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Droplet microfluidic methods have massively increased the throughput of single-cell sequencing campaigns. The benefit of scale-up is, however, accompanied by increased background noise when processing challenging samples and the overall RNA capture efficiency is lower. These drawbacks stem from the lack of strategies to enrich for high-quality material or specific cell types at the moment of cell encapsulation and the absence of implementable multi-step enzymatic processes that increase capture. Here we alleviate both bottlenecks using fluorescence-activated droplet sorting to enrich for droplets that contain single viable cells, intact nuclei, fixed cells or target cell types and use reagent addition to droplets by picoinjection to perform multi-step lysis and reverse transcription. Our methodology increases gene detection rates fivefold, while reducing background noise by up to half. We harness these unique properties to deliver a high-quality molecular atlas of mouse brain development, despite starting with highly damaged input material, and provide an atlas of nascent RNA transcription during mouse organogenesis. Our method is broadly applicable to other droplet-based workflows to deliver sensitive and accurate single-cell profiling at a reduced cost.

Summary Human periimplantation development requires the transformation of the naive pluripotent epiblast into a polarized epithelium. Lumenogenesis plays a critical role in this process, as the epiblast undergoes rosette formation and lumen expansion to form the amniotic cavity. Here, we present a high-throughput in vitro model for epiblast morphogenesis. We established a microfluidic workflow to encapsulate human pluripotent stem cells (hPSCs) into monodisperse agarose microgels. Strikingly, hPSCs self-organized into polarized epiblast spheroids that could be maintained in self-renewing and differentiating conditions. Encapsulated primed hPSCs required Rho-associated kinase inhibition, in contrast to naive hPSCs. We applied microgel suspension culture to examine the lumen-forming capacity of hPSCs and reveal an increase in lumenogenesis during the naive-to-primed transition. Finally, we demonstrate the feasibility of co-encapsulating cell types across different lineages and species. Our work provides a foundation for stem cell-based embryo models to interrogate the critical components of human epiblast self-organization and morphogenesis., Graphical abstract, Highlights • High-throughput platform to encapsulate human PSCs in agarose microgels • Human PSCs self-organize into epiblast spheroids and undergo lumen expansion • Lumen expansion increases in primed versus naive human PSCs • Monodisperse co-culture of embryonic and extraembryonic stem cells, In this article, Boroviak and colleagues present a high-throughput platform to encapsulate human PSCs into agarose microgels. Human PSCs undergo lumenogenesis and self-organize into rosette-like structures, similar to the human peri-implantation embryo. Their microgel suspension culture regime provides a foundation for the controlled assembly of stem cell-based embryo models in our own species.

Data de publicació electrònica: 27-06-2022 Most methods for single-cell transcriptome sequencing amplify the termini of polyadenylated transcripts, capturing only a small fraction of the total cellular transcriptome. This precludes the detection of many long non-coding, short non-coding and non-polyadenylated protein-coding transcripts and hinders alternative splicing analysis. We, therefore, developed VASA-seq to detect the total transcriptome in single cells, which is enabled by fragmenting and tailing all RNA molecules subsequent to cell lysis. The method is compatible with both plate-based formats and droplet microfluidics. We applied VASA-seq to more than 30,000 single cells in the developing mouse embryo during gastrulation and early organogenesis. Analyzing the dynamics of the total single-cell transcriptome, we discovered cell type markers, many based on non-coding RNA, and performed in vivo cell cycle analysis via detection of non-polyadenylated histone genes. RNA velocity characterization was improved, accurately retracing blood maturation trajectories. Moreover, our VASA-seq data provide a comprehensive analysis of alternative splicing during mammalian development, which highlighted substantial rearrangements during blood development and heart morphogenesis. This work was supported by a European Research Council (ERC) Advanced Grant (ERC-AdG 742225-IntScOmics), a Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO) TOP award (NWO-CW 714.016.001) and the Wellcome Trust (WT108438/C/15/Z). This work is part of the Oncode Institute, which is partly financed by the Dutch Cancer Society. J.D.J. received scholarship support from the Biotechnology and Biological Sciences Research Council (BBSRC), T.N.K. from AstraZeneca, A.L.E. from the Cambridge Trusts and the EU H2020 Marie Curie ITN MMBio and T.S.K. from an EU H2020 Marie Skłodowska-Curie Actions Individual Fellowship (MSCA-IF 750772). F.H. is an H2020 ERC Advanced Investigator (69566). M.H. was supported by a core grant from the Wellcome Trust and by funding from the Evergrande Center for Immunologic Diseases. J.N. was funded by the Wellcome Trust (03151/Z/16/Z). For the purpose of open access, the author has applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission. A.Y. was funded by the BBSRC (RG83885), and the mice used in the study are associated with the Wellcome Trust Strategic Grant (105031). Parts of the illustrations were designed using BioRender.

Funder: Wellcome Trust; Id: http://dx.doi.org/10.13039/100010269, Funder: Royal Society; Id: http://dx.doi.org/10.13039/501100000288, Funder: AstraZeneca; Id: http://dx.doi.org/10.13039/100004325, Funder: University of Cambridge; Id: http://dx.doi.org/10.13039/501100000735, The early specification and rapid growth of extraembryonic membranes are distinctive hallmarks of primate embryogenesis. These complex tasks are resolved through an intricate combination of signals controlling the induction of extraembryonic lineages and, at the same time, safeguarding the pluripotent epiblast. Here, we delineate the signals orchestrating primate epiblast and amnion identity. We encapsulated marmoset pluripotent stem cells into agarose microgels and identified culture conditions for the development of epiblast- and amnion-spheroids. Spatial identity mapping authenticated spheroids generated in vitro by comparison to marmoset embryos in vivo. We leveraged the microgel system to functionally interrogate the signalling environment of the postimplantation primate embryo. Single-cell profiling of the resulting spheroids demonstrated that ACTIVIN/NODAL signalling is required for embryonic lineage identity. BMP4 promoted amnion formation and maturation, which was counteracted by FGF-signalling. Our combination of microgel culture, single-cell profiling and spatial identity mapping provides a powerful approach to decipher the essential cues for embryonic and extraembryonic lineage formation in primate embryogenesis.