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Stem Cell Research & Therapy Jan 2019Human mesenchymal stem cells are a strong candidate for cell therapies owing to their regenerative potential, paracrine regulatory effects, and immunomodulatory...
BACKGROUND
Human mesenchymal stem cells are a strong candidate for cell therapies owing to their regenerative potential, paracrine regulatory effects, and immunomodulatory activity. Yet, their scarcity, limited expansion potential, and age-associated functional decline restrict the ability to consistently manufacture large numbers of safe and therapeutically effective mesenchymal stem cells for routine clinical applications. To overcome these limitations and advance stem cell treatments using mesenchymal stem cells, researchers have recently derived mesenchymal progenitors from human-induced pluripotent stem cells. Human-induced pluripotent stem cell-derived progenitors resemble adult mesenchymal stem cells in morphology, global gene expression, surface antigen profile, and multi-differentiation potential, but unlike adult mesenchymal stem cells, it can be produced in large numbers for every patient. For therapeutic applications, however, human-induced pluripotent stem cell-derived progenitors must be produced without animal-derived components (xeno-free) and in accordance with Good Manufacturing Practice guidelines.
METHODS
In the present study we investigate the effects of expanding mesodermal progenitor cells derived from two human-induced pluripotent stem cell lines in xeno-free medium supplemented with human platelet lysates and in a commercial high-performance Good Manufacturing Practice-compatible medium (Unison Medium).
RESULTS
The results show that long-term culture in xeno-free and Good Manufacturing Practice-compatible media somewhat affects the morphology, expansion potential, gene expression, and cytokine profile of human-induced pluripotent stem cell-derived progenitors but supports cell viability and maintenance of a mesenchymal phenotype equally well as medium supplemented with fetal bovine serum.
CONCLUSIONS
The findings support the potential to manufacture large numbers of clinical-grade human-induced pluripotent stem cell-derived mesenchymal progenitors for applications in personalized regenerative medicine.
Topics: Cell Culture Techniques; Cell Differentiation; Cell Line; Cell Proliferation; Culture Media; Gene Expression Regulation, Developmental; Humans; Induced Pluripotent Stem Cells; Mesenchymal Stem Cells; Mesoderm; Regenerative Medicine
PubMed: 30635059
DOI: 10.1186/s13287-018-1119-3 -
Cell Stem Cell Sep 2018The mesoderm arises from pluripotent epiblasts and differentiates into multiple lineages; however, the underlying molecular mechanisms are unclear. Tbx6 is enriched in...
The mesoderm arises from pluripotent epiblasts and differentiates into multiple lineages; however, the underlying molecular mechanisms are unclear. Tbx6 is enriched in the paraxial mesoderm and is implicated in somite formation, but its function in other mesoderms remains elusive. Here, using direct reprogramming-based screening, single-cell RNA-seq in mouse embryos, and directed cardiac differentiation in pluripotent stem cells (PSCs), we demonstrated that Tbx6 induces nascent mesoderm from PSCs and determines cardiovascular and somite lineage specification via its temporal expression. Tbx6 knockout in mouse PSCs using CRISPR/Cas9 technology inhibited mesoderm and cardiovascular differentiation, whereas transient Tbx6 expression induced mesoderm and cardiovascular specification from mouse and human PSCs via direct upregulation of Mesp1, repression of Sox2, and activation of BMP/Nodal/Wnt signaling. Notably, prolonged Tbx6 expression suppressed cardiac differentiation and induced somite lineages, including skeletal muscle and chondrocytes. Thus, Tbx6 is critical for mesoderm induction and subsequent lineage diversification.
Topics: Animals; Cardiovascular System; Cell Differentiation; Cell Lineage; Cells, Cultured; Humans; Male; Mesoderm; Mice; Mice, Inbred ICR; Mice, Transgenic; Pluripotent Stem Cells; Somites; T-Box Domain Proteins; Transcription Factors
PubMed: 30100166
DOI: 10.1016/j.stem.2018.07.001 -
Developmental Biology Jun 2001Endodermally derived organs of the gastrointestinal and respiratory system form at distinct anterioposterior and dorsoventral locations along the vertebrate body axis.... (Review)
Review
Endodermally derived organs of the gastrointestinal and respiratory system form at distinct anterioposterior and dorsoventral locations along the vertebrate body axis. This stereotyped program of organ formation depends on the correct patterning of the endodermal epithelium so that organ differentiation and morphogenesis occur at appropriate positions along the gut tube. Whereas some initial patterning of the endoderm is known to occur early, during germ-layer formation and gastrulation, later signaling events, originating from a number of adjacent tissue layers, are essential for the development of endodermal organs. Previous studies have shown that signals arising from the notochord are important for patterning of the ectodermally derived floor plate of the neural tube and the mesodermally derived somites. This review will discuss recent evidence indicating that signals arising from the notochord also play a role in regulating endoderm development.
