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International Journal of Molecular... Aug 2022The integration of cell metabolism with signalling pathways, transcription factor networks and epigenetic mediators is critical in coordinating molecular and cellular...
H NMR Metabolite Monitoring during the Differentiation of Human Induced Pluripotent Stem Cells Provides New Insights into the Molecular Events That Regulate Embryonic Chondrogenesis.
The integration of cell metabolism with signalling pathways, transcription factor networks and epigenetic mediators is critical in coordinating molecular and cellular events during embryogenesis. Induced pluripotent stem cells (IPSCs) are an established model for embryogenesis, germ layer specification and cell lineage differentiation, advancing the study of human embryonic development and the translation of innovations in drug discovery, disease modelling and cell-based therapies. The metabolic regulation of IPSC pluripotency is mediated by balancing glycolysis and oxidative phosphorylation, but there is a paucity of data regarding the influence of individual metabolite changes during cell lineage differentiation. We used H NMR metabolite fingerprinting and footprinting to monitor metabolite levels as IPSCs are directed in a three-stage protocol through primitive streak/mesendoderm, mesoderm and chondrogenic populations. Metabolite changes were associated with central metabolism, with aerobic glycolysis predominant in IPSC, elevated oxidative phosphorylation during differentiation and fatty acid oxidation and ketone body use in chondrogenic cells. Metabolites were also implicated in the epigenetic regulation of pluripotency, cell signalling and biosynthetic pathways. Our results show that H NMR metabolomics is an effective tool for monitoring metabolite changes during the differentiation of pluripotent cells with implications on optimising media and environmental parameters for the study of embryogenesis and translational applications.
Topics: Cell Differentiation; Chondrogenesis; Epigenesis, Genetic; Humans; Induced Pluripotent Stem Cells; Proton Magnetic Resonance Spectroscopy
PubMed: 36012540
DOI: 10.3390/ijms23169266 -
Development (Cambridge, England) Sep 2022Despite the growing interest in the rabbit model for developmental and stem cell biology, the characterization of embryos at the molecular level is still poorly...
Despite the growing interest in the rabbit model for developmental and stem cell biology, the characterization of embryos at the molecular level is still poorly documented. We conducted a transcriptome analysis of rabbit preimplantation embryos from E2.7 (morula stage) to E6.6 (early primitive streak stage) using bulk and single-cell RNA-sequencing. In parallel, we studied oxidative phosphorylation and glycolysis, and analysed active and repressive epigenetic modifications during blastocyst formation and expansion. We generated a transcriptomic, epigenetic and metabolic map of the pluripotency continuum in rabbit preimplantation embryos, and identified novel markers of naive pluripotency that might be instrumental for deriving naive pluripotent stem cell lines. Although the rabbit is evolutionarily closer to mice than to primates, we found that the transcriptome of rabbit epiblast cells shares common features with those of humans and non-human primates.
Topics: Animals; Blastocyst; Epigenesis, Genetic; Germ Layers; Mice; Pluripotent Stem Cells; Rabbits; Transcriptome
PubMed: 35993311
DOI: 10.1242/dev.200538 -
Advances and Technical Standards in... 2022Recently, advanced knowledge on secondary neurulation and its application to the clinical experience have led to the deeper insight into the pathoembryogenesis of...
Recently, advanced knowledge on secondary neurulation and its application to the clinical experience have led to the deeper insight into the pathoembryogenesis of secondary neurulation with new classifications of the caudal spinal dysraphic entities. Here, we summarize the dynamic changes in the concepts of disordered secondary neurulation over the last two decades. In addition, we suggest our new pathoembryogenetic explanations for a few entities based on the literature and the data from our previous animal research. Disordered secondary neurulation at each phase may cause various corresponding lesions, such as (1) failed junction with the primary neural tube (junctional neural tube defect and segmental spinal dysgenesis), (2) dysgenesis or duplication of the caudal cell mass associated with disturbed activity of caudal mesenchymal tissue (caudal agenesis and caudal duplication syndrome), (3) abnormal continuity of medullary cord to the surrounding layers, namely, failed ingression of the primitive streak to the caudal cell mass (myelomeningocele), focal limited dorsal neurocutaneous nondisjunction (limited dorsal myeloschisis and congenital dermal sinus), and neuro-mesenchymal adhesion (lumbosacral lipomatous malformation), and (4) regression failure spectrum of the medullary cord (thickened filum and filar cyst, retained medullary cord and low-lying conus, terminal myelocele, and terminal myelocystocele). It seems that almost every anomalous entity of the primary neural tube may occur in the area of secondary neurulation. Furthermore, the close association of the caudal cell mass with the activity of caudal mesenchymal tissue involves a wider range of surrounding structures in secondary neurulation than in primary neurulation. Although the majority of the data are from animals and many theories are still conjectural, these changing concepts of normal and disordered secondary neurulation will provoke further advancements in our management strategies as well as in the pathoembryogenetic understanding of anomalous lesions in this area.
