Probing Sporadic and Familial Alzheimerã¢â‚¬â„¢s Disease Using Induced Pluripotent Stem Cells

Abstruse

Our agreement of Alzheimer'due south disease pathogenesis is currently limited by difficulties in obtaining alive neurons from patients and the disability to model the sporadic form of the illness. It may exist possible to overcome these challenges past reprogramming primary cells from patients into induced pluripotent stem cells (iPSCs). Here nosotros reprogrammed primary fibroblasts from two patients with familial Alzheimer's disease, both caused by a duplication of the amyloid-β precursor protein gene¹ (APP; termed APP^Dp), ii with sporadic Alzheimer's illness (termed sAD1, sAD2) and two non-demented control individuals into iPSC lines. Neurons from differentiated cultures were purified with fluorescence-activated cell sorting and characterized. Purified cultures contained more than ninety% neurons, clustered with fetal brain messenger RNA samples by microarray criteria, and could form functional synaptic contacts. Nearly all cells exhibited normal electrophysiological activeness. Relative to controls, iPSC-derived, purified neurons from the 2 APP^Dp patients and patient sAD2 exhibited significantly higher levels of the pathological markers amyloid-β(1–40), phospho-tau(Thr 231) and agile glycogen synthase kinase-3β (aGSK-3β). Neurons from APP^Dp and sAD2 patients also accumulated large RAB5-positive early endosomes compared to controls. Treatment of purified neurons with β-secretase inhibitors, but non γ-secretase inhibitors, caused pregnant reductions in phospho-Tau(Thr 231) and aGSK-3β levels. These results suggest a directly human relationship between APP proteolytic processing, but non amyloid-β, in GSK-3β activation and tau phosphorylation in man neurons. Additionally, nosotros observed that neurons with the genome of ane distressing patient exhibited the phenotypes seen in familial Alzheimer's disease samples. More generally, we demonstrate that iPSC applied science tin be used to observe phenotypes relevant to Alzheimer'due south disease, even though it tin take decades for overt disease to manifest in patients.

Alzheimer'due south illness is a common neurodegenerative disorder, defined mail mortem by the increased presence of amyloid plaques and neurofibrillary tangles in the brain². Amyloid plaques are extracellular deposits consisting primarily of amyloid-β peptides, and neurofibrillary tangles are intraneuronal aggregations of hyperphosphorylated tau, a microtubule-associated protein involved in microtubule stabilization³. The causative human relationship between amyloid plaque/amyloid-β and tau pathologies is unclear in humans. Although the vast majority of Alzheimer's disease is apparently sporadic with significant non-Mendelian genetic contributions⁴, analyses of cellular and animal models of rare, dominantly inherited familial forms of Alzheimer'southward disease have driven nigh ideas well-nigh illness mechanisms. These rare cases have mutations or a duplication of APP, which encodes the amyloid-β precursor poly peptide, or mutations in the presenilin genes, which encode proteolytic enzymes that cleave APP into amyloid-β and other fragments. Mouse models that overexpress familial Alzheimer'due south affliction mutations develop extensive plaque deposition and amyloid-associated pathology, merely neurofibrillary tangles and pregnant neuronal loss are conspicuously absent-minded^5,half-dozen. Fetal human cortical cultures have as well been used to study the APP–tau relationship. For instance, cortical cultures treated with xx μM amyloid-β accept elevated phosphorylated tau (p-tau)⁷. However, information technology is yet unclear whether physiologically relevant levels of amyloid-β directly crusade elevated p-tau and which kinases are straight involved in this aberrant phosphorylation. Additionally, experimental approaches using fetal human neurons are hindered by limited availability of samples and unknown genetic backgrounds. The recent developments in iPSCs and induced neurons take allowed investigation of phenotypes of neurological diseases in vitro ^8,9,10. Still, non all diseases have been successfully modelled using iPSCs¹¹, and information technology is unclear whether iPSCs can be used to study sporadic forms of disease.

