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Waseem K. Raja,

Alison E. Mungenast,

Yuan-Ta Lin,

Tak Ko,

Fatema Abdurrob,

Jinsoo Seo,

Li-Huei Tsai

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Abstract

The dismal success rate of clinical trials for Alzheimer's disease (AD) motivates us to develop model systems of AD pathology that have higher predictive validity. The advent of induced pluripotent stem cells (iPSCs) allows us to model pathology and study disease mechanisms directly in human neural cells from healthy individual as well as AD patients. However, two-dimensional culture systems do not recapitulate the complexity of neural tissue, and phenotypes such as extracellular protein aggregation are difficult to observe. We report brain organoids that use pluripotent stem cells derived from AD patients and recapitulate AD-like pathologies such as amyloid aggregation, hyperphosphorylated tau protein, and endosome abnormalities. These pathologies are observed in an age-dependent manner in organoids derived from multiple familial AD (fAD) patients harboring amyloid precursor protein ( APP) duplication or presenilin1 ( PSEN1) mutation, compared to controls. The incidence of AD pathology was consistent amongst several fAD lines, which carried different mutations. Although these are complex assemblies of neural tissue, they are also highly amenable to experimental manipulation. We find that treatment of patient-derived organoids with β- and γ-secretase inhibitors significantly reduces amyloid and tau pathology. Moreover, these results show the potential of this model system to greatly increase the translatability of pre-clinical drug discovery in AD.

Citation: Raja WK, Mungenast AE, Lin Y-T, Ko T, Abdurrob F, Seo J, et al. (2016) Self-Organizing 3D Human Neural Tissue Derived from Induced Pluripotent Stem Cells Recapitulate Alzheimer's Disease Phenotypes. PLoS ONE 11(9): e0161969. https://doi.org/10.1371/journal.pone.0161969

Editor: Jaya Padmanabhan, USF Health Morsani College of Medicine, UNITED STATES

Received: May 29, 2016; Accepted: August 15, 2016; Published: September 13, 2016

Copyright: © 2016 Raja et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: This work was supported by the National Institutes of Health ( https://www.nih.gov/), Grant numbers: RF1AG042978, RF1AG048029, and R01NS051874 to LHT; Jeffry M. Picower and Barbara Picower (JPB foundation) ( http://www.jpbfoundation.org/), Picower Institute Innovation Fund to LHT; and the Robert A. and Renee E. Belfer Family Foundation and Belfer Neurodegeneration Consortium to LHT. The funder provided support in the form of salaries for authors WKR, AM, YL, FA, and JS, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the 'author contributions' section.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Alzheimer's disease (AD) is an age-related neurodegenerative disorder associated with severe memory impairments, which has become the sixth leading cause of death in the United States ( www.alz.org) [ 1, 2]. Individuals with AD develop brain atrophy and neuronal loss, and the disease is characterized by the presence of dense extracellular deposits of amyloid plaques and neurofibrillary tangles [ 3- 14]. Much of our understanding of the mechanisms underlying AD pathology comes from a population of individuals with early-onset familial AD (fAD). These cases harbor causal mutations involving primarily the Aβ processing enzymes, presenilin 1 and 2 ( PSEN1, PSEN2), which are part of the γ-secretase complex [ 15- 18] [ 15- 18], or mutations within or duplications of the amyloid precursor protein ( APP) gene itself [ 19- 22]. Neurofibrillary tangles are comprised of intraneuronal accumulations of the microtubule-associated protein tau [ 19]. Tau pathology (tauopathy), consisting of aberrantly phosphorylated (pTau) and aggregated tau, is characteristic of a number of neurodegenerative disorders, including AD [ 23- 25]. The extent of tau pathology in human AD has been shown to correlate well to cognitive impairment [ 26]. Many studies have shown that the aberrant phosphorylation and aggregation of tau underlie tauopathy in human brain and animal models.

Neurological conditions are difficult to study because of the limited accessibility to human brain tissue. Animal models, such as transgenic rodents, can recapitulate AD phenotypes to an extent; however, a number of drugs that have shown promising results in mouse models have, failed to prevent cognitive decline in late phase clinical trials [ 27- 30]. There are general concerns on the validity of rodent models [ 31, 32], and a number of genetic variants associated with increased risk for human disease are located in non-coding regions of the genome that are not well-conserved between species [ 33]. Thus, an alternative model for AD that exhibits pathology in human cells may better serve to predict clinical outcomes. In the last decade, the advent of induced pluripotent stem cells (iPSCs) has revolutionized human in vitro models systems [ 34- 37]. Using this technology, we can derive specific neural cell types from patients with sporadic or familial AD, and use these cultures to both study disease mechanisms and develop novel therapies [ 38, 39]. A number of groups have performed functional studies using iPSC-derived neural cells to model various aspects of AD pathology [ 40- 50].

One concern with the use of in vitro systems to model AD pathology, however, is that phenotypes of aberrant extracellular protein aggregation are lost in two-dimensional (2D) cultures simply due to the lack of an interstitial compartment. To overcome this, the next logical approach is to use three-dimensional (3D) culture systems. There exist two main types of 3D culture systems: a scaffold-free 3D culture system, exemplified by self-organizing structures such as organoids or spheroids [ 51- 53] and 3D engineering tissue, in which a variety of support materials (scaffolds or gels) provide a structure within which cells are cultivated [ 54- 57]. One published study in this second category embedded genetically modified human neural precursor cells, which overexpressed mutant APP and PSEN1, into a Matrigel scaffold. After twelve weeks in vitro, these cultures generated AD-like phenotypes including amyloid plaque deposition and hyperphosphorylated tau [ 40, 58]. Another group studied AD-relevant phenotypes in scaffolded 3D culture systems following exogenous amyloid β (Aβ) application [ 59].

While the tissue engineering approaches mentioned above do produce AD-like phenotypes and are highly amenable to experimental manipulation, the process is labor-intensive and requires the exogenous overexpression of disease-relevant mutations or factors. In the current work, we have adapted a scaffold-free culture approach to generate neural organoids from AD patient derived cells. These 3D cultures efficiently produce robust AD phenotypes, without genetic manipulation or exogenous toxins. Although neural organoids have been shown to be extremely useful for the study of neurodevelopmental phenomena and prenatal injury [ 51- 53, 60- 63], no previous study had applied this system to the study of age-related neurodegeneration. We show that, by "aging" these cultures in vitro, we can observe the spontaneous emergence of hallmark AD pathologies such as amyloid aggregates and hyperphosphorylated tau. Importantly, we demonstrate that this model system is amenable to experimental manipulation, such as drug treatment, and that these phenotypes are robust enough to be recapitulated across multiple cells lines derived from different AD patients.

