A high-throughput conditioned-media-based screening system identifies inhibitors of aggregation induced by iPSC-secreted amyloid β

Domain: biology
Published: 2026-03-24
Editorial outcome: major_revision

Abstract
In early drug discovery, in vitro screening is frequently used, but selected candidates often fail in vivo. Induced pluripotent stem cell (iPSC)–based disease models offer improved physiological relevance; however, the high costs of media and differentiation procedures limit large-scale testing. Here, we develop a high-throughput conditioned-media-based screening system—the High-throughput screening technology for Aggregation Inhibitors of Diseased cell-derived Aggregative Proteins (HaiDap) system—to identify inhibitors of aggregation induced by iPSC-secreted amyloid β (Aβ). Using conditioned media derived from differentiated iPSCs of a male Alzheimer’s disease patient, we screen extracts from 22 edible plants. Whereas PBS-based assays showed 40.9% (9/22) apparent selectivity, the HaiDap system demonstrates higher specificity (13.6%; 3/22). All three identified extracts (O. aristatus, S. aromaticum, and G. yesoense) significantly delay Aβ aggregation on neuronal surfaces in an iPSC-based assay. These findings suggest that the HaiDap system enables efficient, accurate, and low-cost screening of amyloid aggregation inhibitors.

Introduction
In recent years, the cost of drug development has skyrocketed. In drug development, candidate substances are screened for disease treatments in vitro at the first stage. Then, during the second stage, promising candidate substances are selected, and their effectiveness is evaluated using an in vivo assay with disease model animals. Recently, disease modeling based on dish technology using induced pluripotent stem cells (iPSCs) and genome editing technology, like CRISPR, are useful tools for developing new drugs and treatment methods for various diseases. Furthermore, the application of iPSCs for screening candidate drugs during drug discovery is also expected to be a tool to eliminate extrapolation to experimental animals and humans, which has been a challenging objective until now. Most screening systems are performed in vitro at an early stage of drug discovery, but a problematic aspect is that candidate substances selected by the in vitro test do not always display an effect in cell-based assays1. Although it is well known that the results of in vitro tests and cell-based assays are not correlated, i.e., there is a problem with the accuracy of the evaluation, the causes are complex, and many aspects remain unknown.

Disease modeling has actively used iPSCs in drug discovery-related research2,3,4. In a previous study, iPSC-derived neurons with familial and sporadic Alzheimer's disease (AD) were generated5. Although AD is classified into different subtypes, it is possible to develop subtype-specific AD drugs using AD patient-specific iPSCs6. However, in a bid to select candidate compounds, it is challenging to apply an iPSC-based assay system for screening drugs in multifactorial diseases such as AD to accommodate genetic and environmental factors since drug development is labor- and time-intensive. By using the supernatant of a cell culture, it is possible to imitate an environment that is closer to that of a living body, as one example, phosphate-buffered saline (PBS), making it easier to discover more effective compounds.

AD, one of the most well-known dementia-related diseases, exhibits amyloidosis7. AD accounts for the majority of dementia-causing diseases8–10. The amyloid cascade hypothesis provides the most promising mechanism for the onset of AD11,12. In this hypothesis, abnormal aggregation and the accumulation of amyloid β (Aβ) cause neurodegeneration in the brain. Aβ molecules begin to aggregate before the onset of AD and exhibit cytotoxicity in neurons12–14. Previously, we reported a real-time imaging method to detect Aβ aggregation using quantum dots (QDs), which are fluorescent semiconductor nanoparticles15. This QD-based imaging method was applied to observe the aggregation of other amyloid proteins, tau, α-synuclein, and serum amyloid A (SAA)16,17. Moreover, using this method, we developed a Microliter-Scale High-Throughput Screening (MSHTS) system for screening Aβ aggregation inhibitors18. The MSHTS system is performed using PBS, which is different from the in vivo environment, except for salt concentration. Separately, living cells contain various proteins, lipids, trace elements, small molecules, and other compounds. Therefore, the identification of materials that exhibit high Aβ aggregate inhibitory activity in the body requires an evaluation of the activity of candidate substances in vivo. However, the complexity of Aβ’s features in living organisms, which contain various biomolecules, causes a dissociation between in vivo and in vitro findings19. To resolve the discrepancy between the results derived from an evaluation that uses in vitro and in vivo assays, in this study we propose a technique that utilizes the culture supernatant of iPSCs, which is a by-product produced by disease modeling in a dish, for screening using the MSHTS system.

In this study, we succeed in the visualization of the Aβ aggregation process in the conditioned media of neurons induced to differentiate iPSCs derived from a male AD patient (i.e., AD-iNeuron) in real-time and evaluation of the aggregation inhibitory activity. We apply this imaging method to develop a High-throughput screening technology for Aggregation Inhibitors of Diseased cell-derived Aggregative Proteins (HaiDap) system (Fig. 1), which uses the supernatant of the neurobasal medium, and used it to evaluate 22 plant extracts. Furthermore, we demonstrate that the HaiDap system could be used to screen materials that prevent the aggregation of various amyloid proteins, tau, α-synuclein, and SAA, which are involved in the pathogenesis of AD, Parkinson's disease (PD), and AA amyloidosis. These results will be useful for highly efficient, accurate, and low-cost screening of disease- and patient-specific drugs involved in protein aggregation.

Results
Examination of Aβ aggregation conditions in the culture supernatant of AD-iNeuron. First, we examined whether it was possible to observe Aβ aggregation by QD-labeled Aβ (QDAβ) in the medium for differentiating iPSCs into neurons. In two types of media, a Neurobasal medium and a DMEM/F12 medium with 0.5% B27 solution20, no aggregates formed (Fig. 2a). The standard deviation (SD) values, which represent the pixel-by-pixel brightness variance of the fluorescence images that are correlated with the amount of Aβ aggregates18, revealed that B27 significantly inhibited aggregation (Fig. 2b). B-27 strongly inhibited Aβ aggregation in PBS (Supplementary Fig. 1A). We confirmed that 0.4-10% B-27 decreased the SD values, indicating that the amount of Aβ aggregates had decreased (Supplementary Fig. 1B). Although the formation of Aβ aggregates was confirmed in DMEM/F12 medium with 10% FBS, the amount was clearly less (Supplementary Fig. 2a) than that in PBS (Fig. 2a). Since serum albumin, which is found in B27 and FBS, inhibit the polymerization of Aβ21–23, we attempted to remove albumin from the medium using a 50 kDa filter device (Supplementary Fig. 2b). Aβ aggregates were observed in filtered medium (Supplementary Fig. 2c), suggesting that albumin present in the medium inhibited Aβ aggregation. Furthermore, we investigated the optimal filter size for removing albumin and found that Amicon Ultra, which removes proteins greater than 50 kDa, was the most appropriate filter for our study (Supplementary Fig. 3). Even when media containing B27 were filtered using this filter setting, Aβ aggregates formed (Fig. 2c). Next, we attempted to establish AD-iNeuron (Supplementary Fig. 4) and observed Aβ in the culture supernatant (Fig. 2d, Table 1). In this case, two media, with or without the culture of AD-iNeuron, were used, resulting in a neurobasal medium and in a supernatant of the neurobasal medium based on AD-iNeuron, which were named NbM and NbM Sup, respectively. As expected, in NbM Sup from which albumin had been removed by filtration (NbM Sup Alb(-)) (Fig. 2e), Aβ aggregation was observed (Fig. 2f). We also assessed the composition of the culture supernatant of AD-iNeuron, as well as the concentration of cell-secreted Aβ, metabolites, and electrolytes in the medium (Supplementary Fig. 4, Table 1). The results revealed that Aβ concentration in the culture medium of AD-iNeuron increased over culture time. We previously demonstrated that 79.3% of the Aβ secreted from AD-iNeuron into NbM Sup remained after filtration using a 50 kDa filter device (see US Patent App. 18/285,090, 2024)24, suggesting that the majority of Aβ secreted from AD-iNeuron remains in NbM Sup Alb(-). To verify whether Aβ fibrils formed in NbM Alb(-) and NbM Sup Alb(-), transmission electron microscopy (TEM) was performed (Fig. 2g). Fibril formation and the incorporation of TQDAβ were observed in all conditions, as had been observed in our previous findings15. These results suggest that Aβ aggregation can be visualized in the culture supernatant by removing albumin from the medium by filtration.

Aggregation of various disease-associated amyloid proteins in the culture supernatant of AD-iNeuron. To date, we have observed the aggregation of various amyloid proteins, Aβ, tau, α-synuclein, and SAA, using QDs16,17,25. In this study, the aggregation of various amyloid proteins in NbM Sup Alb(-) was observed using QD imaging methods using the same protein concentration that was employed in our previous studies16,17,25 (Fig. 3). Tau aggregated slightly after 192 h (Fig. 3 tau). We previously reported that heparin and dithiothreitol (DTT) are required to observe Tau aggregation25. The binding affinity between heparin and Tau was strong in the presence of DTT, resulting in their fibrillization under reducing conditions26. The addition of heparin and DTT may accelerate tau aggregation in this condition. SAA aggregated in NbM Sup Alb(-) (Fig. 3 SAA), as well as in PBS, but the morphology of the aggregates was slightly different17. Interestingly, α-synuclein increased the rate of aggregation in NbM Sup Alb(-) (Fig. 3 α-synuclein) compared to aggregation in PBS16. Aβ plaques are involved in α-synuclein and Tau seeding in the mouse brain27. The culture supernatant of AD-iNeuron contained just over 40 nM of Aβ42 (Supplementary Fig. 4). As mentioned above, it is estimated that approximately 80% of the Aβ remained in NbM Sup Alb(-) after filtration, and it presumably affected the aggregation of α-synuclein. Elucidating the mechanism underlying the increased rate of synuclein aggregation in NbM Sup Alb(-) will provide important insight into the pathological elucidation of many proteinopathies, including PD.

Kinetics of Aβ aggregation in Aβ-containing culture supernatant of AD-iNeuron. When Aβ aggregation in each medium without albumin was observed over time, it was noticed that this process began earlier in NbM Sup Alb (-) than in PBS and NbM Alb(-) [PBS: ~4 h, NbM Alb(-): ~6 h, NbM Sup Alb(-): ~2 h] (Fig. 4a). A logistic regression analysis of Aβ aggregation using the SD value of each image was then performed (Fig. 4b and Table 2). The difference in slope for each sample is caused by different liquid components. The difference between PBS and NbM Alb(-) is mainly due to differences in components such as ions and sugar whereas the difference between NbM Alb(-) and NbM Sup Alb(-) is due to a difference in cell secretory components. As shown in Table 2, the slope of NbM Sup Alb(-) is higher than that of NbM Alb(-), which might be due to low molecular weight cell-secreted components, such as remaining Aβ in the filtrate, that are widely involved in forming Aβ aggregates within a short period of time. In addition, to confirm whether incubation of the medium at 37°C affects Aβ aggregation, we prepared NbM medium without AD-iNeuron, and incubated it overnight at 37°C. Observation of Aβ aggregates in NbM Alb(-) after incubation showed that their morphology was similar to that before incubation (Supplementary Fig. 5a). Moreover, the SD value of brightness did not differ between - incubation and + incubation (Supplementary Fig. 5b). These results suggest that the presence of small molecules or compounds, including Aβ42 secreted from patient-derived iPSCs in the culture supernatant, can accelerate the formation of aggregates and that the QDAβ-based observation methods could detect a variety of Aβ aggregation kinetics. To confirm whether the Aβ in the culture medium can accelerate aggregations, we performed real-time imaging with or without 25 pM of monomer Aβ42 or 25 pM of oligomer Aβ42 in NbM Alb(-) (Supplementary Fig. 6). These concentrations mimicked the concentration of Aβ secreted from AD-iNeuron and remaining after filtration. Interestingly, we confirmed that the addition of Aβ42 oligomer significantly accelerates the aggregation process (Supplementary Fig. 6a and 6b). On the other hand, the morphology of Aβ aggregates formed when oligomers were added (Supplementary Fig. 6a and 6c) was different from that in NbM Alb(-) (Fig. 4a and 4c), suggesting that various molecules secreted from the AD-iNeuron also affect aggregation. The 2D images of the aggregates revealed that the shape of Aβ aggregates differed in each medium, with or without cultured AD-iNeuron. Therefore, morphological differences were investigated as three-dimensional (3D) observations (Fig. 4c). The 3D observations of Aβ aggregation in each medium with a confocal microscope revealed that the manner of deposition of aggregates was dramatically different in each medium. Although aggregated particles formed in NbM Alb(-), they were densely packed, and a very thin aggregate structure resulted. In contrast, in NbM Sup Alb(-), aggregated particles were finer than in NbM Alb(-), and they piled up three-dimensionally. As described above, AD-iNeuron-derived NbM Sup Alb(-) contained cellular Aβ42 as an impurity, suggesting that this reflects Aβ aggregation in vivo. To further verify this, we prepared healthy control (HC)-iNeuron-derived NbM Sup Alb(-) and evaluated the kinetics of Aβ aggregation in these samples (Supplementary Fig. 7). The chemical compositions of HC-iNeuron-derived NbM Sup are displayed in Table 1. The most notable differences in the composition of the HC-iNeuron-derived NbM Sup and AD-iNeuron-derived NbN Sup used in this experiment were the concentrations of Aβ40 and Aβ42, both of which were lower in HC-iNeuron-derived NbM Sup (Table 1). Real time imaging showed Aβ aggregation proceeded more rapidly in AD- than in HC-iNeuron-derived NbM Sup Alb(-) (Supplementary Fig. 7a and 7b). These results supported that the aggregation of 25 µM Aβ was affected by iNeuron-derived Aβ and its oligomers contained in the culture medium at the pM level (Supplementary Fig. 6a and 6b), which promoted aggregation. Intriguingly, the morphology of Aβ aggregates in HC-iNeuron-derived NbM Sup Alb(-) (Supplementary Fig. 7a bottom) was similar to that in PBS control (Fig. 4a top) but different/dissimilar to those in AD-iNeuron-derived NbM Sup Alb(-) (Fig. 4a bottom). These results indicate that differences in culture medium components other than Aβ also affect aggregation, suggesting that the use of patient-derived iPSC culture supernatants may enable the screening of disease- or patient-specific drugs.