Topics: Body Patterning; Ectoderm; Embryonic Induction; Endoderm; Germ Layers; Mesoderm; Notochord; Pancreas
PubMed: 11356015
DOI: 10.1006/dbio.2001.0214 -
Developmental Biology Mar 2015Activation of the Pax2 gene marks the intermediate mesoderm shortly after gastrulation, as the mesoderm becomes compartmentalized into paraxial, intermediate, and...
Activation of the Pax2 gene marks the intermediate mesoderm shortly after gastrulation, as the mesoderm becomes compartmentalized into paraxial, intermediate, and lateral plate. Using an EGFP knock-in allele of Pax2 to identify and sort cells of the intermediate mesodermal lineage, we compared gene expression patterns in EGFP positive cells that were heterozygous or homozygous null for Pax2. Thus, we identified critical regulators of intermediate mesoderm and kidney development whose expression depended on Pax2 function. In cell culture models, Pax2 is thought to recruit epigenetic modifying complex to imprint activating histone methylation marks through interactions with the adaptor protein PTIP. In kidney organ culture, conditional PTIP deletion showed that many Pax2 target genes, which were activated early in renal progenitor cells, remained on once activated, whereas Pax2 target genes expressed later in kidney development were unable to be fully activated without PTIP. In Pax2 mutants, we also identified a set of genes whose expression was up-regulated in EGFP positive cells and whose expression was consistent with a cell fate transformation to paraxial mesoderm and its derivatives. These data provide evidence that Pax2 specifies the intermediate mesoderm and renal epithelial cells through epigenetic mechanisms and in part by repressing paraxial mesodermal fate.
Topics: Animals; Blotting, Western; Carrier Proteins; DNA Primers; DNA-Binding Proteins; Flow Cytometry; Gene Expression Regulation; Gene Knock-In Techniques; Green Fluorescent Proteins; In Situ Hybridization; Kidney; Mesoderm; Mice; Microarray Analysis; Nuclear Proteins; PAX2 Transcription Factor; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Stem Cells
PubMed: 25617721
DOI: 10.1016/j.ydbio.2015.01.005 -
Developmental Biology Nov 2011Possession of paired appendages is regarded as a novelty that defines crown gnathostomes and allows sophisticated behavioral and locomotive patterns. During embryonic... (Comparative Study)
Comparative Study
Possession of paired appendages is regarded as a novelty that defines crown gnathostomes and allows sophisticated behavioral and locomotive patterns. During embryonic development, initiation of limb buds in the lateral plate mesoderm involves several steps. First, the lateral plate mesoderm is regionalized into the cardiac mesoderm (CM) and the posterior lateral plate mesoderm (PLPM). Second, in the PLPM, Hox genes are expressed in a collinear manner to establish positional values along the anterior-posterior axis. The developing PLPM splits into somatic and splanchnic layers. In the presumptive limb field of the somatic layer, expression of limb initiation genes appears. To gain insight into the evolutionary sequence leading to the emergence of paired appendages in ancestral vertebrates, we examined the embryonic development of the ventral mesoderm in the cephalochordate amphioxus Branchiostoma floridae and of the lateral plate mesoderm in the agnathan lamprey Lethenteron japonicum, and studied the expression patterns of cognates of genes known to be expressed in these mesodermal layers during amniote development. We observed that, although the amphioxus ventral mesoderm posterior to the pharynx was not regionalized into CM and posterior ventral mesoderm, the lateral plate mesoderm of lampreys was regionalized into CM and PLPM, as in gnathostomes. We also found nested expression of two Hox genes (LjHox5i and LjHox6w) in the PLPM of lamprey embryos. However, histological examination showed that the PLPM of lampreys was not separated into somatic and splanchnic layers. These findings provide insight into the sequential evolutionary changes that occurred in the ancestral lateral plate mesoderm leading to the emergence of paired appendages.
