摘要
1.研究目的和背景
种子细胞是组织工程化组织、器官构建,干细胞移植的基础和关键环节。心血管领域目前研究应用的种子细胞,以胚胎干细胞(ESC)和骨髓间充质干细胞(BMSC)为主,但前者诱导分化条件复杂、易于成瘤,后者属于成体干细胞的范畴,寿命有限、活力较差;且作为自体来源的细胞,BMSC必须在发现病例后才可进行繁琐的体外细胞培养、扩增,其时效性和有限的生长潜能限制了临床的广泛应用。此外,已有的研究均表明上述两种细胞诱导分化为心血管细胞的比率较低,因此,都不是理想的种子细胞。寻找适宜的种子细胞是开展心血管组织工程、和干细胞移植治疗心血管疾患必须解决的首要问题。
从理论上推测,胚胎心脏干/祖细胞发育时序上更接近心脏的组织细胞,且具有较强的分化增殖能力,因而,诱导分化为心脏的组织细胞更容易。近来研究较多的心脏干/祖细胞主要有三种来源,分别是早期胚胎心脏或心管、成体心脏、ESC所形成类胚体中的心脏原基。后两者来源的心脏干/祖细胞其分离培养和生物学特性的研究已有较多报道,而从早期胚胎心管分离培养的心脏干/祖细胞生物学特性的研究目前则尚未见报道。从心脏发育的角度粗略讲,早期胚胎心管中存在心肌源性祖细胞和心内膜源性的祖细胞,前者构成了心脏的绝大部分,最终形成各种类型的心肌细胞和传导细胞;后者在心脏中只占很小一部分,最终分化形成心内膜、瓣膜、隔膜等组织。本课题拟对胚胎心脏祖细胞的生物学特性进行深入研究,以便为心脏组织工程、细胞移植治疗心肌梗死、生物心脏起搏器的构建等研究寻找一种理想的新型种子细胞。
2.材料和方法
2.1人胚胎心脏祖细胞的生物学特性
2.1.1细胞培养和鉴定
2.1.1.1胚胎心脏祖细胞的培养
取4周胚龄的药物流产人胚胎,在解剖显微镜下分离心管,将心管在0.25%胰酶中消化分散为单细胞悬液;上述得到的单细胞悬液离心并去上清,使用含15%胎牛血清、0.375%碳酸氢钠、10ug/L VEGF、10~6U/L LIF、10~6U/L青霉素、10~6U/L链霉素、2mmol/L谷安酰氨的高糖DMEM培养液重悬;将重悬的单细胞悬液接种于六孔细胞培养板或者十二孔细胞培养板,细胞长满后进行传代扩增。
2.1.1.2胚胎心脏祖细胞的鉴定
人胚胎心脏祖细胞的鉴定包括:①形态学的观察;②采用免疫荧光的方法检测Nkx2.5、Isl-1分子标志物的表达;③采用免疫细胞化学的方法检测平滑肌肌动蛋白、细胞角蛋白、Ⅷ因子,C-kit的表达;④RT-PCR检测GATA-4(心脏特异转录因子)的表达。
2.1.2胚胎心脏祖细胞的生物学特性
2.1.2.1胚胎心脏祖细胞的基本生物学特性
通过倒置相差显微镜和透射电镜观察培养细胞的形态学特征;通过生长曲线和分裂指数曲线的绘制检测细胞的生长特性。
2.1.2.2胚胎心脏祖细胞的分化特性
2.1.2.2.1向心肌细胞的诱导分化
利用5-氮胞苷诱导第15代胚胎心脏祖细胞向心肌细胞的分化,分别在诱导前、诱导后10天、15天三个时相RT-PCR检测心肌源性特异基因GATA-4、α-MHC的表达,免疫荧光细胞化学的方法检测心肌肌动蛋白的表达,倒置相差显微镜下观察细胞形态的改变,Western Blot的方法检测诱导前后细胞心肌肌动蛋白的表达。
2.1.2.2.2向平滑肌细胞的诱导分化
与家犬血管内皮细胞共培养诱导胚胎心脏祖细胞向平滑肌细胞方向分化,对诱导前后的细胞免疫荧光检测α-SMA和MHC-sm的表达,Western Blot的方法检测诱导前后细胞α-SMA的表达。
2.2胚胎心脏祖细胞向心脏起搏细胞的诱导分化
2.2.1大鼠胚胎心脏祖细胞的培养
250g雌雄SD大鼠配对合笼喂养,第二天观察粪盘是否有阴栓,确定雌鼠受孕开始时间;于受孕后10.5天断颈处死母鼠,取出子宫在无菌PBS中洗涤,用镊子撕开子宫和羊膜,分离得到心管;将心管在0.25%胰酶中消化分散为单细胞悬液后,采用与人胚胎心脏祖细胞相同的操作接种、培养、传代扩增大鼠胚胎心脏祖细胞。
2.2.2大鼠胚胎心脏祖细胞的鉴定
大鼠胚胎心脏祖细胞的鉴定包括:①形态学的观察鉴定;②采用免疫荧光的方法检测原代、第5代、第20代细胞Nkx2.5分子标志物的表达;③采用免疫荧光的方法检测原代、第5代、第20代细胞OCT-4分子标志物的表达;④采用免疫荧光的方法检测原代、第5代、第20代细胞阶段特异性胚胎抗原SSEA-4分子的表达。
2.2.3大鼠胚胎心脏祖细胞向起搏细胞的诱导分化
