CN101541954A - 含有人脐带血衍生的间充质干细胞的组合物诱导神经前体细胞或神经干细胞分化和增殖为神经细胞的用途 - Google Patents

含有人脐带血衍生的间充质干细胞的组合物诱导神经前体细胞或神经干细胞分化和增殖为神经细胞的用途 Download PDF

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CN101541954A
CN101541954A CNA2007800440974A CN200780044097A CN101541954A CN 101541954 A CN101541954 A CN 101541954A CN A2007800440974 A CNA2007800440974 A CN A2007800440974A CN 200780044097 A CN200780044097 A CN 200780044097A CN 101541954 A CN101541954 A CN 101541954A
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吴元一
梁允瑄
蒋钟旭
崔秀真
金珠渊
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Abstract

本发明提供了包含脐带血衍生的间充质干细胞的组合物用于诱导神经前体细胞或神经干细胞分化增殖为神经细胞的用途,所述组合物对于治疗神经损伤性疾病是有效的。

Description

含有人脐带血衍生的间充质干细胞的组合物诱导神经前体细胞或神经干细胞分化和增殖为神经细胞的用途
发明领域
本发明涉及包含人脐带血衍生的间充质干细胞的组合物用于诱导神经前体细胞或神经干细胞分化或增殖为神经细胞的用途。
发明背景
中风、帕金森病、阿尔茨海默病、皮克氏病、亨廷顿病、肌萎缩侧索硬化、外伤性中枢神经系统疾病和脊髓损伤疾病涉及由于神经细胞损伤导致的神经机能障碍,它们通常通过可能严重损害正常细胞的药物和外科手术治疗。
最近,发现移植正常细胞来替代被破坏的或损伤的细胞的细胞替代疗法对于这种疾病是有效的,特别地,能够分化和增殖为所需组织的神经细胞正在大力研究中。
干细胞是非特化细胞,其可以在非分化阶段无限增殖并可以在应答特异性刺激时分化为多种组织。
可以形成神经元和/或神经胶质如星形胶质细胞、少突胶质细胞和/或神经膜细胞的神经干细胞也是具有自我繁殖能力的未分化细胞。它们通过神经前体细胞或神经胶质前体细胞而分化为神经细胞,例如神经元或神经胶质。
已知分化为骨、软骨、脂肪组织、肌肉、腱、韧带、神经组织和其他的间充质干细胞对于细胞替代疗法是可行的。间充质干细胞主要自骨髓获得,但是这种间充质干细胞由于其有限的分化和增殖能力而仅提供有限的应用。而且,除了发现与患者具有相同的组织相容性抗原的供体以避免在骨髓移植中的移植物抗宿主反应的问题之外,对于这种细胞替代疗法还需进行包括数个步骤的复杂、通常痛苦的术语。
近年来,脐带血由于其高干细胞浓度而成为研究者的目标。已经进行了许多通过移植脐带血给患者来治疗血液病的试验,而且已经建立了以冷冻形式保存脐带血直至使用的脐带血库以用于自体移植疗法。
与骨髓不同,脐带血可以通过简单的术语从脐带获得而且其几乎不引起移植物抗宿主反应。为此,最近进行了关于脐带血临床应用的世界性研究。
本发明人深入研究了脐带血衍生的间充质干细胞并且发现其能够诱导神经前体细胞或神经干细胞分化增殖为神经细胞。
发明简述
因此,本发明的一个目的是提供包含脐带血衍生的间充质干细胞的组合物诱导神经前体细胞或神经干细胞分化增殖为神经细胞的用途。
