Search Results (12)

The mammalian early embryo possesses totipotency and can be captured as extended pluripotent stem cells (EPSCs). The first two cell differentiations result in epiblast, primitive endoderm, and trophectoderm, with the trilineage coexisting in a unified uterine microenvironment. Nevertheless, the in vitro counterparts—primed PSCs, trophoblast stem cells (TSCs), and extraembryonic endoderm (XEN) cells—require a distinct culture system. In this study, we successfully derived stable porcine EPSCs from fibroblasts at 35% efficiency, and interestingly observed that these EPSCs differentiated in parallel and gave rise to the transient coexistence (36–84 h) of trilineage cells when cultured in a single system (LCDM: hLIF, CHIR99021, DiM, and MiH). Then, XEN cells gradually predominated and eventually became the sole population in prolonged LCDM culture. However, TSCs and primed PSCs had to be differentiated from EPSCs under their respective culture system. EPSCs can differentiate into PGC-like cells independently of genetic modification and contribute to mouse neurula-stage embryos. Collectively, the trilineage coexistence phenomenon may provide novel insight into an early embryogenesis mechanism and strategy for porcine blastoid construction. Full article

Species have different strategies for implantation and placentation. Much can be learned about general molecular and cellular biology through the examination and comparison of these differences. To varying degrees, implantation in all species includes alterations in epithelial polarity, the transformation of the endometrial stroma, the differentiation of the trophoblast, cell-to-cell and tissue-to-tissue signaling through hormones, cytokines, and extracellular vesicles, and the alteration of the maternal immune system. This review focuses on implantation in pigs, sheep, and cows. These species share with mice/rats and humans/primates the key events of early embryonic development, pregnancy recognition, and the establishment of functional placentation. However, there are differences between the pregnancies of livestock and other species that make livestock unique biomedical models for the study of pregnancy and cell biology in general. Pig, sheep, and cow conceptuses (embryo/fetus and associated placental membranes) elongate prior to implantation, displaying central implantation, extended periods of conceptus attachment to the uterus, and epitheliochorial (pigs) and synepitheliochorial (sheep and cows) placentation. This review will discuss what is understood about how the trophoblast and extraembryonic endoderm of pig, sheep, and cow conceptuses elongate, and how a major goal of current in vitro models is to achieve conceptus elongation. It will then examine the adhesion cascade for conceptus implantation that initiates early placental development in pigs, sheep, and cows. Finally, it will conclude with a brief overview of early placental development in pigs, sheep, and cows, with a listing of some important “omics” studies that have been published. Full article

Embryonic stem cells (ESCs) favor glycolysis over oxidative phosphorylation for energy production, and glycolytic metabolism is critical for pluripotency establishment, maintenance, and exit. However, an understanding of how glycolysis regulates the self-renewal and differentiation of ESCs remains elusive. Here, we demonstrated that protein lactylation, regulated by intracellular lactate, contributes to the self-renewal of ESCs. We further showed that Esrrb, an orphan nuclear receptor involved in pluripotency maintenance and extraembryonic endoderm stem cell (XEN) differentiation, is lactylated on K228 and K232. The lactylation of Esrrb enhances its activity in promoting ESC self-renewal in the absence of the LIF and XEN differentiation of ESCs by increasing its binding at target genes. Our studies reveal the importance of protein lactylation in the self-renewal and XEN differentiation of ESCs, and the underlying mechanism of glycolytic metabolism regulating cell fate choice. Full article

During early mammalian embryonic development, fertilized one-cell embryos develop into pre-implantation blastocysts and subsequently establish three germ layers through gastrulation during post-implantation development. In recent years, stem cells have emerged as a powerful tool to study embryogenesis and gastrulation without the need for eggs, allowing for the generation of embryo-like structures known as synthetic embryos or embryoids. These in vitro models closely resemble early embryos in terms of morphology and gene expression and provide a faithful recapitulation of early pre- and post-implantation embryonic development. Synthetic embryos can be generated through a combinatorial culture of three blastocyst-derived stem cell types, such as embryonic stem cells, trophoblast stem cells, and extraembryonic endoderm cells, or totipotent-like stem cells alone. This review provides an overview of the progress and various approaches in studying in vitro embryogenesis and gastrulation in mice and humans using stem cells. Furthermore, recent findings and breakthroughs in synthetic embryos and gastruloids are outlined. Despite ethical considerations, synthetic embryo models hold promise for understanding mammalian (including humans) embryonic development and have potential implications for regenerative medicine and developmental research. Full article