Results

Generation of organoids and analysis

To investigate the utility of such a system, we created scaffold free three-dimensional (3D) human neural organoids, from human iPSCs derived from AD patients and healthy controls and test each line for pluripotency ( S1 Fig). Several protocols have been developed to create neural organoids from human pluripotent stem cells [ 53, 64- 67]. We followed the protocol published by Kadoshima et al. [ 64] with minor modifications (see Methods) and successfully created complex dense 3D neural tissues from a number of human iPSC lines from AD patients and healthy control ( S2A Fig and S1 Table). We subjected the 3D cultures, heretofore referred to as organoids, to immunohistochemistry with antibodies against a neuronal protein (MAP2) as well as SOX2, a marker for neural progenitor cells, to determine the presence of neural cell types. After one month of culture, we observed the emergence of translucent regions of neuroectoderm and immunolabeling for neuronal MAP2 as well as SOX2-positive neural progenitor cells, as previously described [ 64] ( S2 Fig). At 60 days (60d) and 90-100 days (90d) in culture, the organoids demonstrated formation of a rolling morphology structure with rosette-like neuroprogenitor rich regions in which the neuroprogenitor cells are spatially arranged in a spherical format, and unorganized regions that contain dense populations of MAP2-positive neurons ( S2B Fig). With age, immunoreactivity for SOX2 appeared to decrease, while MAP2 immunoreactivity remained ( S2C and S2D Fig). Since we are interested in age-related neural pathology, we found that the large regions of neuron-rich tissue produced by the modified Kadoshima et al protocol [ 64] best suited our modeling needs.

At 60d and 90d of culture, we subjected organoids from a healthy control (Ctrl) and a familial (fAD) patient ( APP duplication, APP Dp1-1, reported in Israel et al., 2012) to a number of assays for AD-relevant phenotypes. We also examined additional fAD lines in ( S1 Table). As the organoid continues to increase in size, the deeper regions of the tissue show evidence of necrosis, which is likely due to the absence of vasculature and lack of nutrients and oxygen penetration in the deeper layers. As the presence of necrosis could confound our measurements of AD-like phenotypes. We used immunohistochemistry for cleaved caspase 3 (CC3; S3A Fig) as an indicator of apoptotic cells and calculated the average distance between the surface and regions of increasing CC3 immunoreactivity. We found that the region of neuron-rich, CC3-sparse tissue extended an average of 250 μm from the surface into the interior of the organoid ( S3A Fig). In addition, we incubated sections with secondary antibody alone (after blocking) to assess the presence of nonspecific binding. We observed that the inner tissue region also gave rise to nonspecific antibody binding compared to the more superficial region ( S3B Fig). Based on these data, we established a limit of 250 μm from the surface of the organoid for our further characterization.

Alzheimer's disease phenotypes in organoids

Amyloid beta.

Neurons derived from the APP Dp1-1 line had previously exhibited increased levels of secreted Aβ ₄₀ and Aβ ₄₂ (Israel et al., 2012). To determine whether this phenotype was perpetuated in 3D culture, we subjected culture media from fAD and control organoids to ELISA. In agreement with the previous study, we detected significantly higher levels of Aβ in the media from fAD organoids culture compared to controls ( Fig 1A). We performed immunohistochemistry on fixed cryosections from control and fAD organoids to examine AD-like pathology using two different antibodies that recognize Aβ. The first antibody, 4G8 (immunoreactive against amino acid residues 17-24 of Aβ) has been widely used to label both soluble and aggregated Aβ [ 68]. In addition, we co-labeled sections with the anti-Aβ antibody D54D2, which recognizes several isoforms of amyloid (Aβ ₃₇, Aβ ₃₈, Aβ ₃₉, Aβ ₄₀, and Aβ ₄₂). In 90d organoids, we detected aggregates that were immunopositive for both Aβ antibodies ( Fig 1B), while 4G8 immunoreactivity appeared against intracellular amyloid that co-localizes with MAP2 as well as putative extracellular aggregates ( Fig 1B, S4A Fig and S1 and S2 Movies). We then used the D54D2 antibody to quantify the size and number of Aβ aggregates. The 90d fAD organoids contained numerous Aβ aggregates compared to the control organoid tissue ( Fig 1C). Analysis at 60d and 90d of culture identified a progressive increase in number and size of Aβ aggregates in the fAD organoids compared to controls ( Fig 1D and S4B Fig). Additionally, we performed Western blot on the 90d whole organoids lysates and observed increased levels of Aβ oligmers in fAD organoids compared to controls (data not shown). These data demonstrate the presence of robust and spontaneous Aβ aggregation in AD patient-derived organoid culture models that appears to develop in a time-dependent manner.

Hyperphosphorylated Tau (pTau).

Another hallmark of AD is the abnormal phosphorylation, mislocalization, and aggregation of the tau protein. Beta-plated sheets of hyperphosphorylated tau (pTau) leads to the disruption of neuronal microtubule assemblies and neurodegeneration [ 19]. To examine control and fAD organoids for the presence of tau pathology (tauopathy), we conducted immunohistochemistry in organoid sections using antibodies against pTau (Ser396 or Thr181). At 90d, the fAD organoids exhibit significantly greater pTau immunoreactivity than did control sections ( Fig 1E and 1F and S4C Fig). Interestingly, there was no significant difference in pTau levels between fAD and control organoids at 60 days ( Fig 1F). This is in contrast to the amyloid phenotype, in which more Aβ aggregates were observed at both 60d and 90d in the fAD organoids ( Fig 1D). The Thioflavin-S dye binds aggregates of β-pleated sheets and is used as an indicator of tau pathology in human brain and mouse models [ 69]. We observed a greater total area covered by Thioflavin-S dye labeling in fAD organoids compared to controls, as well as a higher number of Thioflavin-S positive particles ( S4D and S4E Fig). Together, these various measures demonstrate that the AD patient-derived organoids have the power to recapitulate both amyloid and pTau phenotypes.

Endosome abnormalities.

Endosome abnormalities are another common cellular phenotype in AD. Enlarged RAB5-positive early endosomes have been observed in mouse models and in the brains of sAD and fAD patients [ 70, 71]. Such enlarged endosomes may contain aberrantly phosphorylated APP and aggregated Aβ [ 72]. iPSC-derived neurons created from sAD, fAD, and frontotemporal dementia (FTD) patients also show increases in the number of medium to large RAB5-positive endosomes [ 44] and defects in endosome trafficking [ 43, 48]. To examine endosome phenotypes in iPSC-derived organoids, we labeled control and fAD organoid sections with antibodies against the early endosome marker 1 (EEA1; Fig 1G). The fAD organoids trended towards an increase in the number of small endosomes (<1 μm in diameter) and exhibited a significantly higher number of large (1-2 μm) endosomes compared to controls. Additionally, we observed a trend towards an increased overall size for EEA1-positive endosome particles in the fAD organoids ( Fig 1H). To functionally assess endosome trafficking in the control and fAD organoids, we employed the transferrin endocytosis assay, in which labeled transferrin is taken up by live cells via clathrin-mediated endocytosis [ 73]. Following incubation with Alexa 488-conjugated transferrin, we observed a significant increase in the size of transferrin-containing endosomes in the fAD line, compared to controls ( Fig 1I). Together, these results demonstrate that AD patient-derived organoids display a third hallmark AD: abnormal endosome morphology and recycling.

