ABSTRACT
Chimaeras are both monsters of the ancient imagination and a long-established research tool. Recent advances, particularly those dealing with the identification and generation of various kinds of stem cells, have broadened the repertoire and utility of mammalian interspecies chimaeras and carved out new paths towards understanding fundamental biology as well as potential clinical applications.
Subject(s)
Chimera , Stem Cells/cytology , Animals , Biological Evolution , Blastocyst/cytology , Cell Lineage , Chimera/embryology , Drug Evaluation, Preclinical , Humans , Species Specificity , Stem Cell Research/ethics , Stem Cell Research/legislation & jurisprudenceABSTRACT
Induced pluripotent stem (iPS) cells provide powerful tools for studying disease mechanisms and developing therapies for diseases. The 8p11 myeloproliferative syndrome (EMS) is an aggressive chronic myeloproliferative disorder (MPD) that is caused by constitutive activation of fibroblast growth factor receptor 1. EMS is rare and, consequently, effective treatment for this disease has not been established. Here, iPS cells were generated from an EMS patient (EMS-iPS cells) to assist the development of effective therapies for EMS. When iPS cells were co-cultured with murine embryonic stromal cells, EMS-iPS cells produced more hematopoietic progenitor and hematopoietic cells, and CD34+ cells derived from EMS-iPS cells exhibited 3.2-7.2-fold more macrophage and erythroid colony forming units (CFUs) than those derived from control iPS cells. These data indicate that EMS-iPS cells have an increased hematopoietic differentiation capacity, which is characteristic of MPDs. To determine whether a tyrosine kinase inhibitor (TKI) could suppress the increased number of CFUs formed by EMS-iPS-induced CD34+ cells, cells were treated with one of four TKIs (CHIR258, PKC 412, ponatinib, and imatinib). CHIR258, PKC 412, and ponatinib reduced the number of CFUs formed by EMS-iPS-induced CD34+ cells in a dose-dependent manner, whereas imatinib did not. Similar effects were observed on primary peripheral blood cells (more than 90% of which were blasts) isolated from the patient. This study provides evidence that the EMS-iPS cell line is a useful tool for the screening of drugs to treat EMS and to investigate the mechanism underlying this disease.
Subject(s)
Induced Pluripotent Stem Cells/drug effects , Myeloproliferative Disorders/drug therapy , Myeloproliferative Disorders/genetics , Oncogene Proteins, Fusion/genetics , Protein Kinase Inhibitors/therapeutic use , Receptor, Fibroblast Growth Factor, Type 1/genetics , Translocation, Genetic , Adolescent , Benzimidazoles/therapeutic use , Cells, Cultured , Drug Evaluation, Preclinical , Hematopoiesis , Humans , Imatinib Mesylate/therapeutic use , Imidazoles/therapeutic use , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/pathology , Male , Myeloproliferative Disorders/pathology , Pyridazines/therapeutic use , Quinolones/therapeutic use , Staurosporine/analogs & derivatives , Staurosporine/therapeutic useABSTRACT
This study was undertaken to establish rat embryonic stem (ES) cells from parthenogenetically developing blastocysts. Ten blastocysts were prepared by treatment of ovulated rat oocytes with ionomycin and cycloheximide, and three alkaline phosphatase-positive ES cell lines were established using the N2B27 medium supplemented with mitogen activated protein kinase kinase inhibitor PD0325901, glycogen synthase kinase 3 inhibitor CHIR99021, rat leukemia inhibitory factor, and forskolin. Expression of stem cell marker genes (Oct-4, rNanog, Fgf-4, and Rex-1) was confirmed in all three ES cell lines by reverse transcriptase-polymerase chain reaction (RT-PCR). Combined bisulfite restriction analysis showed that the differentially methylated region locus of five imprinted genes (H19, Meg3IG, Igf2r, Peg5, and Peg10) in these ES cells remained to be demethylated or was hypomethylated, which was similar to that in control ES cells established from normal blastocysts. Characteristics of the parthenogenetic blastocyst-derived ES cells were successfully transmitted to the next generation through a chimeric rat for one of the three ES cell lines. This is the first report on germline-competent (genuine) ES cells derived from parthenogenetically developing rat blastocysts.
Subject(s)
Blastocyst/metabolism , Embryonic Stem Cells/metabolism , Oocytes/metabolism , Parthenogenesis , Adjuvants, Immunologic/pharmacology , Animals , Benzamides/pharmacology , Calcium Ionophores/pharmacology , Cell Differentiation , Cells, Cultured , Colforsin/pharmacology , Cycloheximide/pharmacology , DNA Methylation , Diphenylamine/analogs & derivatives , Diphenylamine/pharmacology , Female , Fibroblast Growth Factor 4/biosynthesis , Genetic Markers , Glycogen Synthase Kinase 3/antagonists & inhibitors , Ionomycin/pharmacology , Leukemia Inhibitory Factor/pharmacology , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Nanog Homeobox Protein , Octamer Transcription Factor-3/biosynthesis , Oocytes/drug effects , Pyridines/pharmacology , Pyrimidines/pharmacology , Rats , Transcription Factors/biosynthesisABSTRACT
UNLABELLED: The molecular mechanisms regulating differentiation of fetal hepatic stem/progenitor cells, called hepatoblasts, which play pivotal roles in liver development, remain obscure. Wnt signaling pathways regulate the development and differentiation of stem cells in various organs. Although a ß-catenin-independent noncanonical Wnt pathway is essential for cell adhesion and polarity, the physiological functions of noncanonical Wnt pathways in liver development are unknown. Here we describe a functional role for Wnt5a, a noncanonical Wnt ligand, in the differentiation of mouse hepatoblasts. Wnt5a was expressed in mesenchymal cells and other cells of wild-type (WT) midgestational fetal liver. We analyzed fetal liver phenotypes in Wnt5a-deficient mice using a combination of histological and molecular techniques. Expression levels of Sox9 and the number of hepatocyte nuclear factor (HNF)1ß(+) HNF4α(-) biliary precursor cells were significantly higher in Wnt5a-deficient liver relative to WT liver. In Wnt5a-deficient fetal liver, in vivo formation of primitive bile ductal structures was significantly enhanced relative to WT littermates. We also investigated the function of Wnt5a protein and downstream signaling molecules using a three-dimensional culture system that included primary hepatoblasts or a hepatic progenitor cell line. In vitro differentiation assays showed that Wnt5a retarded the formation of bile duct-like structures in hepatoblasts, leading instead to hepatic maturation of such cells. Whereas Wnt5a signaling increased steady-state levels of phosphorylated calcium/calmodulin-dependent protein kinase II (CaMKII) in fetal liver, inhibition of CaMKII activity resulted in the formation of significantly more and larger-sized bile duct-like structures in vitro compared with those in vehicle-supplemented controls. CONCLUSION: Wnt5a-mediated signaling in fetal hepatic stem/progenitor cells suppresses biliary differentiation. These findings also suggest that activation of CaMKII by Wnt5a signaling suppresses biliary differentiation. (HEPATOLOGY 2013;).
