Early Postimplantation Development Research Articles

3D-cultured human blastoids model trophoblast expansion, diversification, and invasion during early post-implantation development

3D-cultured human blastoids model trophoblast expansion, diversification, and invasion during early post-implantation development

3D-cultured blastoids model human embryogenesis from pre-implantation to early gastrulation stages.

Naive human pluripotent stem cells have the remarkable ability to self-organize into blastocyst-like structures ("blastoids") that model lineage segregation in the pre-implantation embryo. However, the extent to which blastoids can recapitulate the defining features of human post-implantation development remains unexplored. Here, we report that blastoids cultured on thick three-dimensional (3D) extracellular matrices capture hallmarks of early post-implantation development, including epiblast lumenogenesis, rapid expansion and diversification of trophoblast lineages, and robust invasion of extravillous trophoblast cells by day 14. Extended blastoid culture results in the localized activation of primitive streak marker TBXT and the emergence of embryonic germ layers by day 21. We also show that the modulation of WNT signaling alters the balance between epiblast and trophoblast fates in post-implantation blastoids. This work demonstrates that 3D-cultured blastoids offer a continuous and integrated invitro model system of human embryonic and extraembryonic development from pre-implantation to early gastrulation stages.

Efficient generation of ETX embryoids that recapitulate the entire window of murine egg cylinder development.

The murine embryonic-trophoblast-extra-embryonic endoderm (ETX) model is an integrated stem cell-based model to study early postimplantation development. It is based on the self-assembly potential of embryonic, trophoblast, and hypoblast/primitive/visceral endoderm-type stem cell lines (ESC, TSC, and XEN, respectively) to arrange into postimplantation egg cylinder-like embryoids. Here, we provide an optimized method for reliable and efficient generation of ETX embryoids that develop into late gastrulation in static culture conditions. It is based on transgenic Gata6-overproducing ESCs and modified assembly and culture conditions. Using this method, up to 43% of assembled ETX embryoids exhibited a correct spatial distribution of the three stem cell derivatives at day 4 of culture. Of those, 40% progressed into ETX embryoids that both transcriptionally and morphologically faithfully mimicked in vivo postimplantation mouse development between E5.5 and E7.5. The ETX model system offers the opportunity to study the murine postimplantation egg cylinder stages and could serve as a source of various cell lineage precursors.

Epiblast-like stem cells established by Wnt/β-catenin signaling manifest distinct features of formative pluripotency and germline competence

Different formative pluripotent stem cells harboring similar functional properties have been recently established to be lineage neutral and germline competent yet have distinct molecular identities. Here, we show that WNT/β-catenin signaling activation sustains transient mouse epiblast-like cells as epiblast-like stem cells (EpiLSCs). EpiLSCs display metastable formative pluripotency with bivalent cellular energy metabolism and unique transcriptomic features and chromatin accessibility. We develop single-cell stage label transfer (scSTALT) to study the formative pluripotency continuum and reveal that EpiLSCs recapitulate a unique developmental period invivo, filling the gap of the formative pluripotency continuum between other published formative stem cells. WNT/β-catenin signaling activation counteracts differentiation effects of activin A and bFGF by preventing complete dissolution of naive pluripotency regulatory network. Moreover, EpiLSCs have direct competence toward germline specification, which is further matured by an FGF receptor inhibitor. Our EpiLSCs can serve as an invitro model for mimicking and studying early post-implantation development and pluripotency transition.

Developmental dynamics of chromatin accessibility during post-implantation development of monkey embryos.