Organoids from multiple AD lines recapitulate AD pathology

To ascertain whether the AD phenotypes of Aβ aggregation and Tau hyperphosphorylation are generalizable to fAD organoids from different sources, rather than being a phenomenon of the APP Dp1-1 line, we examined additional organoids created from patient-derived and healthy control iPSCs ( S1 Table). At 90d of culture, we processed and analyzed organoids created from an additional APP duplication line ( APP Dp2-3; [ 44]) two PSEN1 fAD mutant lines ( PSEN1 M146I, PSEN1 A264E; Fig 2A, S1 Table). Organoids derived from fAD patients with APP duplication ( APP Dp2-3) or the PSEN1 A264E mutation exhibited increased numbers of Aβ aggregates verses the control lines ( Fig 2B). While the fAD organoids from PSEN1 M146I patient cells exhibited a trend towards higher amyloid levels, this was not significant. We also examined organoids from these additional fAD lines for the presence of pTau immunoreactivity. Similar to the pattern observed with amyloid aggregation, the APP Dp2-3 and the PSEN1 A264E fAD organoids exhibited increased pTau (Ser396) immunoreactivity at 90 days of culture, while the PSEN1 M146I organoids did not differ from control ( Fig 2C).

(A) Tissue sections from fAD ( APP Dp2-3, ND34732, AG068840) and control (Ctrl; CS-0020-01, AG09173) organoids were processed for immunoreactivity against Aβ (D45D2, white), MAP2 (red), and pTau (S396, green) and labeled with the nuclear dye Hoechst. (B) Quantification of Aβ immunoreactivity in fAD and Ctrl organoids following 90 days of culture. Values between the two control lines did not significantly differ. Number of Aβ-positive aggregates in two size classes (Particle Counts): one-way ANOVA with post-hoc Tukey's multiple comparisons test; F (4,21) = 6.15, **p = 0.0019, R ² = 0.5396 (1-3μm); F (4,21) = 7.95, ***p = 0.0005, R ² = 0.6024 (3-6 μm). (C) Quantification of the average intensity of pTau Ser396 immunoreactivity as a fold change of Ctrl in fAD and Ctrl organoids following 90 days of culture. Values between the two control lines did not significantly differ. (Each data point represent one organoid). One-way ANOVA with post-hoc Tukey's multiple comparisons test; F (4,20) = 9.629, ***p = 0.0002, R ² = 0.6582. On charts: *p < 0.05, **p < 0.01, ***p < 0.001.

Attenuation of AD pathology in neural organoids by β- and γ-secretase inhibitor treatment

These data indicate that organoids created from multiple fAD patient iPSC lines demonstrate robust and relevant AD-like phenotypes. To determine whether these phenotypes are indeed a result of altered Aβ production, we treated the fAD organoids with two compounds well known to reduce amyloid aggregation: the γ-secretase inhibitor Compound E (Comp-E; γ2) and a BACE-1 β-secretase inhibitor (β-Secretase Inhibitor IV, EMD Millipore) [ 40, 74]. These experiments also allow us to determine the feasibility of pharmacologic manipulation in the organoid cultures. Comp-E and β-Secretase Inhibitor IV, or DMSO vehicle, were added to the culture media of 30-day-old fAD organoids and replenished with every media change ( Fig 3A). At day 60 (30 days of treatment) we assessed Aβ and pTau pathology ( Fig 3A) and showed that compound treatment significantly reduced the number of amyloid aggregates in the fAD organoids in dose dependent manner, compared to vehicle treated fAD tissue ( Fig 3B). This reduction was particularly evident in the in 90-day-old fAD organoids treated for 60-days ( Fig 3C). In contrast to the dramatic reduction of Aβ aggregation with compound treatment, immunoreactivity for pTau was unaffected following 30 days of treatment compared to vehicle-treated controls ( Fig 3D). However, following 60 days of treatment, the 90 day-old organoids showed significantly less pTau immunoreactivity compared to vehicle-treated controls in dose dependent manner ( Fig 3D). These results indicate that specific AD-like phenotypes observed in patient iPSC-derived organoids can be ameliorated with drug treatment, suggesting that this system is amenable to compound testing.

(A) Schematic of beta (BACE-1) and gamma (Comp-E) secretase inhibitor treatment (top). At 30 days of culture, fAD ( APP Dp1-1) organoids were treated with low dose (BACE-1, 1μM and Comp-E, 3nM) or high dose (BACE-1, 5 μM and Comp-E 6nM) combined compounds, or equivalent DMSO vehicle. Following 30 or 60 days of culture and drug treatment, organoids at 60 and 90 days of culture, respectively, were processed for immunohistochemistry (IHC). Tissue sections from fAD ( APP Dp1-1) and control (Ctrl; CS-0020-01) organoids were processed for immunoreactivity against Aβ (D45D2, white), pTau (Ser396, green), and MAP2 (red). Examine images are from 90 day organoids. (B) Quantification of Aβ particle number and size in compound treated and fAD organoids following 30 days of administration. Number of Aβ-positive aggregates in two size classes (Particle Counts): one-way ANOVA with Fishers LSD test for multiple comparisons; F (5,24) = 3.58, *p = 0.014, R ² = 0.4296. Particle size: one-way ANOVA with Kruskal-Wallis test for non-normal distribution (α < 0.05), p = 0.475. (C) Quantification of Aβ particle number and size in treated (high dose) and untreated fAD organoids following 60 days of compound administration. Number of Aβ-positive aggregates in three size classes (Particle Counts): one-way ANOVA with Fishers LSD test for multiple comparisons; F (5,19) = 5.02, **p = 0.004, R ² = 0.5691. Particle size: Mann-Whitney two-tailed test for non-normal distribution (α < 0.05), p = 0.09. (D) Quantification of the average intensity of pTau Ser396 immunoreactivity as a fold change of Ctrl in fAD organoids following 30 and 60 days of compound treatment. 30 day treatment. (Each data point represent one organoid). Unpaired two-tailed t-test with equal variance, p = 0.69. 60 day treatment: one-way ANOVA with Tukey's multiple comparisons test, F (2,13) = 19.82, ***p = 0.0001, R ² = 0.7530. On charts: *p < 0.05, **p < 0.01, ***p < 0.001.