Here we report the derivation and neuronal differentiation of iPSCs from patients with familial and sporadic Alzheimer's illness, as well equally from non-demented, age-matched controls. Using purified man neurons we probe three cardinal questions concerning Alzheimer's disease: (1) can iPSC engineering be used to notice phenotypes of patients with Alzheimer's disease, even though it can take decades for overt disease to manifest; (2) is there a causative relationship between APP processing and tau phosphorylation; and (3) can neurons with the genome of a pitiful patient exhibit phenotypes seen in familial Alzheimer's disease samples? Supplementary Fig. one summarizes the experimental approach and findings.

We characterized APP metabolism in fibroblasts before reprogramming to iPSCs (Supplementary Fig. 2). APP expression and amyloid-β secretion were quantified in early-passage primary fibroblasts from two non-demented command (NDC) individuals, two lamentable patients and ii APP^Dp patients (Table one). The presence of the genomic duplication was confirmed in fibroblasts. Relative to NDC and sAD cells, APP^Dp fibroblasts expressed college levels of APP mRNA and secreted 1.five- to twofold college amounts of amyloid-β(1–40) peptides into culture media compared to NDC cells. We did non detect significant increases in amyloid-β(ane–42/one–forty) or amyloid-β(1–38/1–xl) in patient samples versus controls.

Table 1

Summary of patient information

Code	Diagnosis	Gender	Family history	Age at onset	Age at biopsy	MMSE at biopsy	APOE
NDC1	Not-demented control	M	Possible	Due north/A	86	xxx	ii-3
NDC2	Not-demented command	M	N	North/A	86	xxx	3-iii
sAD1	Sporadic AD	F	Northward	78	83	4	3-iii
sAD2	Desultory Advertizing	M	N	78	83	18	3-three
APPDp1	Familial AD, APP duplication	Yard	Y	46	51	21	3-3
APPDp2	Familial AD, APP duplication	F	Y	53	lx	17	3-three

We generated iPSC lines by transducing fibroblasts with retro-viruses encoding OCT4, SOX2, KLF4, c-MYC and, in one-third of cultures, EGFP. Each of the vi individuals was represented by three clonal iPSC lines. All 18 iPSC lines maintained embryonic stem (ES)-cell-like morphology, expressed the pluripotency-associated proteins NANOG and TRA1-81, maintained euploid karyotypes, expressed endogenous locus-derived SOX2, repressed retroviral transgenes, and could differentiate into cells of ectodermal, mesodermal and endodermal lineages in vitro (Fig. 1a–d, Supplementary Figs 3a–e and 4). All lines tested (ane per individual) formed teratomas when injected into nude rats (Supplementary Fig. 5). Supplementary Tabular array 1 provides details of each iPSC line.

Generation of iPSC lines and purified neurons from APP^Dp, pitiful and NDC fibroblasts

a, b, iPSC lines express NANOG and TRA1-81. c, d, iPSC-derived, FACS-purified NPCs limited SOX2 and nestin. e–h, iPSC-derived, FACS-purified neurons limited MAP2 and βIII-tubulin. Scale confined in a–h, 50 μm. i, Representative activeness potentials in response to somatic electric current injections. Information from iPSC line APP^Dptwo.2. j, Spontaneous synaptic activity was detected (voltage clench recording at the reversal potential of sodium (0 mV)) and reversibly blocked by GABA_A receptor adversary SR95531 (x μM). Each console represents ~4 min continuous recordings separated in 25 sweeps (grayness traces) and superimposed for clarity. Black traces represent a unmarried sweep. Data from iPSC line NDC2.1. k, l, No significant deviation was seen betwixt NDCs and any patient's cultures in the ability of iPSCs to generate NPCs at solar day 11 (P = 0.08, north = 9), or the ability of NPCs to class neurons at 3 weeks (P = 0.82, n = nine). Error bars indicate s.e.m.

Variability in differentiation efficiency exists between pluripotent cell lines. To analyse variability in our iPSC lines, we used a fluorescence-activated cell sorting (FACS)-based method of neuronal differentiation and purification (summarized in Supplementary Fig. 6), based on work described previously¹². Briefly, the 18 iPSC lines were first differentiated in to cultures containing neural rosettes (Supplementary Fig. 3f). From these cultures, neural progenitor cells (NPCs) were purified and the efficiency of NPC formation was assessed by CD184⁺CD15⁺CD44⁻CD271⁻ immunoreactivity. These FACS-purified NPCs maintained expression of NPC-associated markers, such equally SOX2 and nestin, over multiple passages (Fig. 1c, d). NPCs were differentiated for iii weeks into heterogeneous cultures containing neurons (Supplementary Fig. 3g, h). APP copy number was faithfully maintained in differentiated cultures (Supplementary Fig. 3i). From these cultures, neurons were purified to near homogeneity, and the efficiency of neuron generation was assessed by CD24⁺CD184⁻CD44⁻ immunoreactivity. No significant differences between any of the individuals in the efficiency of NPC or neuronal differentiation were detected (Fig. 1k, fifty).

