title: Activation of Notch1 signalling promotes multi-lineage differentiation of c-Kit(POS)/NKX2.5(POS) bone marrow stem cells: implication in stem cell translational medicine.
abstract: BACKGROUND
  Transplantation of bone marrow mesenchymal stem cells (BMSCs) can repair injured hearts. However, whether BMSC populations contain cells with cardiac stem cell characteristics is ill-defined. We report here that Notch signalling can promote differentiation of c-Kit(POS)/NKX2.5(POS) BMSCs into cardiomyocyte-like cells.
  
  
  METHODS
  Total BMSCs were isolated from Sprague-Dawley rat femurs and c-Kit(POS) cells were purified. c-Kit(POS)/NKX2.5(POS) cells were isolated by single-cell cloning, and the presence of cardiomyocyte, smooth muscle cell (SMC), and endothelial cell differentiation markers assessed by immunofluorescence staining and semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis. Levels of c-Kit and Notch1-4 in total BMSCs and c-Kit(POS)/NKX2.5(POS) BMSCs were quantitated by flow cytometry. Following infection with an adenovirus over-expressing Notch1 intracellular domain (NICD), total BMSCs and c-Kit(POS)/NKX2.5(POS) cells were assessed for differentiation to cardiomyocyte, SMC, and endothelial cell lineages by immunofluorescence staining and real-time quantitative RT-PCR. Total BMSCs and c-Kit(POS)/NKX2.5(POS) cells were treated with the Notch1 ligand Jagged1 and markers of cardiomyocyte, SMC, and endothelial cell differentiation were examined by immunofluorescence staining and real-time quantitative RT-PCR analysis.
  
  
  RESULTS
  c-Kit(POS)/NKX2.5(POS) cells were present among total BMSC populations, and these cells did not express markers of adult cardiomyocyte, SMC, or endothelial cell lineages. c-Kit(POS)/NKX2.5(POS) BMSCs exhibited a multi-lineage differentiation potential similar to total BMSCs. Following sorting, the c-Kit level in c-Kit(POS)/NKX2.5(POS) BMSCs was 84.4%. Flow cytometry revealed that Notch1 was the predominant Notch receptor present in total BMSCs and c-Kit(POS)/NKX2.5(POS) BMSCs. Total BMSCs and c-Kit(POS)/NKX2.5(POS) BMSCs overexpressing NICD had active Notch1 signalling accompanied by differentiation into cardiomyocyte, SMC, and endothelial cell lineages. Treatment of total BMSCs and c-Kit(POS)/NKX2.5(POS) BMSCs with exogenous Jagged1 activated Notch1 signalling and drove multi-lineage differentiation, with a tendency towards cardiac lineage differentiation in c-Kit(POS)/NKX2.5(POS) BMSCs.
  
  
  CONCLUSIONS
  c-Kit(POS)/NKX2.5(POS) cells exist in total BMSC pools. Activation of Notch1 signalling contributed to multi-lineage differentiation of c-Kit(POS)/NKX2.5(POS) BMSCs, favouring differentiation into cardiomyocytes. These findings suggest that modulation of Notch1 signalling may have potential utility in stem cell translational medicine.
authors.affiliation: Department of Pathology, Guangdong Medical University, Zhanjiang, 524023, China. dingran525@126.com.
authors.forename: Ranran
authors.initials: R
authors.lastname: Ding
authors.affiliation: Department of Pathology, Guangdong Medical University, Zhanjiang, 524023, China. Jxf6295096@163.com.
authors.forename: Xiaofan
authors.initials: X
authors.lastname: Jiang
authors.affiliation: Department of Pathology, Guangdong Medical University, Zhanjiang, 524023, China. 272220175@qq.com.
authors.forename: Yanping
authors.initials: Y
authors.lastname: Ha
authors.affiliation: Department of Pathology, Guangdong Medical University, Zhanjiang, 524023, China. 823501000@qq.com.
authors.forename: Zhenliang
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Cardiovascular Institute of Affiliated Hospital, Hainan Medical College, Haikou, 571199, China. guojl79@163.com.
authors.forename: Junli
authors.initials: J
authors.lastname: Guo
authors.affiliation: Department of Pathology, Guangdong Medical University, Zhanjiang, 524023, China. Jianghg12@163.com.
authors.forename: Hanguo
authors.initials: H
authors.lastname: Jiang
authors.affiliation: Cardiovascular Institute of Affiliated Hospital, Hainan Medical College, Haikou, 571199, China. zhengsj2008@163.com.
authors.forename: Shaojiang
authors.initials: S
authors.lastname: Zheng
authors.affiliation: Department of Pathology, Guangdong Medical University, Zhanjiang, 524023, China. szh75@126.com.
authors.forename: Zhihua
authors.initials: Z
authors.lastname: Shen
authors.affiliation: Department of Pathology, Guangdong Medical University, Zhanjiang, 524023, China. Jiewei74@126.com.
authors.forename: Wei
authors.initials: W
authors.lastname: Jie
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation; D019070:Cell Lineage; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D000072100:Jagged-1 Protein; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D008840:Microfilament Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009124:Muscle Proteins / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000166:cytology; D032389:Myocytes, Smooth Muscle / Q000166:cytology; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D051881:Receptor, Notch1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000072099:Serrate-Jagged Proteins; D015398:Signal Transduction; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D020107:Troponin T / Q000235:genetics / Q000378:metabolism; D014841:von Willebrand Factor / Q000235:genetics / Q000378:metabolism
chemical_list: D002135:Calcium-Binding Proteins; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins; D036341:Intercellular Signaling Peptides and Proteins; C000606275:Jag1 protein, rat; D000072100:Jagged-1 Protein; D008565:Membrane Proteins; D008840:Microfilament Proteins; D009124:Muscle Proteins; C524161:Nkx2-5 protein, rat; C497763:Notch1 protein, rat; D051881:Receptor, Notch1; D000072099:Serrate-Jagged Proteins; D014157:Transcription Factors; D020107:Troponin T; C029417:Von Willebrand antigen; C051615:transgelin; D014841:von Willebrand Factor; D019009:Proto-Oncogene Proteins c-kit