Activation of Notch1 signalling promotes multi-lineage differentiation of c-KitPOS/NKX2.5POS bone marrow stem cells: implication in stem cell translational medicine

Ranran Ding, Xiaofan Jiang, Yanping Ha, Zhenliang Wang, Junli Guo, Hanguo Jiang, Shaojiang Zheng, Zhihua Shen, Wei Jie

Transplantation of bone marrow mesenchymal stem cells (BMSCs) can repair injured hearts. However, whether BMSC populations contain cells with cardiac stem cell characteristics is ill-defined. We report here that Notch signalling can promote differentiation of c-Kit(POS)/NKX2.5(POS) BMSCs into cardiomyocyte-like cells. Total BMSCs were isolated from Sprague-Dawley rat femurs and c-Kit(POS) cells were purified. c-Kit(POS)/NKX2.5(POS) cells were isolated by single-cell cloning, and the presence of cardiomyocyte, smooth muscle cell (SMC), and endothelial cell differentiation markers assessed by immunofluorescence staining and semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis. Levels of c-Kit and Notch1-4 in total BMSCs and c-Kit(POS)/NKX2.5(POS) BMSCs were quantitated by flow cytometry. Following infection with an adenovirus over-expressing Notch1 intracellular domain (NICD), total BMSCs and c-Kit(POS)/NKX2.5(POS) cells were assessed for differentiation to cardiomyocyte, SMC, and endothelial cell lineages by immunofluorescence staining and real-time quantitative RT-PCR. Total BMSCs and c-Kit(POS)/NKX2.5(POS) cells were treated with the Notch1 ligand Jagged1 and markers of cardiomyocyte, SMC, and endothelial cell differentiation were examined by immunofluorescence staining and real-time quantitative RT-PCR analysis. c-Kit(POS)/NKX2.5(POS) cells were present among total BMSC populations, and these cells did not express markers of adult cardiomyocyte, SMC, or endothelial cell lineages. c-Kit(POS)/NKX2.5(POS) BMSCs exhibited a multi-lineage differentiation potential similar to total BMSCs. Following sorting, the c-Kit level in c-Kit(POS)/NKX2.5(POS) BMSCs was 84.4%. Flow cytometry revealed that Notch1 was the predominant Notch receptor present in total BMSCs and c-Kit(POS)/NKX2.5(POS) BMSCs. Total BMSCs and c-Kit(POS)/NKX2.5(POS) BMSCs overexpressing NICD had active Notch1 signalling accompanied by differentiation into cardiomyocyte, SMC, and endothelial cell lineages. Treatment of total BMSCs and c-Kit(POS)/NKX2.5(POS) BMSCs with exogenous Jagged1 activated Notch1 signalling and drove multi-lineage differentiation, with a tendency towards cardiac lineage differentiation in c-Kit(POS)/NKX2.5(POS) BMSCs. c-Kit(POS)/NKX2.5(POS) cells exist in total BMSC pools. Activation of Notch1 signalling contributed to multi-lineage differentiation of c-Kit(POS)/NKX2.5(POS) BMSCs, favouring differentiation into cardiomyocytes. These findings suggest that modulation of Notch1 signalling may have potential utility in stem cell translational medicine.