Generation of a human induced pluripotent stem cell-based ... · Q3 Abstract Tauopathies are...

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Alzheimer’s & Dementia - (2018) 1-20

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Featured Article

Generation of a human induced pluripotent stem cell–based model fortauopathies combining three microtubule-associated protein tau

mutations which displays several phenotypes linked toneurodegeneration

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Juan Antonio Garc�ıa-Le�ona,*,1, Alfredo Cabrera-Socorrob,2, Kristel Eggermonta,2,Ann Swijsend,e, Joke Terryna,e, Raheem Fazala,d,e, FatemehArefeh Namia, Laura Ordov�asa,Ana Quilesa, Frederic Lluisa, Lutgarde Serneelsc, Keimpe Wierdad,e, Annerieke Sierksmac,

Mohamed Kreirb, Francisco Pestanab, Philip Van Dammed,e,f, Bart De Strooperc,g,Lieven Thorrezh, Andreas Ebnethb, Catherine M. Verfailliea,d,**

aDepartment of Development and Regeneration, Stem Cell Biology and Embryology, KU Leuven Stem Cell Institute, Leuven, BelgiumbJanssen Research & Development, a Division of Janssen Pharmaceutica N.V., Beerse, Belgium

cVIB Center for the Biology of Disease, Leuven, Belgium Center for Human Genetics, Universitaire ziekenhuizen and LIND, KU, Leuven, BelgiumdKU Leuven-Department of Neurosciences, Experimental Neurology and Leuven Research Institute for Neuroscience and Disease (LIND), Leuven, Belgium

eVIB Center for Brain and Disease Research, Laboratory of Neurobiology, Leuven, BelgiumfUniversity Hospitals Leuven, Department of Neurology, Leuven, Belgium

gInstitute of Neurology, University College London, London, UKhTissue Engineering Laboratory, Department of Development and Regeneration, KU Leuven, Campus Kulak Kortrijk, Kortrijk, Belgium

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Abstract Tauopathies are neurodegenerative diseases characterized by TAU protein–related pathology,
The authors have1Current address: D

Faculty of Sciences,

University of Malaga,

ter for Networked Bi

(CIBERNED), Madrid

https://doi.org/10.1016

1552-5260/� 2018 T

license (http://creative

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including frontotemporal dementia and Alzheimer’s disease among others. Mutant TAU animalmodels are available, but none of them faithfully recapitulates human pathology and are not suitablefor drug screening. To create a new in vitro tauopathy model, we generated a footprint-free tripleMAPT-mutant human induced pluripotent stem cell line (N279K, P301L, and E10116 mutations) us-ing clustered regularly interspaced short palindromic repeats-FokI and piggyBac transposase technol-ogy.Mutant neurons expressed pathogenic 4R and phosphorylated TAU, endogenously triggered TAUaggregation, and had increased electrophysiological activity. TAU-mutant cells presented deficienciesin neurite outgrowth, aberrant sequence of differentiation to cortical neurons, and a significant activa-tion of stress response pathways. RNA sequencing confirmed stress activation, demonstrated a shifttoward GABAergic identity, and an upregulation of neurodegenerative pathways. In summary, wegenerated a novel in vitro human induced pluripotent stem cell TAU-mutant model displaying neuro-degenerative disease phenotypes that could be used for disease modeling and drug screening.� 2018 The Authors. Published by Elsevier Inc. on behalf of the Alzheimer’s Association. This is anopen access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Keywords: Tauopathies; Frontotemporal dementia; Parkinsonism linked to chromosome 17; Progressive supranuclear palsy; 106 Alzheimer’s disease; CRISPR-Cas; Disease modeling; Drug screening; Neurodegeneration 107
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declared that no conflict of interest exists.

epartment of Cell Biology, Genetics and Physiology,

Research Biomedical Institute of Malaga (IBIMA),

Campus de Teatinos s/n, 29010 Malaga, Spain. Cen-

omedical Research on Neurodegenerative Diseases

, Spain.

2These authors contributed equally.

*Corresponding author. Tel.: ---; Fax: ---. Q2

**Corresponding author. Tel.: ---; Fax: ---.

E-mail addresses: [email protected] or juanantonio.garcialeon@

kuleuven.be (J.A.G-L.), [email protected] (C.M.V.)

/j.jalz.2018.05.007

he Authors. Published by Elsevier Inc. on behalf of the Alzheimer’s Association. This is an open access article under the CC BY-NC-ND

commons.org/licenses/by-nc-nd/4.0/).

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http://creativecommons.org/licenses/by-nc-nd/4.0/


mailto:[email protected]&/elink; or &elink;[email protected]

mailto:[email protected]



https://doi.org/10.1016/j.jalz.2018.05.007



J.A. Garc�ıa-Le�on et al. / Alzheimer’s & Dementia - (2018) 1-202

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1. Introduction

Tauopathies are a group of sporadic and familial neurode-generative disorders, which are characterized by filamentousaccumulations of hyperphosphorylated TAU proteins in neu-rons and glial cells [1]. They are classified into primary orsecondary tauopathies depending on whether TAU pathol-ogy is the major contributing factor to neurodegenerationor if it is associated with other pathologies. Primary tauopa-thies include Pick’s disease, progressive supranuclear palsy,corticobasal degeneration, argyrophilic grain disease, fron-totemporal dementia with parkinsonism linked to chromo-some 17 (FTDP-17), globular glial tauopathy, and others.On the other hand, secondary tauopathies represent a ratherheterogeneous group of disorders of quite diverse etiology,including Down’s syndrome, Lewy body disorders, and,the most prevalent and studied one, Alzheimer’s disease(AD). AD represents the most common cause of dementiain the elderly, clinically characterized by a progressionfrom episodic memory problems to a slow global declineof cognitive function that leaves patients with end-stageAD bedridden and dependent on custodial care, with deathoccurring on average 9 years after diagnosis. Histopatholog-ically, AD is distinguished by the presence of both plaques ofb-amyloid and neurofibrillary tangles composed of hyper-phosphorylated TAU species, with both pathological hall-marks playing a fundamental role in AD pathology [2].

TAU is a microtubule-stabilizing protein encoded by themicrotubule-associated protein TAU (MAPT) gene on chro-mosome 17q21.31 spanning 16 exons, with exons 2, 3, and10 being alternatively spliced. Differential splicing resultsin the expression of six different isoforms present in the adulthuman brain. TAU isoforms differ in the absence or presenceof one or two 29 amino acid inserts (0N, 1N, 2N) in theamino-terminal part (encoded by exons 2 and 3) and in thepresence of three or four highly conserved repetitivemicrotubule-binding domains in the C-terminal part (3R or4R, encoded by exons 9–12) [3]. In both human and mousefetal brain, only the shorter 3R isoforms are detected,whereas 4R isoforms arise during development, and bothisoforms become equally expressed in the adult brain [1].The developmental switch of incorporating exon 10 in theTAU protein results in the addition of microtubule-bindingrepeats, which may stabilize microtubules. Accordingly,expression of 3R isoforms in fetal neurons may support theirhigher plasticity, required for process formation and neuriteelongation, during neuronal development. Aberrant regula-tion of exon 10 splicing in the adult brain has been identifiedas a major cause of tauopathies, resulting in an imbalance of3R and 4R TAU isoforms [4].

More than 50 mutations in the MAPT gene have beendescribed so far [5]. MAPT mutations are typically associ-ated with FTDP-17 but have also been observed for Pick’sdisease, progressive supranuclear palsy, corticobasal degen-eration, and globular glial tauopathies. Phenotypes causedby MAPT mutations are heterogeneous, even within family

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relatives carrying the same mutation(s) [6]. Most mutationsare located in exons 9–12 encoding for the repeat regions andadjacent introns and affect either protein levels or the alter-native splicing of the pre-mRNA [6]. Several of these muta-tions cause a decrease in the affinity of TAU for microtubulesand a reduced ability to promote microtubule assembly.Other mutations affect exon 10 splicing capacity, resultingin a shift in the physiological ratio between 3R and 4R iso-forms [7]. In addition, some mutations facilitate TAU phos-phorylation, the main and most diverse post-translationalmodification of TAU, causing a significant heterogeneityof different TAUmolecules. Increased TAU phosphorylationis associated with its aggregation into neurofibrillary tanglesin AD and other tauopathies, likely affecting synaptic activ-ity, neuronal physiology, and underlying neurodegeneration[8].

In an attempt to reproduce the pathology present in ADand other tauopathies, numerous animal models have beengenerated based on the (over)expression of wild-type(WT) or mutant forms of human TAU. Depending on theTAU variant over-expressed and the genetic background ofthe animal, TAU aggregation and phosphorylation, neuronaland synaptic activity impairments, behavioral deficits, andmotor disturbances have been described [9]. With the adventof induced pluripotent stem cell (iPSC) technology, iPSCshave been generated from individuals carrying differentMAPT mutations and differentiated into neurons. Althoughdifferent aspects of tauopathy have been reported, findingsare heterogeneous, possibly due to the presence of differentMAPTmutations, in methods used and/or the phenotypic as-pects that were evaluated [10–13].