Automation of the HaiDap system and screening of plant extracts. We next endeavored to establish an automated MSHTS system25 for Aβ aggregation inhibitors in NbM Sup Alb(-) (Supplementary Fig. 8). In automated MSHTS, the variation in brightness of each pixel in the central part of the image, i.e., the SD value, was used as an index of the amount of aggregates25. To accurately calculate SD values, histogram data needs to be normally distributed. As shown in Supplementary Fig. 9a and b, the SD value of each boxed region was measured. If the selection range is too wide or too narrow, the SD value cannot be measured accurately, so the same measurement range, as was suggested in our previous report25, was adopted. Rosmarinic acid (RA), a polyphenol, is a popular inhibitor of Aβ aggregation that decreases Aβ deposition in AD model mice28–30. Thus far, RA has been used as a positive control in MSHTS using PBS. Therefore, we tested whether the Aβ aggregation inhibitory effects of RA could be evaluated, even in NbM Sup Alb(-), by automated MSHTS (Fig. 5a and b). When the concentration of RA was 60 and 300 µM, Aβ aggregation in NbM Sup Alb(-) was inhibited, similar to the PBS results. In addition, after creating an inhibition curve, the EC50 value was calculated (PBS: 20.1±2.2 µM; NbM Sup Alb(-): 44.3±8.8 µM). Interestingly, the inhibitory activity of RA against Aβ aggregation in PBS decreased to less than half in NbM Sup Alb(-), indicating that the evaluation results change depending on the composition of the solvent used. On the basis of these results, we developed the HaiDap system, as indicated in Fig. 6. Briefly, iPSCs derived from AD patients differentiated to neuron cells, which are AD model cells. Then, culture supernatants of disease model cells, including extracellular Aβ, were collected. Excessive albumin was removed using a filtering device. Various concentrations of candidate inhibitors were incubated with 25 nM QDAβ and 25 µM Aβ42 in a 1536-well plate at 37℃ for 24 h. The aggregation images of each well of the 1536-well plate were captured using an automatic imaging system (Supplementary Fig. 10). After observation, the SD values of fluorescence intensity in each image were analyzed. EC50 values of the inhibitors were estimated from inhibition curves.

Using the HaiDap system, we evaluated the Aβ aggregation inhibitory activity of 22 plant extracts (Supplementary Fig. 11, Supplementary Table 1). The plants that were selected have been used in human diets and are relatively safe for long-term consumption. Orthosiphon aristatus exhibited high Aβ aggregation inhibitory activity in NbM Sup Alb(-) (1/EC50 value in the HaiDap system: 76.938±7.588 mL/mg). We then compared the Aβ aggregation inhibitory activity of Hydrangea macrophylla var. thunbergia and O. aristatus between the previous MSHTS system (in PBS) and the HaiDap system (Fig. 7a and b). Interestingly, Aβ aggregation inhibitory activity of H. macrophylla was not detected by the HaiDap system, and only by the MSHTS system (1/EC50 value in the MSHTS system: 11.572 mL/mg). Although O. aristatus also showed Aβ aggregation inhibitory activity when using the MSHTS system (1/EC50 value = 13.507 mL/mg), the activity obtained with the MSHTS system was less than that obtained with the HaiDap system (1/EC50 value = 76.938 mL/mg). These results indicate that the inhibitory activity in PBS and in NbM Sup Alb(-) differed. In other words, the efficacy of Aβ aggregation was inhibited depending on the nature of the solvent.

Validation of the HaiDap system by iPSC-based screening. Finally, we verified the efficacy of Aβ aggregation inhibitory activity evaluated by the HaiDap system using iPSC-based screening (Fig. 7c). To measure the Aβ aggregation-suppressing effect of the plant extracts on AD-iNeuron, the neurons were cultured in a Neurobasal medium supplemented separately with the extract of either H. macrophylla, O. aristatus, S. aromaticum, or G. yesoense (Supplementary Fig. 12). Aβ aggregation on the surface of neurons was visualized using QDAβ31, and then Aβ counts, Aβ aggregation size and Aβ intensity were measured using the IncuCyte Live Imaging System (Fig. 7d and Supplementary Fig. 13). In the DMSO control sample, the amount of Aβ aggregation increased around neurons. RA, which showed high activity in the MSTHS system using PBS (Fig. 5a, PBS), showed low aggregation inhibitory activity in the HaiDap system (Fig. 5a, NbM Sup Alb(-)) and iPSC-based assay (Fig. 7d and Supplementary Fig. 13). Furthermore, the H. macrophylla extract that showed no detectable activity in the HaiDap system also failed to inhibit Aβ aggregation in the iPSC-based assay (Fig. 7d and Supplementary Fig. 13). In conclusion, the extracts of three plants (O. aristatus, S. aromaticum, and G. yesoense) exhibited Aβ aggregation-inhibitory activity in both the HaiDap system and the iPSC-based assay. On the other hand, the results of cell-based assays are also strongly influenced by the cytotoxicity of the samples. To address this possible concern, we evaluated the cytotoxicity of the three plant extracts used in the experiments (Supplementary Fig. 14a). We then performed a dose-dependent iPSC-based assay using S. aromaticum extract, which showed no cytotoxicity even at the maximum concentration used in this experiment, i.e., 500 µg/mL (Supplementary Fig. 14a). The S. aromaticum extract showed a dose-dependent response in the iPSC-based assay (Supplementary Fig. 14b and c). The results of the iPSC-based assay were in good agreement with those of the HaiDap system. Although RA was effective in both the MSHTS and HaiDap systems, its effect in the cell-based assay was limited, which left us concerned about the reproducibility of the HaiDap system. Therefore, we searched for a positive control that showed significant effects in all three (MSHTS, HaiDap and cell-based) assays, and found that epigallocatechin gallate (EGCG) was the appropriate control (Supplementary Fig. 15). EGCG is a catechin found in a variety of plants, including green tea and black tea, and has been reported to be an effective ingredient in inhibiting Aβ aggregation32,33. As shown in Supplementary Fig. 15a and b, EGCG could inhibit Aβ aggregation in both of MSHTS and HaiDap systems. Further, EGCG exhibited Aβ aggregation-suppressing effect in a dose-dependent manner in the iPSC-based assay (Supplementary Fig. 15c). These results indicate that EGCG can be a positive control to inhibit Aβ aggregation across different evaluation systems. We thus succeeded in developing a method for more effectively screening Aβ aggregation inhibitory activity in an environment closer to that in living cells than currently available screening systems.

Methods
Ethics statement.
Human iPSCs and iPSC-derived NPCs used in this study were obtained from tissues collected by a third-party organization after written informed consent had been appropriately obtained from patients and healthy donors. These cells were purchased and used in accordance with all required procedures. All procedures related to the acquisition and handling of human tissues and cells, as well as all associated research protocols, were approved by the Ethics Committee of Kaneka Corporation (Approval No: 2025-5). The research was conducted in compliance with all applicable regulations, including the required Material Transfer Agreements (MTAs) and relevant licensing requirements. Race and ethnicity information was obtained solely from publicly available cell line metadata provided by RIKEN BRC and StemCell Technologies. The study used one iPSC line (HPS0854) derived from a male donor with Alzheimer’s disease, and NPCs derived from an iPSC line (SCTi003-A) originating from a healthy female donor. No additional personal or socially relevant information was collected, accessed, or used. Socially sensitive attributes such as race, ethnicity, socioeconomic status, or related characteristics were not used to guide the study design, data analyses, or interpretation.

Reagents. Synthetic peptides of human Aβ42 purified by reversed-phase column chromatography (#4349-v; Peptide Institute, purity based on HPLC: 97.4%) and Cys-conjugated Aβ40 (#23519; Anaspec, purity based on HPLC: 97%) were purchased commercially. These synthetic peptides were monomerized using 1,1,1,3,3,3-hexafluoro-2-propanol (#083-04231, Wako Pure Chemical Industries) and stored at −20°C as previously reported18. Aβ oligomerization was performed according to a reported protocol45, and 1 mM Aβ was diluted with ultra-pure water to 100 µM and incubated for 12 h at 37°C. The solution was then centrifuged at 16,900 ×g for 5 min at 4°C. The supernatant was collected and stored in a deep freezer (−80°C).

Preparation of QDAβ nanoprobes. QDAβ nanoprobes were prepared using QD-PEG-NH2 (Qdot 605 ITK Amino (PEG) Quantum dot; #Q21501MP, Thermo Fisher Scientific) according to our previous reports18,25, as follows: 10 µM QD-PEG-NH2 was first reacted with 1 mM sulfo-EMCS (#22307; Pierce/Thermo Fisher Scientific) in PBS for 1 h at room temperature. After quenching and eliminating unreacted sulfo-EMCS, the QD-PEG-NH2-bound sulfo-EMCS was reacted with 74 μM of Cys-conjugated Aβ40 for 1 h at room temperature. QDAβ concentration was determined by comparing absorbance at 350 nm to unlabeled QD-PEG-NH2.

Preparation of tau, α-synuclein, and SAA. Bacterial expression and purification of the tau MBD fragment were carried out as described previously25. Briefly, the expression plasmids were transformed into Escherichia coli (Rosetta (DE3) pLys, Novagen, Burlington), and plasmid expression was induced by 1 mM isopropyl-1-thio-β-D-galactopyranoside. The heat-stable fraction of each extract was subjected to successive column chromatographies using a Bio-Scale Mini UNOsphereTM S (Bio-Rad) and a TOYOPEARL butyl column (Tosoh). Protein concentration was estimated using the method described by Lowry et al.46, using BSA as the standard. SDS-PAGE was carried out according to the method of Laemmli et al.47.

The preparation of α-synuclein was performed as described previously48, with some modifications. Briefly, Rosetta (DE3) cells harboring pT7-7 asyn WT plasmid (Addgene plasmid #36046; Watertown) were grown in Luria-Bertani medium supplemented with 100 µg/mL ampicillin and 34 µg/mL chloramphenicol at 37℃. When OD600 of the culture reached 0.6, the cells were induced with 0.5-mM isopropyl thio-β-D-galactoside for 3 h. Harvested cells were suspended in a buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride), then lysed by sonication for 10 s. After centrifugation at 10,000×g, the resultant supernatant was boiled for 20 min and centrifuged once again at 16,000×g. Ammonium sulfate was gently added to the supernatant until it reached a saturation of 35%. After centrifugation at 13,500×g, additional ammonium sulfate was added until the supernatant reached 50% saturation. After further centrifuging at 13,500×g, the pellet was resuspended in 10 mM Tris-HCl (pH 7.4) and dialyzed overnight against 10 mM Tris-HCl (pH 7.4). The dialysate was applied to a HiTrap Q HP column (GE Healthcare Life Sciences), and the protein was eluted using a linear gradient of NaCl (0–500 mM) in 10 mM Tris-HCl (pH 7.4). The fraction containing α-synuclein was desalted using a PD Midi trap G-25 gel filtration column (GE Healthcare Life Sciences). The concentration of α-synuclein was determined using an extinction coefficient of 5,600 M-1·cm-2. Purified α-synuclein was snap-frozen in liquid N2 and stored at −80℃.

The preparation of mouse SAA (mSAA) was performed as described previously with some modifications. Briefly, mSAA cDNA was inserted into the pET-15b vector, expressed in E. coli Rosetta cells and purified49. E. coli was grown in Luria-Bertani medium supplemented with 25 µg/mL chloramphenicol and 100 µg/mL ampicillin at 37℃. When OD600 of the culture reached 0.6, cells were induced by 1 mM isopropyl thio-β-D-galactoside for 5 h. After centrifugation at 3,000×g, the pellet was suspended in a 10 mM HEPES buffer (pH 7.4) and washed twice. After washing, the pellet was resuspended in buffer A (10 mM HEPES (pH 7.4), 500 mM NaCl, 0.5% TritonX-100), sonicated (5 kHz) for 5 min, and centrifuged at 38,000×g for 30 min. The supernatant was applied to a Ni SepharoseTM 6 Fast Flow column (GE Healthcare Life Sciences) then washed with buffer B (10 mM HEPES, 500 mM NaCl, pH 7.4, 0.5% TritonX-100, 50 mM imidazole). mSAA protein was eluted using buffer C (10 mM HEPES (pH 7.4), 100 mM NaCl, 500 mM imidazole), dialyzed for 8 h in 10 mM HEPES buffer, then centrifuged at 27,000×g for 30 min at 4℃. The Lowry method was used to determine mSAA protein concentration in the supernatant. The prepared mSAA was diluted with PBS (pH 7.4) to a concentration of 1 mg/mL and stored at −80℃ until use.