Although we observed variability in differentiation among lines from each individual, the extent of inter-individual variation was less than observed intra-individual variability. These results advise that any observed biochemical aberrations in neurons, if present in multiple lines derived from the aforementioned patient, are probably caused by features of that patient'southward genotype. Purified neurons were plated at a density of 2 × x^v cells per well of a 96-well plate and cultured for an boosted v days. More than ninety% of cells in these cultures were neurons, equally judged by the presence of βIII-tubulin⁺, MAP2⁺ projections (Fig. 1e–h). Genome-wide mRNA expression profiles of v representative purified neuronal cultures were compared to the parental iPSC lines and samples from fetal brain, centre, liver and lung (Supplementary Fig. 7 and Supplementary Tabular array 2). Unsupervised hierarchical clustering analysis revealed that purified neurons most closely resembled fetal encephalon samples, in part due to a global upregulation of neuronal genes. Interestingly, the largest departure between fetal brain samples and purified neurons was downregulation in purified neurons of the hippo signalling cascade (~6.one fold), which regulates proliferation of cells such every bit NPCs and glia^{thirteen,fourteen}.

We determined multiple electrophysiological properties of purified neurons to assess passive membrane properties and synaptic connectivity (Fig. 1i, j, Supplementary Tabular array iii and Supplementary Fig. eight). Notably, virtually all neurons tested generated voltage-dependent action potentials and currents (Fig. 1i), which were blocked by tetrodotoxin (Supplementary Fig. 8). Transient bathroom application of ionotropic receptor agonists (25 μM muscimol or 10 μM AMPA) evoked transient currents, showing that purified neurons expressed functional GABA and AMPA receptors, respectively (Supplementary Table iii). To determine whether neurons were as well able to form functional synaptic contacts, nosotros analysed continuous whole-cell voltage clench recordings. We detected spontaneous inhibitory and/or excitatory synaptic currents in a subset of cells (~40%). Analysis of the kinetics of those events combined with reversible occludent using GABA_A or AMPA receptor antagonists demonstrated that the neurons not just fire activeness potentials only also made functional synaptic contacts (Supplementary Table iii). The electrophysiological results were supported by analysis of expression of protein markers of glutamatergic and GABAergic neuronal subtypes (VGluT1 and GABA, respectively), which were detected by immunofluorescence, with approximately 15% of cells staining brightly for VGluT1 and 8% for GABA, and most remaining neurons staining dimly for ane of the markers (Supplementary Fig. 9a). RNAs indicative of glutamatergic, GABAergic and cholinergic subtypes (that is, VGLUT1, GAD67 and CHAT, respectively) were detected by quantitative polymerase concatenation reaction (qPCR). Importantly, no significant differences in neuronal subtypes were detected between patients and controls (Supplementary Fig. 9b–f).

Elevated or altered secretion of amyloid-β peptides by fibroblasts is a feature common to all familial Alzheimer'due south affliction mutations identified so far^15,xvi. It is not known if iPSC-derived neurons from familial Alzheimer's disease patients maintain the elevated amyloid-β product seen in the parental fibroblasts. In sorry fibroblasts and other peripheral cells, APP expression and amyloid-β secretion are non consistently altered¹⁷. To determine if iPSC-derived neurons from APP^Dp and sAD patients showroom elevated amyloid-β secretion, amyloid-β levels in neuron-conditioned media were measured and normalized to total protein levels of cell lysates. Purified neurons from patients APP^Dp1 and APP^Dp2, each represented by three independently derived iPSC lines, secreted significantly higher levels of amyloid-β(1–xl) compared to mean NDC levels (Fig. 2a). Neurons from patient sAD2 too had significantly higher amyloid-β(1–40) levels compared to NDC neurons, even though no difference was observed between the fibroblasts of sAD2 and NDC individuals. We found that amyloid-β(ane–42) and amyloid-β(1–38) levels in these purified neuronal cultures were oft below the detection range of our assay, owing to the relatively modest number of neurons purified. Past cell type, neurons exhibited a larger departure in amyloid-β levels between APP^Dp and NDC than fibroblasts, further suggesting that fibroblasts are not fully predictive of neuronal phenotypes (Fig. 2b).

