The Korea Excellence Animal Laboratory Facility of Korea Food and Drug Administration was accredited by the Institutional Animal Care and Use Committee (IACUC) and the Department of Laboratory Animal (DOLA) in the Catholic University of Korea, Songeui Campus in 2017, and acquired AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care) complete international accreditation in 2018. All procedures of animal research were conducted in accordance with the Laboratory Animals Welfare Act, the Guide for the Care and Use of Laboratory Animals, and the Guidelines and Policies for Rodent Experiment provided by the IACUC School of Medicine at the Catholic University of Korea. The animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of School of Medicine, The Catholic University of Korea (Approval number: CUMS-2020-0217-08).

The B6.CG-Tg (APPSwFILon on PSEN1*M146L*L286V) 6799Vas/J transgenic (5×FAD) mice which have high amyloid deposition from 2 months of age [34] and C57BL/6 (wild-type, WT) mice were purchased from Jackson Laboratory (Bar Harbor, MN). All mice were maintained under specific pathogen-free conditions. A previous study by Santamaria et al. reported that a single intranasal dose of MSC secretome resulted in sustained memory recovery in AD mice for 7 days [27]. Based on this, we administered iPSC-derived CNSC-SE (5 μg/g) in 12-week-old male mice once a week for 4 weeks (4 times in total) by inserting a 26-G intravenous catheter (Poly Medicure POLYPEN IV Catheter, 26 G/19 mm) along the nasal cavity.

The following four groups were set in this study: (i) WT mice as controls (WT group); (ii) 5×FAD AD mouse model (AD group); (iii) CNSC-SE-treated 5×FAD mice (CNSC-SE-5×FAD group); and (iv) MSC-treated 5×FAD mice (MSC-SE-5×FAD group).

The Barnes maze was used to assess spatial learning and memory [35]. The maze consisted of an elevated circular platform (92 cm in diameter), containing 18 equally spaced holes around the circumference, each 5 cm in diameter. Visual cues were placed around the walls (periphery) during the test. For training, a mouse was placed in the center of the maze under a transparent beaker for 1 min, and it was then guided to the target hole, which contained sweet-flavored chips, for 1 min. The mouse was trained for 2 consecutive days followed by testing on day 3. On the test day, the mouse was placed in the center of the maze and observed for 3 min to record the following parameters: (i) distances from the target hole; (ii) the resting time in the target hole; (iii) the first latency to reach the target hole; and (iv) the error rate, i.e., the number of times the mouse entered a zone other than the target zone. After all trials, the maze was completely cleaned with 70% alcohol solution. All experimental tests were recorded by a video camera and analyzed with SMART 3.0 (Panlab, Harvard Apparatus, Barcelona, Spain).

All hiPSC lines used in this study were generated through our previous research using cord blood cells isolated from a healthy individual [36]. Human iPSCs were seeded on cell culture dishes coated with vitronectin (Gibco, Waltham, MA; #A14700), and cultured in Essential 8 media (Gibco #A1517001) in a 10% CO2 environment at 37 °C.