Cell culture. In this study, we used one iPS cell line HPS0854 (RIKEN BRC). Information about the cells is provided next. Cell line: HPS0854; classification: iPSCs; gender: male; tissue: skin; disease name: AD. AD-iPSCs were maintained under feeder-free conditions using mTeSR1 medium (Stem Cell Technologies, Vancouver, Canada) on an hES-Matrigel®-coated plate. Medium was changed daily. The passaging method was described previously50. Human iPSC-derived neural progenitor cells (NPCs; catalog #200-0620, StemCell Technologies) were generated from the SCTi003-A induced pluripotent stem cell line, which was established from peripheral blood mononuclear cells (PBMCs) obtained from a healthy female donor. These NPCs exhibit multipotency and were maintained in STEMdiff™ Neural Progenitor Medium (catalog #05833, Stem Cell Technologies) according to the manufacturer’s instructions.

Collection of culture supernatant containing cell-derived Aβ. To collect the culture supernatant containing secreted Aβ with AD-iNeuron, 1.8-3.5×105 cells/cm2 iPSCs were seeded on a Matrigel (#354277, Corning) coated 6-well plate in mTeSR1 medium (#85850, Stem Cell Technologies) with 10 μM Y-27632 (#18190, Nacalai Tesque). Then, the STEMdiff™ SMADi neural induction kit (#08582, Stem Cell Technologies) was used with the monolayer culture protocol to promote efficient conversion of iPSCs to CNS-type neural progenitor cells (NPCs). Moreover, in order to optimize the subculture and maintenance of the NPCs, STEMdiff™ Neural Progenitor Medium (#05833, Stem Cell Technologies) was utilized. A total of 1.9-2.3×10⁵ cells/cm² NPCs were seeded on Growth Factor Reduced Matrigel (GFRM)-coated 6-well plates. During passage, Accutase (Innovative Cell Technologies) was used for cell detachment, and the medium was exchanged every three days. NPCs were cryopreserved using STEMdiff™ Neural Induction Medium (#05838, Stem Cell Technologies). To generate induced AD-iNeuron derived from NPCs, 1.0×105 cells/cm² NPCs seeded on GFRM-coated 96-well plates using phenol-red-free Neurobasal® medium (NbM) (Thermo Fisher Scientific) with B27 (×50) (#A1895601, Thermo Fisher Scientific) plus 100 ng/mL of recombinant human sonic hedgehog51–53 (#78065, Stem Cell Technologies) (Supplemental Fig. 4a). In this AD-iNeuron induction step, glutamine-free Neurobasal medium was used. Cells were incubated at 37℃ in a 5% CO2 atmosphere. One day after plating NPCs and while collecting half the culture supernatant every 3 days, NbM with B27 medium was added and cultured for 21 days to allow for neural differentiation. The changes in cell morphology during each culture process were confirmed by images (Supplemental Fig. 4b). The culture supernatant was centrifuged at 2,000×g for 3 min, and the supernatant was collected in a tube and stored at −20°C. The generation of healthy control neurons (HC-iNeuron) was described previously54. The differentiated HC-iNeuron were cultured for 28 days in NbM supplemented with B27, as in the case of AD-iNeuron. The culture supernatant was centrifuged at 2,000 × g for 3 min, and the resulting supernatant was stored at −20°C.

Immunocytochemistry. AD-iNeuron was labeled with NeuroFluor™ NeuO (green) (Fig. 2d). The other iPSCs, NPCs, and neurons were also confirmed by immunocytochemistry (Supplemental Fig. 4c). These cells were washed once with D-PBS then fixed using 4% paraformaldehyde for 30 min at 4℃. Fixed cells were permeabilized and blocked with PBS containing 0.1% TritonX-100 and 1% BSA for 1 h at room temperature. Samples were incubated with primary antibody diluted in PBS containing 1% BSA at 4℃ overnight. After washing with PBS three times, samples were incubated with secondary antibody diluted in PBS, and cells were observed using a BZ-X710 microscope (Keyence). The antibodies used in this study were: anti-Nanog antibody (dilution ×250, host: rabbit, ab109250), anti-SSEA4 (dilution ×500, host: mouse, #MA1-021), anti-NESTIN (dilution ×150, host: mouse, #A24345), rabbit anti-PAX6 (dilution ×150, host: rabbit, #A24340), anti-SOX2 (dilution ×150, host: rabbit, #ab137358), anti-MAP2 (dilution ×250, host: mouse, #ab254144), Alexa 488 donkey anti-mouse (dilution ×250, #A24350), Alexa 594 donkey anti-rabbit (dilution ×250, #A24343), and Alexa 594 goat anti-mouse (dilution ×100, #A21155). Cell nuclei were stained blue with DAPI (19178-91, Nacalai Tesque).

Exclusion of albumin using the filtering device. To exclude albumin derived from the B-27 solution, a commercial filtering device, Amicon Ultra (#UFC505096, UFC905024, Millipore), was used (for details, see US Patent App. 18/285,090, 2024). The time and speed of centrifugation were set according to the manufacturer’s procedure. Before filtering the medium, the filtering device was cleaned by washing it twice with water in a centrifuge. Then, 0.1% Tween-20 solution was applied to the filtering device. After incubation for 2 h at room temperature, the filtering device was centrifuged three times to remove the 0.1% Tween-20 solution. Finally, the culture supernatant obtained from AD-iNeuron was filtered.

Aβ aggregation model. The Richards growth curve, which is a generalized formulation of the logistic function, was used to model time evolution of the intensity SD, which represents Aβ aggregation. The growth curve is defined by:

f(t,α,β,γ,θ) = α / [1+βe(−γ(t−θ))]1/β (1)

where α is the amplitude and γ describes the growth rate. β and θ together represent the growth rate r(β,θ). Here, the growth rate (r) describes the steepest slope and can be written as r = max(df/dt). Least square optimization was used to fit the curve f(t,α,β,γ,θ) to the data and to compute its characteristic properties.

Analysis of the culture supernatant. Analyses of the collected culture supernatant (on days 1, 7, 14, and 21) were performed using BioProfile® FLEX2™ (Nova Biomedical). The culture supernatant medium was analysed and evaluated for pH, glucose, lactate and electrolytes (calcium, sodium, potassium). Aβ secreted from neurons was quantitatively analyzed by the Molecular Devices SpectraMax i3X using an ELISA kit (Human βAmyloid (1-42); FUJIFILM 298-62401, Human βAmyloid (1-40) Wako II; FUJIFILM 298-64601).

Preparation of plant extracts. One g of dry plant material (Hydrangea macrophylla var. thunbergia, Orthosiphon aristatu, Ocimum americanum, Glebionis coronaria, Stevia rebaudiana, Cycas revoluta, Citrus tankan, Ipomoea batatas, Canavalia gladiata, Azadirachta indica, Ocimum basilicum, Clitoria ternatea, Coix lacryma-jobi var. mayuen, Senna occidentalis, Eriobotrya japonica, Lablab purpureus, Syzygium aromaticum, Fagopyrum esculentum, immature Pisum sativum (green), mature P. sativum (yellow)) was immersed in 90% ethanol, and stirred at 10×g at 28°C for 20 h in the dark to separate the extracts. The parts of each plant used in the experiment are shown in Supplementary Table 1. Extracts were centrifuged (2,000×g) for 20 min and concentrated with an evaporator after filtration. Each extract was transferred to a glass vial, weighed after lyophilization, and dissolved in DMSO to a final concentration of 100 mg/mL each.

MSHTS and HaiDap systems. The SD values were determined by a modified MSHTS system, which was previously described18,25. More specifically, various concentrations of aggregation inhibitors, 25 μM Aβ42 and 25 nM QDAβ in PBS or NbM Sup Alb(-), were incubated in a 1536-well plate (#782096, Greiner Bio-One) at 37°C for 24 h. The QDAβ-Aβ42 aggregates that formed in each well were observed by an inverted fluorescence microscope (TE2000, Nikon) equipped with a color CCD camera (DP72, Olympus) and an objective lens (Plan Fluor 4×/0.13 PhL DL, Nikon). SD values of fluorescence intensities of 40,000 pixels (200 × 200 pixels) around the central region of each well were measured by ImageJ software (NIH). EC50 values were estimated using Prism software (GraphPad Software).

Automated MSHTS and HaiDap systems. The automated MSHTS system used was described in our previous report25. Briefly, sample preparation was carried out using automated workstation JANUS G3 (Perkin Elmer). To create a series of dilution samples, dilution buffer (10% EtOH, 1×PBS or NbM Sup Alb(-)) were aspirated using a conductive chip that can detect the level of liquid and inject it into each well of a 384-well plate (#HSP3801, BIO-RAD). Stock sample solutions were injected manually into vacant wells of the 384-well plate. This dilution step was repeated five times so that a six-dilution series was prepared for each sample. The diluted samples in the 384-well plate were aspirated using a 384-chip head and injected into a 1536-well plate. The Aβ solution, which is a mixture of 50 nM QDAβ and 50 µM Aβ42, was prepared in a microcentrifuge tube at 4°C, aspirated by a conductive chip that can detect the level of liquid, and injected into a cold 384-well plate placed at 4°C. Finally, the Aβ and sample solutions were mixed by pipetting three times into a 1536-well plate. The 1536-well plate was sealed with a plate seal to prevent evaporation. In order to remove bubbles and flatten the liquid surface, the 1536-well plate was centrifuged at 1530×g for 5 min in a multi-well plate centrifuge (PlateSpin II, KUBOTA). After centrifugation, the 1536-well plate was observed by an inverted fluorescence microscope system (ECLIPSE Ti-E, Nikon) equipped with a color CMOS camera (DS-Ri2, Nikon). QD was imaged using a 4× objective lens (Plan Apo λ 4×/0.20, Nikon) and a TRITC filter set (TRITC-A-Basic-NTE, Semrock).

TEM observations. A total of 10 µL of amyloid fibrils were prepared in PBS or NbM Sup Alb(-). Each sample was transferred onto a 200-mesh copper grid (Nisshin) and incubated for 5 min at room temperature. Grids were then washed twice with water. Samples were negatively stained twice with 1% phosphotungstic acid. Specimens were examined under an H-7600 transmission electron microscope (Hitachi) at 60 kV.

Confocal laser scanning microscopy. Z-stacks images of Aβ aggregates were captured using an inverted microscope (Ti-E) and a confocal laser microscope system (C2 Plus; Nikon) equipped with an objective lens (PlanApo λ 20×/0.75 NA; Nikon). Images were captured and oversampled by taking 2 µm z-steps between the acquired images, which were analyzed with NIS-Elements software (Nikon).

