title: Rho-associated kinase inhibitors promote the cardiac differentiation of embryonic and induced pluripotent stem cells.
abstract: BACKGROUND
  Rho-associated kinase (ROCK) plays an important role in maintaining embryonic stem (ES) cell pluripotency. To determine whether ROCK is involved in ES cell differentiation into cardiac and hematopoietic lineages, we evaluated the effect of ROCK inhibitors, Y-27632 and fasudil on murine ES and induced pluripotent stem (iPS) cell differentiation.
  
  
  METHODS
  Gene expression levels were determined by real-time PCR, Western blot analysis and immunofluorescent confocal microscopy. Cell transplantation of induced differentiated cells were assessed in vivo in a mouse model (three groups, n=8/group) of acute myocardial infarction (MI). The cell engraftment was examined by immunohistochemical staining and the outcome was analyzed by echocardiography.
  
  
  RESULTS
  Cells were cultured in hematopoietic differentiation medium in the presence or absence of ROCK inhibitor and colony formation as well as markers of ES, hematopoietic stem cells (HSC) and cells of cardiac lineages were analyzed. ROCK inhibition resulted in a drastic change in colony morphology accompanied by loss of hematopoietic markers (GATA-1, CD41 and β-Major) and expressed markers of cardiac lineages (GATA-4, Isl-1, Tbx-5, Tbx-20, MLC-2a, MLC-2v, α-MHC, cTnI and cTnT) in murine ES and iPS cells. Fasudil-induced cardiac progenitor (Mesp-1 expressing) cells were infused into a murine MI model. They engrafted into the peri-infarct and infarct regions and preserved left ventricular function.
  
  
  CONCLUSIONS
  These findings provide new insights into the signaling required for ES cell differentiation into hematopoietic as well as cardiac lineages and suggest that ROCK inhibitors are useful in directing iPS cell differentiation into cardiac progenitor cells for cell therapy of cardiovascular diseases.
authors: Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Taiwan.
authors: Ya-Ting
authors: YT
authors: Cheng
authors: Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan; Department of Internal Medicine, Cardinal Tien Hospital, New Taipei City, Taiwan; Department of Internal Medicine, School of Medicine, Fu-Jen Catholic University, New Taipei City, Taiwan.
authors: Dong-Feng
authors: DF
authors: Yeih
authors: Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Taiwan.
authors: Shu-Man
authors: SM
authors: Liang
authors: Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Taiwan.
authors: Chia-Ying
authors: CY
authors: Chien
authors: Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Taiwan.
authors: Yen-Ling
authors: YL
authors: Yu
authors: Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan.
authors: Bor-Sheng
authors: BS
authors: Ko
authors: Department of Pathology and Laboratory Medicine, Taichung Veterans General Hospital, Taichung, Taiwan.
authors: Yee-Jee
authors: YJ
authors: Jan
authors: Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Taiwan.
authors: Cheng-Chin
authors: CC
authors: Kuo
authors: Institute of Biotechnology, National Taiwan University, Taipei, Taiwan.
authors: Li-Ying
authors: LY
authors: Sung
authors: Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.
authors: Song-Kun
authors: SK
authors: Shyue
authors: Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan.
authors: Ming-Fong
authors: MF
authors: Chen
authors: Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Taiwan.
authors: Shaw-Fang
authors: SF
authors: Yet
authors: Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Taiwan; Metabolomic Medicine Research Center, China Medical University, Taichung, Taiwan.
authors: Kenneth K
authors: KK
authors: Wu
authors: Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Taiwan; Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan. Electronic address: jliou@nhri.org.tw.
authors: Jun-Yang
authors: JY
authors: Liou
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D000577:Amides / Q000494:pharmacology; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D004452:Echocardiography; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D015870:Gene Expression; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000187:drug effects*; D008297:Male; D051379:Mice; D009203:Myocardial Infarction / Q000628:therapy; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects*; D040201:Platelet Membrane Glycoprotein IIb / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011725:Pyridines / Q000494:pharmacology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D033581:Stem Cell Transplantation / Q000379:methods; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000577:Amides; D051792:Basic Helix-Loop-Helix Proteins; D015415:Biomarkers; D040201:Platelet Membrane Glycoprotein IIb; D047428:Protein Kinase Inhibitors; D011725:Pyridines; D054460:rho-Associated Kinases; C101538:Mesp1 protein, mouse; C108830:Y 27632; C049347:fasudil
keywords: Cardiac differentiation; Embryonic stem cells; Hematopoietic differentiation; Induced pluripotent stem cells; Rho-associated kinase