To induce differentiation of iPSCs into cortical neurons, we referred to the protocol of Shi et al. [37]. For neural induction, iPSCs were seeded on vitronectin-coated 60-mm dishes at 4.6×106 cells/dish with Essential 8 media and 10 µM rho-associated kinase inhibitor for 24 h. After 24 h of incubation, the culture medium was changed to a neuronal differentiation medium (NDM), which is a 1:1 mixture of N-2 (consisting of Dulbecco’s Modified Eagle Medium, Nutrient Mixture F12 GlutaMAX, 1 × N-2, 5 μg/ml insulin, 1 mM L-glutamine, 100 μm nonessential amino acids, 100 μM 2-mercaptoethanol, and 10% penicillin/streptomycin) and B-27 (consisting of Neural basal, 1 × B-27, 200 mM L-glutamine and 10% penicillin/streptomycin, with 10 µM SB431542 [Tocris Bioscience, Bristol, UK; #1614]) with 1 µM Dorsomorphin (Tocris Bioscience, #BML-275), and incubated for 10 days. Neuronal progenitor cells were collected by Gentle Cell Dissociation Reagent (STEMCELL Technologies, Vancouver, BC, Canada; #100-0485) for NSC expansion. The cells were seeded on a laminin-coated (Biolamina, Stockholm, Sweden; #LN521-05) 60-mm dish (9 × 106 cells per dish) and cultured with NDM for 6 days, followed by maintenance in NDM supplemented with 20 ng/ml FGF2 for an additional 2 days. After 2 days, the NSCs were gently dissociated with accutase (Innovative Cell Technologies, San Diego, CA; #AT-104) for CNSC expansion and seeded on laminin-coated (Biolamina, #LN111) dishes under the same condition as for NSCs for 6 days. CNSCs, called cortical neurons, were cultured for 5 days and were passaged once more with accutase under the same conditions and maintained for up to 90 days.

Bone marrow-derived human MSCs were purchased from the Catholic Institute of Cell Therapy, South Korea. Human MSCs were maintained in a 10% CO2 environment at 37 °C and were cultured in DMEM (Gibco) with 20% FBS (Gibco) and 10% penicillin (Gibco).

After 10 days of neuroepithelial differentiation from iPSCs, 8 days of NSC differentiation, and subsequent CNSC differentiation by the previously described cell culture method, the culture medium was changed every 48 h, and the culture supernatants of the last 6 days were used as the material for CNSC-SE. For MSC-SE, the same conditions as that for CNSC-SE were used; cells were seeded and culture supernatant was collected after 3 days of culture. The collected culture supernatants were filtered through a 0.2-μm filter to remove cell debris. The filtered supernatants were stored at − 80 °C for 3 days, and the frozen samples were lyophilized for 7 days. The solid samples were stored at − 20 °C.

iPSCs and differentiated cells (iPSC-derived CNSCs) used for in-vitro experiments were fixed with 4% paraformaldehyde (PFA), permeabilized with 0.1% Triton X-100, and blocked with 2% bovine serum albumin (BSA) for 40 min, before being incubated overnight with primary antibodies. The primary antibodies used are given in Table 1. Secondary antibodies were then treated for 1 h. DAPI was used for nuclear staining. Confocal microscopy was performed on LSM900 w/Airyscan (Carl Zeiss, Oberkochen, Baden-Württemberg, Germany). Images represent confocal Z-stack taken with identical laser and detection settings.

The iPSC-derived cortical neurons were treated with 130 μg/ml CNSC-SE for 20 days. After treatment, 1.3×107 cells were seeded on a poly-L-ornithine/laminin-coated CytoView 24-well plate (Axion Biosystems, Atlanta, GA; #M384-tMEA-24W), and iPSCs and non-treated (0 μg/ml group) iPSC-derived cortical neurons were used as controls.

The AxIS software was used to measure the spontaneous potential activity of cortical neurons. Cells were seeded at a density of 1.3 × 107 per well in a CytoView 24-well plate (Axion Biosystems, #M384-tMEA-24W) and cultured in 5% CO2 at 37 °C. The spontaneous activity was recorded for 15 min at the same time every day, and temperatures were maintained during all experiments.

RNA was extracted with Trizol reagent (Invitrogen, Waltham, MA). The RevertAid First strand cDNA Synthesis kit (Invitrogen, #K1622) was used for cDNA synthesis. Real-time PCR was performed with a PowerSYBR Green PCR Master Mix (Applied Biosystems, Waltham, MA; #436759) using the Real-Time PCR System (Applied Biosystems). The primer sequences are shown in Table 2.

