Research ArticleAlzheimer’s Disease

A Human Stem Cell Model of Early Alzheimer’s Disease Pathology in Down Syndrome

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Science Translational Medicine  07 Mar 2012:
Vol. 4, Issue 124, pp. 124ra29
DOI: 10.1126/scitranslmed.3003771

Abstract

Human cellular models of Alzheimer’s disease (AD) pathogenesis would enable the investigation of candidate pathogenic mechanisms in AD and the testing and developing of new therapeutic strategies. We report the development of AD pathologies in cortical neurons generated from human induced pluripotent stem (iPS) cells derived from patients with Down syndrome. Adults with Down syndrome (caused by trisomy of chromosome 21) develop early-onset AD, probably due to increased expression of a gene on chromosome 21 that encodes the amyloid precursor protein (APP). We found that cortical neurons generated from iPS cells and embryonic stem cells from Down syndrome patients developed AD pathologies over months in culture, rather than years in vivo. These cortical neurons processed the transmembrane APP protein, resulting in secretion of the pathogenic peptide fragment amyloid-β42 (Aβ42), which formed insoluble intracellular and extracellular amyloid aggregates. Production of Aβ peptides was blocked by a γ-secretase inhibitor. Finally, hyperphosphorylated tau protein, a pathological hallmark of AD, was found to be localized to cell bodies and dendrites in iPS cell–derived cortical neurons from Down syndrome patients, recapitulating later stages of the AD pathogenic process.

Introduction

Alzheimer’s disease (AD) is a major global health problem for which there are no disease-modifying treatments. A key challenge for developing effective treatments for AD is that the etiology and pathogenesis of the sporadic form of the disease are not well understood. The classic pathological hallmarks of AD are amyloid plaques composed of amyloid-β (Aβ) peptides, which are products of the transmembrane amyloid precursor protein (APP), and intracellular neurofibrillary tangles composed of hyperphosphorylated forms of the microtubule-associated protein tau (1). There is an ongoing debate as to the pathogenic process in AD and the relative contributions of Aβ peptides and tau (2). Support for a causal role for Aβ peptides in AD pathogenesis comes from studies of autosomal dominant forms of familial AD. Mutations that cause the rare familial forms of AD are all found either in APP or in components of the enzyme complex that processes APP; all of these mutations result in an increase in Aβ peptide production and aggregation (3). In contrast, mutations in the tau protein lead to neurodegenerative disorders that are phenotypically distinct from AD including frontal temporal dementia and progressive supranuclear palsy (4).

Animal models, although useful for modeling aspects of APP processing in AD, have been of limited use in developing treatments for AD (5). Rodent models do not capture many key aspects of the disease process, and only triple transgenic mice that express mutant forms of human APP, presenilin, and tau develop both plaque and tangle pathology in brain tissue (5). Therefore, it has been argued that these models are useful for studying the initiation of AD, but not the disease process itself (5). A human cellular model of AD would enable detailed functional studies of AD pathogenesis. An effective human cellular model would use the appropriate cell type, in this case, glutamatergic projection neurons of the human cerebral cortex, would develop relevant molecular pathology (altered APP processing, Aβ aggregation, and tau hyperphosphorylation), and would do so in a reproducible manner over a time scale short enough for practical use. A pressing question for the usefulness of this approach is whether neurological diseases that take decades to become manifest in humans can be successfully modeled over a reasonable time scale (6, 7).

Here, we report an in vitro human cellular model of AD pathogenesis in Down syndrome. We applied a process that we developed for directed differentiation of human induced pluripotent stem (iPS) cells into cerebral cortex projection neurons (8). We generated cortical neurons from iPS cells derived from patients with Down syndrome caused by trisomy of chromosome 21. Down syndrome is the commonest genetic cause of mental retardation, occurring in about 1 in 700 to 800 live births (9). Individuals with Down syndrome also have a very high incidence of AD (10), attributed in part to the presence of three copies of the gene on chromosome 21 encoding the APP (11, 12). Duplication of the APP gene in humans results in autosomal dominant, early-onset dementia (13), and mice with increased APP gene dosage develop amyloid plaques and the neuropathological hallmarks of Alzheimer-type dementia (14). A number of other genes on chromosome 21 may also contribute to the greatly increased risk of dementia in Down syndrome patients including that encoding the Dyrk1A kinase that phosphorylates tau (15).

Increased production of Aβ peptides from processing of APP has been observed in children with Down syndrome, with plaques characteristic of AD pathology present in the nervous system as early as the teen years (16). Given the early onset of amyloid pathology in Down syndrome, we hypothesized that cortical neurons generated from iPS cells derived from these patients could potentially develop phenotypes typical of AD quickly in vitro.

