CN1852973A - 从活检分离和扩充心脏干细胞的方法 - Google Patents
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Abstract
从人或动物组织活检样品分离、扩充和保存心脏干细胞以用于心肌或其它器官的细胞移植和功能修复的方法。细胞也可以在基因治疗中用于治疗遗传性心肌病,用于治疗缺血性心脏病和用于建立体外模型以研究药物。
Description
技术领域
本发明涉及分离和扩充源自出生后的心脏组织活检的心脏干细胞的方法。
本发明涉及从人或动物组织活检样品中分离、扩充和保存心脏干细胞以用于心肌或其它器官的细胞移植和功能修复的方法。
该细胞也可以用在基因治疗中,通过在来自具有遗传缺陷的对象的活检的细胞中表达健康基因、在体外繁殖该细胞和然后将它们移植进患者,用于治疗遗传性心肌病;通过诱导移植的细胞释放血管生成性生长因子,用于治疗缺血性心脏病;和用于建立研究药物的体外模型。
现有技术
干细胞(SC)能对适当的信号作出响应,进行复制和分化,从而形成或再生特化组织。
认为心肌细胞是终末分化的细胞;但是,新出现的证据已经显示出这些细胞在动物模型和心脏移植患者中增殖的适度潜力(1-4)。
成年心肌细胞在损伤后经历有丝分裂和再生心肌的能力有限,导致有功能细胞数目的永久不足,心功能不全发生和进展。在疾病的晚期,移植的替代治疗是,将SC植入受损的心肌(心肌成形术)。该方法已经在动物模型中产生了有希望的结果,还已经在人中进行了实验。但是,仍然存在SC的来源和可用性的问题(5-7)。
尽管胚胎SC(来自胚胎的未分化的细胞,其可以产生多种特化细胞,且可以源自胚泡内的细胞团,在人中,所述的胚泡在卵子受精后4-5天形成)具有显著的增殖和分化能力,它们的潜在的免疫原性、致心律失常性(arrhythmogenicity)和尤其是伦理问题已经限制了它们的应用。而且,胚胎SC是多能性的,因而它们的应用具有产生畸胎瘤的潜在风险(如在动物模型中发生的)。因此,在这些细胞可以使用之前,它们需要在体外分化成心肌细胞。
存在多种类型的心肌细胞(心室的,心房的,窦房结,浦肯野,具有起搏功能,等)。胚胎SC具有在体外产生这些心肌细胞表型的潜在能力,但是产量是不足的。另外,源自胚胎SC的心肌细胞的体内增殖能力似乎受到多核细胞的生长的限制。
一个替代方案是使用成年SC(在分化的组织中发现的未分化的细胞,其能增殖、再生和分化成它们从其中分离的组织的特化细胞类型),优选地从同一患者得到,这会提供允许自体移植而无需免疫抑制疗法的优点。为此,已经使用了成骨骼肌细胞(卫星细胞);但是,它们分化成的骨骼肌细胞具有不同于心肌的形态和功能性质。成骨骼肌细胞不能转分化成心肌细胞并与它们偶联,可造成心律失常或其它异常。
源自骨髓的SC提供有吸引力的替代方案。骨髓的间充质SC(MSC)可以在体外(用DNA-脱甲基化试剂处理)和体内分化成心肌细胞,但是,在体内时,在有纤维变性存在下,它们主要形成成纤维细胞样细胞。骨髓的造血SC(HSC)(所谓的旁群细胞[SP细胞])是多能性的,在于它们可以产生血管上皮、平滑肌细胞和心肌细胞。但是,尚未充分表征HSC-和MSC-衍生的心肌细胞的功能和电生理学性质,且未分化的细胞替代心肌细胞的应用可造成在体内分化成成纤维细胞,而不是肌细胞,或者造成肿瘤的发生。
虽然已经常规地将人心肌细胞视作终末分化的细胞(即不能重新进入细胞周期和分裂),过去2年来积累的间接证据已经暗示在心脏中存在成年SC。这些细胞是心脏成形术(cardioplasty)的理想候选品,因为它们不需要改编程序,仅仅产生存在于心脏中的细胞(即心肌细胞)和血管(内皮细胞和平滑肌),且因为这是它们的生理功能而可以在移植患者中存活,整合进周围组织中,并长期执行它们的功能,而不会造成任何损伤。
专利申请WO 03/008535和WO 03/006950涉及从胚胎SC衍生心肌细胞的方法。专利申请WO 02/13760和WO 02/09650大体上涉及成年SC(尤其是造血和/或心脏细胞,没有指出分离它们的方法,组合亦然)在修复心脏损伤或治疗心血管疾病中的应用。
专利申请WO 99/49015涉及成年p53-/-小鼠的多能性心脏SC的分离。更具体地,说明书涉及心脏-衍生的多能SC,其分化和增殖产生许多细胞类型,包括心肌细胞,成纤维细胞,平滑肌细胞,骨骼肌细胞,角质形成细胞,成骨细胞和软骨细胞。这些细胞可以用于治疗患有心脏组织坏死的患者的方法中。SC能增殖和分化,产生能替代坏死的组织的心肌细胞。
但是,与本发明的方法不同,该方法是基于一个假设,即心肌细胞、横纹肌和平滑肌细胞源自共同的前体-成肌细胞。另外,没有来自心肌病动物的体内证据能够支持该方法的可应用性。最后,该方法实质上不同。在专利WO 99/49015所述的方法中,粉碎了成年p53-/-小鼠心脏,用脱氧核糖核酸酶和胶原酶解离。离心后,在不连续梯度(Percoll)上分离沉淀肌细胞,并铺到含有5%FBS的培养基上,再在20天后,铺到含有15%FBS的培养基上。在第20-26天之间,在悬浮液中形成小的(<5m)圆形的、非粘附的、缓慢生长的、相差显微镜下透亮的(phase-bright)、具有高核质比的细胞。在有10%马血清存在的情况下,这些细胞继续悬浮生长约1.5个月。然后,在不加入马血清的情况下,细胞保持悬浮。非粘附的SC不在甲基纤维素中形成菌落,且在有血清,SCF,aFGF,bFGF,和cFGF存在的情况下增殖。在没有马血清存在的情况下,非粘附的细胞分化成不同外观的粘附细胞,发明人已经根据主要的形态标准,将它们鉴别为心肌细胞,软骨细胞,成纤维细胞,平滑肌细胞,骨骼肌成肌细胞,周细胞,和发明人称作粘附SC的其它细胞。这些细胞中的约四分之一至五分之一是碱性磷酸酶阳性的(成骨细胞和内皮细胞);所有细胞都是乙酰化LDL(没有内皮细胞)和肌球蛋白重链(MF20)阴性的。当用bFGF,aFGF和cFGF刺激时,细胞经历有丝分裂。在没有血清的情况下,它们分化成类似于油煎蛋的细胞(肌细胞)。在用抗坏血酸/α-GP处理后,它们分化成软骨细胞样的细胞。
