Agarose microgel culture delineates lumenogenesis in naive and primed human pluripotent stem cells.

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

Perceptions and experiences of medical student first responders: a mixed methods study

Context Medical Student First Responders (MSFRs) are volunteers who respond to emergency calls, managing patients before ambulance staff attend. The MSFR role provides opportunities to manage acutely unwell patients in the prehospital environment, not usually offered as part of formal undergraduate medical education. There are few previous studies describing activities or experiences of MSFRs or exploring the potential educational benefits. We aimed to investigate the activity of MSFRs and explore their experiences, particularly from an educational perspective. Methods We used a mixed methods design, combining quantitative analysis of ambulance dispatch data with qualitative semi-structured interviews of MSFRs. Dispatch data were from South Central and East Midlands Ambulance Service NHS Trusts from 1st January to 31st December 2019. Using propensity score matching, we compared incidents attended by MSFRs with those attended by other Community First Responders (CFRs) and ambulance staff. We interviewed MSFRs from five English (UK) medical schools in those regions about their experiences and perceptions and undertook thematic analysis supported by NVivo 12. Results We included 1,939 patients (median age 58.0 years, 51% female) attended by MSFRs. Incidents attended were more urgent category calls (category 1 n = 299, 14.9% and category 2 n = 1,504, 77.6%), most commonly for chest pain (n = 275, 14.2%) and shortness of breath (n = 273, 14.1%). MSFRs were less likely to attend patients of white ethnicity compared to CFRs and ambulance staff, and more likely to attend incidents in areas of higher socioeconomic deprivation (IMD – index of multiple deprivation) (p < 0.05). Interviewees (n = 16) consistently described positive experiences which improved their clinical and communication skills. Conclusion MSFRs’ attendance at serious medical emergencies provide a range of reported educational experiences and benefits. Further studies are needed to explore whether MSFR work confers demonstrable improvements in educational or clinical performance

Trophoblast specification and differentiation potential in early human and non-human primate embryogenesis

Implantation is mediated by trophoblast, the outer layer of the early embryo. Trophoblast is an extraembryonic tissue that gives rise to the placenta, which sustains fetal growth and development. Human embryo implantation and early trophoblast development have remained elusive due to the inaccessibility of early implantation stages. The conservation of trophoblast development between humans and non-human primates provides an exciting opportunity to understanding of the pathophysiology of implantation and related pregnancy disorders. This thesis examines trophoblast development in the common marmoset (Callithrix jacchus) to investigate the molecular regulation of (1) trophoblast specification and (2) trophoblast differentiation, and ultimately the conservation of these processes in human. To delineate the molecular framework of marmoset trophoblast development, a single-cell RNA sequencing dataset was generated from zygote to postimplantation embryos at Carnegie stage (CS) 7. Postimplantation samples were isolated from in utero embryos using laser capture dissection, enabling spatial reconstruction of trophoblast expression patterns and virtual reconstruction of implanted marmoset embryos. I identify and molecularly characterize three distinct trophoblast populations: preimplantation trophectoderm, postimplantation luminal cytotrophoblast, and postimplantation syncytiotrophoblast. Marmoset trophectoderm differentiated into syncytiotrophoblast upon implantation but did not form invasive extravillous trophoblast. Staining of in utero embryos demonstrated extensive cytotrophoblast proliferation but superficial invasion of syncytiotrophoblast by CS7. To determine if these molecular regulators were conserved in human trophoblast development, I generated a continuous pseudotime trajectory of human trophoblast development from zygote to mid-gestation using publicly available single cell RNA sequencing datasets. The results from this analysis showed that core transcription factors controlling trophectoderm, cytotrophoblast and early syncytiotrophoblast identity are conserved between marmoset and human. To interrogate regulators of trophoblast differentiation, I developed a protocol to derive marmoset trophoblast stem cells (TSCs) from pluripotent stem cells (PSCs) in a naïve (preimplantation) like state. Screening of culture conditions for trophoblast derivation revealed that human TSC medium (OKAE) did not promote trophoblast from embryos and naïve PSCs, in contrast to human. However, OKAE conditions with MEK inhibition generated marmoset TSCs. Single-cell RNA sequencing of marmoset TSCs suggested a postimplantation trophoblast identity. In contrast, OKAE conditions promoted an extraembryonic mesenchyme (ExMes) phenotype. WNT signalling induced ExMes fates in the absence of MEK and TGFβ inhibition. Marmoset TSCs form syncytiotrophoblast by endoreduplication as determined by live imaging. Removal of EGF promotes a peri-implantation like phenotype that can form correctly polarized trophoblast spheroids. Moreover, implantation of marmoset TSC spheroids into extracellular matrix marmoset form syncytiotrophoblast. Together, this study established a non-human model system to investigate non-human primate trophoblast development. The study of conserved and divergent regulators of early trophoblast differentiation represents an exciting opportunity to delineate the molecular mechanisms underlying pathophysiological changes in early human implantation and placental development

Evolutionary divergence of embryo implantation in primates.

