Biology subjects

Horii, M.

Publications and source records attributed to Horii, M..

4 recordsLinked to original sources

bioRxiv2025

Derivation of functional early gestation decidual natural killer cell subtypes from induced pluripotent stem cells

Abnormal decidual natural killer cell (dNK) function is linked to pregnancy complications occurring in both early and late gestation, including recurrent pregnancy loss, preeclampsia, and preterm birth. Exploration of dNK heterogeneity as it relates to function is an active area of research; however, most of this work has focused on early gestation. Using flow cytometric and transcriptomic single-cell definitions of dNK subtypes, we characterized dNK heterogeneity in term dNK within both chorioamniotic membranes and basal plate. We also applied aptamer-based secretome profiling to first trimester and term dNK, and found dNK-specific proteins - VEGF and PLGF - to be reduced at term. We further determined that, compared to first trimester dNK, term dNK have reduced cytoxicity against target cells. Finally, we applied this knowledge to establish a protocol for differentiation of induced pluripotent stem cells (iPSC) into functional dNK. We found that treatment with TGF{beta} enriched for dNK2 subtype, while inducing dNK markers, CD9 and CD103. We evaluated function using cytokine and degranulation assays, aptamer-based secretome profiling, and cytotoxicity assays. We found that iPSC-dNK are functionally most similar to primary dNK. Further, TGF{beta} iPSC-dNK had reduced GM-CSF in response to PMA/I and increased secretion of VEGF and other first trimester-specific proteins - supportive of a shift towards an early gestation, dNK2-dominant, phenotype. We conclude that changes in dNK function across gestation reflect shifts in dNK subtypes that can be reproducibly derived from iPSC, providing a new method for modeling dNK and laying the foundation for cell-based therapeutics for reproductive disease. Significance StatementAlterations in maternal decidual natural killer cells (dNK) are associated with pregnancy complications - from recurrent pregnancy loss to preeclampsia and preterm birth. We found dNK from different regions of the term placenta to be distinct from peripheral blood NK and early gestation dNK, based on gene and surface marker expression, subtype composition, secretome, and cytotoxicity. We report a novel, reproducible protocol to generate dNK resembling the most abundant dNK subtype in early gestation from induced pluripotent stem cells. Our study lays the foundation for in vitro modeling of the maternal-fetal interface and therapeutic development for reproductive disease.

cell biology↗

bioRxiv2025

WHEP Domain of Glycyl-tRNA Synthetase Regulates Neuropilin 1 Binding and Vascular Permeability

AbstractAminoacyl-tRNA synthetases (aaRSs) charge tRNAs with their cognate amino acids, ensuring accurate translation of the genetic code from mRNA to protein. During eukaryotic evolution, aaRSs acquired additional domains with unclear functions, including the WHEP domain, a two-helix bundle found in several eukaryotic aaRSs such as glycyl-tRNA synthetase (GlyRS, encoded by GARS1). We generated Gars1{Delta}WHEP mutant mice lacking exon 2, which disrupts most of the WHEP domain. Homozygous Gars1{Delta}WHEP/{Delta}WHEP mice exhibited late embryonic or neonatal lethality, with delayed lung development, characterized by reduced airway dilation (inflatability), and increased vascular leakage. Disruption of the WHEP domain did not impair tRNA aminoacylation but inhibited the free release of GlyRS from cells. Instead, GlyRS{Delta}WHEP was found in the membrane fractions and showed a stronger interaction with the extracellular region of neuropilin 1 (Nrp1) receptor compared to full-length GlyRS. This aberrant interaction enhanced Nrp1s endocytic activity and significantly reduced the localization of the Nrp1 interactor VE-cadherin at the adherens junctions of endothelial cells. A heterozygous knockout of Nrp1 in the Gars1 {Delta}WHEP/{Delta}WHEP mice partially rescued body weight and vascular permeability defects. This study establishes a physiological role for the GlyRS WHEP domain in lung development and its regulation of GlyRS-Nrp1 interaction and vascular permeability.