胚胎心脏祖细胞接种培养两天后,更换培养基为含有内皮素—1的DMEM,送孵箱进行诱导培养,三天后换回正常培养液继续培养,倒置相差显微镜下观察细胞的搏动情况并录像。诱导前后的细胞通过免疫荧光的方法检测HCN2、HCN4、Connexin43、Connexin45、心肌肌动蛋白、Nkx2.5、OCT—4的表达,通过HE染色和电镜观察检测其形态结构的改变,通过膜片钳技术检测其电生理情况。
3.结果
3.1人胚胎心脏祖细胞的生物学特性
3.1.1生长情况和鉴定
原代培养的人胚胎心脏祖细胞接种后六个小时贴壁生长,4-8天后融合生长,镜下主要呈圆形、长梭形、三角形,传代后的胚胎心脏祖细胞呈长梭形、三角形、和少量圆形;原代和传代的细胞免疫荧光染色Nkx2.5、Isl-1呈阳性,免疫细胞化学染色平滑肌肌动蛋白、细胞角蛋白、Ⅷ因子、C-kit呈阴性;GATA-4mRNA的表达高于对照组胚胎肢芽间充质细胞。
3.1.2胚胎心脏祖细胞的生物学特性
3.1.2.1胚胎心脏祖细胞基本生物学特性
第八代细胞透射电镜显示典型的幼稚细胞亚微结构特征;生长曲线和分裂指数曲线显示细胞生长迅速扩增能力强,并计算出群体倍增时间约为56个小时;目前细胞已传至20代并能在体外活跃增殖。
3.1.2.2胚胎心脏祖细胞的分化特性
3.1.2.2.1向为心肌细胞的诱导分化
5-氮胞苷处理后,经RT-PCR检测α-MHC、GATA-4的表达随着诱导时程的延长而增高,心肌肌动蛋白的免疫荧光染色在诱导后变为阳性,Western Blot检测心肌肌动蛋白的表达也随着诱导增高,诱导后细胞的形态亦发生了明显的改变,出现了典型的肌节和闰盘结构。
3.1.2.2.2向平滑肌细胞的诱导分化
胚胎心脏祖细胞与家犬内皮细胞混合共培养诱导后,α-SMA免疫荧光染色变为为阳性,Western Blot检测其表达亦随着诱导的进行而增高,但未检测到MHC-sm的表达。
3.2胚胎心脏祖细胞向心脏起搏细胞的诱导分化
3.2.1大鼠胚胎心脏祖细胞生长情况及鉴定
原代培养的大鼠胚胎心脏祖细胞生长情况和基本形态与上述人的细胞类似,分子标志物鉴定原代、第5代、第20代细胞Nkx2.5、OCT-4为阳性,SSEA-4为阴性。
3.2.2向心脏起搏细胞的诱导分化
胚胎心脏祖细胞诱导前观察较少见单个细胞的搏动,融合生长后仅见局部细胞团簇的搏动,经内皮素-1诱导4天后可见大量单个细胞的搏动,融合生长后成同步性收缩;诱导前后细胞的基本形状HE染色观察无明显变化,但诱导后出现少量的线粒体、尚不发达的高尔基体和较不完整的肌丝样结构,细胞之间可见有连接样的结构增多,总体看仍为幼稚型细胞结构。
胚胎心脏祖细胞经过内皮素—1诱导后,利用膜片钳技术检测单细胞动作电位,结果表明所检测细胞均出现自发放电现象,其中大量细胞产生了起搏动作电位。在诱导前的胚胎心脏祖细胞中,免疫荧光检测缝隙连接蛋白43、缝隙连接蛋白45、HCN2、HCN4、心肌肌动蛋白的表达是成阴性的;在细胞经过诱导后,五种蛋白的表达由阴性变为了阳性;Nkx2.5、OCT-4在诱导前细胞中成阳性表达,随着诱导的进行,表达无明显变化。
4.讨论
4.1胚胎心脏祖细胞的鉴定
在早期胚胎中,心脏是最早形成的组织器官之一。早在胚胎管状心脏形成之初,主要存在两类心脏的祖细胞谱系;一是心肌层细胞谱系,另一类是心内膜细胞谱系。其中前者构成了心脏的绝大部分,最终形成各种类型的心肌细胞和传导细胞。后者在心脏中只占很小一部分,最终分化形成心内膜、瓣膜、隔膜等组织。在本研究中培养的胚胎心脏祖细胞分离自胚胎心管形成之初,主要以心肌源性祖细胞为主;该细胞经检测表达心脏早期祖细胞的特异转录因子Nkx2.5,这与Hidaka等从鼠ES细胞形成的类胚体中分离培养的心脏祖细胞类似,提示本研究中分离培养的胚胎心脏祖细胞具有分化为各种类型心肌细胞的潜力。
除了Nkx2.5之外,对于人胚胎心脏祖细胞,尚进行了C-kit和Isl-1心脏干/祖细胞分子标志物检测鉴定,结果显示Isl-1是阳性的,这与国外学者Cai等的研究结果类似。α-SMA、CK、Ⅷ因子的免疫细胞化学检测为阴性,表明该细胞群中不存在成熟肌细胞、内皮细胞、上皮细胞等不同类型的成熟杂细胞。
OCT—4是胚胎早期发育的一个关键基因,一般作为具有多向分化潜能的细胞的标记物,在大鼠胚胎心脏祖细胞鉴定中,OCT—4的阳性进一步说明了该细胞的多向分化潜能。SSEA—4是阶段特异性胚胎抗原,其与早期胚胎发育全能性细胞之间的识别和相互作用有关,大鼠胚胎心脏祖细胞分离自早期胚胎心管,该细胞在发育时序上已经向特定组织的祖细胞方向分化,故SSEA-4的表达是阴性的。
