CN101128578B - 骨骼肌细胞的诱导分化方法 - Google Patents
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Abstract
通过本发明可提供一种方法,是可以有效且简便、再现性好地从骨髓细胞诱导分化为骨骼肌细胞的方法,其包含以下步骤:(a)在骨髓基质细胞(其中,该骨髓基质细胞为未经过脱甲基化剂处理的细胞)的培养物中,添加选自环状AMP增强作用性药物、cAMP类似物、及细胞分化生长作用性因子中的1种或以上的物质进行培养的步骤;(b)在所述(a)步骤中得到的细胞中导入Notch基因及/或Notch信号传导相关基因进行培养以获得成肌细胞培养物的步骤(其中,所述培养不包含导入基因的细胞和未导入细胞的共同培养);以及(c)在所述步骤(b)中得到的成肌细胞培养物中添加Notch配体而进行培养的步骤。
Description
技术领域
本发明涉及一种从骨髓基质细胞(骨髓間質細胞)诱导分化为骨骼肌细胞的方法。
背景技术
目前针对肌肉疾病、尤其是作为骨骼肌肉病变性疾病的肌肉营养不良症等尚无有效治疗方法。如果可以由患者本身的骨髓基质细胞制造骨骼肌细胞,便有可能进行自身移植,并成为一种有效治疗方法。另外,由患者自身的骨髓基质细胞生成的骨骼肌细胞的用途不仅仅局限于上述再生性疗法的治疗方面,还可以利用于推测将在未来开发的人工器官等工程方面的用途中。由于在培养水平可容易的制备肌肉细胞,因此还可以被用于制造杂交型的人工器官等。
由骨髓基质细胞诱导分化为骨骼肌的方法,已知有特开2003-144155号公报中记载的方法。该方法包括以下几个必需的步骤:(1)从骨髓分离培养骨髓基质细胞的步骤;(2)添加脱甲基化剂(5-氮杂胞嘧啶核苷)的步骤;(3)添加cAMP增强作用性药物或者cAMP类似物(弗司扣林(forskolin))、及/或细胞分化生长作用性因子(bFGF、PDGF-AA、Heregulin)的步骤;(4)在细胞中导入Notch基因及/或Notch信号传导相关基因进行培养的步骤;(5)将导入基因的细胞与未导入基因的细胞共同培养的步骤;及(6)添加cAMP增强作用性药物或cAMP类似物(弗司扣林)的步骤。
但是,上述方法含有6个步骤,存在操作非常繁琐的问题。因操作繁琐要进行大量步骤而增加了不确定性因素,导致诱导率降低。另外,该方法是作为基本的诱导分化神经细胞的方法而提出的,并非最佳的用于选择性的诱导分化骨骼肌的方法。此外,在该方法中还使用含有肌肉细胞之外的要素的混合体系,因此从安全性考虑,该方法诱导的骨骼肌细胞在临床应用时存在问题。
发明内容
本发明的目的为提供一种从骨髓基质细胞诱导分化为骨骼肌细胞的方法。更具体而言,本发明的目的为提供一种相比在特开2003-144155号公报中公开的方法更加有效和方便、且再现性好的从骨髓基质细胞诱导分化为骨骼肌细胞的方法。
本发明人为了解决上述课题进行了深入的研究,结果发现在从骨髓基质细胞诱导分化为骨骼肌细胞的过程中,可以通过省略作为在特开2003-144155号公报中公开的方法中所必需的步骤的(2)添加脱甲基化(5-氮杂胞嘧啶核苷)的步骤、(5)将导入基因的细胞与未导入基因的细胞共同培养的步骤、及(6)添加cAMP增强作用性药物或cAMP类似物(弗司扣林)的步骤,以及如上所述地对全部步骤的操作进行简化,能够飞跃性地提高诱导分化的效率,从而有效的制备骨骼肌细胞。本发明是基于上述发现而完成的。
也即,通过本发明可提供一种方法,是从骨髓基质细胞诱导分化为骨骼肌细胞的方法,其包含下述步骤:
(a)在骨髓基质细胞(其中,该骨髓基质细胞为未经过脱甲基化剂处理的细胞)的培养物中,添加选自环状AMP(以下也简称为“cAMP”)增强作用性药物、cAMP类似物、及细胞分化生长作用性因子中的1种或以上的物质进行培养的步骤;
(b)在上述(a)步骤中得到的细胞中导入Notch基因及/或Notch信号传导相关基因进行培养以获得成肌细胞培养物的步骤(其中,上述培养物不包含导入基因的细胞和未导入细胞的共同培养物);
以及(c)在上述步骤(b)中得到的成肌细胞培养物中添加Notch配体而进行培养的步骤。
通过上述发明的优选的方案,可提供一种在上述步骤(c)中使用Jagged1蛋白作为Notch配体的上述方法、以及将上述步骤(c)在无血清培养基中进行操作的上述方法。
