title: Human cardiac stem cells isolated from atrial appendages stably express c-kit.
abstract: The in vivo studies of myocardial infarct using c-ki⁺/Lin⁻ cardiac stem cells (CSCs) are still in the early stage with margin or no beneficial effects for cardiac function. One of the potential reasons may be related to the absence of fully understanding the properties of these cells both in vitro and in vivo. In the present study, we aimed to systematically examine how CSCs adapted to in vitro cell processes and whether there is any cell contamination after long-term culture. Human CSCs were enzymatically isolated from the atrial appendages of patients. The fixed tissue sections, freshly isolated or cultured CSCs were then used for identification of c-kit⁺/Lin⁻ cells, detection of cell contamination, or differentiation of cardiac lineages. By specific antibody staining, we demonstrated that tissue sections from atrial appendages contained less than 0.036% c-kit⁺/Lin⁻ cells. For the first time, we noted that without magnetic activated cell sorting (MACS), the percentages of c-kit⁺/Lin⁻ cells gradually increased up to ∼40% during continuously culture between passage 2 to 8, but could not exceed >80% unless c-kit MACS was carried out. The resulting c-kit⁺/Lin⁻ cells were negative for CD34, CD45, CD133, and Lin markers, but positive for KDR and CD31 in few patients after c-kit MACS. Lin depletion seemed unnecessary for enrichment of c-kit⁺/Lin⁻ cell population. Following induced differentiation, c-kit⁺/Lin⁻ CSCs demonstrated strong differentiation towards cardiomyocytes but less towards smooth and endothelial cells. We concluded that by using an enzymatic dissociation method, a large number, or higher percentage, of relative pure human CSCs with stable expression of c-kit⁺ could be obtained from atrial appendage specimens within ∼4 weeks following c-kit MACS without Lin depletion. This simple but cost-effective approach can be used to obtain enough numbers of stably-expressed c-kit⁺/Lin⁻ cells for clinical trials in repairing myocardial infarction.
authors.affiliation: Institute of Molecular Cardiology, University of Louisville, Louisville, Kentucky, United States of America.
authors.forename: Jia-Qiang
authors.initials: JQ
authors.lastname: He
authors.forename: Duc Minh
authors.initials: DM
authors.lastname: Vu
authors.forename: Greg
authors.initials: G
authors.lastname: Hunt
authors.forename: Atul
authors.initials: A
authors.lastname: Chugh
authors.forename: Aruni
authors.initials: A
authors.lastname: Bhatnagar
authors.forename: Roberto
authors.initials: R
authors.lastname: Bolli
mesh_terms: D000918:Antibody Specificity / Q000187:drug effects; D020517:Atrial Appendage / Q000166:cytology*; D001374:Azacitidine / Q000494:pharmacology; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D019070:Cell Lineage / Q000187:drug effects; D002469:Cell Separation / Q000379:methods*; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005434:Flow Cytometry; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D008280:Magnetics; D008407:Mast Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009206:Myocardium / Q000166:cytology*; D019009:Proto-Oncogene Proteins c-kit / Q000378:metabolism*; D013194:Staining and Labeling; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D015415:Biomarkers; D014157:Transcription Factors; D019009:Proto-Oncogene Proteins c-kit; D001374:Azacitidine