Results

Cortical neurons from Down syndrome iPS cells

To investigate the potential for modeling AD pathology in Down syndrome, we differentiated human healthy control (17) and Down syndrome iPS cells (DS1-iPS4) (18) into cortical neural stem and progenitor cells (Fig. 1, A to H). Control and Down syndrome iPS cells (referred to here as DS-iPS cells; fig. S1) generate cortical neuronal stem and progenitor cells at high efficiency, defined by expression of Pax6, vimentin, and Otx1/2 (Fig. 1, A to D). As described for human iPS cells and embryonic stem (ES) cells (8), control and DS-iPS cell–derived cortical neural stem cells develop into polarized neuroepithelial rosettes (Fig. 1, E and F), with a subpopulation of basal progenitor cells that express the basal progenitor cell–specific transcription factor Tbr2/Eomes (Fig. 1, G and H).

Fig. 1

Directed differentiation of iPS cells into cortical neurons. Directed differentiation of healthy control and DS-iPS cells into cerebral cortex projection neurons. (A to F) iPS cells from healthy individuals (control) and Down syndrome patients were induced to differentiate into cortical neural stem and progenitor cells expressing Pax6 over 15 days. The stem and progenitor cells formed polarized neuroepithelial rosettes expressing Pax6 (green; A and B), vimentin (red; A and B), Otx1/2 (green; C and D), and CD133 (A to F). No difference in differentiation efficiency was noted between the control and the DS-iPS cell lines. Scale bars, 50 μm. (G and H) Within the neuroepithelial rosettes generated from control (G) and DS-iPS (H) cells was a subpopulation of proliferating basal progenitor cells (Ki67-positive; turquoise) expressing the transcription factor Tbr2 (red), in addition to a population of newly born neurons that expressed Tbr2 and doublecortin (Dcx, green). Scale bar, 50 μm. (I and J) Differentiation of early-born, layer 6 glutamatergic cortical projection neurons expressing Tbr1 (red) from healthy control (I) and DS-iPS cell–derived cortical neural stem cells (J). Scale bar, 50 μm. (K to N) Control (K and M) and DS (L and N) iPS cells generate later-born neurons of upper cortical layers defined by expression of the layer-specific transcription factors Cux1 (turquoise), Brn2 (red), and Satb2 (green). Scale bar, 50 μm. (O and P) Astrocytes expressing S100 (red) were generated last in both control (O) and DS-iPS cell–derived cortical neuronal cultures (P). Scale bar, 50 μm. DAPI, 4′,6-diamidino-2-phenylindole. (Q) About equal numbers of deep and upper layer cortical neurons were generated in both control and DS-iPS–cell derived cortical neuronal cultures. Deep layer transcription factors: Tbr1 and Satb2; upper layer transcription factors: Brn2 and Satb2. For each cell-specific marker, a minimum of n = 3 experiments were counted. Error bars, SEM.

Under neuronal differentiation conditions, control and DS-iPS cell–derived cortical neural stem cells efficiently differentiate into glutamatergic projection neurons of each cortical layer (Fig. 1, I to N and Q) and then generate astrocytes (Fig. 1, O and P). As previously described (8), all neurons generated by control and DS-iPS cells using this process are glutamatergic (fig. S2). Mouse models of Down syndrome have suggested that DS cortical neural stem and progenitor cells produce proportionally fewer upper layer cortical neurons (19, 20), although it is not clear that this occurs in humans with trisomy 21. DS-iPS cells did not demonstrate a significant neurogenic or neuronal differentiation phenotype. In particular, no difference was observed in the numbers of neurons produced or in the relative numbers of different classes of projection neurons generated by DS-iPS cells (Fig. 1Q).

Synapse formation by cortical neurons derived from DS-iPS cells

DS-iPS cell–derived cortical neurons mature in culture, as shown by their acquisition of the ability to fire trains of action potentials upon stimulation with a current (Fig. 2, A and B). These neurons also form functional synapses, as evidenced by the presence of miniature excitatory postsynaptic currents in cultures 45 to 100 days old (Fig. 2, C and D). The formation of physical synapses among cortical projection neurons derived from DS-iPS cells was confirmed using super-resolution (structured illumination) microscopy to visualize the localization of pre- and postsynaptic proteins. The localization of postsynaptic proteins on or within a few 100 nm of the dendritic shaft, as well as closely juxtaposed pre- and postsynaptic proteins, was used to define the synapses (Fig. 2, E to J). The glutamatergic postsynaptic density protein PSD95 was found to be localized close to MAP2-positive dendrites in neurons derived from DS-iPS and control iPS cells (Fig. 2, E and F). In addition, both PSD95 and Homer1, another protein enriched in excitatory postsynaptic compartments, were frequently found in close association with the presynaptic proteins synaptophysin and Munc13-1 (Fig. 2, E to J). No difference in the density of synapses per unit length of dendrite was found between DS-iPS cell–derived neurons and neurons generated from four different control iPS cell lines (Fig. 2K).