另外,从其它角度出发,通过本发明可提供一种方法,是从骨髓基质细胞诱导分化为成肌细胞的方法,其包含以下步骤:(a)在骨髓基质细胞(其中,该骨髓基质细胞为未经脱甲基化剂处理的细胞)的培养物中,添加cAMP增强作用性药物、cAMP类似物、及细胞分化生长作用性因子中的1种或以上的物质进行培养的步骤;以及(b)在上述步骤(a)中得到的细胞中导入Notch基因及/或Notch信号传 导相关基因进行培养的步骤(其中,上述培养物不包含导入基因的细胞和未导入细胞的共同培养物)。
进一步从其它角度出发,通过本发明可提供一种方法,是从成肌细胞诱导分化为骨骼肌细胞的方法,其包含在成肌细胞培养物中添加Notch配体进行培养的步骤。
另外,通过本发明可提供通过上述方法得到的骨骼肌细胞或成肌细胞、以及含有该骨骼肌细胞或成肌细胞的用于肌肉疾病的治疗的的医药组合物、以及用于上述医药组合物的制造的上述骨骼肌细胞或成肌细胞的使用用途。进一步,通过本发明可提供一种方法,是治疗肌肉疾病的方法,其包含针对必须进行骨骼肌细胞或成肌细胞的给药的患者,将通过上述方法得到的骨骼肌细胞或成肌细胞以有效剂量进行局部给药或全身性给药的步骤;还提供一种上面所述的方法,其骨骼肌细胞或成肌细胞为由患者自身或其他来源的骨髓基质细胞诱导分化而成的细胞。
具体实施方式
本发明的方法为从骨髓基质细胞诱导分化为骨骼肌细胞的方法,其包含下述步骤:(a)在骨髓基质细胞(其中,该骨髓基质细胞为未经脱甲基化剂处理的细胞)的培养物中,添加cAMP增强作用性药物、cAMP类似物、及细胞分化生长作用性因子中的1种或以上的物质进行培养的步骤;以及(b)在上述步骤(a)中得到的细胞中导入Notch基因及/或Notch信号传导相关基因进行培养的步骤(其中,上述培养物不包含导入基因的细胞和未导入细胞的共同培养物);以及(c)在上述步骤(b)中得到的成肌细胞培养物中添加Notch配体进行培养的步骤。
本发明的方法的特征为,不含有在已记载的骨骼肌细胞诱导分化方法(含有同公报第[0022]段落等中记载的6步骤的方法)中所必需的步骤的(2)添加脱甲基化剂(5-氮杂胞嘧啶核苷)的步骤;(5)将导入基因的细胞与未导入基因的细胞共同培养的步骤、及(6)添加cAMP增强作用性药物或cAMP类似物(弗司扣林)的步骤。特别是不含有将导入基因的细胞与未导入基因的细胞共同培养的步骤是本发明的主要特征。本发明的方法可用于包含人在内的哺乳动物的骨 骼肌细胞的制备,优选的对象为人。本发明的个步骤的细节可通过参照上述特开2003-144155号公报,得到更深入的理解。上述公报中公开的全部参照内容被包含于本说明书所公开的内容中。
本发明的方法中使用的骨髓基质细胞意指存在于骨髓中的除造血系统以外的间充质细胞,可被认为是可以分化为骨骼、软骨、或脂肪细胞等的细胞。骨髓基质细胞可通过Thy1及2为“+”,或者β1-交联为“+”、以及CD34为“-”等标准进行识别。骨髓基质细胞的制备方法在特开2003-144155号公报中有详细且具体的说明,本领域人员可以容易的获得骨髓基质细胞。例如,从骨髓提取骨髓基质细胞,通过在标准的基础培养基中加入血清的培养基中对上述细胞进行培养可制备骨髓基质细胞的培养物。例如,优选对骨髓基质细胞进行3~4代传代培养后,制备例如细胞密度调节为1700细胞/cm2左右的培养物。作为标准的培养基,例如可使用Eagle’sα改进的极限必需培养基(α-MEM)等,可使用胎牛血清作为血清,或如果是人的情况下使用人血清。本发明的方法中使用的骨髓基质细胞没有必要进行特开2003-144155号公报中记载的方法中采用的用脱甲基化剂进行处理的操作。本发明的特征之一为发现了可省略从来被认为是必须的用脱甲基化剂进行处理的步骤,从而可以有效地分化诱导骨骼肌细胞。