通过有限稀释而克隆的粘附细胞产生间充质细胞,包括成骨细胞,软骨细胞,脂肪细胞和肌细胞,尽管由于经常不适当的形态标准和标记而不能清楚地鉴别它们。所有细胞经测试都是乙酰化LDL阴性的(没有内皮细胞)。11个分离的克隆都不能被诱导向单一间充质系分化。
还描述了新生小鼠(1-4天)的心脏-衍生的SC的分离,其中加入了在人纤连蛋白上的肌细胞传代,以消除成纤维细胞。但是,没有给出关于分离的SC的特征的数据。另外,用前述方法分离的细胞不能形成心脏组织的必需组分,即血管和内皮。
发明简述
本发明的方法使用心脏活检组织作为原料,由此是不能用于专利申请WO 99/49015所述的方法中的选择材料,因为该材料是不足的。破碎活检标本后,且可能使用解离剂(例如胰蛋白酶,EDTA和胶原酶),将碎片平板接种并加入含有10%FBS的培养基;10-30天后,从外植块生长出成纤维细胞样粘附细胞,小圆形的、透亮的细胞在该外植块上迁移,其趋向于聚团,但是不粘附或仅仅较弱地粘附。通过洗涤和温和的解离(例如EDTA,胰蛋白酶-EDTA,2-3分钟),分离细胞。然后,将细胞平板接种在聚赖氨酸处理过的细胞基质上,在与在先前技术中使用的培养基不同的适当培养基中,在于所述培养基没有马血清,且含有其它的生长因子;2-3天后,细胞聚集体(心球体(cardiosphere))出现,其倾向于作为漂浮结构生长。发明人已经发现,心脏形成细胞是出生后的SC,其可以有利地用于重新植入心肌。
这些细胞能够倍增,同时维持它们的原始特征一段时间(至少60天),其长度足以显著地富集细胞群体。在至少前20天,通过每3天重复移液和更换培养基来机械解聚心球体(CS),从而增加CS的数目(每10天约100-倍)。考虑到可以从活检衍生的SC的数目和它们在体外倍增的能力,认为它们可以用于替代比取出的组织更大量的组织。
CS中的某些细胞呈现干细胞标记(ckit,sca-1,CD34),其能向心肌的主要组分(心肌细胞和血管)分化。如通过免疫组织化学和/或RT-PCR评价的,某些细胞自发地表达(尤其是在CS的边缘)心肌细胞的标记(肌钙蛋白I,ANP,肌球蛋白重链)和内皮细胞的标记(vonWillebrand因子和Ve-钙粘蛋白)。在与大鼠肌细胞的共培养中,人CS自发地搏动。当在SCID小鼠中皮下接种时,鼠CS在几天内产生含有心肌组织和血管的生长。
发明人因而已经证实,SC可以以可再现的方式从1个月至70岁年龄的人对象的心房、心室和心耳的活检组织中衍生。可以冷藏属于本发明的CS,它们在融化后维持它们的功能特征。
也可以从小鼠分离出具有类似特征的成年心脏SC。更具体地,为了更好地理解CS中的细胞分化,研究了几个转基因小鼠品种;结果证实了用人细胞得到的结果。
最后,发明人已经在动物模型中显示了人CS可以用于心脏成形术。当接种在SCID小鼠的梗塞区域(经胸廓的烧灼或LAD结扎)时,所述细胞形成心脏组织,其呈现与宿主组织的良好整合,如通过形态学和免疫组织化学研究所观察到的。
因此,与先前技术所述的相比,就样品来源、衍生的细胞的分离和扩充方法以及形态和功能特征而言,通过本发明的方法分离和扩充CS是新的且有利的。
发明详述
该方法包括下述步骤:在无菌条件下得到活检样品,并运送到实验室;制备碎片,其大小足以允许培养基中存在的营养物的扩散;将碎片分布到培养平板上,在适于细胞存活和生长的条件下温育;对培养基和细胞取样,并转移到处于适合细胞扩充的条件下的其它培养平板。
本发明的一个目的是,获得能修复受损的心肌组织的干细胞的方法,其包括下述步骤:
a)取心脏组织的活检标本,并将其保持在适当的培养基中;
b)在适当的条件下,用温和的机械的和/或化学的和/或酶促技术处理标本,得到组织碎片,其大小足以允许培养基中存在的营养物的扩散;
c)使组织碎片粘附到适当的固相支持体上,并将它们维持在含有合适的血清和/或生长因子的培养基中;
d)使细胞生长,部分地或全部地更换培养基,直到形成多细胞结构,其弱粘附或不粘附到支持体上;
e)从剩余的培养物中分离所述的多细胞结构;
f)通过温和的解离处理所述的多细胞结构,直到大部分小的透亮的球形细胞脱附,但是维持它们的形态和功能特征;
g)将细胞平板接种在用聚赖氨酸或其它促进培养物向支持体粘附的试剂处理的培养基质上,在至少含有用于哺乳动物细胞生长的极限必需组分的培养基中;
h)可能重复步骤d)-g)至少一次;
i)选择聚集成透亮的球形体结构(心球体)的细胞;
l)通过它们的温和解离和新的形成,有选择地促进新心球体的形成;
m)最后冷藏心球体,准备在融化后使用。
优选地,干细胞源自非-胚胎的心脏组织活检。
在本发明的一个实施方案中,至少一个步骤是在用不同于大气中正常存在的氧浓度处理以改变培养物的生物学特征之后。
本领域的专家能够明白,源自本发明的方法的CS能够在自发转化或由化学的、物理的或生物的活性因子诱导的转化后,产生连续细胞系。
在另一个实施方案中,将产生和/或构成心球体的细胞与其它细胞融合。
在另一个实施方案中,将产生和/或构成心球体的细胞用于向和从其它细胞核转移。
在另一个实施方案中,使产生和/或构成心球体的细胞在至少一个阶段中生长在可生物降解的和/或生物相容的支持体上。
在另一个实施方案中,在生物反应器和/或发酵罐中培养产生和/或构成心球体的细胞。
本发明的另一个目的是,可以根据前述权利要求的方法得到的产生和/或构成心球体的能修复心肌组织的细胞。
优选地,所述的细胞将要用于基因治疗中。优选地,所述的细胞将要用于向和从其它细胞进行核转移。源自本发明的方法的CS可以不同地用于修复心肌组织,用于从和向其它细胞核转移,在基因疗法中用于遗传起源的心脏病。
附图简述