Normal developmental progression relies on close interactions between the embryonic and extraembryonic lineages in the pre- and peri-gastrulation stage conceptus. For example, mouse epiblast-derived FGF and NODAL signals are required to maintain a stem-like state in trophoblast cells of the extraembryonic ectoderm, while visceral endoderm signals are pivotal to pattern the anterior region of the epiblast. These developmental stages also coincide with the specification of the first heart precursors. Here, we established a robust differentiation protocol of mouse embryonic stem cells (ESCs) into cardiomyocyte-containing embryoid bodies that we used to test the impact of trophoblast on this key developmental process. Using trophoblast stem cells (TSCs) to produce trophoblast-conditioned medium (TCM), we show that TCM profoundly slows down the cardiomyocyte differentiation dynamics and specifically delays the emergence of cardiac mesoderm progenitors. TCM also strongly promotes the retention of pluripotency transcription factors, thereby sustaining the stem cell state of ESCs. By applying TCM from various mutant TSCs, we further show that those mutations that cause a trophoblast-mediated effect on early heart development in vivo alter the normal cardiomyocyte differentiation trajectory. Our approaches provide a meaningful deconstruction of the intricate crosstalk between the embryonic and the extraembryonic compartments. They demonstrate that trophoblast helps prolong a pluripotent state in embryonic cells and delays early differentiative processes, likely through production of leukemia inhibitory factor (LIF). These data expand our knowledge of the multifaceted signaling interactions among distinct compartments of the early conceptus that ensure normal embryogenesis, insights that will be of significance for the field of synthetic embryo research. Full article

In vitro modeling of human peri-gastrulation development is a valuable tool for understanding embryogenetic mechanisms. The extraembryonic mesoderm (ExM) is crucial in supporting embryonic development by forming tissues such as the yolk sac, allantois, and chorionic villi. However, the origin of human ExM remains only partially understood. While evidence suggests a primitive endoderm (PrE) origin based on morphological findings, current in vitro models use epiblast-like cells. To address this gap, we developed a protocol to generate ExM-like cells from PrE-like cell line called naïve extraembryonic endoderm (nEnd). We identified the ExM-like cells by specific markers (LUM and ANXA1). Moreover, these in vitro-produced ExM cells displayed angiogenic potential on a soft matrix, mirroring their physiological role in vasculogenesis. By integrating single-cell RNA sequencing (scRNAseq) data, we found that the ExM-like cells clustered with the LUM/ANXA1-rich cell populations of the gastrulating embryo, indicating similarity between in vitro and ex utero cell populations. This study confirms the derivation of ExM from PrE and establishes a cell culture system that can be utilized to investigate ExM during human peri-gastrulation development, both in monolayer cultures and more complex models. Full article

Progesterone treatment is commonly employed to promote and support pregnancy. While maternal tissues are the main progesterone targets in humans and mice, its receptor (PGR) is expressed in the murine embryo, questioning its function during embryonic development. Progesterone has been previously associated with murine blastocyst development. Whether it contributes to lineage specification is largely unknown. Gastrulation initiates lineage specification and generation of the progenitors contributing to all organs. Cells passing through the primitive streak (PS) will give rise to the mesoderm and endoderm. Cells emerging posteriorly will form the extraembryonic mesodermal tissues supporting embryonic growth. Cells arising anteriorly will contribute to the embryonic heart in two sets of distinct progenitors, first (FHF) and second heart field (SHF). We found that PGR is expressed in a posterior–anterior gradient in the PS of gastrulating embryos. We established in vitro differentiation systems inducing posterior (extraembryonic) and anterior (cardiac) mesoderm to unravel PGR function. We discovered that PGR specifically modulates extraembryonic and cardiac mesoderm. Overexpression experiments revealed that PGR safeguards cardiac differentiation, blocking premature SHF progenitor specification and sustaining the FHF progenitor pool. This role of PGR in heart development indicates that progesterone administration should be closely monitored in potential early-pregnancy patients undergoing infertility treatment. Full article

The regulation of proteins through the addition and removal of O-linked β-N-acetylglucosamine (O-GlcNAc) plays a role in many signaling events, specifically in stem cell pluripotency and the regulation of differentiation. However, these post-translational modifications have not been explored in extraembryonic endoderm (XEN) differentiation. Of the plethora of proteins regulated through O-GlcNAc, we explored galectin-3 as a candidate protein known to have various intracellular and extracellular functions. Based on other studies, we predicted a reduction in global O-GlcNAcylation levels and a distinct galectin expression profile in XEN cells relative to embryonic stem (ES) cells. By conducting dot blot analysis, XEN cells had decreased levels of global O-GlcNAc than ES cells, which reflected a disbalance in the expression of genes encoding O-GlcNAc cycle enzymes. Immunoassays (Western blot and ELISA) revealed that although XEN cells (low O-GlcNAc) had lower concentrations of both intracellular and extracellular galectin-3 than ES cells (high O-GlcNAc), the relative secretion of galectin-3 was significantly increased by XEN cells. Inducing ES cells toward XEN in the presence of an O-GlcNAcase inhibitor was not sufficient to inhibit XEN differentiation. However, global O-GlcNAcylation was found to decrease in differentiated cells and the extracellular localization of galectin-3 accompanies these changes. Inhibiting global O-GlcNAcylation status does not, however, impact pluripotency and the ability of ES cells to differentiate to the XEN lineage. Full article

