Subpopulation Analysis of Sca-1, Nanog, and Islet-1 Positive Cells in Myocardial Tissue

Background: Since cells in myocardial tissue do not only express a single marker, several different stem cell markers may be colocalized on the same type of cell simultaneously. Therefore, using only one stem cell marker protein to classify myocardial cell populations may not be accurate. There are few reports on the co-expression of various marker proteins in the heart. Therefore, to investigate the co-expression between different stem-cell or embryonic markers in myocardial tissue, we selected three typical molecular markers, namely stem cell antigen-1 (Sca-1), an embryonic transcription factor (Nanog) and Insulin gene enhancer protein-1 (Islet-1).Methods: We obtained the hearts of newborn, 1-week-old, 3-week-old, and 5-week-old mice, and separated the cells using density gradient centrifugation. The expression of Sca-1, Nanog, and Islet-1 in cardiac cells of different age groups was detected by immunofluorescence staining, myocardial differentiation potential identification, and Western blotting. Immunofluorescence analysis was used to co-express Sca-1, Nanog, and Islet-1. Single cell RNA sequencing was used to analyze cardiac tissue cell type and stem cell gene localization.Results: The results showed that Sca-1+ and Islet-1+ cells had a stronger potential to express cardiac troponin T (cTnT) than Nanog+ cells (p < 0.05). Western blotting results showed that with the increase in age, the expression of Sca-1 first increased and then decreased, while the expression of Nanog steadily increased (p < 0.05). The expression of Islet-1 did not change significantly (p > 0.05). Immunofluorescence results showed that the co-expression rates of Sca-1 and Islet-1 increased from birth to the 3rd week, and then gradually decreased (p < 0.05). The co-expression rate of Sca-1 and Nanog continued to increase from birth, and showed a downward trend at the 5th week (p < 0.05). The co-expression rate of Islet-1 and Nanog gradually increased from 1 week after birth (p < 0.05). Single-cell RNA sequencing analysis identified eight major cell populations, including cardiomyocytes. Further analysis showed that Sca-1 positive cells had the highest proportion and were mainly concentrated in fibroblasts and endothelial cells.Conclusion: Sca-1, Nanog, and Islet-1 positive cells vary dynamically at different stages of cardiac development. Therefore, a single stem cell marker cannot be used to classify cardiac stem cell subpopulations.