Isolation And Characterization Of Adult Cardiac Stem Cells From Human And Murine Heart

Background:The heart has been traditionally regarded as terminally differentiated organ that adapt to increased work and compensate for disease exclusively through hypertrophy.Recent studies showed that intrinsic cardiac stem cells exist in the mammalian heart which have potent proliferation and differentiation to cardiac cells,endothelial cells and smooth muscle cells.Recent evidence suggested that cardiac stem cells have been considered to have most potential clinical application to treat patients with heart failure.Aims:This present study describes the isolation and preliminary characterization of cells from the adult human and murine heart.Methods:1.tissue samples:human tissue was derived from right atrial belonging to patients undergoing heart surgery and right septum by biopsy.2.cell isolation and primary cell culture:isolated myocardial tissue was cut into 1 to 2 mm3 pieces, washed with Ca2+-Mg2+-free phosphate-buffered solution and digested two times for 20 minutes at 37℃ with 0.2% or 0.4% collagenase Ⅱ.The obtained cells were cultured with medium supplemented with 10%FBS.3 secondary cell culture:CSCs were passaged every 8-10 days.4 idenfication:hCSCs were labeled with the following antibodies: CD29、CD90、CD105、CD45、Stro-land HLA-DR. Cell events were collected by FACS Calibur flow cytometer and data were analyzed by Cell Quest.Total RNA was exacted from cells using TaKaRa RNA PCR Kit and RT-PCR was performed with a SuperScript Ⅲ First Strand Synthesis Systerm.Results:The cells obstained became loosely adherent after seeded in dish about 1-2 days later,and became confluent about 7-9 days later. They can expand in vitro to reach enough cell number for cell transplantation.FACS analysis revealed that hCSCs did not express the hematopoietic progenitor cell -specific surface antigens:CD45 and HLA-DR, while they were positive for typical mesenchymal stem cells surface antigens: CD29、CD90、CD105 .RT-PCR showed that hCSCs expressed Rexl, Nanog, Sox2 and Oct4, suggesting that hCSCs express the embryonic stem cell markers and contain the mesenchymal cell-like population.Conculsions:We developed a stable and efficient method to isolate and expand CSCs successfully.More importantly, our data confirmed that it is possible to isolate cells from very small fragments of human myocardium and expand these cells in vitro in a month to reach numbers that would be appropriate for in vivo transplantation in patients.