Self‐Emergent Protocells Generated in an Aqueous Solution with Binary Macromolecules through Liquid‐Liquid Phase Separation

生体内の高分子混雑に着目した新規の細胞モデルの創成に成功. 京都大学プレスリリース. 2020-12-16.Recently, liquid–liquid phase separation (LLPS) has attracted considerable attention among researchers in the life sciences as a plausible mechanism for the generation of microstructures inside cells. LLPS occurs through multiple nonspecific interactions and does not always require a lock‐and‐key interaction with a binary macromolecular solution. The remarkable features of LLPS include the non‐uniform localization and concentration of solutes, resulting in the ability to isolate certain chemical systems and thereby parallelize multiple chemical reactions within the limited space of a living cell. We report that, by using the macromolecules, poly(ethylene glycol) (PEG) and dextran, that exhibit LLPS in an aqueous solution, cell‐sized liposomes are spontaneously formed therein in the presence of phospholipids. In this system, LLPS is generated through the depletion effect of macromolecules. The results showed that cell‐like microdroplets entrapping DNA wrapped by a phospholipid layer emerge in a self‐organized manner

Human artificial chromosome with a conditional centromere for gene delivery and gene expression.

Human artificial chromosomes (HACs), which carry a fully functional centromere and are maintained as a single-copy episome, are not associated with random mutagenesis and offer greater control over expression of ectopic genes on the HAC. Recently, we generated a HAC with a conditional centromere, which includes the tetracycline operator (tet-O) sequence embedded in the alphoid DNA array. This conditional centromere can be inactivated, loss of the alphoid(tet-O) (tet-O HAC) by expression of tet-repressor fusion proteins. In this report, we describe adaptation of the tet-O HAC vector for gene delivery and gene expression in human cells. A loxP cassette was inserted into the tet-O HAC by homologous recombination in chicken DT40 cells following a microcell-mediated chromosome transfer (MMCT). The tet-O HAC with the loxP cassette was then transferred into Chinese hamster ovary cells, and EGFP transgene was efficiently and accurately incorporated into the tet-O HAC vector. The EGFP transgene was stably expressed in human cells after transfer via MMCT. Because the transgenes inserted on the tet-O HAC can be eliminated from cells by HAC loss due to centromere inactivation, this HAC vector system provides important novel features and has potential applications for gene expression studies and gene therapy

A clinically applicable and scalable method to regenerate T-cells from iPSCs for off-the-shelf T-cell immunotherapy

動物由来の成分を含まないより安全な製法でiPS細胞から大量の再生T細胞を培養する方法の開発 --T細胞を使ったがん免疫療法での利用も--. 京都大学プレスリリース. 2021-01-18.Clinical successes demonstrated by chimeric antigen receptor T-cell immunotherapy have facilitated further development of T-cell immunotherapy against wide variety of diseases. One approach is the development of “off-the-shelf” T-cell sources. Technologies to generate T-cells from pluripotent stem cells (PSCs) may offer platforms to produce “off-the-shelf” and synthetic allogeneic T-cells. However, low differentiation efficiency and poor scalability of current methods may compromise their utilities. Here we show improved differentiation efficiency of T-cells from induced PSCs (iPSCs) derived from an antigen-specific cytotoxic T-cell clone, or from T-cell receptor (TCR)-transduced iPSCs, as starting materials. We additionally describe feeder-free differentiation culture systems that span from iPSC maintenance to T-cell proliferation phases, enabling large-scale regenerated T-cell production. Moreover, simultaneous addition of SDF1α and a p38 inhibitor during T-cell differentiation enhances T-cell commitment. The regenerated T-cells show TCR-dependent functions in vitro and are capable of in vivo anti-tumor activity. This system provides a platform to generate a large number of regenerated T-cells for clinical application and investigate human T-cell differentiation and biology

The Role of Robo3 in the Development of Cortical Interneurons

A number of studies in recent years have shown that members of the Roundabout (Robo) receptor family, Robo1 and Robo2, play significant roles in the formation of axonal tracks in the developing forebrain and in the migration and morphological differentiation of cortical interneurons. Here, we investigated the expression and function of Robo3 in the developing cortex. We found that this receptor is strongly expressed in the preplate layer and cortical hem of the early cortex where it colocalizes with markers of Cajal–Retzius cells and interneurons. Analysis of Robo3 mutant mice at early (embryonic day [E] 13.5) and late (E18.5) stages of corticogenesis revealed no significant change in the number of interneurons, but a change in their morphology at E13.5. However, preliminary analysis on a small number of mice that lacked all 3 Robo receptors indicated a marked reduction in the number of cortical interneurons, but only a limited effect on their morphology. These observations and the results of other recent studies suggest a complex interplay between the 3 Robo receptors in regulating the number, migration and morphological differentiation of cortical interneurons

Patterns of Neurogenesis and Amplitude of Reelin Expression Are Essential for Making a Mammalian-Type Cortex

The mammalian neocortex is characterized as a six-layered laminar structure, in which distinct types of pyramidal neurons are distributed coordinately during embryogenesis. In contrast, no other vertebrate class possesses a brain region that is strictly analogous to the neocortical structure. Although it is widely accepted that the pallium, a dorsal forebrain region, is specified in all vertebrate species, little is known of the differential mechanisms underlying laminated or non-laminated structures in the pallium. Here we show that differences in patterns of neuronal specification and migration provide the pallial architectonic diversity. We compared the neurogenesis in mammalian and avian pallium, focusing on subtype-specific gene expression, and found that the avian pallium generates distinct types of neurons in a spatially restricted manner. Furthermore, expression of Reelin gene is hardly detected in the developing avian pallium, and an experimental increase in Reelin-positive cells in the avian pallium modified radial fiber organization, which resulted in dramatic changes in the morphology of migrating neurons. Our results demonstrate that distinct mechanisms govern the patterns of neuronal specification in mammalian and avian pallial development, and that Reelin-dependent neuronal migration plays a critical role in mammalian type corticogenesis. These lines of evidence shed light on the developmental programs underlying the evolution of the mammalian specific laminated cortex

Chitosan Modification and Pharmaceutical/Biomedical Applications

Chitosan has received much attention as a functional biopolymer for diverse applications, especially in pharmaceutics and medicine. Our recent efforts focused on the chemical and biological modification of chitosan in order to increase its solubility in aqueous solutions and absorbability in the in vivo system, thus for a better use of chitosan. This review summarizes chitosan modification and its pharmaceutical/biomedical applications based on our achievements as well as the domestic and overseas developments: (1) enzymatic preparation of low molecular weight chitosans/chitooligosaccharides with their hypocholesterolemic and immuno-modulating effects; (2) the effects of chitin, chitosan and their derivatives on blood hemostasis; and (3) synthesis of a non-toxic ion ligand—D-Glucosaminic acid from Oxidation of D-Glucosamine for cancer and diabetes therapy

A Novel Role for Dbx1-Derived Cajal-Retzius Cells in Early Regionalization of the Cerebral Cortical Neuroepithelium

Patterning of the cerebral cortex during embryogenesis depends not only on passive diffusion of morphogens but also on signal delivery by Cajal-Retzius neurons that migrate over long distances