Topics: Animals; Base Sequence; Biological Evolution; Chordata, Nonvertebrate; DNA Primers; Genes, Homeobox; In Situ Hybridization; Lampreys; Mesoderm; Reverse Transcriptase Polymerase Chain Reaction
PubMed: 21864524
DOI: 10.1016/j.ydbio.2011.08.003 -
Journal of Cellular Biochemistry Oct 2015The concentration, location, and timing of bone morphogenetic protein 2 (BMP2, HGNC:1069, GeneID: 650) gene expression must be precisely regulated. Abnormal BMP2 levels... (Review)
Review
The concentration, location, and timing of bone morphogenetic protein 2 (BMP2, HGNC:1069, GeneID: 650) gene expression must be precisely regulated. Abnormal BMP2 levels cause congenital anomalies and diseases involving the mesenchymal cells that differentiate into muscle, fat, cartilage, and bone. The molecules and conditions that influence BMP2 synthesis are diverse. Understandably, complex mechanisms control Bmp2 gene expression. This review includes a compilation of agents and conditions that can induce Bmp2. The currently known trans-regulatory factors and cis-regulatory elements that modulate Bmp2 expression are summarized and discussed. Bone morphogenetic protein 2 (BMP2, HGNC:1069, GeneID: 650) is a classical morphogen; a molecule that acts at a distance and whose concentration influences cell behavior. In mesenchymal cells, the concentration of BMP2 influences myogenesis, adipogenesis, chondrogenesis, and osteogenesis. Because the amount, timing, and location of BMP2 synthesis influence the allocation of cells to muscle, fat, cartilage, and bone, the mechanisms that regulate the Bmp2 gene are crucial. Key early mesodermal events that require precise Bmp2 regulation include heart specification and morphogenesis. Originally named for its osteoinductive properties, healing fractures requires BMP2. The human Bmp2 gene also has been linked to osteoporosis and osteoarthritis. In addition, all forms of pathological calcification in the vasculature and in cardiac valves involve the pro-osteogenic BMP2. The diverse tissues, mechanisms, and diseases influenced by BMP2 are too numerous to list here (see OMIM: 112261). However, in all BMP2-influenced pathologies, changes in the behavior and differentiation of pluripotent mesenchymal cells are a recurring theme. Consequently, much effort has been devoted to identifying the molecules and conditions that influence BMP2 synthesis and the complex mechanisms that control Bmp2 gene expression. This review begins with an overview of the Bmp2 gene's chromosomal neighborhood and then summarizes and evaluates known regulatory mechanisms and inducers.
Topics: Adipogenesis; Bone Morphogenetic Protein 2; Calcinosis; Chondrogenesis; Gene Expression Regulation; Humans; Mesoderm; Muscle Development; Osteoarthritis; Osteogenesis; Osteoporosis; Regulatory Sequences, Nucleic Acid
PubMed: 25776852
DOI: 10.1002/jcb.25164 -
Cell Cycle (Georgetown, Tex.) Mar 2013Identification of sequential progenitors leading to blood formation from pluripotent stem cells (PSCs) will be essential for understanding the molecular mechanisms of... (Review)
Review
Identification of sequential progenitors leading to blood formation from pluripotent stem cells (PSCs) will be essential for understanding the molecular mechanisms of hematopoietic lineage specification and for development of technologies for in vitro production of hematopoietic stem cells (HSCs). It is well established that during development, blood and endothelial cells in the extraembryonic and embryonic compartments are formed in parallel from precursors with angiogenic and hematopoietic potentials. However, the identity and hierarchy of these precursors in human PSC (hPSC) cultures remain obscure. Using developmental stage-specific mesodermal and endothelial markers and functional assays, we recently identified discrete populations of angiohematopoietic progenitors from hPSCs, including mesodermal precursors and hemogenic endothelial cells with primitive and definitive hematopoietic potentials. In addition, we discovered a novel population of multipotent hematopoietic progenitors with an erythroid phenotype, which retain angiogenic potential. Here we introduce our recent findings and discuss their implication for defining putative HSC precursor and factors required for activation of self-renewal potential in hematopoietic cells emerging from endothelium.
Topics: Cell Differentiation; Cell Lineage; Endothelial Cells; Hematopoietic Stem Cells; Humans; Mesoderm; Pluripotent Stem Cells
PubMed: 23388453
DOI: 10.4161/cc.23823 -
Developmental Biology Oct 2021To date, the role of miRNAs on pluripotency and differentiation of ESCs into specific lineages has been studied extensively. However, the specific role of miRNAs during...