4.2胚胎心脏祖细胞的分化潜能和其他生物学特性
4.2.1胚胎心脏祖细胞基本生物学特性
未分化的幼稚型细胞一般具备如下形态结构特征:细胞体积小,核大胞浆少,有一个或几个核仁,常染色质多,异染色质少,胞浆细胞器少而简单,滑面内质网较粗面内质网多,游离核糖体密集有序排列等。本实验培养的胚胎心脏祖细胞细胞超微结构观察显示:细胞核大,为圆形或椭圆形,核内常染色质区域大,异染色质区域小,除内质网和核糖体外基本上无其他胞浆细胞器,具有较为幼稚的细胞结构特点。生长曲线和分裂指数曲线亦显示该细胞生长迅速,扩增能力强,符合原始幼稚细胞的特点。
4.2.2胚胎心脏祖细胞的分化特性
4.2.2.1胚胎心脏祖细胞向心肌细胞分化的潜能
5—氮胞苷是目前常用的诱导干细胞向心肌细胞转化的诱导剂,MSCs、脐血间充质干细胞和成体心脏干细胞均可被一定剂量的5—氮胞苷诱导为心肌细胞。本实验利用5—氮胞苷诱导人胚胎心脏祖细胞后,细胞形态发生了改变;横纹肌肌动蛋白免疫荧光检测由阴性变为阳性;Western Blot检测表达明显升高:RT-PCR检测心肌特异分子标志物心肌肌球蛋白重链、GATA—4的表达升高;超微结构观察出现了肌节和闰盘的结构;证明胚胎心脏祖细胞已经被诱导为心肌样细胞。上述研究结果提示,来源于人胚胎心管的心脏祖细胞有潜力分化为成熟心肌细胞。
4.2.2.2胚胎心脏祖细胞向平滑肌分化的潜能
在本实验的研究当中,采用与内皮细胞共培养的方法诱导胚胎心脏祖细胞向平滑肌方向分化,检测诱导后的胚胎心脏祖细胞平滑肌肌动蛋白免疫荧光细胞化学染色由阴性变为阳性,Western Blot检测其表达亦随着诱导的进行而增高,表明已向平滑肌方向发生转化;但在实验中未检测到平滑肌肌球蛋白重链的表达,表明胚胎心脏祖细胞完全转化为平滑肌细胞尚需进一步的诱导。
4.3胚胎心脏祖细胞向心脏起搏细胞分化的潜能
目前生物心脏起搏器构建的研究十分活跃,利用细胞的方法构建生物心脏起搏器是将具有起博活性的细胞移植到心脏,代替和修复宿主缺损的起博细胞,达到重新恢复心脏正常起搏能力的目的。本研究在明确了人胚胎心脏祖细胞的基本生物学特性和分化特性后,欲进一步检测其构建生物心脏起搏器和向心脏起搏细胞方向分化的可能性。
人胚胎心脏祖细胞移植到动物心脏进行构建生物心脏起搏器的实验研究中,存在异种间免疫反应的问题,虽然可以考虑施用免疫抑制剂,但对于窦房结毁损的动物模型而言,无疑极大的增加了实验动物的死亡率。此外,人类和实验动物心脏起搏细胞的起搏节律并不一致,所以不宜直接利用人胚胎心脏祖细胞进行构建生物心脏起搏器的动物实验研究。为了检测胚胎心脏祖细胞向起搏细胞分化的潜能及方便利用其进行构建生物心脏起搏器的动物实验研究,我们从与人四周龄胚胎同期的大鼠胚胎心管中分离培养了胚胎心脏祖细胞,并对其进行了向心脏起搏细胞方向的诱导分化。
心脏中起搏细胞早期发育分化的过程和机理目前知之甚少,最初的研究显示胚胎早期心脏传导系统是通过接受到某种旁分泌信号,开始募集周围的多潜能心肌细胞(心脏祖细胞)形成的。随后的研究表明内皮—肌信号在这个过程中起到了关键的作用,邻近内皮细胞产生的内皮素—1和神经调节蛋白—1等旁分泌因子诱导周围的心脏祖细胞募集并向传导系统组织细胞转变,这一结果在体外培养的细胞模型(胚胎干细胞来源的心肌细胞)中也得到了证实。提示内皮素—1和神经调节蛋白—1等旁分泌因子可以促使原始心肌细胞(心脏祖细胞)转变为起搏细胞。
在本研究中,胚胎心脏祖细胞经内皮素-1诱导五天后,光镜下直接观察或HE染色观察其基本形状与诱导前相比无明显改变;但透射电镜观察超微结构证实诱导后细胞有了较为明显的分化;证据之一是诱导后出现较诱导前为多的简单细胞器结构,包括少量的线粒体、尚不发达的高尔基体和较不完整的肌丝样结构,证据之二是诱导后细胞之间可见有连接样的结构增多。这一超微结构上的变化在免疫荧光染色上得到了进一步的证实,心肌肌动蛋白(细肌丝的主要组成成分)的染色在诱导前后由阴性变为了阳性,缝隙连接蛋白43和45在诱导后也出现了多量的表达。肌动蛋白的表达或肌丝样结构的出现是诱导后细胞搏动更为明显的结构基础,而缝隙连接蛋白的表达则表明诱导后细胞之间更易建立肌电偶联。总之,搏动情况和形态结构上的变化为诱导后细胞已经向起搏细胞方向发生转变给出了初步的证据。