本发明另一方面提供了诱导神经前体细胞或神经干细胞分化增殖为神经细胞的方法,其包括共同培养脐带血衍生的间充质干细胞和神经前体细胞或神经干细胞。
本发明再一方面通提供了诱导神经前体细胞或神经干细胞分化增殖为神经细胞的组合物,其包含作为活性成分的脐带血衍生的间充质干细胞。
本发明再一方面通提供了治疗神经损伤疾病的方法,包括将所述组合物给予需要治疗所述神经损伤疾病的对象的神经细胞损伤区域。
附图简述
本发明的上述和其他目的和特征根据如下发明描述以及结合所述附图将会变得清楚,其分别示出:
图1:用于共同培养脐带血衍生的间充质干细胞和神经前体细胞的transwell室(chamber)的示意图;
图2:示出单独培养NG108-15或与脐带血衍生的间充质干细胞共同培养后4天和7天,用相差显微镜(×100)观测到的NG108-15(NG108)的分化增殖图;
图3:示出单独培养NG108-15或与脐带血衍生的间充质干细胞共同培养后7天神经分化的早期标记微管蛋白βIII的免疫染色结果图;
图4:示出在补加cAMP单独培养NG108-15细胞或者与得自两个不同个体(hUCB-MSC-1和hUCB-MSC-2)的脐带血衍生的间充质干细胞共同培养后7天,用相差显微镜(×100)观测到的NG108-15的分化增殖图;
图5:示出在神经干细胞分别与不同浓度的hUCB-MSC-1和hUCB-MSC-2共同培养后7天用相差显微镜(×100)观测到的衍生自胎鼠脑皮层的神经干细胞的分化增殖图;
图6:示出单独培养衍生自胎鼠脑皮层的神经干细胞或与脐带血衍生的间充质干细胞共同培养后7天神经分化的早期标记微管蛋白βIII和微管相关蛋白2(MAP2)的免疫染色结果图;
图7:示出NG108-15与不同浓度的脐带血衍生的间充质干细胞共同培养后7天通过台盼蓝染色评估的存活细胞数的示意图;
图8:示出衍生自胎鼠脑皮层的神经干细胞与不同浓度的脐带血衍生的间充质干细胞共同培养后7天通过台盼蓝染色评估的存活细胞数的示意图。
发明详述
本发明诱导神经前体细胞或神经干细胞分化增殖为神经细胞的组合物特征在于包含作为活性成分的脐带血衍生的间充质干细胞。
如本文所用,术语“脐带血”指取自联系母亲与新生儿的胎盘的脐带静脉的血液。
如本文所用,术语“脐带血衍生的间充质干细胞”指分离自哺乳动物优选人的脐带血的间充质干细胞。
如本文所用,术语“神经损伤疾病”指伴随由于受损的运动或感觉神经导致的行为功能障碍的疾病。示例的神经损伤疾病包括中风、帕金森病、阿尔茨海默病、皮克氏病、亨廷顿病、肌萎缩侧索硬化、外伤性中枢神经系统疾病和脊髓损伤疾病。
术语“治疗”指:预防动物、优选哺乳动物、最优选人中尚未确诊的疾病或病症的表现,动物倾向于患有所述疾病或病症;或者抑制神经损伤疾病的进展。
如本文所用,术语“神经细胞”指中枢或周围神经系统的神经元和/或神经胶质如星形胶质细胞、少突胶质细胞和/或神经膜细胞。
分离包含来自脐带血的间充质干细胞的单核细胞时可以使用通常方法如Ficoll-Hypaque密度梯度方法。特别地,所述方法包括如下步骤:在分娩直至胎盘脱落后自脐带静脉采集脐带血;用Ficoll-Hypaque梯度离心所述脐带血以获得单核细胞;及从中去除污染物。获得的单核细胞可以从中分离间充质干细胞或者超低温冷冻以长期保管直至使用。
可以通过Yang SE et al.(Yang SE et al.,Cytotherapy,6(5):476-486,2004)的方法从脐带血衍生的单核细胞分离间充质干细胞。特别地,单核细胞悬浮在含有5-30重量%、优选5-15重量%的胎牛血清(FBS)的培养基中,所述培养基包括常规培养基如DMEM、α-DMEM、Eagle′s基础培养基RPMI1640。然后所述悬浮液中的细胞被分到具有如上所述相同组成的培养基中并在5%CO2培养箱、37℃培养。当培养的细胞形成单层时,观测到具有纺锤形的间充质干细胞。然后将间充质干细胞重复传代培养直至细胞充分扩增。