图1-CS增殖。a,源自人心房活检样品的原代培养物的漂浮CS(从<24小时至>48小时培养)的相差显微照片。b,在有(左图)和没有(右图)3.5%血清存在的情况下,人和小鼠CS[分别源自8名不同的对象(上图)和源自出生前的和出生后的心脏(下图)]的增殖曲线。球体的数目是指每孔的平均数目,在每个时间点从孔中取90%球体用于进一步分析。注意到人和小鼠CS之间不同的增殖方式,和曲线的快速升高,然后是在无血清的条件下的不可逆的下降。c,当通过有限稀释铺板到丝裂霉素-处理过的STO-成纤维细胞包被的96孔平板上CGM中时,在GFP-标记的克隆的产生过程中,单细胞的荧光分析(右上图)(从解离的表达GFP的CS得到)。该克隆可以在聚-D-赖氨酸涂层上传代和扩充(左下图)。d,暴露于无生长因子的培养基48小时后,eGFP/MLC3F克隆(作为那些人的得到)的x-Gal染色:在这些条件下,克隆中的细胞变得更扁平,许多核显示出蓝色,这证实发生了分化过程。
图2-CS表征。a,对BrdU-标记的人CS进行的心脏分化标记的荧光-共焦分析:6μm扫描(从球体的外周到中心)和最终的图片(分别是小图和大图)。BrdU(绿色),cTnI和ANP(红色)。b,培养12小时后人CS的共焦分析:产生CS的细胞在开始形成球体时的CD-34,CD-31,KDR和c-Kit标记。c,人CS(冷冻切片)的荧光表型分析:cTnI(红色),肌节肌球蛋白和vWf(绿色)。d-d1,在CEM中胶原涂层上培养4天后,人的部分解离的-CS的荧光表型分析:cTnl(红色)表达出现在人细胞(从球体迁移)的细胞质中,显示出三角形及行排列)。e,与大鼠心肌细胞共培养96小时时,部分解离的eGFP-标记的人CS的荧光分析:表现出与心肌细胞同步收缩的相同的绿色细胞表达cTnI。f,与大鼠心肌细胞共培养的eGFP-标记的人CS中的连接蛋白-43表达的荧光分析(红色)(如在小图e中):加强红色荧光存在于人细胞的细胞膜中。g,来自MLC3F-nlacZ和cTnI-nlacZ小鼠的CS的相差显微照片:在距它们的形成较短的时间后(插图)和在培养几天后(右图和中间图),核lacZ表达主要定位于胚胎和成年CS的外层。细胞(源自部分解离的CS,在胶原-包被的表面上培养了5天)的核也被蓝染。h,自发地分化的小鼠CS的荧光分析:如在培养物中显示出的同步收缩所暗示的,cTnI(红色)在球体和迁移的细胞中表达;最后,肌节也是明显的。i,来自GFP-cKit,GFP-cKit/MLC3F-nLacZ和GFP-cKt/cTnI-nlacZ小鼠的CS的荧光和相差分析。在将它们接种到含有CGM的培养物中后几分钟,在开始形成CS时,随后在它们的内部块中,和在它们从最老的粘附球体(箭头)迁移出来后,存在GFP-标记的细胞(上图、下图和左图和中间图)。GFP-标记的细胞不与染成蓝色的细胞(箭头)共同定位在来自GFP-cKit/MLC3F-nLacZ和GFP-cKt/cTnI-nlacZ小鼠的CS中;荧光细胞也存在于CS的生长区(箭头)(右侧的上图和下图)。荧光,相差(小的)和融合的(大的)图像。1,出生后小鼠CS-衍生的细胞的FACS分析。进行了在0和6天的时间进程,并分析了CD34,cKit,Cd31和sca-1表达的表型特征,显示为阳性事件的百分比。将数据表达为平均值±SD(n=3)。*指示着与T0的统计学显著差异。
图3-体内分析。a,来自MLC3F-nlacZ/B5-eGFP小鼠的CS在SCID小鼠中的异位移植(左上图)。来自皮下背部接种物(第17天)的未固定冷冻切片的荧光分析(左上小图,右上和左下大图):GFP-细胞似乎已经从球体迁移,而血管样结构簇主要在外部区域观察到(插图)。对这些冷冻切片之一进行SMA染色,显示了球体和接种物内的一些细胞的阳性免疫反应。b,来自MLC3F-nlacZ/CD-1和cTnI-lacZ/CD-1小鼠的CS的背部接种物的固定的和免疫染色的冷冻切片的荧光(右图)和相差分析(左图,融合的):对管状结构进行肌节肌球蛋白和cTnI染色(分别是中图和下图)。X-Gal染色标记了在CS内的和从CS迁移的细胞(右上图)。内皮标记(SMA和Ve-钙粘蛋白)染色了脉管系统(″黑洞″)(小图)。c,在SCID-bg小鼠上,将冷藏的人CS同位移植进新产生的梗塞边界处的活心肌中。自冠脉结扎18天后,冷冻切片的左心室心脏的共焦分析表明(左上小图)在再生心肌(特别是用2个中心箭头指示的那些)中表达MHC(红色)的心肌细胞对于核纤层蛋白A/C(一种特异性的人核标记)也呈染色阳性(绿色)。在这些细胞中,MHC表达主要在核周区域明显。核纤层蛋白A/C-标记的细胞(红色)存在于新产生的毛细血管中,后者对平滑肌α-肌动蛋白(右上图)和PECAM(左下图)染色;连接蛋白-43(红色)(右下高放大率图),如在共培养实验中,衬在再生心肌中的一些人细胞(绿色)的细胞质膜。表1.人CS同位移植对心肌性能的超声心动图指数的影响。将数据表达为平均值±SD。缩写:LVIDd,舒张末期左心室内部尺寸;AWThd,前壁厚度;FS,缩短分数;EF,射血分数。*:vs CAL+CS p<0.05,§:vs CALp<0.05
图4-a)(左图)来自少儿(pCS)、成年(aCS)对象的人CS和心脏碎片(H)的RT-PCR分析(ANF,NKx2.5,Ve-钙粘蛋白,GAPDH),和b)(右图)鼠CS(mCS)和小鼠心脏碎片(H)的RT-PCR分析(α-MHC,TnC,心脏α-肌动蛋白,GAPDH)。
方法和材料
组织样品
人组织取自成年人或经历了开放式心脏手术(主动脉冠状动脉分流术,心脏瓣膜置换,法洛四联症,室间隔缺损)或由于晚期扩张性心肌病或梗塞后慢性充血性心肌病而进行了心脏移植的其它患者的心肌活检。鼠组织取自先前表征为纯合MLC3F-nLacZ小鼠(8)纯合肌钙蛋白-I-nLacZ(9)和EGFP/ckit(10)CD1-杂交小鼠的心脏。小鼠分别表现出肌球蛋白轻链启动子的β-半乳糖苷酶转基因的局限定位性核表达(心脏和骨骼肌),肌钙蛋白-I转基因的组织特异性核表达(唯一地在心脏)和ckit启动子的EGFP转基因(在这些细胞实验中的基因)的细胞质表达。使用B5-EGFP小鼠(11)作为基本株,其表现出普遍的细胞质GFP表达。根据实验方法,培育繁殖杂交的MLC3F-nLacZ/EGFP,MLC3F-nLac-Z/EGFP-ckit,Tn-I-nLac-Z/EGFP-ckit小鼠。将人心脏组织活检保藏在0℃无血清的IMDM(Euroclone)中,并维持在该条件,直到到达实验室(在2小时内)。
