Biology subjects

Azami, T.

Publications and source records attributed to Azami, T..

7 recordsLinked to original sources

bioRxiv2025

Simplified In Vitro Generation of Human Gastruloids for Modelling Early Development

The purpose of this study was to optimize the efficiency and cost of human gastruloid formation by testing and adjusting individual parameters using as examples two distinct human pluripotent stem cell lines, both available from the UK Stem Cell Bank. For the first step, commercially sourced culture medium was replaced with a home-made defined recipe, known as N2B27, into which specific reagents can be titrated. By reducing the concentration of Activin A to 15% of the original protocol, efficient elongation of aggregated embryoid bodies was achieved. Also, titrating initial cell density and delaying the brief culture in GSK3 inhibitor until the onset of cell aggregation in individual wells was advantageous. Efficiency of formation of early gastruloids exhibiting the expected regionalization of the three embryonic germ layers was further enhanced by addition of TGF{beta}-inhibitor. The optimization steps presented here thus provide a simplified, robust and relatively economical protocol for consistent generation of elongated gastruloids from human pluripotent stem cells. This streamlined method improves accessibility and reproducibility, also providing a standardized platform to investigate fundamental principles of early human development. Summary StatementWe present an optimized protocol for human gastruloid production that should enhance efficiency, reproducibility and affordability for future in vitro studies into early human post-implantation development.

developmental biology↗

bioRxiv2025

A new resource of clonal pluripotent human stem cell lines exhibiting inter- and intra-embryo consistency and variability

Human naive pluripotent stem cells can generate all somatic tissues and extra-embryonic components of the blastocyst. We derived multiple clonal naive pluripotent stem cell lines from individual embryos by physical separation of inner cell mass cells and subsequent individual expansion of each resulting dome-shaped colony, providing the foundation for a resource to investigate intra- and inter-embryo variation. Twenty lines were derived from ten embryos donated from nine couples. While differences between lines are observed, the overarching pluripotency circuitry is preserved in each. They can differentiate into extra-embryonic lineages and readily acquire post-implantation pluripotent identity when exposed to culture conditions driving in vitro capacitation, subsequently to generate derivatives of the three germ layers: ectoderm, mesoderm and endoderm. Some lines exhibit intra-chromosomal amplification and deletions and are therefore anticipated to provide a valuable, accessible system for modelling chromosomal mosaicism and its potential consequences using chimeric organoids, such as blastoids and gastruloids.

developmental biology↗

bioRxiv2024

WNT-mediating TCF/LEF transcription factor gene expression in early human pluripotency and cell lineages differs from the rodent paradigm

Embryonic stem cell research has uncovered different requirements for WNT/{beta}-catenin signalling in human naive pluripotent cells compared to the mouse paradigm. It is therefore important to study WNT/{beta}-catenin signalling directly in models of early human development. Since TCF/LEF factors mediate the regulation of target genes downstream of WNT/{beta}-catenin signalling, we studied the expression and protein localisation of the four TCF/LEF genes by analysing in vitro "snapshots" of human development, leveraging naive and primed pluripotent cells as well as extraembryonic and early embryonic cell lineages. Strikingly, we comprehensively confirm clear differences between mouse and human pluripotent stem cells, suggesting species-specific requirements for WNT signalling that may reflect differences in states of pluripotency. Human naive ES cells express very low TCF7L1, unlike their mouse counterparts. TCF7L2 is robustly expressed in human naive ES-derived trophectoderm cells. In human primed pluripotent stem cells, activation of WNT/{beta}-Catenin signalling is required to induce expression of both TCF7 and LEF1, concomitant with hallmark gastrulation markers. This expression of human TCF/LEF genes benchmarks differential requirements for WNT/{beta}-catenin signalling throughout early human embryo development that requires further investigation.

developmental biology↗

bioRxiv2024

STAT3 signalling enhances tissue expansion during postimplantation mouse development