根据本发明,神经前体细胞或神经干细胞向神经细胞的分化增殖均可以通过脐带血衍生的间充质干细胞和神经前体细胞或神经干细胞的共同培养而诱导。即脐带血衍生的间充质干细胞对于诱导神经前体细胞或神经干细胞分化为神经细胞以及对于通过增加神经细胞数来维持和强化这种作用以增强其治疗作用是同时有效的。
因此,本发明提供了脐带血衍生的间充质干细胞或包含所述细胞的组合物用于诱导神经前体细胞或神经干细胞分化为神经细胞以及增殖所得神经细胞的用途。
脐带血衍生的间充质干细胞或包含所述细胞的组合物可以用于患有神经损伤疾病例如中风、帕金森病、阿尔茨海默病、皮克氏病、亨廷顿病、肌萎缩侧索硬化、外伤性中枢神经系统疾病和脊髓损伤疾病、优选中风和脊髓损伤疾病的患者的细胞疗法。
本发明的组合物还可包含药物可接受的添加剂。
可以根据本领域的常规方法使用本发明的组合物制备单位剂量形式的药物制剂。优选用于胃肠外施用的制剂例如注射或局部剂量形式。本发明的药物制剂还可包括药物可接受的添加剂,例如填充剂、膨胀剂、粘合剂、湿润剂、崩解剂、稀释剂如表面活性剂和其他赋形剂。
本发明的药物制剂可以根据本领域常规方法胃肠外施用,例如通过注射进入损伤区域以及注射进入脑脊液例如腰椎穿刺术和实质注射(parenchymal injection)、静脉或动脉。优选地,其可以通过直接注射进脑或脊髓损伤区域的周围或相对区域而施用。另外,可以使用DouglasKondziolka(Douglas Kondziolka,Pittsburgh,1998)的临床方法将本发明的药物制剂施用给损伤区域。特别地,将对象的颅骨切开一个直径1cm的孔,将于HBSS(Hank’s平衡盐溶液)中的间充质干细胞的悬浮液通过长针注射器和立体定位架而注射进该孔。
间充质干细胞的典型剂量范围从1×105到1×107细胞/kg体重/注射,优选从5×105到5×106细胞/kg体重/注射,其可以单剂量或分份剂量施用。另外,应理解实际施用给某个患者的有效成分的量应根据多种相关因素包括被分化增殖的神经细胞的量、选择的施用途径和个体患者的体重、年龄和性别确定。
本发明还提供了诱导神经前体细胞或神经干细胞分化增殖为神经细胞的方法,其包括共同培养脐带血衍生的间充质干细胞和神经前体细胞或神经干细胞。可以通过基于细胞数以1∶0.1至1∶10、优选1∶1至1∶2的比率混合脐带血衍生的间充质干细胞和神经前体细胞或神经干细胞向神经细胞并且在常规细胞培养基如DMEM、α-DMEM、α-MEM、Eagle′s基础培养基和RPMI 1640中剪切所述细胞混合物而进行所述共同培养。所述培养基还可包含抗生素,例如庆大霉素,和/或5到15重量%的FBS。培养时间从5到10天。
本发明还提供了治疗神经损伤疾病的方法,所述方法包括将脐带血衍生的间充质干细胞或包含其的组合物施用给需要治疗所述神经损伤疾病的对象的神经细胞损伤区域。所述对象可以是哺乳动物,包括人。
当以治疗有效量施用时,脐带血衍生的间充质干细胞不但包括中枢或周围神经系统的神经前体细胞或神经干细胞向神经细胞的分化,还包括所得神经细胞的增殖,从而导致神经功能的恢复以及治疗这种神经损伤疾病。脐带血衍生的间充质干细胞的治疗作用通过其增殖再生的神经细胞的能力而大大增强并且长时间持续。
给出以下实施例和测试例仅为说明目的,不是为了限制本发明的范围。
实施例1:分离和培养脐带血衍生的间充质干细胞
(步骤1)获得脐带血(UCB)
获得分娩妇女的同意,UCB样品得自分娩妇女的脐静脉。特别地,将含有44ml的CPDA-1抗凝剂的UCB收集袋(GREEN CROSS)的16-号针插入脐静脉以使UCB流入袋中。在48小时内处理收集的血液,细胞存活率高于90%。