Increased amyloid-β, p-tau and aGSK-3β in sAD2 and APP^Dp neuronal cultures

a, Purified neurons from sAD2, APP^Dpi and APP^Dpii secrete increased amyloid-β(ane–40) (Aβ(1–40)) compared to NDC samples (P = 0.0012, 0.0014 and < 0.0001, respectively). b, Amyloid-β differences between patients and controls are larger in neurons versus fibroblasts. Data sets are relative to NDC mean. c, d, Neurons from sAD2, APP^Dpane and APP^Dp2 have increased aGSK-3β (per centum not-phospho-Ser nine) and p-tau/total tau (p-tau/t-tau) compared to NDC samples (aGSK-3β, P < 0.0001, 0.0005 and 0.0001; p-tau/full tau, P < 0.0001, 0.0001 and 0.0002). In a–d, n values on graphs bespeak the number of biological replicates per patient, contributed equally by iii iPSC lines. e, sAD2 findings verified in two additional iPSC lines (sAD2.4 and sAD2.v). sAD2(one-3) indicates findings from initial sAD2 iPSC lines. For amyloid-β, aGSK-3β and p-tau/total tau, sAD2 remained significantly higher than controls (P < 0.0001). No significant difference was found between original and secondary sAD2 lines (P = 0.14, 0.44, 0.63). f, Strong positive correlations between amyloid-β(1–40), aGSK-3β and p-tau/total tau in purified neurons. Pearson R = 0.94, 0.91 and 0.83, respectively. thou, Twenty-four hour treatment with β- and γ-secretase inhibitors reduced secreted amyloid-β(1–xl) compared to command treatment. β-Secretase inhibitors partially rescued aGSK-3β and p-tau/total tau in sAD2 and APP^Dpii neurons (P < 0.01 for aGSK-3β, P < 0.03 for p-tau). γ-Secretase inhibition did not significantly bear on aGSK-3β and p-tau/total tau. In chiliad, number of handling sets is indicated on the graph (n), NDCs are represented by two iPSC lines each and sAD2 and APP^Dptwo are represented by three. Error bars indicate s.e.1000.

Genetic evidence implicates altered or elevated APP processing and amyloid-β levels as the driving agents behind familial Alzheimer'due south illness^two and, because of identical neuropathology, desultory Alzheimer's disease. However, tau, although non genetically linked to Alzheimer's illness, forms neurofibrillary tangles, which correlate better with disease severity than plaque numbers¹⁸. The mechanism past which contradistinct APP processing might cause elevated p-tau and neurofibrillary tangle pathology is unclear. Tau phosphorylation at Thr 231, one of several tau phosphoepitopes, regulates microtubule stability^nineteen and correlates with both neurofibrillary tangle number and degree of cognitive reject^xx,21. To determine if tau phosphorylation at Thr 231 is elevated in APP^Dp and sAD neurons, nosotros measured the amount of p-tau(Thr 231) relative to total tau levels in lysates from purified neurons from three iPSC lines from each of the NDC, sAD and APP^Dp patients. Neurons from both APP^Dp patients had significantly college p-tau/total tau ratios than neurons from NDC lines (Fig. 2c). p-Tau/total tau in the two deplorable patients mirrored the amyloid-β findings: no difference was observed betwixt sAD1 and NDC neurons whereas sAD2 neurons had significantly increased p-tau/total tau.