Discussion

In diseases such as AD that are characterized by protein aggregation, the presence of a true interstitial compartment is important for modeling pathology. Previous three-dimensional (3D) tissue engineering approaches have embedded neural progenitors or cell types of interest in a matrix or a scaffold [ 40, 58]. While these ingenious approaches can model AD phenotypes, they do not recapitulate spontaneous pathology resulting from endogenous cellular characteristics, but rather necessitate the overexpression of fAD genes. In the current work, we took advantage of scaffold-free 3D tissue culture protocols to create neural organoids using iPSCs from fAD patients and healthy controls. The dense nature of these cultures likely facilitates protein aggregation, while remaining amenable to experimental manipulation such as compound treatment.

The ease with which large numbers of these organoids can be created, and their ability to respond to compound treatment, open the possibility for phenotypic and mechanistic compound screening in complex human tissue models of multiple neurodegenerative diseases. With iPSC technology, we can access patient-derived cells carrying AD-associated variants in a number of genes, which are difficult to model as many are non-coding and/or associated with more than one significant single-nucleotide polymorphism (SNP) [ 75]. Moreover, rapid improvements in gene editing technology allows researchers to create isogenic iPSC and ESC lines that carry disease-associated coding variants, both well-known (such as in the APOE gene, [ 76, 77] or emerging (i.e. SORL1 [ 50, 78], TREM2, [ 79]). Together, these advancements allow us to conceive of systems in which we can test candidate therapies on complex neural tissue systems targeted to defined subpopulations of late-onset or sporadic AD patients.

One powerful aspect of the current model is the spontaneous appearance of both [ 1] amyloid and tau pathology, and the distinct timeline on which these phenotypes appear. Modeling both these facets of AD has been challenging in mouse models. Most mouse AD models must carry multiple transgenes to achieve robust amyloid phenotypes, and rarely have significant tau pathology or neuronal loss (for review see [ 31]). Mouse models of tauopathy overexpress mutated human MAPT (which is causal for frontotemporal dementia, not AD) to induce tau pathology [ 80, 81], and do not exhibit amyloid aggregation. Using the organoid model, we observed that amyloid pathology emerges prior to significant tau hyperphosphorylation in neural tissue derived from fAD patients carrying a duplication of the APP gene. While the sequential emergence of amyloid and tau pathology in human AD remains somewhat controversial [ 82- 85], this timing is in close agreement with that observed in the scaffolded 3D cultures of Choi et al. 2014; [ 40]. Additionally, the inhibition of Aβ production using β and γ-secretase inhibitors reduced tau hyperphosphorylation only at the later time point of treatment, after Aβ reduction was observed. Thus, AD-relevant phenotypes of Aβ accumulation emerge prior to tauopathy in this model. Moreover, the reductions in Aβ accumulation that occur from the inhibition of APP processing lead to a dose- and time-dependent amelioration of tauopathy in the fAD organoids, suggesting a causal relationship between these relevant pathologies in the neural organoid model.

In the current work, we focused our efforts on determining whether or not these organoid systems could model age-related AD-like pathology. The pioneering works that we drew our techniques from used the organoid system as a means to study neurodevelopment [ 53, 64, 66, 67]. Since work by several groups has suggested that iPSC reprogramming "re-sets" the epigenome, and that other phenotypes associated with cellular aging, such as mitochondrial function and telomere length, are returned to a "juvenile-like" state [ 86, 87], the obvious question is: to what extent can we model phenotypes associated with aging in human neural cells? While we observe robust AD-like phenotypes that increase with "age" in the organoids, the extent to which the organoid tissue represents the aged human brain has not been examined. We believe that the scaffold-free three dimensional model has good potential for studying neurological diseases. This will be important for future works to use this model system to examine other AD related phenotypes, such as neuroinflammation, gliosis, DNA damage, U1 tangles [ 88] and synaptic dysfunction. Also, without a means of tissue perfusion, the organoid suffers from the same issues as primary slice culture, in that the distance from the culture medium interface is correlated with tissue necrosis. There is currently great interest in the combination of three-dimensional neural culture systems with artificial blood-brain barrier technology [ 89- 91], to address this issue.

Experimental Procedures

Maintenance of PSC and 3D culture differentiation

Induced pluripotent stem cells (iPSCs) were created from human fibroblasts ( S1 Table). Two of the iPSC lines carrying duplications in the gene for Amyloid Precursor Protein ( APP; APP Dp1-1 and APP Dp2-3) were provided by Dr. Lawrence Goldstein at the University of California, San Diego, and have been described previously [ 44]. One control iPSC line (AG09173) was kindly provided by Dr. Bruce A. Yankner at McLean Hospital and Harvard Medical School. The other lines were generated from fibroblasts at the Picower institute of Learning and Memory, iPSC core facility at the Massachusetts Institute of Technology (MIT) using Sendai virus to overexpress OCT4, SOX2, KLF4, and c-MYC. S1 Table. details the sources and attributes of the cells used in this study. Pluripotency was confirmed by immunocytochemistry for TRA-1-81 and TRA-1-60 ( S1 Fig). All reagents were purchased from Life Technologies Corporation, Grand Island, NY, unless mentioned otherwise. iPSCs were cultured on irradiated mouse embryonic fibroblasts (MEFs, MTI-GlobalStem, Gaithersburg, MD) in DMEM/F12 media supplemented with knockout serum replacement (KSR, 20% v/v), non-essential amino acids (NEAA-1X), GlutaMAX (1X), beta-Fibroblast Growth Factor (FGF2, PeproTech, Inc, Rocky Hill, NJ) and 2-mercaptoethanol (0.1 mM). The quality of cells was monitored daily and differentiated cells were mechanically removed under a light microscope in a biosafety hood. iPSCs were culture up to 80% confluence and dissociated into single cell suspension after treated with Accutase (diluted in PBS (1.5:1) containing Rock inhibitor (Y-27632 dihydrochloride, Tocris Biosciences, Minneapolis, MN) to improve cell survival. The MEFs and iPSCs were separated by plating the single cell suspension onto 0.1% gelatin (0.1%, EMD Millipore, Billerica, MA) coated dishes for 45 min, after which time the MEFs attach to the substrate and the non-adherent iPSCs were collected.