(步骤2)分离和扩增间充质干细胞
步骤1中获得的UCB使用Ficoll-Hypaque梯度(密度:1.077g/ml,Sigma)离心以获得单核细胞。然后单核细胞洗涤几次以除去杂质并悬浮在含有5-15重量%FBS(HyClone)极限基础培养基(α-MEM,Gibco BRL)中。随后,将预定量的所述悬浮液加入与上述相同的培养基中并在5%C02培养箱在37℃培养,每周两次用新鲜批次的培养基更换培养基。当培养的细胞形成单层时,通过显微镜证实产生具有纺锤形的间充质干细胞。将如此形成的间充质干细胞重复传代培养直至细胞被充分扩增(Yang SE et al.,Cytotherapy,6(5):476-486,2004)。
实施例2:培养NG108-15(NG108)
具有与神经前体细胞相似的生理学和形态学特征的小鼠脑衍生的细胞NG108-15(神经母细胞瘤X神经胶质瘤杂种)(ATCC,Cat.No.ATCC-CRL-HB-12317)在DMEM(Dulbecco’s modified Eagle′s medium)(4mM/L谷氨酰胺,4.5g/L葡萄糖、4.0mg/L吡哆醇-HCl、0.1mM次黄嘌呤-鸟嘌呤、400nM氨基蝶呤、0.016mM胸苷、5-15重量%FBS)中培养。
实施例3:培养神经干细胞
衍生自胎鼠脑皮层的神经干细胞(Chemicon,Cat.No.SCR029)在神经干细胞基础培养基中培养(20ng/ml FGF-2,20ng/ml EGF和2mg/ml肝素)。
实施例4:共同培养脐带血衍生的间充质干细胞和NG108-15(I)
将实施例1的人脐带血衍生的间充质干细胞(hUCB-MSCs)与实施例2的NG108-15(hUCB-MSCs∶NG108-15=1∶1)使用transwell室(图1)和实施例2的培养基共同培养。作为对照组,NG108-15单独在实施例2的培养基中培养。如图1所示,transwell室由被具有1μm-孔的微孔膜彼此分开的下室(lower compartment)和上室(upper compartment)组成。hUCB-MSC置于上室,NG108-15在下室中。
在4天和7天用相差显微镜(×100)观测NG108-15的分化。如图2所示,与hUCB-MSC共同培养的NG108-15分化为典型的成熟神经元样细胞的形式,其中细胞伸展出长分支并且分化具有纺锤形。
在7天使用神经发育的早期标记微管蛋白βIII的免疫染色进一步证实分化的细胞是神经元样细胞。特别地,hUCB-MSCs、NG108-15及其混合物分别在盖片上培养,通过将其在室温加入含有0.3%的Triton X-100的10%正常山羊血清中1小时而封闭。免疫染色中使用的第一抗体与藻红蛋白缀合的抗微管蛋白βIII小鼠单克隆抗体(Chemicon)用山羊血清稀释100倍并加入其中。然后将所述混合物在4℃保持过夜,所得混合物用0.01M PBS洗涤3次(每次5分钟)。
如图3所示,与实施例1的间充质干细胞共同培养的NG108-15对于微管蛋白βIII的免疫染色显示独特应答,证实分化的是神经元样细胞。
实施例5:共同培养脐带血衍生的间充质干细胞和NG108-15(II)
从两个不同个体通过实施例1的方法获得人脐带血衍生的间充质干细胞(hUCB-MSCs-1和hUCB-MSCs-2)。实施例2的NG108-15单独培养或者与hUCB-MSCs-1或者hUCB-MSCs-2根据实施例4的方法共同培养7天。用相差显微镜(×100)观测细胞的分化增殖。
作为比较组,1mM的诱导NG108-15分化为神经元样细胞的cAMP(NeuroReport 9,1261-1265,1998)加入NG108-15培养基中。