Early post-implantation development, especially gastrulation in primates, is accompanied by extensive drastic chromatin reorganization, which remains largely elusive. To delineate the global chromatin landscape and understand the molecular dynamics during this period, a single-cell assay for transposase accessible chromatin sequencing (scATAC-seq) was applied to in vitro cultured cynomolgus monkey (Macaca fascicularis, hereafter referred to as monkey) embryos to investigate the chromatin status. First, we delineated the cis-regulatory interactions and identified the regulatory networks and critical transcription factors involved in the epiblast (EPI), hypoblast, and trophectoderm/trophoblast (TE) lineage specification. Second, we observed that the chromatin opening of some genome regions preceded the gene expression during EPI and trophoblast specification. Third, we identified the opposing roles of FGF and BMP signaling in pluripotency regulation during EPI specification. Finally, we revealed the similarity between EPI and TE in gene expression profiles and demonstrated that PATZ1 and NR2F2 were involved in EPI and trophoblast specification during monkey post-implantation development. Our findings provide a useful resource and insights into dissecting the transcriptional regulatory machinery during primate post-implantation development.

Contemporary Notions On The Role Of 5'-Nucleotidase In Pregnancy

The review aimed at pooling together available information on 5'-nucleotidase – an enzyme hydrolyzing ribo- and deoxyribonucleoside-5'-monophosphates. The most important product of 5'-nucleotidase activity is adenosine. This enzyme is, in fact, involved in most aspects of normal physiology, along with numerous pathological processes. The article discusses the role of the enzyme in pregnancy, its involvement in early post-implantation development, proliferation, migration/invasion, trophoblast differentiation, decidualization, angiogenesis, vasculogenesis, modulation of cell growth during embryonic development, regulation of hemodynamics, and control of myometrial contractions. A large section of this review is dedicated to the contribution of 5'-nucleotidase to the development of gestational complications, such as preeclampsia. We conducted our study via searching through various databases until October 30, 2021, using the following keywords: 5'-nucleotidase, adenosine, pregnancy, and the combinations of those. All reviewed articles were published in English.

Nhlrc2 is crucial during mouse gastrulation.

SummaryThe loss of NHL repeat containing 2 (Nhlrc2) leads to early embryonic lethality in mice, but the exact timing is currently unknown. In this study, we determined the time of lethality for Nhlrc2 knockout (KO), C57BL/6NCrl‐Nhlrc2 tm1a(KOMP)Wtsi/Oulu, embryos and the in situ expression pattern of Nhlrc2 based on LacZ reporter gene expression during this period. Nhlrc2 KO preimplantation mouse embryos developed normally after in vitro fertilization. Embryonic stem (ES) cells established from KO blastocysts proliferated normally despite a complete loss of the NHLRC2 protein. Nhlrc2 KO embryos from timed matings implanted and were indistinguishable from their wildtype littermates on embryonic day (E) 6.5. On E7.5, Nhlrc2 KO embryo development was arrested, and on E8.5, only 6% of the genotyped embryos were homozygous for the Nhlrc2 tm1a(KOMP)Wtsi allele. Nhlrc2 KO E8.5 embryos showed limited embryonic or extraembryonic tissue differentiation and remained at the cylinder stage. Nhlrc2 expression was ubiquitous but strongest in the epiblast/ectoderm and extraembryonic ectoderm on E6.5 and E7.5. NHLRC2 is essential for early postimplantation development, and its loss leads to failed gastrulation and amniotic folding in mice. Future studies on the evolutionarily conserved NHLRC2 will provide new insights into the molecular pathways involved in the early steps of postimplantation development.

Human embryonic development: from peri-implantation to gastrulation.

The basic body plan of the mammalian embryo is established through gastrulation, a pivotal early postimplantation event during which the three major germ layers (endoderm, ectoderm, and mesoderm) are specified with cellular and spatial diversity. Despite its basic and clinical importance, human embryo development from peri-implantation to gastrulation remains shrouded in mystery. Recent advances in the elongated in vitro culture of rodent and non-primate embryos and the construction of embryo-like structures have helped to improve understanding of the mechanisms of human early embryonic development. Here, we review the recent advances and possible future directions in the development of in vitro models to better understand human embryogenesis from peri-implantation to gastrulation.

