Biology subjects

Minegishi, K.

Publications and source records attributed to Minegishi, K..

6 recordsLinked to original sources

bioRxiv2025

Scalable expansion and hepatic zone maturation of hepatic progenitor cells from human pluripotent stem cells

Scalable generation of functionally mature hepatocytes from human pluripotent stem cells (hPSCs) is vital for regenerative medicine, disease modeling, and drug screening. We present a modular differentiation platform that enables serial expansion and cryopreservation of hepatic progenitors (hepatoblasts; HBs), supporting efficient downstream maturation. By modulating bile acid signaling with FGF19 and culturing under hypoxia, we preserved HB proliferation and bipotency. Subsequent thyroid hormone treatment induced alpha-fetoprotein-negative, albumin-positive hepatocytes, while WNT pathway modulation promoted zonal identity, mimicking in vivo hepatic metabolic heterogeneity. Zonation was confirmed by differential drug-metabolizing enzyme activity, mitochondrial function, and enhanced engraftment in a liver injury mouse model. To evaluate translational relevance, mature hepatocytes were incorporated into extrusion-based 3D bioprinted constructs, which maintained hepatic viability and function. This integrated strategy of combining progenitor cell banking, zonation control, and tissue engineering offers a scalable and clinically relevant approach for generating functional human liver tissue suitable for therapeutic development.

cell biology↗

bioRxiv2022

Resolution of fibrosis in mdx dystrophic mouse after oral consumption of N-163 strain of Aureobasidium pullulans produced biological response modifier β-glucan (BRMG)`

Recent advances in the management of Duchenne Muscular Dystrophy (DMD), such as exon skipping therapy, have reached a clinical stage, and although gene therapy is in clinical trials, the outcome at its best is still considered suboptimal to yield clinically discernible progress. In this study, we evaluated a novel N-163 strain of Aureobasidium pullulans produced biological response modifier {beta}-glucan (BRMG) for its potential as an adjuvant to slow down the progression of the disease by anti-inflammatory and anti-fibrotic effects. This N-163 {beta}-glucan is a safe and orally consumable food supplement with similar effects that have been previously proven in pre-clinical studies of organ fibrosis, and their beneficial effects have been proven in DMD clinical studies through blood parameters as well. In this study, 45 mice in the three groups, 15 each in a group; Gr. 1 normal mice, Gr.2 mdx mice as vehicle, and Gr.3 mdx mice administered the N-163 strain produced {beta}-glucan for 45 days. Blood biochemical parameters, body weight, muscle weight, inflammation score, and fibrosis score were evaluated using H&E and Massons trichrome staining. The N-163 {beta}-glucan group showed a significant decrease in the plasma ALT, AST, and LDH levels (126 {+/-} 69, 634 {+/-} 371, 3335 {+/-} 1258 U/l) compared with the vehicle group (177 {+/-} 27 U/l, 912 {+/-} 126 U/l, 4186 {+/-} 398 U/l). Plasma TGF-{beta} levels increased, and plasma IL-13 levels decreased in the N-163 group. The inflammation score of HE-stained muscle sections in the N-163 group (1.5 {+/-} 0.8) was lower than that in the vehicle group (2.0 {+/-} 0.8). The percentage of centrally nucleated fibres (CNF) evaluated by Massons trichrome staining was 0 in the normal group, while it increased to 80% in the vehicle group and 76.8% in the N-163 group. The N-163 {beta}-glucan group (24.22 {+/-} 4.80) showed a significant decrease in the fibrosis area (Massons trichrome-positive area). The N-163 {beta}-glucan thus, demonstrated its anti-fibrotic effect in this study. Considering their safety and easy oral consumption, this BRMG could be worth large multicentre clinical studies as adjuvant in slowing down the progress of DMD.

genetics↗

bioRxiv2022

Immotile cilia of the mouse node sense a fluid flow-induced mechanical force for left-right symmetry breaking

Immotile cilia of crown cells at the node of mouse embryos are required for sensing of a leftward fluid flow1 that gives rise to the breaking of left-right (L-R) symmetry2. The flow-sensing mechanism has long remained elusive, however, with both mechanosensing and chemosensing models having been proposed1, 3-5. Here we show that immotile cilia at the mouse node respond to mechanical force. In the presence of a leftward flow, immotile cilia on the left side of the node bend toward the ventral side whereas those on the right side bend toward the dorsal side. Application of mechanical stimuli to immotile cilia along the dorsoventral axis by optical tweezers induced Ca2+ transients and degradation of Dand5 mRNA--the first known L-R asymmetric molecular events--in the targeted cells. The Pkd2 channel protein was found to be preferentially localized to the dorsal side of immotile cilia on both left and right sides of the node, and the observed induction of Ca2+ transients preferentially by mechanical stimuli directed toward the ventral side could explain the differential response of immotile cilia to the directional flow. Our results thus suggest that immotile cilia at the node sense the direction of fluid flow in a manner dependent on a flow-generated mechanical force.