Fig. 2

Synapse formation by DS-iPS cell–derived cortical neurons. (A and B) Control and DS-iPS cell–derived cortical neurons become functionally mature in vitro, firing trains of action potentials upon stimulation with a current (control, n = 15 neurons; Down syndrome, n = 23 neurons). A representative example of a single neuron’s response to step current stimulation is shown. Voltage and time scales are as shown. (C and D) Detection of miniature excitatory postsynaptic currents by whole-cell recordings from control (C) or DS-iPS cell–derived cortical neurons (D) (average of n = 4 neurons). Representative recordings from single neurons are shown. (E and F) Super-resolution microscopic images of dendrites (MAP2, green) from iPS cell–derived cortical neurons showing localization of foci of PSD95 (red), an excitatory synapse-specific protein. (G to J) Physical synapses were identified as containing juxtaposed pre- and postsynaptic protein complexes of >100 nm in width containing either synaptophysin (red) and PSD95 (green) (G and H) or Munc13 (red) and Homer (green) (I and J). Scale bar, 1 μm. (K) Synapse density was measured by the number of PSD95+ foci per unit dendrite length. No difference was found between cortical neurons derived from DS-iPS cells and from four different control iPS cell lines (control 1, 2F8; control 2, BBHX; control 3, JRO; control 4, CRL).

Increased Aβ peptide generation in DS-iPS cell cortical neurons

A key stage in AD pathogenesis in vivo is the increased generation of short Aβ peptides (38 to 43 amino acids in length) from APP by glutamatergic neurons in the cerebral cortex. These peptides form soluble and insoluble aggregates, or amyloid plaques (12), that are deposited in brain tissue. We monitored the time course of the extracellular accumulation of Aβ40 and Aβ42 peptides by cortical neurons derived from stage- and cell density–matched control iPS and DS-iPS cell cultures over a period of 2 weeks from the onset of neuronal differentiation (Fig. 3A). Healthy cortical neurons typically produce considerably more Aβ40 than Aβ42 in vivo (21). Extracellular concentrations of Aβ40 peptides are low in both control and DS cortical neuronal cultures at the onset of neuronal differentiation (Fig. 3A). However, DS cortical neurons increased their production of Aβ40 to high levels over the subsequent 10 days, reaching an average of 233 pg/ml by day 28 of differentiation (n = 3 for each 48-hour window of production and collection), whereas control neuron production of Aβ40 remained consistently low at less than 100 pg/ml (n = 3 for each time point; Fig. 3A; P < 0.01).

Fig. 3

Increased Aβ peptide production by DS-iPS cell–derived cortical neurons. (A) Secretion of Aβ40 peptides by control and DS-iPS cell–derived cortical neurons (n = 3 cultures for each cell line). Cell culture media were collected every 48 hours to measure Aβ40 peptide concentrations using a sandwich ELISA assay. Cell culture media were completely refreshed every 48 hours such that Aβ40 concentrations reflect secretion and accumulation over a 48-hour period. The green arrowhead indicates the onset of overt neuronal differentiation in these cultures. Asterisks indicate significant differences (Student’s t test) in Aβ40 concentrations between control and DS-iPS cell–derived cortical neurons for time points between 14 and 28 days of differentiation. (B) Cortical neurons derived from DS-iPS cells produce large amounts of soluble extracellular Aβ40 and Aβ42 peptides by day 70 of differentiation, in contrast to fibroblasts from Down syndrome patients and cultures of cortical neurons derived from healthy control iPS cells and matched for developmental stage and cell density. Asterisks indicate statistically significant differences (Student’s t test) in production of both Aβ40 and Aβ42 between control and DS-iPS cell–derived cortical neurons. Inhibition of the γ-secretase protease with DAPT for 4 days reduced secretion of both Aβ40 and Aβ42 peptides by about half in cortical neurons derived from DS-iPS cells. Inhibition of γ-secretase for 21 days reduced production of both Aβ peptides to undetectable levels (P < 0.01, Student’s t test).

Extracellular accumulation of pathogenic Aβ42 peptide from control or DS cortical neurons was not detected at this early stage of neuronal differentiation. However, analysis of the extracellular medium from older (day 70) cultures of DS and control iPS cell–derived cortical neuron cultures confirmed that older DS neurons produce large amounts of both Aβ40 (mean of 843 pg/ml, n = 3) and Aβ42 (mean of 183 pg/ml, n = 3) in a 48-hour period (Fig. 3B), whereas control cortical neurons continue to generate low concentrations of Aβ40 (control iPS cell–derived neurons, mean of 124 pg/ml, n = 3) and Aβ42 (control iPS cell–derived neurons, mean of 32 pg/ml, n = 3; Fig. 3B; P < 0.01). High-level production of Aβ peptides was specific to DS cortical neurons, because the production of Aβ40 and Aβ42 peptides by fibroblasts from Down syndrome patients was low and undetectable, respectively (Fig. 3B).

