple brain regions. However, the early sequence of histological events that manifest in region- and cell-specific manner has not been well characterized. Here we use a high-content histological approach to precisely monitor changes in HTT expression and characterize deposition dynamics of mHTT protein inclusion bodies in the recently characterized zQ175 knock-in mouse line. We carried out an automated multi-parameter quantitative analysis of individual cortical and striatal cells in tissue slices from mice aged 2-12 months and confirmed biochemical reports of an age-associated increase in mHTT inclusions in this model. We also found distinct regional and subregional dynamics for inclusion number, size and distribution with subcellular resolution. We used viral-mediated suppression of total HTT in the striatum of zQ175 mice as an example of a therapeutically-relevant but heterogeneously transducing strategy to demonstrate successful application of this platform to quantitatively assess target engagement and outcome on a cellular basis.