Embryotoxic effects of tribromophenol on early post-implantation development of mouse embryos in vitro

Embryotoxic effects of tribromophenol on early post-implantation development of mouse embryos in vitro

P–803 Novel culture conditions for the improvement of the in vitro expansion of human male fetal germ cells

Abstract Study question Do different ECMs/substrates and growth media culture conditions improve in vitro male human primordial germ cell (hPGC) expansion? Summary answer We achieved in vitro expansion improvement of male hPGCs with specific growth factors such as LIF, EGF, FGF2 and GDNF on gelatin- and vitronectin-coating cultures. What is known already PGCs are the precursors of male and female gametes, which are specified during early mammalian post-implantation embryonic development. PGCs undergo sequential cell divisions to differentiate into pro-spermatogonia (pSPG). In vitro propagation of pSPG could be important to understand the transition to spermatogonial stem cells (SSCs), important for fertility preservation in patients with infertility. Here, we aimed at performing a comparative analysis on in vitro feeder-free culture systems, based on different extracellular matrix (ECM) and growth media culture conditions, to support the expansion of the male germline stem cell populations using second trimester human male gonads as primary material. Study design, size, duration We collected human 2nd trimester male fetal gonads from elective abortions. Male gonads were dissected in saline solution (0.9% NaCl) and were either fixed overnight in 4% paraformaldehyde (PFA) for immunohistochemistry or disaggregated by enzymatic digestion for in vitro culture. Participants/materials, setting, methods After differential plating, fetal cells were cultured for 6 days. Disaggregated gonads were cultured with two different growing media (Medium 1 supplemented with LIF, EGF, FGF–2 and GDNF and Medium 2 supplemented with RA, BMP 4 and Activin A) on gelatin, laminin, vitronectin or matrigel coated plates. Cultured cells were immunostained, quantified for the expression of DDX4 and POU5F1 after 3 days (D3) and 6 days (D6) of culture. Main results and the role of chance We pursued to evaluate whether germ cells dissociated from a pool of male fetal gonads could propagate in vitro when cultured for D6 in different conditions. We observed that expansion of POU5F1-positive early PGCs and DDX4-positive late PGCs was only observed when cells were plated on gelatin or vitronectin and cultured with Medium 1, containing the growth factors LIF, EGF, FGF2 and GDNF. However, a reduced percentage of PGCs was observed in all four different coatings when grown with Medium 2, containing RA, BMP4 and Activin A. We analyzed the relative expression of the PGC markers POU5F1, DDX4 and MAGEA4 in histological sections of gonads from embryos at 18.5 weeks of gestation. Two populations of hPGCs were observed: ∼10–30% of the gonadal cells expressed solely DDX4 (late PGCs), whereas less than 10% of gonadal cells expressed POU5F1 (early PGCs). SOX9 and STARD1 expression was evaluated, confirming the presence of Sertoli cells and Leydig cells, respectively. Limitations, reasons for caution Due to the limited and difficulty to obtain human fetal tissue, a limited number of samples were used. Wider implications of the findings: We expanded human male fetal germ cells in vitro for D6 on gelatin and vitronectin coated plates with Medium 1, containing growth factors LIF, EGF, FGF2 and GDNF. Our findings provide a 2D culture system to expand hPGCs that could be useful to study propagation to pSPGs and eventually SSCs. Trial registration number Not applicable

Human embryo research, stem cell-derived embryo models and invitro gametogenesis: Considerations leading to the revised ISSCR guidelines.

SummaryThe ISSCR Guidelines for Stem Cell Research and Clinical Translation were last revised in 2016. Since then, rapid progress has been made in research areas related to in vitro culture of human embryos, creation of stem cell-based embryo models, and in vitro gametogenesis. Therefore, a working group of international experts was convened to review the oversight process and provide an update to the guidelines. This report captures the discussion and summarizes the major recommendations made by this working group, with a specific emphasis on updating the categories of review and engagement with the specialized scientific and ethical oversight process.

