title: Enhancement of Anti-Hypoxic Activity and Differentiation of Cardiac Stem Cells by Supernatant Fluids from Cultured Macrophages that Phagocytized Dead Mesenchymal Stem Cells.
abstract: BACKGROUND
  Most mesenchymal stem cells (MSCs) die shortly after transplantation into a myocardial infarcted area. Dead MSCs (dMSCs) are phagocytized by macrophages (pMΦ) in vivo and in vitro; however, the effects of pMΦ on cardiac stem cells (CSCs) remain unknown.
  
  
  METHODS
  MSCs, CSCs, and macrophages were obtained from bone marrow, hearts, and peritoneal cavity of mice, respectively. dMSCs were harvested after hypoxia for 24 h, and incubated with macrophages (2:1) for another 2 days with or without lipopolysaccharide (LPS, 50 ng/mL) and sorted by flow cytometry to obtain pMΦ. Viability and apoptosis of CSCs were respectively evaluated with the cell counting kit-8 (CCk-8) assay and Annexin V-PE/7-AAD staining at 0, 6, 12, and 24 h of culture with supernatant fluids from macrophages (MΦ), LPS-stimulated macrophages (LPS-pMΦ), pMΦ, and MSCs. GATA-4 and c-TnI expression was measured by flow cytometry on the seventh day. Expression of inflammation and growth factors was assessed by real-time polymerase chain reaction (RT-PCR) in MΦ, LPS-pMΦ, and pMΦ cells.
  
  
  RESULTS
  pMΦ expressed higher levels of interleukin-10 (IL-10) and transforming growth factor-β (TGF-β)and lower levels of tumor necrosis factor-α(TNF-α)and IL-6 than LPS-pMΦ, higher levels of growth factors and of GATA-4 and c-TnI at the 7th day, which were similar to those in MSCs. CSCs cultured with supernatant fluids of pMΦ exhibited higher proliferative, anti-hypoxic, and differentiation activities.
  
  
  CONCLUSIONS
  The supernatant fluids of macrophages that had phagocytized dead MSCs encouraged changes in phenotype and growth factor expression, enhanced proliferation, differentiation, and anti-hypoxic activity of CSCs, which is relevant to understanding the persistent therapeutic effect of MSCs after their massive demise upon transplantation in myocardial infarction. Furthermore, some miRNAs or proteins which were extracted from the supernatant fluids may give us a new insight into the treatment of myocardial infarction in the future.
authors.affiliation: Department of Cardiology, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, 1665 Kongjiang Road, Shanghai 200092, China. liuliangsuda@163.com.
authors.forename: Liang
authors.initials: L
authors.lastname: Liu
authors.affiliation: Department of Cardiology, Central Hospital of Minhang District, 170 Xinsong Road, Shanghai 201199, China. jinxianian@126.com.
authors.forename: Xian
authors.initials: X
authors.lastname: Jin
authors.affiliation: Department of Cardiology, Central Hospital of Minhang District, 170 Xinsong Road, Shanghai 201199, China. zhouzhonge2006@126.com.
authors.forename: Zhong'e
authors.initials: Z
authors.lastname: Zhou
authors.affiliation: Department of Cardiology, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, 1665 Kongjiang Road, Shanghai 200092, China. shencx@sjtu.edu.cn.
authors.forename: Chengxing
authors.initials: C
authors.lastname: Shen
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects*; D015687:Cell Hypoxia / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000494:pharmacology; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D006321:Heart / Q000187:drug effects / Q000502:physiology*; D017737:Macrophages, Peritoneal / Q000378:metabolism / Q000473:pathology*; D008297:Male; D059630:Mesenchymal Stem Cells / Q000378:metabolism / Q000473:pathology*; D051379:Mice; D008810:Mice, Inbred C57BL; D010587:Phagocytosis; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology*
chemical_list: D017077:Culture Media, Conditioned; D016207:Cytokines; D012333:RNA, Messenger
keywords: CSCs; dMSC; differentiation; growth factors; macrophages