RRC ID 67692
Author Nishimura T, Hatoya S, Kanegi R, Sugiura K, Wijewardana V, Kuwamura M, Tanaka M, Yamate J, Izawa T, Takahashi M, Kawate N, Tamada H, Imai H, Inaba T.
Title Generation of functional platelets from canine induced pluripotent stem cells.
Journal Stem Cells Dev
Abstract Thrombocytopenia (TTP) is a blood disease common to canines and human beings. Currently, there is no valid therapy for this disease except blood transfusion. In this study, we report the generation of canine induced pluripotent stem cells (ciPSCs) from canine embryonic fibroblasts, and a novel protocol for creating mature megakaryocytes (MKs) and functional platelets from ciPSCs. The ciPSCs were generated using lentiviral vectors, and differentiated into MKs and platelets on OP9 stromal cells supplemented with growth factors. Our ciPSCs presented in a tightly domed shape and showed expression of a critical pluripotency marker, REX1, and normal karyotype. Additionally, ciPSCs differentiated into cells derived from three germ layers via the formation of an embryoid body. The MKs derived from ciPSCs had hyperploidy and transformed into proplatelets. The proplatelets released platelets early on that expressed specific MK and platelet marker CD41/61. Interestingly, these platelets, when activated with adenosine diphosphate or thrombin, bind to fibrinogen. Moreover, electron microscopy showed that the platelets had the same ultrastructure as peripheral platelets. Thus, we have demonstrated for the first time the generation of ciPSCs that are capable of differentiating into MKs and release functional platelets in vitro. Our system for differentiating ciPSCs into MKs and platelets promises a critical therapy for canine TTP and appears to be extensible in principle to resolve human TTP.
Volume 22(14)
Pages 2026-35
Published 2013-7-15
DOI 10.1089/scd.2012.0701
PMID 23409943
MeSH Adenosine Diphosphate / pharmacology Animals Blood Platelets / cytology Blood Platelets / drug effects Blood Platelets / metabolism* Cell Differentiation Cells, Cultured Dogs Embryo, Mammalian Embryoid Bodies Fibrinogen / chemistry Fibroblasts / cytology Fibroblasts / drug effects Fibroblasts / metabolism* Gene Expression Genetic Vectors Induced Pluripotent Stem Cells / cytology Induced Pluripotent Stem Cells / drug effects Induced Pluripotent Stem Cells / metabolism* Intercellular Signaling Peptides and Proteins / pharmacology Kruppel-Like Transcription Factors / genetics Kruppel-Like Transcription Factors / metabolism Lentivirus Megakaryocytes / cytology Megakaryocytes / drug effects Megakaryocytes / metabolism* Platelet Membrane Glycoprotein IIb / genetics Platelet Membrane Glycoprotein IIb / metabolism Protein Binding Thrombin / pharmacology
IF 3.153
DNA material CSIV-CMV-hOct3/4-IRES2-Venus (RDB08323) CSIV-CMV-hKlf4-IRES2-Venus (RDB08325) CSIV-CMV-hSox2-IRES2-Venus (RDB08324) CSIV-CMV-hc-Myc-IRES2-Venus (RDB08326)
Human and Animal Cells OP9(RCB1124)