To validate the DS-iPS cell–based model of the early stages of AD pathogenesis, we asked whether Aβ40 and Aβ42 peptide generation by DS-iPS cell–derived cortical neurons could be reduced by pharmacological inhibition of the γ-secretase complex, one of the two essential protease complexes that process APP to generate Aβ peptides. Inhibition of γ-secretase also inhibits Notch signaling in neural stem cells, resulting in cell cycle exit and differentiation (22). Therefore, the γ-secretase inhibitor DAPT (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester) was administered at a stage (day 50; as we have shown in multiple iPS cell lines) when about 70% of cells in culture are postmitotic neurons (8). DAPT inhibition of γ-secretase over 4 days reduced Aβ40 and Aβ42 peptide production from DS cortical neurons by almost half, whereas longer-term treatment (21 days) reduced secretion of both Aβ peptides to below detectable levels (Fig. 3B).

DS-iPS cell–derived cortical neurons generate Aβ aggregates

Given the increased generation of Aβ40 and Aβ42 peptides by DS-iPS cell–derived cortical neurons over time, we assayed the development of amyloid plaques in these neurons in culture by live staining of intracellular and extracellular aggregates of amyloid with the thioflavin T analog BTA1 (23) (Fig. 4, A and B). Over a 2-month period after the initiation of cortical differentiation, BTA1-positive neurons or extracellular aggregates were not observed in cultures of control iPS cell–derived cortical neurons (Fig. 4A). However, DS-iPS cell–derived cortical neurons generated both intracellular and extracellular aggregates of BTA1-labeled amyloid over the same time period (Fig. 4B). Polyclonal and monoclonal antibody staining and confocal microscopy demonstrated the presence of numerous Aβ42-containing aggregates both within and outside DS-iPS cell–derived cortical neurons (Fig. 4, C to F, and fig. S3), with extracellular Aβ42-positive aggregates often found around neurites (Fig. 4F), confirming that Aβ42 is produced by DS cortical neurons and forms aggregates over time. Aβ42-positive aggregates were rarely found in cultures of control iPS cell–derived cortical neurons (Fig. 4C and fig. S3) or in cultures of DS fibroblasts (fig. S3).

Fig. 4

Formation of amyloid aggregates by DS-iPS cell–derived cortical neurons. (A and B) Live staining of amyloid aggregates in control iPS (A) and DS-iPS–cell derived cortical neurons (B) after 90 days in culture using the thioflavin analog BTA1. White arrows in (B) indicate BTA1-positive aggregates in the DS-iPS cell–derived cortical neuronal cultures. Scale bar, 100 μm. (C and D) Aβ42-positive staining (green) is infrequently observed in cultured cortical neurons derived from control iPS cells (C). In contrast, large numbers of intracellular and extracellular deposits of Aβ42 (green) were found in cultures of DS-iPS cell–derived cortical neurons between 60 and 90 days of culture (D). Scale bars, 50 μm [(C) and (D)]. (E) Ninety-day-old cultures of DS-iPS cell–derived cortical neurons showed extracellular amyloid aggregates (green) indicated by white arrows. Some amyloid aggregates were found outside neurons in regions lacking DAPI-positive nuclei and Tuj1-positive neurites, as can be seen in the X-Z and Y-Z projections shown on the top and left sides of the main image. Scale bar, 20 μm. (F) A three-dimensional rendering of a series of confocal images of 90-day cultures of DS-iPS cell–derived cortical neurons showing extracellular amyloid plaques (green; indicated by white arrows) around cortical neurites. Scale bar, 20 μm.

AD pathogenesis is recapitulated by Down syndrome ES cells

Variation among iPS cell lines at the epigenetic level, together with the occurrence of genetic mutations during reprogramming from adult fibroblasts, has been suggested to be an important factor in interpreting the reliability of disease-related phenotypes observed in iPS cell systems (2426). To investigate the reproducibility of the AD phenotypes observed from DS-iPS cells and their association with the iPS cell state, we also generated cortical neurons from Down syndrome and control (H9) human ES cells (hESCs) (fig. S4).