Tau can be phosphorylated by multiple kinases. The kinase GSK-3β can phosphorylate tau at Thr 231 in vitro and co-localizes with neurofibrillary tangles and pre-tangle phosphorylated tau in deplorable postmortem neurons²². GSK-3β is thought to be constitutively agile but is inactivated when phosphorylated at Ser 9 (ref. 23). To determine if iPSC-derived neurons with elevated p-tau have increased GSK-3β action, the proportion of aGSK-3β in purified neurons was calculated by measuring the amount of GSK-3β lacking phosphorylation at Ser ix relative to total GSK-3β levels. Nosotros observed that neurons from patients APP^Dp1, APP^Dpii and sAD2 had significantly higher aGSK-3β than NDC neurons (Fig. second). The amyloid-β, GSK-3β and tau findings of sAD2 were verified by analysing an additional ii iPSC lines (sAD2.4 and sAD2.5; characterization in Supplementary Fig. 10), and we observed that levels remained consistently elevated (Fig. 2e). Results are detailed per patient in Supplementary Table 4a, per cell line in Supplementary Fig. 11, and per cell culture in Supplementary Table 5.

Although amyloid-β, p-tau and GSK-3β clearly have roles in Alzheimer's disease pathogenesis, their relationship is unclear. We observed that iPSC-derived neurons exhibited potent or very strong correlations between amyloid-β(1–40), p-tau/full tau and aGSK-3β levels (Fig. 2f and Supplementary Table 4b). We reasoned that if APP proteolytic products, such as amyloid-β or carboxy-terminal fragments (CTFs), have a causative role in p-tau and aGSK-3β elevation, then inhibiting γ- or β-secretase activity could reduce p-tau and aGSK-3β. We treated purified neurons from NDC1, NDC2, sAD2 and APP^Dp2 (2–3 iPSC lines each) with γ-secretase inhibitors (CPD-E and DAPT) or β-secretase inhibitors (βSi-II and OM99-2) for 24 h and measured amyloid-β, GSK-3β and p-tau/full tau compared to vehicle-treated samples. All inhibitors reduced amyloid-β(1–40) past similar levels (32–45% in patient samples) (Fig. 2g). Intriguingly, for both sAD2 and APP^Dp2 neurons, nosotros observed that β-secretase inhibitors significantly reduced aGSK-3β and p-tau/total tau (Fig. 2g, and shown per iPSC line in Supplementary Fig. 12). Neither γ-secretase inhibitor significantly differed from control-treated samples for aGSK-3β levels and p-tau/total tau.

We extended phenotypic characterization of sAD2 and APP^Dp by analysing endosomal and synaptic markers in FACS-purified neurons co-cultured with astrocytes for 12 days. Aggregating of large RAB5⁺ early endosomes in neurons has been observed in autopsies from desultory Alzheimer'due south affliction and some forms of familial Alzheimer's disease^24,25. As β-secretase is localized to endosomes and has an acidic pH optimum, it has been proposed that early endosomes potentially mediate the furnishings of APP processing on downstream pathologies such as increased p-tau, neurofibrillary tangles, synaptic loss and apoptosis²⁶; however, these hypotheses have been hard to exam straight without live, patient-specific neurons. To determine if early endosome phenotypes are present in iPSC-derived neurons from Alzheimer's affliction patients, purified neurons from NDC1, NDC2, sAD2 and APP^Dp2 (2 iPSC lines each) co-cultured with astrocytes were harvested and large and very large Rab5⁺ early on endosomes (1–2.ane μm³ and 2.1–seven μm^three) in neuronal soma were counted. Whereas control neurons more often than not had few Rab5⁺ structures >1 μmⁱⁱⁱ, neurons from both sAD2 and APP^Dp2 oftentimes had Rab5⁺ early on endosomes highly similar in volume, morphology and localization to what has been observed in autopsy samples (Fig. 3a–c). When compared, the neurons from both sAD2 and APP^Dp2 had significantly increased numbers of both large and very big early endosomes relative to controls (Fig. 3d). We sought to decide if neuronal cultures from sAD2 and APP^Dp2 likewise contained reduced levels of the presynaptic marker synapsin I. In Alzheimer's disease autopsies, synaptic loss is one of the strongest pathological correlates with dementia severity, and in regions of the encephalon affected past Alzheimer'south disease, the presynaptic marker synapsin I is decreased in patients versus controls^27,28. To analyse synapsin I levels in iPSC-derived neurons co-cultured with astrocytes, we quantified synapsin I⁺ puncta on MAP2⁺ dendrites (Fig. 3e). We found no significant difference betwixt NDCs and either sAD2 or APP^Dpii in the number of puncta per μm dendrite (Fig. 3f). Extended civilisation periods may be required to study Alzheimer's affliction-associated loss of synaptic proteins.

