Biology subjects

Matsui, T. K.

Publications and source records attributed to Matsui, T. K..

6 recordsLinked to original sources

bioRxiv2020

Comparative analysis of human brain organoids of brainstem and midbrain at single-cell resolution

Human brain organoids provide us the means to investigate human brain development and neurological diseases, and single-cell RNA-sequencing (scRNA-seq) technologies allow us to identify homologous cell types and the molecular heterogeneity between individual cells. Previously established human brain organoids of brainstem (hBSOs) and midbrain (hMBOs) were analyzed by scRNA-seq, but the difference in cellular composition between these organoids remains unclear. Here, we integrated and compared the single-cell transcriptome of hBSOs and hMBOs. Our analysis demonstrated that the hBSOs and hMBOs contain some unique cell types, including inflammatory and mesenchymal cells. Further comparison of the hBSOs and hMBOs with publicly available scRNA-seq dataset of human fetal midbrain (hMB) showed high similarity in their neuronal components. These results provide new insights into human brain organoid technologies.

bioinformatics↗

bioRxiv2020

RT-qPCR analyses on the osteogenic differentiation from human iPS cells: An investigation of reference genes

Reverse transcription quantitative PCR (RT-qPCR) is used to quantify gene expression and require standardization with reference genes. We sought to identify the reference genes best suited for experiments that induce osteogenic differentiation from human induced pluripotent stem (iPS) cells. They were cultured in an undifferentiated maintenance medium and after confluence, further cultured in an osteogenic differentiation medium for 28 days. RT-qPCR was performed on undifferentiation markers, osteoblast and osteocyte differentiation markers, and reference gene candidates. The expression stability of each reference gene candidate was ranked using four algorithms. General rankings identified TATA box binding protein (TBP) in the first place, followed by transferrin receptor (TFRC), ribosomal protein large P0 (RPLP0), and finally, beta-2-microglobulin (B2M), which was revealed as the least stable. Interestingly, universally used GAPDH and ACTB were found to be unsuitable. Our findings strongly suggest a need to evaluate the expression stability of reference gene candidates for each experiment.

cell biology↗

bioRxiv2020

ATR inhibition enhances 5-fluorouracil sensitivity independent of non-homologous end-joining and homologous recombination repair pathway

The anticancer agent, 5-fluorouracil (5-FU), is typically applied in the treatment of various types of cancers because of its properties. Thought to be an inhibitor of the enzyme thymidylate synthase which plays a role in nucleotide synthesis, 5-FU has been found to induce single- and double-strand DNA breaks. The activation of ATR occurs as a reaction to UV- and chemotherapeutic drug-induced replication stress. In this study, we examined the effect of ATR inhibition on 5-FU sensitivity. Using western blotting, we found that 5-FU treatment led to the phosphorylation of ATR. Surviving fractions were remarkably decreased in 5-FU with ATR inhibitor (ATRi) compared to 5-FU with other major DNA repair kinases inhibitors. ATR inhibition enhanced induction of DNA double-strand breaks and apoptosis in 5-FU-treated cells. Using gene expression analysis, we found that 5-FU could induce the activation of intra-S checkpoint. Surprisingly, BRCA2-deficient cells were sensitive to 5-FU in the presence of ATRi. In addition, ATR inhibition enhanced the efficacy of 5-FU treatment, independent of non-homologous end-joining and homologous recombination repair pathways. Findings from the present study suggest ATR as a potential therapeutic target for 5-FU chemotherapy.

cancer biology↗

bioRxiv2020

Oxygen-glucose deprivation and reoxygenation on human cerebral organoids alters expression related to lipid metabolism

Ischemic stroke is one of the most common neurological disease. However, the impact of ischemic stroke on human cerebral tissue remains largely unknown; due to a lack of ischemic human brain samples. In this study, we used cerebral organoids derived from human induced pluripotent stem cells to evaluate the effect of oxygen-glucose deprivation/reoxygenation (OGD/R). We identified 15 differentially expressed genes (DEGs); and found that all the DEGs were downregulated. Pathway analysis showed the relationship of vitamin digestion and absorption, fat digestion and absorption, peroxisome proliferator-activated receptor signaling pathway, and complement and coagulation cascades. These findings indicate the mechanisms underlying ischemic injury in human cerebral tissue.

neuroscience↗

bioRxiv2019

Human URAT1/SLC22A12 gene promoter is regulated by 27-hydroxycholesterol through estrogen response elements

Elevated levels of uric acid, a metabolite of purine in humans, is related to various diseases, such as gout, atherosclerosis and renal dysfunction. The excretion and reabsorption of uric acid to/from urine is tightly regulated by uric acid transporters. The amino acid sequences of uric acid reabsorption transporters, URAT1/SLC22A12, OAT4/SLC22A11, and OAT10/SLC22A13, share closer phylogenic relationship, whereas the gene promoter sequences are distant phylogenic relationship. Through the single-cell RNA-sequencing analysis of an adult human kidney, we found that only a small number of cells express these transporters, despite their role in the regulation of serum uric acid levels. Transcriptional motif analysis on these transporter genes, revealed that the URAT1/SLC22A12 gene promoter displayed the most conserved estrogen response elements (EREs) among the three transporters. The endogenous selective estrogen receptor modulator (SERM) 27-hydroxycholesterol (27HC) had positive effects on the transcriptional activity of URAT1/SLC22A12. We also found that 27HC increased the protein and gene expression of URAT1/SLC22A12 in mouse kidneys and human kidney organoids, respectively. These results strongly suggest the role of 27HC for URAT1/SLC22A12 expression in renal proximal tubules and upregulation of serum uric acid levels and also show the relationship between cholesterol metabolism and serum uric acid regulation. Significance StatementThe elevated levels of serum uric acid cause various diseases, and the excretion/reabsorption of uric acid to/from urine is tightly regulated by the uric acid transporters. We found that despite the role in serum uric acid regulation, only a small number of cells express URAT1/SLC22A12. We also found that URAT1/SLC22A12 gene promoter region has effective estrogen response elements, and endogenous selective estrogen receptor (ER) modulator 27-hydroxycholesterol (27HC) increased URAT1/SLC22A12 expression in the mice kidneys and human kidney organoids. These suggest that 27HC increases URAT1/SLC22A12 expression and upregulate serum uric acid levels. Since 27HC connects cholesterol metabolism, our study indicates the important link between cholesterol metabolism and serum uric acid regulation, and also provides a novel therapeutic approach to hyperuricemia.

biochemistry↗

bioRxiv2019

Brainstem organoids from human pluripotent stem cells contain neural crest population

The brainstem controls heartbeat, blood pressure and respiration, which are life-sustaining functions, therefore, disorders of the brainstem can be lethal. Brain organoids derived from human pluripotent stem cells recapitulate the course of human brain development and are expected to be useful for medical research on central nervous system disorders. However, existing organoid models have limitations, hampering the elucidation of diseases affecting specific components of the brain. Here, we developed a method to generate human brainstem organoids (hBSOs), containing neural crest stem cells as well as midbrain/hindbrain progenitors, noradrenergic and cholinergic neurons, and dopaminergic neurons, demonstrated by specific electrophysiological signatures. Single-cell RNA sequence analysis, together with proteomics and electrophysiology, revealed that the cellular population in these organoids was similar to that of the human brainstem and neural crest, which raises the possibility of making use of hBSOs in grafting for transplantation, efficient drug screenings and modeling the neural crest diseases.

developmental biology↗