如图4所示,与间充质干细胞共同培养的NG108-15分化为成熟的神经元样细胞的形式。hUCB-MSCs-1和hUCB-MSCs-2在分化诱导活性上没有显著差异。
实施例6:共同培养脐带血衍生的间充质干细胞和神经干细胞(I)
实施例3的神经干细胞单独培养(对照组)或与实施例5的hUCB-MSCs-1或hUCB-MSCs-2使用transwell室共同培养在实施例3的培养基中。hUCB-MSCs置于上室,神经干细胞在下室中。
hUCB-MSCs-1和hUCB-MSCs-2分别以500、1000、2000、4000和6000细胞/cm2的浓度加入培养基中,神经干细胞以2000细胞/cm2的浓度共同培养。在7天用相差显微镜(×100)观测细胞的分化增殖(图5)。
如图5所示,与间充质干细胞共同培养的神经干细胞分化为成熟神经元的形式。hUCB-MSCs-1和hUCB-MSCs-2在分化诱导活性上没有显著差异。另外,神经干细胞的分化增殖程度与共同培养的间充质干细胞的浓度成正比。
实施例7:共同培养脐带血衍生的间充质干细胞和神经干细胞(II)
实施例3的神经干细胞单独培养(对照组)或者与实施例1的hUCB-MSCs使用transwell室共同培养(hUCB-MSCs∶神经干细胞=1∶1)在实施例3的培养基中。hUCB-MSCs置于上室,神经干细胞于下室中。
在4天和7天使用实施例4的方法对神经元发育的早期标记微管蛋白βIII和微管相关蛋白2(MAP2)进行免疫染色,以便证实分化的是神经元。
如图6所示,与实施例1的间充质干细胞共同培养的神经干细胞对于微管蛋白βIII和MAP2的免疫染色显示独特的应答,证实分化的是神经元。
实施例8:共同培养脐带血衍生的间充质干细胞和神经前体细胞或神 经干细胞
根据实施例4和6的方法,分别将实施例2的NG108-15单独培养(对照组)或与实施例1的hUCB-MSCs共同培养及将实施例3的神经干细胞单独培养(对照组)或与实施例5的hUCB-MSCs-1或hUCB-MSCs-2共同培养。使用台盼蓝染色在7天计数存活细胞数(图7和8)。
如图7和8所示,增加的NG108-15和神经干细胞的数目和与之共同培养的间充质干细胞的浓度成比例,表示脐带血衍生的间充质干细胞可以同时有效地诱导神经前体细胞或神经干细胞分化为神经细胞以及通过增加神经细胞数来维持和强化这种作用。
尽管本发明根据上述特异实施方案进行了描述,应知道本领域技术人员可以对本发明进行不同的修改和变化,这也落在由所附的权利要求书限定的本发明的保护范围中。

Claims (7)

1.包含脐带血衍生的间充质干细胞的组合物用于诱导神经前体细胞或神经干细胞分化增殖为神经细胞的用途。
2.诱导神经前体细胞或神经干细胞分化增殖为神经细胞的方法,包括共同培养脐带血衍生的间充质干细胞和神经前体细胞或神经干细胞。
3.权利要求2的方法,其中使用的脐带血衍生的间充质干细胞与神经前体细胞或神经干细胞的比例为1∶0.1至1∶10。
4.治疗神经损伤疾病的方法,包括将UCB-MSC施用给需要治疗所述神经损伤疾病的对象的神经细胞损伤区域。
5.权利要求4的方法,其中所述神经损伤疾病是中风、帕金森病、阿尔茨海默病、皮克氏病、亨廷顿病、肌萎缩侧索硬化、外伤性中枢神经系统疾病或脊髓损伤疾病。
6.权利要求4的方法,其中所述对象是人。
7.诱导神经前体细胞或神经干细胞分化增殖为神经细胞的组合物,包含作为活性成分的脐带血衍生的间充质干细胞。
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AU2007326171A1 (en) 2008-06-05
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