Ding Ranran, Jiang Xiaofan, Ha Yanping, Wang Zhenliang, Guo Junli, Jiang Hanguo, Zheng Shaojiang, Shen Zhihua, Jie Wei
Department of Pathology, Guangdong Medical University, Zhanjiang, 524023, China.
Cardiovascular Institute of Affiliated Hospital, Hainan Medical College, Haikou, 571199, China.
Stem Cell Res Ther. 2015 May 9;6(1):91. doi: 10.1186/s13287-015-0085-2.
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.
骨髓间充质干细胞（BMSC）移植可修复受损心脏。然而，BMSC群体中是否含有具有心脏干细胞特征的细胞尚不清楚。我们在此报告，Notch信号可促进c-Kit(POS)/NKX2.5(POS) BMSC分化为心肌样细胞。
从Sprague-Dawley大鼠股骨中分离出总BMSC，并纯化c-Kit(POS)细胞。通过单细胞克隆分离出c-Kit(POS)/NKX2.5(POS)细胞，并通过免疫荧光染色和半定量逆转录聚合酶链反应（RT-PCR）分析评估心肌细胞、平滑肌细胞（SMC）和内皮细胞分化标志物的存在情况。通过流式细胞术定量总BMSC和c-Kit(POS)/NKX2.5(POS) BMSC中c-Kit和Notch1-4的水平。在用过表达Notch1细胞内结构域（NICD）的腺病毒感染后，通过免疫荧光染色和实时定量RT-PCR评估总BMSC和c-Kit(POS)/NKX2.5(POS)细胞向心肌细胞、SMC和内皮细胞谱系的分化情况。用Notch1配体Jagged1处理总BMSC和c-Kit(POS)/NKX2.5(POS)细胞，并通过免疫荧光染色和实时定量RT-PCR分析检测心肌细胞、SMC和内皮细胞分化标志物。
c-Kit(POS)/NKX2.5(POS)细胞存在于总BMSC群体中，这些细胞不表达成年心肌细胞、SMC或内皮细胞谱系的标志物。c-Kit(POS)/NKX2.5(POS) BMSC表现出与总BMSC相似的多谱系分化潜能。分选后，c-Kit(POS)/NKX2.5(POS) BMSC中的c-Kit水平为84.4%。流式细胞术显示，Notch1是总BMSC和c-Kit(POS)/NKX2.5(POS) BMSC中存在的主要Notch受体。过表达NICD的总BMSC和c-Kit(POS)/NKX2.5(POS) BMSC具有活跃的Notch1信号，并伴有向心肌细胞、SMC和内皮细胞谱系的分化。用外源性Jagged1处理总BMSC和c-Kit(POS)/NKX2.5(POS) BMSC可激活Notch1信号并驱动多谱系分化，c-Kit(POS)/NKX2.5(POS) BMSC有向心脏谱系分化的趋势。
c-Kit(POS)/NKX2.5(POS)细胞存在于总BMSC库中。Notch1信号的激活有助于c-Kit(POS)/NKX2.5(POS) BMSC的多谱系分化，有利于分化为心肌细胞。这些发现表明，Notch1信号的调节在干细胞转化医学中可能具有潜在用途。
