Pluripotent stem cell models of early mammalian development.

Pluripotent stem cells derived from the early mammalian embryo offer a convenient model system for studying cell fate decisions in embryogenesis. The last 10 years have seen a boom in the popularity of two-dimensional micropatterns and three-dimensional stem cell culture systems as a way to recreate the architecture and interactions of particular cell populations during development. These methods enable the controlled exploration of cellular organization and patterning during development, using cell lines instead of embryos. They have established a new class of in vitro model system for pre-implantation and peri-implantation embryogenesis, ranging from models of the blastocyst stage, through gastrulation and toward early organogenesis. This review aims to set these systems in context and to highlight the strengths and suitability of each approach in modelling early mammalian development.This work was supported by the European Research Council (Advanced Grant to A.M.A., number 834580) and the Leverhulme Trust (RPG-2018-356). N.M. holds the Constance Work Junior Research Fellowship at Newnham College, Cambridge

Gastruloids: Embryonic Organoids from Mouse Embryonic Stem Cells to Study Patterning and Development in Early Mammalian Embryos

Gastruloids are embryonic organoids made from small, defined numbers of mouse embryonic stem cells (mESCs) aggregated in suspension culture, which over time form 3D structures that mimic many of the features of early mammalian development. Unlike embryoid bodies that are usually disorganized when grown over several days, gastruloids display distinct, well-organized gene expression domains demarcating the emergence of the three body axes, anteroposterior axial elongation, and implementation of collinear Hox transcriptional patterns over 5–7 days of culture. As such gastruloids represent a useful experimental system that is complementary to in vivo approaches in studying early developmental patterning mechanisms regulating the acquisition of cell fates. In this protocol, we describe the most recent method for generating gastruloids with high reproducibility, and provide a comprehensive list of possible challenges as well as steps for protocol optimization

Convalescent plasma in patients admitted to hospital with COVID-19 (RECOVERY): a randomised controlled, open-label, platform trial

SummaryBackground Azithromycin has been proposed as a treatment for COVID-19 on the basis of its immunomodulatoryactions. We aimed to evaluate the safety and efficacy of azithromycin in patients admitted to hospital with COVID-19.Methods In this randomised, controlled, open-label, adaptive platform trial (Randomised Evaluation of COVID-19Therapy [RECOVERY]), several possible treatments were compared with usual care in patients admitted to hospitalwith COVID-19 in the UK. The trial is underway at 176 hospitals in the UK. Eligible and consenting patients wererandomly allocated to either usual standard of care alone or usual standard of care plus azithromycin 500 mg once perday by mouth or intravenously for 10 days or until discharge (or allocation to one of the other RECOVERY treatmentgroups). Patients were assigned via web-based simple (unstratified) randomisation with allocation concealment andwere twice as likely to be randomly assigned to usual care than to any of the active treatment groups. Participants andlocal study staff were not masked to the allocated treatment, but all others involved in the trial were masked to theoutcome data during the trial. The primary outcome was 28-day all-cause mortality, assessed in the intention-to-treatpopulation. The trial is registered with ISRCTN, 50189673, and ClinicalTrials.gov, NCT04381936.Findings Between April 7 and Nov 27, 2020, of 16 442 patients enrolled in the RECOVERY trial, 9433 (57%) wereeligible and 7763 were included in the assessment of azithromycin. The mean age of these study participants was65·3 years (SD 15·7) and approximately a third were women (2944 [38%] of 7763). 2582 patients were randomlyallocated to receive azithromycin and 5181 patients were randomly allocated to usual care alone. Overall,561 (22%) patients allocated to azithromycin and 1162 (22%) patients allocated to usual care died within 28 days(rate ratio 0·97, 95% CI 0·87–1·07; p=0·50). No significant difference was seen in duration of hospital stay (median10 days [IQR 5 to >28] vs 11 days [5 to >28]) or the proportion of patients discharged from hospital alive within 28 days(rate ratio 1·04, 95% CI 0·98–1·10; p=0·19). Among those not on invasive mechanical ventilation at baseline, nosignificant difference was seen in the proportion meeting the composite endpoint of invasive mechanical ventilationor death (risk ratio 0·95, 95% CI 0·87–1·03; p=0·24).Interpretation In patients admitted to hospital with COVID-19, azithromycin did not improve survival or otherprespecified clinical outcomes. Azithromycin use in patients admitted to hospital with COVID-19 should be restrictedto patients in whom there is a clear antimicrobial indication

Dimethyl fumarate in patients admitted to hospital with COVID-19 (RECOVERY): a randomised, controlled, open-label, platform trial