球体-形成细胞的加工、分离和冷藏
小心地切离肉眼可见的结缔组织后,将样品切成1-2mm3碎块,用无Ca++/Mg++的磷酸盐缓冲溶液(PBS,Invitrogen)洗涤3次,相继用0.2%胰蛋白酶(Gibco)和0.1%胶原酶IV(Sigma)在37℃消化3次,每次5分钟。抛弃得到的细胞,其大部分是污染血液的成分,用完全外植块培养基(CEM)[IMDM,其添加了10%胎牛血清(FCS)(Hyclone),100mg/ml青霉素,100U/ml链霉素(Gibco),2mM L-谷氨酰胺(Gibco),0.1mM 2-巯基乙醇(Sigma)]洗涤剩余的组织碎片。然后,通过用解剖刀在塑料表面上轻刮,将组织碎块固定到培养皿(Falcon)上。在37℃、5%CO2,在完全IMDM中培养外植块。类似地处理鼠心脏组织,例外是对于胚胎心脏,省略了外植块消化之前的酶消化,用25号针部分解离器官。1-3周后(根据样品的来源,即对于胚胎组织更短的时间,对于成年组织更长的时间),形成一层成纤维细胞样细胞,其源自或围绕着外植块。然后定期处理外植块(每6-10天,最多4次),以分离球体-形成细胞。为了仅仅取出透亮的细胞-其从外植块向外细胞层迁移,取出培养基,如下收集材料:通过用无Ca++-Mg++的PBS洗涤2次、并用0.53mM EDTA(Versene,Gibco)洗涤1-2分钟1次,随后,在肉眼显微镜控制下,在室温用0.5g/L-0.53mM胰蛋白酶-EDTA(Gibco)温和地胰蛋白酶消化再2-3分钟。收集细胞后,将完全培养基加入外植块,通过离心(1200rpm,7分钟)收集经洗涤和酶处理得到的细胞,并重悬于心球体-生长培养基(CGM)(35%完全IMDM/65%DMEM-Ham′s F-12混合物,含有2%B27[Gibco],0.1mM 2-巯基乙醇,10ng/ml EGF(Prepotek EC,Ltd.),40ng/ml bFGF(Prepotek EC,Ltd.),4nM cardiotrophin-1(RD),40nM凝血酶(Sigma)(终浓度),抗生素和L-Glu,如在完全培养基中的。根据得到的细胞的数目(104-4×105细胞/外植块),通过重复吸取它们,然后以约2×105细胞/ml平板接种到聚-D-赖氨酸(BD)包被的多孔平板上,重悬细胞。12-24小时后,若干细胞开始分裂,48小时后,形成细胞团,其经常被薄膜围绕,且可以作为漂浮的球体和粘附的球体生长。每2-3天,部分地更换生长培养基,使用吸管或1ml针头,机械地捣碎球体。为了冷藏,将球体(在无Ca++-Mg++的PBS和Versene中洗涤)重悬在冷冻培养基(完全IMDM/DMEM-Ham-F-12 50∶50,5%B27,10%DMSO)中。为了计算生长曲线,在生长的第一周中,计数所有的球体,然后在指定的时间取出90%球体(并用于RT-PCR或免疫组织化学分析);加入CGM和机械地捣碎剩余的球体后,使它们增殖,直到下一次取样,那时对它们重新计数。如(Roche)所述,对新产生的球体进行BrdU标记12小时,并在指定的时间在其它球体中进行标记。为了克隆分析,如别处所述(12),用表达绿色荧光蛋白(GFP)的第3代慢病毒载体pRRLsin.PPT-PGK.GFP感染了人CS。洗涤2次后,通过依次在无Ca++-Mg++的PBS、Versene和1×胰蛋白酶-EDTA溶液中捣碎,将GFP-标记的CS解离成单个细胞,重悬在CGM中,然后以假定的1细胞/孔的浓度接种到用丝裂霉素-C处理的STO成纤维细胞(2ug/ml)的滋养层包被的96-孔平板中。为了在基质-包被的表面上分化,重复吸取经无Ca++/Mg++的PBS洗涤的、离心的和部分解离的CS,然后在小体积的CEM(200-300μl)中接种到I型胶原-(Sigma)或Matrigel-(Falcon)包被的培养皿上,培养3-6天。
体内分析
为了异位移植,将从出生前的和出生后的EGFP/MLC3F-nLacZ或EGFP/TnI-nLacZ或MLC3F/nLacZ,TnI-nLacZ小鼠得到的约60个混合CS在PBS中洗2次,并悬浮在100μl Matrigel(BD)中,皮下注射到麻醉的(氯胺酮,35mg/kg,肌肉内地)成年NOD-SCID小鼠的背部。透过皮肤直接触摸搏动,监视移植的心球体的存活和功能。约3周后,处死小鼠,将分离的接种物包埋入OCT,用于免疫细胞化学分析。融化后,将源自成年对象的心室和心房心脏外植块的冷藏的人CS的10天培养物用于同位移植。将约20个经洗涤的和部分解离的CS悬浮在3μl PBS中,并使用27号针头和Hamilton注射器,注射进梗塞的心肌区域。如别处所述(13),略作改动,诱导了心肌梗塞。简而言之,用通过心脏前表面上第4肋间隙内的肋间内肌插入的改进的电灼探头,使受体NOD-SCID小鼠(用氯胺酮[35mg/kg]+甲苯噻嗪[5mg/kg]i.p.麻醉)接受经胸廓烧灼(Surgitron 140v)。在注射CS之前,以切割模式应用电灼术(ca.40W)2次1秒(将相同体积的PBS注射进对照小鼠中)。在一些小鼠中,还已经通过LAD结扎诱发了心肌梗塞。约3周后,处死小鼠,在PBS中泛泛洗涤并用在PBS pH 7.4中的低聚甲醛(4%)固定后,将分离的心脏包埋入OCT中。
免疫细胞化学