molecular biology↗

bioRxiv2025

Chlamydomonas γ-tubulin mutations reveal a critical role of γ-TuRC in maintaining the stability of centriolar microtubules

The centriolar triplet microtubule consists of an A-tubule with 13 protofilaments, and B-and C-tubules, each with 10 protofilaments. Although the formation of the triplets has been shown to require {gamma}-tubulin, its specific role in the formation of each tubule remains elusive. We isolated two novel Chlamydomonas reinhardtii mutants, bld13-1 and bld13-2, each expressing {gamma}-tubulin with a single amino-acid substitution (T292I or E89D). Similar to known centriole-deficient mutants, both mutants exhibited defects in ciliary assembly, nuclear number, as well as the number and orientation of cytoplasmic microtubules. Genetic analyses of the mutants, along with the expression of the mutant {gamma}-tubulins in the wild-type cells, suggested that both mutants exert dominant-negative effects over wild-type {gamma}-tubulin. Interestingly, although the centrioles in these mutants retained the typical nine triplet structure, their triplets frequently lacked several protofilaments in specific regions of the A- and C-tubules. The protofilament loss occurs more frequently in the proximal region of the centriole. These structural defects suggest a critical role for {gamma}-tubulin in maintaining the stability of the A- and C-tubules of centriolar triplets. Summary statementNovel Chlamydomonas {gamma}-tubulin mutations cause a partial loss of protofilaments in centriolar microtubules, indicating a critical role of {gamma}-tubulin in structural stabilization of triplet microtubules.

cell biology↗

bioRxiv2024

Single-cell transcriptomics reveal differences between chorionic and basal plate cytotrophoblasts and trophoblast stem cells

Cytotrophoblast (CTB) of the early gestation human placenta are bipotent progenitor epithelial cells, which can differentiate into invasive extravillous trophoblast (EVT) and multinucleated syncytiotrophoblast (STB). Trophoblast stem cells (TSC), derived from early first trimester placentae, have also been shown to be bipotential. In this study, we set out to probe the transcriptional diversity of first trimester CTB and compare TSC to various subgroups of CTB. We performed single-cell RNA sequencing on six normal placentae, four from early (6-8 weeks) and two from late (12-14 weeks) first trimester, of which two of the early first trimester cases were separated into basal (maternal) and chorionic (fetal) fractions prior to sequencing. We also sequenced three TSC lines, derived from 6-8 week placentae, to evaluate similarities and differences between primary CTB and TSC. CTB clusters displayed notable distinctions based on gestational age, with early first trimester placentae showing enrichment for specific CTB subtypes, further influenced by origin from the basal or chorionic plate. Differential expression analysis of CTB from basal versus chorionic plate highlighted pathways associated with proliferation, unfolded protein response, and oxidative phosphorylation. We identified trophoblast states representing initial progenitor CTB, precursor STB, precursor and mature EVT, and multiple CTB subtypes. CTB progenitors were enriched in early first trimester placentae, with basal plate cells biased toward EVT, and chorionic plate cells toward STB, precursors. Clustering and trajectory inference analysis indicated that TSC were most like EVT precursor cells, with only a small percentage of TSC on the pre-STB differentiation trajectory. This was confirmed by flow cytometric analysis of 6 different TSC lines, which showed uniform expression of proximal column markers ITGA2 and ITGA5. Additionally, we found that ITGA5+ CTB could be plated in 2D, forming only EVT upon spontaneous differentiation, but failed to form self-renewing organoids; conversely, ITGA5-CTB could not be plated in 2D, but readily formed organoids. Our findings suggest that distinct CTB states exist in different regions of the placenta as early as six weeks gestation and that current TSC lines most closely resemble ITGA5+ CTB, biased toward the EVT lineage.

developmental biology↗