Silencing of p53 and CDKN1A establishes sustainable immortalized megakaryocyte progenitor cells from human iPSCs

iPS細胞を用いた人工血小板の作製の効率化に成功 血小板のテイラーメイド医療に向けた一歩. 京都大学プレスリリース. 2021-12-03.Platelet transfusions are critical for severe thrombocytopenia but depend on blood donors. The shortage of donors and the potential of universal HLA-null platelet products have stimulated research on the ex vivo differentiation of human pluripotent stem cells (hPSCs) to platelets. We recently established expandable immortalized megakaryocyte cell lines (imMKCLs) from hPSCs by transducing MYC, BMI1, and BCL-XL (MBX). imMKCLs can act as cryopreservable master cells to supply platelet concentrates. However, the proliferation rates of the imMKCLs vary with the starting hPSC clone. In this study, we reveal from the gene expression profiles of several MKCL clones that the proliferation arrest is correlated with the expression levels of specific cyclin-dependent kinase inhibitors. Silencing CDKN1A and p53 with the overexpression of MBX was effective at stably inducing imMKCLs that generate functional platelets irrespective of the hPSC clone. Collectively, this improvement in generating imMKCLs should contribute to platelet industrialization and platelet biology

A pathogenic C terminus-truncated polycystin-2 mutant enhances receptor-activated Ca2+ entry via association with TRPC3 and TRPC7.

Mutations in PKD2 gene result in autosomal dominant polycystic kidney disease (ADPKD). PKD2 encodes polycystin-2 (TRPP2), which is a homologue of transient receptor potential (TRP) cation channel proteins. Here we identify a novel PKD2 mutation that generates a C-terminal tail-truncated TRPP2 mutant 697fsX with a frameshift resulting in an aberrant 17-amino acid addition after glutamic acid residue 697 from a family showing mild ADPKD symptoms. When recombinantly expressed in HEK293 cells, wild-type (WT) TRPP2 localized at the endoplasmic reticulum (ER) membrane significantly enhanced Ca2+ release from the ER upon muscarinic acetylcholine receptor (mAChR) stimulation. In contrast, 697fsX, which showed a predominant plasma membrane localization characteristic of TRPP2 mutants with C terminus deletion, prominently increased mAChR-activated influx in cells expressing TRPC3 or TRPC7. Coimmunoprecipitation, pulldown assay, and cross-linking experiments revealed a physical association between 697fsX and TRPC3 or TRPC7. 697fsX but not WT TRPP2 elicited a depolarizing shift of reversal potentials and an enhancement of single-channel conductance indicative of altered ion-permeating pore properties of mAChR-activated currents. Importantly, in kidney epithelial LLC-PK1 cells the recombinant 679fsX construct was codistributed with native TRPC3 proteins at the apical membrane area, but the WT construct was distributed in the basolateral membrane and adjacent intracellular areas. Our results suggest that heteromeric cation channels comprised of the TRPP2 mutant and the TRPC3 or TRPC7 protein induce enhanced receptor-activated Ca2+ influx that may lead to dysregulated cell growth in ADPKD. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.Publisher\u27s version/PDF may be used after 12 months embarg

Geological background of the Kairei and Edmond hydrothermal fields along the Central Indian Ridge : Implications for the distinct chemistry between their vent fluids

Hydrogen-rich hydrothermal areas, such as those in the Indian Ocean, may have had an influence on early evolution of life on Earth and thus have attracted interest because they may be a proxy for ancient ecosystems. The Kairei and Edmond hydrothermal fields in the Indian Ocean are separated by 160 km, but exhibit distinct fluid chemistry: Kairei fluids are hydrogen-rich; Edmond fluids are hydrogen-poor. At this region, the Central Indian Ridge shows an intermediate spreading rate, 48 mm year−1 as full rate, where the hydrothemal fields occur. Kairei field vent fluids show persistently high concentrations of H2. The Kairei field seems to be unique among hydrogen-enriched hydrothermal regions: most similar hydrogen-rich hydrothermal activity occurs along slowly spreading ridge, <40 mm year−1. The geological and tectonic aspects of the Kairei and Edmond hydrothermal fields were studied to try to elucidate geological constraints on hydrogen production. Visual observations of the seafloor near Kairei from a submersible revealed olivine-rich plutonic rocks with olivine gabbro-troctolite-dunite assemblages exposed within 15 km of the vent field, with serpentinized ultramafic mantle rocks on the Oceanic Core Complex (OCC). The OCC area might be a recharge zone of Kairei hydrothermal activity producing H2-rich vent fluids. The chaotic seafloor within 30 km of the Kairei field reflects a magma-starved condition persisting there for 1 Myr. Asymmetric geomagnetic and gravity anomalies near the Kairei field can be used to infer that patchy olivine-rich intrusions are scattered within mantle ultramafics, where infiltrated seawater reacts with magma and ultramafic rocks or olivine-rich rocks. The heterogeneous uppermost lithosphere containing shallow olivine-rich rock facies surrounding the Kairei field provides abundant H2 into the vent fluid through serpentinization. The hydrogen-rich Kairei field is hosted by basalt, with mafic-ultramafic olivine-rich lithology; the ordinary, hydrogen-poor Edmond field is hosted by a normal basaltic lithology. The contrasting geochemical signatures of the two fields reported here can also be found in ancient rocks from a juvenile Earth. This suggests that lithology-controlled generation of hydrogen may have operated for a long time and be relevant to the origin of life on Earth