Isolation,Culture,Identification And Physiological Characteristics Of Sca-1~+ Cardiac Stem Cells In Newborn Mice

Background:Conventional methods for treating ischemic heart disease include drugs to improve clinical symptoms,cardiac interventional therapy or surgical bypass operation to improve blood supply.In recent years,people have begun to explore the possibility and methods of stem cell transplantation to repair myocardium,especially cardiac stem cells?CSCs?.It has been confirmed that sca-1⁺CSCs are the largest number of stem cell types in the heart.It can be differentiation into independent beating heart muscle cells both in vivo and in vitro.Other types of cardiac stem cells?such as c-kit⁺CSCs?after induction is more a preference for differentiation into vascular endothelial cells^[1-2].Thus,sca-1⁺cells are most closely related to myocardial origin in types of cardiac stem cell.Although sca-1⁺CSCs have been harvested from animals and human hearts,the improvement of heart function has been observed through cell transplantation experiments.However,the acquisition and cultivation methods of sca-1⁺CSCs are not mature yet.And the understanding of its physiological characteristics is not enough to apply it to clinical treatment.Objectives:1.Isolation and identification of sca-1⁺CSCs in newborn mice,culture and amplification in vitro.2.To study the surface markers of stem cell and the myocardial specific markers on the sca-1⁺CSCs.Methods:1.The heart of the Kunming mice born within 7 days was cut up into tissue pieces,and digested with 0.25%trypsin+0.1%type?collagenase repeatedly.After 10-14 days of tissues culture,the sca-1⁺cardiac stem cells were selected by MASC.The cells were cultured by CGM medium.The contents of sca-1⁺CSCs were determined by flow cytometry before and after separation.2.The expression of CD31,CD34 and c-kit were determined by flow cytometry.3.The expression of sca-1 and myocardial specific markers Nkx-2.5 and GATA-4were measured by immunofluorescence.Results:1.The Sca-1⁺CSCs are low in content before separation,only accounts for 5.44%of the proportion of the cells.After separation by MASC and send to the third generation,the Sca-1⁺CSCs content accounts for 86.73%.The first generation of Sca-1⁺CSCs were slow growth,and reach to 80%after 10 days.Cells have different shapes,the morphology is given priority to with spindle and triangle,gathered to grow.The cells can proliferate rapidly in CGM culture medium after passed down,can be passage again after about 6 days.2.The expression of CD31,CD34 and c-kit of Sca-1⁺CSCs were detected by flow cytometry.The results:cells don't express CD31 and CD34,and lower express c-kit?4.78%?.3.The expression of sca-1 and cardiogenic transcription factor Nkx-2.5 and GATA-4 of Sca-1⁺CSCs were measured by immunofluorescence.The results show that almost all of the cells express green fluorescence of FITC-Sca-1,and almost all of the cells express green fluorescence of FITC-GATA-4 and red fluorescence of PE-Nkx-2.5 at the same time.Conclusion:1.With the method of tissues culture combined with MASC,a considerable amount of high-purity sca-1⁺CSCs can be obtained from the heart of newborn Kunming mice.The CGM culture medium was used to develop the sca-1⁺CSCs after separation,and the cells were stable and the stem cell characteristics were maintained.The first generation of cells is slow to grow and can proliferate rapidly after passed down.2.The sca-1⁺CSCs of mouse do not express CD31 and CD34,which could eliminate the possibility of vascular endothelial stem cells and hematopoietic stem/progenitor cells.Cells low express c-kit,which means that c-kit⁺CSCs and Sca-1⁺CSCs can be obtained from the same individual heart tissue by cell surface markers.3.Sca-1⁺CSCs express the related proteins of myocardial specific markers Nkx-2.5 and GATA-4simultaneously,which proves that the Sca-1+CSCs are cardiogenic stem cells and have the potential to differentiate into cardiogenic cells.