Assay of early endosome and synapsin levels in purified neurons co-cultured with astrocytes

a–c, Extended focus images of Rab5-stained neuronal soma from NDC1, sAD2 and APP^Dptwo. Arrowhead in b marks a one–ii.1 μm³ early endosome, and the arrow marks a 2.1–7 μm³ early endosome. Scale bars, 10 μm. d, Neurons from sAD2 and APP^Dp2 have significantly increased numbers of big and very large early on endosomes compared to NDC neurons (P < 0.0001, north = 40 neurons from two iPSC lines per individual). due east, Representative paradigm of synapsin I (green) on a MAP2⁺ dendrite (reddish). Arrowhead marks a synapsin I⁺ punctum. Scale bar, 3 μm. f, No significant difference between patients and controls in the number of synapsin⁺ structures per μm dendrite (P = one.00, north = twoscore dendrites from two iPSC lines per individual). Neurons were scored blinded to genotype. Fault confined indicate south.due east.m.

The results of this study provide potent evidence that iPSC engineering tin be used in concert with post-mortem samples and animate being models to study early pathogenesis and drug response in sporadic and familial Alzheimer'due south disease. In purified, electrophysiologically active neurons from one sporadic Alzheimer'south disease and ii APP^Dp patients, each represented by at least three clonally derived iPSC lines, nosotros observed significantly increased levels of three major biochemical markers of Alzheimer's disease: amyloid-β(i–40), aGSK-3β and p-tau/total tau. Increased sAD2 amyloid-β levels were not observed in the parental fibroblasts, suggesting a prison cell-blazon-specific phenotype. Among the individuals in this study, not only did potent correlations exist between amyloid-β(1–40), p-tau/total tau and aGSK-3β, just both p-tau/total tau and aGSK-3β levels were also partially rescued in neurons from sAD2 and APP^Dp following treatment with β-secretase inhibitors, suggesting that the APP processing pathway has a causative role in tau Thr 231 phosphorylation in homo neurons. Because γ-secretase inhibition did not cause a significant effect, products of APP processing other than amyloid-β may take a role in induction of GSK-3β action and p-tau. One potential culprit is the β-CTF, the levels of which correlate with axonopathies in mouse models harbouring APP duplications²⁹ and mediate early endosome accumulation in man Down syndrome fibroblasts³⁰. The observation that neurons from patients sAD2 and APP^Dp2 have early endosome phenotypes raises the question of how aberrant early endosomes relate to other phenotypes of Alzheimer'southward disease, such as axonopathies, synaptic loss and prison cell death, in homo neurons. Neurons and synapses rely heavily on endocytic pathways, and thus iPSC technology can now be used to written report the role of this dynamic process in alive patient-specific neurons. Ane bespeak of caution is that it is possible that the cultures of purified neurons that we generated and studied may not have been fully mature, every bit they lacked repetitive action potentials and had limited spontaneous activeness. Although some types of mature neurons likewise take these properties, it is conceivable that the phenotypes we observed might be modified past duration of in vitro culture. In this context, while there is contend about when Alzheimer's disease phenotypes initiate, bear witness exists that Alzheimer's affliction-similar pathology can occur in Down's syndrome fetuses as early as 28 weeks of gestation²⁴.

Our finding that the genome of patient sAD2, but non patient sAD1, generates pregnant Alzheimer'south disease phenotypes in purified neurons has important implications. Outset, this finding suggests that an unknown frequency of sporadic Alzheimer's affliction patients volition have genomes that generate strong neuronal phenotypes. The frequency of such genomes in the sporadic Alzheimer'south disease population cannot be adamant from the small sample size we written report and will require a larger sample size to ask how frequent such genomes are in the clinical population diagnosed with sporadic Alzheimer's disease. 2d, the genome of sAD2 clearly harbours one or more than variants that generate Alzheimer's disease phenotypes, which can thus be elucidated by future molecular genetic studies. Third, we speculate that desultory Alzheimer's illness might be sub-divided depending on whether neurons themselves are contradistinct, equally in the case of sAD2, equally opposed to other cell types such as astrocytes, which could be altered in other cases, for example, sAD1. Thus, time to come iPSC studies examining larger numbers of patients and controls have the potential to provide nifty insight into the mechanisms behind the observed heterogeneity in sporadic Alzheimer'southward affliction pathogenesis, the function of different cell types, patient-specific drug responses, and prospective diagnostics.