To generate a robust model for tauopathies also suitablefor drug discovery screenings, we generated a footprint-free triple MAPT-mutant human iPSC line that whendifferentiated to cortical neurons reproduces several neuro-degenerative aspects, including modified TAU isoformexpression, TAU aggregation, altered electrophysiologicalactivity, aberrant cortical neuron differentiation, and an acti-vation of pathways related to inflammation, oxidative stress,endoplasmic reticulum/unfolded protein response (ER/UPR), and apoptosis. RNAseq analysis of WT and MAPT-mutant cells confirmed these findings and further revealeda shift of differentiation into GABAergic neurons in themutant cells, as well as an increased expression of pathwayspreviously linked to neurodegeneration.

2. Results

2.1. Selection ofMAPTmutations and generation of tripleTAU-mutant iPSC lines

To generate a robust model reproducing different charac-teristics of tauopathies, we introduced three mutations(N279K, P301L, and E10116) in and next to exon 10 oftheMAPT gene in WT donor healthy-derived human iPSCs.The N279K and P301L mutations are among the most

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prevalent FTDP-17–linked mutations, causing early onsetand aggressive disease progression. These mutations arecharacterized by the presence of phosphorylated TAU spe-cies in both neurons and glia and an elevated prevalence of4R TAU isoforms [14]. The E10116 mutation is located inthe intron between exons 10 and 11, resulting in an alterationof a stem-loop structure affecting splicing of the pre-mRNA,and increasing incorporation of exon 10 in TAU, thusincreasing the expression of 4R TAU isoforms [15].Together, these three mutations account for up to 60% ofall FTDP-17 cases [16].

We used clustered regularly interspaced short palin-dromic repeats (CRISPR)-FokI nucleases to introduce themutations, as they are known to cause less off-target effectsthan the CRISPR-Cas9 system [17]. The donor plasmidused for homology-directed recombination (gently pro-vided by Dr. Kosuke Yusa, Wellcome Trust Sanger Insti-tute, UK [18]) included a positive (hygromycin resistance[HYGR]) and a negative (thymidine kinase [TK]) selectioncassette, with a 30 homology arm containing the threeselected point mutations (Fig. 1A). After HYG selectionand clonal expansion, the recombinant clones were evalu-ated for in situ integration (Fig. 1B and C) and presenceof the three selected mutations. To excise the HYG/TK se-lection cassette without any additional modification in thegenome, the piggyBac transposase system was used, by us-ing piggyBac inverted terminal repeats flanking the HYG/TK cassette. An endogenous TTAA site was present next tothe N279K mutation, allowing the employment of thisstrategy (Fig. 1A) [18]. Following transient expression ofthe piggyBac transposase, cells were cultured with 1-(2-deoxy-2-fluoro-1-D-arabinofuranosyl)-5-iodouracil (FIAU)to select for cells from which the TK-containing selectioncassette was removed [18] (see Fig. 1 for a scheme aboutthe generation of the mutant cells). This resulted in thegeneration of heterozygous triple MAPT-mutant hiPSCsdisplaying a pluripotent phenotype (Fig. 1G), which re-tained their capacity to differentiate into the three germinallayers on embryoid body formation (Fig. 1H). The arraycomparative genome hybridization analysis confirmedthat genome integrity was preserved after CRISPR-FokItreatment (Supplementary Fig. 1).

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2.2. Neuronal differentiation and TAU expression

To evaluate the effect of MAPT mutations, we differenti-ated both WT and mutant (triple TAU-mutant) iPSCs tocortical neurons for up to 200 days, using a protocol devel-oped by Shi et al. [19]. We first evaluated the expressionof MAPT isoforms by qRT-PCR based on the presence/absence of exon 10 (3R- vs. 4R-MAPT isoforms) during dif-ferentiation. We observed a significant alteration of the 3R/4R ratio in triple TAU-mutant neurons, as 4R-MAPT mRNAwas already highly expressed in mutant cells on day in vitro(DIV) 32 (neural progenitor stage, neural precursor [NPC]).Four repeat-MAPT RNA could only be detected from


DIV160 onward in WT neurons (Fig. 2A–F), consistentwith the notion that 4R-TAU expression is only found inmature neurons. The expression of 4R TAU protein wasalso confirmed by Western blotting (Fig. 2G).

On DIV65 and DIV94, we assessed the protein levels oftotal TAU and phosphorylated TAU isoforms by Westernblotting. As shown in Fig. 2H–J, more total and phosphory-lated TAU isoforms (determined by the use of the AT270 andAT8 antibodies) protein was present at both time points. Wealso assessed the presence of detergent-insoluble TAU iso-forms, finding little if any presence of insoluble TAU iso-forms within the mutant neurons (data not shown).

As expression of aberrant TAU isoforms has been re-ported to cause TAU disassembly and accumulation inneuronal soma [14], we assessed TAU distribution withinneuronal cells by visualization of total TAU by confocal mi-croscopy. As can be clearly observed (Fig. 2K), differentialpattern of TAU distribution was seen between WT andmutant neurons. In WT cells, TAU was homogeneouslydistributed along neuronal prolongations, whereas in mutantneurons, TAU was clearly more concentrated within the cellsoma, consistent with an abnormal physiology in the main-tenance and/or functioning of TAU in the mutant neurons.

2.3. TAU aggregation

We next explored the aggregation potential of TAU in thetriple mutant-TAU neurons by amplified luminescent prox-imity homogeneous assay (alpha-LISA technology). Theassay uses antibodies recognizing human TAU (hTAU10)and labeled with either a donor or acceptor bead. WhenTAU proteins are aggregated, the proximity of the donor-and acceptor-labeled antibodies leads to the emission oflight at 615 nm. The assay hence allows the quantitativedetection of aggregated TAU (hTAU10/hTAU10 signal) ina high-throughput-compatible setting [20].

As we previously reported [20], in WT neurons, no TAUaggregation can be observed unless the cells are transducedwith an adeno-associated viral vector encoding P301Lmutant TAU and are simultaneously seeded with K18 (ag-gregates of recombinant 4R TAU produced in vitro). K18triggers aggregation of overexpressed but not endogenouslyproduced TAU in control cells, which was used as a positivecontrol in our assays. Similarly, WT cells did not show an in-crease in hTAU10/hTAU10 signal when only seeded ortransduced. In contrast, we observed a twofold increase inhTAU10/hTAU10 signal in triple TAU-mutant neuronsfollowing either P301L transduction or K18 seeding sepa-rately (Fig. 2L and M). Interestingly, we quantified a 15-fold increase in hTAU10/hTAU10 signal when P301L wasoverexpressed in TAU-mutant neurons on DIV60, increasingup to 50-fold 10 days later (DIV70, Fig. 2L). We also eval-uated if aggregated TAU was also phosphorylated in mutantneurons by quantifying AT8 detection using AlphaLISA.The signal for AT81/phosphorylated TAU in untreated orseeded cells with K18 was significantly higher in mutant

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Fig. 1. Design and generation of the triple TAU-mutant hiPSC lines. (A) General overview of the experimental design and strategy used to generate the template

DNA including the double selection cassette and the introduction of the selected mutations into the genome through direct genome targeting mediated by

tailored CRISPR-FokI nucleases and homologous recombination (HR) between highly similar sequences. (B) Layout of the nucleofection (NF) with

CRISPR-FokI nucleases plus template DNA and HYG selection to generate iPSC clones harboring the GFP1 selection cassette. (C) Example of junction assay

(JA) PCRs to discriminate the presence or absence of the selection cassette in the MAPT gene in different clones. (D) Experimental design for excision of se-

lection cassette byNF of the piggyBac Transposase followed by FIAU negative selection. (E) JA PCRs demonstrating correct integration of the selection cassette

before and after the excision of the piggyBac Transposase; and random integration (RI) PCRs demonstrating absence of random cassette integration in the

genome. (F) Sequencing of theMAPT gene following genetic modification demonstrating presence of the three mutations in heterozygosity. (G)MAPT-mutant

hiPSCs express typical pluripotency markers as SOX2, SEEA4, NANOG, TRA1-81, OCT4, and TRA1-60. Hoechst 33258 (blue) was used as nuclear marker.

Scale bars: 50 mm. (H) Results of embryoid body formation and characterization using the score card assay (three germ layer differentiation and loss of plu-

ripotency marker gene expression). Abbreviations: bef., before; Tem., template; lad., ladder Q13.



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neurons (Fig. 2N), suggesting that TAU aggregates detectedare also hyperphosphorylated, similar to the neurofibrillarytangles found in AD affected brains [3]. Overall, thesestudies indicate that endogenously expressed triple mutant-TAU may be sufficient to seed for TAU aggregation in vitro.

