title: A highly sensitive and accurate method to quantify absolute numbers of c-kit+ cardiac stem cells following transplantation in mice.
abstract: Although transplantation of c-kit+ cardiac stem cells (CSCs) alleviates post-myocardial infarction left ventricular dysfunction, there are no reliable methods that enable measurement of the absolute number of CSCs that persist in the recipient heart. To overcome this limitation, we developed a highly sensitive and accurate method to quantify the absolute number of murine CSCs after transplantation. This method has two unique features: (1) real-time PCR-based detection of a novel male-specific, multiple-copy gene, Rbmy, which significantly increases the sensitivity of detection of male donor cells in a female recipient, and (2) an internal standard, which permits quantification of the absolute number of CSCs as well as the total number of cells in the recipient organ. Female C57BL/6 mice underwent coronary occlusion and reperfusion; 2 days later, 10(5) male mouse CSCs were injected intramyocardially. Tissues were analyzed by real-time PCR at serial time points. In the risk region, >75 % of CSCs present at 5 min were lost in the ensuing 24 h; only 7.6 ± 2.1 % of the CSCs present at 5 min could still be found at 7 days after transplantation and only 2.8 ± 0.5 % (i.e., 1,224 ± 230 cells/heart) at 35 days. Thus, even after direct intramyocardial injection, the total number of CSCs that remain in the murine heart is minimal (at 24 h, ~10 % of the cells injected; at 35 days, ~1 %). This new quantitative method of stem cell detection, which enables measurement of absolute cell number, should be useful to optimize cell-based therapies, not only for CSCs but also for other stem cells and other organs.
authors.affiliation: Department of Medicine, Institute of Molecular Cardiology, University of Louisville, Louisville, KY 40202, USA.
authors.forename: Kyung U
authors.initials: KU
authors.lastname: Hong
authors.forename: Qian-Hong
authors.initials: QH
authors.lastname: Li
authors.forename: Yiru
authors.initials: Y
authors.lastname: Guo
authors.forename: Nikita S
authors.initials: NS
authors.lastname: Patton
authors.forename: Afsoon
authors.initials: A
authors.lastname: Moktar
authors.forename: Aruni
authors.initials: A
authors.lastname: Bhatnagar
authors.forename: Roberto
authors.initials: R
authors.lastname: Bolli
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002452:Cell Count; D058948:Cell Tracking / Q000379:methods* / Q000592:standards; D002478:Cells, Cultured; D004195:Disease Models, Animal; D005260:Female; D005786:Gene Expression Regulation; D005819:Genetic Markers; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009203:Myocardial Infarction / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000601:surgery*; D032383:Myocytes, Cardiac / Q000378:metabolism / Q000637:transplantation*; D009687:Nuclear Proteins / Q000235:genetics*; D019009:Proto-Oncogene Proteins c-kit / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards; D012015:Reference Standards; D015203:Reproducibility of Results; D033581:Stem Cell Transplantation; D013997:Time Factors
chemical_list: D015415:Biomarkers; D005819:Genetic Markers; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; C501500:Rbmy protein, mouse; D019009:Proto-Oncogene Proteins c-kit
references.citation: Nature. 2003 Jun 19;423(6942):825-37
references.pmid: 12815422
references.citation: Proc Natl Acad Sci U S A. 2004 Nov 30;101(48):16891-6
references.pmid: 15548607
references.citation: Circulation. 2009 Sep 29;120(13):1222-30
references.pmid: 19752329
references.citation: Am J Pathol. 2005 Sep;167(3):663-71
references.pmid: 16127147
references.citation: J Assist Reprod Genet. 2008 Jul;25(7):323-31
references.pmid: 18677557
references.citation: Nat Biotechnol. 2007 Sep;25(9):1015-24
references.pmid: 17721512
references.citation: Xenotransplantation. 2009 May-Jun;16(3):145-51
references.pmid: 19566654
references.citation: Basic Res Cardiol. 2011 Sep;106(5):849-64
references.pmid: 21516491
references.citation: Proc Natl Acad Sci U S A. 2007 Aug 28;104(35):14068-73
references.pmid: 17709737
references.citation: J Mol Cell Cardiol. 2008 Oct;45(4):567-81
references.pmid: 18466917
references.citation: Circulation. 2002 Jan 1;105(1):93-8
references.pmid: 11772882
references.citation: Nature. 1990 Jul 19;346(6281):240-4
references.pmid: 1695712
references.citation: Circulation. 2008 Sep 30;118(14):1425-32
references.pmid: 18794392
references.citation: Genomics. 1998 Apr 15;49(2):283-9
references.pmid: 9598316
references.citation: Circ Res. 2008 Jun 20;102(12):1471-82
references.pmid: 18566313
references.citation: Cardiovasc Res. 2010 Jun 1;86(3):365-73
references.pmid: 20071355
references.citation: J Neurosci Methods. 2006 Oct 30;157(2):225-9
references.pmid: 16735065
references.citation: Nature. 1990 Nov 29;348(6300):448-50
references.pmid: 2247149
references.citation: J Mol Cell Cardiol. 2002 Feb;34(2):107-16
references.pmid: 11851351
references.citation: J Mol Cell Cardiol. 2008 Oct;45(4):554-66
references.pmid: 18561945
references.citation: Nat Genet. 1999 Jul;22(3):223-4
references.pmid: 10391206
references.citation: Cell. 1993 Dec 31;75(7):1287-95
references.pmid: 8269511
references.citation: J Nucl Med. 2010 May 1;51 Suppl 1:128S-136S
references.pmid: 20395348
references.citation: Proc Natl Acad Sci U S A. 1999 Sep 28;96(20):11507-12
references.pmid: 10500207
references.citation: Circulation. 2003 Mar 11;107(9):1247-9
references.pmid: 12628942
references.citation: Nature. 1990 Jul 19;346(6281):245-50
references.pmid: 2374589
references.citation: Nucleic Acids Res. 2011 Jan;39(Database issue):D800-6
references.pmid: 21045057
references.citation: Am J Cardiol. 2007 Mar 1;99(5):732-8
references.pmid: 17317382
references.citation: Cardiovasc Res. 2005 Aug 1;67(2):301-7
references.pmid: 15907822
references.citation: Liver Transpl. 2002 Sep;8(9):822-8
references.pmid: 12200785
references.citation: Stem Cells. 2008 Jun;26(6):1646-55
references.pmid: 18420834
references.citation: Circulation. 2010 Jan 19;121(2):293-305
references.pmid: 20048209
references.citation: Cell Transplant. 2001 Jan-Feb;10(1):91-9
references.pmid: 11294476
references.citation: Hepatology. 1996 Apr;23(4):828-34
references.pmid: 8666338
references.citation: J Am Coll Cardiol. 2005 Oct 4;46(7):1339-50
references.pmid: 16198853
references.citation: J Mol Cell Cardiol. 1996 Aug;28(8):1737-46
references.pmid: 8877783
references.citation: Cell. 2003 Sep 19;114(6):763-76
references.pmid: 14505575
references.citation: Circulation. 1999 Jul 13;100(2):193-202
references.pmid: 10402450