Dimethyl fumarate (DMF) inhibits inflammasome-mediated inflammation and has been proposed as a treatment for patients hospitalised with COVID-19. This randomised, controlled, open-label platform trial (Randomised Evaluation of COVID-19 Therapy [RECOVERY]), is assessing multiple treatments in patients hospitalised for COVID-19 (NCT04381936, ISRCTN50189673). In this assessment of DMF performed at 27 UK hospitals, adults were randomly allocated (1:1) to either usual standard of care alone or usual standard of care plus DMF. The primary outcome was clinical status on day 5 measured on a seven-point ordinal scale. Secondary outcomes were time to sustained improvement in clinical status, time to discharge, day 5 peripheral blood oxygenation, day 5 C-reactive protein, and improvement in day 10 clinical status. Between 2 March 2021 and 18 November 2021, 713 patients were enroled in the DMF evaluation, of whom 356 were randomly allocated to receive usual care plus DMF, and 357 to usual care alone. 95% of patients received corticosteroids as part of routine care. There was no evidence of a beneficial effect of DMF on clinical status at day 5 (common odds ratio of unfavourable outcome 1.12; 95% CI 0.86-1.47; p = 0.40). There was no significant effect of DMF on any secondary outcome

Pluripotent stem cell models of early mammalian development

Pluripotent stem cells derived from the early mammalian embryo offer a convenient model system for studying cell fate decisions in embryogenesis. The last 10 years have seen a boom in the popularity of two-dimensional micropatterns and three-dimensional stem cell culture systems as a way to recreate the architecture and interactions of particular cell populations during development. These methods enable the controlled exploration of cellular organization and patterning during development, using cell lines instead of embryos. They have established a new class of in vitro model system for pre-implantation and peri-implantation embryogenesis, ranging from models of the blastocyst stage, through gastrulation and toward early organogenesis. This review aims to set these systems in context and to highlight the strengths and suitability of each approach in modelling early mammalian development.This work was supported by the European Research Council (advanced grant to AMA, number 834580) and the Leverhulme Trust (RPG-2018-356). NM holds the Constance Work Junior Research Fellowship at Newnham College, Cambridge

Gastruloid-derived primordial germ cell-like cells develop dynamically within integrated tissues.

Peer reviewed: TrueAcknowledgements: We would like to thank Azim Surani, Mitinori Saitou, Christian Schroeter and Tristan Rodriquez for kindly providing cell lines used in this manuscript. We also thank Alfonso Martinez Arias for his support, advice and feedback throughout this project, as well as Federica Cermola for feedback on preliminary experiments, Carla Mulas and Ken Jones for their advice and expertise in CRISPR/Cas9 gene editing, and Ignacio Rodriquez Polo for feedback on the manuscript. In addition, the authors gratefully acknowledge the Francis Crick Light Microscopy, Flow Cytometry, Advanced Sequencing and Bioinformatics and Biostatistics Science and Technology Platforms (STPs) for their support and assistance in this work.Funder: Francis Crick Institute; doi: http://dx.doi.org/10.13039/100010438Primordial germ cells (PGCs) are the early embryonic precursors of gametes - sperm and egg cells. PGC-like cells (PGCLCs) can currently be derived in vitro from pluripotent cells exposed to signalling cocktails and aggregated into large embryonic bodies, but these do not recapitulate the native embryonic environment during PGC formation. Here, we show that mouse gastruloids, a three-dimensional in vitro model of gastrulation, contain a population of gastruloid-derived PGCLCs (Gld-PGCLCs) that resemble early PGCs in vivo. Importantly, the conserved organisation of mouse gastruloids leads to coordinated spatial and temporal localisation of Gld-PGCLCs relative to surrounding somatic cells, even in the absence of specific exogenous PGC-specific signalling or extra-embryonic tissues. In gastruloids, self-organised interactions between cells and tissues, including the endodermal epithelium, enables the specification and subsequent maturation of a pool of Gld-PGCLCs. As such, mouse gastruloids represent a new source of PGCLCs in vitro and, owing to their inherent co-development, serve as a novel model to study the dynamics of PGC development within integrated tissue environments

In vitro teratogenicity testing using a 3D, embryo-like gastruloid system

Pharmaceuticals intended for use in patients of childbearing potential need to be tested for teratogenicity before marketing. Several pharmaceutical companies use animal-free in vitro models which allow a more rapid selection of lead compounds and contribute to 3Rs principles ('replace, reduce and refine') by streamlining the selection of promising compounds submitted to further regulatory studies in animals. Currently available in vitro models typically rely on adherent monolayer cultures or disorganized 3D structures, both of which lack the spatiotemporal and morphological context of the developing embryo. A newly developed 3D 'gastruloid' model has the potential to achieve a more reliable prediction of teratogenicity by providing a robust recapitulation of gastrulation-like events alongside morphological coordination at relatively high-throughput. In this first proof-of-concept study, we used both mouse and human gastruloids to examine a panel of seven reference compounds, with associated in vivo data and known teratogenic risk, to quantitatively assess in vitro teratogenicity. We observed several gross morphological effects, including significantly reduced elongation or decreased size of the gastruloids, upon exposure to several of the reference compounds. We also observed aberrant gene expression using fluorescent reporters, including SOX2, BRA, and SOX17, suggestive of multi-lineage differentiation defects and disrupted axial patterning. Finally, we saw that gastruloids recapitulated some of the known in vivo species-specific susceptibilities between their mouse and human counterparts. We therefore suggest that gastruloids represent a powerful tool for teratogenicity assessment by enabling relevant physiological recapitulation of early embryonic development, demonstrating their use as a novel in vitro teratogenic model system.This work was supported by grants from Medical Research Council (MRC) (MR/R017190/1 and MR/V005367/1) to A.M.A. and N.M., a Leverhulme Trust grant (RPG-2018-356) to AMA, as well as a Gates Cambridge Scholarship awarded to V.M.. The mouse part was supported by an ERC Advanced investigator grant (AdG 834580) to A.M.A. supporting P.B.B. Data derived from human iPSC in vitro model based compound assessment has been supported by the BMBF, EFSA, and the DK-EPA (MST-667-00205) and received funding from the European Union's Horizon 2020 research and innovation program under grant agreements No. 681002 (EU-ToxRisk) and No. 825759 (ENDpoiNTs

Regulation of long-range BMP gradients and embryonic polarity by propagation of local calcium-firing activity.