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2.4. Triple TAU-mutant neuronal progeny displaysincreased electrophysiological activity

Patient iPSC-derived neurons with MAPT mutationsaffecting exon 10 splicing have been previously described todisplay a faster maturation phenotype as defined by their elec-trophysiological properties [21]. We hypothesized that thisphenotype would also be reproduced in the triple mutant-TAU iPSC progeny. We applied whole-cell patch-clamp oniPSC-derived neurons to record action potentials and voltage-gated currents on an early (DIV70) and a late (.DIV90) stageduring neuronal differentiation. At both time points, WT andTAU-mutant cells displayed a comparable percentage of cellsfiring spontaneous (Fig. 3D and F) and evoked (Fig. 3E andG) action potentials. Quantification of the number of sponta-neous (Fig. 3H and J) and evoked (Fig. 3I and K) action poten-tials revealed a similar firing frequency between WT andTAU-mutant cells on DIV70 and on .DIV90. TAU-mutantneurons displayed slightly decreased inward and outwardvoltage-gated current densities compared with WT neuronson DIV70 (P , .05; Fig. 3L and M), whereas no differenceswere observed at.DIV90 (Fig. 3N and O).

Basic electrophysiological membrane properties (capaci-tance and potential) were similar in WT and TAU-mutantcells on DIV70 and.DIV90, except that the membrane po-tential on .DIV90 was more depolarized in TAU-mutantthan that in WT cells (WT: 255 6 2 mV, mutants: 248 62 mV; P 5 .0172).

To exclude the intrinsic bias associated with single-cellpatch-clamping, wherein only a limited number of individ-ual cells are evaluated and without considering the overallneuronal network activity, we assessed electrophysiologicalproperties of the neurons using multielectrode arrays(MEAs). Co-culture of iPSC-derived neurons with murineprimary astrocytes (1:1 ratio) was used, as this hastens elec-trophysiological maturation of iPSC neurons [22]. MEAsprovide information on the electrical properties of the wholeneuronal population by measuring extracellular potentialsacross electrodes distributed throughout the culture wells.Using this experimental setup, we observed a significant in-crease in the number of spikes, bursts, and burst frequency intriple TAU-mutant neurons compared with WTs (on DIV60,Fig. 3P–S), suggesting an increase in the electric activity dueto the TAU mutations.

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2.5. Aberrant differentiation of TAU-mutant iPSCs tocortical neurons

We next assessed if presence of the three MAPT muta-tions affected the differentiation of iPSC toward cortical


neurons. We analyzed neurons at different time points forexpression of cortical layer markers both at the mRNA andprotein levels (Fig. 4). As shown in Fig. 4A–L, early duringdifferentiation (DIV32–45), mutant cells displayedincreased levels of early neuronal and deeper cortical layermarkers, including FOXG1, CTIP2, and BRN2 comparedwith WT cells, suggesting an accelerated maturation ofmutant cells. By contrast, mutant cells expressed lowerlevels of transcripts and/or proteins of markers for superfi-cial layer cortical neurons, such as SATB2, while theselevels progressively increased from DIV65 onward duringthe differentiation ofWT cells. A similar trend was observedfor TBR1 (Fig. 4C), which in addition to be a marker fordeep cortical layers, also plays a role in glutamatergic pro-jection and, therefore, in the development of cortical layers[23]. Differences in cell proliferation (EdU1 cells) wereobserved as well betweenWTandmutant cells, with a higherprevalence of EdU1 WT neurons at early time points(DIV40) but a greater number of EdU1TAU-mutant neuronslater (DIV68, Fig. 4M).

To further address the impact of the triple MAPT muta-tions on the neuronal progeny, we performed RNAseq anal-ysis on WTand mutant iPSC neuronal progeny harvested onDIV70 and DIV110. Unsupervised principal componentanalysis demonstrated that WT and triple TAU-mutant neu-rons, at both time points, clustered separately (Fig. 5A).1490 (DIV70) and 1868 (DIV110) genes were differentiallyexpressed between both genotypes (adjusted P-value , .05& log twofold change), 395 of which were shared for bothtime points (Fig. 5B). We next analyzed the different cellpopulations using a set of markers that define differentneuronal populations, developmental stages, and differentforebrain areas [24]. This demonstrated that, as expected,DIV70 and DIV110 progeny clustered together, with a cleardistinction between WT and mutant progeny at both timepoints (Fig. 5C). Further analysis also demonstrated thatexpression of typical glutamatergic and GABAergic markergenes was significantly different. Mutant cells expressedlower levels of glutamatergic markers and increased levelsof GABAergic markers compared withWT cells, differencesthat were more pronounced on DIV110 (Fig. 5D). This wasfurther substantiated by ingenuity pathway analysis, demon-strating significantly lower expression of glutamatergicsignaling pathway as well as genes involved in synapticlong-term potentiation (LTP) in TAU-mutant neurons(Fig. 5E and F).

As differences in the balance between excitatory andinhibitory transcripts were observed at the transcriptomelevel betweenWTand triple TAU-mutant cells, we immuno-stained DIV88 progeny with antibodies against the gluta-mate (excitatory) and GABA and glycine (inhibitory)neurotransmitter transporters, vGlut1 and vGat, respectively.Quantification was performed based on the intensity thresh-olds and normalized to the number of DAPI1 nuclei orthe expression of the neuronal marker b-3-tubulin(Supplementary Fig. 2A–F). The area and integrated density

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Fig. 2. Increased Q144RTAU expression and TAU aggregation in the triple-mutant TAU cortical neurons. (A–F) qRT-PCR analysis showing expression of 3R and

4R TAU isoforms during the neuronal differentiation of both WT and triple TAU-mutant cells. (A) Relative expression to glyceraldehyde 3-phosphate



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(product of the area by the intensity) of vGat-positive stain-ing related to the number of nuclei was higher in TAU-mutant cells than in WT cells (even if this did not reachstatistical significance, Supplementary Fig. 2B and C).Nevertheless, when we compared the area of vGat-positivestaining to the total area of b-3-tubulin, these differenceswere not obvious, with presence of 640% of positive areafor both vGlut and vGat markers referred to the b-3-tubulin positive area. This suggests that it may be necessaryto analyze many more markers for excitatory vs. inhibitoryneurons and that single marker analysis might not revealthe differences present between WT and mutant cultures.

We also evaluated the presence of GFAP1 cells in thecultures (Supplementary Fig. 2E–G). On DIV65,12.01 6 1.82% of the cells present in the culture expressedthe GFAP antigen, consistent with previous reports [19].Subtle differences were found in the percentage of GFAP1

cells between WT and mutant cells (WT: 9.05 6 2.29%vs. mutant: 14.97 6 1.61% of GFAP1 cells), but this didnot reach statistical significance. Notably, most of theseGFAP1 cells (82.19 6 5.23% in total) presented a bipolarelongated phenotype, which would resemble radial-glial–like cells, present in the developing brain [25].

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2.6. Mutant cells display a stress gene signature

Several reports indicated the presence of mitochondrialand ER/UPR stress in MAPT mutant iPSC-derived neuronsand in neurons from tauopathy mouse models and patients[26]. As we observed a progressive cell loss in mutantneuronal cultures compared with WT, we evaluated theRNAseq data to determine if different stress pathwayswere more highly expressed in triple TAU-mutant progenycompared with WT cells. Gene ontology pathway analysisrevealed increased expression of gene ontology terms asso-ciated with cellular stress as, for example, “response to inter-feron-g”, “positive regulation of reactive oxygen speciesmetabolic process”, “regulation of extrinsic apoptoticsignaling pathway”, “activation of signaling protein activityinvolved in unfolded protein response,” and “positive regula-

dehydrogenase (GAPDH) of 4RMAPT isoforms during the differentiation ofWT (b

expression ratio during differentiation of both cell types. (C) 4R MAPT isoform e

neurons. (D) 4R/3RMAPT isoform ratio expression levels during differentiation of

levels during differentiation of TAUmutant cells relative toWT neurons. (F) TotalM

WT neurons. Data are represented as mean 6 standard error of mean (SEM) of

analysis for total TAU expression inWTand triple TAUmutant neurons following i

of 4R TAU expression only in mutant cells (arrow). (H–J) Western blot analysis o

tibodies) of protein extracts from WT and mutant neuronal progeny on DIV 65 (H

sented as mean6 SEM of N5 3 independent experiments. (K) Immunocytochem

and mutant neurons on DIV65. Hoechst 33258 (blue) was used as nuclear mark

measured in both WT and triple TAU-mutant neuronal progeny in nontreated neu

cultivated in the presence of the K18 seed and transduced to overexpress the P301L

control. (I) hTAU10/hTAU10 alphaLISA values measured in both WT and triple T

DIV60 and DIV70, and alphaLISA levels measured on DIV70 in both cell types tra

values referred to phosphorylated TAU aggregation from WT and mutant cells see

licates, One-way analysis of variance. *P , .05, **P , .01, ****P , .0001 (Dunn

relative fluorescence units; A.U., arbitrary units.


tion of NFkB signaling”, in triple TAU-mutant cellscompared with WT progeny at both time points analyzed(DIV70 and 110, Supplementary Tables 1 and 2). Ingenuitypathway analysis performed on selected pathways alsodemonstrated increased expression of the genes involvedin these pathways in mutant cells, as exemplified by theNFkB signaling pathway, represented in Fig. 6Q.The increased activation of stress pathway genes withinthe TAU-mutant neurons was confirmed by qRT-PCR formarker genes of the oxidative stress, ER/UPR stress, and in-flammatory response pathways, on different time-pointsthroughout neuronal differentiation (Fig. 6A–M). This wasconfirmed at the protein level because the expression ofthe tumor necrosis factor receptor 1 (implicated in inflam-matory response) and CHOP (involved in ER/UPR) wassignificantly upregulated within the mutant neurons(Fig. 6N–P).