起搏细胞之所以具有自动兴奋性,是由于在动作电位四期激活产生一种称为If的内向离子流,导致了细胞的自动除极。编码If离子流的通道分子称为超级化激活的环核苷酸门控的离子通道(hyperpolarization-activated cyclicnucleotide-gated channel,HCN),由于HCN与心脏起搏的产生和调节关系密切,所以被称为起搏基因。在本研究中,我们通过免疫荧光染色和RT-PCR检测了诱导后细胞起搏基因HCN2和HCN4的表达,结果证明了诱导后细胞存在上述两种起搏离子通道,具备了起搏细胞产生自动兴奋的结构基础。进一步我们利用单细胞膜片钳实验检测诱导后胚胎心脏祖细胞的电生理情况,显示诱导后细胞出现了连续的自发性动作电位(窦房结样起搏动作电位和心房肌样起搏动作电位),这一结果最终证明了诱导后细胞可以自发产生起搏动作电位(自动兴奋性),具备了起搏细胞最基本的电生理特征。
5.结论
人早期胚胎心管来源的心脏祖细胞具有幼稚型的细胞结构和旺盛的生长特性,诱导分化为心肌样细胞和平滑肌样细胞的诱导率高、诱导分化条件简单,是心血管组织工程和干细胞移植研究较理想的种子细胞。
大鼠胚胎心管来源的胚胎心脏祖细胞易于分离、培养、扩增,利用内皮素-1可将其诱导成为具有稳定起搏活性的心脏起搏细胞,是生物心脏起搏器构建较理想的细胞来源。
1.Objective and background
It is critical to seek ideal seeding cells for the development of cardiovascular tissue engineering(CvTE).Currently autologous vascular wall cells(AVWCs) and marrow stromal cells(MSCs) represent established cell sources for CvTE.However,the invasive harvesting of vessel segments or bone marrow,a wound brought to body,are required duing cells isolation.Furthermore,these autologous cells were greatly limited in clinical applications,because the fussy experiment in vitro culture can be performed only with necessitating the appearance of a case.Additionally,it is controversial about the differentiatal potential and proliferative ability of MSCs.So both AVWCs and MSCs are not preferable cell sources for tissue engineer,whereas stem cells derived from embryos are better than the two types of cells.
In early embryos,there are two cardiac progenitor cell lineages,cardiomyogenic cells and endocardial endothelial cells.The cardiomyogenic cells occupy 95 percent of cardiac progenitor cells.The cells with potential of differentiate into cardiomyocytes,could express cardiac transcription factors gata-4,nkx2.5 and so on, which were considered as molecular markers of cardiac progenitor cells.It has been reported that nkx2.5 positive cells were isolated from embryoid bodies and proved to be capable of differentiating into various cardiac cell types.This suggests the possibility that cardiac stem cells can be cultured from embryos.In this study,we isolated embryonic cardiac progenitor cells as an alternative cell source for cardiovascular tissue engineering.
