Simple modeling of familial Alzheimer's disease using human pluripotent stem cell-derived cerebral organoid technology.

BACKGROUND: Cerebral organoids (COs) are the most advanced in vitro models that resemble the human brain. The use of COs as a model for Alzheimer's disease (AD), as well as other brain diseases, has recently gained attention. This study aimed to develop a human AD CO model using normal human pluripotent stem cells (hPSCs) that recapitulates the pathological phenotypes of AD and to determine the usefulness of this model for drug screening. METHODS: We established AD hPSC lines from normal hPSCs by introducing genes that harbor familial AD mutations, and the COs were generated using these hPSC lines. The pathological features of AD, including extensive amyloid-ß (Aß) accumulation, tauopathy, and neurodegeneration, were analyzed using enzyme-linked immunosorbent assay, Amylo-Glo staining, thioflavin-S staining, immunohistochemistry, Bielschowsky's staining, and western blot analysis. RESULTS: The AD COs exhibited extensive Aß accumulation. The levels of paired helical filament tau and neurofibrillary tangle-like silver deposits were highly increased in the AD COs. The number of cells immunoreactive for cleaved caspase-3 was significantly increased in the AD COs. In addition, treatment of AD COs with BACE1 inhibitor IV, a ß-secretase inhibitor, and compound E, a γ-secretase inhibitor, significantly attenuated the AD pathological features. CONCLUSION: Our model effectively recapitulates AD pathology. Hence, it is a valuable platform for understanding the mechanisms underlying AD pathogenesis and can be used to test the efficacy of anti-AD drugs.

RFX4 is an intrinsic factor for neuronal differentiation through induction of proneural genes POU3F2 and NEUROD1.

Proneural genes play a crucial role in neuronal differentiation. However, our understanding of the regulatory mechanisms governing proneural genes during neuronal differentiation remains limited. RFX4, identified as a candidate regulator of proneural genes, has been reported to be associated with the development of neuropsychiatric disorders. To uncover the regulatory relationship, we utilized a combination of multi-omics data, including ATAC-seq, ChIP-seq, Hi-C, and RNA-seq, to identify RFX4 as an upstream regulator of proneural genes. We further validated the role of RFX4 using an in vitro model of neuronal differentiation with RFX4 knock-in and a CRISPR-Cas9 knock-out system. As a result, we found that RFX4 directly interacts with the promoters of POU3F2 and NEUROD1. Transcriptomic analysis revealed a set of genes associated with neuronal development, which are highly implicated in the development of neuropsychiatric disorders, including schizophrenia. Notably, ectopic expression of RFX4 can drive human embryonic stem cells toward a neuronal fate. Our results strongly indicate that RFX4 serves as a direct upstream regulator of proneural genes, a role that is essential for normal neuronal development. Impairments in RFX4 function could potentially be related to the development of various neuropsychiatric disorders. However, understanding the precise mechanisms by which the RFX4 gene influences the onset of neuropsychiatric disorders requires further investigation through human genetic studies.

Human embryonic stem cell-specific role of YAP in maintenance of self-renewal and survival.

Human embryonic stem cells (hESCs) have unique characteristics, such as self-renewal and pluripotency, which are distinct from those of other cell types. These characteristics of hESCs are tightly regulated by complex signaling mechanisms. In this study, we demonstrate that yes-associated protein (YAP) functions in an hESC-specific manner to maintain self-renewal and survival in hESCs. hESCs were highly sensitive to YAP downregulation to promote cell survival. Interestingly, hESCs displayed dynamic changes in YAP expression in response to YAP downregulation. YAP was critical for the maintenance of self-renewal. Additionally, the function of YAP in maintenance of self-renewal and cell survival was hESC-specific. Doxycycline upregulated YAP in hESCs and attenuated the decreased cell survival induced by YAP downregulation. However, decreased expression of self-renewal markers triggered by YAP downregulation and neural/cardiac differentiation were affected by doxycycline treatment. Collectively, the results reveal the mechanism underlying the role of YAP and the novel function of doxycycline in hESCs.