Topics: Animals; Magnetic Resonance Imaging; Meningomyelocele; Neural Tube Defects; Neurulation; Spinal Dysraphism; Spinal Neoplasms
PubMed: 35976454
DOI: 10.1007/978-3-030-99166-1_9 -
Stem Cell Research & Therapy Jul 2022In diabetic kidney disease, high glucose damages specialized cells called podocytes that filter blood in the glomerulus. In vitro culture of podocytes is crucial for...
BACKGROUND
In diabetic kidney disease, high glucose damages specialized cells called podocytes that filter blood in the glomerulus. In vitro culture of podocytes is crucial for modeling of diabetic nephropathy and genetic podocytopathies and to complement animal studies. Recently, several methods have been published to derive podocytes from human-induced pluripotent stem cells (iPSCs) by directed differentiation. However, these methods have major variations in media composition and have not been compared.
METHODS
We characterized our accelerated protocol by guiding the cells through differentiation with four different medias into MIXL1+ primitive streak cells with Activin A and CHIR for Wnt activation, intermediate mesoderm PAX8+ cells via increasing the CHIR concentration, nephron progenitors with FGF9 and Heparin for stabilization, and finally into differentiated podocytes with Activin A, BMP-7, VEGF, reduced CHIR, and retinoic acid. The podocyte morphology was characterized by scanning and transmission electron microscopy and by flow cytometry analysis for podocyte markers. To confirm cellular identity and niche localization, we performed cell recombination assays combining iPSC-podocytes with dissociated mouse embryonic kidney cells. Finally, to test iPSC-derived podocytes for the modeling of diabetic kidney disease, human podocytes were exposed to high glucose.
RESULTS
Podocyte markers were expressed at similar or higher levels for our accelerated protocol as compared to previously published protocols that require longer periods of tissue culture. We confirmed that the human podocytes derived from induced pluripotent stem cells in twelve days integrated into murine glomerular structures formed following seven days of culture of cellular recombinations. We found that the high glucose-treated human podocytes displayed actin rearrangement, increased cytotoxicity, and decreased viability.
CONCLUSIONS
We found that our accelerated 12-day method for the differentiation of podocytes from human-induced pluripotent stem cells yields podocytes with comparable marker expression to longer podocytes. We also demonstrated that podocytes created with this protocol have typical morphology by electron microscopy. The podocytes have utility for diabetes modeling as evidenced by lower viability and increased cytotoxicity when treated with high glucose. We found that multiple, diverse methods may be utilized to create iPSC-podocytes, but closely mimicking developmental cues shortened the time frame required for differentiation.
Topics: Animals; Diabetes Mellitus; Diabetic Nephropathies; Glucose; Humans; Induced Pluripotent Stem Cells; Kidney Glomerulus; Mice; Podocytes
PubMed: 35883199
DOI: 10.1186/s13287-022-03040-6 -
Seminars in Cell & Developmental Biology May 2023Recent advances in pluripotent stem cell culture allow researchers to generate not only most embryonic cell types, but also morphologies of many embryonic structures,... (Review)
Review
Recent advances in pluripotent stem cell culture allow researchers to generate not only most embryonic cell types, but also morphologies of many embryonic structures, entirely in vitro. This recreation of embryonic form from naïve cells, known as synthetic morphogenesis, has important implications for both developmental biology and regenerative medicine. However, the capacity of stem cell-based models to recapitulate the morphogenetic cell behaviors that shape natural embryos remains unclear. In this review, we explore several examples of synthetic morphogenesis, with a focus on models of gastrulation and surrounding stages. By varying cell types, source species, and culture conditions, researchers have recreated aspects of primitive streak formation, emergence and elongation of the primary embryonic axis, neural tube closure, and more. Here, we describe cell behaviors within in vitro/ex vivo systems that mimic in vivo morphogenesis and highlight opportunities for more complete models of early development.
Topics: Gastrulation; Morphogenesis
PubMed: 35817656
DOI: 10.1016/j.semcdb.2022.07.002 -
Cells Jun 2022Fibronectin is essential for somite formation in the vertebrate embryo. Fibronectin matrix assembly starts as cells emerge from the primitive streak and ingress in the...
Fibronectin is essential for somite formation in the vertebrate embryo. Fibronectin matrix assembly starts as cells emerge from the primitive streak and ingress in the unsegmented presomitic mesoderm (PSM). PSM cells undergo cyclic waves of segmentation clock gene expression, followed by Notch-dependent upregulation of in the rostral PSM which induces somite cleft formation. However, the relevance of the fibronectin matrix for these molecular processes remains unknown. Here, we assessed the role of the PSM fibronectin matrix in the spatio-temporal regulation of chick embryo somitogenesis by perturbing (1) extracellular fibronectin matrix assembly, (2) integrin-fibronectin binding, (3) Rho-associated protein kinase (ROCK) activity and (4) non-muscle myosin II (NM II) function. We found that integrin-fibronectin engagement and NM II activity are required for cell polarization in the nascent somite. All treatments resulted in defective somitic clefts and significantly perturbed and segmentation clock gene expression in the PSM. Importantly, inhibition of actomyosin-mediated contractility increased the period of oscillations from 90 to 120 min. Together, our work strongly suggests that the fibronectin-integrin-ROCK-NM II axis regulates segmentation clock dynamics and dictates the spatio-temporal localization of somitic clefts.