如别处所述(14),使用下面的抗体,对组织切片和细胞培养物进行了免疫细胞化学分析:单克隆的抗-人-cTnI,抗-人-心脏-MHC,抗-人核和多克隆的(pAb)抗-人ANP(Chemicon);mAb抗-CD-31,CD-34(BD Biosciences),mAb抗-人Cripto-1(RD),单克隆的抗-Ve-钙粘蛋白,抗-sca-1,mAb抗-小鼠-cKit(Pharmigen),mAb抗-人-c-Kit(DAKO);pAb抗-人-von-Willebrand-因子和mAb抗-人-KDR(Sigma);mAb MF20和pAb抗-小鼠/人MHC(14),抗-desmine和抗-平滑肌-肌动蛋白(Sigma),mAb抗-人/小鼠-cTnI(15),由S.Schiaffino(Dept.of Pathology,Univ.of Padua)捐赠,pAb抗-小鼠-flk-1(Santa Cruz,USA)。如别处所述(14),通过光学显微镜检测了β-半乳糖苷酶活性。
反向-PCR转录分析
如别处所述(16),进行了反向-PCR转录分析。用于扩增源自少儿(pCS)、成年对象(aCS)的CS和心脏碎片(H)的基因的寡核苷酸如下:
hNkx2,5(150bp)正向5′-CTCCCAACATGACCCTGAGT-3′和
反向5′-GAGCTCAGTCCCAGTTCCAA-3′,
hANF(350bp)正向5′-AATCAAGTTCAGAGGATGGG-3′和
反向5′-AATGCATGGGGTGGGAGAGG-3′,
hVe-Cad(330bp)正向5′-TCTCTGTCCTCTGCACAA-3′和
反向5′-ATGCAGAGGCTCATGATG-3′,
hGAPDH正向5′-GAAGAGCCAAGGACAGGTAC-3′和
反向5′-CTGCACCACCAACTGCTTAG-3;
用于扩增鼠CS和心脏碎片(H)的基因的寡核苷酸如下:
mMHC(302bp)正向5′-GAAGAGTGAGCGGCGCATCAAGGA-3′和
反向5′-TCTGCTGGAGAGGTTATTCCTCG-3′,
正向5′-TGTTACGTCGCCTTGGATTTTGAG-3′和
反向5′-AAGAGAGAGACATATCAGAAGC-3′,
m心脏TnC(410bp)正向5′-AATGGATGACATCTACAAAG-3′和
反向5′-TGAGCTCTTCAATGTCATCT-3′.
mGAPDH正向5′-CCTCTGGAAAGCTGTGGCGT-3′和
反向5′-TTGGAGGCCATGTAGGCCAT-3′。
结果
CS的分离和扩充
通过温和地酶消化外植的人心房或心室活检和胎儿、胚胎和出生后小鼠的心脏,获得球体-产生细胞。从较好粘附的外植块产生一层成纤维细胞样细胞后不久,小的、圆形的透亮的细胞开始在该层上迁移。通过EDTA处理和温和的胰蛋白酶消化,可以定期地收获这些细胞,并使其在低血清(3.5%FCS)培养基(添加了血清替代物(B27),生长因子(EGF和bFGF),cardiothrophin-1(CT-1)(17)和凝血酶(18))中在聚-D-赖氨酸-包被的培养表面上生长,在培养的第一周,与使用单独地或组合地添加了其它因子的培养基得到的结果相比,这使球体数目增加了7倍。对源自人和鼠外植块的细胞的时间进程观察表明,它们接种后早期(30分钟),这些细胞中的一些开始分裂,同时仍然悬浮;大多数细胞变成松散粘附的,其它的保持悬浮,且一些污染性成纤维细胞样细胞紧紧地附着在聚-D-赖氨酸层上。从松散粘附的细胞群也明显地观察到了细胞分裂,并在10-12小时后,产生了小的、圆形的透亮的细胞团[我们将其称作心球体(CS)](图1a)。自它们出现24-36小时内,CS的大小增加,它们中的一些从培养表面脱附;48-72小时后,大多数CS是20-150μm大小,当不进行机械解离时,最大的在它们的内部团块中含有黑色区(图1a)。
鼠CS在它们产生后不久开始自发的节律性收缩活性,并在它们的寿命过程中维持该功能,而人CS仅仅在与大鼠心肌细胞共培养时才这样。为了确认收缩是cs细胞获得的新性状,将gfp-标记的人CS(部分地或完全地解离的)与用或未用dil预染色的心肌细胞共培养。在共培养48小时后,观察到了收缩的gfp-标记的细胞;另外,从该时间起,绿色荧光细胞也染上了红色,表明人CS和大鼠心肌细胞之间建立了联系。实际上,用cx-43(主要的心室间隙连接蛋白)标记人cs/大鼠-心肌细胞共培养物(其中仅人细胞通过慢病毒感染预标记了gfp),证实了沿着细胞质膜的典型的加强荧光图案(图2f),表明在这2个细胞群体之间建立了功能联系。
发现CS由克隆衍生的细胞组成,且并不简单地代表细胞聚集体。实际上,当将人CS[在感染了表达报告基因的慢病毒载体后,表达绿色荧光蛋白(GFP)]或鼠CS(源自eGFP/MLC3F或eGFP/cTrI-小鼠)解离并作为单个细胞平板接种在经丝裂霉素-处理的STO-成纤维细胞-包被的96-孔上或以克隆稀释接种到10cm直径的培养皿上时,以1-10%效率产生了可以亚克隆到聚-D-赖氨酸-包被的表面上的荧光球体(图1c)。这些亚克隆衍生的CS在培养中表现出相同的功能和表型行为:自它们出现3天后,一些鼠克隆开始表现,并在用CEM培养48小时后,它们中的大多数(6/7)表现出核内的lac-Z转基因的表达(在特异性的组织化学染色后)(图1d)。同样地,源自单个GFP-标记的细胞的人克隆在与大鼠心肌细胞共培养48小时后开始同步搏动并表达cTnI。
另外,当将BrdU加入培养基中时,实际上小CS中的几乎所有细胞和最大CS内部的那些细胞被标记(图2a),表明这些细胞是新产生的。
人CS-产生细胞能自我更新。利用定期的解离,以及每2-3天部分地更换生长培养基,得到了球体的对数期扩充(图1b)。小鼠CS的生长较慢(可能是由于培养中承担的更为分化的特征,例如搏动),且如同人的一样是血清-依赖性的(图1b)。