A simple method to improve the quality and yield of human pluripotent stem cell-derived cerebral organoids.

The development of cerebral organoid technology has allowed the human neural tissue to be collected for studying human brain development and neurological diseases. Human pluripotent stem cell-derived cerebral organoids (hCOs) are a theoretically infinite source of fresh human brain tissue for various research purposes. However, hCOs have limitations, including core necrotic cell death. To solve this problem, we tested a simple method, which has been previously overlooked. In this study, we mechanically cut 70-day-old hCOs with a scalpel blade into 2 to 4 pieces, each depending on their original size. After culturing cut hCOs for additional 7 days, their size was less variable and smaller than uncut hCOs and there were no histological differences between uncut and cut hCOs. Note that hypoxia-inducible factor (HIF)-1α was expressed in the central area of uncut hCOs but not in cut hCOs. Uncut hCOs, therefore, showed broad core areas stained with terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL), whereas cut hCOs did not. In conclusion, this simple mechanical cutting method allowed us to acquire a larger number of hCOs without a necrotic core.

Effects of Dipsacus asperoides Extract on Monosodium Iodoacetate-Induced Osteoarthritis in Rats Based on Gene Expression Profiling.

The root of Dipsacus asperoides C. Y. Cheng et T. M. Ai is traditionally used as an analgesic and anti-inflammatory agent to treat pain, rheumatoid arthritis, and bone fractures. However, neither its effects on osteoarthritis (OA) nor its effects on the arthritic cartilage tissue transcriptome have not been fully investigated. In this study, we used a rat model of monosodium iodoacetate- (MIA-) induced OA to investigate the therapeutic effects of a Dipsacus asperoides ethanolic extract (DAE, 200 mg/kg for 21 days). The study first assessed joint diameter, micro-CT scans, and histopathological analysis and then conducted gene expression profiling using RNA sequencing in articular cartilage tissue. We found that DAE treatment ameliorates OA disease phenotypes; it reduced the knee joint diameter and prevented changes in the structural and histological features of the joint, thereby showing that DAE has a protective effect against OA. Based on the results of gene expression profiling and subsequent pathway analysis, we found that several canonical pathways were linked to DAE treatment, including WNT/ß-catenin signaling. Taken together, the present results suggest molecular mechanism, involving gene expression changes, by which DAE has a protective effect in a rat model of MIA-induced OA.

Protective effects of Phlomis umbrosa extract on a monosodium iodoacetate-induced osteoarthritis model and prediction of molecular mechanisms using transcriptomics.

BACKGROUND: Phlomis umbrosa Turczaninow root has been traditionally used to treat fractures, rheumatoid arthritis, and arthralgia. However, the effects and mechanisms of P. umbrosa on osteoarthritis (OA) remain poorly understood and a functional genomic approach has not been investigated. AIM: The purpose of this study was to investigate the effects and mechanisms of P. umbrosa extract (PUE) on OA using transcriptomic analysis. METHODS: We performed joint diameter measurements, micro computed tomography, and histopathological analysis of monosodium iodoacetate (MIA)-induced OA rats treated with PUE (200 mg/kg) for 3 weeks. Gene expression profiling in articular cartilage tissue was then performed using RNA sequencing (RNA-seq) followed by signaling pathway analysis of regulatory genes. RESULTS: PUE treatment improved OA based on decreased joint diameter, increased joint morphological parameters, and histopathological features. Many genes involved in multiple signal transduction pathway and collagen activation in OA were differentially regulated by PUE. These included genes related to Wnt/ß-catenin, OA pathway, and sonic hedgehog signaling activity. Furthermore, PUE treatment downregulated cartilage damage factors (MMP-9, MMP-13, ADAMTs4, and ADMATs5) and upregulated chondrogenesis (COL2A1 and SOX-9) by regulating the transcription factors SOX-9, Ctnnb1, and Epas1. CONCLUSION: Based on the results of gene expression profiling, this study highlighted the molecular mechanisms underlying the effects of PUE in MIA-induced OA rats. The findings provide novel insight into the mechanisms by which PUE treatment-induced gene expression changes may influence OA disease progression. Taken together, the results suggest that PUE may be used as a source of therapeutic agents for OA.