To create 3D cultures, or neural organoids, we followed a published protocol [ 64] with some modifications. Embryoid bodies (EBs) were formed by loading 12,000 iPSCs per well into 96-well plates with cone-shaped wells (Nunc ® 96-well Conical Bottom plates, VWR International, Bridgeport, NJ) pre-coated with Pluronic acid (F-127, 1%, Sigma-Aldrich, Natick, MA). The 96 well plates were transferred to an incubator at 37 C° with 95% relative humidity and 5% CO ₂. Media used in EB culture consisted of Glasgow-MEM supplement with KSR (20% v/v), Sodium Pyruvate (1X), NEAA (1x), 2-mercaptoethanol (0.1mM), Rock inhibitor (20 μM), TGFβ-inhibitor (SB431532 compound, Tocris Biosciences, Minneapolis, MN; 5 μM), Wnt-inhibitor (IWRe1 compound, Tocris Biosciences, Minneapolis, MN; 3 μM). The EBs maintained in this medium for 18-20 days, with Dorsomorphin (BMP signal inhibitor, Tocris Biosciences, Minneapolis, MN; 2 μM) added to the culture for the first three days to promote a neuronal lineage. Media was replaced every other day. The edges of the EBs began to appear translucent around day 10, and the tissue grew to be large than 0.6 mm in diameter by day 18. The cell aggregates, or organoids, were transferred to non-adherent petri-dishes (EZsphere dish) to prevent fusion of separate organoids, and cultured in a medium designed to promote neuroepithelial formation, which consisted of DMEM/F12 supplemented with Chemically Defined Lipid Concentrate (1X) and N2-supplement (1X) and maintained in an incubator with 5% CO ₂ and 40% Oxygen. The aggregates were kept in this media for 15 to 20 days, at which time heparin (5 μM, Sigma-Aldrich, Natick, MA), FBS (10% v/v, Gemini Bio-Products, West Sacramento CA), and Matrigel (final 1% v/v, Corning Incorporated-Life Sciences, Oneonta, NY) were added to the medium. On day 70 the amount of Matrigel was increased to 2% and B27 supplement (1X) was added to the medium. The aggregates were maintained in this final medium for the remained of the culture period. Medium in the culture dishes was replaced every 4 to 5 days.

Supporting Information

S1 Fig. Immunoreactivity for markers of pluripotency.

iPSC colonies were plated onto MEF-covered glass coverslips in hES maintenance media and subjected to live immunocytochemistry for the pluripotency markers Tra-1-60 and Tra-1-81 three days after plating. Cells were then fixed and stained with the nuclear dye Hoechst (blue) and imaged using a confocal microscope. All lines tested were immunopositive for Tra-1-60 (left, green) and Tra-1-81 (right, green).
https://doi.org/10.1371/journal.pone.0161969.s001
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S2 Fig. Work flow for three-dimensional (3D) neural organoid culture and immunohistochemistry with cell type-specific markers.

(A) The process of 3D culture protocol begins with iPSCs that are dissociated into a single-cell suspension and induced to form embryoid bodies (EBs) in a 96 well format (one EB per well). EBs were then differentiated over a period of weeks to months. (B) Images from organoids cultured for fewer than 60 days: phase contrast images (left) and sections immunostained using antibodies against markers of neural progenitor cells (SOX2, white) and neurons (MAP2, red), as well as the nuclear stain Hoechst (blue). In the images, one can see the (i) rolling morphology structure with organized translucent layers previously reported by the Sasai group [ 64], (ii) regions of the organoid with a rosette-like structure dense in neural progenitors, and (iii) unorganized translucent regions. (C and D) At 60 and 90 days in culture, organoids exhibit SOX2 (white) and MAP2 (red) immunoreactivity that is variable by region.
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S3 Fig. Immunoreactivity for cleaved-caspase 3 (CC3) and non-specific antibody binding define deep regions of necrotic tissue in the organoid.

(A) A quantitative analysis of CC3 immunoreactivity leads us to limit our analyses to the peripheral 250 μm of MAP2-rich (red) and CC3-poor (white) region of the organoid. CC3- and Hoechst (nuclei)-positive cells were counted in bins of 30μm from the surface of the organoid in three replicates. (B) Organoid sections were blocked and incubated with secondary antibodies alone to determine the degree of non-specific labeling (left column = 90d whole organoid images). Regions of interest within the first 250 μm (right column) and deep (middle column) in the organoid tissue were analyzed for signal from secondary antibodies (anti-mouse-Cy2, green; anti-rabbit-Cy3, red; anti-chicken, white).
https://doi.org/10.1371/journal.pone.0161969.s003
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S4 Fig. Immunohistochemistry for amyloid beta and pTau at Threonine 181 (Thr181) and quantification for Thioflavin-S labeling in Ctrl and fAD organoids.

(A) Additional images from tissue sections from fAD ( APP Dp1-1) and control (Ctrl; CS-0020-01) organoids processed for immunoreactivity against amyloid β (Aβ) using two antibodies (D54D2: white, 4G8: green), as well as antibodies against the neuronal marker MAP2 (red) and stained with the nuclear dye Hoechst (blue). (B) Table of average particle number observed per 250 x 250 μm ² area in control and fAD organoids. (C) Tissue sections from fAD ( APP Dp1-1) and control (Ctrl; CS-0020-01) organoids were processed for immunoreactivity against phosphorylated Tau (pTau, green) at Thr181 and MAP2 (red) following 90 days of culture. Hoechst (blue) labels cell nuclei. (D) Representative images of fAD ( APP Dp1-1) and control (Ctrl; CS-0020-01) organoids section stained with Thioflavin-s dye for beta-pated sheet structure. (E) Quantification of fluorescent Thioflavin-S in tissue sections from fAD ( APP Dp 1-1) and control (Ctrl; CS-0020-01) organoids after 90 days of culture (Each data point represent one organoid). Measurements consist of the percent of the visual field covered by Thioflavin-S signal (left) and the number of distinct Thioflavin S-positive particles (right). Unpaired two-tail t-test with equal variance, **p = 0.0074 (% Area Covered), *p = 0.022 (Particle Count). On charts: *p < 0.05, **p < 0.01.
https://doi.org/10.1371/journal.pone.0161969.s004
(TIF)

S1 Movie. Extracellular amyloid aggregates.

30 μm thick (90d) fAD organoids (made from App Dp 1-1) were stained for amyloid beta using two different antibodies; 4G8 (green) and D54D2 (White) along with neuronal marker MAP2 and Hoechst for nuclear staining. The images were converted into movie (2 frames per second) using imageJ (NIH).
https://doi.org/10.1371/journal.pone.0161969.s005
(AVI)

S2 Movie. Extracellular amyloid aggregates.

90d fAD organoids (made from App Dp 1-1) were immunolabeled using antibodies against amyloid beta (D54D2, White) along with neuronal marker MAP2 and Hoechst for nuclear staining. The images were converted into movie (6 frames per second) using imageJ (NIH).
https://doi.org/10.1371/journal.pone.0161969.s006
(AVI)

Acknowledgments

We would like to thank; Ashley Watson from Li-Huei Tsai Lab at MIT for the inputs and proof reading of this manuscript, Professor Bruce A. Yankner at Harvard Medical School (HMS) and Professor Larry Goldstein at University of California San Diego (UCSD) for providing induced pluripotent stem cells and Professor Bruce M. Cohen at the McLean Hospital HMS for providing the fibroblast cells.

