体外诱导人脐血间充质干细胞未能向心肌细胞转分化

Human mesenchymal stem cells derived from umbilical cord blood fail to transdifferentiate toward cardiomyocytes in vitro

目的选择从人脐带血中分离出的间充质干细胞(UCB1-MSCs),通过5-氮胞苷(5-aza)进行诱导,对其体外向心肌细胞转分化的能力做初步探讨。方法培养第11代 hMSCs,加入6、9、12 μmol/L 的5-aza 分别作用24h 和48h,相差显微镜观察细胞形态直到3周后实验结束。以未经处理的细胞为对照组。采用 RT-PCR 检测心肌转录因子 MEF2C、GATA4、Nkx2.5和心肌特异性基因 MLC-2v、MLC-2a、Connexin 43及α-actinin 的表达;免疫荧光染色检测 Connexin 43和α-actinin 的表达,以共聚焦显微镜成像。结果经5-aza 诱导后观察3周未见细胞的自发搏动。诱导前后的 hMSC 均可不同程度的表达 MEF2C、Connexin 43及α-actinin。在12 μmol/L 水平诱导24h 和6、9、12 μmol/L 水平诱导48h 共4个组中可见 MLC-2a 的表达。GATA4、Nkx 2.5和 MLC-2v 在诱导前后均未见表达。免疫荧光染色显示诱导前后 hMSCs 的胞浆内存在散在排列无序的α-actinin 和 Connexin 43荧光,而始终未见肌小节结构。结论经5-aza 诱导 UCB 来源 hMSCs 在基因水平可以表达部分心肌特异性基因,但是在诱导后的一个月时间内未见相应的心肌特异性结构出现,此类细胞向心肌细胞转分化的能力需再证实。

Objective The aim of this work was to assess in vitro the transdifferentiation potential of human mesenchymal stem cells （hMSCs） derived from umbilical cord blood （UCB） （called UCB1-MSC） into cardiomyocytes by treatment with 5-azacytidine （5-aza）. Methods UCB1-MSCs in culture （passage 11） were treated with different concentrations of 5-aza （6, 9, 12μmol/L） for 24 and 48 hours respectively, and their phenotypes were assessed after 3 weeks of culture. The expressions of cardiac-specific transcription factors （MEF2C, GATA4 and Nkx2.5） and structure genes [ventricular and atrial myosin light chains （MLC-2v and MLC- 2 a）, connexin 43 （Cx 43） and α-actinin] were analyzed at the mRNA level by reverse transcription polymerase chain reaction （RT-PCR） , at the protein level by westernblot or by indirect immunofluorescence （Cx and α-actinin） using confocal microscopy. Results Over 3 weeks observation, both untreated and treated UCB1-MSCs did not display cardiac-like or myogenic-like phenotype. RT-PCR analysis revealed the expressions of MEF2C, α-actinin and connexin 43 in both untreated and treated hMSCs. In contrast, 5-aza treatment was able to induce the mRNA expression of MLC2a, at a concentration of 12 μmol/L applied for 24 hours and at all. concentrations when applied for 48 hours. By immunolocalization, the distribution of α-actinin and Cx 43 in both treated and untreated hMSCs was disorganized and sarcomeres were never observed. Conclusions Human MSC from UCB （UCBI-hMSC） could be induced to express some cardiac-specific genes at the mRNA level by 5-aza, but coherent cardiac myofibrillogenesis does not take place over a period of almost 1 month. Therefore, the effective transdifferentiationpotential of these cells toward eardiomyoeytes remains to be demonstrated.