2.Materials and Methods
2.1 Biological characteristics of human embryonic cardiac progenitor cells
2.1.1 Embryonic cardiac progenitor cells culture and identification
2.1.1.1 Human embryonic cardiac progenitor cells culture
Heart tubes were isolated under a dissecting microscope by surgical nippers,and then incubated in 0.25%trypsin at 37℃.Cell suspensions obtained in this way were pelleted by centrifugation at 800×g for 5 min.The cells were then resuspended in normal growth medium at 10~5/ml.The cell suspensions was seeded into 12-well cluster dishes.The cultures were maintained at 37℃,95%humidity,and 5%CO_2. The normal growth medium was DMEM supplemented with 15%fetal calf serum, 10ug/L VEGF,10~6U/L LIF,0.375%NaHCO_3,100 U/mL penicillin G,100μg/mL streptomycin,2 mmol/L L-glutamine.After 5 or 8-day primary culture,with routine passage methods cell suspension was obtained and the cells were passaged.
2.1.1.2 Identification of Human embryonic cardiac progenitor cells
Human embryonic cardiac progenitor cells at passage 5 were detected for the expression of Nkx2.5,Isl-1,α-smooth muscle actin,cytokeratin,factorⅧ,C-kit by immunocytochemistry,and gata-4 mRNA by reverse transcriptase polymerase chain reaction(RT-PCR).
2.1.2 Biological characteristics of embryonic cardiac progenitor cells
2.1.2.1 Hasic biological characteristics embryonic cardiac progenitor cells
Morphological characters of cultured cells were investigated by transmission electron microscope(TEM) and inverted phasecontrast microscope.To check growth characteristics,the growth curves were plotted and the mitotic index was tested.
