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Cytometry. Part a : the Journal of the... Aug 2022Mouse embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) are both pluripotent stem cells from early embryos. Another type of pluripotent stem cells, which are...
Mouse embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) are both pluripotent stem cells from early embryos. Another type of pluripotent stem cells, which are similar with EpiSCs and derive from pre-implantation embryos in feeder-free and chemically defined medium containing Activin A and basic fibroblast growth factors (bFGF), is termed as AFSCs. The pluripotency and self-renewal maintenance of ESCs rely on Leukemia inhibitory factor (LIF)/STAT/BMP4/SMAD signaling, while the pluripotency and self-renewal maintenance of EpiSCs and AFSCs rely on bFGF and Activin/Nodal signaling. However, the establishment efficiency of AFSCs lines is low. In this study, we stimulated early embryos by 2i/LIF (CHIR99021 + PD0325901 + LIF) and Activin A + bFGF respectively, to change the cell fate in inner cell mass (ICM). The "fate changed embryos" by 2i/LIF can efficiently produce AFSCs in feeder-free and chemically defined medium, but the efficiency of embryos treated with Activin A + bFGF were poor. The AFSCs from fate-changed embryos share similar molecular characteristics with conventional AFSCs and EpiSCs. Our results suggest that the advanced stimulation of 2i/LIF and the premature stimulation of Activin A + bFGF contribute to capturing the pluripotent stem cells in early embryos, and the FGF/MAPK signaling dominate early embryo development. Our study provides a new approach to capturing pluripotency from pre-implantation embryos.
Topics: Animals; Cell Differentiation; Embryonic Stem Cells; Germ Layers; Mice; Pluripotent Stem Cells; Signal Transduction
PubMed: 35332996
DOI: 10.1002/cyto.a.24551 -
Cold Spring Harbor Protocols Nov 2022Mesendoderm mantle closure completes the gastrulation movements of the embryo and provides an unparalleled opportunity to study collective cell behaviors within a...
Mesendoderm mantle closure completes the gastrulation movements of the embryo and provides an unparalleled opportunity to study collective cell behaviors within a mesenchymal tissue. Free-edge sheet-like collective movements of these tissues contrast with movements of epithelial tissues in that mesendodermal cells are not constrained by tight junctions or adherens junctions, yet migrate in a coherent and persistent mode over several hours. Mesendoderm cells are the largest motile cells in the embryo and complete a 500-µm migratory path. When mesendoderm is cultured on rigid glass substrates, these cells can exceed 100 µm in length and show a highly persistent leading lamellipodia that can exceed 20 µm from tip to base. These large collectively migrating cells provide a unique imaging opportunity to visualize polarized adhesive and cytoskeletal structures with high-numerical-aperture objectives. Mesendodermal cells in the early embryo originate from around the entirety of the marginal zone and may also be distinguished by their source along the animal-vegetal axis. Here we use the term mesendoderm but note alternative terms for these cells can include head mesoderm, endomesoderm, and prechordal mesoderm. This protocol summarizes microsurgical preparation of mesendoderm tissue explants and "windowed" embryos. Skills needed to dissect fragments of the mesendoderm mantle are marginally greater than those needed to isolate animal cap ectoderm and can be mastered within 2 weeks; skills needed to isolate the mesendoderm "donut" or "ring" or to prepare windowed embryos are significantly greater and may require several additional weeks of training.
Topics: Animals; Gastrulation; Xenopus laevis; Mesoderm; Ectoderm; Pseudopodia
PubMed: 35577524
DOI: 10.1101/pdb.prot097378 -
The Keio Journal of Medicine Sep 2022Pluripotent stem cells (PSCs), which include embryonic stem cells and induced pluripotent stem cells, have the potential for unlimited self-renewal and proliferation and... (Review)
Review
Pluripotent stem cells (PSCs), which include embryonic stem cells and induced pluripotent stem cells, have the potential for unlimited self-renewal and proliferation and the ability to differentiate into all three embryonic germ layers. Human PSCs (hPSCs) are used in drug discovery screening, disease models, and regenerative medicine. These cells maintain a transcriptional regulatory network based on a set of unique transcription factors to maintain their stem cell properties. Downstream of such transcriptional regulatory networks, various stem cell-specific metabolic programs are used to produce energy and metabolites as necessary. hPSCs and differentiated cells utilize different metabolic programs for self-renewal ability and maintenance of quiescence. Understanding the different metabolic features of hPSCs and differentiated cells can contribute to the development of technologies that are useful for regenerative medicine, such as the purification of differentiated cells. This review describes the unique metabolic programs active in hPSCs and their differences from somatic cells, with a focus on cardiomyocytes.