The inner cell mass of the pre-implantation blastocyst consists of the epiblast and hypoblast from which embryonic stem cells (ESCs) and extra-embryonic endoderm (XEN) stem cells, respectively, can be derived. Importantly, each stem cell type retains the defining properties and lineage restriction of its in vivo tissue origin. We have developed a novel approach for deriving porcine XEN (pXEN) cells via culturing the blastocysts with a chemical cocktail culture system. The pXEN cells were positive for XEN markers, including Gata4, Gata6, Sox17, and Sall4, but not for pluripotent markers Oct4, Sox2, and Nanog. The pXEN cells also retained the ability to undergo visceral endoderm (VE) and parietal endoderm (PE) differentiation in vitro. The maintenance of pXEN required FGF/MEK+TGFβ signaling pathways. The pXEN cells showed a stable phenotype through more than 50 passages in culture and could be established repeatedly from blastocysts or converted from the naïve-like ESCs established in our lab. These cells provide a new tool for exploring the pathways of porcine embryo development and differentiation and providing further reference to the establishment of porcine ESCs with potency of germline chimerism and gamete development. Full article

Background and Objectives: Medullary thyroid carcinoma (MTC) accounts for 1–2% of all thyroid malignancies, and it originates from parafollicular “C” cells. Carcinoembryonic antigen (CEA) is a tumor marker, mainly for gastrointestinal malignancies. There are references in literature where elevated CEA levels may be the first finding in MTC. The aim of this study is to determine the importance of measuring preoperative and postoperative CEA values in patients with MTC and to define the clinical significance of the correlation between CEA and the origin of C cells. Materials and Methods: The existing and relevant literature was reviewed by searching for articles and specific keywords in the scientific databases of PubMedCentraland Google Scholar (till December 2020). Results: CEA has found its place, especially at the preoperative level, in the diagnostic approach of MTC. Preoperative CEA values >30 ng/mL indicate extra-thyroid disease, while CEA values >100 ng/mL are associated with lymph node involvement and distant metastases. The increase in CEA values preoperatively is associated with larger size of primary tumor, presence of lymph nodes, distant metastases and a poorer prognosis. The clinical significance of CEA values for the surgeon is the optimal planning of surgical treatment. In the recent literature, C cells seem to originate from the endoderm of the primitive anterior gut at the ultimobranchial bodies’ level. Conclusions: Although CEA is not a specific biomarker of the disease in MTC, itsmeasurement is useful in assessing the progression of the disease. The embryonic origin of C cells could explain the increased CEA values in MTC. Full article

Early embryonic development in mammals, from fertilization to implantation, can be viewed as a process in which stem cells alternate between self-renewal and differentiation. During this process, the fates of stem cells in embryos are gradually specified, from the totipotent state, through the segregation of embryonic and extraembryonic lineages, to the molecular and cellular defined progenitors. Most of those stem cells with different potencies in vivo can be propagated in vitro and recapitulate their differentiation abilities. Complex and coordinated regulations, such as epigenetic reprogramming, maternal RNA clearance, transcriptional and translational landscape changes, as well as the signal transduction, are required for the proper development of early embryos. Accumulated studies suggest that Dicer-dependent noncoding RNAs, including microRNAs (miRNAs) and endogenous small-interfering RNAs (endo-siRNAs), are involved in those regulations and therefore modulate biological properties of stem cells in vitro and in vivo. Elucidating roles of these noncoding RNAs will give us a more comprehensive picture of mammalian embryonic development and enable us to modulate stem cell potencies. In this review, we will discuss roles of miRNAs in regulating the maintenance and cell fate potential of stem cells in/from mouse and human early embryos. Full article

Retinoic acid (RA) is an important signaling molecule in the development of the endoderm and an important molecule in protocols used to generate endodermal cell types from stem cells. In this review, we describe the RA signaling pathway and its role in the patterning and specification of the extra embryonic endoderm and different endodermal organs. The formation of endoderm is an ancient evolutionary feature and RA signaling appears to have coevolved with the vertebrate lineage. Towards that end, we describe how RA participates in many regulatory networks required for the formation of extraembryonic structures as well as the organs of the embryo proper. Full article