The whole brain of each mouse was homogenized with a protein extraction reagent (Thermo Fisher Scientific, Waltham, MA) supplemented with 1 protease inhibitor cocktail tablet (Roche, Basel, Switzerland) and 1 mM phenylmethylsulfonyl fluoride. The homogenate was sonicated and centrifuged at 4 °C for 20 min at 16,000 rpm. Proteins were quantified using the Bradford dye (Bio-Rad, Hercules, CA) with BSA as a standard. Next, 30 μg of total proteins was separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membrane was blocked with 3% BSA for 1 h at room temperature (RT) and incubated at 4 °C overnight with primary antibodies (Table 3). After washing with TBST, the membrane was incubated with the secondary antibody for 1 h at RT. Protein bands were visualized using enhanced chemiluminescence. Band intensities were normalized to β-actin.

Mice were intracardially perfused with saline, and their brains were collected and fixed in 4% PFA at 4 °C, followed by dehydration in 15% sucrose and then 30% sucrose. The brains were then embedded in OCT Compound (Sakura Finetek, Torrance, CA) and sectioned (9 μm) with a microtome (Thermo Fisher Scientific) onto gelatin-coated slides (Masterflex, Gelsenkirchen, Germany; #HV-75955-51). These brain section slides were stored at − 80 °C.

For immunohistochemistry, the slides were dried overnight at RT and fixed with cold acetone for 10 min. Endogenous peroxidase activity was blocked by treating all slides with 0.3% H2O2. The slides were incubated for 1 h with 10% normal goat serum and thereafter overnight at 4 °C with anti-Aβ (1:500, Abcam, Cambridge, UK, #ab201060). After incubation with biotinylated secondary antibodies (1:200) for 40 min, the slides were incubated with the ABC HRP reagent (Vector Laboratories, Newark, CA; #PK-7100) for 10 min and DAB (Vector Laboratories) for 1 min. The slides were counterstained with Mayer’s hematoxylin.

The brain sections were dried overnight at RT and fixed with cold acetone for 10 min. Endogenous peroxidase activity was blocked by treating the slides with 0.3% H2O2. Then the sections were blocked with Mouse on Mouse (M.O.M.) IgG blocking reagent (M.O.M. Fluorescein Kit, Vector Laboratories, #FMK-2201) diluted in 1% BSA for 5 min, followed by incubation overnight at 4 °C with 6E10 (1:1000, BioLegend, #803001). The next day, the slides were incubated with M.O.M. Biotinylated anti-mouse IgG and with the secondary antibody attached to fluorescein Avidin DCS.

For GFAP and IBa-1 immunofluorescence staining, the sections were incubated with anti-GFAP (1:1000, Abcam, #ab7260) or anti-IBa-1 (1:1000, Fujifilm, #019-19741) in 10% normal goat serum at 4 °C overnight. Then the sections were incubated with secondary antibodies such as goat anti-rabbit IgG Alexa 594 (1:200, Thermo Fisher Scienctific) for 1 h. Confocal microscopy was performed on LSM900 w/Airyscan, Carl Zeiss. Confocal Z-stack images were taken with identical laser and detection settings.

The brain sections were dried overnight at RT, fixed with cold acetone for 10 min, rehydrated in 70% and 95% alcohol for 2 min each, and stained with cresyl violet solution (Abcam, ab246816) for 1 min. Then they were washed in 70% and 95% alcohol for 2 min each, dehydrated in 100% ethanol for 30 s, cleared with xylene twice and mounted.

Metabolomics analysis for iPSC secretomes and CNSC-SE was performed using capillary electrophoresis mass spectrometry (Human Metabolome Technologies, Inc, Yamagata, Japan). Samples were prepared to allow for metabolite extraction. To perform metabolomics analysis, we used three samples of iPSC secretomes and CNSC-SE each, and Essential 8 media and neural basal media were used as a control.