Measurement of Aβ aggregation in the AD-iNeuron. Aβ aggregation on the cell surface in indicated conditions was measured using the IncuCyte Live Imaging System (Sartorius). Neurons co-incubated with 5 μM Aβ42, 30 nM QDAβ, and plant extracts were measured by Incucyte every hour. The Aβ aggregation process (Aβ counts, Aβ aggregation size, and Aβ aggregation integrated intensity), when surrounded by yellow lines, indicate the perforated areas in the cell monolayer. For apoptosis assays, the IncuCyte Caspase 3/7 green dye kit (#4440, Sartorius) was used at a final concentration of 1:1000. Final concentrations of toxic control compounds added to the plate were: 5 µM staurosporin while the extracts of four plants (H. macrophylla var. thumbergii, O. aristatu, S. aromaticum, and G. yesoense var. yesoense), were tested at five concentrations (0.8, 4, 20, 100, and 500 ng/µL: n = 20 samples). Treated plates were incubated at 37℃ for 24 h, imaged at 20× magnification using phase and green channels at nine images per well, and analyzed using IncuCyte software.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In the manuscript entitled “High-throughput screening technology for aggregation inhibitors of diseased cell-derived\naggregative proteins (HaiDap) system,” the authors have developed and refined a screening system for amyloid aggregation\ninhibitors using iPSCs derived from Alzheimer’s disease patients. The goal of the paper is to study aggregation inhibitors in\na more disease relevant context. This paper reports on the aggregation of Aβ42, and other disease-associated amyloid\nproteins, in supernatant of the differentiated iPSC cell line, in addition to the kinetics of aggregation. The proposed\nmethodology has optimized neural cells derived from an AD patient and reports the aggregation in presence of\ncharacterized culture media obtained from AD-derived neural cells. Cleverly, quantum dot labeled Ab40 with unlabeled\nAb42 were used to define the shape, morphology and the texture of the aggregates at the scale of several hundreds of\nmicrometers.\nThe scale to which the authors can screen compounds is impressive, and markedly higher throughput than most assays. I\nappreciate their efforts to do aggregation studies that are more biologically relevant to pathology, as this is important.\nHowever, a number of issues are noted below that in my opinion limit the probability that this system recapitulates key\naspects of neurodegenerative disease.\n1) The model system was established using cells derived from one AD patient (n=1). Beyond the limited n value,\nbackground information about the patient is missing, such as the genotype, if the patient was a familial or sporadic case of\nAD, age of onset, MMSE scores, etc.\n2) Filtration also removes other proteins in solution than albumin (Fig. 2e), so it is possible that species beyond albumin are\nremoved and likely are relevant to the aggregation kinetics of Ab42 since this is so dependent on solution conditions. While\nthe secretion of species from the AD cells makes this system more relevant than standard immortalized cell lines, it is not\nknown that this reaction mixture actually recapitulates key aspects of AD.\n3) The concentrations of amyloidogenic proteins used (25 µM for Aβ42) to test for aggregation in NbM sup Alb- medium are\nquite a bit higher than those used in typical in vitro aggregation studies (purified Ab42 typically aggregates with a half-time of\n2 h at a concentration of 2 µM). One would predict their system would show faster aggregation at such concentrations (eg a\nfew µM), considering the crowded nature of their media relative to a typical simple buffer system. It is unsurprising that\naggregation begins immediately (Fig 7) considering the very high concentration of Ab42 and the crowded nature of the\nmedia. As a result, mechanistic insight into the microscopic mechanisms impacted by candidate compounds is limited in this\nsystem. Since the purpose of this system is to be more physiologically relevant, using at least 50-100x greater\nconcentrations of Ab42 than are necessary to observe cytotoxicity is an issue.\n4) The rationale for using plant extracts for screening is not clear. The authors should consider elaborating on the plant\nextracts that were screened and provide additional background information on the significance of deploying specific plant\nextracts for this advanced screening methodology. Using very well characterized inhibitors of Ab42 aggregation would be\nhighly helpful, for example EGCG, bexarotene, specific chaperones (many additional examples are published in the\nliterature with known mechanisms for targeting primary nucleation, secondary nucleation, elongation, or other aspects of\nAb42 deposition). Seeing how inhibitors with well-characterized mechanisms change the Ab42 aggregation rate in their\nsystem would be very helpful for showing its utility.\n5) How did the authors assess the cost comparison between the ThT system and the HaiDAP? The costs of the ThT assay\nseems overstated, especially considering 384-well plates are commonplace.\n6) The scale at which the Ab42 aggregates are imaged in this study is typically on the order of hundreds of microns. As such,\nlimited structural insight into the types of Ab42 fibrils that are formed with and without inhibitors can be gained. The degree to\nwhich these Ab42 aggregates recapitulate those observed in AD at the structural level is not investigated, especially\nconsidering they contain Ab40 labeled with QDs. The TEM images are helpful, but do not resolve the above issues.\n7) Details on how Ab42 was prepared are absent. All that seems listed in the methods is that human Ab42 was bought from\nrPeptide, and searching the provided catalog number did not readily show the kind of Ab42 used. Presumably it is\nrecombinant, but with what counterion. How was it solubilized and monomerized? Was it size excluded or purified before\nuse? Etc.\n8) The rationale for the success rate difference of the plant extracts in PBS versus their system is lacking.\n9) Further details on how B27 works to inhibit aggregation would be helpful.", "reviewer_2": "In the manuscript entitled „ High-throughput screening technology for aggregation inhibitors of diseased cell-derived\naggregative proteins (HaiDap) system”, Kuragano and colleagues present interesting data on the establishment of a higher\nthroughput-compatible assay allowing to screen natural compounds for their ability to inhibit aggregation of Ab42 in an in\nvitro assay. This assay is facilitated by cell culture supernatant of hiPSC – derived neurons and can be evaluated in high\nthroughput formats. Compared to previously presented assay systems that rely on PBS as buffer, this assay is more stringent\nin identifying crude plant extracts with such action. The assay system appears scalable and reproducible, as many\nexperiments were performed with at least 3 replicate experiments.\n- Although the Assay system presented identifies less active compounds, it is still somewhat surprising that more than 10%\nof the crude extracts showed activity and could be tested in a cellular assay system. A major point of concern is the cell-\nbased assay system presented in the end of the manuscript. Important information (such as dose-dependency and / or\ntoxicity readouts are not shown, even though they are mentioned in the text. Also, the HaiDap assay is supposed to “bridge”\nthe less complex biochemical in vitro assays (such as the MSHTS) and more complex assays (such as the neuron-based\nsystem), and finally the effect in an animal model. Surprisingly, the assay fails to do so in the case of the only positive control\nsubstance (Rosmarinic Acid – RA) presented here: The authors show that RA is active in both assays, MSHTS, and HaiDap\nassay. It is stated that it shows a lower activity in HaiDap, but differences shown in Figure 5 are low, and as stated in the\nresults section, HaiDap shows an approximately double EC50 compared to the PBS based system. Nevertheless, RA did\nnot show any activity in the cell-based assay (Figure 7, Supplementary Figure 8 and 9). Also, authors noted / cited that RA is\nthe only substance of the tested set that has repeatedly shown efficacy in animal models for amyloid deposition. In the eyes of\nthis reviewer, this is worrisome, as the system was initially presented to be a better selector for efficacy in more\ncomplicated assays. If RA shows (albeit reduced) efficacy in HaiDap, why does it not show efficacy in the cell based model,\nbut has so before in other studies in vivo?\nOther specific comments / questions are listed below:\n- Various experimental details are unknown / could not be easily evaluated by this reviewer. Please see below for some\nspecific examples.\n- One hiPSC line was used in this study. Therefore, saying that the HaiDap system is allowing a patient-specific screening is\npossibly overstated, this should be toned down.\n- What is the rationale for including SHH protein in the differentiation protocol?\n- There is a discrepancy between the materials & methods section and the legend of SupFigure 3 in regard to the medium\nchanging / feeding procedure. That needs to be checked for consistency. Also, certain possibly important experimental\ndetails are missing, such as the coating procedure used for the cell culture vessels. Is the given cell seeding density at time\nof replating per well or cm^2 (see materials and methods)?\n- The authors reported that aggregation in PBS (MSHTS) is faster and leads to a stronger signal than in NbM Alb(-). Figure 4\nindicates that NbM Sup Alb(-) leads to an aggregation behaviour that is similar to the one obtained in PBS. This is\ninteresting, as MSHTS is not as predictable as HaiDap according to authors. But what about the effect of the NbM Alb(-) on\nthis selectivity? It is not clear to this reviewer that the “selectivity effect” of the HaiDap system is introduced by the culture\nmedium, or by factors secreted by cells. Even though the aggregation in NbM Alb(-) is lower than in PBS, this selectivity\neffect should be checked in the cell-free system, but with the medium incubated on the culture vessel coating for the same\ntime as with the cells. Adding B27 supplement to PBS, or just using Neurobasal medium would be also appropriate controls.\n- It is not proven to this reviewer that the accelerated and increased aggregation is caused by Ab42 in the culture medium,\nas this is presented as an important factor. Medium cultured on cells and, for example, immunodepleted to remove Ab,\nwould be an appropriate control. This is possibly even more relevant, as – to the understanding of this reviewer – the cell\nsupernatant is used in the HaiDap assay with several orders of magnitude higher concentration of recombinant Ab42 added\nin the assay.\n- Is the medium supernatant from the hiPSC-derived neurons collected from several medium changes? If so, is it pooled\nafterwards? This is potentially relevant, as the authors state in the discussion (page 8 – top part) that one benefit of the\nHaiDap system is to be more efficient than complicated hiPSC-based systems. If the medium is only collected once, one\nhiPSC culture would be needed for one harvest, largely reducing this effect.\n- It is not easily understandable how the plant extracts were dissolved and applied in the assay system. Is it ensured that the\ndilution series is performed in the solvent, thus adding always the same amount of solvent into each dilution / the control\nwells? Also, in the cellular assay, DMSO is used as solvent, but it is not clear which concentrations are shown in the\nquantified figures, as well as what the replication of this data was (generating the error bars).\n- Also, it is difficult to impossible to compare a purified substance (as RA) directly with a crude plant extract, such as done\nin the cell-based assay. Only one concentration is shown in the time-course of Figure 5, a dilution series would be needed to\nfurther specify the activity (see also below).\n- Did the authors also evaluate other known compounds to inhibit aggregation, such as “beta sheet-breakers”? These would\nbe an appropriate positive control for a revised manuscript.\n- What do the cells in the cell-based assay system add to the assay? The selectivity for the compounds could come from the\nmedium composition used, one possible benefit, using the cells for assessing toxicity of plant extracts, is briefly mentioned,\nbut not shown. For the cell-based assays, it is therefore necessary to further elude on that, also how survival / toxicity was\nassessed.\n- There are instances of figure duplication observed, as in Supplementary Figure 7: all plant extracts that do not show an\neffect in either system have always the same panels used for both MSHTS and HaiDap.\n- Figure 4 is missing the legend for subpanel c.\nEven though the system presented here is of interest to a scientific community, especially in the protein aggregation and\nAlzheimer’s Disease field, it is questionable if it is suitable for the very broad audience targeted by the journal in the current\nform. This in particular, as controls are missing to understand the origin of the higher selectivity of the HaiDap system and\nthe disconnect of the positive control substance efficacy in two important assays presented here.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We are honored that you have highly evaluated the HaiDap system. We carefully considered all comments, conducted\nadditional experiments, and revised the manuscript. The point-by-point responses to all comments are listed below. We\nbelieve that this revision has significantly improved the manuscript and addressed your concerns.\n1) The model system was established using cells derived from one AD patient (n=1). Beyond the limited n value, background\ninformation about the patient is missing, such as the genotype, if the patient was a familial or sporadic case of AD, age of\nonset, MMSE scores, etc.\nWe have added the following information about the iPS cell line derived from an AD patient to the \"Cell culture\" section\nof the Methods (Page 11, Lines 379-381); In this study we used one iPS cell line HPS0854 (RIKEN BRC, Tsukuba, Japan).\nCell information; Cell line: HPS0854, Classification: iPSCs, Race: Japanese, Gender: Male, Age at sampling: 50s, Tissue:\nskin, Disease name: Alzheimer's disease.\nIn this study, we aimed to establish the basis of the HaiDap system by using the culture supernatant of iPSCs, and first used\none iPS cell line. We believe that the usefulness of the HaiDap system developed in this study will be established by\naccumulating further screening knowledge using culture supernatants of various iPS cell lines. We added this information\nto the Discussion section as a limitation of this study and future prospects (Page 9, Lines 303-306).\n2) Filtration also removes other proteins in solution than albumin (Fig. 2e), so it is possible that species beyond albumin\nare removed and likely are relevant to the aggregation kinetics of Ab42 since this is so dependent on solution conditions.\nWhile the secretion of species from the AD cells makes this system more relevant than standard immortalized cell lines, it\nis not known that this reaction mixture actually recapitulates key aspects of AD.\nAs the reviewer pointed out, filtration may remove molecules other than albumin, and it is not fully clear whether AD-\niNeouron culture supernatants actually recapitulate important aspects of AD. On the other hand, we confirmed that\napproximately 80% of the Aβ secreted into the AD-iNeuron culture supernatant remains in the culture supernatant after\n42\nfiltration by optimizing the filtration technology (see US Patent App. 18/285,090, 2024). This study also demonstrated that\nscreening with culture supernatant (HaiDap system) showed a higher correlation with subsequent iPSC-based assays than\nprevious screening in PBS (MSHTS system). Based on the above, although it remains unclear to what extent the HaiDap\nsystem actually recapitulates key aspects of AD, we believe that it is more selective than previous screening in PBS.