Subject(s)
Bile Ducts, Intrahepatic/embryology , Cell Differentiation , Fetal Stem Cells/physiology , Wnt Proteins/metabolism , Animals , Bile Ducts, Intrahepatic/cytology , Bile Ducts, Intrahepatic/metabolism , Biomarkers/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Cell Line , Frizzled Receptors/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Phosphorylation , Wnt-5a ProteinABSTRACT
Plasminogen activator inhibitor-1 (PAI-1), an endogenous inhibitor of a major fibrinolytic factor, tissue-type plasminogen activator, can both promote and inhibit angiogenesis. However, the physiologic role and the precise mechanisms underlying the angiogenic effects of PAI-1 remain unclear. In the present study, we report that pharmacologic inhibition of PAI-1 promoted angiogenesis and prevented tissue necrosis in a mouse model of hind-limb ischemia. Improved tissue regeneration was due to an expansion of circulating and tissue-resident granulocyte-1 marker (Gr-1(+)) neutrophils and to increased release of the angiogenic factor VEGF-A, the hematopoietic growth factor kit ligand, and G-CSF. Immunohistochemical analysis indicated increased amounts of fibroblast growth factor-2 (FGF-2) in ischemic gastrocnemius muscle tissues of PAI-1 inhibitor-treated animals. Ab neutralization and genetic knockout studies indicated that both the improved tissue regeneration and the increase in circulating and ischemic tissue-resident Gr-1(+) neutrophils depended on the activation of tissue-type plasminogen activator and matrix metalloproteinase-9 and on VEGF-A and FGF-2. These results suggest that pharmacologic PAI-1 inhibition activates the proangiogenic FGF-2 and VEGF-A pathways, which orchestrates neutrophil-driven angiogenesis and induces cell-driven revascularization and is therefore a potential therapy for ischemic diseases.
Subject(s)
Angiogenesis Inducing Agents/metabolism , Neovascularization, Physiologic/drug effects , Neutrophils/drug effects , Piperazines/pharmacology , Regeneration/drug effects , Serpin E2/antagonists & inhibitors , para-Aminobenzoates , 4-Aminobenzoic Acid/pharmacology , Animals , Cells, Cultured , Drug Evaluation, Preclinical , Fibrinolytic Agents/pharmacology , Humans , Matrix Metalloproteinase 9/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Neutrophils/immunology , Neutrophils/metabolism , Neutrophils/physiology , Regeneration/physiology , Tissue Plasminogen Activator/genetics , Up-Regulation/drug effectsABSTRACT
OBJECTIVE: The signaling by thrombopoietin (TPO) via its receptor, c-MPL, plays a crucial role in the maintenance of hematopoietic stem cells (HSCs). Small-molecule c-MPL agonists have recently been shown to be beneficial in the treatment of thrombocytopenia. However, their effects on HSCs have not yet been explored. In this study, we evaluated the effects of NR-101, a novel small-molecule c-MPL agonist, on the ex vivo expansion of human cord blood (hCB) HSCs. MATERIALS AND METHODS: hCB CD34(+) or CD34(+)CD38(-) hematopoietic stem and progenitor cells were cultured for 7 days in the presence of thrombopoietin (TPO) or NR-101, and then subjected to flow cytometric analyses, colony-forming cell assays, and severe combined immunodeficiency-repopulating cell assays. RESULTS: During a 7-day culture of CD34(+) or CD34(+)CD38(-) hematopoietic stem and progenitor cells, NR-101 efficiently increased their numbers, with a greater than twofold increase compared to TPO, although its effect on megakaryocytopoiesis was comparable to that of TPO. Correspondingly, severe combined immunodeficiency-repopulating cells were increased 2.9-fold during a 7-day culture with NR-101 compared to freshly isolated CD34(+) cells, and 2.3-fold compared to that with TPO. Of note, NR-101 persistently activated signal transducer and activator of transcription (STAT) 5 but not signal transducer and activator of transcription 3. Furthermore, NR-101 induced a long-term accumulation of hypoxia-inducible factor-1alpha protein and enhanced activation of its downstream target genes. CONCLUSION: This is the first time that a small-molecule c-MPL agonist has been demonstrated to promote net expansion of HSCs. NR-101 is more efficient in ex vivo expansion of HSCs than TPO. NR-101 could be a useful tool for the therapeutic manipulation of human HSCs.