A simple metastatic brain cancer model using human embryonic stem cell-derived cerebral organoids.

Every year, hundreds of thousands of people die because of metastatic brain cancer. Most metastatic cancer research uses 2D cell culture or animal models, but they have a few limitations, such as difficulty reproducing human tissue structures. This study developed a simple 3D in vitro model to better replicate brain metastasis using human cancer cells and human embryonic stem cell-derived cerebral organoids (metastatic brain cancer cerebral organoid [MBCCO]). The MBCCO model successfully reproduced metastatic cancer processes, including cell adhesion, proliferation, and migration, in addition to cell-cell interactions. Using the MBCCO model, we demonstrated that lung-specific X protein (LUNX) plays an important role in cell proliferation and migration or invasion. We also observed astrocyte accumulation around and their interaction with cancer cells through connexin 43 in the MBCCO model. We analyzed whether the MBCCO model can be used to screen drugs by measuring the effects of gefitinib, a well-known anticancer agent. We also examined the toxicity of gefitinib using normal cerebral organoids (COs). Therefore, the MBCCO model is a powerful tool for modeling human metastatic brain cancer in vitro and can also be used to screen drugs.

Trolox-induced cardiac differentiation is mediated by the inhibition of Wnt/ß-catenin signaling in human embryonic stem cells.

Cardiac differentiation of human pluripotent stem cells may be induced under chemically defined conditions, wherein the regulation of Wnt/ß-catenin pathway is often desirable. Here, we examined the effect of trolox, a vitamin E analog, on the cardiac differentiation of human embryonic stem cells (hESCs). 6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) significantly enhanced cardiac differentiation in a time- and dose-dependent manner after the mesodermal differentiation of hESCs. Trolox promoted hESC cardiac differentiation through its inhibitory activity against the Wnt/ß-catenin pathway. This study demonstrates an efficient cardiac differentiation method and reveals a novel Wnt/ß-catenin regulator.

Comparative Proteomic Analysis Reveals the Upregulation of Ketogenesis in Cardiomyocytes Differentiated from Induced Pluripotent Stem Cells.

Diverse metabolic pathways, such as the tricarboxylic acid cycle, pyruvate metabolism, and oxidative phosphorylation, regulate the differentiation of induced pluripotent stem cells (iPSCs) to cells of specific lineages and organs. Here, the protein dynamics during cardiac differentiation of human iPSCs into cardiomyocytes (CMs) are characterized. The differentiation is induced by N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]-acetamide, a Wnt signaling inhibitor, and confirmed by the mRNA and protein expression of cTnT and MLC2A in CMs. For comparative proteomics, cells from three stages, namely, hiPSCs, cardiac progenitor cells, and CMs, are prepared using the three-plex tandem mass tag labeling approach. In total, 3970 proteins in triplicate analysis are identified. As the result, the upregulation of proteins associated with branched chain amino acid degradation and ketogenesis by the Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis are observed. The levels of 3-hydroxymethyl-3-methylglutaryl-CoA lyase, 3-hydroxymethyl-3-methylglutaryl-CoA synthase 2, and 3-hydroxybutyrate dehydrogenase 1, involved in ketone body metabolism, are determined using western blotting, and the level of acetoacetate, the final product of ketogenesis, is higher in CMs. Taken together, these observations indicate that proteins required for the production of diverse energy sources are naturally self-expressed during cardiomyogenic differentiation. Furthermore, acetoacetate concentration might act as a regulator of this differentiation.