Topics: Cell Differentiation; Germ Layers; Humans; Induced Pluripotent Stem Cells; Myocytes, Cardiac; Pluripotent Stem Cells; Transcription Factors
PubMed: 35082186
DOI: 10.2302/kjm.2021-0015-IR -
Nature Communications Jul 2023The molecular mechanisms that coordinate patterning of the embryonic ectoderm into spatially distinct lineages to form the nervous system, epidermis, and neural...
The molecular mechanisms that coordinate patterning of the embryonic ectoderm into spatially distinct lineages to form the nervous system, epidermis, and neural crest-derived craniofacial structures are unclear. Here, biochemical disease-variant profiling reveals a posttranslational pathway that drives early ectodermal differentiation in the vertebrate head. The anteriorly expressed ubiquitin ligase CRL3-KLHL4 restricts signaling of the ubiquitous cytoskeletal regulator CDC42. This regulation relies on the CDC42-activating complex GIT1-βPIX, which CRL3-KLHL4 exploits as a substrate-specific co-adaptor to recognize and monoubiquitylate PAK1. Surprisingly, we find that ubiquitylation converts the canonical CDC42 effector PAK1 into a CDC42 inhibitor. Loss of CRL3-KLHL4 or a disease-associated KLHL4 variant reduce PAK1 ubiquitylation causing overactivation of CDC42 signaling and defective ectodermal patterning and neurulation. Thus, tissue-specific restriction of CDC42 signaling by a ubiquitin-based effector-to-inhibitor is essential for early face, brain, and skin formation, revealing how cell-fate and morphometric changes are coordinated to ensure faithful organ development.
Topics: Brain; Ectoderm; Neural Crest; Signal Transduction; Ubiquitin
PubMed: 37495603
DOI: 10.1038/s41467-023-40223-y -
Aging Cell Oct 2023Recent research revealed a rejuvenation event during early development of mice. Here, by examining epigenetic age dynamics of human embryogenesis, we tested whether a...
Recent research revealed a rejuvenation event during early development of mice. Here, by examining epigenetic age dynamics of human embryogenesis, we tested whether a similar event exists in humans. For this purpose, we developed an epigenetic clock method, the intersection clock, that utilizes bisulfite sequencing in a way that maximizes the use of informative CpG sites with no missing clock CpG sites in test samples and applied it to human embryo development data. We observed no changes in the predicted epigenetic age between cleavage stage and blastocyst stage embryos; however, a significant decrease was observed between blastocysts and cells representing the epiblast. Additionally, by applying the intersection clock to datasets spanning pre and postimplantation, we found no significant change in the epigenetic age during preimplantation stages; however, the epigenetic age of postimplantation samples was lower compared to the preimplantation stages. We further investigated the epigenetic age of primed (representing early postimplantation) and naïve (representing preimplantation) pluripotent stem cells and observed that in all cases the epigenetic age of primed cells was significantly lower than that of naïve cells. Together, our data suggest that human embryos are rejuvenated during early embryogenesis. Hence, the rejuvenation event is conserved between the mouse and human, and it occurs around the gastrulation stage in both species. Beyond this advance, the intersection clock opens the way for other epigenetic age studies based on human bisulfite sequencing datasets as opposed to methylation arrays.