Author Contributions

1. Conceptualization: WKR AEM LHT.

2. Formal analysis: WKR AEM.

3. Funding acquisition: LHT.

4. Methodology: WKR YL.

5. Writing - original draft: WKR AEM.

6. Writing - review & editing: WKR AEM YT TK JS LHT.

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Self-Organizing 3D Human Neural Tissue Derived from Induced Pluripotent Stem Cells Recapitulate Alzheimer's Disease Phenotypes

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The dismal success rate of clinical trials for Alzheimer's disease (AD) motivates us to develop model systems of AD pathology that have higher predictive validity. The advent of induced pluripotent stem cells (iPSCs) allows us to model pathology and study disease mechanisms directly in human neural cells from healthy individual as well as AD patients.

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Tender Rose Dementia Care Specialists, Mar 2010: https://www.youtube.com/watch?v=Eq_Er-tqPsA

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Understand Alzheimer's Disease in 3 Minutes

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Amyloid Beta accumulation (Aβ40 and Aβ42 for example) into plaques (Ballatore et al, 2007; Goate , 2006)

Tauopathy: Aberrant phosphorylation leading to  aggregation of hyperphosphorylated tau protein (pTau) into neurofibrillary tangles. (Ballatore et al, 2007; Buée et al, 2000; Feany et al. 1996; Lee et al, 2001).

Endosome abnormalities: enlarged endosomes (Cataldo et al, 2000; Cataldo et al, 2001) and defects in endosome trafficking (Iovino et al, 2015; Wren et al, 2015)

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Many different invertebrate and vertebrate species are used for modelling: drosophila (Dissel et al, 2017), zebrafish (Bhattarai et al, 2012), and rodents (Elder et al, 2010; Do Carmo, Cuello, 2013) where mouse models are the most prevalent

Advantages:

Mouse models successfully recapitulate many molecular mechanisms involved in AD listed above (Elder et al, 2010; Webster et al, 2014; Guo, Zhou, 2017)

Use of genetic (ex: mutations in genes controlling amyloid beta deposition) and non-genetic (ex: transverse aortic constriction to induce hypertension) models can be used to model both fAD and sAD (Elder et al, 2010; Carnevale et al, 2012)

Evaluation of behaviour with cognitive and memory tasks with findings that are translatable to human AD (Webster et al, 2014).

Disadvantages:

Genetic models often require multiple mutations or copies of mutations to create a phenotype that resembles human AD (Elder et al, 2010; Webster et al, 2014). The observable molecular and behavioural phenotype measured in these models therefore does not rely on the same molecular etiologies as human AD.

Many drug therapies developed from mouse models fail in clinical trials, indicating a lack of efficiency in representing human AD phenotypes. (DeMattos et al, 2001; Cedernaes et al, 2014; Raja et al, 2016)

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Hypothesis: Scaffold-free brain organoids grown from fAD patient iPSCs can recapitulate the molecular mechanisms of AD phenotype and will be responsive to AD treatments.

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This paper has successfully recapitulated AD phenotypes in organoids, which importantly are derived from human patient cells and not animals. Hence, it could be a promising model to retry therapies that failed translation from mouse models to human clinical trials (Doody et al, 2012; Doody et al, 2014; Green et al, 2009; Salloway et al, 2014). Mouse models have supported the therapeutic benefit of treatment with monoclonal antibodies specific for Aβ to resolve the AD phenotype (DeMattos et al, 2001). When this research was translated into clinical trials, many forms of anti-Aβ drugs were attempted that did not show therapeutic benefit. One such drug, called bapineuzumab, showed a significant decrease in CSF levels of pTau, but no difference in levels of Aβ and no improvement in cognitive symptoms (Blennow et al., 2012). It would be of interest to apply bapineuzumab to these organoids to assess its efficacy of resolving AD phenotypes.

If bapineuzumab is successful in resolving the Aβ and the pTau phenotypes, this would be significant in demonstrating this model's responsiveness to treatments. It would further indicate that there are differences between the organoid model and human fAD that prevent these models from responding in the same fashion as human AD would.

If bapineuzumab is successful in resolving only the pTau phenotype, this would be an indication that the fAD organoids hold high representational power of human AD. As anti-Aβ would be inducing a decrease in pTau, it would also support the hypothesis that the hyperphosphrylation of tau protein is causally connected to increased Aβ deposition.

If bapineuzumab does not resolve the Aβ or pTau phenotypes, this could indicate a lack of responsiveness of the organoid, reflecting poorly on its representational abilities, indicating that the organoid is less sensitive than human AD.

Due to its closer resemblance to humans, this is a model that can serve as an additional step in the translation process to test the potential of proposed therapies before these therapies move on to the more costly and time-consuming clinical trials.

Because organoids can be generated by cells from specific patients, there is also potential to develop patient specific therapies using this model. Patients can donate fibroblasts to make organoids, and for each of patient's organoids we can try a set of different drugs to see which drug works best for that patient's organoid, and consequently decide which is the drug best given to the actual patient.

When it comes to sAD, genetic factors only partially explain the risk of AD. One widely accepted notion is that the onset of AD is most likely the consequence of complicated interactions of multiple genetic and non-genetic factors (Gatz, 2006; Yang et al, 2016). Several non-genetic risk factors have been proposed, including aging, cerebro-cardiovascular diseases, metabolic disorders, traumatic brain injury, sleep disorders, chronic hypoxia, environmental toxins and high cholesterol (Yang et al, 2016; Grant et al, 2002; Styczyńska et al, 2008; Dosunmu et al, 2007). One model has even looked at sAD iPSC cells grown in culture clustered with fetal brain messenger RNA samples, and showed AD phenotypes to appear in these sAD patient-derived iPSC cells (Israel et al. 2012). However, the mechanisms underlying them still remain largely unknown, due to the lack of non-postmortem and AD patient-specific research models (Yang et al, 2016). Although the organoids produced in this paper are based on fAD mutations, they have the possibility of being used to model sAD as well. For example, researchers could take cells from sAD patients, derive iPSCs, and grow multiple organoids under some environmental conditions that may induce AD, so AD will arise in these organoids sporadically, similarly to sAD patients. Specifically, this means that for a specific sAD patient whose disease arose in a way that cannot be fully accounted for by genetic mutation, we can take these cells and grow multiple organoids, some in neutral conditions as control, and others subjected to some environment such as high cholesterol. This will allow us to better observe how the proposed environmental factors can influence disease progression in sAD patient organoids, if they are an influence at all. In addition to studying sAD disease progression, we may even be able to take one step further and use this organoid model to help design sAD patient-specific therapy.

