www.cellulardynamics.com Madison, WI USA +1 (608) 310-5100 Characterization of an Isogenic Disease Model of Alzheimer’s Disease from Human iPSC-derived Neurons Coby Carlson , Jun Wang, Natsuyo Aoyama, Rachel Llanas, Kile Mangan, Michael McLachlan, Tom Burke, Susan DeLaura, and Eugenia Jones AD-model Generation Amyloid Beta (1-40) -11 -10 -9 -8 -7 -6 -5 -4 1000 1500 2000 2500 3000 3500 4000 Log [DAPT] (M) Alpha Counts Genotype Phenotype iPSC line # Control (WT) Healthy; normal 01279.107 APP A673T (homo) Isogenic AD model 01279.A27 01279.A32 APP A673V (het) “Hot Topics” Introduction The development of therapies for neurodegenerative diseases such as Alzheimer’s disease (AD) has been hindered by limited availability of relevant human cells for research and drug discovery. Using induced pluripotent stem (iPS) cell technology, we have created an unlimited source of human neurons that incorporate specific genetic profiles associated with AD pathology. Researchers are using these cells to understand the fundamental mechanisms underlying AD and to identify novel drug treatments for this disease. AD is a progressive neurodegenerative disease that results in gradual memory loss and impairment in the ability to learn or carry out daily tasks. Recent studies estimate that AD may contribute to as many deaths as heart disease and cancer combined. Development of therapies for the treatment of AD is lengthy, costly and has been largely unsuccessful, with drug attrition rates of >90%. As a result, there are no cures and few treatment options. A hallmark of AD pathology is the development of toxic plaques in the brain that contain beta amyloid (Aβ), which is produced from amyloid precursor protein (APP). To build a cellular model that could help to better understand the mechanisms by which Aβ is produced, we used iPSC technology and genetic engineering to create human neurons that exhibit AD-specific profiles. Our studies with these human iPSC-derived neurons highlights the “disease-in-a-dish” approach for AD drug development. iPS Cells Genetic Engineering Human Neurons Cortical Neuron Production Electrical Activity in APP Variants Correlates with Allele: A673T > WT > A673V 5 sec A673T is the first variant in APP associated with protection against amyloid pathology and AD. A673T was identified in a whole genome sequencing project of approx. 1,800 people from Iceland. (Jonsson Nature 2012) The A673V variant is also near the APP beta-secretase cleavage site and the mutation contributes to AD pathology not only by increasing Aβ production, but also by enhancing aggregation and toxicity. (Di Fede Science 2009) (B) Real-time qPCR analysis of a focus gene panel reveals modest but significant differences in gene expression; notably protein kinase C delta (PRKCD) – involved in learning and memory – and the AD-associated protein alpha-2-macro- globulin (A2M) WT control βIII-Tubulin A673T A673V Nestin (A) Characterization of neuron purity by flow cytometry. Using the expression markers of βIII- Tubulin (TUJ1) and Nestin, we observed >98% pure neurons. WT control (98.6%), A673T (99.6%), and A673V (98.9%). Day 7 Day 14 Day 21 WT control A673T A673V MAP2 / Synaptophysin / DAPI (C) ICC staining of neurons in culture A 40/42 Ratio -11 -10 -9 -8 -7 -6 -5 -4 1 2 3 4 5 Log [DAPT] (M) A 40/42 Ratio (A) Measurement of AD-relevant biomarkers by AlphaLISA: Aβ (1-40), Tau, and sAPPα in iPSC-derived neurons produced from a healthy donor. (B) Modulation of APP processing with the -secretase inhibitor (DAPT) shows a change in the amount of Aβ (1-40) and the Aβ 40/42 ratio. (C) Comparison of Aβ (1-40) levels in neurons produced from a healthy donor (WT, gray), an isogenic APP A673T variant (blue), and an isogenic APP A673V variant (red) shows differences in the amounts detected (HTRF assay). Treatment with small-molecule inhibitors of ‒secretase (ie. BMS 299897 and DAPT) resulted in decreased assay signal (AlphaLISA) in all three APP backgrounds. AD Biomarker Levels Differ in APP Mutant and Normal iPSC-derived Neurons Amyloid Beta (1-40) Levels To introduce these mutations in human iPSC-derived neurons, we genetically engineered a “control” iPS cell line (01279.107) from an apparently healthy normal Caucasian male donor (with no family history of neurological disorders) to engineer either the A673T or A673V alleles. ; • The A673T and A673V mutations were introduced in human iPSCs using TALENS. • The APP A673T variant is homozygous, whereas the APP A673V variant is heterozygous. Both are clonal. • Cortical neurons were differentiated from these three unique isogenic iPSC lines using CDI’s proprietary differentiation protocol outlined in the next panel. These lines can be purchased from CDI. β-secretase -secretase Aβ APP Ala→Thr = protective and Ala→Val = causative A673 Day 6 Day 7 Day 15 Day 8 Bursting Rate (BPM) Firing Rate (Hz) WT A673T A673V WT A673T A673V WT A673T A673V WT A673T A673V (A) Real-time heat map of activity electrical activity recorded on the 48-well Maestro MEA system from Axion BioSystems. (B) Display of action potentials captured from a 4x4 grid of electrodes in a single well (top) and the raster plot depicting action potential ticks across time for all 16 electrodes (bottom). Blue tick marks indicate a “Poisson surprise”-defined burst. (C) Mean firing rate (Hz; red) and bursting rate (BPM; blue) for WT, A673T, and A673V neurons (n≥12) over different DIV. Notice that as the cultures mature, bursting behaviors are enhanced for A673T and decreased for A673V. Summary Human iPS cells were genome-edited to create isogenic cell lines for modeling Alzheimer disease (AD) . Cortical neurons can be derived from human iPS cells with different genetic backgrounds at high purity. Wild-type APP, A673T, and A673V neurons express Tau, APP, and amyloid beta isoforms. Treatment with drugs modulated Aβ levels in the expected manner. Protocols for handling iPS cell-derived neurons from CDI, as well as performing various applications (ie. ICC, MEA) are available online. Please contact CDI with any questions at: [email protected] Future Directions A B C Schematic of cortical neuron differentiation process Post-thaw MyCell Neurons iPS Cell Expansion Neuron Maturation Day 0 Day 28-30 βIII-Tubulin-(+) Nestin-(-) Cell Cryopreservation Neuronal Differentiation A B C Bursting electrical activity in neurons carrying the Icelandic protective APP variant (A673T) is higher than WT neurons, which are more active than AD-associated A673V variant. These data here are consistent with previously reported AD models of transfected HEK 293 cells and primary rodent neurons (Jonsson Nature 2012 & Benilova J Biol Chem 2014). The advantage of using Multi-Electrode Array (MEA) technology to assess neuronal cultures is that it is a non-invasive, label-free method that enables the direct sensing of voltage to characterize network-level phenotypes. Recently published work from Genentech using these neurons purchased from CDI shows similar differences in APP processing (Maloney J Biol Chem 2014). MyCell Neurons (WT control) Leveraging the power of iPSC technology, CDI is building out a panel of MyCell Disease & Diversity Products, which includes human neurons from donors of diverse ethnic and disease- specific populations. Not only will there be more “controls”, but also other innate disease models for ALS, Rett Syndrome, Parkinson’s Disease, and epilepsy are being generated. Abstract Control Number: 1983 A B C