Dissecting the hierarchy and lineage of mesenchymal stem cells by mouse genetics

Determination of stem cell hierarchy/lineage is indispensable for a better understanding and augmentation of many aspects of medical sciences, including the mechanisms of tissue development and maintenance of tissue homeostasis, as well as disease development. It also has implications in the field of tissue regeneration for medical treatments and disease modeling for drug discovery using iPS technology. Mesenchymal stem cells are multipotent stem cell that can differentiate into various type of cells including osteoblasts, adipocytes, myocytes and chondrocytes. Runt-related transcription factor 2 (Runx2) is an essential transcriptional regulator of osteoblast differentiation. Runx2 deficiency in Prx1+-derived cells (Runx2prx1−/− mice) resulted in defective intramembranous ossification. Double-positive cells for Prx1-GFP and stem cell antigen-1 (Sca1) (Prx1+Sca1+ cells) in the calvaria expressed Runx2 at lower levels and were more homogeneous and primitive as compared with Prx1+Sca1− cells. Our results suggest that osteoblast differentiation in vivo may begin at the Prx1+Sca1+ MSC stage with sequential progression to Prx1+Sca1−cells, then Osx+Prx1−Sca1− osteoblast precursors, which eventually form mature α1(I)-collagen+ osteoblasts

Shared Renewable Resource and International Trade: Technical Measures for Resource Management

We examine trade and strategic interaction between countries that enforce technical measures for resource management which restricts capacity of exploitation to protect an internationally shared renewable resource. The technical measures are common management tools in fisheries (e.g., restrictions on gears, vessels, areas and time). We show that under bilateral resource management, the resource exporting country gains from trade, whereas trade causes steady state utility to fall in the resource importing country because the resource exporting country implements non-cooperative management when the demand for the harvest is not so high. Under sufficiently high demand for the harvest, maximum sustainable yield (MSY) can be attained after trade by what we call cooperative management and both countries are better off. Under low demand for the harvest, trade benefits the resource importing country but may harm the resource exporting country although it implements strict resource management which leads to MSY.

A protocol to induce expandable limb-bud mesenchymal cells from human pluripotent stem cells

Here, we present a protocol for the selective differentiation of human pluripotent stem cells mimicking human developmental processes into expandable PRRX1+ limb-bud mesenchymal (ExpLBM) cells. This approach enables expansion through serial passage while maintaining capacity for chondrogenic differentiation. For complete details on the use and execution of this protocol, please refer to Yamada et al. (2021, 2022)

Mouse Model for Optogenetic Genome Engineering

Optogenetics, a technology to manipulate biological phenomena thorough light, has attracted much attention in neuroscience. Recently, the Magnet System, a photo-inducible protein dimerization system which can control the intracellular behavior of various biomolecules with high accuracy using light was developed. Furthermore, photoactivation systems for controlling biological phenomena are being developed by combining this technique with genome-editing technology (CRISPR/Cas9 System) or DNA recombination technology (Cre-loxP system). Herein, we review the history of optogenetics and the latest Magnet System technology and introduce our recently developed photoactivatable Cre knock-in mice with temporal-, spatial-, and cell-specific accuracy

Continuous drawing of poly(ethylene terephthalate) in ethanol and agile functionalization through infusion

To explore the possibility of applying organic-solvent drawing technique to accomplish a process of industrial significance, continuous drawing of as-spun PET fibers in ethanol was performed. The formation of multiple necks, infusion of ethanol into filament along with the uptake of dissolved dye was observed during the continuous drawing of PET filaments. The propagation speed of individual neck increased as the draw ratio increased, while effect of revolution speed of roller on the neck propagation speed was minimal because of the increase of the number density of neck along with the increase in processing speed. Through the monitoring of the drawing tension, it was found that the fluctuation of drawing tension is a source of instability of the drawing process. The installation of a drawing pin was discovered to be useful for the stabilization of the drawing process while increasing the amount of infused ethanol and dye uptake

