-
Cells Apr 2021The endoderm, differentiated from human induced pluripotent stem cells (iPSCs), can differentiate into the small intestine and liver, which are vital for drug absorption...
The endoderm, differentiated from human induced pluripotent stem cells (iPSCs), can differentiate into the small intestine and liver, which are vital for drug absorption and metabolism. The development of human iPSC-derived enterocytes (HiEnts) and hepatocytes (HiHeps) has been reported. However, pharmacokinetic function-deficiency of these cells remains to be elucidated. Here, we aimed to develop an efficient differentiation method to induce endoderm formation from human iPSCs. Cells treated with activin A for 168 h expressed higher levels of endodermal genes than those treated for 72 h. Using activin A (days 0-7), CHIR99021 and PI-103 (days 0-2), and FGF2 (days 3-7), the hiPSC-derived endoderm (HiEnd) showed 97.97% CD-117 and CD-184 double-positive cells. Moreover, HiEnts derived from the human iPSC line Windy had similar or higher expression of small intestine-specific genes than adult human small intestine. Activities of the drug transporter P-glycoprotein and drug-metabolizing enzyme cytochrome P450 (CYP) 3A4/5 were confirmed. Additionally, Windy-derived HiHeps expressed higher levels of hepatocyte- and pharmacokinetics-related genes and proteins and showed higher CYP3A4/5 activity than those derived through the conventional differentiation method. Thus, using this novel method, the differentiated HiEnts and HiHeps with pharmacokinetic functions could be used for drug development.
Topics: Activins; Bone Morphogenetic Protein 4; Cell Culture Techniques; Cell Differentiation; Cell Line; Cells, Cultured; Dimethyl Sulfoxide; Endoderm; Enterocytes; Feeder Cells; Fibroblast Growth Factor 2; Furans; Hepatocytes; Humans; Induced Pluripotent Stem Cells; Intestine, Small; Primitive Streak; Pyridines; Pyrimidines; Reproducibility of Results
PubMed: 33917333
DOI: 10.3390/cells10040812 -
Journal of Korean Neurosurgical Society May 2021Recent advancements in basic research on the process of secondary neurulation and increased clinical experience with caudal spinal anomalies with associated...
Recent advancements in basic research on the process of secondary neurulation and increased clinical experience with caudal spinal anomalies with associated abnormalities in the surrounding and distal structures shed light on further understanding of the pathoembryogenesis of the lesions and led to the new classification of these dysraphic entities. We summarized the changing concepts of lesions developed from the disordered secondary neurulation shown during the last decade. In addition, we suggested 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 of development may cause corresponding lesions, such as failed junction with the primary neural tube (junctional neural tube defect and segmental spinal dysgenesis), dysgenesis or duplication of the caudal cell mass associated with disturbed activity of caudal mesenchymal tissue (caudal agenesis and caudal duplication syndrome), failed ingression of the primitive streak to the caudal cell mass (myelomeningocele), focal limited dorsal neuro-cutaneous nondisjunction (limited dorsal myeloschisis and congenital dermal sinus), neuro-mesenchymal adhesion (lumbosacral lipomatous malformation), and regression failure spectrum of the medullary cord (thickened filum and filar cyst, low-lying conus, retained medullary cord, 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 with the activity of caudal mesenchymal tissue in secondary neurulation involves a wider range of surrounding structures than in primary neurulation. Although the majority of the data are from animals, not from humans 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.
PubMed: 33906343
DOI: 10.3340/jkns.2021.0023 -
Stem Cell Reports May 2021Human embryonic stem cells cultured in 2D micropatterns with BMP4 differentiate into a radial arrangement of germ layers and extraembryonic cells. Single-cell...
Human embryonic stem cells cultured in 2D micropatterns with BMP4 differentiate into a radial arrangement of germ layers and extraembryonic cells. Single-cell transcriptomes demonstrate generation of cell types transcriptionally similar to their in vivo counterparts in Carnegie stage 7 human gastrula. Time-course analyses indicate sequential differentiation, where the epiblast arises by 12 h between the prospective ectoderm in the center and the cells initiating differentiation toward extraembryonic fates at the edge. Extraembryonic and mesendoderm precursors arise from the epiblast by 24 h, while nascent mesoderm, endoderm, and primordial germ cell-like cells form by 44 h. Dynamic changes in transcripts encoding signaling components support a BMP, WNT, and Nodal hierarchy underlying germ-layer specification conserved across mammals, and FGF and HIPPO pathways being active throughout differentiation. This work also provides a resource for mining genes and pathways expressed in a stereotyped 2D gastruloid model, common with other species or unique to human gastrulation.
Topics: Amnion; Cell Culture Techniques; Cell Differentiation; Cell Lineage; Gastrula; Gastrulation; Gene Expression Regulation, Developmental; Germ Cells; Germ Layers; Human Embryonic Stem Cells; Humans; Mesoderm; Primitive Streak; Signal Transduction; Time Factors; Transcription, Genetic
PubMed: 33891870
DOI: 10.1016/j.stemcr.2021.03.031 -
Journal of Visualized Experiments : JoVE Mar 2021Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial cells, which...
Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial cells, which line blood vessel lumens, from human pluripotent stem cells is crucial for a multitude of tissue engineering and regeneration applications. In vivo, primordial endothelial cells are derived from the mesodermal lineage and are specified toward specific subtypes, including arterial, venous, capillary, hemogenic, and lymphatic. Hemogenic endothelial cells are of particular interest because, during development, they give rise to hematopoietic stem and progenitor cells, which then generate all blood lineages throughout life. Thus, creating a system to generate hemogenic endothelial cells in vitro would provide an opportunity to study endothelial-to-hematopoietic transition, and may lead to ex vivo production of human blood products and reduced reliance on human donors. While several protocols exist for the derivation of progenitor and primordial endothelial cells, generation of well-characterized hemogenic endothelial cells from human stem cells has not been described. Here, a method for the derivation of hemogenic endothelial cells from human embryonic stem cells in approximately 1 week is presented: a differentiation protocol with primitive streak cells formed in response to GSK3β inhibitor (CHIR99021), then mesoderm lineage induction mediated by bFGF, followed by primordial endothelial cell development promoted by BMP4 and VEGF-A, and finally hemogenic endothelial cell specification induced by retinoic acid. This protocol yields a well-defined population of hemogenic endothelial cells that can be used to further understand their molecular regulation and endothelial-to-hematopoietic transition, which has the potential to be applied to downstream therapeutic applications.
Topics: Cell Differentiation; Endothelial Cells; Humans; Pluripotent Stem Cells
PubMed: 33871448
DOI: 10.3791/62391 -
Stem Cells International 2021Mouse embryonic stem cells (mESCs) and mouse epiblast stem cells (mEpiSCs) are the pluripotent stem cells (PSCs), derived from the inner cell mass (ICM) of...
Mouse embryonic stem cells (mESCs) and mouse epiblast stem cells (mEpiSCs) are the pluripotent stem cells (PSCs), derived from the inner cell mass (ICM) of preimplantation embryos at embryonic day 3.5 (E3.5) and postimplantation embryos at E5.5-E7.5, respectively. Depending on their environment, PSCs can exist in the so-called naïve (ESCs) or primed (EpiSCs) states. Exposure to EpiSC or human ESC (hESC) culture condition can convert mESCs towards an EpiSC-like state. Here, we show that the undifferentiated epiblast state is however not stabilized in a sustained manner when exposing mESCs to hESC or EpiSC culture condition. Rather, prolonged exposure to EpiSC condition promotes a transition to a primitive streak- (PS-) like state via an unbiased epiblast-like intermediate. We show that the Brachyury-positive PS-like state is likely promoted by endogenous WNT signaling, highlighting a possible species difference between mouse epiblast-like stem cells and human Embryonic Stem Cells.
PubMed: 33828592
DOI: 10.1155/2021/8818356 -
Stem Cell Reports May 2021Stem cell-based embryo models open an unprecedented avenue for modeling embryogenesis, cell lineage differentiation, tissue morphogenesis, and organogenesis in mammalian... (Review)
Review
Stem cell-based embryo models open an unprecedented avenue for modeling embryogenesis, cell lineage differentiation, tissue morphogenesis, and organogenesis in mammalian development. Experimentation on these embryo models can lead to a better understanding of the mechanisms of development and offers opportunities for functional genomic studies of disease-causing mechanisms, identification of therapeutic targets, and preclinical modeling of advanced therapeutics for precision medicine. An immediate challenge is to create embryo models of high fidelity to embryogenesis and organogenesis in vivo, to ensure that the knowledge gleaned is biologically meaningful and clinically relevant.
Topics: Animals; Body Patterning; Embryo, Mammalian; Embryonic Development; Embryonic Stem Cells; Humans; Models, Biological; Terminology as Topic
PubMed: 33667412
DOI: 10.1016/j.stemcr.2021.02.002 -
Development (Cambridge, England) Feb 2021The generation of the components that make up the embryonic body axis, such as the spinal cord and vertebral column, takes place in an anterior-to-posterior... (Review)
Review
The generation of the components that make up the embryonic body axis, such as the spinal cord and vertebral column, takes place in an anterior-to-posterior (head-to-tail) direction. This process is driven by the coordinated production of various cell types from a pool of posteriorly-located axial progenitors. Here, we review the key features of this process and the biology of axial progenitors, including neuromesodermal progenitors, the common precursors of the spinal cord and trunk musculature. We discuss recent developments in the production of axial progenitors and their potential implications in disease modelling and regenerative medicine.