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In terms of Aβ aggregates, Organoids derived from the APP Dp2-3 (blue bar) and the PSEN1 A264E (red bar) lines exhibited increased numbers of Aβ aggregates verses the controls. Organoids from the PSEN1 M146I (green bar) patient cell line exhibited a trend towards higher amyloid levels, but this was not significant. Values between the two control lines did not significantly differ.

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Figure 3: fAD organoids respond to treatment, resolving the AD phenotype

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Figure 3: fAD organoids respond to treatment, resolving the AD phenotype

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fAD organoids are treated with β-secretase and γ-secretase inhibitors which inhibit the formation of the complex that cleaves amyloid precursor protein into Aβ. The immunohistochemical stains below show that this treatment reduces Aβ and pTau deposition, two strong molecular indicators of the AD phenotype

These results indicate responsiveness of the model to treatments, meaning that this could be a possible model for screening prospective clinical therapies. There is some debate as to the timeline of emergence of human AD molecular mechanisms, but the time-dependent change in Aβ and pTau in these organoids in response to treatment also mimic human AD progression (Jack, 2014) with the appearance of Aβ plaques preceding neurofibrillary tangle formation.

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Can Brain ORGANOIDS help to unlock human brain cells. (2016, March 7). http://technologyinfoo.com/724/can-brain-organoids-help-to-unlock-human-brain- cells/

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Induced Pluripotent Stem Cell Models of AD

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Induced Pluripotent Stem Cell Models of AD

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Induced pluripotent stem cells (iPSCs) cultured from fAD patients can be differentiated into neurons to model human AD in vitro in 2D cultures

Advantages:

Successfully produces the hallmarks of AD pathology (Yahata, 2011; Sproul, 2014; Hossini, 2015; Lullo, Kriegstein, 2017)

Because of their closeness to human AD, once they become more well established, these models may have the capacity to produce findings pertaining to the molecular phenotype of AD that hold more translational power than previous models.

Disadvantages

Due to the nature of 2D models, the phenotypes of aberrant extracellular protein aggregation are lost.

As for 3D models, previous methods of organoid production require a scaffold, are labor-intensive, and require exogenous over-expression of disease-relevant mutations or factors in order to express AD phenotype (Zhang et al, 2014).

While not yet well characterized, papers such as this one by Raja et al (2016) aim to develop high fidelity, well-characterized iPSC-derived 3D organoid models of fAD.

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The authors conducted three sets of experiments. The experiments in Figure 1 demonstrate that the organoids were able to recapitulate hallmark symptoms of AD. Figure 2 demonstrates that the hallmark AD phenotypes generated by the organoid could be generated from multiple cell lines with different AD-inducing mutations. Finally, Figure 3 demonstrates that the fAD organoids were responsive to common AD treatments

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Advantages of the fAD organoids proposed in this article

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Advantages of the fAD organoids proposed in this article

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These organoids were able to develop and respond to AD therapies within 90 days. Previously, scientists only insights into human AD pathology were from behavioural assessments, imaging techniques, and postmortem analysis. This will allow scientists to access live, human-derived tissue that strongly represents the human AD phenotype.

The recapitulation of the phenotype is evidenced by the induction of Aβ and pTau deposition, as well as endosomal abnormalities. The response to therapy is also evidenced by a reduction in Aβ and pTau deposition.

fAD derived organoids may be more representative of human AD pathology than traditional animal models. While many mouse models of fAD rely on genetic manipulation to produce an AD-like phenotype, fAD organoids are directly derived from human AD patients. The artificial mutagenesis required to create these animal models may create a pathology that does not replicate the course of human disease, and is therefore unsuitable for developing human therapies. By directly recreating human AD pathology, fAD organoids may provide researchers with better tools to develop AD therapies.

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Disadvantages of fAD Organoids proposed in this article

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Disadvantages of fAD Organoids proposed in this article

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While organoids may be better able to replicated the molecular etiology of AD than animals models, organoids cannot show behavioural responses. Murine AD models can be subjected to behavioural tests, allowing researchers to identify specific behavioural deficits. Researchers testing a drug in a murine AD model can examine both behavioural and molecular rescue of phenotype. While drugs may affect the molecular pathology or improve biomarkers, this does not guarantee that they will have any functional impact (Salloway et al 2014). As functional recovery cannot be measured with fAD organoids, their scope as a model system is limited. Furthermore, AD is a heterogenous pathology affecting multiple cognitive domains. Without behaviour, researchers cannot identify how molecular pathologies and treatments relate to specific cognitive deficits (Ex attentional vs memory deficits).

While this model can replicate several strains of human fAD, it does not represent sAD. Familial cases of AD are much rarer than sAD. As such, it is critical the better models of sAD be developed. The scope of fAD organoids is somewhat limited as they only represent a minority of AD cases.

As these organoids are constructed without a scaffold there is significant tissue necrosis in the centre. To combat the potential influence of the necrotic tissue the authors only examined the peripheral 250um of the organoids. However, this is a rather arbitrary decision. The figure below (supplemental figure 3) shows cleaved cascade 3 staining (white) for apoptosis, and Hoechst staining (blue) for nuclear material. It is obvious that even in the peripheral 250um there is tissue necrosis. While this may have no effect on the organoids AD relevance, the proinflammatory environment may influence the disease progression, especially given the growing epidemiological findings indicating that immune activation plays an important role in AD pathogenesis (Carnevale et al, 2012).

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All figures adapted from Raja et al. 2016, unless otherwise cited

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While iPSC induction erases epigenetic factors, direct reprogramming has been shown to maintain epigenetic memory (Hou, Lu, 2016). With more research being conducted on the epigenetics of AD, one question in the field of AD organoinds going forward could be whether it would be feasible and practical to differentiate a brain organoid through direct reprogramming as opposed to IPSC induction. By direct reporgramming brain organoids from sAD patient cells, this may provide a possible model for studying the epigenetic factors contributing to sAD.

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(AD) is an age-related, progressive neurodegenerative disorder associated with cognitive decline, severe memory impairments, anxiety, circadian rhythm disturbances, activity disturbances, and aggression. It is associated with brain atrophy, neuronal losses, dense extracellular deposits of amyloid plaques and neurofibrillary tangles (Webster et al, 2014).

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It is currently the 6th leading cause of death in the United States. With medical advances increasing life expectancies and the growing population of seniors, the incidence of AD in the US is said to increase from 5.3 million patients to 13.8 million by 2050 (Alzheimer's Association, 2015). This reflects an urgent need for reserach to be conducted with models that can yield relevant results that can be translated to a clinical setting.

