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

Abstract

Introduction: 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-KitPOS/NKX2.5POS BMSCs into cardiomyocyte-like cells. Methods: Total BMSCs were isolated from Sprague-Dawley rat femurs and c-KitPOS cells were purified. c-KitPOS/NKX2.5POS 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-KitPOS/NKX2.5POS BMSCs were quantitated by flow cytometry. Following infection with an adenovirus over-expressing Notch1 intracellular domain (NICD), total BMSCs and c-KitPOS/NKX2.5POS 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-KitPOS/NKX2.5POS 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. Results: c-KitPOS/NKX2.5POS cells were present among total BMSC populations, and these cells did not express markers of adult cardiomyocyte, SMC, or endothelial cell lineages. c-KitPOS/NKX2.5POS BMSCs exhibited a multi-lineage differentiation potential similar to total BMSCs. Following sorting, the c-Kit level in c-KitPOS/NKX2.5POS BMSCs was 84.4%. Flow cytometry revealed that Notch1 was the predominant Notch receptor present in total BMSCs and c-KitPOS/NKX2.5POS BMSCs. Total BMSCs and c-KitPOS/NKX2.5POS BMSCs overexpressing NICD had active Notch1 signalling accompanied by differentiation into cardiomyocyte, SMC, and endothelial cell lineages. Treatment of total BMSCs and c-KitPOS/NKX2.5POS BMSCs with exogenous Jagged1 activated Notch1 signalling and drove multi-lineage differentiation, with a tendency towards cardiac lineage differentiation in c-KitPOS/NKX2.5POS BMSCs. Conclusions: c-KitPOS/NKX2.5POS cells exist in total BMSC pools. Activation of Notch1 signalling contributed to multi-lineage differentiation of c-KitPOS/NKX2.5POS BMSCs, favouring differentiation into cardiomyocytes. These findings suggest that modulation of Notch1 signalling may have potential utility in stem cell translational medicine.