Study on cardiac stem cell niche and its biological characteristics in vitro / 中华胸心血管外科杂志

ABSTRACT

Objective Endogenous cadiac stem cells become the famous star in the cardiac regeneration field in recent years.But the biological behavior of cardiac stem cells has not been fully explained clearly.This experiment intends to research the cardiac stem cell niche and its characteristics,as well as the succinct method for cardiac stem cells (Cardiospheres derived cells) culture in vitro.Methods Hearts from four 8-week-old SD rats were cut into pieces less than I mm3.Then,the small tissues were digested by trypsogen and collagenase in turn.And the undigested myocardium was collected and cultured in media until the de novo cells bespread the pertri-dish bottom.The 3rd generation of cardiac stem cells were harvested and prepared for immunofluorescence to detect the expression of related molecular markers,so as to identify the differentiation of these stem cells.Then,the cultural tissues were collected and embedded in paraffin,and the H-E staining,Masson staining,immunoflnorescence and TUNEL apoptosis assay were carried out.In addition,EdU was added into the medium and cuhured for 72 hours before the cultural tissues were harvested so as to label the proliferative cells.Results After 7 ～ 10 days in culture,some small,round and phase-bright cells aresed from the adherent plated tissue.These cells had the spontaneous differentiation potency in vitro culture.The immunofluorescence shows mitotic phase cells were c-kit positive,and most of passage cells were α-sarcomeric actin 、cardiac troponin T、flk-1 and vemintin positive while CD90 negative,but few of them were α-smooth muscle actin 、conexin-43 、CD31 and CD45 positive.There were lots of newborn cells grow exuberantly while accompanied apoptosis of myocardial cells in the paraffin section.The myocardial collagen remodels in the cultural myocardial tissues at the same time.The proliferative cells in the cultural tissues were EdU positive.Throe myocardium within the cultural tissues were MMP-2、MMP-9 and TGF-β1 positive,On the contrary,the proliferative cells were almost negative when stained with those antibodies.Conclusion Conclusions It's a convenient way to harvest cardiac stem cells by the use of myocarlial? tissues cultured in vitro.Those stem cells grow and proliferate in the cultural myocardial tissues accompanied with the degradation of extracellular matrix.Cardiospheres derived stem cells have the spontaneous differentiation potency when cultured in vito,and should be a promising? seed cells for stem cell therapy.