In addition, to substantiate the observation that tripleTAU-mutant progeny were progressively lost from cultureover time, we assessed the frequency of apoptotic cells inculture. We observed significantly more CASPASE31 neu-rons in the triple TAU-mutant iPSC progeny comparedwith WT (Fig. 6R and S). Moreover, apoptotic nuclei (char-acterized by a reduction of nuclear size and compaction ofgenomic material) were also frequently observed at thebeginning of neuronal maturation (wDIV40), but this onlyin triple TAU-mutant progeny (Fig. 6T and U). Thus, tripleTAU-mutant neuronal progeny, at least under these cultureconditions, suffered from oxidative, protein folding, and in-flammatory stress, which might underlie the increase forapoptosis.

2.7. Neurite outgrowth

TAU is a microtubule-stabilizing protein implicated inneurite formation, stabilization, and maintenance, as part ofits cytoskeletal plasticity functions [26]. Because the MAPTmutations altered the ratio between 3R- and 4R-TAU iso-forms (Fig. 2), we hypothesized that differences in neuriteformation and outgrowth might be present between WT and

lack) and triple TAU-mutant (Mut, gray) neurons. (B) 4R/3RMAPT isoform

xpression levels during differentiation of TAU-mutant cells relative to WT

TAUmutant cells relative to WT neurons. (E) 3RMAPT isoform expression

APTexpression levels during differentiation of TAUmutant cells relative to

N 5 3–4 independent experiments. *P , .05, **P , .01. (G) Western blot

ncubation with or without l-phosphatase (1/2), demonstrating the presence

f total TAU (HT7 antibody) and phosphorylated forms (AT270 and AT8 an-

) and DIV 94 (I), quantified based on intensity signal in (J). Data are repre-

istry showing cellular localization of TAU expression (HT7 antibody) in WT

er. Scale bar: 50 mm. (L) hTAU10/hTAU10 alphaLISA values on DIV60

rons, in neurons cultivated in the presence of the K18 seed and in neurons

mutant TAU. Cells that were seeded and transduced were used as a positive

AU-mutant transduced neurons without the presence of the K18 seeding on

nsduced and in the presence of the K18 seeds. (N) AT8/hTAU10 alphaLISA

ded in the absence or presence of the K18 seed. N 5 3–6 independent rep-

ett’s multiple comparison test vs. nonseeded control). Abbreviations: RFU,

� 19 July 2018 � 8:16 pm � ce

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Fig. 3. Altered electrophysiological activity of TAU-mutant iPSC-derived cortical neurons. (A) Representative recording (WT neuron on DIV70) of sponta-

neous action potentials. No current injection was applied. (B) Experimental current pulse step protocol (top panel) and representative traces (triple TAU-

mutant neuron at .DIV90) of membrane potential responses to current injections (lower panel). The membrane potential was clamped at approximately



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triple TAU-mutant neuronal progeny (Supplementary Fig. 3).Progeny from WT and TAU-mutant iPSCs on DIV40 wereplated and allowed to extend neurites for 3 days before fixa-tion.We used high-content imaging to assess cell morphologyand neurite frequency and length following stainingwith anti-TUJI antibodies.

Compared with WT iPSC progeny, significantly fewerTAU-mutant neurons displayed a multi-branching pheno-type, with most of TAU-mutant cells having a bi-polarmorphology (Supplementary Fig. 3H and I). We found analmost 50% reduction in the number of branching pointsin the mutant compared with WT cells (SupplementaryFig. 3B). We also found a reduction in the total area coveredby neurites, the total neurite length, and the length of all neu-rites per cell (Supplementary Fig. 3C–E) in mutantcompared with WT cells, even if the cell density was thesame in both cultures (Supplementary Fig. 3G). On the otherhand, the length of each neurite branch was slightlyincreased in mutant cells (Supplementary Fig. 3F).

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2.8. Confirmation of phenotypic features in a secondindependent triple TAU-mutant cell line

We evaluated if the findings obtained for the triplemutant-TAU neurons could be reproduced using a secondand independently generated genome-engineered line. Thisindependent mutant line reproduced the features displayedby the first mutant line, including increased expression of4R-TAU isoforms already on DIV32 (altering therefore the3R/4R ratio; Supplementary Fig. 4). Action potential pro-portions and frequencies obtained from the second indepen-dent mutant line on DIV70 were also similar to the actionpotential properties of the first mutant line (SupplementaryFig. 5A–F). The second independently generated tripleTAU-mutant line also showed altered cortical markerexpression (by qRT-PCR and immunostaining) similar tothe data from the first line (Supplementary Figs. 6 and 7);a significant increased expression of genes within the oxida-tive stress, ER/UPR and inflammatory pathways and ahigher prevalence of apoptotic cells (SupplementaryFig. 7); and aberrant neurite outgrowth (SupplementaryFig. 8).

265 mV between current pulses. The maximal amount of action potentials (red trac

Experimental voltage pulse step protocol (top panel) and representative traces (W

and outward currents. The amplitude of the peak inward current (red trace in gra

quantified at 130 mV during the time course indicated by the red dashed box. T

F) and evoked (E and G) action potentials is similar, on DIV70 (WT: n 5 30; mut

frequency of spontaneous (H and J) and evoked (I and K) action potentials is comp

mutant: n 5 8; evoked, WT: n 5 10, mutant: n 5 7) as well as on DIV90 (spontan

Inward and outward current densities are different between WT (n5 24) and TAU

similar on DIV90 (N and O; inward, WT: n5 20, Mut: n5 22; outward, WT: n5were sampled from four neuronal differentiations. Data are represented as median

Whitney test. *P , .05. (P–S) Electrophysiological activity measured by multi-el

astrocytes from DIV40. (P) Depicts the number of spikes (note that astrocytes cul

(.5 spikes per second) and burst frequency detected. (S) Depicts synchronicity.

**P , .01.


These results confirm that the phenotypic changes identi-fied in the mutant cells are specific for the MAPT mutationsintroduced and are not restricted to cell clone-specific ef-fects.

3. Discussion

Tauopathies are a heterogeneous group of diseases char-acterized by hyperphosphorylation and accumulation ofTAU protein in the brain. Some of these diseases are directlylinked to specific MAPT gene mutations that alter the phys-iological ratio between 3R- and 4R-TAU isoforms and/orrenders TAU protein more susceptible to aggregation.Nevertheless, the mechanisms underlying TAU-mediatedneurodegeneration are only partially understood [27].

Although mouse models have been generated (over)ex-pressing different human TAU variants, these only partiallyreproduce human TAU pathology with significant variabilitybetween models depending on the TAU variant expressedand/or promoter used [9]. In addition, murine neuronsappear to be less sensitive to neurodegeneration comparedwith human neurons [28].

The advent of human iPSCs has created the possibility tostudy mutations in human cell models. A number of studieshave generated iPSC-derived cells from individuals carryingdifferent MAPT mutations. These in vitro models reproducesome aspects of tauopathies, including the presence ofaltered TAU isoforms expression, TAU hyperphosphoryla-tion, activation of ER/UPR stress pathways, acceleratedelectrophysiological maturation, and/or impaired neuriteoutgrowth [10–13,21]. However, depending on the linesstudied, some but not all of these phenotypes weredescribed simultaneously.

Ideally, drug discovery and validation platforms shoulduse cell models wherein most of these TAU-associated phe-notypes are present and can be compared with WT controllines, to prove the causal relation of the phenotype. Wetherefore created an iPSC-derived model that reproducesmost of the phenotypes described elsewhere by introducingthe three mutations found in .60% FTDP-17 patients(N279K, P301L, and E10116) [16] in a single iPSC line.

We introduced the three mutations in a footprint-freemanner in hiPSCs, combining the CRISPR-FokI

e) in response to a depolarizing current pulse was counted for every cell. (C)

T neuron on DIV70) of current responses showing voltage-activated inward

y dashed box) was determined for every cell. Outward peak currents were

he percentage of WT and TAU-mutant neurons firing spontaneous (D and

ant: n 5 23) as well as on .90 DIV (WT: n 5 26; Mutant: n 5 35). FiringQ16arable in WTand TAU-mutant neurons, on DIV70 (spontaneous, WT: n5 7;

eous, WT: n 5 22, mutant: n 5 16; evoked, WT: n 5 21, mutant: n 5 22).