\nInformation on the residual Aβ fraction after filtration has been added to the Results section (Page 4, Lines 130-132), while\na reflection on whether this reaction mixture actually recapitulates key aspects of AD has been added to the Discussion\nsection (Page 9, Lines 299-302).\n3) The concentrations of amyloidogenic proteins used (25 µM for Aβ42) to test for aggregation in NbM sup Alb- medium\nare quite a bit higher than those used in typical in vitro aggregation studies (purified Ab42 typically aggregates with a\nhalf-time of 2 h at a concentration of 2 µM). One would predict their system would show faster aggregation at such\nconcentrations (eg a few µM), considering the crowded nature of their media relative to a typical simple buffer system. It\nis unsurprising that aggregation begins immediately (Fig 7) considering the very high concentration of Ab42 and the\ncrowded nature of the media. As a result, mechanistic insight into the microscopic mechanisms impacted by candidate\ncompounds is limited in this system. Since the purpose of this system is to be more physiologically relevant, using at least\n50-100x greater concentrations of Ab42 than are necessary to observe cytotoxicity is an issue.\nAs the reviewer pointed out, drug screening should be performed under physiological conditions as much as possible. On\nthe other hand, the concentration of Aβ in cerebrospinal fluid is 0.186 nM, which is much lower than the 2 µM used in\ngeneral cell tests, as the reviewer pointed out. It is also unclear whether 2 µM is optimal. We recently reported the kinetic\nverification of Aβ aggregation under viscous physiological conditions, kinetically demonstrating that it takes about 10 years\nfor Aβ aggregates to grow to a diameter of 1 µm when used at 0.186 nM (Kuragano et al., 2022;\nhttps://doi.org/10.1016/j.colsurfb.2022.112449). In fact, the size of amyloid plaques can be tens to hundreds of µm in\ndiameter, and this result is consistent with the knowledge that it takes several decades from Aβ aggregation to the onset of\nAD. Screening Aβ aggregation inhibitors over decades would take too long, so some form of acceleration is necessary. The\nsimplest way to accelerate the aggregation reaction is to increase the reaction temperature or the concentration of molecules.\nHowever, since proteins are thermally denatured, we believe that the most appropriate way to accelerate the reaction is to\nincrease protein concentration. In our previous paper on the development of an automated MSHTS system, we confirmed\nthat 25 µM Aβ aggregation saturated in approximately 16 h (Sasaki et al., 2019; https://doi.org/10.1038/s41598-019-38958-\n0). We also reported that in the presence of model neuronal PC12 cells, 20 µM Aβ aggregated and deposited around the\ncells within 16 h, causing cell death (Kuragano et al., 2020; https://doi.org/10.1038/s41598-020-66129-z). As our objective\nwas to establish a screening system that bridges in vitro screening to cell-based screening, Aβ concentration was set at 25\nµM because at this concentration, the effect of Aβ aggregation on cells could be detected within 24 h.\n4) The rationale for using plant extracts for screening is not clear. The authors should consider elaborating on the plant\nextracts that were screened and provide additional background information on the significance of deploying specific plant\nextracts for this advanced screening methodology. Using very well characterized inhibitors of Ab42 aggregation would be\nhighly helpful, for example EGCG, bexarotene, specific chaperones (many additional examples are published in the\nliterature with known mechanisms for targeting primary nucleation, secondary nucleation, elongation, or other aspects of\nAb42 deposition). Seeing how inhibitors with well-characterized mechanisms change the Ab42 aggregation rate in their\nsystem would be very helpful for showing its utility.\nWe wanted to screen for substances that could potentially be ingested over a long period of time, so we screened 22 plant\nspecies in this study. These plant species were selected because they have a known history in the human diet and are\nrelatively easy to obtain. Relevant statements have been added to the abstract (Page 2, Lines 38-39) and the Results section\n(Page 7, Lines 211-212).\nUnfortunately, at this time we have not been able to identify any pure compounds that assayed positively in all three\nscreening systems: PBS screening (MSHTS system), AD-iNeuron culture supernatant screening (HaiDap system), and the\ncell-based assay. The only compounds that were positive in all three screening systems were from the three plant extracts\nreported in this paper (O. aristatus, S. aromaticum, and G. yesoense). As the reviewer pointed out, we performed a cell-\nbased assay using AD-iNeuron with two compounds, morin and quercetin, whose aggregation inhibition mechanisms are\nwell known. However, as with RA, they were not as effective as the three plant extracts.\nThe results of the experiment are shown below:\nThe initial compound utilized was morin. Morin is a compound employed as a control reagent for Thioflavin T assays and\nis recognized for its capacity to impede seed formation from Aβ monomers. The other compound employed was quercetin,\nwhich is a compound that robustly inhibits Aβ fibril formation. In other words, it has been hypothesized that morin impedes\nearly Aβ aggregation, while quercetin hinders later Aβ fibril formation to a greater extent than morin.\nAs shown below, the kinetics of Aβ aggregation inhibition were confirmed using these two compounds in a cell-based\nassay. It was determined that morin inhibits the early stage of Aβ aggregation (up to 6 h), while quercetin inhibits later Aβ\nfibril formation (after 19 h) to a greater extent than morin. However, the effect was not as dramatic as that of the three plant\nextracts.\nThus, it is expected that the HaiDap system will enable the evaluation of the inhibitory effects at each step of Aβ aggregation,\nsuch as seed formation and fibril elongation. However, no compounds used so far have shown dramatic effects, and at this\nstage, we were unable to explicitly mention these results in this paper.\n5) How did the authors assess the cost comparison between the ThT system and the HaiDAP? The costs of the ThT assay\nseems overstated, especially considering 384-well plates are commonplace.\nAs the reviewer pointed out, it is also possible to perform the ThT assay using 384-well plates, so we clearly stated in the\ntext that the cost calculations are for 96-well plates (Page 8, Line 271). We also mentioned the assay using 384-well plates\nin the legend of Supplementary Fig. 13.\n6) The scale at which the Ab42 aggregates are imaged in this study is typically on the order of hundreds of microns. As\nsuch, limited structural insight into the types of Ab42 fibrils that are formed with and without inhibitors can be gained. The\ndegree to which these Ab42 aggregates recapitulate those observed in AD at the structural level is not investigated,\nespecially considering they contain Ab40 labeled with QDs. The TEM images are helpful, but do not resolve the above\nissues.\nAs the reviewer pointed out, we used QD-labeled Aβ as a fluorescent probe, but the amount was 0.1% of Aβ . In our\n40 42\nfirst paper in which this fluorescent probe was used, we confirmed that QDAβ , when added at a ratio of 0.1% to Aβ, was\n40\nincorporated into Aβ fibrils at a ratio of 0.1% and did not significantly affect the aggregation rate of Aβ (Tokuraku et\n42 42\nal., 2009; https://doi.org/10.1371/journal.pone.0008492). On the other hand, we also reported that QDAβ alone cannot\nform amyloid fibrils, probably due to steric hindrance by the QDs (Tokuraku and Ikezu, 2014;\nhttps://doi.org/10.1016/B978-0-12-394431-3.00011-0). Based on these prior results, we believe that the current imaging\nconditions using 0.1% QDAβ do not significantly affect Aβ aggregation. Understandably, because this is an accelerated\n42\ntest in which Aβ concentrations are far higher than physiological conditions, concerns remain as to whether Aβ aggregates\n42\nrecapitulate those observed in AD, as stated in response to comment 3 above. This is a dilemma when employing in vitro\nscreening, and this concern can only be resolved by analyzing in detail the mechanism of inhibition of screened inhibitors\nin vivo, as in other screening methods.\n7) Details on how Ab42 was prepared are absent. All that seems listed in the methods is that human Ab42 was bought from\nrPeptide, and searching the provided catalog number did not readily show the kind of Ab42 used. Presumably it is\nrecombinant, but with what counterion. How was it solubilized and monomerized? Was it size excluded or purified before\nuse? Etc.\nThe Aβ that was used can be easily searched for, and identified, when using the company name and product number\n(https://www.peptide.co.jp/en/catalog/f-cat?k_code=4349-v). To assist readers, we have added a brief explanation of the\nmethods, as suggested by the reviewer (Page 10, Lines 325-328).\n8) The rationale for the success rate difference of the plant extracts in PBS versus their system is lacking.\nFollowing the reviewer's suggestion, we added a discussion of the differences in success rates between the MSHTS and\nHaiDap systems (Page 9, Lines 285-292).\n9) Further details on how B27 works to inhibit aggregation would be helpful.\nWe performed additional experiments with B27 (Supplementary Fig. 1) and have added the results to the manuscript (Page\n4, Lines 110-112).\nReviewer #2 (Remarks to the Author):\nIn the manuscript entitled „ High-throughput screening technology for aggregation inhibitors of diseased cell-derived\naggregative proteins (HaiDap) system”, Kuragano and colleagues present interesting data on the establishment of a higher\nthroughput-compatible assay allowing to screen natural compounds for their ability to inhibit aggregation of Ab42 in an\nin vitro assay. This assay is facilitated by cell culture supernatant of hiPSC – derived neurons and can be evaluated in high\nthroughput formats. Compared to previously presented assay systems that rely on PBS as buffer, this assay is more stringent\nin identifying crude plant extracts with such action. The assay system appears scalable and reproducible, as many\nexperiments were performed with at least 3 replicate experiments.\nWe thank you for your interest in our manuscript. We have carefully considered all of your comments, added some\nexperiments to address your concerns, and revised the manuscript extensively. We believe that this revision has significantly\nimproved the manuscript and addressed your concerns.\n- Although the Assay system presented identifies less active compounds, it is still somewhat surprising that more than 10%\nof the crude extracts showed activity and could be tested in a cellular assay system. A major point of concern is the cell-\nbased assay system presented in the end of the manuscript. Important information (such as dose-dependency and / or\ntoxicity readouts are not shown, even though they are mentioned in the text. Also, the HaiDap assay is supposed to “bridge”\nthe less complex biochemical in vitro assays (such as the MSHTS) and more complex assays (such as the neuron-based\nsystem), and finally the effect in an animal model.\nAs the reviewer pointed out, cell-based assays are more complicated than in vitro assays. In particular, cytotoxicity in the\nsamples can significantly impact the results. To address the reviewer's concerns, we performed dose-dependent iPSC-based\nassays for the plant extracts that showed significant inhibitory activity (Supplementary Fig. 12) and added a corresponding\nexplanation (Page 7-8, Lines 236-244). We believe that these experiments demonstrate that at least one plant extract, S.\naromaticum extract, was effective in both the HaiDap system and cell-based assays.\nSurprisingly, the assay fails to do so in the case of the only positive control substance (Rosmarinic Acid – RA) presented\nhere: The authors show that RA is active in both assays, MSHTS, and HaiDap\nassay. It is stated that it shows a lower activity in HaiDap, but differences shown in Figure 5 are low, and as stated in the\nresults section, HaiDap shows an approximately double EC50 compared to the PBS based system. Nevertheless, RA did\nnot show any activity in the cell-based assay (Figure 7, Supplementary Figure 8 and 9). Also, authors noted / cited that RA is\nthe only substance of the tested set that has repeatedly shown efficacy in animal models for amyloid deposition. In the eyes of\nthis reviewer, this is worrisome, as the system was initially presented to be a better selector for efficacy in more\ncomplicated assays. If RA shows (albeit reduced) efficacy in HaiDap, why does it not show efficacy in the cell based model,\nbut has so before in other studies in vivo?\nAs stated in response to comment 4 of reviewer #1, we have not been able to identify a pure compound that shows clear\npositive results in the MSHTS system, the HaiDap system, and the iPSC-based assay. RA, morin, and quercetin all showed\na slight effect in iPSC-based assays, although the effects were not as great as those of the three plant extracts used in this\nstudy. In iPSC-based assays, factors such as inhibition of Aβ aggregation as well as deposition on the cell surface are\ninvolved, and it is believed that a mixture containing multiple active compounds, such as a plant extract, would be more\nadvantageous than a single pure compound. The HaiDap system using QD imaging is also suitable for screening crude\nextracts containing various molecules, such as plant extracts (see the following paper for details; Sasaki et al., 2019;\nhttps://doi.org/10.1038/s41598-019-38958-0), and may be suitable for screening mixtures that effectively inhibit Aβ\naggregation and deposition on neuronal surfaces.\nAs mentioned in the manuscript (Page 6, Lines 193-194), RA is a positive control in the MSHTS system using PBS, so we\ndo not believe that it is a positive control that will be effective in all tests. In fact, the paper by Hase et al. (Ref. No. 29)\ncited in this paper reported that Aβ aggregation was suppressed by monoamines that increased in the brain following RA\nintake, and that RA does not directly suppress Aβ aggregation in the brain. To avoid any confusion to readers, we added\nsome discussion about the experimental results using RA (Page 9, Lines 294-298).\nOther specific comments / questions are listed below:\n- Various experimental details are unknown / could not be easily evaluated by this reviewer. Please see below for some\nspecific examples.\nFollowing the reviewer's suggestions, we increased and fortified the descriptions of the experimental conditions in the\nMethods section (indicated in red text) and added an Excel file containing the source data for all experiments.\n- One hiPSC line was used in this study. Therefore, saying that the HaiDap system is allowing a patient-specific screening\nis possibly overstated, this should be toned down.\nWe agree with your comment. A similar point was made by reviewer #1's comment (1), so kindly refer to that response.\n- What is the rationale for including SHH protein in the differentiation protocol?\nSHH was added to induce AD-iNeuron derived from NPCs. Please see the revised Method section for more details (Page12,\nLines 397-401).\n- There is a discrepancy between the materials & methods section and the legend of SupFigure 3 in regard to the medium\nchanging / feeding procedure. That needs to be checked for consistency. Also, certain possibly important experimental\ndetails are missing, such as the coating procedure used for the cell culture vessels. Is the given cell seeding density at time\nof replating per well or cm^2 (see materials and methods)?