Down syndrome ES cells (DS-ES cells) differentiate efficiently into cortical projection neurons in numbers equivalent to those observed for iPS cells (Fig. 5, A and B). Control and DS-ES cell–derived cortical neurons do not display marked differences in the expression or cellular localization of full-length APP protein (Fig. 5, C and D). However, extracellular and intracellular aggregates of Aβ42 peptides are abundant in DS-ES cell cortical neuronal cultures (Fig. 5, E, F, and I) and have the same distribution as seen for DS-iPS cell–derived cortical neuronal cultures. DS-ES cell– and DS-iPS cell–derived cortical neurons produce similar amounts of Aβ40 and Aβ42 peptides over a 48-hour collection period (Fig. 5G). These amounts are more than fourfold higher than the amounts produced by control ES cell– and iPS cell–derived neurons (Fig. 5G). Notably, in addition to the increased secretion of both Aβ40 and Aβ42 peptides, there was also a marked reduction in the Aβ40/Aβ42 peptide ratio in both DS-ES cell– and DS-iPS cell–derived cortical neuronal cultures (from 6.6 and 6.9 in control ES and iPS cell cultures to 4.7 and 4.4 in DS-ES and DS-iPS cell cultures). This demonstrates that DS cortical neurons do not simply increase their overall production of Aβ peptides but also disproportionately increase their secretion of the pathogenic Aβ42 peptide. The increased production of Aβ peptides is accompanied by an increase in release of the soluble APPβ (sAPPβ) fragment of APP into the extracellular medium (Fig. 5H), as would be predicted for increased processing of APP proteins by the β-secretase protease (27).

Fig. 5

DS-ES cell–derived cortical neurons recapitulate AD phenotypes. (A and B) ES cells from Down syndrome patients differentiate into (A) deep layer cortical projection neurons expressing Tbr1 (red) and CTIP2 (green), and (B) upper layer cortical projection neurons expressing Brn2 (red) (a few also express Satb2, green). Representative images of day 55 cultures are shown. Scale bar, 50 μm. (C and D) Similar expression and localization patterns for APP protein (green) were seen in control (H9) and DS-ES cell–derived cortical neurons. Scale bar, 50 μm. (E and F) Intracellular and extracellular deposits of Aβ42 (green; white arrows) are found in cultures of DS-ES cell–derived cortical neurons (F) but not control ES cell–derived cortical neurons (E) at day 55. Scale bar, 50 μm. (G) Both DS-ES cell– and iPS cell–derived neurons produced about fourfold more Aβ40 and Aβ42 peptides (measured by sandwich ELISA) over a 48-hour period in culture compared to age-matched control ES cell– and iPS cell–derived neurons. Differences between matched ES cell– and iPS cell–derived cortical neuronal cultures were significant (P < 0.01, Student’s t test). Aβ40 and Aβ42 concentrations are expressed relative to the total amount of cellular protein in each culture to control for cell number. Numbers above each set of samples denote the Aβ40/Aβ42 ratio for each cell type (calculated from n = 3 cultures for each line). (H) β-Secretase cleavage of APP was measured by release of sAPPβ into the extracellular medium. The amount of sAPPβ produced was significantly higher in DS-ES cell– and iPS cell–derived cortical neurons compared with their respective control iPS neurons (P < 0.01, Student’s t test). (I) Aβ42 aggregates in control and DS-ES cell– and iPS cell–derived cortical neuronal cultures were calculated as a function of cell number (day 62; n = 3 for each cell line). The number of aggregates was greater in both DS-ES cell– and iPS cell–derived neuronal cultures compared to their respective controls (P < 0.05, Student’s t test).

Hyperphosphorylation and redistribution of tau in DS-iPS cell–derived cortical neurons

Hyperphosphorylation of the microtubule-associated protein tau, dissociation of tau from axonal microtubules, and redistribution of hyperphosphorylated tau to the neuronal soma and dendritic tree are well-described later-stage pathologies in AD (28). Detection of tau phosphorylated at Ser202 and Thr205 using the AT8 antibody and immunofluorescence and confocal microscopy (29) in control and DS-iPS cell– and DS-ES cell–derived cortical neurons revealed abnormal localization in the neurons derived from Down syndrome patients (Fig. 6, A to L). Costaining with axonally localized Tuj1 and dendritically localized MAP2 was used to define the subcellular localization of tau staining positively with the AT8 antibody (AT8+ tau) in control and DS cultures. AT8+ tau was found in both DS and control neurons, but it was aberrantly localized into linear foci in the cell bodies and dendrites of DS-iPS and DS-ES cortical neurons (Fig. 6, K and L) compared with a diffuse localization primarily to axons in control cultures (Fig. 6, A to F).