Modulation by static magnetism of neuronal activity

In neurons, the signal propagation involves both the conduction mediated by local electric currents through voltage-sensitive cation channels in axons and the transmission mediated by the exocytotic release of neurotransmitters from nerve endings into synaptic clefts. A great number of desperate efforts have been dedicated to biochemical, pharmacological and molecular biological studies on the elucidation of mechanisms underlying the neurotransmission at synapses, while relatively little attention has been paid to the comprehensive evaluation of the conduction except for local anesthetics. According to a physical theorem, exposure to magnetism should lead to the generation of a certain mechanical force in neurons with concomitant electric currents in a particular situation. In particular, repetitive transcranial magnetic stimulation is beneficial for the treatment of selected patients suffering from depression, bipolar affective disorder and schizophrenia as a possible alternative to the electroconvulsive therapy for refractory depression. In this review, therefore, we will summarize our recent advances made on the neurochemical and molecular biological elucidation in cultured rat hippocampal neurons toward better understanding by the readers of different disciplines of mechanisms associated with the modulation by magnetism of the neuronal activity in the brain.Biomedical Reviews 2004; 15: 21-35

Establishment of a human pluripotent stem cell-derived MKX-td Tomato reporter system

Tendon regeneration is difficult because detailed knowledge about tendon progenitor cells (TPCs), which produce tenocytes to repair tendon tissue, has not been revealed. Mohawk homeobox (MKX) is a marker of TPCs or tenocytes, but a human pluripotent stem cell (hPSC)-based reporter system that visualizes MKX+ cells has not been developed. Here, we established an hPSC-derived MKX-tdTomato reporter cell line and tested the induction ratio of MKX-tdTomato(+) cells using our stepwise/xeno-free differentiation protocol. MKX-tdTomato(+) cells were generated with high efficiency and expressed tendon-specific markers, including MKX, SCX, TNMD, and COL1A1. Our MKX-tdTomato hPSC line would be a useful tool for studying the development or regeneration of tendon tissue

骨関節系細胞に発現するグルタメイトシグナル装置に関する分子薬理学的研究

取得学位：博士(薬学)，学位授与番号：博乙第322号，学位授与年月日：平成20年3月22

Glial assembly: a new regulatory machinery of brain function and disorders

金沢大学医薬保健研究域薬学系グリア病である神経障害性疼痛は、精神的ストレスや精神疾患との関連性が臨床上指摘されている（うつを伴う慢性痛、統合失調症や自閉スペクトラム症での痛覚鈍麻など）。しかし、この「精神と疼痛(痛み)」の関連性（連関）の分子基盤は未解明である。我々の解析結果より、睡眠障害等の精神疾患との関連性が深い体内時計システムが破綻したマウスでは、行動・疼痛機能の異常とともに、脳・脊髄組織でのアストロサイトでの異常活性化が認められる。これにより、血管周囲に存在するアストロサイト-ペリサイトアセンブリが異常をきたす可能性を提唱した。つまり、BBB恒常性は体内時計システムによるグリアネットワークの上に成り立つことを示唆するものである。研究課題/領域番号:26117507, 研究期間(年度):2014-04-01 – 2016-03-3

Identification of Surface Antigens That Define Human Pluripotent Stem Cell-Derived PRRX1+Limb-Bud-like Mesenchymal Cells

Stem cell-based therapies and experimental methods rely on efficient induction of human pluripotent stem cells (hPSCs). During limb development, the lateral plate mesoderm (LPM) produces limb-bud mesenchymal (LBM) cells that differentiate into osteochondroprogenitor cells and form cartilage tissues in the appendicular skeleton. Previously, we generated PRRX1-tdTomato reporter hPSCs to establish the protocol for inducing the hPSC-derived PRRX1(+) LBM-like cells. However, surface antigens that assess the induction efficiency of hPSC-derived PRRX1(+) LBM-like cells from LPM have not been identified. Here, we used PRRX1-tdTomato reporter hPSCs and found that high pluripotent cell density suppressed the expression of PRRX1 mRNA and tdTomato after LBM-like induction. RNA sequencing and flow cytometry suggested that PRRX1-tdTomato(+) LBM-like cells are defined as CD44(high) CD140B(high) CD49f(-). Importantly, other hPSC lines, including four human induced pluripotent stem cell lines (414C2, 1383D2, HPS1042, HPS1043) and two human embryonic stem cell lines (SEES4, SEES7), showed the same results. Thus, an appropriate cell density of hPSCs before differentiation is a prerequisite for inducing the CD44(high) CD140B(high) CD49f(-) PRRX1(+) LBM-like cells