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The full etiology of AD is unknown. It can be broadly divided into sAD (sporadic) and fAD (familial). fAD has been attributed to a series of mutations in the amyloid pathway. Epidemiological studies have considered risk factors for AD that may result in sAD.

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Immunohistochemisty using an Aβ specific antibody on 60 and 90 day old cultures shows a time dependant increase in Aβ deposition. Organoids created from fAD cells showed significantly higher levels of Aβ deposition compared to controls. Staining for MAP2, a neuronal protein, suggests both intracellular and extracellular plaques. This suggests that fAD organoids mayrecapitulate the degenerative Aβ pathology central to alzheimer's disease.

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Using an antibody specific for pTau, the authors show increased pTau deposition in fAD organoids compared to controls. It is important to note that this increase is only significant at the 90 day time point, suggesting that Tau pathology may be antecedent to Aβ pathology. This finding is replicated using a second antibody specific to pTau, although this data is not displayed here. This suggests that fAD organoids may recapitulate AD Tau pathology.

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Immunohistochemical staining for EEA1, an early end-somalia marker, shows a significant increase in the number of large endosomes in fAD organoids compared to controls. The authors also measured functional endocytosis by allowing cells to take up labelled transferrin. They noted that cells from fAD organoids had significantly larger endosomes compared to controls. This suggests that fAD organoids may model the endosomal abnormalities associated with AD.

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Organoids were produced using donated fibroblasts from patients with fAD resulting from the APP Dp1-1 mutation, and healthy age matched controls. Donated cells were induced to pluripotency, which was validated by staining cells for TRA-1-60 and TRA-1-81. Pluripotent cells were then plated to form embryoid bodies (EB) in a growth medium promoting neural lineage differentiation. After 18–20 days the EBs were separated and allowed to mature for a further 15–20 days in a growth medium with factors promoting neural epithelial formation. Following this, cells were moved to a growth medium containing 2% matrigel for the remainder of the experiment (supplemental figure 2).

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Immunohistochemical staining for Sox2, a marker of neural progenitor cells, and MAP2, a marker of mature neurons, shows that organoids develop stratified layers (supplemental figure 2). Further, organoids display characteristic neural rosettes. Based on cleaved caspase expression, all data was collected from the outer 250um of tissue so as to exclude any necrotic tissue in the centre of the organoid.

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The authors were able to create scaffold-free organoids from multiple lines of fAD iPSCs that both recapitulated the major symptoms of AD pathology, and responded to common AD treatments. The evidence presented supports the authors hypothesis, and suggests that fAD organoids may be a useful model system. The ease of organoid growth could allow for high throughput screening of novel AD therapies.

The authors were able to demonstrate that fAD organoids show the major molecular symptoms of AD (Figure 1). Without this, these organoids would show little promise as a model system.

Organoids grown using tissue from multiple different fAD patients shows that these organoids are a useful model for studying multiple forms of fAD (Figure 2). The potential for performing experiments on cells with different fAD causing mutations will increase the robustness of any experimental findings.

By culturing organoids in growth media containing common AD therapeutics, the authors were able to reduce the severity of AD symptomology (Figure 3). This demonstrates the usefulness of fAD organoids as a model system for drug development.

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Despite the stated ease of growing fAD organoids, the authors include very small samples sizes in many experiments. While the authors do manage to achieve statistical significance, there is extremely high variability in some groups. For example, the vehicle treated groups in figure 3 show very high variability, with some vehicle treated organoids showing no pathology. In conjunction with the small sample size, this raises the possibility that the observed results are merely an artifact of a small highly variable sample.

While the paper was broadly well constructed, there were a number of significant errors in figure design. For example, Figure 1 B in the original paper is missing an arrow head which should indicate intracellular amyloid beta. These omissions are compounded by inconsistencies in font size and colour coding throughout. While this does not directly impact on the validity of the author's results, it reduces the readability and credibility of the paper.

The authors examined endosomal abnormalities as a characteristic phenotype of AD in their effort to recapitulate AD phenotypes in the initial organoids derived from the APP Dp1-1 line. But when they showed the results of recapitulating AD pathology in organoids derived from additional lines, there was no mentioning of any efforts to analyze whether there was presence of endosomal abnormalities in this section. This is an inconsistency they should address.

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Immunohistochemical staining for Serine 396, a phosphotylated tau residue, shows significant decrease in pTau deposition in the treated organoids than the vehicle-treated controls. This signifcant difference appeared 60 days after treatment began, indicating that there is a temporal difference in resolution of the pTau phenotype and the Aβ phenotype.

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Immunohistochemical staining for D54D2, indicative of Aβ deposition, indicates that at 30 days, there are significantly fewer 3-6um plaques in the treated organoids than in the vehicle-treated controls. At 60 days, there are significantly fewer 1-3um, 3-6um, and 6um< plaques in the treated organoids than in the control group. There are no significant differences in plaque size between the treatment and control groups

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Figure 2: Organoids from multiple AD lines recapitulate AD pathology

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Figure 2: Organoids from multiple AD lines recapitulate AD pathology

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To confirm that observed pathologies were not limited to the APP Dp1-1  line , but rather generalizable to fAD, the researchers created organoids from 3 other lines of patient-derived iPSC with different mutations. They saw similar significant phenotypes in 2 of the 3 lines after 90d of culture. Specifically, the 3 lines are an additional APP duplication line APP Dp2-3, and two PSEN1 fAD mutant lines PSEN1 M146I, PSEN1 A264E. Below is the immunohistochemistry of these 3 lines compared to 2 lines of control. Visually, staining of D45D2 and MAP2 suggests higher levels of Aβ aggregation, and staining of Ser396 suggests higher levels of pTau, compared to the controls.

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Examining the presence of pTau immunoreactivity, again the APP Dp2-3 and the PSEN1 A264E organoids (blue and red) exhibited increased pTau (Ser396) immunoreactivity. However, the PSEN1 M146I organoids (green) did not differ from control. Each data point represent one organoid, and values between the two control lines did not significantly differ.

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Much of the current understanding comes from studying mutation of fAD patients

For example, mutations involving the Aβ processing enzymes presenilin 1 and 2 (PSEN1, PSEN2), which are part of the γ-secretase complex that cuts APP into Aβ (Lamere et al, 1996;  Gerrish et al, 2012)

Or, mutations/duplications of the amyloid precursor protein (APP) gene itself (Rovelet-Lecrux et al, 2006; Kosaka et al, 1996).

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The mouse: the scientist’s best friend. (n.d.) https://www.medicalnewstoday.com/articles/277169.php

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By Ce Shang, David Oliver, and Saige Power, demonstratingthe experimental protocol by Raja et al, 2016