Topics: Actomyosin; Animals; Biological Clocks; Chick Embryo; Fibronectins; Integrins; Somites
PubMed: 35805087
DOI: 10.3390/cells11132003 -
Metabolic Engineering Sep 2022CRISPR-based systems have fundamentally transformed our ability to study and manipulate stem cells. We explored the possibility of using catalytically dead Cas9 (dCas9)...
CRISPR-based systems have fundamentally transformed our ability to study and manipulate stem cells. We explored the possibility of using catalytically dead Cas9 (dCas9) from S. pyogenes as a platform for targeted epigenetic editing in stem cells to enhance the expression of the eomesodermin gene (EOMES) during differentiation. We observed, however, that the dCas9 protein itself exerts a potential non-specific effect in hiPSCs, affecting the cell's phenotype and gene expression patterns during subsequent directed differentiation. We show that this effect is specific to the condition when cells are cultured in medium that does not actively maintain the pluripotency network, and that the sgRNA-free apo-dCas9 protein itself influences endogenous gene expression. Transcriptomics analysis revealed that a significant number of genes involved in developmental processes and various other genes with non-overlapping biological functions are affected by dCas9 overexpression. This suggests a potential adverse phenotypic effect of dCas9 itself in hiPSCs, which could have implications for when and how CRISPR/Cas9-based tools can be used reliably and safely in pluripotent stem cells.
Topics: CRISPR-Cas Systems; Gene Expression; Humans; Induced Pluripotent Stem Cells; Primitive Streak
PubMed: 35724832
DOI: 10.1016/j.ymben.2022.06.003 -
Development (Cambridge, England) Jun 2022Classical studies have established that the marginal zone, a ring of extra-embryonic epiblast immediately surrounding the embryonic epiblast (area pellucida) of the...
Classical studies have established that the marginal zone, a ring of extra-embryonic epiblast immediately surrounding the embryonic epiblast (area pellucida) of the chick embryo, is important in setting embryonic polarity by positioning the primitive streak, the site of gastrulation. The more external extra-embryonic region (area opaca) was thought to have only nutritive and support functions. Using experimental embryology approaches, this study reveals three separable functions for this outer region. First, juxtaposition of the area opaca directly onto the area pellucida induces a new marginal zone from the latter; this induced domain is entirely posterior in character. Second, ablation and grafting experiments using an isolated anterior half of the blastoderm and pieces of area opaca suggest that the area opaca can influence the polarity of the adjacent marginal zone. Finally, we show that the loss of the ability of such isolated anterior half-embryos to regulate (re-establish polarity spontaneously) at the early primitive streak stage can be rescued by replacing the area opaca by one from a younger stage. These results uncover new roles of chick extra-embryonic tissues in early development.
Topics: Animals; Blastoderm; Chick Embryo; Gastrula; Primitive Streak
PubMed: 35723262
DOI: 10.1242/dev.200303 -
Stem Cell Reports Jul 2022Embryo studies have established that the patterning of the mouse gastrula depends on a regulatory network in which the WNT, BMP, and NODAL signaling pathways cooperate,...
Embryo studies have established that the patterning of the mouse gastrula depends on a regulatory network in which the WNT, BMP, and NODAL signaling pathways cooperate, but aspects of their respective contributions remain unclear. Studying their impact on the spatial organization and developmental trajectories of micropatterned epiblast-like cell (EpiLC) colonies, we show that NODAL is required prior to BMP action to establish the mesoderm and endoderm lineages. The presence of BMP then forces NODAL and WNT to support the formation of posterior primitive streak (PS) derivatives, while its absence allows them to promote that of anterior PS derivatives. Also, a Nodal mutation elicits more severe patterning defects in vitro than in the embryo, suggesting that ligands of extra-embryonic origin can rescue them. These results support the implication of a combinatorial process in PS patterning and illustrate how the study of micropatterned EpiLC colonies can complement that of embryos.
Topics: Animals; Body Patterning; Endoderm; Gastrula; Germ Layers; Mesoderm; Mice; Primitive Streak; Transforming Growth Factor beta
PubMed: 35714597
DOI: 10.1016/j.stemcr.2022.05.009 -
Nature Sep 2022Gastrulation controls the emergence of cellular diversity and axis patterning in the early embryo. In mammals, this transformation is orchestrated by dynamic signalling...
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 tissues. Elucidating the molecular framework of axis formation in vivo is fundamental for our understanding of human development and to advance stem-cell-based regenerative approaches. 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.
Topics: Animals; Callithrix; Cell Differentiation; Embryo, Mammalian; Endoderm; Female; Gastrulation; Gene Expression Profiling; Germ Layers; Humans; Pluripotent Stem Cells; Uterus
PubMed: 35709828
DOI: 10.1038/s41586-022-04953-1