The Expression Of Cited2 In Cardiac Stem Cells

Objectives:Cited2, a member of the transcriptional co-factors family, is critically involved in the regulation of cell senescence and apoptosis. Recently, a number of studies have demonstrated that Cited2 plays an important role in the maintenance of tissue-specifc stem cells, and is essential for development of the heart. Based on these observations, we hypothesize that Cited2 might be a key regulator of cardiac stem cell function. The purpose of this study is to characterize the expression pattern of transcriptional co-factor Cited2 in cardiac stem cells of different age mice, which may serve as a entry into further dessecting the regulatory roles and molecular mechanisms of Cited2 in cardiac stem cell maintenance and aging.Methods:(1) The isolation of mouse cardiac cells:C57BL/6J mice were used in the present study. Mice were killed by cervical dislocation and the heart was collected. Heart single cell suspension was prepared by mechanical shear method and collagenase Ⅱ digestion. (2) Flow cytometry analysis and sorting of mouse cardiac cells:Freshly isolated cells were first stained with different combination of fluorescent-labeled antibodies against a series of surface markers, and then analyzed or sorted by flow cytometry analysis. (3) Immunefluorescence assays:Cells were seeded onto coverslips. The cells were fixed with 4% paraformaldehyde and blocked with bovine serum albumin. After, incubating with specific primary antibodies overnight, the cells were washed and incubated with secondary antibodies. Nucleus were stained with DAPI. Stained cells were observed under a fluorescence microscope. (4) In vitro culture of cardiac stem cells:FACS-sorted cells were seeded into 96-well culture plates at a density of 3×105-cells/well, and cultured in growth medium. The medium was replaced half with fresh medium every two to three days. Cells were monitored for sphere formation or morphological changes. (5) RT-PCR analysis of gene expression:Total RNA was extracted, and the first strand cDNA of each gene was synthezied and used, as template for PCR reaction. The PCR products were examined by agarose gel electrophoresis, and analyze by a gel imager.Results:(1) Characterization of mouse cardiac stem cells:By lebeling freshly isolated cardiac cells with anti mouse Sca-1 antibody and other anti-surface marker antibodies, we observed that mouse heart harbors a Lin- CD45-Sca-1+ cell population. The percentage of these cells among total cardiac cells was 2.98±1.23%. In vitro, Lin-CD45-Sca-1+ cells were able to proliferate and form cardiac spheres. Work from our laboratory and others have also showed that Lin-CD45-Sca-1+ cells can differentiate into myocardial cells, smooth muscle cells and endothelial cells in vitro. Take together, the Lin-CD45-Sca-1+ cell population, which is endowed with typical properties of stem cells such as self-renewal and differentiation, may represent the prime cardiac stem cells.(2)Age-related changes in abundance of mouse cardiac stem cells:No apperent age-dependent changes were detecteded in the total cardiac cell numbers of mice. However, the number of Lin-CD45-Sca-1+ cells appeared to increase gradually as aging. The percentage of Lin-CD45-Sca-1+ cells among total cardiac cells in old mice (13.11 ±1.14%) was significantly higher than that of either young or middle age mice (P<0.05). The content of Lin-CD45-Sca-1+ cells was also increased in middle age mice (9.96±0.49%) compared to young mice (10.44±0.25%), although the change was not significant. We further analysed the cell cycle distribution of Lin-CD45-Sca-1+ cells from different age mice. In consistent with cell contents, Lin-CD45-Sca-1+ cells in aged mice contained significant higher number of cells in S phase than young mice (12.58±4.31%, elderly VS 11.98±2.33%, youth,*P< 0.05), suggesing that old cardiac stem cells become more active in proliferation, and consequently lead to an increase in the cardiac stem cells size.(3) Expression of Cited2 gene in cardiac cells:We first analyed Cited2 expression in total cardiac cells of different age mice. The results showed that the expression of Cited2 decreased gradually with increasing age, but there is no significant difference (youth VS middle-aged VS old:0.4033±0.0782 VS 0.3490±0.1756 VS 0.2490±0.0515). Then we measured the expression of Cited2 in the specific Lin-CD45-Sca-1+ cells isolated from mice of different ages. Similar to the expression pattern observed with cardiac tissues, Cited2 expression of Lin-CD45-Sca-1+ cell also decreased with age, and increasingly, the Cited2 mRNA level in cells of aged mice was significant higher that young mice(0.7232±0.0741, youth VS 0.3637±0.1583, old,*P< 0.05). These results strongly suggest that Cited2 is expressed mainly in cardiac stem cells, and its expression was larely diminished in aged mice.Conclusion:The number of mouse cardiac stem cells (Lin-CD45-Sca-1+ cells) increases as age, which may result from an increased proliferative ablity. The transcription co-factor Cited2 is expressed primarily in the cardiac stem cell compartment of mouse heart, and its expression leveles gradually decrease with increasing age. Based on these findings, we speculate that Cited2 may play a fundermental role in cardiac stem cell self-renewal.