title: Generation of functional platelets from canine induced pluripotent stem cells.
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.
authors: Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Japan.
authors: Toshiya
authors: T
authors: Nishimura
authors: Shingo
authors: S
authors: Hatoya
authors: Ryoji
authors: R
authors: Kanegi
authors: Kikuya
authors: K
authors: Sugiura
authors: Viskam
authors: V
authors: Wijewardana
authors: Mitsuru
authors: M
authors: Kuwamura
authors: Miyuu
authors: M
authors: Tanaka
authors: Jyoji
authors: J
authors: Yamate
authors: Takeshi
authors: T
authors: Izawa
authors: Masahiro
authors: M
authors: Takahashi
authors: Noritoshi
authors: N
authors: Kawate
authors: Hiromichi
authors: H
authors: Tamada
authors: Hiroshi
authors: H
authors: Imai
authors: Toshio
authors: T
authors: Inaba
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D001792:Blood Platelets / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002454:Cell Differentiation; D002478:Cells, Cultured; D004285:Dogs; D004622:Embryo, Mammalian; D058732:Embryoid Bodies; D005340:Fibrinogen / Q000737:chemistry; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism; D016086:Lentivirus; D008533:Megakaryocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D040201:Platelet Membrane Glycoprotein IIb / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D013917:Thrombin / Q000494:pharmacology
chemical_list: D036341:Intercellular Signaling Peptides and Proteins; D051741:Kruppel-Like Transcription Factors; D040201:Platelet Membrane Glycoprotein IIb; D000244:Adenosine Diphosphate; D005340:Fibrinogen; D013917:Thrombin