Topics: Animals; Biology; Body Patterning; Ectoderm; Endoderm; Gastrulation; Gene Expression Regulation, Developmental; Germ Layers; Humans; In Vitro Techniques; Mesoderm; Muscle, Skeletal; Stem Cells
PubMed: 33593754
DOI: 10.1242/dev.180612 -
Nature Cell Biology Jan 2021Extra-embryonic mesoderm (ExM)-composed of the earliest cells that traverse the primitive streak-gives rise to the endothelium as well as haematopoietic progenitors in...
Extra-embryonic mesoderm (ExM)-composed of the earliest cells that traverse the primitive streak-gives rise to the endothelium as well as haematopoietic progenitors in the developing yolk sac. How a specific subset of ExM becomes committed to a haematopoietic fate remains unclear. Here we demonstrate using an embryonic stem cell model that transient expression of the T-box transcription factor Eomesodermin (Eomes) governs haemogenic competency of ExM. Eomes regulates the accessibility of enhancers that the transcription factor stem cell leukaemia (SCL) normally utilizes to specify primitive erythrocytes and is essential for the normal development of Runx1 haemogenic endothelium. Single-cell RNA sequencing suggests that Eomes loss of function profoundly blocks the formation of blood progenitors but not specification of Flk-1 haematoendothelial progenitors. Our findings place Eomes at the top of the transcriptional hierarchy regulating early blood formation and suggest that haemogenic competence is endowed earlier during embryonic development than was previously appreciated.
Topics: Animals; Core Binding Factor Alpha 2 Subunit; Embryonic Stem Cells; Female; Hemangioblasts; Male; Mesoderm; Mice, Knockout; Pregnancy; RNA-Seq; Single-Cell Analysis; T-Box Domain Proteins; T-Cell Acute Lymphocytic Leukemia Protein 1; Vascular Endothelial Growth Factor Receptor-2; Yolk Sac; Mice
PubMed: 33420489
DOI: 10.1038/s41556-020-00611-8 -
Developmental Cell Feb 2021The development of mouse embryos can be partially recapitulated by combining embryonic stem cells (ESCs), trophoblast stem cells (TS), and extra-embryonic endoderm (XEN)...
The development of mouse embryos can be partially recapitulated by combining embryonic stem cells (ESCs), trophoblast stem cells (TS), and extra-embryonic endoderm (XEN) stem cells to generate embryo-like structures called ETX embryos. Although ETX embryos transcriptionally capture the mouse gastrula, their ability to recapitulate complex morphogenic events such as gastrulation is limited, possibly due to the limited potential of XEN cells. To address this, we generated ESCs transiently expressing transcription factor Gata4, which drives the extra-embryonic endoderm fate, and combined them with ESCs and TS cells to generate induced ETX embryos (iETX embryos). We show that iETX embryos establish a robust anterior signaling center that migrates unilaterally to break embryo symmetry. Furthermore, iETX embryos gastrulate generating embryonic and extra-embryonic mesoderm and definitive endoderm. Our findings reveal that replacement of XEN cells with ESCs transiently expressing Gata4 endows iETX embryos with greater developmental potential, thus enabling the study of the establishment of anterior-posterior patterning and gastrulation in an in vitro system.
Topics: Animals; Biomarkers; Cell Line; Cell Lineage; Embryo, Mammalian; Embryonic Stem Cells; Endoderm; Epithelial-Mesenchymal Transition; GATA4 Transcription Factor; Gastrulation; Induced Pluripotent Stem Cells; Mice; Morphogenesis; Primitive Streak; Signal Transduction
PubMed: 33378662
DOI: 10.1016/j.devcel.2020.12.004 -
Selective activation of FZD7 promotes mesendodermal differentiation of human pluripotent stem cells.ELife Dec 2020WNT proteins are secreted symmetry breaking signals that interact with cell surface receptors of the FZD family to regulate a multitude of developmental processes....
WNT proteins are secreted symmetry breaking signals that interact with cell surface receptors of the FZD family to regulate a multitude of developmental processes. Studying selectivity between WNTs and FZDs has been hampered by the paucity of purified WNT proteins and by their apparent non-selective interactions with the FZD receptors. Here, we describe an engineered protein, called F7L6, comprised of antibody-derived single-chain variable fragments, that selectively binds to human FZD7 and the co-receptor LRP6. F7L6 potently activates WNT/β-catenin signaling in a manner similar to Wnt3a. In contrast to Wnt3a, F7L6 engages only FZD7 and none of the other FZD proteins. Treatment of human pluripotent stem (hPS) cells with F7L6 initiates transcriptional programs similar to those observed during primitive streak formation and subsequent gastrulation in the mammalian embryo. This demonstrates that selective engagement and activation of FZD7 signaling is sufficient to promote mesendodermal differentiation of hPS cells.
Topics: Blotting, Western; Cell Differentiation; Frizzled Receptors; Gene Expression Regulation; Humans; Mesoderm; Pluripotent Stem Cells; Real-Time Polymerase Chain Reaction; Recombinant Proteins; Wnt Signaling Pathway
PubMed: 33331818
DOI: 10.7554/eLife.63060