-mutant (n5 14) neurons on DIV70 (P, .05; L and M), whereas they were

19; Mut: n5 22). The number of patched cells is represented by n; all cells

with interquartile range. Statistical analysis was performed using a Mann-

ectrode arrays (MEAs) on DIV65 neural progeny co-cultured with primary

tured alone do not display any activity). Q and R show the number of bursts

N 5 3 independent replicates. All data are means 6 SD, t test. *P , .05,

� 19 July 2018 � 8:16 pm � ce

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Fig. 4. Altered cortical neuron differentiation from TAU-mutant iPSCs. (A–G) qRT-PCR analysis for deep and superficial cortical layer neuron marker gene

expression during differentiation of WT (black) and TAU-mutant iPSC (gray) progeny (B3TUB, FOXG1, TBR1, CTIP2, BRN2, CUX1, and SATB2). Gene

expression levels are shown as fold changes relative toWTNPCs fromDIV32 (gene expression was normalized to the housekeeping geneGAPDH). (H) Graph-

ical representation of different cortical layer marker genes. (I–L) Representative pictures for immunostaining of TUJ1 together with FOXG1, TBR1, CITP2, and

SATB2 in WT (upper row) and TAU-mutant progeny at the specified time points (middle row). Quantification levels of each marker during differentiation for

both cell types (WT in black and TAU-mutants in gray) depicted in the lower row. (M) DIV40 and DIV68 differentiating cells were incubated with EdU for

3 hours, 24 hours before fixation, followed by staining with an antibody against EdU and TUJ1. A representative picture of both WTand TAU-mutant neuronal

progeny onDIV40 is shown, with quantification of the percentages of EdU1 cells at both time points ofWT (black) and TAU-mutant cells (gray). Hoechst 33258

(blue) was used as nuclear marker. Scale bar: 50 mm. Data are represented as mean 6 SEM of N 5 3–4 independent experiments. *P , .05, **P , .01.



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Fig. 5. Genome-wide transcriptome studies demonstrate significant differences between triple TAU-mutant and WT neurons. RNA sequencing (seq) was per-

formed on WT and TAU-mutant iPSC progeny on DIV70 and DIV110. (A) Principal component analysis (PCA) of WT and TAU-mutant iPSC progeny on

DIV70 and DIV110. (B) Volcano plots at DIV70 (left) and DIV110 (right) illustrating the number of differentially expressed genes (adjusted P-value , .05



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nuclease–mediated homology-directed recombination withthe piggyBac transposase–mediated excision of a select-able cassette, introduced during the homology–directedrecombination [18]. Because of the use of two guideRNAs, this approach has been shown to cause less off-target cuts compared with the CRISPR-Cas9 system [17].

We describe that knock-in of the three mutations in theMAPT gene resulted in an altered 3R/4R-TAU isoformexpression, with high levels of 4R-TAU already found asearly as DIV32 in iPSC neural progeny. The preciseknock-in strategy used, together with the observation ofsimilar phenotypes in two independently generated lines,demonstrates conclusively that the MAPT-mutations areresponsible for this observation.

The three introduced mutations have been reported (indi-vidually) to lead to the incorporation of the exon 10 in themRNA (leading to the expression of 4R-TAU isoforms)and/or expression of pathogenic TAU. Because single orcombinations of dual mutation insertions have not been per-formed, we cannot firmly state which of the mutations isresponsible for the phenotypes observed. Nonetheless, dueto the wide range of phenotypes, we describe encompassingmost of the phenotypes observed by other studies using sin-gle TAU-mutated neurons [10–13,21], we hypothesize thatthe phenotypes we obtained are the consequence of boththe incorporation of 4R-TAU isoforms and the synergisticassociation of the different mutations introduced.

Expression of TAU protein isoforms is brain region anddevelopmental stage-specific [29]. During fetal develop-ment, only 3R-TAU isoforms are present, which is believedto be important to allow dynamic changes in neurites andsynapses formation. Continued presence of 3R-TAU in post-natal neurons is likely required for the establishment of newsynapses [29]. By contrast, 4R-TAU, which binds microtu-bules with higher affinity than 3R-TAU, become expressedin postnatal cortex [29]. The altered ratio between the twogroups of TAU isoforms in the triple mutant neurons maybe responsible for the aberrant neurite outgrowth weobserved. As the assay can be done using a (semi)-automaticimaging platform, this phenotype would be amenable to(semi)high-throughput screening to test compounds that cor-rect mutant TAU–mediated neurite outgrowth abnormalities.This model might help to understand the role of TAU in neu-rite outgrowth, branching, or synapse formation and bepotentially useful to test compounds aiming to correct theobserved phenotype.

TAU aggregation is the pathological hallmark that bestcorrelates with the progression of AD. Although it isaccepted that TAU aggregates are rather a cause than a

and log2 fold change.1) between WTand TAU-mutant neurons at DIV70 (1490 g

both stages. (C) Supervised clustering analysis of a selected list of cortical neurona

(D) Supervised clustering analysis for glutamatergic vs. GABAergic marker expre

genuity pathway analysis (IPA) of the glutamate receptor signaling pathway betw

green: highly expressed genes on TAU-mutant neurons). (F) IPA analysis of th

iPSC progeny on DIV 110 (red: highly expressed genes on WT cells; green: high


consequence of neurodegeneration, the precise molecularmechanisms by which TAU pathology is triggered and pro-gresses throughout brain areas are not yet well understood. Itis hypothesized that interfering with TAU expression, aggre-gation, and/or clearance may be a potential strategy for treat-ing AD and other tauopathies [30], highlighting the need forin vitro TAU aggregation assays. Both in animal models andin vitro assays, physiological levels of TAU expression donot lead to TAU aggregation and requires not only the addi-tion of recombinant TAU (seeds) but also the expression ofnonphysiological levels of TAU expression (e.g., by overex-pression of P301LTAU) [20]. In the triple TAU-mutant iPSCneurons on DIV70, we observed TAU aggregation only byseeding with K18 fibrils and without TAU overexpression.Similarly, we also observed a fivefold increase in aggrega-tion by simply overexpressing TAU and in the absence ofk18 fibrils. Importantly, we observe that TAU aggregates de-tected in mutant neurons are hyperphosphorylated, in anal-ogy with which occurs in the brain of patients with AD[3]. Taken together, our findings suggest that the tripleMAPT mutation is sufficient to seed for TAU aggregation,with a signal that is directly proportional to the amount ofmonomeric TAU availablewithin the cell. As the seeding po-tential of endogenously expressed TAU has never beenobserved in any other in vitro assay, we suggest that tripleTAU-mutant neurons may be a more clinically relevantmodel to screen for drugs interfering with TAU aggregationand/or propagation.

We found, based on RNAseq, qRT-PCR, Western blots,and functional studies, that triple TAU-mutant neuronalprogeny displayed considerably increased levels of oxida-tive stress, ER/UPR stress, and activation of inflammatory-related response marker genes, which were also associatedwith an increased number of apoptotic cells. TAU abnormal-ities have been reported to lead to mitochondrial dysfunctionand increased oxidative stress [31]. In addition, genetic andbiochemical studies have shown that the UPR is activated atearly stages in tauopathy brains [32]. Thus, the tripleMAPT-mutant iPSC neuronal progeny recreate the TAU-mediatedactivation of the oxidative stress, ER/UPR, and inflamma-tory pathways; possibly also representing targetable pheno-types.

Transcriptome analysis demonstrated significant de-creases in the levels of genes involved in the glutamatesignaling pathway and a shift toward the expression of genesinvolved in GABAergic signaling in the triple mutant-TAUcells. Dysfunction of TAU is believed to cause an impairedmicrotubule organization that affects synapse organization,which underlies neurotoxicity and neurodegeneration. In

enes) and DIV110 (1868 genes, green), 395 of which (red) were common at

l markers in normal and TAU-mutant iPSC progeny on DIV70 and DIV110.

ssion in WT and TAU-mutant iPSC progeny on DIV70 and DIV110. (E) In-

een both cell types on DIV110 (red: highly expressed genes on WT cells;

e synaptic long-term potentiation (LTP) pathway in WT and TAU-mutant

ly expressed genes on mutant neurons).

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addition to this mechanism, accumulation of TAU withinintact dendritic spines results in synaptic abnormalities,where it disrupts synaptic function by impairing glutamatereceptor trafficking or synaptic anchoring [33]. Consistentwith this notion, we also observed a downregulation ofmarkers of LTP pathway in the mutant cells. LTP is a formof synaptic plasticity believed to be involved in memory for-mation required for learning and memory [34]. LTP deficitshave been described mostly for amyloidogenic rather thanTAU-mutant models of AD [35]. However, our resultssuggest that this phenomenon might also be linked toTAU-mediated pathogenesis and suggest that the tripleMAPT-mutant model might be a good platform to interrogateTAU-based disease mechanisms.