developmental biology↗

bioRxiv2020

Fluid flow-induced left-right asymmetric decay of Dand5 mRNA in the mouse embryo requires Bicc1-Ccr4 RNA degradation complex

Molecular left-right (L-R) asymmetry is established at the node of the mouse embryo as a result of the sensing of a leftward fluid flow by immotile cilia of perinodal crown cells and the consequent degradation of Dand5 mRNA on the left side. We here examined how the fluid flow induces Dand5 mRNA decay. We found that the 3 untranslated region (3-UTR) of Dand5 mRNA is necessary and sufficient for the left-sided decay and is responsive to the flow direction, loss of the cation channel Pkd2, and Ca2+. The 200-nucleotide proximal-most portion of the 3-UTR, which is conserved among mammals, is essential for the asymmetric mRNA decay and binds Bicc1, an RNA binding protein specifically expressed at the node. Bicc1 preferentially recognizes GAC and GACR sequences in RNA, and these motifs are enriched in the 200-nucleotide region of the Dand5 3-UTR. The Cnot3 component of the Ccr4-Not deadenylase complex interacts with Bicc1 and is also required for Dand5 mRNA decay at the node. Our results thus suggest that leftward fluid flow induces binding of Bicc1 to the 3-UTR of Dand5 mRNA in crown cells on the left side of the node, and that consequent recruitment of Ccr4-Not mediates mRNA degradation.

developmental biology↗

bioRxiv2020

Bicc1 and dicer regulate left-right patterning through posttranscriptionalcontrol of the Nodal-inhibitor dand5

Rotating cilia at the vertebrate left-right organizer (LRO) generate an asymmetric leftward flow, which is sensed by cells at the left LRO margin. How the flow signal is processed and relayed to the laterality-determining Nodal cascade in the left lateral plate mesoderm (LPM) is largely unknown. We previously showed that flow down-regulates mRNA expression of the Nodal inhibitor Dand5 in left sensory cells. De-repression of the co-expressed Nodal drives LPM Nodal cascade induction. Here, we identify the mechanism of dand5 downregulation, finding that its posttranscriptional repression is a central process in symmetry breaking. Specifically, the RNA binding protein Bicc1 interacts with a proximal element in the 3-UTR of dand5 to repress translation in a dicer1-dependent manner. The bicc1/dicer1 module acts downstream of flow, as LRO ciliation was not affected upon its loss. Loss of bicc1 or dicer1 was rescued by parallel knockdown of dand5, placing both genes in the process of flow sensing.

developmental biology↗

bioRxiv2019

PIN FORMED 2 facilitates the transport of Arsenite in Arabidopsis thaliana

Arsenic contamination is a major environmental issue as it may lead to serious health hazard. Reduced trivalent form of inorganic arsenic, arsenite, is in general more toxic to plants compared with the fully oxidized pentavalent arsenate. The uptake of arsenite in plants has been shown to be mediated through a large subfamily of plant aquaglyceroporins, nodulin 26-like intrinsic proteins (NIPs). However, the efflux mechanisms, as well as the mechanism of arsenite-induced root growth inhibition, remain poorly understood. Using molecular physiology, synchrotron imaging, and root transport assay approaches, we show that the cellular transport of trivalent arsenicals in Arabidopsis thaliana is strongly modulated by PIN FORMED 2 (PIN2) auxin efflux transporter. Direct transport assay using radioactive arsenite, X-ray fluorescence imaging (XFI) coupled with X-ray absorption spectroscopy (XAS), and ICP-MS analysis revealed that pin2 plants accumulate higher concentrations of arsenite in root compared to wild-type. At the cellular level, arsenite specifically targets intracellular cycling of PIN2 and thereby alters the cellular auxin homeostasis. Consistently, loss of PIN2 results in aresenite hypersensitivity in root. XFI coupled with XAS further revealed that loss of PIN2 results in specific accumulation of arsenical species, but not the other metals like iron, zinc or calcium in the root tip. Collectively, these results demonstrate that PIN2 serves as a putative transporter of arsenical species in planta.

plant biology↗