Implantation of the conceptus into the uterus is absolutely essential for successful embryo development. In humans, our understanding of this process has remained rudimentary owing to the inaccessibility of early implantation stages. Non-human primates recapitulate many aspects of human embryo development and provide crucial insights into trophoblast development, uterine receptivity and embryo invasion. Moreover, primate species exhibit a variety of implantation strategies and differ in embryo invasion depths. This review examines conservation and divergence of the key processes required for embryo implantation in different primates and in comparison with the canonical rodent model. We discuss trophectoderm compartmentalization, endometrial remodelling and embryo adhesion and invasion. Finally, we propose that studying the mechanism controlling invasion depth between different primate species may provide new insights and treatment strategies for placentation disorders in humans. This article is part of the theme issue 'Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom'

An integrated atlas of human placental development delineates essential regulators of trophoblast stem cells

Funder: Centre for Trophoblast Research, University of CambridgeFunder: Cambridge Philosophical SocietyFunder: Pathological Society of Great Britain and Ireland; Id: http://dx.doi.org/10.13039/501100000672Funder: Royal Society; Id: http://dx.doi.org/10.13039/501100000288Funder: University of Cambridge; Id: http://dx.doi.org/10.13039/501100000735ABSTRACT: The trophoblast lineage safeguards fetal development by mediating embryo implantation, immune tolerance, nutritional supply and gas exchange. Human trophoblast stem cells (hTSCs) provide a platform to study lineage specification of placental tissues; however, the regulatory network controlling self-renewal remains elusive. Here, we present a single-cell atlas of human trophoblast development from zygote to mid-gestation together with single-cell profiling of hTSCs. We determine the transcriptional networks of trophoblast lineages in vivo and leverage probabilistic modelling to identify a role for MAPK signalling in trophoblast differentiation. Placenta- and blastoid-derived hTSCs consistently map between late trophectoderm and early cytotrophoblast, in contrast to blastoid-trophoblast, which correspond to trophectoderm. We functionally assess the requirement of the predicted cytotrophoblast network in an siRNA-screen and reveal 15 essential regulators for hTSC self-renewal, including MAZ, NFE2L3, TFAP2C, NR2F2 and CTNNB1. Our human trophoblast atlas provides a powerful analytical resource to delineate trophoblast cell fate acquisition, to elucidate transcription factors required for hTSC self-renewal and to gauge the developmental stage of in vitro cultured cells

Spatial profiling of early primate gastrulation in utero.

Gastrulation controls the emergence of cellular diversity and axis patterning in the early embryo. In mammals, this transformation is orchestrated by dynamic signalling centres at the interface of embryonic and extraembryonic tissues1-3. Elucidating the molecular framework of axis formation in vivo is fundamental for our understanding of human development4-6 and to advance stem-cell-based regenerative approaches7. Here we illuminate early gastrulation of marmoset embryos in utero using spatial transcriptomics and stem-cell-based embryo models. Gaussian process regression-based 3D transcriptomes delineate the emergence of the anterior visceral endoderm, which is hallmarked by conserved (HHEX, LEFTY2, LHX1) and primate-specific (POSTN, SDC4, FZD5) factors. WNT signalling spatially coordinates the formation of the primitive streak in the embryonic disc and is counteracted by SFRP1 and SFRP2 to sustain pluripotency in the anterior domain. Amnion specification occurs at the boundaries of the embryonic disc through ID1, ID2 and ID3 in response to BMP signalling, providing a developmental rationale for amnion differentiation of primate pluripotent stem cells (PSCs). Spatial identity mapping demonstrates that primed marmoset PSCs exhibit the highest similarity to the anterior embryonic disc, whereas naive PSCs resemble the preimplantation epiblast. Our 3D transcriptome models reveal the molecular code of lineage specification in the primate embryo and provide an in vivo reference to decipher human development

Reproducibility of fluorescent expression from engineered biological constructs in E. coli

We present results of the first large-scale interlaboratory study carried out in synthetic biology, as part of the 2014 and 2015 International Genetically Engineered Machine (iGEM) competitions. Participants at 88 institutions around the world measured fluorescence from three engineered constitutive constructs in E. coli. Few participants were able to measure absolute fluorescence, so data was analyzed in terms of ratios. Precision was strongly related to fluorescent strength, ranging from 1.54-fold standard deviation for the ratio between strong promoters to 5.75-fold for the ratio between the strongest and weakest promoter, and while host strain did not affect expression ratios, choice of instrument did. This result shows that high quantitative precision and reproducibility of results is possible, while at the same time indicating areas needing improved laboratory practices.Peer reviewe