Machine learning-assisted high-content analysis of pluripotent stem cell-derived embryos in vitro

SummaryStem cell-based embryo models by cultured pluripotent and extra-embryonic lineage stem cells are novel platforms to model early postimplantation development. We showed that induced pluripotent stem cells (iPSCs) could form ITS (iPSCs and trophectoderm stem cells) and ITX (iPSCs, trophectoderm stem cells, and XEN cells) embryos, resembling the early gastrula embryo developed in vivo. To facilitate the efficient and unbiased analysis of the stem cell-based embryo model, we set up a machine learning workflow to extract multi-dimensional features and perform quantification of ITS embryos using 3D images collected from a high-content screening system. We found that different PSC lines differ in their ability to form embryo-like structures. Through high-content screening of small molecules and cytokines, we identified that BMP4 best promoted the morphogenesis of the ITS embryo. Our study established an innovative strategy to analyze stem cell-based embryo models and uncovered new roles of BMP4 in stem cell-based embryo models.

High levels estradiol affect blastocyst implantation and post-implantation development directly in mice

BackgroundPrevious studies have demonstrated that high levels of estradiol (E2) impair blastocyst implantation through effects on the endometrium; however, whether high E2 directly affects blastocysts is not well established. The present study sought to clarify the direct impacts of high E2 levels on blastocysts in vitro. MethodsICR virgin albino mice were used. Using an in-vitro 8-day blastocyst culture model, immunofluorescence staining for the estrogen receptor (ER), blastocyst outgrowth assays, differential staining and TUNEL assays of blastocysts, and embryo transfer, we investigated the main outcomes of exposure to different E2 concentrations (10−7 to 10−4 M) in vitro and in vivo. ResultsERα and ERβ expression were detected in pre-implantation stage embryos. In vitro exposure of blastocysts to 10−4 M E2 for 24 h followed by 7 days culture in the absence of E2 caused severe inhibition of implantation and post-implantation development. The late adverse effects of E2 on post-implantation development still occurred at concentrations of 10−7 to 10−5 M. In addition, blastocyst proliferation was reduced and apoptotic cells were increased following exposure to 10−4 M E2. Using an in vivo embryo-transfer model, we also showed that treatment with high E2 resulted in fewer implantation sites (38% vs. 72% in control) and greater resorption of implanted blastocysts (81% vs. 38% in control). ConclusionExposure to high E2 concentrations in vitro is deleterious to blastocyst implantation and early post-implantation development, mainly owing to direct impacts of E2 on implanting blastocysts. In clinical assisted reproductive technique (ART), high serum E2 concentrations not only affects the endometrium, but also affects blastocysts directly at the period of implantation.

Modeling human embryo development with embryonic and extra-embryonic stem cells

Early human post-implantation development involves extensive growth combined with a series of complex morphogenetic events. The lack of precise spatial and temporal control over these processes leads to pregnancy loss. Given the ethical and technical limitations in studying the natural human embryo, alternative approaches are needed to investigate mechanisms underlying this critical stage of human development. Here, we present an overview of the different stem cells and stem cell-derived models which serve as useful, albeit imperfect, tools in understanding human embryogenesis. Current models include stem cells that represent each of the three earliest lineages: human embryonic stem cells corresponding to the epiblast, hypoblast-like stem cells and trophoblast stem cells. We also review the use of human embryonic stem cells to model complex aspects of epiblast morphogenesis and differentiation. Additionally, we propose that the combination of both embryonic and extra-embryonic stem cells to form three-dimensional embryo models will provide valuable insights into cell-cell chemical and mechanical interactions that are essential for natural embryogenesis.