Fig. 6

Phosphorylation and redistribution of tau in DS-ES cell– and DS-iPS cell–derived cortical neurons. (A to L) Confocal microscopic images for tau phosphorylated at Ser202 and Thr205 detected using AT8 antibody (green) (29) with counterstaining for the dendritic protein MAP2 (red) in age-matched control and DS-ES cell– and iPS cell–derived cortical neuronal cultures. Dual-color (A, D, G, and J) and single-channel images (B, C, E, F, H, I, K, and L) are shown for clarity. Linear foci of phosphorylated tau (Ser202 and Thr205; AT8+) were observed in dendrites (MAP2-positive) in DS-ES cell– and DS-iPS cell–derived cortical neurons [white arrow in (H)], but not in control neurons (B and E). Phosphorylated tau (AT8+) is also found in the cell body, axon, and dendrites of neurons in DS-ES cell– and DS-iPS cell–derived neuronal cultures [white arrowheads in (K) and (L)], but is found at much lower levels and not in the cell body and dendrites of control neurons. Scale bar, 50 μm. (M to O) Soluble tau protein is found in the extracellular medium of DS-ES cell– and DS-iPS cell–derived neurons at higher levels than in control cultures (I) (P < 0.01, Student’s t test). Two different phosphorylated tau epitopes, pSer396 and pThr231, were detected in the extracellular medium of DS-ES cell– and DS-iPS cell–derived cortical neurons (J and K). Neither form of tau was found in the culture medium of control ES cell– and iPS cell–derived cortical neurons (I) (P < 0.01, Student’s t test). In each case, the total amount of each form of tau is expressed relative to the total amount of cellular protein in each culture to control for cell number. (P) Quantification of cell death in control and DS-ES cell– and iPS cell–derived cortical neuronal cultures (day 62; n = 3 for each line). Apoptotic cells with pyknotic condensed nuclei associated with cleaved caspase-3 (aCasp3+) were counted as a percentage of all cells. The proportion of apoptotic cells was increased (P < 0.05, Student’s t test) in both DS-ES cell– and iPS cell–derived cortical neuronal cultures compared to their respective controls.

Given that soluble phosphorylated tau is commonly found in the cerebrospinal fluid (CSF) of AD patients (30), potentially due to synaptic dysfunction or cell death, we performed enzyme-linked immunosorbent assays (ELISAs) to assess the concentrations of total and phosphorylated tau in the culture medium of DS-iPS cell– and DS-ES cell–derived compared to age-matched control cortical neurons (day 55). Total extracellular tau was three- to fourfold higher in medium collected over a 48-hour window from DS-iPS and DS-ES cortical neurons compared to control cortical neurons (P < 0.01; Fig. 6M). In addition, two forms of phosphorylated tau, pSer396 and pThr231, were only detectable in the medium from DS neurons and not from matched control cultures (Fig. 6, N and O). In age-matched cultures, neuronal cell death was increased about twofold in DS-iPS cell– and DS-ES cell–derived cortical neurons (Fig. 6P and fig. S5), suggesting that apoptosis and cell lysis of neurons are major contributors to the appearance of tau in the extracellular medium.

Discussion

We report here the reproducible development of AD pathology in cortical neurons generated from iPS cells derived from Down syndrome patients, including neuron-specific Aβ peptide production, Aβ42 aggregate formation, and altered tau protein phosphorylation and localization. These pathologies were developed by both DS-iPS cell– and DS-ES cell–derived cortical neurons, demonstrating that these pathologies are reproducible and are not influenced by the variations and mutations introduced by the cellular reprogramming strategy. The increased Aβ peptide production by DS-iPS cell–derived cortical neurons can be reversed by γ-secretase inhibition, demonstrating the usefulness of this system for testing new disease intervention strategies and for drug screening.

A marked finding in this study is that high-level secretion of Aβ peptides and the formation of amyloid aggregates are specific to DS neurons; production by DS fibroblasts, DS-iPS cells, and DS cortical neural stem cells of Aβ40 peptide is low and of Aβ42 peptide is undetectable. This is particularly noteworthy given the ubiquitous expression of APP in all tissues. AD pathology only occurs in the brain and provides a point of entry for studying both the nervous system specificity of AD and the selective vulnerability of different parts of the brain to AD.

Recent reports have shown that neurons generated from individuals with autosomal dominant, early-onset AD also display increased Aβ peptide secretion at a relatively early stage (3133), suggesting that this early aspect of AD pathogenesis is a general property of neurons. Using iPS cells generated from two patients with familial AD, a recent study has shown that heterogeneous cultures of neurons of a number of different types increase production of Aβ40 peptides and alter tau phosphorylation (32). Using DS-iPS cells differentiated into cerebral cortex neurons, the major cell type affected in the disease, we report here increased production of both Aβ40 and the more pathogenic Aβ42 peptide. This was accompanied by a change in the Aβ40/Aβ42 ratio, demonstrating that DS cortical neurons disproportionately increase Aβ42 production, as seen in AD in vivo. Increased phosphorylation of tau is a feature common to the familial AD model (32) and the Down syndrome model reported here. In addition, we observed relocalization of phosphorylated tau to the cell body and dendrites of Down syndrome cortical neurons, as occurs in the AD brain.