METHODS SUMMARY

iPSC generation and differentiation

Primary fibroblast cultures were established from dermal dial biopsies taken from individuals following informed consent and Institutional Review Board approval. To generate iPSCs, fibroblasts were transduced with MMLV vectors containing the complementary DNAs for OCT4, SOX2, KLF4, c-MYC and ± EGFP. IPSC-derived NPCs were differentiated for iii weeks, neurons were purified by FACS, and amyloid-β, p-tau/total tau and aGSK-3β were measured on purified control and mutant neurons from multiple lines cultured in parallel for an additional 5 days by multi-spot electrochemiluminescence assays (Meso Calibration Diagnostics). Early endosomes were analysed past confocal microscopy on purified neurons co-cultured with man astrocytes (Lonza) for 12 days. To ensure reproducible and consistent information, we found that it is of import to differentiate and evaluate neurons from full sets of mutant and control iPSC lines together.

Statistics

P < 0.05 was considered statistically meaning. Individuals were statistically compared to the total NDC pool by Tukey's test. Drug responses were compared to controls by Dunnett's method. Due north values signify the total number of dissever cultures analysed, with each iPSC line contributing equally to the total.

Supplementary Material

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Acknowledgments

We thank D. Galasko, M. Sundsmo, J. Rivera, J. Fontaine, C. Gigliotti and B. Yu at the Academy of California, San Diego (UCSD) Alzheimer'southward Disease Research Center for patient samples and data (grant AGO 5131); S. Dowdy and North. Yoshioka for viral vectors; B. Balderas at BD Biosciences for antibodies; C. Santucci and S. Nguyen for teratoma assay assistance; the UCSD Neuroscience Microscopy Shared Facility (grant P30 NS047101); and Planned Parenthood of the Pacific Southwest for fetal brain specimens. Funding was from California Institute of Regenerative Medicine (CIRM) comprehensive grants (M.Chiliad., F.H.K., L.S.B.M.), CIRM predoctoral fellowship (G.A.I.), FP7 Marie Curie IOF (C.B.), Weatherstone Foundation fellowship (K.Fifty.N.), National Institutes of Wellness K12 HD001259, the Hartwell Foundation (Fifty.C.L., F.Due south.B.), the Sentinel Fund and the McDonnell Foundation (F.H.Thou.). L.S.B.One thousand. is an investigator with the Howard Hughes Medical Plant.

Footnotes

Writer Contributions G.A.I. and L.S.B.M. conceived the project; K.A.I. and Fifty.S.B.Chiliad. designed the experiments; M.A.I., S.H.Y., C.B., S.M.R., Y.One thousand., C.H., G.P.H., South.V.Thou., K.Chiliad., Yard.Fifty.North. and F.S.B. performed the experiments; Thou.A.I., S.H.Y. and C.T.C. developed differentiation methods; A.Yard.R. and East.H.K. provided APP^Dp patient samples and information; F.H.G. supervised C.B. and Y.M.; K.K. supervised M.P.H. and Due south.V.G.; L.C.50. supervised K.L.N. and F.S.B.; M.A.I. and 50.S.B.G. wrote the manuscript; F.H.Thousand., Due east.H.One thousand. and A.1000.R. edited the manuscript.

Data have been deposited in the Gene Expression Autobus under accretion {"type":"entrez-geo","attrs":{"text":"GSE34879","term_id":"34879"}}GSE34879. Reprints and permissions information is available at world wide web.nature.com/reprints.

The authors declare no competing financial interests.

Readers are welcome to comment on the online version of this commodity at www.nature.com/nature.

Supplementary Data is linked to the online version of the paper at www.nature.com/nature.

Full Methods and whatsoever associated references are available in the online version of the paper at world wide web.nature.com/nature.

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Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3338985/

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