Different studies have reported an abnormally enhancedelectrophysiological activity in TAU-mutant animal models[36,37] and in tauopathy patient-derived neurons [21]. Themechanisms by which aberrant TAU causes neuronal hyper-excitability are not yet elucidated, but it is believed that thismay contribute to neuronal dysfunction at the onset of AD[36]. In our study, co-cultures of iPSC-derived neuronswith primary astrocytes in MEAs showed an increase inelectrophysiological activity for TAU-mutant cells in com-parison withWT. This result, in principle, appears contradic-tory with the transcriptome data indicating the prevalence ofa GABAergic inhibitory phenotype within mutant cells.Nevertheless, during development, there is developmentalshift in GABAergic populations from immature excitatoryto mature inhibitory synaptic transmission [38]. ImmatureGABAergic neurons are not able to maintain a properreversal potential for the ion chloride (Cl2), resulting in ahigher intracellular Cl2 concentration compared withmature neurons. This is because the chloride exporterKCC2 (SLC12A5) is not yet expressed in immature GA-BAergic neurons. After GABA synaptic transmission, thisderegulated Cl2 potential results into an outward Cl2 fluxand hence, an activating depolarization [39]. We thereforequeried the RNAseq data and found that the expression ofKCC2 (SLC12A5) was substantially decreased in mutantneurons compared with WT cells mainly at later stages(DIV110, Supplementary Fig. 5G), reconciling the observa-tion of a higher electrophysiological activity with a predom-inant GABAergic phenotype within TAU-mutant neurons.Recently, it has been described that amyloid precursor pro-tein deficiency leads to KCC2 degradation, causing impairedchloride concentrations inside the neurons. This resulted indeficient GABAergic inhibition in the hippocampus of pa-tients with AD [40]. Our results might suggest a role foraberrant TAU as well in this phenomenon.

The increased electrophysiological activity of TAU-mutant cells in MEAs was not reproduced in whole cellpatch-clamps experiments. Possible explanations for thismight be as follows: (1) considerable fewer neurons aresampled in patch-clamp studies compared with MEA anal-ysis; (2) MEA experiments allow to determine neuronalnetwork signaling, in contrast to patch-clamp where only in-


dividual cell electrophysiological activities can be evalu-ated; and (3) astrocytes are essential to support neuronalactivity both in vivo and in vitro and the absence of astrocytesin the patch-clamp experiments might have had a negativeimpact on the neuronal activity assessed [22].

Finally, we found defective cortical maturation in vitrofrom triple TAU-mutant cells, with a notable impairmentin the capacity to differentiate toward SATB21 superficialcortical layer neurons and a relative increase in cells withdeeper cortical layer markers. Concomitantly, we observeda decreased frequency of TBR1-positive cells, a transcriptionfactor governing cortical layer formation with alteredexpression in patients with AD [23]. Decreased cortical neu-rogenesis have been described as well for haploinsufficientprogranulin mutant neuronal progeny derived from iPSCsof frontotemporal dementia patients [41], which might sug-gest that aberrant mechanisms convey in similar neurode-generative consequences. The reason for the failure togenerate superficial layer cortical neurons observed in tripleTAU-mutant neurons is not clear, but one possibility is thatthese cells might be more sensitive to (oxidative, ER/UPR,and/or inflammatory) stress, as has been reported in patientswith AD [42], with therefore a more specific disappearanceof these cells from the culture. The loss of the predominantlyglutamatergic superficial layer neurons would result in animbalance of excitatory vs. inhibitory signaling [43], as wehave observed at the transcriptome level in our cultures.

In conclusion, we generated a hiPSC-derived TAU-mutant model that reproduces key several neurodegenerativephenotypes associated with tauopathies, such as altered TAUexpression, including phosphorylated isoforms, TAU aggre-gation, defective neurite conformation, altered neuronalmaturation, enhanced electrophysiological excitability, andupregulation of stress pathways. The robustness of thefootprint-free and nonadditional mutation-generated line,together with the advantages inherent to iPSC-derived sys-tems, makes the generated model an ideal candidate plat-form for the identification of therapeutic targetscounteracting TAU pathology excluding the variability asso-ciated with patient-derived cells.

4. Experimental procedures

4.1. Human iPSC lines culture conditions, gene editing,and selection

The hiPSC ChiPS6b healthy donor-derived WT cells(purchased from Takara Bio Inc.) were maintained infeeder-free conditions using E8medium (Life Technologies)on QhESC-qualified matrigel (Becton Dickinson) and weresplit twice a week using EDTA (Lonza).

4.2. Selection of TAU mutations

We aimed to generate a cell model that reproduces severalaspects of tauopathy-related neurodegeneration. Therefore,we selected the N279K, P301L, and E10116 mutations

� 19 July 2018 � 8:16 pm � ce

web4C=FPO

Fig. 6. qRT-PCR confirms increased stress pathway gene expression in TAU-mutant cortical progeny. (A–M) qRT-PCR analysis for genes implicated in oxida-

tive stress (A–D), ER/UPR stress (E–I), and inflammatory responses (WT: black and TAU-mutant iPSC: gray, J–M). Gene expression levels are shown as fold

changes relative to WT NPCs on DIV32 (gene expression was normalized to the housekeeping gene GAPDH). (N–P) Western blot experiments and



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present within the exon 10 or next intron of theMAPT gene.These three mutations are the most prevalent in FTDP-17,accounting for up to 60% of all cases [16]. Presence of theN279K or P301L mutations causes early onset and aggres-sive disease course, increased TAU-phosphorylated speciesboth in neurons and glia, and a higher prevalence of the path-ogenic 4R TAU isoforms, altering the 3R/4R ratio [14]. TheE10116 mutation is located in the intron between exons 10and 11, causing alterations in the stem-loop structure andaffecting splicing capacity of pre-mRNA, with increasedexon 10 incorporation as a result and, therefore, a higherexpression of the 4R TAU isoforms [15].

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4.3. Design of gene-editing strategy

To accurately and efficiently target the exon 10 of theMAPT gene, we developed a combination of specific target-ing using CRISPR-FokI nucleases [17] together with theconstruction of a template DNA vector harboring the threeselected mutations and a dual HYGR (positive)/TK (nega-tive) selection cassette (backbone vector provided by Dr. Ko-suke Yusa, Wellcome Trust Sanger Institute, UK [18]). Thistemplate incorporated as well a 700 bp left and right homol-ogy arms to allow homologous recombination of the DNAtemplate into the genome, which should result, in mostcases, in the incorporation of the three selected mutations.To generate footprint-free mutant cell lines, we combinedthis strategy with the use of the piggyBac transposase tech-nology, which allows excision by recombining TTAA se-quences contained in their integration sites (invertedterminal repeats) [44]. Therefore, we introduced the selec-tion cassette next to an endogenous TTAA sequence, suchthat following cassette excision the TTAA remained,without altering the genomic sequence, therefore generatingcell lines without any additional genetic alterations [18].CRISPR-FokI nucleases were designed using the ZincFinger Consortium (ZiFiT) algorithm [45] covering thetarget sequence. Target site 1: TTATTAATTATCTG-CACCTT, target site 2: AGCAACGTCCAGTCCAAGTG.The efficiency of the selected nucleases was tested and vali-dated on HEK293T cells (not shown).

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4.4. Nucleofection and selection

Nucleofection was performed as previously described[46]. Briefly, two million hiPSCs were transfected with5 mg donor vector, 3 mg of pSQT1601 plasmid encodingfor dCas9-FokI and 1 mg of each plasmid encoding for

quantification of protein levels for the inflammatory response (tumor necrosis facto

DIV65. (Q) IPA analysis, based on RNA-seq analysis depicted in Fig. 5, of the NFk

expressed genes on TAU-mutant neurons). (R and S) Representative immunostaini

cells stained with antibodies against activated CASPASE3 and TUJ1. (T–U) Anal

DIV40 andDIV68 (a representative picture of TAU-mutant neurons at DIV40 is sho

were defined as those nuclei with smaller size and more concentrated Hoechst sign

dent differentiations. Hoechst 33258 (blue) was used as nuclear marker. Scale bar

periments. *P , .05, **P , .01.


gRNAs directed toward left and right target sites using theAmaxa Nucleofector, and the hESC Nucleofector SolutionKit 2 (Amaxa, Lonza) using the program F16. Cells werecultured in feeder-free conditions as previously describedin the presence of a ROCK inhibitor analog (Revitacell�,Life Technologies). Selection was initiated after 2–3 dayswith 25–50 mg/mL of Hygromycin B (Sigma-Aldrich) andmaintained for 10–15 days. Recombinant colonies weremanually picked and expanded for further characterization.

Removal of the selection cassette from selected cloneswas performed by nucleofection of 5 mg of the PiggyBacTransposon. Two to 3 days after the transfection, cellswere treated with 0.5 mM 1-(2-deoxy-2-fluoro-beta-D-arabi-nofuranosyl)-5-iodouracil (FIAU) for 7 days. GFP expres-sion was monitored by immunofluorescence Qand FACSduring the selection process with both HYG and FIAU.