Notably, the development of AD pathologies in Down syndrome cortical neurons is accelerated in culture; for example, the formation of Aβ42 aggregates occurs in this system over a period of months. Why this aspect of the disease process is accelerated in culture is a focus of ongoing work but may be due to the increased production of reactive oxygen species by Down syndrome neurons in culture (34). In addition, children with Down syndrome already have significant increases in soluble Aβ42 in early life (35), and amyloid plaques can be found in teenagers with Down syndrome (16). Therefore, individuals with Down syndrome show a predisposition to the early development of amyloid phenotypes compared with the age of onset of dementia, which typically occurs in middle age in these individuals (36). The combination of the lack of clearance mechanisms for Aβ peptides in this culture system, together with the rapid increase in Aβ peptide production from DS-iPS cell–derived cortical neurons, may account for the relatively rapid appearance of Aβ aggregates in this system. Similarly, whereas it is possible that the changes in tau phosphorylation and localization observed here are solely dependent on the increased Aβ peptide secretion by DS cortical neurons, increased expression of the Dyrk1A kinase may accelerate that process (15), underlining the complexity of AD pathogenesis and dementia development in Down syndrome.

A key question arising from this work is whether the development of Aβ peptide secretion and aggregation phenotypes that we have reported here are a general property of iPS cells from Down syndrome individuals. We demonstrate here that both DS-iPS cell– and DS-ES cell–derived cortical neurons can specifically, robustly, and reproducibly develop the Aβ phenotype compared with DS fibroblasts and control iPS cell– and hESC–derived cortical neurons. In that respect, the DS-iPS cell–derived cortical neurons reproduce the observed in vivo phenotypes (16). However, the incidence of overt dementia in the Down syndrome community is the subject of debate, because it has historically been challenging to diagnose dementia in severely learning disabled people. A range of incidence rates has been reported up to as high as 100% in Down syndrome patients older than 50 years (36, 37). It will be of interest in future work to investigate the penetrance of Aβ phenotypes in cortical neurons generated from iPS cells derived from cohorts of individuals with Down syndrome with and without confirmed diagnoses of dementia in later life.

In conclusion, we report here the use of iPS cell– and ES cell–derived cortical neurons to model AD pathogenesis in Down syndrome. This approach provides a potentially powerful in vitro system for functional analyses of pathways regulating Aβ42 peptide production in human cortical neurons, cellular mechanisms regulating disease pathogenesis, and the identification and testing of candidate disease-modifying compounds.

Materials and Methods

Culture of human iPS cells

hESC research was approved by the Steering Committee for the UK Stem Cell Bank and for the Use of Stem Cell Lines and carried out in accordance with the UK Code of Practice for the Use of Human Stem Cell Lines. Human DS-ES cells (SC-321 cell line) were derived by the Reproductive Genetics Institute (Chicago) under their Institutional Review Board approval for the Orkin laboratory, and the use of cells was approved by Children’s Hospital Embryonic Stem Cell Research Oversight Committee (ESCRO; January 2007). Culture of hESCs (H9, WiCell Research Institute, and SC-321, DS-ES cells) and hiPS cell lines [DS1-iPS4, Harvard Stem Cell Institute (18); BBHX and CRL healthy control iPS lines, provided by L. Vallier, MRC Laboratory for Regenerative Medicine, Cambridge (17)] was carried out on mitomycin-treated mouse embryonic fibroblasts (MEFs) according to standard methods (38). Briefly, cells were maintained in hESC medium (all components were from Invitrogen unless otherwise stated): Dulbecco’s modified Eagle’s medium/F12 containing 20% knockout serum replacement, fibroblast growth factor 2 (FGF2) (6 ng/ml) (PeproTech), 1 mM l-glutamine, 100 μM nonessential amino acids, 100 μM 2-mercaptoethanol, penicillin (50 U/ml), and streptomycin (50 mg/ml).

Directed differentiation of human ES and iPS cells

Directed differentiation of hESCs and iPSCs to cerebral cortex was carried out as described (8). Briefly, dissociated pluripotent stem cells were plated on Matrigel (BD)–coated 12-well plates in MEF-conditioned hESC medium with FGF2 (10 ng/ml). Neural induction was initiated by changing the culture medium to a culture medium that supports neural induction, neurogenesis, and neuronal differentiation, a 1:1 mixture of N2- and B27-containing media (8). Culture media were supplemented with mouse Noggin-CF chimera (500 ng/ml) (R&D Systems) and 10 μM SB431542 (Tocris) to inhibit transforming growth factor–β (TGFβ) signaling during neural induction (38). Neuroepithelial cells were harvested by dissociation with Dispase and replated in 3N medium including FGF2 (20 ng/ml) on polyornithine- and laminin-coated plastic plates. FGF2 was withdrawn to promote differentiation. Cultures were passaged once more with Accutase, replated at 50,000 cells/cm2 on polyornithine- and laminin-coated plastic plates, and maintained for up to 100 days with a medium change every other day.