如图2a所示,用抗-BrdU(绿色)和心脏-肌钙蛋白i(ctni)或心房利钠肽(anp)(红色)共焦免疫荧光分析BrdU标记的人CSS,揭示了BrdU-阳性的细胞特别地在球体内部,而ctni-或anp-阳性的细胞主要定位于外层。另外,若干cs-细胞表达心脏分化标记(ctni,anp),同时仍在分裂,如BrdU掺入所表明的(图2a),这表明在对数期生长的过程中,已经发生了早期的心脏分化;一般地,在2-3周内,一些球体变成粘附的,表现出更扁平的形态。一些小细胞最终以能产生新球体的粘附的(分化的)或小的圆形细胞的形式,从这些″太阳样″球体迁移出来。从冷藏融化后,CS再次增殖,维持它们搏动的倾向。
新发生的人和小鼠CS的表型分析揭示了内皮(KDR(人)/flk-1(小鼠),CD-31)和干-细胞(CD-34,ckit,sca-1)标记物的表达。如图2b所示,在2-10细胞阶段的CS能强烈地与针对这些抗原的抗体反应。在更大的球体中,这些标记物中的一些(尤其是cKit)的表达模式类似于BrdU标记(在中心和在一些产生卫星球体的周围区域的阳性染色)。
通过FACS分析,用这些干细胞和内皮标记进行了CS细胞的定量表征的时间进程(0和6天)(图21:如所示的,在它们开始形成时(T0),这些细胞的表型似乎反映epi-荧光显微分析,对所有4种表型有约10%阳性染色。但是,在第6天(T6),cKit作为唯一保守的标记物出现,表明cKit+细胞可能是有助于维持增殖的主要细胞,而对其它标记的初始阳性可能反映出早期活化状态,如已经在几个系统中关于CD-34所指明的(19)。对冷冻切片的人CS进行的荧光显微术分析揭示了心脏-分化标记(cTnI,MHC)和内皮标记[von Willebrand因子(vWf)]的表达(图2c)。当完全地或部分解离成单细胞并在胶原-包被的培养皿上在与外植块相同的培养基中培养时,小鼠和人CS-衍生的细胞呈现出了典型的心肌细胞形态学,表型(图d-d1,h)和功能(通过自发性收缩证实(仅仅在小鼠中))。
如前所述,人CS不能自发地搏动;但是,当与出生后的大鼠心肌细胞共培养时,它们在24小时内开始搏动,在该时刻之后,丧失它们的球体形状,并呈现出″太阳样″外观。在人标记的CS-细胞中,心脏分化的标记物与GFP共表达(图2e)。
为了追踪在出生前和出生后年龄过程中CS的分化过程,使用了MLC3F-nlacZ和cTnI-nlacZ小鼠(8,9)。这些小鼠表达定位于核内的一种形式的lacZ转基因,该转基因分别在骨骼肌和心肌肌球蛋白轻链或心脏肌钙蛋白I启动子的控制下。从胚胎第9-12天、胎儿第17-18天、新生的和成年的小鼠得到的CS,表现出了报告基因的自发表达,在采用的不同培养条件下,以可变的球体百分比(10-60%)(图2e);另外,如同人的,来自小鼠的CS产生细胞表达干细胞(CD-34,sca-1,cKit)和内皮细胞标记物(flk-1,CD-31)。
在此基础上,我们使用表达在c-kit启动子控制下的绿色荧光蛋白(GFP)的转基因小鼠(10),进一步阐明这些球体的细胞来源,并追踪它们的生长过程的模式。如图2i所示,从开始形成CS时,就存在GFP-阳性的细胞,且虽然具有减低的荧光强度,以后也在CS的细胞团块内和在从老的″太阳样″粘附CS迁移的细胞中。另外,如人CS的生长模式所暗示的,当卫星二代CS表现出从原代CS脱附时,GFP-阳性细胞定位于后者的边缘和在前者的内部。
我们在由GFP-cKit/MLC3F-nlacZ或GFP-cKit/cTnI-nLacZ杂交得到的双-杂合小鼠中研究了该过程:如图2i所示,β-Gal-阳性没有与GFP共同定位于存在于生长区内的细胞中。
总之,CS似乎是心脏干细胞、分化祖细胞和甚至自发分化的心肌细胞的混合物。取决于球体的大小和培养的时间,也可以存在血管细胞。关于神经球体(20),分化中的/分化了的细胞可能停止分裂和/或死亡,而干细胞以明显非对称的方式继续增殖,产生许多二代球体,并在体外指数生长。机械的解离有利于该过程。在CS内的不同细胞的死亡、分化和对生长因子的反应性,可取决于它的三维结构和在CS内的定位(21-22)。球体的自发形成是神经干细胞、一些肿瘤细胞系(LIM)(22)、内皮细胞(23)和胎鸡心肌细胞(24)的已知特权。所有这些模拟组织的真实三维结构的模型(包括我们的),由聚集细胞的球形体组成,所述聚集细胞发展成由分化细胞表层和无组织细胞核心组成的双区室系统,所述无组织细胞首先增殖,此后随时间消失(可能通过凋亡性细胞死亡)。如在胎鸡心肌细胞和内皮细胞球形体培养中充分证实的,三维结构影响细胞对存活和生长因子的敏感性(22,23)。更具体地,中心的球形体细胞不分化,且依赖于存活因子而防止细胞凋亡,而表层细胞的分化似乎超过了可以在二维培养中得到的程度,且变得不依赖于存活因子的活性。另外,如″生态位″假说所提出的(26),根据干细胞只在适当的环境中保持它们的多能性的观点,已知细胞-细胞接触和膜-相关因子对于神经前体细胞的分裂是重要的(25)。
为了研究CS在体内的存活和形态-功能潜力,进行了2组实验:在第一组中,将CS细胞注射进SCID小鼠的背部皮下区域;在第二组中,将它们注射进心肌梗塞后急性期的SCID-浅褐色小鼠的心脏。异位移植实验的目的是研究CS在中性环境(即没有特殊心脏诱导的环境)中的生长模式和行为,以验证它们产生主要心脏细胞类型的独特潜力,并排除致瘤性转化的可能性。为了这些实验,使用了来自出生前的和出生后的MLC3F-nlacZ/B5-eGFP TnI-nlacZ/B5-eGFP小鼠或MLC3F-nlacZ/CD-1和cTnI-nlacZ/CD-1小鼠的约60混合球体/接种物/小鼠。在前10天,通过远离大血管的注射部位上方的皮肤,可感知搏动。在第17天,处死动物,识别接种物为半透明的结构、谷粒样大小、包在有分支的血管样结构中。通过荧光显微术观察未固定的冷冻切片,揭示了开放球体的存在,细胞似乎已从该球体迁移;″黑洞″簇,尤其是在该结构的周边,是明显的。该组织包含管状结构,被核围绕(Hoechst-阳性),鉴定为心肌节,因为它们是cTnI和肌节肌球蛋白阳性的(图3a)。α-SMA-阳性结构(已知在心肌发生过程中瞬时表达(27))存在于球体的剩余部分中,且与脉管系统(″黑洞″簇)相关:这呈现出具有表达Ve-钙粘蛋白的薄内皮的良好分化的结构(图3a)和相对较大的含有红细胞的内腔,表明宿主建立了成功的灌注。X-Gal染色后,通过光学显微镜观察接种物,显示出横纹肌-特异性的lacZ在球体的剩余部分和靠近它们的一些细胞中强烈的核表达。没有观察到提示存在肿瘤形成的多分化结构。