2.1.2.2 Embryonic cardiac progenitor cells differentiation characteristics
2.1.2.2.1 Differentiation of embryonic cardiac progenitor cells into cardiomyocytes
To investigate the differentiation of embryonic cardiac progenitor cells into cardiomyocytes,the cells at passage 15 were treated with 5-azacytidine.The expression of cardiogenic and myogenic specific genes(gata-4,α-MHC) were detected by RT-PCR at day 14,and the expression ofα-sarcomeric actin was analyzed by the immune fluorescence assay and Western Blot.
2.1.2.2.2 Differentiation of embryonic cardiac progenitor cells into smooth muscle cells
Embryonic cardiac progenitor cells were cocultured with canine vascular endothelial cells(CVECs) in direct cell-cell contact to find out the possibility of their differentiation into vascular smooth muscle cells(VSMCs).α-smooth muscle actin and smooth muscle-myosin heavy chain were detected in induced Embryonic cardiac progenitor cells.
2.1.3 Differentiation of embryonic cardiac progenitor cells into pacemaking cells
2.1.3.1 Rat embryonic cardiac progenitor cells culture
Male and female rats were mated.After 10.5 days heart tubes were isolated under a dissecting microscope.In succession,the other performation refered to 2.1.1.1
2.1.3.2 Rat embryonic cardiac progenitor cells identification
Rat embryonic cardiac progenitor cells at primary passage,5 passageand 20 passage,were detected for the expression of Nkx2.5,OCT-4 and SSEA-4 by immunocytochemistry.
2.1.3.3 Differentiation into pacemaking cells
To induce differentiation of cardiac progenitor cells towards cardiac pacemaking cells,endothelin-1(10~(-7)M) was added to the cells in the growth medium without LIF. After inducement culture,the cells was detected for morphological characters, electrophysiology and expression of molecule marker.
3.Results
3.1 Biological characteristics of human embryonic cardiac progenitor cell
3.1.1 Culture and identification of human embryonic cardiac progenitor cells
The primary cultured embryonic cardiac progenitor cells are mainly round and long spindle-shaped,however,the passaged cells were long spindle and triangon-shaped. Embryonic cardiac progenitor cells stained positive for Nkx2.5,Isl-1 and negative forα-smooth muscle actin,cytokeratin,factorⅧ,C-kit.GATA-4 expression of the cells was higher than that of the control group embryonic limb bud mesenchymal cells (p＜0.05).
3.1.2 Biological characteristics of human embryonic cardiac progenitor cells
3.1.2.1 Basic biological characteristics
TEM showed that the cells had elements of typical fetal-type cells.The cell population doubling time was 56 hours.The cells had been passaged continuously for more than 2 months(20 passages),and could proliferate actively in vitro.Growth curves and mitotic index curves were plotted.
3.1.2.2 Differentiation of embryonic cardiac progenitor cells
3.1.2.2.1 Differentiation into cardiomyocytes
The expressions ofα-MHC and GATA-4 were increased in embryonic cardiac progenitor cells treated with 5-azacytidine in contrast to the untreated.The fluorescence intensity ofα-sarcomeric actin of cells induced by 5-azacytidine was stronger than that of the control group.Similarly the protein level ofα-sarcomeric actin was higher in cardiomyogenic differentiation cells than the control group (p＜0.05).The isolated cells exhibited sarcomeres and intercalated discs after being treated with 5-azacytidine.So it was confirmed that the isolated Nkx2.5 positive cells could differentiate into cardiomyocytes by 5-azacytidine.
3.1.2.2.2 Differentiation into smooth muscle cells
Embryonic cardiac progenitor cells exhibited positive staining ofα-smooth muscle actin when cocultured with CVECs,however,smooth muscle-myosin heavy chain was not detected in the induced cells.Similarly the protein level ofα-smooth muscle actin was higher in the induced cells than the control group(p＜0.05).
3.2 Differentiation into cardiac pacemaking cells
3.2.1 Rat embryonic cardiac progenitor cells culture and identification
Embryonic cardiac progenitor cells derived from rat stained positive for Nkx2.5, OCT-4 and negative for SSEA-4 at primary,5 and 20 passage.The shape of the cells were the same as human embryonic cardiac progenitor cells.