Immunocytochemistry and imaging

Cultures were fixed in 4% paraformaldehyde in phosphate-buffered saline or in ice-cold methanol and processed for immunofluorescence staining and confocal microscopy. Antibodies used for this study are as follows: Tbr1 (Abcam), Tbr2 (Millipore), CTIP2 (Abcam), prominin/CD133 (Abcam), phosphorylated histone H3 (Abcam), γ-tubulin (Abcam), Pax6 (Chemicon), Oct4 (Abcam), Ki67 (BD), vimentin (Abcam), Otx1/2 (Millipore), doublecortin (Santa Cruz), β-tubulin III (Chemicon), β-tubulin III (Covance), vGlut1 (Synaptic Systems), Cux1 (Santa Cruz), Brn2 (Santa Cruz), Satb2 (Abcam), cleaved caspase 3 (Cell Signaling), C terminus of Aβ42 polyclonal (Chemicon) and monoclonal (Covance), phosphorylated tau (AT8, Pierce), Homer1 (Synaptic Systems), Munc13 (Synaptic Systems), PSD-95 (Abcam), synaptophysin (Abcam), and MAP2 (Abcam). Secondary antibodies used for primary antibody detection were species-specific Alexa dye conjugates (Invitrogen).

For quantifying numbers of cells expressing specific cortical neuronal markers, cell cultures were dissociated into single cells with Accutase and resuspended at a density of 100,000 cells/ml. Cells (20,000) were plated onto each polylysine-coated glass slide with a Cytospin Centrifuge (Thermo Scientific) and fixed and stained for confocal microscopy. For live staining of amyloid in neuronal cultures, BTA1 (Sigma) in dimethyl sulfoxide was added to a final concentration of 100 nM for 20 min before washing and imaging. Super-resolution microscopy imaging of synaptic proteins was carried out with standard fixation and staining techniques and visualized with a Deltavision OMX system (Applied Precision).

Quantifications of Aβ40, Aβ42, sAPPβ, total tau, pT231-Tau, and pS396-tau were carried out with commercial sandwich ELISAs (Millipore, Covance, and Invitrogen) using 50 μl of cell culture supernatant from cultures of DS-iPS, DS-ES, iPS control, and H9 hES cortical neurons. Inhibition of γ-secretase was carried out in DS cortical cultures by addition of 10 μM DAPT (Calbiochem) every 48 hours from day 50 of differentiation onward.

Electrophysiology

Whole-cell current clamp recordings were performed at room temperature in artificial cerebral spinal fluid containing 125 mM NaCl, 25 mM NaHCO3, 1.25 mM NaH2PO4, 3 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 25 mM glucose, and 3 mM pyruvic acid, bubbled with 95% O2 and 5% CO2. Borosilicate glass electrodes (resistance, 6 to 10 megohms) were filled with an intracellular solution containing 135 mM K-gluconate, 7 mM NaCl, 10 mM Hepes, 2 mM Na2ATP, 0.3 mM Na2GTP, and 2 mM MgCl2. Cells were viewed with a BW50WI microscope (Olympus) with infrared differential interference contrast optics. Recordings were made with a Multiclamp 700A amplifier (Molecular Devices). Signals were filtered at 6 kHz, sampled at 20 kHz with 16-bit resolution, and analyzed with Matlab (MathWorks).

Supplementary Materials

www.sciencetranslationalmedicine.org/cgi/content/full/4/124/124ra29/DC1

Fig. S1. Characterization of human ES cells and human iPS cells.

Fig. S2. Human induced pluripotent stem cell–derived cortical neurons are exclusively glutamatergic excitatory neurons.

Fig. S3. Aβ aggregate formation.

Fig. S4. Characterization of Down syndrome ES cells (SC-321 cell line).

Fig. S5. Cell death in control and Down syndrome cortical neurons.

References and Notes

  1. Acknowledgments: We thank H. Robinson for electrophysiology support, G. Daley for providing the DS-iPS cell line, and L. Vallier for providing the control human iPS cell lines. We thank A. Surani, A. Smith, and D. Crowther for critical reading of the manuscript. We also thank the members of the Livesey lab for their comments. Funding: This research benefits from core support to the Gurdon Institute from the Wellcome Trust and Cancer Research UK and grants to F.J.L. from the Wellcome Trust and Alzheimer’s Research UK. Author contributions: Y.S., P.K., and F.J.L. designed the experiments. Y.S. generated cortical neurons from pluripotent stem cells and carried out immunohistochemistry and confocal microscopy studies. P.K. carried out electrophysiology and super-resolution microscopy studies. Y.S. and J.S. carried out ELISA assays. G.M. and S.H.O. generated and characterized the DS-ES cell line. Y.S., P.K., J.S., and F.J.L. analyzed the data. Y.S., P.K., and F.J.L. wrote the manuscript. Competing interests: Y.S. and F.J.L. are authors on a patent associated with this work PCT/GB2011/001144 entitled “Corticogenesis from human stem cells” filed by the University of Cambridge. The other authors declare that they have no competing interests.
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