为了测试当激发到梗塞的心肌中时CS的功能能力和心脏再生潜力的获得,用人CS进行了同位移植实验。为此,将融化的(冷藏的)取自三个心房(1名男性和2名女性)和一个心室(1名女性)的活检的成年人CS注射进新鲜产生的梗塞边缘处的活心肌。每只小鼠接受了来自单一传代外植块(源自单一对象)的CS。给4只对照梗塞动物注射了等体积的PBS。自干预起18天后,处死动物,测量梗塞大小。在CS-处理组和PBS-注射组中,梗塞大小分别是34.9±7.1(3.6)和31.9±6.9(3.5)(p=n.s.)。但是,超声心动图显示了与PBS-注射组相比,在CS-处理组中更好地保存了梗塞的前壁厚度(0.80±0.29(0.15)vs.0.60±0.20(0.08)p=n.s.),特别是FS%(36.85±16.43(8.21)vs.17.87±5.95(2.43)p<0.05)(图3-表1)。
在评价时,如用苏木精-伊红组织化学和MHC免疫荧光所评价的,在大部分梗塞区中,存在再生心肌带(具有不同程度的组构和厚度)(图3c)。在再生心肌中,表达核纤层蛋白A/C(一种特异性的人核标记物)的细胞也与MHC染色阳性的心肌细胞、平滑肌α-肌动蛋白和PECAM染色的新产生的毛细血管(图3c)和表达连接蛋白-43的细胞(如在共培养实验中,其定义了人细胞和再生心肌之间的关联)共定位。
因而,可以将CS视作成年干细胞的克隆,在冷藏后也能维持它们在体外和体内的功能性质。
在准备本原稿的同时,已经公开了2篇文章,它们涉及从成年哺乳动物心脏分离心脏干细胞或祖细胞(28,29)。这些细胞的分离仅仅基于干细胞-相关表面抗原的表达:第一篇文章中的c-kit和第二篇文章中的Sca-1。在第1项研究中(28),发现从大鼠心脏新鲜分离的c-kit阳性Lin-细胞是自我更新的、可形成克隆的和多能性的,表现出向生肌的平滑肌细胞或内皮细胞系的生化分化,但是与在本文所述的条件下生长的细胞不同,不能自发地收缩。当注射进缺血的心脏中时,这些细胞再生功能性心肌。在第2项研究中(29),在体外诱导来自小鼠心脏的Sca-1+ cKit阴性细胞,以对5′-氮杂胞苷作出响应而向生心肌系分化。当在缺血/再灌注后静脉内地施用时,这些细胞归巢至受损的心肌,并分化成与和不与宿主细胞融合的心肌细胞。我们在GFP-cKit转基因小鼠上得到的数据也表明,该成年心脏干细胞是cKit阳性。可能CS包封混合的细胞群,后者作为生态位可促进cKit祖细胞的生存力,并有助于它们的增殖。在本文章中得到的数据,证实了成年心脏干细胞的存在。更重要地,它们首次证实了可以从非常小的人心肌碎片分离细胞,并在体外扩充这些细胞许多倍(达到适于在患者中进行体内移植的数目),而不丧失它们的分化潜力,从而开辟以前未预见到的心肌修复的机会。
转基因小鼠
为了追踪在出生前和出生后的年龄过程中CS的分化过程,使用了MLC3F-nlacZ和TnI-nLacZ小鼠。这些小鼠表达定位于核内的一种形式的lacZ转基因,该转基因分别在骨骼肌和心肌肌球蛋白轻链或心脏肌钙蛋白I启动子的控制下(8,9)。从胚胎第9-12天、胎儿第17-18天、新生的和成年的小鼠得到的CS,表现出了报告基因的自发表达,在采用的不同培养条件下以可变的球体百分比(10-60%)(图4a);另外,小鼠CS在开始形成时(特别是在胚胎中)就开始搏动,并在它们的寿命过程中持续搏动。人CS表达干细胞(CD-34,sca-1,cKit)和内皮细胞标记物(flk-1,CD-31)。
为了进一步阐明这些球体的细胞来源,并追踪它们的生长过程的模式,我们使用了在c-kit启动子控制下表达绿色荧光蛋白(GFP)的转基因小鼠(10)。从开始形成CS时,就存在GFP-阳性的细胞,且虽然具有减低的荧光强度,以后也是如此。另外,如人CS的生长模式所提示的,当卫星二代CS开始从原代CS脱附时,GFP-阳性的细胞定位于后者的边缘和在前者的内部。我们在通过EGFP-cKit/MLC3F-nlacZ或TnI-nLacZ杂交得到的双-杂合的小鼠中研究了该过程。如图4b所示,β-Gal-阳性没有与EGFP共同定位在存在于生长区内的细胞中。
遗传表型
图5显示了在鼠或人CS RNA提取物上建立的RT-PCR图。与鼠样品相比,心脏祖细胞的更典型的特征似乎是人样品的(在对数生长期),其中增殖和分化更容易一起发生。
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Claims (11)
1.获得能修复受损的心肌组织的干细胞的方法,其包括下述步骤:
a)取心脏组织的活检标本,并将其保持在适当的培养基中;
b)在适当的条件下,用温和的机械的和/或化学的和/或酶促技术处理标本,得到组织碎片,其大小足以允许培养基中存在的营养物的扩散;
c)使组织碎片粘附到适当的固相支持体上,并将它们维持在含有合适的血清和/或生长因子的培养基中;
d)使细胞生长,部分地或全部地更换培养基,直到形成多细胞结构,其弱粘附或不粘附到所述支持体上;
e)从剩余的培养物中分离所述的多细胞结构;
f)通过温和的解离处理所述的多细胞结构,直到大部分小的透亮的球形细胞脱附,但是维持它们的形态和功能特征;
g)将细胞平板接种在用聚赖氨酸或其它促进培养物向支持体粘附的试剂处理的培养基质上,在至少含有用于哺乳动物细胞生长的极限必需组分的培养基中;
h)可能重复步骤d)-g)至少一次;
i)选择聚集成透亮的球形体结构(心球体)的细胞;
l)通过它们的温和解离和新的形成,有选择地促进新心球体的形成;