Topics: Humans; Animals; Mice; Rejuvenation; Blastocyst; Germ Layers; Embryonic Development; DNA Methylation
PubMed: 37786333
DOI: 10.1111/acel.13922 -
BMC Molecular and Cell Biology May 2020Members of the T-box family of DNA-binding proteins play a prominent role in the differentiation of the three primary germ layers. VegT, Brachyury, and Eomesodermin...
BACKGROUND
Members of the T-box family of DNA-binding proteins play a prominent role in the differentiation of the three primary germ layers. VegT, Brachyury, and Eomesodermin function as transcriptional activators and, in addition to directly activating the transcription of endoderm- and mesoderm-specific genes, serve as regulators of growth factor signaling during induction of these germ layers. In contrast, the T-box gene, tbx2, is expressed in the embryonic ectoderm, where Tbx2 functions as a transcriptional repressor and inhibits mesendodermal differentiation by the TGFβ ligand Activin. Tbx2 misexpression also promotes dorsal ectodermal fate via inhibition of the BMP branch of the TGFβ signaling network.
RESULTS
Here, we report a physical association between Tbx2 and both Smad1 and Smad2, mediators of BMP and Activin/Nodal signaling, respectively. We perform structure/function analysis of Tbx2 to elucidate the roles of both Tbx2-Smad interaction and Tbx2 DNA-binding in germ layer suppression.
CONCLUSION
Our studies demonstrate that Tbx2 associates with intracellular mediators of the Activin/Nodal and BMP/GDF pathways. We identify a novel repressor domain within Tbx2, and have determined that Tbx2 DNA-binding activity is required for repression of TGFβ signaling. Finally, our data also point to overlapping yet distinct mechanisms for Tbx2-mediated repression of Activin/Nodal and BMP/GDF signaling.
Topics: Activins; Animals; Body Patterning; Bone Morphogenetic Proteins; Ectoderm; Gene Expression Regulation, Developmental; Germ Layers; Growth Differentiation Factors; Phosphorylation; Protein Domains; Signal Transduction; Smad1 Protein; Smad2 Protein; T-Box Domain Proteins; Transforming Growth Factor beta; Xenopus Proteins; Xenopus laevis
PubMed: 32466750
DOI: 10.1186/s12860-020-00282-1 -
Current Topics in Developmental Biology 2022Ectodermal organs originate from the outermost germ layer of the developing embryo and include the skin, hair, tooth, nails, and exocrine glands. These organs develop...
Ectodermal organs originate from the outermost germ layer of the developing embryo and include the skin, hair, tooth, nails, and exocrine glands. These organs develop through tightly regulated, sequential and reciprocal epithelial-mesenchymal crosstalk, and they eventually assume various morphologies and functions while retaining the ability to regenerate. As with many other tissues in the body, the development and morphogenesis of these organs are regulated by a set of common signaling pathways, such as Shh, Wnt, Bmp, Notch, Tgf-β, and Eda. However, subtle differences in the temporal activation, the multiple possible combinations of ligand-receptor activation, the various cofactors, as well as the underlying epigenetic modulation determine how each organ develops into its adult form. Although each organ has been studied separately in considerable detail, the mechanisms underlying the parallels and differences in signaling that regulate their development have rarely been investigated. First, we will use the tooth, the hair follicle, and the mammary gland as representative ectodermal organs to explore how the development of signaling centers and establishment of stem cell populations influence overall growth and morphogenesis. Then we will compare how some of the major signaling pathways (Shh, Wnt, Notch and Yap/Taz) differentially regulate developmental events. Finally, we will discuss how signaling regulates regenerative processes in all three.