Cytokines in CNSC-SE were assayed using a human XL cytokine array kit (ARY022B, R&D Systems, Minneapolis, MN) according to the manufacturer's protocol. The membranes of the kit were blocked at RT for 1 h, and then incubated with culture medium overnight at 4 °C. After 3 days of culture, the medium was debris removed with a 0.22 um syringe filter and used for experiments. The detection antibody cocktail diluted in the assay buffer was added and incubated at 25 °C for 1 h, and 1 × Streptavidin-HRP was added and incubated at RT for 30 min. All experiments were performed on a shaker. Images were acquired using a bio-image analysis system (Amersham Imager 600), and quantitative evaluation was performed using the Image J software.

The human growth factor antibody array (ab134002; Abcam) was performed according to the manufacturer’s protocol. The lyophilized CNSC-SE and MSC-SE samples used in the experiments were diluted to 0.1 g/ml in blocking buffer. Membranes blocked at RT for 1 h were incubated with samples overnight at 4 °C. The biotin-conjugated anti-cytokines cocktail, diluted 1000 × , was added and incubated for 2 h. After washing, HRP-Conjugated Streptavidin was added and incubated for 1 h at RT. The membrane was then exposed using a bio-image analysis system (Amersham Imager 600).

All experiments were repeated at least three times, and results are expressed as the mean and standard error of the mean (shown as error bars). Statistical analysis was performed using GraphPad Prism 9.0. Comparison of multiple groups was performed by one-way analysis of variance (ANOVA) followed by post-hoc Dunnett’s multiple comparison test. A t-test was used to analyze nonparametric quantitative datasets, and one-tailed P values were calculated. Differences between groups were examined for statistical significance using Welch’s t-test, which was also used for analyzing metabolomics. Kruskal-Wallis and Mann-Whitney analyses were performed for intergroup comparison. Statistical significance was set at P < 0.05.

In this study, we used the iPSC-derived cortical neuron differentiation protocol based on a previously reported study (Additional file 1: Fig. S1a) [37]. The cortical identity of the neural tissue formed was validated by the expression of genes expressed in cortical neuronal cells during differentiation and by the absence of expression of other regionally expressed genes (Additional file 1: Fig. S1b). The protein expression of specific markers was confirmed by immunofluorescence staining (Additional file 1: Fig. S1c). To determine if CNSC-SE promotes cortical neuron differentiation, CNSC-SE was treated at a range of concentrations (0 μg/ml, 50 μg/ml, 70 μg/ml, 90 μg/ml, 100 μg/ml, and 130 μg/ml) over a period of 7 days (Fig. 1a). The cortical neurons were confirmed with the expression of neural markers, such as neuronal nuclei (NEUN), vesicular glutamate transporter (vGLUT), and microtubule-associated protein 2 (MAP2), and growth factors, such as brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), and vascular endothelial growth factor (VEGF). In cortical neurons treated with 130 μg/ml CNSC-SE, the mRNA expression of neural markers and growth factors was significantly increased compared to that of other concentrations (Fig. 1b). Therefore, this concentration was selected for in vitro administration.

In the immunofluorescence assay, the 130 μg/ml CNSC-SE-treated cortical neurons showed higher expression of markers and growth factors compared to the cortical neurons treated with 0 μg/ml, such as MAP2, which is a predominant cytoskeletal regulator within neuronal dendrites (Fig. 1c), BDNF, VEGF (Fig. 1d-e), and vGLUT, a marker of mature neurons (Fig. 1f). Taken together, we confirmed that the iPSC-derived CNSC-SE increased neuronal differentiation in vitro and possibly improved dendritic structure by increasing the expression of MAP2.