4.5. Flow cytometry

To determine the frequency of GFP1 cells during theinitial HYG and subsequent FIAU selection procedure, cellswere enzymatically harvested using accutase, centrifuged,and resuspended in 100 mL FACS buffer (PBS 1!, 2% fetalbovine serum and 0.02% sodium azide). Cells were analyzedon a FACSCanto flow cytometer using the FACS DIVA soft-ware (Becton &Dickinson). After exclusion of cell doublets,GFP fluorescence was determined on FL1-FITC channel,with gates established based on fluorescence of untargeted(WT) cells.

All results were analyzed using Flow Jo (FlowJo, LLC,USA) and FACS DIVA software (Becton & Dickinson).Flow cytometry and FACS sorting was performed at theKU Leuven Flow Cytometry Facility.

4.6. PCR genotyping

PCR genotyping was done using 40 ng of genomic DNAwith Go Taq DNA polymerase (Promega) in 10 mL reactions.Primer sequences and PCR program conditions aredescribed in Supplementary Table 1. PCR products wereloaded on 1.5% agarose (Sigma) gels and visualized withSYBR Safe (Invitrogen) on a Gel Doc XR1 System (Bio-Rad).

4.7. Pluripotency characterization

Both WT and triple TAU-mutant cells were subjected tospontaneous differentiation mediated by embryoid body for-mation and subsequently analyzed for the three-lineage

r receptor 1 (TNFR1)) and ER/UPR stress (CHOP) markers respectively, on

B signaling pathway (red: highly expressed genes onWT cells; green: highly

ng images and quantification on DIV40 and DIV68 of WTand TAU-mutant

ysis of apoptotic nuclei in WT and TAU-mutant cortical neuron progeny on

wn for immunostainingwith TUJI antibodies andHoechst). Apoptotic nuclei

al, lacking expression of TUJI. Representative images of N5 3–4 indepen-

: 50 mm. Data are represented as mean 6 SEM of N 5 3–4 independent ex-

� 19 July 2018 � 8:16 pm � ce

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differentiation using the ScoreCard methodology [47](Thermo Fisher Scientific), according to manufacturer in-structions.

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4.8. Neuronal differentiation protocol

Cortical neurons were generated from both WTand tripleTAU mutant hiPSCs as previously described by Shi et al.[19]. Briefly, single-cell hiPSCs were collected and platedon Matrigel-coated (BD) well plates in E8 medium supple-mented with ROCK inhibitor analog (Revitacell, Life Tech-nologies). Once the cell culture reached 95% confluence,neural induction was initiated by changing the culture me-dium to N2B27 medium, consisting of a 1:1 mixture ofN2- and B27-containing media. N2 medium consisted ofDMEM/F12, N2, 5 mg/mL insulin, 1! GlutaMax, 1!nonessential amino acids, 1! 2-mercaptoethanol, 1! peni-cillin/streptomycin. B27 medium consisted of Neurobasal,B27 without retinoic acid, 1! glutamax, 1 ! 200 mMglutamine and 1! penicillin/streptomycin (all from LifeTechnologies). N2B27 medium was supplemented withthe small molecules SB431542 10 mM (Tocris) andLDN193189 1 mM (Miltenyi Biotec), and daily mediumchanges were performed till day 12. Then, neuroepithelialcells were collected by dissociation with dispase andreplated in N2B27 medium, including 20 ng/mL bFGF (Pe-protech) on matrigel-coated dishes. Rosette-forming neuro-epithelial cells were purified two more times (one each after4–5 days), after which single-cell splitting with accutase wasperformed twice before cryopreserving NPCs (whichoccurred on DIV32–33). For terminal neuronal differentia-tion, NPCs were thawed, cultured in N2B27 medium for afew days, and replated on DIV40-42 at 15–25.000 cellsper cm2 on poly-ornithine and laminin-coated plastic dishesin N2B27 medium and maintained for up to DIV200 withpartial medium changes twice a week.

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4.9. RNA extraction, cDNA synthesis, and gene expression(QPCR)

Total RNA was purified using the GenElute� Mamma-lian Total RNA Miniprep Kit (Sigma-Aldrich, Saint Louis,MO, USA) and ZR RNA MicroPrep (Zymo Research, CA,USA). After concentration and integrity validation (Nano-Drop 1000, Thermo Fisher Scientific, MA USA), cDNAwas generated using 0.5–1 mg of RNA with SuperScript�

III First-Strand Synthesis SuperMix for qRT-PCR kit (Invi-trogen, CA, USA), and qRT-PCRwas performed in technicaltriplicates on a ViiA� 7 Real-Time PCR System with 384-well plate (Applied Biosystems, Carlsbad, CA, USA) with aPlatinum� SYBR� Green qPCR SuperMix-UDG w/ROX(Invitrogen, CA, USA) and primers mix at final concentra-tion of 250 nM.

Gene expression (cycle threshold) values were normal-ized based on the GAPDH (glyceraldehyde 3-phosphate de-hydrogenase) housekeeping gene, and the delta CT


calculated. Gene-specific primers were designed in exon-exon spanning regions in common domains of all isoformsdescribed for a given gene. The efficiency of primers wastested by serial dilutions of cDNA and by calculating the co-efficient of regression (R2). An efficiency of 90%–105%with an R2 � 95% was accepted. See SupplementaryTable 2 for a list of all qRT-PCR primers used in this study.

Identification of the 3R or 4R species was done usingthe following TaqMan assays (Thermofisher): glyceralde-hyde 3-phosphate dehydrogenase (Hs99999905_m1),MAPT all isoforms (Hs00902194_m1), MAPT 3R iso-forms (Hs00902192_m1), and MAPT 4R isoforms(Hs00902312_m1).

4.10. Transcriptome analysis by RNA sequencing

Total RNA was purified using the GenElute� Mamma-lian Total RNA Miniprep Kit (Sigma). RNA concentrationand purity were determined spectrophotometrically usingthe NanoDrop ND-1000 (NanoDrop Technologies), andRNA integrity was assessed using a Bioanalyzer 2100 (Agi-lent). Per sample, an amount of 100 ng of total RNA wasused as input. Using the Illumina TruSeq� StrandedmRNA Sample Prep Kit (protocol 15031047 Rev.E“October 2013”), poly-A–containing mRNA moleculeswere purified from the total RNA input using poly-T oligo-attached magnetic beads. In a reverse transcription reactionusing random primers, RNA was converted into first strandcDNA and subsequently converted into double-strandedcDNA in a second strand cDNA synthesis reaction usingDNA Polymerase I and RNAse H. The cDNA fragmentswere extended with a single deoxyadenosine to the 30 endsof the blunt-ended cDNA fragments after which multiple in-dexing adapters were ligated, introducing different barcodesfor each sample. Finally, enrichment PCR was carried out toenrich DNA fragments that had adapter molecules on bothends and to amplify the DNA in the library. Sequence li-braries of each sample were equimolar pooled andsequenced on an Illumina NextSeq 500 instrument (HighOutput, 75 bp, Single Reads, v2) at the VIB Nucleomicscore (www.nucleomics.be).

Low-quality ends and adapter sequences were trimmedoff from the Illumina reads. Subsequently, small reads(length , 35 bp), ambiguous reads (containing N), andlow-quality reads (more than 50% of the bases , Q25)were filtered. Processed reads were aligned with Tophat,v2.0.8b, to the human reference genome (GRCh38), asdownloaded from the Genome Reference Consortium(https://www.ncbi.nlm.nih.gov/grc/human/data). DefaultTophat parameter settings were used, except for “min-intron-length 5 50”, “max-intron-length 5 500,000”, “nocoverage-search” and “read-realign-edit-dist 5 3”.

Principal component analysis and volcano plots weregenerated in the statistical software R, version 3.4.1. Clus-tering was performed with ClustVis [48]. Differentially ex-pressed genes were identified based on P . .05, and a fold

� 19 July 2018 � 8:16 pm � ce

http://www.nucleomics.be

https://www.ncbi.nlm.nih.gov/grc/human/data

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change of two or more in gene expression among thedifferent pair-wise comparison was used to identify thedifferentially expressed genes. Gene ontology ofthe different differentially expressed genes was carried outusing the Gene Ontology Consortium (http://www.geneontology.org/). Ingenuity pathway analysis was per-formed with build 456367, content version 39480507.

4.11. Western blot

Cells were lysed and sonicated in RIPA buffer (Sigma),containing phosphatase and protease inhibitors. Lysate wascleared by 15 min centrifugation at 12,000! g, and superna-tant was recovered for protein quantification. Twenty micro-grams of protein extract was then loaded per lane andresolved on SDS-PAGE before transferring to nitrocellulosemembranes. Blocking was done for 1 h with 5% nonfat milksolution in PBST before incubation with primary antibodies.