Topics: Ectoderm; Hair Follicle; Morphogenesis; Signal Transduction; Transforming Growth Factor beta
PubMed: 35606061
DOI: 10.1016/bs.ctdb.2022.02.006 -
Seminars in Cell & Developmental Biology Mar 2023Of all the cell types arising from the neural crest, ectomesenchyme is likely the most unusual. In contrast to the neuroglial cells generated by neural crest throughout... (Review)
Review
Of all the cell types arising from the neural crest, ectomesenchyme is likely the most unusual. In contrast to the neuroglial cells generated by neural crest throughout the embryo, consistent with its ectodermal origin, cranial neural crest-derived cells (CNCCs) generate many connective tissue and skeletal cell types in common with mesoderm. Whether this ectoderm-derived mesenchyme (ectomesenchyme) potential reflects a distinct developmental origin from other CNCC lineages, and/or epigenetic reprogramming of the ectoderm, remains debated. Whereas decades of lineage tracing studies have defined the potential of CNCC ectomesenchyme, these are being revisited by modern genetic techniques. Recent work is also shedding light on the extent to which intrinsic and extrinsic cues determine ectomesenchyme potential, and whether maintenance or reacquisition of CNCC multipotency influences craniofacial repair.
Topics: Neural Crest; Mesoderm; Ectoderm; Embryo, Mammalian
PubMed: 35331627
DOI: 10.1016/j.semcdb.2022.03.018 -
Lab on a Chip Nov 2021Human induced pluripotent stem cells (hiPSCs) can serve as an unlimited source to rebuild organotypic tissues . Successful engineering of functional cell types and...
Human induced pluripotent stem cells (hiPSCs) can serve as an unlimited source to rebuild organotypic tissues . Successful engineering of functional cell types and complex organ structures outside the human body requires knowledge of the chemical, temporal, and spatial microenvironment of their counterparts. Despite an increased understanding of mouse and human embryonic development, screening approaches are still required for the optimization of stem cell differentiation protocols to gain more functional mature cell types. The liver, lung, pancreas, and digestive tract originate from the endoderm germ layer. Optimization and specification of the earliest differentiation step, which is the definitive endoderm (DE), is of central importance for generating cell types of these organs because off-target cell types will propagate during month-long cultivation steps and reduce yields. Here, we developed a microfluidic large-scale integration (mLSI) chip platform for combined automated three-dimensional (3D) cell culturing and high-throughput imaging to investigate anterior/posterior patterns occurring during hiPSC differentiation into DE cells. Integration of 3D cell cultures with a diameter of 150 μm was achieved using a U-shaped pneumatic membrane valve, which was geometrically optimized and fluidically characterized. Upon parallelization of 32 fluidically individually addressable cell culture unit cells with a total of 128 3D cell cultures, complex and long-term DE differentiation protocols could be automated. Real-time bright-field imaging was used to analyze cell growth during DE differentiation, and immunofluorescence imaging on optically cleared 3D cell cultures was used to determine the DE differentiation yield. By systematically alternating transforming growth factor β (TGF-β) and WNT signaling agonist concentrations and temporal stimulation, we showed that even under similar DE differentiation yields, there were patterning differences in the 3D cell cultures, indicating possible differentiation differences between established DE protocols. The automated mLSI chip platform with the general analytical workflow for 3D stem cell cultures offers the optimization of generation of various cell types for cell replacement therapies.
Topics: Cell Culture Techniques; Cell Differentiation; Endoderm; Humans; Induced Pluripotent Stem Cells
PubMed: 34751293
DOI: 10.1039/d1lc00565k -
Philosophical Transactions of the Royal... Dec 2022The first few days of embryonic development in eutherian mammals are dedicated to the specification and elaboration of the extraembryonic tissues. However, where the... (Review)
Review
The first few days of embryonic development in eutherian mammals are dedicated to the specification and elaboration of the extraembryonic tissues. However, where the fetus ends and its adnexa begins is not always as self-evident during the early stages of development, when the definitive body axes are still being laid down, the germ layers being specified and a discrete form or bodyplan is yet to emerge. Function, anatomy, histomorphology and molecular identities have been used through the history of embryology, to make this distinction. In this review, we explore them individually by using specific examples from the early embryo. While highlighting the challenges of drawing discrete boundaries between embryonic and extraembryonic tissues and the limitations of a binary categorization, we discuss how basing such identity on fate is the most universal and conceptually consistent. This article is part of the theme issue 'Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom'.
Topics: Animals; Embryo, Mammalian; Embryonic Development; Germ Layers; Mammals; Models, Biological
PubMed: 36252217
DOI: 10.1098/rstb.2021.0255