iPSC-derived mature cortical neurons were treated with CNSC-SE once every 2 days, for 20 days, to test the effects of CNSC-SE on electrical network activity. After 20 days of treatment, the cells were attached to a multiwell microelectrode array (MEA) plate, and MEA was measured for a week from day 55 (Fig. 2a). Interestingly, the electromagnetic signal was detected in the CNSC-SE-treated cortical neurons from the first day of attachment. The electrical signals increased gradually over time, suggesting that the CNSC-SE-treated neurons acquired synchronous network activity (Fig. 2b). The spontaneous activity at day 60 was significantly increased in the CNSC-SE-treated cortical neurons (Fig. 2c-e), especially in the roaster plot, showing a single-channel burst (blue line) pattern (Fig. 2d). Heat map activity showed that the CNSC-SE-treated cortical neurons had increased spike density and area within the cortical networks (Fig. 2e). The iPSC-derived cortical neuron differentiation protocol was reported to exhibit mature electrical signals on day 80 after differentiation [37]; however, in our study, the CNSC-SE-treated cortical neurons showed electrical signals on day 55, confirming that CNSC-SE had potential effects on the development of electrical network.

The ultimate goal of AD treatment is to improve memory. For more efficient CNSC-SE delivery, we used the intranasal route, which is non-invasive and provides the benefits of brain delivery via the olfactory pathway. In addition, to confirm the efficiency of CNSC-SE as a targeted treatment for AD, MSCs, which are known to be specifically effective in neuro-inflammatory processes and in memory recovery, were used as a comparison group. The 12-week-old 5×FAD mice were administered with the secretome intranasally at the dose of 5 μg/g body weight (Additional File 2: Fig. S2) once a week, for 4 weeks, and were tested 1 week later in the Barnes maze (Fig. 3a, b). The CNSC-SE-treated group showed significant improvement in behavioral performance based on the measurements of the following parameters: distance from target zone, resting time in target zone, and error rate. In particular, similar to the control group (WT), mice in the CNSC-SE-5×FAD group stayed in the target hole for a significantly longer time compared to the AD group (Fig. 3c). There was no significant difference between the two groups with respect to the first latency. In addition, regarding the error rate, the CNSC-SE-5×FAD mice showed the next lowest error rate after WT (Fig. 3c). The distinct efficacy of CNSC-SE treatment in improving cognition and memory recovery can also be seen from the tracks (Fig. 3d) and the videos (Additional files 2, 3, 4, and 5). The 3-min camera tracking system showed that the AD model mice did not find the target zone easily. On the contrary, the CNSC-SE-5×FAD mice showed a similar pattern to that of the WT mice and had fewer erroneous entries than the MSC-SE-5×FAD mice. These results suggest that intranasal injection of CNSC-SE may ameliorate cognitive impairment and increase spatial cognition of AD mice.

To investigate the anti-amyloid and anti-inflammatory effects of CNSC-SE in AD, we performed Western blotting for AD-specific markers in mouse brain. Amyloid precursor protein (APP) was significantly decreased in CNSC-SE-5×FAD mouse brains compared to the AD group (Fig. 4a, b). The expression of phosphorylated tau (pTau) was significantly higher in AD and MSC-SE-5×FAD compared to WT, while CNSC-SE-5×FAD did not show any significant difference from WT. We found that beta-site APP cleaving enzyme (BACE), which is an enzyme essential for Aβ generation, was significantly reduced in the CNSC-SE-5×FAD mice (Fig. 4a, b). These results demonstrated a potential protective effect of CNSC-SE in modulating the rate of AD progression, not only by reducing the expression of Aβ precursor APP, but also by regulating the expression of BACE, which initiates Aβ production. To confirm the effect of CNSC-SE on the excessive immune-inflammatory response following amyloid plaque infiltration in AD brain, we investigated the expression of Iba-1, a specific marker for microglial cells (Fig. 4a, b). The expression of Iba-1 was reduced in the secretome-treated groups (CNSC-SE and MSC-SE) compared to the AD model group. Interestingly, a significant reduction of Iba-1 was seen in the CNSC-SE-5×FAD mice compared to both WT and AD (Fig. 4b). Expression of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNFα) was decreased in the 5×FAD mouse brains treated with secretomes (CNSC-SE and MSC-SE). Immunohistochemistry showed pronounced plaque deposition in AD mouse brain, especially in the subiculum location, whereas significantly less accumulation was found in the CNSC-SE-5×FAD mice compared to the AD mice (Fig. 4c, d). When we further checked plaque deposition in the ventral hippocampus, we found a trend of plaque reduction in the CNSC-SE mice compared to the AD and the MSC-SE mice, although there was no significant difference (Additional file 1: Fig. S3).