For TAU WBs, cells were lysed in RIPA buffer (150 mMNaCl, 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mMEDTA, 1% NP-40, 0.25% sodium deoxycholate supple-mented with complete protease inhibitors (Roche) and Phos-STOP (Sigma). The homogenate was ultra-centrifuged at100,000! g for 60 min at 4�C. Equal amounts of proteinwere taken and analyzed by SDS–PAGE and semi-quantitativeWestern immunoblot. Bands were visualized us-ing a cooled charge-coupled device camera (ImageQuantLAS-4000; GE Healthcare) and quantified by AIDA ImageAnalyzer software.

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5. AlphaLISA immunoassay

Cell lysis was performed using RIPA buffer containingphosphatase (PhosSTOP, Roche, Mannheim, Germany)and protease inhibitors (cOmplete, Roche) in culture platesfor 1 h at room temperature (RT) on an orbital shaker. ForhTAU10/hTAU10 TAU aggregation assays, we performedmeasurements in duplicates using 5 mL of lysate eachtime. Five microliters of the remaining lysate were usedfor the relative quantification of total TAU and for measuringcell viability, respectively. Each sample was transferred to a384-well assay plate for AlphaLISA reaction, in which 5 mLof cell extracts were incubated for 2 h at RTwith a mixture ofbiotinylated antibody and acceptor beads. Subsequently,donor beads were added to the wells and incubated at RTfor 30 min before reading at 615 nm (on illumination at680 nm) on the EnVision plate reader (PerkinElmer, Wal-tham, MA). Output measurement is quantified as relativefluorescent units and compared versus control conditions.

5.1. Immunostainings

Cells were washed with PBS 1! and fixed for 15 min atRT with 4% formaldehyde solution and washed with PBS.Preparations were permeabilized in 0.1% Triton-X-100(Sigma) and blocked in 10% donkey or goat serum (Dako)in PBS for 1 h at RT. Primary antibodies were incubated


overnight at 4�C in 5% in donkey or goat serum in PBS,washed 3 times, and incubated for 1 h at RT with fluores-cently labeled secondary antibodies diluted to 1:500. Sam-ples were incubated for 15 min at RT with Hoechst 33258(1:2000 dilution) for nuclear staining and mounted withProLong� Gold Antifade Mountant (Thermo Fisher Scienti-fic). The list of primary and secondary antibodies and the di-lutions used can be found in Supplementary Tables 3 and 4.

5.2. Image acquisition and analysis

The stained cells were examined using an Axioimager.Z1microscope (Carl Zeiss). For quantification purposes, at leastfive independent fields per condition and per experimentwere obtained. For neurite outgrowth experiments, imageswere acquired on an In Cell Analyzer 2000 High ContentImager (GEHealthcare), acquiring sufficient numbers of im-ages to cover at least 70% of the well area. Data analysis wasperformed using the In Cell image analysis software (GEHealthcare), with the specific software package of neuriteoutgrowth.

5.3. EdU incorporation assays

Cells were pulse-labeled with 10 mM 5-ethynyl-20-deox-yuridine (EdU, Life Technologies) for 3 hours, and severalmedium washes were performed afterward. Twenty-fourhours after onset of EdU incubation, cells were fixed with4% PFA for 15 minutes, washed with PBS 1 2% BSA, fol-lowed Qby 300 permeabilization with PBS 1 0.5% Triton.Cells were then incubated for 30 min at RTwith freshly pre-pared reaction buffer (per mL: 976 mL PBS 1 10 mLCuSO4 1 10 mL Sodium Ascorbic 1 4 mL of 1/100Azide-AF647 antibody), washed for three times, and stainedwith b-3-tubulin and Hoechst. The presence of EdU1 cellswas evaluated using an Axioimager.Z1 microscope (CarlZeiss).

5.4. Electrophysiological recordings

Whole-cell patch-clamp recordings of hIPSC-derivedcortical neurons were carried out in a recording chamberwhich was continuously perfused with artificial cerebrospi-nal fluid at RT. Artificial cerebrospinal fluid contained145 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2,10 mM Hepes, and 12 mM glucose (pH adjusted to 7.4with NaOH; w310 mOsm). Cells were visualized by an in-verted Olympus IX73 microscope equipped with a 40!objective. Recording patch pipettes were pulled from boro-silicate glass using a vertical PIP6 micropipette puller(HEKA Elektronik, Lambrecht/Pfalz, Germany) and filledwith an internal solution containing (in mM): 120 K-gluco-nate, 20 KCl, 1 MgCl2, 10 Hepes, 0.2 EGTA, 0.3 Na-GTP, 5NaCl, 4 Mg-ATP (pH adjusted to 7.3 with KOH; w290mOsm). Current-clamp mode was applied to measure actionpotentials, while voltage-gated Na1 and K1 currents wererecorded in voltage-clamp mode. An online P4 leak

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subtraction protocol was applied during the acquisition of allvoltage-gated currents. Details of the performed current- andvoltage-clamp protocols are described in the results section.Signals were acquired, filtered (at 2.8 kHz), and digitized (at20 kHz) using an EPC10 USB amplifier and PatchMastersoftware (HEKA Elektronik). The liquid junction potentialof 14 mV was corrected off-line. Action potential detectionwas performed using Stimfit software; current amplitudeswere measured by PatchMaster software.

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5.5. Multi-electrode arrays

Human iPSC-derived NPCs (DIV30) were co-culturedwith rat primary astrocytes in 1:1 ratio and plated onto 48-well MEA plates (Axion BioSystems) coated with polyethy-leneimine and laminin. Plating was done using drop seedingmethod, where 15 mL of mixed cells (60,000 cells in total) ina 10% dilution of N2B27media: Matrigel (BD Biosciences,Cat#356230) was placed onto each well. Cells were thencultured in neuronal differentiation media for up to 40 addi-tional days (DIV70), with partial medium replacementsevery 2–3 days. Spontaneous network activity of the matu-rating neurons was recorded for 5 minutes on specific timepoints using the Axion Biosystems Maestro MEA at 37�Cand 5% CO2.

Data analysis was performed using AxIs software (AxionBiosystems) to obtain from the recording the data concern-ing the spikes, bursts, and network activity. Quality criteriafor the assays were defined as follows: active electrodeswere defined as an electrode having an average of morethan five spikes per minute. Wells in which less than 30%of electrodes were active were considered inactive andremoved from analysis. For synchronous network activity,at least 25% of the total electrode in a well was required toparticipate in a network event in order for the network tobe qualified as network spike or bursts.

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5.6. Statistical analysis

Comparisons between two groups were analyzed usingunpaired or paired two-tailed Student’s t test. P-values, .05 were considered significant (*). Data are shown asmean, and error bars represent standard error of mean of aminimum three independent experiments.

For patch-clamp data, a Mann-Whitney test or a Kruskal-Wallis with post hoc Dunn’s multiple comparison’s test wereused to compare respectively two or three groups as patchclamp data were not normally distributed. Results wereplotted and analyzed using GraphPad Prism 6 software.

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Acknowledgments

The authors would like to thank all members of the Stellarproject consortium for fruitful scientific discussions and col-laborations.


The authors would like to thank Prof. F.L. and Anchel De-Jaime (Stem Cell Institute, KU Leuven) for their assistancewith Western blotting of stress marker proteins.J.A.G.L. has been supported by a fellowship from AlfonsoMart�ın Escudero Foundation (Madrid, Spain). P.V.D. holdsa senior clinical investigatorship of FWO-Vlaanderen. Thework was supported by the Stellar project iPSC-NeDi (Jans-sen Pharmaceutica), IWT-iPSCAF grant (#150031), IWT-VIND project and the KUL-PF Stem Cells (#PFO3) toC.M.V.Authors’ contributions: C.M.V. and A.E. conceived thestudy. J.A.G.L. and C.M.V. designed the project and exper-iments. J.A.G.L. performedmost of the experiments and per-formed analysis of the data. A.C.S. provided expertise andperformed TAU aggregation, MEAs, and some WB experi-ments and actively contributed to the writing of the manu-script. K.E. actively assisted and performed neuronaldifferentiations and immunostainings. A.S. performedpatch-clamp experiments and analysis. J.T. assisted withneurite outgrowth and transcriptome analysis. R.F. andA.Q. helped with gene-editing and iPSC maintenance.L.O. and A.N. helped with the gene-editing strategy. K.W.assisted with electrophysiology experiments and data inter-pretation. L.S. performed WBs for TAU protein isoforms.A.S. and B.D.S. assisted with data interpretation. M.K. andF.P. assisted with MEAs experiments. P.V.D. provided sup-port for electrophysiology experiments. L.T. performedmost of the transcriptome data. J.A.G.L., and C.M.V. wrotethe manuscript. C.M.V. provided scientific guidance andsupport. All the authors have read the manuscript.

Supplementary data

Supplementary data related to this article can be found athttps://doi.org/10.1016/j.jalz.2018.05.007.

RESEARCH IN CONTEXTQ

1. Systematic review: ---.

2. Interpretation: ---.

3. Future directions: ---.

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