3.2.2 Differentiation into cardiac pacemaking cells
Endothelin-1 was added to induce differentiation of the cells towards cardiac pacemaking cells.After the inducement the cells exhibited more spontaneous beating. There was Connexin-45 and Connexin-43 staining at interfaces between the cells treated with endothelin-1.In contrast,no staining for the both protein was seen between undifferentiated embryonic cardiac progenitor cells.The induced cells exhibited positive staining of HCN2,HCN4 andα-sarcomeric actin.
After being treated with endothelin-1,the cells all displayed spontaneously electrical activity(a slow diastolic depolarization phenomenon).Ultrastructural observation showed that the induced cells exhibited some new elements.
4.Discussion
4.1 Biological characteristics of human embryonic cardiac progenitor cell
4.1.1 Identification of embryonic cardiac progenitor cells
It was reported that murine cardiac cell progenitors can express cardiac transcription factors GATA-4 and Nkx2-5 which were considered as molecular markers of cardiac progenitor cells.In our study cardiac progenitor cells derived from embryonic hearts tubes expressed a high level of Nkx2-5,Isl-1,OCT-4 and GATA-4. This characterization of the cells is similar to that of Isl1~+ cardiac progenitors from postnatal hearts and Nkx2-5~+ cardiac precursor cells from murine embryonic stem cells which have been reported.It is demonstrated that the cells mainly consisted of cardiogenic progenitors.These results suggested that the cells might differentiate into cardiomyocytes more easily than ESCs and MSCs do.
C-Kit is expressed in adult cardiac progenitor cells and some cardiac stem cells are isolated using c-kit as a marker(Antonio et al.,2003).However,C-Kit was stained negative in hCPCs.Immunocytochemistry of the cells revealed negative staining for several differentiated cell markers such as factorⅧ,α-SMA,CK andα-sarcomeric actin.These results confirmed that there are no endothelial cells,mature myocytes and epithelia in the cells.
4.1.2 Biological characteristics of human embryonic cardiac progenitor cells
4.1.2.1 Basic biological characteristics
Being isolated from early embryos,cardiac cell progenitors had elements of typical fetal-type cells which ultrastructural observation have showed.These primitive and fetal cells were capable of self-renewal and continuous passage.From the growth curve and mitotic index curve,it can be seen that cardiac cell progenitors grew vigorously and proliferate actively.The growth properties of cardiac cell progenitors were consistent from 8 passage to 20 passage.These results imply that cardiac cell progenitors could be expanded to a large quantity which is required for CTE.
4.1.2.2 Differentiation of embryonic cardiac progenitor cells
After cardiac cell progenitors were induced toward myocytes,some myocytes markers(α-MHC,α-smooth muscle actin andα-sarcomeric actin) were expressed. These results indicated that hCPCs possess potential to differentiate into cardiac muscle-like cells and smooth muscle-like cells.It is suggested that cardiac cell progenitors may be a new source of cardiac muscle cells for CTE.But whether hCPCs differentiate into functional mature cardiomyocytes with a high efficiency or not and how many subpopulations of cardiac progenitor cells there are in this cell line await further study.
4.2 Differentiation into cardiac pacemaking cells
After cardiac progenitor cells were treated with endothelin-1,the cells all displayed spontaneously electrical activityand and spontaneous beating.These cells with spontaneous depolarization generated sinus nodal and atrial-like pacemaking action potentials.The markers of sinus node cells,including Connexin-45,Connexin-43, HCN2,HCN4,were stained positive in the induced cells.It is confirmed that cardiac progenitor cells treated with endothelin-1 can differentiate into cardiac pacemaking cells.So cardiac progenitor cells can be used as a cell source of creating biological cardiac pacemakers and tissue engineered sinus node.
5.Conclusion
In this study,embryonic cardiac progenitor cells have been isolated,cultured, identificatified.Our results suggest that these cells be able to differentiate into cardiomyocytes,smooth muscle cells and pacemaking cells.The cells,which have the ability to undergo self-renew,belong to cardiac progenitor cells and are multipotent. These cells maybe have enormous potential in the application to cardiovascular tissue engineering.
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