\nThe Methods section has been revised according to the reviewer’s comments.\n- The authors reported that aggregation in PBS (MSHTS) is faster and leads to a stronger signal than in NbM Alb(-). Figure\n4 indicates that NbM Sup Alb(-) leads to an aggregation behaviour that is similar to the one obtained in PBS. This is\ninteresting, as MSHTS is not as predictable as HaiDap according to authors. But what about the effect of the NbM Alb(-)\non this selectivity? It is not clear to this reviewer that the “selectivity effect” of the HaiDap system is introduced by the\nculture medium, or by factors secreted by cells. Even though the aggregation in NbM Alb(-) is lower than in PBS, this\nselectivity effect should be checked in the cell-free system, but with the medium incubated on the culture vessel coating for\nthe same time as with the cells. Adding B27 supplement to PBS, or just using Neurobasal medium would be also appropriate\ncontrols.\nFollowing the reviewer’s suggestions, we added the two experiments described next:\n(1) Effects of 37 ℃ incubation in Aβ aggregation using NbM Alb(-)\nTo confirm whether the incubation without iPS cells affects Aβ aggregation, we observed Aβ aggregates in NbM Alb(-)\nwith or without incubation, noting that the SD values (amount of Aβ) were not different between these conditions.\nWe mentioned our new results in the revised manuscript (Page 5, Lines 166-171). In addition, we added images and a graph as\nSupplementary Fig. 5 with an accompanying figure legend in the revised Supplementary Information.\n(2) Effects of B-27 solution on Aβ aggregation\nAs the reviewer suggested, we verified the effect of B-27 solution in PBS. We observed Aβ aggregates in various\nconcentrations of B-27 solution in PBS, finding that 0.4-10% B-27 solution inhibited Aβ aggregates, indicating that Aβ\naggregates might be inhibited in the general medium condition (i.e., 0.5% B-27 solution). We added these new results to\nthe revised manuscript (Page 4, Lines 110-112). In addition, we added corresponding images and a graph as Supplementary\nFig. 1 with an accompanying figure legend in the revised Supplementary Information.\n- It is not proven to this reviewer that the accelerated and increased aggregation is caused by Ab42 in the culture medium,\nas this is presented as an important factor. Medium cultured on cells and, for example, immunodepleted to remove Ab,\nwould be an appropriate control. This is possibly even more relevant, as – to the understanding of this reviewer – the cell\nsupernatant is used in the HaiDap assay with several orders of magnitude higher concentration of recombinant Ab42 added\nin the assay.\nWe believe that various low molecular weight compounds, including Aβ derived from AD-iNeuron, affect Aβ aggregation.\n42\nthe culture supernatant affect Aβ aggregation, it is necessary to quantify the concentrations of various molecules present in\nthe culture supernatant, which is a future challenge. We have revised the statement that only Aβ secreted into the culture\nsupernatant affects aggregation to avoid any misunderstanding by readers (Page 6, Line 172).\n- Is the medium supernatant from the hiPSC-derived neurons collected from several medium changes? If so, is it pooled\nafterwards? This is potentially relevant, as the authors state in the discussion (page 8 – top part) that one benefit of the\nHaiDap system is to be more efficient than complicated hiPSC-based systems. If the medium is only collected once, one\nhiPSC culture would be needed for one harvest, largely reducing this effect.\nAs suggested by the reviewer, detailed information regarding the collection of AD-iNeuron culture supernatants has been\nadded to the Methods section (Page 12, Lines 389-406).\n- It is not easily understandable how the plant extracts were dissolved and applied in the assay system. Is it ensured that\nthe dilution series is performed in the solvent, thus adding always the same amount of solvent into each dilution / the\ncontrol wells? Also, in the cellular assay, DMSO is used as solvent, but it is not clear which concentrations are shown in\nthe quantified figures, as well as what the replication of this data was (generating the error bars).\nAs for general screening, samples were prepared in our experiments so that each dilution/control well always contained\nthe same solvent composition. We added information about the error bars of the cell-based assays to the figure legends.\n- Also, it is difficult to impossible to compare a purified substance (as RA) directly with a crude plant extract, such as done\nin the cell-based assay. Only one concentration is shown in the time-course of Figure 5, a dilution series would be needed\nto further specify the activity (see also below).\nIn cell-based assays, cytotoxicity can have a significant effect on the results. We performed dose-dependent experiments\nin the range where cytotoxicity was not an issue and have added the results to the main text (Page 7, Lines 238-244) and\nSupplementary Fig. 12.\n- Did the authors also evaluate other known compounds to inhibit aggregation, such as “beta sheet-breakers”? These\nwould be an appropriate positive control for a revised manuscript.\nWe confirmed that the chaperones CRT, Erp57, and PDI show Aβ aggregation inhibitory activity in PBS, although these\nare chaperones that function intracellularly. Since our target in this study was inhibition of Aβ aggregation outside cells,\nwe decided that they would not be suitable as controls. As stated in response to Reviewer #1's comment No. 4, we also\nexamined morin and quercetin, but were unable to find any clear positive results in all screenings to date.\n- What do the cells in the cell-based assay system add to the assay? The selectivity for the compounds could come from the\nmedium composition used, one possible benefit, using the cells for assessing toxicity of plant extracts, is briefly mentioned,\nbut not shown. For the cell-based assays, it is therefore necessary to further elude on that, also how survival / toxicity was\nassessed.\nAs mentioned above, we added a new experiment on cytotoxicity (Supplementary Fig. 12).\n- There are instances of figure duplication observed, as in Supplementary Figure 7: all plant extracts that do not show an\neffect in either system have always the same panels used for both MSHTS and HaiDap.\nAs the reviewer pointed out, we made a mistake during the presentation of these images. We apologize and have revised\nthem accordingly.\n- Figure 4 is missing the legend for subpanel c.\nAs the reviewer indicated, we had made a mistake in the Fig. 4 legend. We have now mentioned panel c in the revised\nmanuscript.\nEven though the system presented here is of interest to a scientific community, especially in the protein aggregation and\nAlzheimer’s Disease field, it is questionable if it is suitable for the very broad audience targeted by the journal in the current\nform. This in particular, as controls are missing to understand the origin of the higher selectivity of the HaiDap system and\nthe disconnect of the positive control substance efficacy in two important assays presented here.\nAs mentioned above, we have not yet found a pure compound that is clearly positive in all assays, although RA has shown\na weak correlation. This is because various factors may be involved in the inhibition of aggregation and deposition of the\naggregates in a complex system where cells exist. In this study, we found a plant extract that showed strong activity in both\nthe HaiDap system and the cell-based assay. We believe that this concern will be alleviated by screening various iPS cell\nlines using the HaiDap system and clarifying the inhibitory mechanism of the obtained compounds in future studies. We\nacknowledge the limitations of the current method and stated them in the Discussion section of the revised manuscript\n(Page 9, Lines 299-309)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have undertaken a solid revision with detailed rebuttals. Several questions remain, and I appreciate that the\nauthors noted many of these limitations in the revised text. I remain concerned about not thoroughly addressing several of\nthe limitations. In particular:\n1. The concentrations of amyloidogenic proteins used (25 µM for Aβ42) are very high. The authors rationalized that “Aβ\nconcentration was set at 25 µM because at this concentration, the effect of Aβ aggregation on cells could be detected within\n24 h.” I understand and appreciate the necessity to use higher concentrations of Aβ in vitro than in CSF, but cytotoxicity can\nusually be readily observed for cells treated with >1-2 µM oligomers or fibrils after 15’ for single cell assays and easily in 20\nh using bulk assays of cytotoxicity, and aggregation can monitored at the low micromolar concentration readily in many\nbiophysical assays. The fact remains that the concentration of Aβ is quite high, which also limits potential ratios of Aβ to\ndrugs (since the drugs will become cytotoxic).\n2. I appreciate the authors “wanted to screen for substances that could potentially be ingested over a long period of time, so\nwe screened 22 plant species in this study. These plant species were selected because they have a known history in the\nhuman diet and are relatively easy to obtain.” To me, the critical point is to prove the system reproducibly works, not to find a\ntranslational therapeutic from extracts. The fact that the authors are unable to show a molecule that works in the assays,\ntherein adding confidence that the system works better and also comparing efficacies across the different methods, is\nconcerning, especially considering very potent inhibitors/disaggregators exist (monoclonal antibodies, EGCG, beta sheet\nbreakers as the other reviewer mentioned, many well characterized in vitro aggregation inhibitors that are commercially\navailable like bexarotene).\n3. The authors also write “Thus, it is expected that the HaiDap system will enable the evaluation of the inhibitory effects at\neach step of Aβ aggregation, such as seed formation and fibril elongation.” It is not clear how the authors would model\nmolecular mechanisms like these microscopic steps with reproducibility from the total aggregated Ab data that are provided.\nFinding positive hits in their assays that have precise molecular mechanisms known in vitro would be helpful (some of which\nare known to have efficacy in vivo, eg monoclonal antibodies).\n4. The authors clarify that the source of their Ab42 is synthetic, which adds to previous concerns that key aspect of this\nsystem likely do not recapitulate AD. This concern is amplified by point 1 above.\n5. It remains unclear that the ab42 in the culture medium is what is accelerating/driving aggregation. Immunodepleting is a\ngreat suggestion from the other reviewer.\n6. Minor point: Does G yesoense interfere with the QD signal, considering it starts muck lower than all the other samples in\nFig 7D?\nConsidering the broad audience of the journal and that key aspects to validate the system remain unclear, I recommend this\nuseful system be published in a more specialized journal.", "reviewer_2": "Thank you to the authors for providing detailed responses to the previous questions and comments. Especially for clarifying\nthat the effects of in vivo of Rosmarinic Acid are rather secondary in nature and this can explain that RA is not identified as\nhit in this assay system presented here, even though it had shown in vivo efficacy. Most of the previous questions were\naddressed in sufficient detail, thank you for that. The following remaining points should be briefly addressed, though:\n- It appears that the system or parts of the system were described in a patent application by some of the authors. If this patent\nis referenced in this revised version of the manuscript, it should be either listed in the references (if compatible with journal\nguidelines), or the data should be added as (supplementary) data to the manuscript. The patent application probably needs\nto be disclosed in the competing interests?\n- Thank you for providing more detail on the differentiation of hiPSCs into neurons. Still, there is no reference given for\nincluding Shh protein in the maturation medium of the cells. Shh is a ventralizing patterning factor and will influence the\noutcome of the differentiation (regionalization) to a more ventral fate. If this differentiation procedure is based on a previously\npublished procedure, it should be cited.\n- If this reviewer understands the procedure correctly, no Glutamine was added in the Neurobasal medium (See also Table\n1)? As the base medium formulation from the manufacturer is “(-) L-Glutamine”, indicating that this needs to be added, as\nalso stated as recommended concentration of 0.5mM in the final medium. If Glutamine is not added, this should be briefly\nstated in the methods section to avoid confusion.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We agree with the reviewer’s comments that some limitations were not sufficiently addressed in\nthe previous version of the manuscript. After carefully considering the reviewer’s comments, we\nconducted additional experiments and revised the manuscript accordingly. We believe that the\nrevised version fully addresses all of the reviewer’s concerns. Thanks to the reviewers’\ninsightful suggestions, we feel that the novelty and robustness of our paper have been greatly\nenhanced, and we are sincerely grateful for the constructive feedback.\n1. The concentrations of amyloidogenic proteins used (25 µM for Aβ42) are very high. The\nauthors rationalized that “Aβ concentration was set at 25 µM because at this concentration, the\neffect of Aβ aggregation on cells could be detected within 24 h.” I understand and appreciate\nthe necessity to use higher concentrations of Aβ in vitro than in CSF, but cytotoxicity can\nusually be readily observed for cells treated with >1-2 µM oligomers or fibrils after 15’ for\nsingle cell assays and easily in 20 h using bulk assays of cytotoxicity, and aggregation can\nmonitored at the low micromolar concentration readily in many biophysical assays. The fact\nremains that the concentration of Aβ is quite high, which also limits potential ratios of Aβ to\ndrugs (since the drugs will become cytotoxic).\nAs pointed out by the reviewer, we used an Aβ concentration of 25 µM in both the MSHTS and\nHaiDap systems because this can shorten the screening time and improve the sensitivity (Sasaki\net al., 2019). On the other hand, in the cell-based assay, we used a concentration of 5 µM Aβ,\nwhich is in the same low micromolar range as recommended by the reviewer. In our previous\nmanuscript, the Aβ concentrations used in the cell-based assays were only listed in the methods\nsection (line 539). To make it clear to readers, we have now added this concentration\ninformation to the figure legends (lines 775-776) as well. Since both the 5 µM Aβ concentration\nused in the cell-based assay and the 25 µM concentration used in the HaiDap system are very\nhigh concentrations compared to physiological conditions, we have added a related discussion\nin the limitations section (lines 335-341). As we performed a dose-dependent iPSC-based assay\nwith S. aromatica extract (Supplementary Fig. 13), we believe that samples screened with the\nHaiDap system should be subjected to a careful second screening, including evaluation of\ncytotoxic activity in a subsequent iPSC-based assay. Therefore, the highly selective HaiDap\nsystem could be one of the excellent initial screening methods for rapidly finding useful\nsubstances that are effective in vivo.