STAT3 signalling has been studied extensively in the context of self-renewal and differentiation of mouse embryonic stem cells. Zygotic STAT3 is required for normal postimplantation development. On an outbred genetic background, Stat3 null embryos consistently lagged behind their littermates, beginning with significant reduction of epiblast cells at implantation. Remarkably, mutants closely resemble non-affected embryos from the previous day at all postimplantation stages examined. We pinpoint this phenotype to loss of the serine-phosphorylated form of STAT3 which predominates in postimplantation embryonic tissues. Bulk RNA-sequencing analysis of isolated mouse epiblasts confirmed Stat3 null embryos exhibited developmental delay transcriptionally. Single cell RNA sequencing of mid gestation chimaeras containing STAT3 null embryonic stem cells revealed exclusion of mutant cells exclusively from the erythroid lineage. Although Stat3 null embryonic stem cells can differentiate into erythroid and hematopoietic lineages in vitro, they are out-competed when mixed with wild type cells. Combined with the reduced size of STAT3 null epiblasts after implantation, our results implicate a role for STAT3 in cell proliferation affecting temporal control of embryonic progression and rapid differentiation. For the purpose of Open Access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission.

developmental biology↗

bioRxiv2023

A single-cell atlas of pig gastrulation as a resource for comparative embryology

Early mammalian gastrulations cell-fate decisions are poorly understood due to difficulties obtaining non-rodent embryos. The bilaminar disc of pig embryos mirrors humans, making them a useful proxy for studying gastrulation. Here we present a single-cell transcriptomic atlas of pig gastrulation, revealing cell-fate emergence dynamics, as well as conserved and divergent gene programs governing early porcine, primate, and murine development. We highlight heterochronicity in extraembryonic cell-type development, despite the broad conservation of cell-type-specific transcriptional programs. We apply these findings in combination with functional investigations, to outline conserved spatial, molecular, and temporal events during definitive endoderm (DE). We find early FOXA2+/TBXT-embryonic disc cells directly from DE, contrasting later-emerging FOXA2/TBXT+ node/notochord progenitors. Unlike mesoderm, none of these progenitors undergo epithelial-to-mesenchymal transition. DE/Node fate hinges on balanced WNT and hypoblast-derived NODAL, which is extinguished upon DE differentiation. These findings emphasise the interplay between temporal and topological signalling in early fate decisions during gastrulation.

developmental biology↗

bioRxiv2022

Requirement for STAT3 and its target, TFCP2L1 in self-renewal of naïve pluripotent stem cells in vivo and in vitro

We previously demonstrated gradual loss of epiblast during diapause in embryos lacking components of the LIF/IL6 receptor. Here we explore requirement for the downstream signalling transducer and activator of transcription, STAT3 and its target, TFCP2L1, in maintenance of naive pluripotency. Unlike conventional markers, such as NANOG, which remains high in epiblast until implantation, both STAT3 and TFCP2L1 proteins decline during blastocyst expansion, but intensify in the embryonic region after induction of diapause, as observed visually and confirmed using our novel image analysis tool, consistent with our previous transcriptional expression data. Embryos lacking STAT3 or TFCP2L1, underwent catastrophic loss of most of the inner cell mass during the first few days of diapause, implicating involvement of signals in addition to LIF/IL6 for sustaining naive pluripotency in vivo. By blocking MEK/ERK signalling from the morula stage we could derive embryonic stem cells with high efficiency from STAT3 null embryos, but not those lacking TFCP2L1, suggesting a hitherto unknown additional role for this essential STAT3 target in transition from embryo to embryonic stem cells in vitro. Summary StatementInducing diapause in mouse embryos demonstrates that STAT3 and TFCP2L1 are essential for self-renewal of the epiblast, but only TFCP2L1 is required for derivation of embryonic stem cells.

developmental biology↗

bioRxiv2022

Distinct phospho-variants of STAT3 regulate naïve pluripotency and developmental pace in vivo

STAT3 has been studied extensively in the context of self-renewal of naive pluripotent mouse embryonic stem cells. We uncovered acute roles for STAT3 and its target, TFCP2L1, in maintenance of epiblast and primitive endoderm during in vivo diapause. On an outbred genetic background, we observed consistent developmental retardation from implantation until embryonic day 11.5, beginning with significant reduction of epiblast cells at implantation in Stat3 null embryos. Remarkably, mutants closely resemble non-affected embryos from the previous day at all postimplantation stages examined. We attribute this phenotype to loss of the active serine phosphorylated form of STAT3 required for neural differentiation and implicated in growth defects in mice and humans. Bulk RNA-sequencing analysis of isolated epiblasts revealed compromised lipid metabolism in Stat3 null embryos by embryonic day 6.5. Furthermore, we demonstrate that gastruloids generated from Stat3 null ESCs failed to extend the posterior axis or maintain BRACHYURY expression and were underrepresented in this region when mixed with wild type cells in chimaeric gastruloids. Our study implicates a role for STAT3 in temporal control of embryonic progression and metabolic mechanisms.

developmental biology↗