P7 La adición de ácidos grasos poliinsaturados en el período de postimplantación temprana previene reabsorciones embrionarias y anomalías deciduales en ratas gestantes con diabetes

Introducción: la diabetes pregestacional incrementa el riesgo de reabsorciones embrionarias y afecta el desarrollo embrionario, de impacto en la programación fetal. La función decidual es crucial en el desarrollo postimplantatorio temprano y sus posibles anomalías son poco conocidas en la diabetes materna. Previamente evidenciamos que la vía de señalización de mTOR en la decidua es relevante para el crecimiento y desarrollo embrionario, y que interactúa con la vía de los PPAR, cuyos agonistas son nutrientes de naturaleza lipídica como los ácidos grasos poliinsaturados (PUFAs).Objetivos: determinar el índice de reabsorciones y evaluar la regulación de la vía de mTOR y marcadores de la función, y remodelación de la decidua en ratas con diabetes pregestacional que recibieron o no dietas enriquecidas en PUFAs exclusivamente en el período de postimplantación temprana.Materiales y métodos: ratas sanas o con diabetes (inducidas preconcepcionalmente con estreptozotocina, 55 mg/kg) fueron apareadas con machos sanos. En los días 7,8 y 9 de preñez recibieron una dieta enriquecida o no en 6% aceite de girasol o de chía (enriquecidas en PUFAs n-6 o n-3, respectivamente). En el día 9 de preñez se estudió el índice de reabsorción embrionaria y se explantó la decidua para estudiar la vía de mTOR, la expresión de su regulador FoxO1 y del microRNA-21 (miR-21) que regula a FoxO1, como así también marcadores de función y remodelación decidual (expresión de LIF y IGFBP1 y actividad gelatinasa de metaloproteasas (MMPs).

In Vitro Modeling of the Early Development of Mouse and Human Embryos

Mammalian embryonic development includes preimplantation, implantation and post-implantation development, during which the embryo is attached to the uterus wall. The modeling of embryo development in culture allows to study the process of ontogenesis in its dynamic development, which is especially valuable for studying early post-implantation development. We reviewed existing cultivation systems for mouse and human embryos. Also we considered a new experimental approach for studying early mammalian embryogenesis—embryo-like cell constructs. Using these methods, the most important results were obtained about the formation of embryonic germ layers, which provided insights into the molecular and cellular mechanisms of morphogenesis of the mouse and human embryos. The purpose of this review is to generalize the possibilities and limitations of such systems for studying mammalian embryogenesis and to find directions for further development of this approach.

Modelling human embryogenesis: embryo-like structures spark ethical and policy debate.

Studying the human peri-implantation period remains hindered by the limited accessibility of the in vivo environment and scarcity of research material. As such, continuing efforts have been directed towards developing embryo-like structures (ELS) from pluripotent stem cells (PSCs) that recapitulate aspects of embryogenesis in vitro. While the creation of such models offers immense potential for studying fundamental processes in both pre- and early post-implantation development, it also proves ethically contentious due to wide-ranging views on the moral and legal reverence due to human embryos. Lack of clarity on how to qualify and regulate research with ELS thus presents a challenge in that it may either limit this new field of research without valid grounds or allow it to develop without policies that reflect justified ethical concerns. The aim of this article is to provide a comprehensive overview of the existing scientific approaches to generate ELS from mouse and human PSCs, as well as discuss future strategies towards innovation in the context of human development. Concurrently, we aim to set the agenda for the ethical and policy issues surrounding research on human ELS. The PubMed database was used to search peer-reviewed articles and reviews using the following terms: 'stem cells', 'pluripotency', 'implantation', 'preimplantation', 'post-implantation', 'blastocyst', 'embryoid bodies', 'synthetic embryos', 'embryo models', 'self-assembly', 'human embryo-like structures', 'artificial embryos' in combination with other keywords related to the subject area. The PubMed and Web of Science databases were also used to systematically search publications on the ethics of ELS and human embryo research by using the aforementioned keywords in combination with 'ethics', 'law', 'regulation' and equivalent terms. All relevant publications until December 2019 were critically evaluated and discussed. In vitro systems provide a promising way forward for uncovering early human development. Current platforms utilize PSCs in both two- and three-dimensional settings to mimic various early developmental stages, including epiblast, trophoblast and amniotic cavity formation, in addition to axis development and gastrulation. Nevertheless, much hinges on the term 'embryo-like'. Extension of traditional embryo frameworks to research with ELS reveals that (i) current embryo definitions require reconsideration, (ii) cellular convertibility challenges the attribution of moral standing on the basis of 'active potentiality' and (iii) meaningful application of embryo protective directives will require rethinking of the 14-day culture limit and moral weight attributed to (non-)viability. Many conceptual and normative (dis)similarities between ELS and embryos thus remain to be thoroughly elucidated. Modelling embryogenesis holds vast potential for both human developmental biology and understanding various etiologies associated with infertility. To date, ELS have been shown to recapitulate several aspects of peri-implantation development, but critically, cannot develop into a fetus. Yet, concurrent to scientific innovation, considering the extent to which the use of ELS may raise moral concerns typical of human embryo research remains paramount. This will be crucial for harnessing the potential of ELS as a valuable research tool, whilst remaining within a robust moral and legal framework of professionally acceptable practices.