In AD, activated glial cells promote the removal of amyloid plaques while simultaneously causing excessive neurotoxicity and memory impairment. In Fig. 5a and c, the number of plaques in the AD brain was greater than that of CNSC-SE-5×FAD mice. By quantifying the plaques in the whole brain, we also confirmed that the expression of glial fibrillary acidic protein (GFAP), an astrocyte marker, was three times higher in AD mouse brain (Fig. 5c). Astrocytes in AD are known to have reduced expression of synaptic proteins, suggesting an excessive immune response, in contrast to healthy astrocytes that protect against synapse loss [38,39]. While the morphology of astrocytes in the CNSC-SE-5×FAD mouse brain was not that different from AD mice brain (Fig. 5b), the number of GFAP-positive cells was significantly decreased in the whole brain as well as in the cortex (Fig. 5c).

To determine the extent of microglial and astrocyte activation, we characterized their morphology and distribution by fluorescent staining and quantified their numbers (Figs. 5 and 6). Microglia in AD brains exposed to high inflammatory conditions are known to have enhanced phagocytosis, accelerating the progression of the disease [40]. Therefore, we also assessed the degree of inflammatory activation of microglia (Iba-1) (Fig. 6). In the AD mouse brain, many round-shaped activated microglia (Fig. 6b, white arrow) were observed especially around the amyloid plaques, whereas in the CNSC-SE mouse brain, dormant microglia (yellow arrows) could be found, and only microglia around the plaques were activated (Fig. 6b). These results suggest that intranasal administration of CNSC-SE potentially reduced the activation of abnormal microglia. Taken together, our results suggest that CNSC-SE treatment not only reduced the deposition of amyloid plaques in the AD brain, but also reduced the excessive inflammatory response by inhibiting the abnormal activation of glial cells.

To identify the possible cytokines and growth factors in the CNSC-SE, we performed a cytokine array using the conditioned medium of CNSCs and iPSCs collected on day 3 of culture (Fig. 7a). We identified 11 cytokines in the conditioned medium of CNSCs and 7 cytokines in that of iPSCs. Most cytokines were highly expressed in CNSCs, especially IGFBP-2, FGF-19, and osteopontin, which were significantly higher than those in iPSC medium. IGFBP-2 showed a fourfold increase in the CNSC medium compared to that in the iPSC medium. We also performed a human growth factor array for CNSC-SE and MSC-SE. Interestingly, only IGFBP-2 in CNSC-SE was confirmed, while no signal was expressed in MSC-SE (Fig. 7b). These results indicate that CNSC-conditioned media and CNSC-SE uniquely retain IGFBP-2.