\n2. I appreciate the authors “wanted to screen for substances that could potentially be ingested over a long period of time, so\nwe screened 22 plant species in this study. These plant species were selected because they have a known history in the\nhuman diet and are relatively easy to obtain.” To me, the critical point is to prove the system reproducibly works, not to find a\ntranslational therapeutic from extracts. The fact that the authors are unable to show a molecule that works in the assays,\ntherein adding confidence that the system works better and also comparing efficacies across the different methods, is\nconcerning, especially considering very potent inhibitors/disaggregators exist (monoclonal antibodies, EGCG, beta sheet\nbreakers as the other reviewer mentioned, many well characterized in vitro aggregation inhibitors that are commercially\navailable like bexarotene).\nAmong the inhibitors recommended by the reviewer, we found EGCG to be a positive control\nthat showed significant effects in all of the MSHTS system, HaiDap system, and cell-based\nassay. We have summarized the results in Supplementary Fig. 14 and added the relevant text at\nthe end of the results (lines 258-268). We believe that this proves that the system works\nreproducibly.\n3. The authors also write “Thus, it is expected that the HaiDap system will enable the evaluation of the inhibitory effects at\neach step of Aβ aggregation, such as seed formation and fibril elongation.” It is not clear how the authors would model\nmolecular mechanisms like these microscopic steps with reproducibility from the total aggregated Ab data that are provided.\nFinding positive hits in their assays that have precise molecular mechanisms known in vitro would be helpful (some of which\nare known to have efficacy in vivo, eg monoclonal antibodies).\nRegarding comments 3 and 5, we carefully considered the reviewer's comments and added a\nnew experiment to respond to them. Specifically, we added Aβ monomers or oligomers at a\nconcentration of 25 pM to NBM Alb(-) and observed Aβ aggregation (Supplementary Fig. 6).\nThe Aβ concentration of 25 pM is similar to the concentration of Aβ secreted from AD-iNeuron\nin NBM Sup and is also a physiological Aβ concentration. Interestingly, it was revealed that the\naddition of oligomeric Aβ accelerated the Aβ aggregation rate to the same extent as aggregation\nin NbM Sup Alb(-). These results indicate that Aβ secreted from the culture cell supernatant\nmay form oligomers, which accelerate Aβ aggregation, and we believe that they represent part\nof the molecular mechanism of Aβ aggregation in NbM Sup Alb(-). We summarized the\nrelevant results in Supplementary Fig. 6 and added explanatory text (lines 176-184).\n4. The authors clarify that the source of their Ab42 is synthetic, which adds to previous\nconcerns that key aspect of this system likely do not recapitulate AD. This concern is amplified\nby point 1 above.\nFollowing reviewer comments, we added a limitation regarding the use of synthetic peptides\n(lines 331–335).\n5. It remains unclear that the ab42 in the culture medium is what is accelerating/driving\naggregation. Immunodepleting is a great suggestion from the other reviewer.\nIt is difficult to completely remove the target substance with the immunodepleting method, and\nnon-specific adsorption may remove substances other than the target substance. Therefore, this\ntime, Aβ was added in the form of monomers or oligomers at the concentration contained in the\nculture supernatant (NbM Sup Alb(-)) to the medium (NbM Alb(-)) and the effect on the\naggregation rate and morphology was analyzed. For detailed results, please refer to the response\nto comment 3. These results showed that Aβ oligomers accelerate/drive the aggregation of Aβ .\n42\n6. Minor point: Does G yesoense interfere with the QD signal, considering it starts muck lower than all the other samples in\nFig 7D?\nAs the reviewer pointed out, I believe there is a possibility of interference, but even after\nsubtracting this, it is clear that it inhibits aggregation.\nConsidering the broad audience of the journal and that key aspects to validate the system remain unclear, I recommend this\nuseful system be published in a more specialized journal.\nWe have carefully addressed your comments, which have allowed us to better clarify key\naspects for validating our system. Protein-degenerative diseases, including AD, are increasing\nworldwide with the aging population. We believe this work will be of interest to the readers of\nNature Communications, which targets a wide readership.\nReviewer #2 (Remarks to the Author):\nThank you to the authors for providing detailed responses to the previous questions and\ncomments. Especially for clarifying that the effects of in vivo of Rosmarinic Acid are rather\nsecondary in nature and this can explain that RA is not identified as hit in this assay system\npresented here, even though it had shown in vivo efficacy. Most of the previous questions were\naddressed in sufficient detail, thank you for that. The following remaining points should be\nbriefly addressed, though:\nThank you very much for appreciating the last revision. We have added some experiments to\naddress the further comments of Reviewer #1, and we believe that the paper is now more robust.\nWe appreciate your helpful comments. We have addressed your comments as follows:\n- It appears that the system or parts of the system were described in a patent application by\nsome of the authors. If this patent is referenced in this revised version of the manuscript, it\nshould be either listed in the references (if compatible with journal guidelines), or the data\nshould be added as (supplementary) data to the manuscript. The patent application probably\nneeds to be disclosed in the competing interests?\nAs suggested by the reviewers, we have added the relevant patent to the reference, and it has\nbeen disclosed in the competing interests.\n- Thank you for providing more detail on the differentiation of hiPSCs into neurons. Still, there\nis no reference given for including Shh protein in the maturation medium of the cells. Shh is a\nventralizing patterning factor and will influence the outcome of the differentiation\n(regionalization) to a more ventral fate. If this differentiation procedure is based on a previously\npublished procedure, it should be cited.\nFollowing reviewers’ comment, we have added papers 51-53 to the Methods section (line 435).\n- If this reviewer understands the procedure correctly, no Glutamine was added in the\nNeurobasal medium (See also Table 1)? As the base medium formulation from the manufacturer\nis “(-) L-Glutamine”, indicating that this needs to be added, as also stated as recommended\nconcentration of 0.5mM in the final medium. If Glutamine is not added, this should be briefly\nstated in the methods section to avoid confusion.\nBecause glutamine-free Neurobasal medium was used in this AD-iNeuron induction step, we\nadded a description of this in the Methods section (lines 436-437).\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nThank you for your review. Your help has significantly improved the manuscript and made it\nsuitable for publication in Nature Communications."}, {"round": 3, "reviewer_comments": {"reviewer_1": "The authors have added experiments to address the feedback of the reviewers. I remain concerned on the\nfollowing points:\n1. 25 uM Ab42 is high as used in the MSHTS, and 5 uM Ab42 is still high as used in the iPSC-based\nscreening assay. More importantly, the use of synthetic peptide raises unanswered concerns. I don’t think\nadding a limitation note is sufficient. Moreover, looking at the manufacturer’s website, I cannot find purity\ndetails for item 4349-v (Ab42), and it does not appear this peptide was further purified (for example with\nsize exclusion) by the authors beyond adding HFIP. Adding in the consideration that they are using\nAb40QDs, the extent to which their aggregation reaction recapitulates key features of AD is unknown, which\nis a major claim of the paper. It would have been good if the authors had tested at least recombinant Ab42\nof high purity for comparison (for example with EGCG or an extract as a positive control). Moreover, the use\nof only one male AD patient may further limit the potential relevance/utility/reproducibility in other labs of\nthis study.\n2. The experiments where 25 pM monomer or oligomer are added to NbM Alb(-) and accelerate aggregation (Fig S6)\nsupport that Ab is relevant, but do not address the fundamental question of what in the iPSC supernatant is actually\nspeeding up aggregation. The authors did not characterize their monomer or oligomer before testing it, so it is hard to be\nsure what species of Ab42 were actually added in that assay. This is further confounded by the point above. Gaining this\nclarity on what actually speeds up aggregation is a challenging point to address in the context of this paper, as the\nsupernatant is necessarily complex, but it remains unclear what is secreted that dominates the observed effect (if it is even\njust Ab42 or some aggregated form of Ab42 - it likely has complex lipids/other metabolites co-aggregated, as the authors\nfairly suggest). Nonetheless, the lack of mechanistic insight remains an understandable limitation.\n3. It would have been pragmatic to use the EGCG positive control to show dose-dependent inhibition in the iPSC-based\nscreening assay.\n4. An EC50 for EGCG in NbM Sup Alb(-) of 146.3 µM is very high. IC50 values in the literature suggest it is in the low µM\nrange (1-10 µM) against Ab42 in most in vitro assays. This discrepancy should be addressed. It also doesn’t seem to agree\nwith the potency reported in iPSC-based screening. Evidence rationalizing its efficacy in the various assays benchmarked to\nthe literature is needed.\n5. Minor: it is odd that S13b lacks the negative control without Syzygium aromaticum extract.\nI commend the authors for their robust study and appreciate the challenges of working with iPSCs, both time and financial.\nTheir concept and use of automation are sound and impressive, respectively. Despite these advances, the above concerns\nremain."}, "author_response": "To address the concerns raised by reviewer #1, we have conducted new experiments and attained additional\nverifications, including the use of recombinant Aβ, assessment using iPS cells derived from a healthy\nsubject, as well as an EGCG concentration-dependent analysis. We would like to express our sincere\ngratitude to reviewer #1 for their valuable advice.\n1. 25 uM Ab42 is high as used in the MSHTS, and 5 uM Ab42 is still high as used in the iPSC-based\nscreening assay. More importantly, the use of synthetic peptide raises unanswered concerns. I don’t think\nadding a limitation note is sufficient. Moreover, looking at the manufacturer’s website, I cannot find purity\ndetails for item 4349-v (Ab42), and it does not appear this peptide was further purified (for example with\nsize exclusion) by the authors beyond adding HFIP. Adding in the consideration that they are using\nAb40QDs, the extent to which their aggregation reaction recapitulates key features of AD is unknown, which\nis a major claim of the paper. It would have been good if the authors had tested at least recombinant Ab42\nof high purity for comparison (for example with EGCG or an extract as a positive control). Moreover, the use\nof only one male AD patient may further limit the potential relevance/utility/reproducibility in other labs of\nthis study.\nReviewer #1 asserts that the synthetic peptides used in our experiments “do not appear to have been further\npurified,” based on the absence of purity information on the manufacturer's website. We inferred that\nreviewer #1 assumed the cytotoxicity of Aβ was weak because low-purity Aβ peptide was used. To clarify\n42\nthis point, we contacted Peptide Institute, Inc. for details regarding the purity of the synthetic Aβ (4349-v)\nemployed in our study. Although the purity of each lot is not disclosed on the company’s website, we\nobtained detailed analytical data for the peptide shown in Fig. R1. According to this information, the peptide\nused in this study was purified using reverse-phase chromatography, and its purity was confirmed by HPLC\nto be 97.4%. We have added peptide purification method and purity information to the Methods section\n(lines 378-380).\nFig. R1 Analytical data of synthetic Aβ42 (#4349-v) purchased from Peptide Institute, Inc.\nTo scientifically verify reviewer #1’s concern that 5 μM Aβ is too high for cell-based assays due to its\n42\ncytotoxicity, we purchased recombinant human Aβ (#AG968, Sigma-Aldrich, MO, USA), which has been\n42\nwidely used in previous studies, as suggested by reviewer #1, and performed a cell-based assay. The purity\nof the purchased recombinant human Aβ was >97.0% (Fig. R2), comparable to the synthetic Aβ used in this\nstudy.\nFig. R2 Certificate of analysis of recombinant Aβ42 (#AG968) purchased from Sigma-Aldrich.\nThe results indicate that, as reviewer #1 pointed out, recombinant Aβ was indeed more toxic than the\nsynthetic Aβ used in our study (Fig. R3). Quantitative comparisons showed that the cytotoxicity of 0.5 µM\nrecombinant Aβ was comparable to that of 7.5 µM synthetic Aβ, and that 2.0 μM recombinant Aβ exhibited\nsignificantly higher cytotoxicity than these samples. These results support the previous comment of reviewer\n#1 that a 5 µM Aβ concentration may be too high when using recombinant Aβ in iPSC-based assays. Finder\net al. (2010, https://www.sciencedirect.com/science/article/pii/S0022283609015216?via%3Dihub) suggested\nthat this difference in cytotoxicity may be due to impurities present during Aβ synthesis. However, according\nto a review by Varshavskaya et al. (2022, https://www.mdpi.com/1422-0067/23/23/15036), none of the\nreviewed studies, including Finder’s work, examined the aggregation and structure of more than two Aβ\nspecies simultaneously, and a comprehensive comparison of these properties within a single study has yet to\nbe conducted. Based on these findings, we considered it necessary to compare the biochemical properties of\nsynthetic and recombinant Aβ with similar levels of purification, and therefore performed additional\nverification (Fig. R4).\nFig. R3 Evaluation of cytotoxicity of synthetic and recombinant Aβ peptide. For apoptosis assays of synthetic Aβ\nand recombinant Aβ; 1.8-2.2×105 cells/cm2 of AD-iNeurons were incubated for 24 h in a NbM plus B27 medium\ncontaining synthetic or recombinant Aβ (0.5 μM, 2 μM, 7.5 μM). Apoptotic cells (in green) were subsequently detected\nusing a fluorogenic substrate specific for Caspase-3/7 activity (Incucyte Caspase 3/7 green dye kit (#4440, Sartorius)).\nTreated plates were imaged at 20× magnification using phase and green channels, at nine images per well, and analyzed\nusing Incucyte software.\na, Caspase 3/7 mediated apoptosis measured by fluorescence intensity. b, Merged images of brightfield and\nfluorescent field.\nWe performed SDS-PAGE to compare the biochemical properties of these Aβ species (Fig. R4a and R4b). In\nboth samples, a major band corresponding to monomeric Aβ was observed (Fig. R4a). However, in the\nrecombinant Aβ sample, additional bands were detected at molecular weights higher than that of the Aβ\nmonomer, which appeared to originate from E. coli or Aβ oligomers (Fig. R4b). Notably, the recombinant Aβ\nband exhibited vertical streaks and appeared blurred—a common phenomenon observed during recombinant\nprotein expression and purification, likely due to contaminating host DNA. When absorbance was measured\nusing a NanoDrop 2000c spectrophotometer, recombinant Aβ showed absorption at 260 nm, indicative of\nnucleic acids (Fig. R5c). The A260/A280 ratio of recombinant Aβ was higher than that of synthetic Aβ (Fig.