m)最后冷藏心球体,准备在融化后使用。
2.根据权利要求1的方法,其中干细胞源自非-胚胎的心脏组织活检。
3.根据权利要求1的方法,其特征在于,至少一个步骤是在用不同于正常情况存在于大气中的氧浓度处理以改变培养物的生物学特征之后。
4.根据权利要求1的方法,其特征在于,衍生的心球体能在自发转化或由化学的、物理的或生物的活性因子诱导的转化后,产生连续细胞系。
5.根据权利要求1的方法,其特征在于,产生和/或构成心球体的细胞进一步与其它细胞融合。
6.根据权利要求1的方法,其特征在于,产生和/或构成心球体的细胞用于向和从其它细胞核转移。
7.根据权利要求1的方法,其中使产生和/或构成心球体的细胞在至少一个阶段生长在可生物降解的和/或生物相容的支持体上。
8.根据权利要求1的方法,其中在生物反应器和/或发酵罐中培养产生和/或构成心球体的细胞。
9.可以根据前述权利要求的方法得到的产生和/或构成心球体的能修复心肌组织的细胞。
10.根据权利要求9的产生和/或构成心球体的细胞用于基因治疗。
11.根据权利要求9的产生和/或构成心球体的细胞用于向和从其它细胞核转移。
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- 2004-07-29 CA CA2793046A patent/CA2793046C/en not_active Expired - Lifetime
- 2004-07-29 WO PCT/IT2004/000421 patent/WO2005012510A1/en active Application Filing
- 2004-07-29 EP EP04770731.0A patent/EP1649012B1/en not_active Expired - Lifetime
- 2004-07-29 US US10/567,008 patent/US8268619B2/en active Active
- 2004-07-29 CN CNA200480026720XA patent/CN1852973A/zh active Pending
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2011
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- 2011-09-26 US US13/245,788 patent/US8772030B2/en not_active Expired - Lifetime
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2016
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Cited By (5)
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CN101215547B (zh) * | 2008-01-15 | 2011-06-08 | 刘岱良 | 一种分离、纯化及提取脂肪间充质干细胞的方法 |
CN108179133A (zh) * | 2018-02-06 | 2018-06-19 | 广州大学 | c-kit+心脏干细胞聚集体及分泌合成培养液在制备药物中应用 |
CN108359635A (zh) * | 2018-02-06 | 2018-08-03 | 广州大学 | 一种用于富集纯化c-kit+心脏干细胞的复合涂层及其制备方法 |
CN113207298A (zh) * | 2019-12-02 | 2021-08-03 | T&R碧欧法博有限公司 | 提高在冷冻保存及低氧条件下的存活率的心肌细胞聚集体制备技术 |
CN113207298B (zh) * | 2019-12-02 | 2024-05-28 | T&R碧欧法博有限公司 | 提高在冷冻保存及低氧条件下的存活率的心肌细胞聚集体制备技术 |
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CA2793046A1 (en) | 2005-02-10 |
CA2793046C (en) | 2016-02-16 |
US20120021019A1 (en) | 2012-01-26 |
CA2534378A1 (en) | 2005-02-10 |
US20120020935A1 (en) | 2012-01-26 |
US8268619B2 (en) | 2012-09-18 |
US20120045421A1 (en) | 2012-02-23 |
ITRM20030376A1 (it) | 2005-02-01 |
US20120093879A1 (en) | 2012-04-19 |
EP1649012A1 (en) | 2006-04-26 |
EP2465921A3 (en) | 2013-06-05 |
US20070020758A1 (en) | 2007-01-25 |
ITRM20030376A0 (it) | 2003-07-31 |
CA2534378C (en) | 2013-12-17 |
WO2005012510A1 (en) | 2005-02-10 |
EP2465921A2 (en) | 2012-06-20 |
EP1649012B1 (en) | 2017-03-29 |
US8772030B2 (en) | 2014-07-08 |
US8846396B2 (en) | 2014-09-30 |
US20160244723A1 (en) | 2016-08-25 |
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