小鼠胚胎干细胞来源平滑肌细胞的鉴定及其功能分析

Identification and function assessment of smooth muscle cells derived from mouse embryonic stem cells

背景:胚胎干细胞来源的平滑肌细胞是血管组织工程潜在的细胞来源之一,但这些细胞是否具有成熟平滑肌细胞的表型和功能仍不清楚。目的:对小鼠胚胎干细胞分化的平滑肌细胞进行表型鉴定及功能分析。设计、时间及地点:细胞学体外观察,于2007-05/2008-12在解放军沈阳军区总医院全军心血管病研究所完成。材料:清洁级孕12.5d昆明小鼠1只用于制备饲养层细胞,SD大鼠1只用于制备主动脉平滑肌细胞。小鼠胚胎干细胞系R1、小鼠微血管内皮细胞系由美国ATCC公司提供,转染pSPIE载体的胚胎干细胞R1由本实验室制备。方法:①诱导分化及筛选:用转染pSPIE载体的胚胎干细胞R1制备拟胚体,拟胚体悬浮培养5d后贴壁培养1d,第7天加入全反维甲酸诱导分化4d,第11天用10mg/L嘌呤霉素对诱导分化后的细胞筛选2d。②表型鉴定:对筛选到的平滑肌细胞进行SMα-actin、SM22α、SM-MHC免疫荧光染色,以大鼠原代主动脉平滑肌细胞、未经筛选的第10天分化细胞作为对照,进行WesternBlot分析。③收缩功能:10-5mol/L卡巴可处理筛选到的平滑肌细胞30min。④体外成血管功能:将等量的内皮细胞和筛选到的平滑肌细胞混合,在Matrigel上培养。主要观察指标:平滑肌细胞标志物的表达,平滑肌细胞的收缩,平滑肌细胞向内皮管腔样结构的募集。结果:分化的平滑肌细胞主要分布于贴壁生长的拟胚体的外周部位,胚胎干细胞来源的平滑肌细胞3种标志物SMα-actin、SM22α、SM-MHC免疫荧光染色均呈阳性表达,WesternBlot结果显示其SMα-actin、SM22α表达水平与大鼠原代主动脉平滑肌细胞相似,高于未经筛选的第10天分化细胞。筛选到的平滑肌细胞在10-5mol/L卡巴可刺激下出现明显收缩,且能够募集到内皮管腔样结构周围。结论:小鼠胚胎干细胞来源的平滑肌细胞具有与成熟平滑肌细胞相似的表型和功能。

BACKGROUND： Smooth muscle cells （SMCs） derived from embryonic stem cells （ESCs） are a potential cell source for tissue-engineered blood vessels, but whether these cells have similarity to mature SMCs in phenotype and function is unclear yet. OBJECTIVE： To investigate the phenotype and function of ESCs-derived SMCs. DESIGN, TIME AND SETTING： The in vitro cytological observational experiment was performed at the Cardiovascular Institute of Chinese PLA, General Hospital of Shenyang Military Area Command of Chinese PLA from May 2007 to December 2008. MATERIALS： One clean Kunming mouse at gestational day 12.5 was used to prepare feeder layer cetls. One Sprague Dawley rat was selected to prepare SMCs in the aorta. Mouse ESC line R1 and mouse vascular endothelial cell line were supplied by ATCC, USA. ESC R1 （transfection pSPIE vector） was prepared by this laboratory. METHODS：①lnduced differentiation and selection： Embryoid bodies were made from pSPIE-ESCs and cultured suspendedly for 5 days. On day 6, embryoid bodies were attached to plates. On day 7, all-trans retinoic acid was used to induce differentiation for 4 days. On day 11, we performed puromycin （10 mg/L） selection for 2 days;②Phenotype identification： Selected SMCs were stained by SM α-actin, SM22α, SM-MHC immunofluorescence. Rat primary cultured aorta SMCs and unselected differentiated cells on day 10 served as controls. Western blotting was performed.③Contractility： Carbachol （10^-5mol/L） treatment was conducted in selected SMCs for 30 minutes.④In vitro vasculogenesis： Selected SMCs was mixed with an equal amount of endothelial cells and then cultured on Matrigel. MAIN OUTCOME MEASURES： Expression of SMC markers; SMC contraction; SMC recruitment were measured. RESULTS： Differentiated SMCs were mainly distributed in periphery of embryoid body. Selected SMCs were positive for SM α-actin, SM22α, SM-MHC staining. Western blotting showed that the expression of SM α-actin and SM22α was comparable to rat primary aortic SMCs, and greater than unselected differentiated cells on day 10. Selected SMCs showed contractility stimulated by 10^-5 mol/L carbachol and were recruited to endothelial lumen-like structure. CONCLUSION： SMCs derived from mouse ESCs are similar to mature SMCs in phenotype and function.