Acknowledgements: We are grateful to Sandip Patel, Anant Parekh, Andrea Streit and Octavian Voiculescu for advice on experiments and helpful comments on the manuscript. We also acknowledge the Cell Services Science Technology Platform at the Francis Crick Institute for providing regular Mycoplasma screening of the hESC line, the Zoology Imaging Facility of Cambridge University for assistance and support with microscopy on Drosophila embryos, and Sophie Brumm for providing a pSMAD staining protocol. This study was funded by a Wellcome Trust Investigator Award (107055/Z/15/Z) to C.D.S., which supported H.C.L., H.-C.L. and N.M.M.O. H.C.L. was also supported by a fellowship from the Basic Science Research Program through the National Research Foundation of Korea (NRF) (2014R1A6A3A03053468). N.M.M.O. was also funded by UCL. C.H. was funded by a Medical Research Council Doctoral Training Programme studentship (MR/N013867/1). P.B.-B. and N.M. were funded by the Francis Crick Institute which receives its core funding from Cancer Research UK (CC2186), the UK Medical Research Council (CC2186), and the Wellcome Trust (CC2186). A.A.M. was funded by a studentship from the Anatomical Society of Great Britain and Ireland in C.D.S.’s lab. Y.Z. and J.F. were funded by the Michigan-Cambridge Research Initiative, the US National Institutes of Health (R21 HD100931), and the National Science Foundation (CMMI 1917304). E.L.W. was funded by a BBSRC DTP scholarship. T.T.W. was funded by the University of Cambridge ISSF (097814) and the Wellcome Trust (200734/Z/16/Z).Funder: Anatomical Society of Great Britain and Ireland. PhD studentshipFunder: Michigan-Cambridge Research InitiativeFunder: U.S. Department of Health & Human Services | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)Many amniote vertebrate species including humans can form identical twins from a single embryo, but this only occurs rarely. It has been suggested that the primitive-streak-forming embryonic region emits signals that inhibit streak formation elsewhere but the signals involved, how they are transmitted and how they act has not been elucidated. Here we show that short tracks of calcium firing activity propagate through extraembryonic tissue via gap junctions and prevent ectopic primitive streak formation in chick embryos. Cross-regulation of calcium activity and an inhibitor of primitive streak formation (Bone Morphogenetic Protein, BMP) via NF-κB and NFAT establishes a long-range BMP gradient spanning the embryo. This mechanism explains how embryos of widely different sizes can maintain positional information that determines embryo polarity. We provide evidence for similar mechanisms in two different human embryo models and in Drosophila, suggesting an ancient evolutionary origin

Regulation of long-range BMP gradients and embryonic polarity by propagation of local calcium-firing activity

Abstract Many amniote vertebrate species including humans can form identical twins from a single embryo, but this only occurs rarely. It has been suggested that the primitive-streak-forming embryonic region emits signals that inhibit streak formation elsewhere but the signals involved, how they are transmitted and how they act has not been elucidated. Here we show that short tracks of calcium firing activity propagate through extraembryonic tissue via gap junctions and prevent ectopic primitive streak formation in chick embryos. Cross-regulation of calcium activity and an inhibitor of primitive streak formation (Bone Morphogenetic Protein, BMP) via NF-κB and NFAT establishes a long-range BMP gradient spanning the embryo. This mechanism explains how embryos of widely different sizes can maintain positional information that determines embryo polarity. We provide evidence for similar mechanisms in two different human embryo models and in Drosophila, suggesting an ancient evolutionary origin

Regulation of long-range BMP gradients and embryonic polarity by propagation of local calcium-firing activity.

Many amniote vertebrate species including humans can form identical twins from a single embryo, but this only occurs rarely. It has been suggested that the primitive-streak-forming embryonic region emits signals that inhibit streak formation elsewhere but the signals involved, how they are transmitted and how they act has not been elucidated. Here we show that short tracks of calcium firing activity propagate through extraembryonic tissue via gap junctions and prevent ectopic primitive streak formation in chick embryos. Cross-regulation of calcium activity and an inhibitor of primitive streak formation (Bone Morphogenetic Protein, BMP) via NF-κB and NFAT establishes a long-range BMP gradient spanning the embryo. This mechanism explains how embryos of widely different sizes can maintain positional information that determines embryo polarity. We provide evidence for similar mechanisms in two different human embryo models and in Drosophila, suggesting an ancient evolutionary origin