Dissecting primate early post-implantation development using long-term in vitro embryo culture.

The transition from peri-implantation to gastrulation in mammals entails the specification and organization of the lineage progenitors into a body plan. Technical and ethical challenges have limited understanding of the cellular and molecular mechanisms that underlie this transition. We established a culture system that enabled the development of cynomolgus monkey embryos in vitro for up to 20 days. Cultured embryos underwent key primate developmental stages, including lineage segregation, bilaminar disc formation, amniotic and yolk sac cavitation, and primordial germ cell-like cell (PGCLC) differentiation. Single-cell RNA-sequencing analysis revealed development trajectories of primitive endoderm, trophectoderm, epiblast lineages, and PGCLCs. Analysis of single-cell chromatin accessibility identified transcription factors specifying each cell type. Our results reveal critical developmental events and complex molecular mechanisms underlying nonhuman primate embryogenesis in the early postimplantation period, with possible relevance to human development.

Generation of Blastocyst-like Structures from Mouse Embryonic and Adult Cell Cultures

A single mouse blastomere from an embryo until the 8-cell stage can generate an entire blastocyst. Whether laboratory-cultured cells retain a similar generative capacity remains unknown. Starting from a single stem cell type, extended pluripotent stem (EPS) cells, we established a 3D differentiation system that enabled the generation of blastocyst-like structures (EPS-blastoids) through lineage segregation and self-organization. EPS-blastoids resembled blastocysts in morphology and cell-lineage allocation and recapitulated key morphogenetic events during preimplantation and early postimplantation development invitro. Upon transfer, some EPS-blastoids underwent implantation, induced decidualization, and generated live, albeit disorganized, tissues in utero. Single-cell and bulk RNA-sequencing analysis revealed that EPS-blastoids contained all three blastocyst cell lineages and shared transcriptional similarity with natural blastocysts. We also provide proof of concept that EPS-blastoids can be generated from adult cells via cellular reprogramming. EPS-blastoids provide a unique platform for studying early embryogenesis and pave the way to creating viable synthetic embryos by using cultured cells.

Single-cell transcriptional profiling: a window into embryonic cell-type specification.

During mammalian embryonic development, a single fertilized egg cell will proliferate and differentiate into all the cell lineages and cell types that eventually form the adult organism. Cell lineage diversification involves repeated cell fate choices that ultimately occur at the level of the individual cell rather than at the cell-population level. Improvements in single-cell technologies are transforming our understanding of mammalian development, not only by overcoming the limitations presented by the extremely low cell numbers of early embryos but also by enabling the study of cell fate specification in greater detail. In this Review, we first discuss recent advances in single-cell transcriptomics and imaging and provide a brief outline of current bioinformatics methods available to analyse the resulting data. We then discuss how these techniques have contributed to our understanding of pre-implantation and early post-implantation development and of in vitro pluripotency. Finally, we overview the current challenges facing single-cell research and highlight the latest advances and potential future avenues.