\nR5d), further suggesting that recombinant Aβ contains nucleic acids derived from E. coli. Although synthetic\nAβ may also contain impurities that cannot be completely removed during purification, this study revealed\nthat recombinant Aβ includes host-derived proteins and nucleic acids. These contaminants may influence\ncytotoxicity and aggregation under certain conditions, indicating that recombinant Aβ is not necessarily\nsuperior to synthetic Aβ for screening purposes. As summarized in the review by Varshavskaya et al., we\nbelieve it is essential to better understand differences in property based on peptide origin before use.\nConsidering all these findings, the cytotoxicity of 5 μM synthetic Aβ used in our iPC-based assay is\nequivalent to that of 0.5 μM recombinant Aβ and is not as high as reviewer #1 feared. Furthermore, the\nsignificant differences observed between materials in the iPC-based assay using 5 μM synthetic Aβ do not\nundermine the validity of our experimental conditions. Therefore, in this paper we have decided to focus on\nresults using synthetic Aβ, with limitations regarding the peptides used for screening added in the Discussion\nsection (lines 348-355).\nFig. R4 Biochemical validation of synthetic and recombinant Aβ peptides.a and b, SDS-PAGE of synthetic and\nrecombinant Aβ . To assess the purity of synthetic (S) and recombinant (R) Aβ peptides, samples prepared at a\n42 42\nconcentration of 100 µM were separated by SDS-PAGE using a 16.5% acrylamide gel. After electrophoresis, bands\nwere visualized in the gel by Coomassie Brilliant Blue (CBB) staining (a) and silver staining (b). Silver staining was\nperformed using 2D-Silver Stain Reagent II (423413; Cosmo Bio Co., Ltd., Tokyo, Japan) according to the\nmanufacturer’s instructions. c and d, Absorbance measurements of synthetic and recombinant Aβ peptides. To\n42\nevaluate the purity of synthetic and recombinant Aβ , absorbance measurements were performed for both samples\n42\nprepared at a concentration of 500 µM (n = 3) using a NanoDrop 2000c Spectrophotometer (ND-2000c; Thermo Fisher\nScientific Inc., Waltham, MA, USA) (c). The absorbance values at 260 nm and 280 nm were used to calculate the\nA260/A280 ratio, which was employed as an indicator of sample purity (d).\nThere are numerous examples of in vitro screening and cell-based assays using synthetic Aβ at\nconcentrations of approximately 25 μM and 5 μM, respectively. Below, we present a selection of recent\nexamples from reputable Springer Nature journals that employed synthetic Aβ peptides at similar\nconcentrations. These examples demonstrate that our experimental conditions align with widely accepted\npractices in the field.\n(1) Xiuhua Yin et al., Nature Communications, 14, 5718 (2023)\nhttps://www.nature.com/articles/s41467-023-41489-y?utm_source=chatgpt.com\nSynthetic Aβ peptides\nIn vitro aggregation inhibition assay：Aβ concentration 50 μM\nCell based assay：Aβ concentration 50 μM\n(2) Qiang Luo et al., Nature Communications, 9, 1802 (2018)\nhttps://www.nature.com/articles/s41467-018-04255-z?utm_source=chatgpt.com\nSynthetic Aβ peptides\nIn vitro aggregation inhibition assay：Aβ concentration 20 μM\nCell based assay：Aβ concentration 20 μM\n(3) K. Rajasekhar et al., Scientific Reports, 5, 8139 (2015)\nhttps://www.nature.com/articles/srep08139?utm_source=chatgpt.com\nSynthetic Aβ peptides\nIn vitro aggregation inhibition assay：Aβ concentration 20 μM\n(4) Si-Cong Bai et al., Communications Chemistry, 8, 196 (2025)\nhttps://www.nature.com/articles/s42004-025-01587-y?utm_source=chatgpt.com\nSynthetic Aβ peptides\nIn vitro aggregation inhibition assay：Aβ concentration 5 μM\nCell based assay：Aβ concentration 5 μM (10-fold dilution after incubation)\nTable R1 Summary of the experimental conditions in the above papers.\nAβ concentration\nPaper name In vitro aggregation Aβ type\nCell based assay\ninhibition assay\nXiuhua Yin et al., 2023 50 μM 50 μM Synthetic Aβ peptides\nQiang Luo et al., 2018 20 μM 20 μM Synthetic Aβ peptides\nK. Rajasekhar et al., 2015 20 μM ー Synthetic Aβ peptides\n5 μM (10-fold dilution\nSi-Cong Bai et al., 2025 5 μM Synthetic Aβ peptides\nafter incubation)\nOur simulations suggest that if Aβ aggregates at picomolar concentrations, it would take decades for the\naggregates to reach an in vivo size of several micrometers, the size of senile plaques (Kuragano et al., 2022;\nhttps://www.sciencedirect.com/science/article/pii/S0927776522001321). It is well established that protein\naggregation associated with protein-denaturing diseases occurs over very long timescales. Therefore, many\nresearchers accelerate aggregation using high concentrations (Table R1), ultrasound, or other methods.\nConsidering these reports and the experimental results described above, we believe that our experimental\nconditions fall within an acceptable range for the initial step of in vitro screening.\nReviewer #1 also expressed concern about the use of Ab40QD, although quantification of aggregation requires\nsome kind of probe, such as ThT or an antibody. As mentioned in our initial response, we used QD-labeled\nAβ40 as a fluorescent probe; however, its concentration was only 0.1% relative to Aβ . This is an extremely\n42\nsmall amount, especially compared to the concentrations of ThT commonly used in amyloid aggregation assays,\nwhich are often equal to or greater than the peptide concentration. We also confirmed that adding QD-labeled\nAβ at a 0.1% ratio did not significantly affect the aggregation kinetics of Aβ42, as demonstrated in our\n40\nprevious study (Tokuraku et al., 2009; https://doi.org/10.1371/journal.pone.0008492). Therefore, the use of\ntrace amounts (0.1%) of QD-labeled Aβ40 is not considered to be particularly inferior to the use of ThT and\nanti-Aβ antibody, which has been reported to affect Aβ aggregation, and we believe that the use of trace\namounts (0.1%) of QD-labeled Aβ40 does not impair the validity of the in vitro screening results. The reasons\nfor not using QD-labeled Aβ42 and the advantages of using QDs for screening, such as the inner filter effect,\nthe lack of fixation required for electron microscopy, and 3D analysis, are described in detail in the cited\nreferences (Ishigaki et al., 2013; https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0072992,\nSasaki et al. 2019, https://www.nature.com/articles/s41598-019-38958-0), including those mentioned above.\nFollowing the comments of reviewer #1, we have also added a dose-dependent analysis using EGCG and\nexperiments using different iPC cell culture supernatants to the revised manuscript. We address these results\nin our response to reviewer #1's comment below.\n2. The experiments where 25 pM monomer or oligomer are added to NbM Alb(-) and accelerate aggregation\n(Fig S6) support that Ab is relevant, but do not address the fundamental question of what in the iPSC\nsupernatant is actually speeding up aggregation. The authors did not characterize their monomer or\noligomer before testing it, so it is hard to be sure what species of Ab42 were actually added in that assay.\nThis is further confounded by the point above. Gaining this clarity on what actually speeds up aggregation is\na challenging point to address in the context of this paper, as the supernatant is necessarily complex, but it\nremains unclear what is secreted that dominates the observed effect (if it is even just Ab42 or some\naggregated form of Ab42 - it likely has complex lipids/other metabolites co-aggregated, as the authors\nfairly suggest). Nonetheless, the lack of mechanistic insight remains an understandable limitation.\nFollowing reviewer #1’s suggestion, we have added results characterizing the monomers and oligomers used\nin the experiment (Supplementary Fig. 6d and 6e). Furthermore, we prepared healthy control (HC) iNeuron-\nderived NbM Sup Alb(-) and conducted a comparative study (Supplementary Fig. 7 and Table 1). The results\nrevealed that the Aβ aggregation rate in HC iNeuron-derived NbM Alb(-), which contains lower levels of\nboth Aβ42 and Aβ40 compared to the culture supernatant derived from AD iNeuron-derived NbM Alb(-)\n(Table 1), was slower than that observed in AD iNeuron-derived NbM Alb(-) (Supplementary Fig. 7). These\nfindings suggest that iNeuron-derived Aβ and its oligomerization state influence Aβ aggregation. We have\nadded these results to lines 192–206 in the Results section.\nAs reviewer #1 commented and as stated in the manuscript, it is possible that various molecules present in\nthe culture supernatant affect Aβ aggregation. However, the addition of new experiments using HC iNeuron-\nderived NbM Alb(-), which has a relatively similar composition except for Aβ content, suggests that at least\nthe amount and state of Aβ secreted from iNeurons derived from AD patients affect aggregation. We believe\nthis strengthens our mechanistic insight.\n3. It would have been pragmatic to use the EGCG positive control to show dose-dependent inhibition in the\niPSC-based screening assay.\nFollowing reviewer #1’s instructions, we performed a dose-dependent iPSC-based screening assay using\nEGCG, and the results confirmed that the effect of EGCG was indeed dose-dependent (Supplementary Fig.\n15d). These results have been added to line 279 of the Results section.\n4. An EC50 for EGCG in NbM Sup Alb(-) of 146.3 µM is very high. IC50 values in the literature suggest it is\nin the low µM range (1-10 µM) against Ab42 in most in vitro assays. This discrepancy should be addressed.\nIt also doesn’t seem to agree with the potency reported in iPSC-based screening. Evidence rationalizing its\nefficacy in the various assays benchmarked to the literature is needed.\nAs reviewer #1 pointed out, the magnitude of activity obtained by the HaiDap system and iPSC-based\nscreening is not completely comparable, although samples in which activity was detected by the HaiDap\nsystem were also detected by iPSC-based screening. This is presumably due to direct interaction with the cell\nmembrane and/or sample cytotoxicity, which is not affected by the HaiDap system. We have added this as a\nlimitation of this study in the Discussion section (lines 335-340).\n5. Minor: it is odd that S13b lacks the negative control without Syzygium aromaticum extract.\nThe negative control without extract in S13b (S14b in the new manuscript) is identical to the DMSO in the\ngraph on the far right of Fig. 7d (Aβ intensity). Since it has already been presented, it is not displayed in\nS14b. However, for clarity, this information has been noted in the figure legend of S14b.\nI commend the authors for their robust study and appreciate the challenges of working with iPSCs, both time\nand financial. Their concept and use of automation are sound and impressive, respectively. Despite these\nadvances, the above concerns remain.\nWe thank you for recognizing our efforts. We believe that the HaiDap system has the potential to become a\nnovel method that could dramatically accelerate the screening process for substances effective against\namyloidosis—a disease whose prevalence is increasing due to aging of the global population. To achieve\nthis, further research involving a wide variety of iPS cell types and diverse peptides will be necessary;\nhowever, we believe this paper represents an important first step toward achieving that goal."}, {"round": 4, "reviewer_comments": {"reviewer_1": "The author rebuttal is detailed and includes substantial new data (purification details of Aβ, HC controls, EGCG dose‑response, and biochemical analyses). The work remains rigorous and addresses a major limitation in early‑stage AD drug discovery. The contribution is of considerable technical novelty and creative. The paper is very well written.\nThe central thesis, that HiDap “will be useful for highly efficient, accurate, and low-cost screening of disease- and patient-specific drugs involved in protein aggregation,” remains undermined by the use of only one AD donor iPSC line. Although the authors acknowledge this limitation and include HC‑iNeuron controls, the universality of the platform is unclear. The inclusion of the HC controls and their chemical compositions in Table 1 is very useful. I was surprised that aggregation was only modestly accelerated in Fig S7 in AD vs HC samples despite an approximately 8-fold increase in Ab40 and almost 4-fold increase in Ab42 from Table 1. The heterogeneity of the HC sample seems considerable, too, given the large error bars. The work runs the risk that the observed response is patient-specific or varies considerably from patient to patient, since they only tested one AD iPSC donor.\nThe main concern with synthetic Ab42, and recombinant for that matter, is that it likely contained significant impurities and, as I wrote in the last revision, it was not purified further by the authors in their lab using a technique like size-exclusion FPLC before kinetic assays were initiated. The recombinant 97% pure Ab42 used by the authors can contain significant impurities, including pre-formed Ab42 aggregates, without further purification, which does seem to be the case for both synthetic and recombinant peptides looking at Fig. R4 by SDS and silver staining. This, alongside using a very high concentration of Ab42, limits the conclusions that can be drawn as to mechanisms of action of hit molecules, but, the authors have not made over-stated claims on this point. Hopefully in future work, they can use lower concentrations of Ab42 that is of a purity sufficient for dedicated kinetic analyses.\nAlthough the authors add HC vs AD supernatant comparisons and further Aβ characterizations, the supernatant remains complex and the mechanism of aggregation acceleration, whether caused some specific conformer or cell-modified version Aβ, lipids, metabolites, differences in pH, ionic strength, other uncharacterized biomolecules, or if it is due only to differences in the total secreted concentration of cellular Ab, remains uncertain. In their samples, ionic strength seems conserved and pH does seem to change only a small amount. I think there is enough evidence that the secreted Ab is important in light of their new experiments.\nThe work represents a positive step towards doing more relevant AD kinetic research. I hope they find their results universal in multiple AD donors, gain better understanding into how aggregation is accelerated and by what precise species, and resolve the reason for the differences in positivity/selectivity in the different assays (PBS, MSHTS, and HiDap)."}, "author_response": "We appreciate your acceptance of our rebuttal, including the new data such as the purification details of Aβ, HC controls, EGCG dose–response experiments, and biochemical analyses. We also thank you for recognizing the technical novelty and creativity of this study.\nIn response to this comment, we have incorporated the concerns raised by Reviewer #1 regarding the use of only one AD donor iPSC line into the Discussion section (lines 346–351). We have also revised the title and other parts of the manuscript to remove claims suggesting universal applicability.\nWe have included a more detailed discussion of our concerns regarding synthetic and recombinant Aβ42 in the Discussion section (lines 332–339), and we appreciate the reviewer’s acknowledgement of these limitations. We will address these issues carefully in future work.\nWe have added a discussion of the potential mechanisms underlying aggregation promotion in the Discussion section (lines 351–354).\nWe have also added a discussion (lines 354–358) addressing concerns regarding differences in positivity/selectivity among the different assays (PBS, MSHTS, and HiDap). As Reviewer #1 noted, this study represents a positive step toward more relevant research on AD aggregation dynamics. We hope to address the remaining concerns carefully in future studies and further enhance the platform’s usefulness for low-cost, high-throughput screening."}]

Decision letter