IGFBP-2 is a polypeptide that is most abundant in the cerebrospinal fluid, the developing brain, the hippocampus and the cortex, and functions as an autocrine and/or paracrine growth factor. While insulin-like growth factor 2 (IGF-2) can be regulated by IGFBP-2, only IGF-2 expression correlated with the expression of IGFBP-2 throughout the central nervous system (CNS) development, suggesting that IGFBP-2 may regulate the function of IGF-2 in the CNS [41]. Here, we confirmed the expression of IGFBP-2 and IGF2/IGF-1r in SE-treated mouse brains (Fig. 7c). IGFBP-2, a neuronal proliferation growth factor responsible for cognition and information processing in the brain, was highly expressed in AD and was about three times higher in CNSC-SE treated mice brain compared to trace amounts in MSC-SE-treated mice. IGF-2 has been reported to be decreased in AD patients and mouse models, and amyloid plaques are reduced in the hippocampus of transgenic mice overexpressing IGF-2 [42,43]. Here, IGF-2, which is known to improve brain function and reduce amyloid plaques, was significantly increased in CNSC-SE-5×FAD compared to AD (Fig. 7c). IGF-1r, on the other hand, showed the highest expression in AD and was significantly reduced in MSC-SE-5×FAD and CNSC-SE-5×FAD (Fig. 7c). IGF-1r is involved in several key biological pathways related to the aging process, and IGF-2 binds to IGF-1r with high affinity [44]. It is reported that IGF-1r is involved in disease exacerbation [45]. However, recent studies have reported that IGFBP-2 is highly expressed in the serum of AD patients, and higher IGFBP-2 levels are only observed in amyloid-negative individuals with smaller hippocampal volumes. Therefore, cresyl violet staining was performed to measure the size of the hippocampus (Fig. 7d). We found an average hippocampal area of 0.23 mm2 in WT and CNSC-SE-5×FAD. In addition, the hippocampus of the 5×FAD mice was not shrunk after CNSC-SE treatment. These results suggest that CNSC-SE, which contains high IGFBP-2, functions as a paracrine growth factor to repair the AD brain by promoting IGF-2 and reducing IGF-1r in the AD environment.

Metabolomics array was performed to determine which components of CNSC-SE promote neuronal differentiation and show disease-relieving effects in AD model mice [46]. As CNSC-SE was differentiated from iPSCs, the iPSC secretome (iPSC-SE) was used as a comparison group. In addition, the neural basal medium contained in the secretome was further analyzed. Comparison of the normalized semi-quantitative metabolite profiles of CNSC-SE with iPSC-SE yielded different trends (Fig. 8a). Comparison of the normalized semi-quantitative metabolite profile of CNSC-SE versus the neural basal medium by principal component analysis highlighted a clear difference between them, further demonstrating that the CNSC-SE and neural basal medium are biochemically distinct (Fig. 8b). Metabolites showing a significant difference between CNSC-SE and iPSC-SE were classified by the Human Metabolome Database physical property classification. In the essential amino acid classification, six metabolites, except “Trp”, were significantly present in CNSC-SE (Fig. 8c). In the vitamin metabolism classification, metabolites such as “nicotinamide,” which has antioxidant effect, were expressed to a higher extent in CNSC-SE (Fig. 8c). A total of 130 metabolites were identified. Interestingly, 47 metabolites were only detected in CNSC-SE (Fig. 8d), demonstrating the unique presence of many metabolites in CNSC-SE compared to iPSC-SE and neural basal medium. Among the 47 metabolites, sarcosine has been reported to improve behavioral defects by activating N-methyl-D-aspartate receptor (NMDAR)-induced electrophysiological activation in the NMDAR dysfunctional mouse model [47]; uridine is used as a component of Fortasyn Connect, a specially prepared drink that contains certain nutrients that are susceptible to deficiency in cognitive impairment and early dementia [48]. In addition, glutathione (GSH) is reported as a metabolic product that plays a major role as an antioxidant, an enzyme aid, and a neuromodulator in the CNS [49]. Cystathione is a rate-limiting step for GSH synthesis [50]. Creatine [51,52] plays an important role in the energy homeostasis of neurons [53], and thiamine [54,55,56] plays an important coenzymatic role and is involved in biochemical reactions; both of them can improve brain function. Thus, it can be stated that the CNSC-SE-unique metabolites included several components that have various nerve-protecting functions or have confirmed effects of improving cognitive function recovery. In particular, GSH, which has antioxidant functions in the brain against oxidative stress, was also expressed higher than iPSC-SE alone.

All animal experiments were performed in accordance with the Laboratory Animals Welfare Act, the Guide for the Care and Use of Laboratory Animals, and the Guidelines and Policies for Rodent Experiments provided by the Institutional Animal Care and Use Committee (IACUC) of the School of Medicine, The Catholic University of Korea.

Not applicable.

The authors have declared that no competing interests exist.