To date, the role of miRNAs on pluripotency and differentiation of ESCs into specific lineages has been studied extensively. However, the specific role of miRNAs during lateral and paraxial mesoderm cell fate decision is still unclear. To address this, we firstly determined miRNA profile of mouse ESCs differentiating towards lateral and paraxial lineages which were detected using Flk1 and PDGFαR antibodies, and of myogenic and hematopoietic differentiation potential of purified paraxial and lateral mesodermal cells within these populations. miRNAs associated with lateral and paraxial mesoderm, and their targets were identified using bioinformatics tools. The targets of the corresponding miRNAs were validated after transfection into mouse ESCs. The roles of the selected miRNAs in lateral, and paraxial mesoderm formation were assessed along with hematopoietic and myogenic differentiation capacity. Among the miRNAs, mmu-miR-126a-3p, mmu-miR-335-5p and mmu-miR-672-5p, upregulated in lateral mesoderm cells, and mmu-miR-10b-5p, mmu-miR-196a-5p and mmu-miR-615-3p, upregulated in paraxial mesoderm cells. While transient co-transfection of mmu-miR-126a-3p, mmu-miR-335-5p and mmu-miR-672-5p increased the number of lateral mesodermal cells, co-transfection of mmu-miR-10b-5p, mmu-miR-196a-5p and mmu-miR-615-3p increased the number of paraxial mesodermal cells. Moreover, differentiation potential of the lateral mesodermal cells into hematopoietic cell lineage increased upon co-transfection of mmu-miR-126a-3p, mmu-miR-335-5p and mmu-miR-672-5p and differentiation potential of the paraxial mesodermal cells into skeletal muscle lineage were increased upon co-transfection of mmu-miR-10b-5p, mmu-miR-196a-5p and mmu-miR-615-3p. In conclusion, we determined the miRNA profile of lateral and paraxial mesodermal cells and co-transfection of miRNAs increased differentiation potential of both lateral and paraxial mesodermal cells transiently.
Topics: Animals; Cell Differentiation; Computational Biology; Embryoid Bodies; Embryonic Stem Cells; Hematopoiesis; Mesoderm; Mice; MicroRNAs; Muscle Development; Transfection; Up-Regulation
PubMed: 34245726
DOI: 10.1016/j.ydbio.2021.07.002 -
Genes & Development Sep 1991twist and snail are members of the helix-loop-helix and zinc-finger protein families, respectively, and determine the development of the mesoderm in Drosophila. This...
twist and snail are members of the helix-loop-helix and zinc-finger protein families, respectively, and determine the development of the mesoderm in Drosophila. This paper analyzes their role in mesoderm development by examining how they affect the expression of downstream genes. twist and snail act by regulating gene expression in the mesoderm and in neighboring regions, and have distinct roles in this process. snail prevents expression in the mesoderm of genes that are destined to be active only in more lateral or dorsal regions. twist is required for the activation of downstream mesodermal genes. twist is also required for the full expression of snail and for the maintenance of its own expression. Only the absence of both twist and snail results in the complete loss of all mesodermal characteristics.
Topics: Animals; Drosophila; Drosophila Proteins; Gene Expression Regulation; Genes; Mesoderm; Mutation; Nuclear Proteins; Nucleic Acid Hybridization; Transcription Factors; Twist-Related Protein 1; Zinc Fingers
PubMed: 1884999
DOI: 10.1101/gad.5.9.1568 -
Proceedings of the National Academy of... Jun 2021Markers for the endoderm and mesoderm germ layers are commonly expressed together in the early embryo, potentially reflecting cells' ability to explore potential fates...
Markers for the endoderm and mesoderm germ layers are commonly expressed together in the early embryo, potentially reflecting cells' ability to explore potential fates before fully committing. It remains unclear when commitment to a single-germ layer is reached and how it is impacted by external signals. Here, we address this important question in , a convenient model system in which mesodermal and endodermal fates are associated with distinct cellular movements during gastrulation. Systematically applying endoderm-inducing extracellular signal-regulated kinase (ERK) signals to the ventral medial embryo-which normally only receives a mesoderm-inducing cue-reveals a critical time window during which mesodermal cell movements and gene expression are suppressed by proendoderm signaling. We identify the ERK target gene () as the main cause of the ventral furrow suppression and use computational modeling to show that Hkb repression of the mesoderm-associated gene is sufficient to account for a broad range of transcriptional and morphogenetic effects. Our approach, pairing precise signaling perturbations with observation of transcriptional dynamics and cell movements, provides a general framework for dissecting the complexities of combinatorial tissue patterning.
Topics: Animals; DNA-Binding Proteins; Drosophila Proteins; Drosophila melanogaster; Endoderm; Gastrula; Gastrulation; MAP Kinase Signaling System; Mesoderm; Models, Biological
PubMed: 34083443
DOI: 10.1073/pnas.2102691118