例如可以使用弗司扣林作为cAMP增强作用性药物或cAMP类似物,但并无限定,可适当的采用1种或2种以上的cAMP增强作用性药物或cAMP类似物。cAMP增强作用性药物或cAMP类似物的浓度并无特别限定,例如可以为0.001nM~100μM左右,优选为500nM~50μM。可以用例如碱性成纤维细胞生长因子(basic-Fibroblast growthfactor(bFGF))、血小板来源生长因子(platelet-derived growthfactor-AA(PDGF-AA))、或神经调节蛋白(Neuregulin,商品名Heregulin)等作为细胞分化生长作用性因子,还可以组合使用它们的2种或2种以上的组合。细胞分化生长作用性因子的浓度并无特别限定,例如可为0.001ng/ml~100μg/ml左右,优选为0.5ng/ml~2μg/ml左右。还优选组合使用cAMP增强作用性药物或cAMP类似物与细胞分化生存作用性因子。例如,将弗司扣林(5μM)、bFGF(10ng/ml)、PDGF-AA(5ng/ml)、Neuregulin(200ng/ml)添加至 含有10%胎牛血清(FBS)的MEM Eagle Modification培养基中进行骨髓细胞的培养。此阶段做为骨骼肌干细胞的标记的Pax7被表达,以此为指标进行上述药物的添加及培养即可。
Notch基因及/或Notch信号传导相关基因的导入,可以通过例如使用哺乳动物表达载体的脂质体转染法进行,但并不限于该方法,还可采用其它适当的基因导入方法。例如,还可导入含有Notch细胞质结构域(NICD)cDNA的pCI-neo-NICD质粒(特开2003-144155号公报的实施例1中记载的质粒)。进行上述基因导入以后,优选对导入了基因的细胞进行选择。该选择例如可基于通过添加硫酸G418引入的新霉素耐性进行选择,通常经过10~14天左右完成。选择的细胞优选培养至细胞达到100%汇合的状态。如上所述获得的细胞为成肌细胞集落,细胞的形态发生变化,作为骨骼肌标记的MyoD、Myogenin等的转录因子将变得可被检出。即在于特开2003-144155号公报中的方法中,采用了在导入基因或进行上述选择以后,导入基因后的细胞与未导入基因的细胞共同培养的步骤,但在本发明的方法中不进行上述共同培养。本发明的另一个特征为,省略了从前认为是必须的上述共同培养的步骤,从而可以有效的诱导分化骨骼肌细胞。因此,本发明的方法中,在进行上述基因导入或上述选择以后,不进行共同培养而进行步骤(c)。
接下来,根据步骤(c),进行用于从得到的成肌细胞诱导分化成熟骨骼肌细胞的融合诱导。该步骤可通过在成肌细胞的培养物中添加Notch配体进行培养而进行。Notch配体可使用例如Jagged1蛋白(Lindsell,C.E.et.al.,Cell,80,pp.909-917,1995)。Jagged1蛋白的浓度并无特别限定,例如可为1~20μg/ml左右,优选为5μg/ml左右。培养基的种类并无特别限定,可使用常用的基础培养基,例如MEM Eagle Modification培养基等。可在培养基中添加10%左右的FBS等血清,可优选使用无血清培养基。例如通过使用TTS-serumfree medium(Yoshida,N.et.al.,J.Cell Sci.,111,pp.769-779,1998)等无血清培养基可以制备适用于临床的骨骼肌细胞。通过上述融合诱导,Myf6/MRF4等成熟标记被表达,且诱导分化了肌球蛋白重链(Myosin heavy chain)或骨骼肌球蛋白(skeletal myosin)呈阳性的多核的骨骼肌细胞。该骨骼肌细胞在培养下显示自发的收缩运动。在 特开2003-144155号公报中记载的方法中,将导入基因后的细胞与未导入基因的细胞进行共同培养后,在培养物中加入cAMP增强作用性药物或cAMP类似物以进行成熟骨骼肌细胞的分化诱导,在本发明的方法中,不必在培养物中添加cAMP增强作用性药物或cAMP类似物。本发明的另一特征为,省略了从来认为必需的cAMP增强作用性药物或cAMP类似物的添加,仍然能够充分的诱导分化功能成熟的骨骼肌细胞。
然后,通过将得到的骨骼肌细胞培养物以每1孔1细胞的比例进行有限稀释可进行骨骼肌细胞的克隆化,可以从通常约90%左右的生长克隆中再次得到具有收缩功能的骨骼肌细胞集落。该细胞集落为不含 有其它要素的基本上纯粹的肌肉细胞所构成的集落,可特别适用于治疗肌肉疾病等用途。进一步,得到的细胞集落含有骨骼肌干细胞,即使进行多次传代培养也可以稳定的制备肌肉细胞。
通过本发明的方法得到的骨骼肌细胞,可用于以下的治疗用途:例如患有肌肉营养不良等肌肉疾病的患者的局部或全身性给药;交通外伤或烫伤等引起的肌肉缺损或损伤时的肌肉填补;脑血管障碍、脊髓损伤、神经病变性疾病引起的废用性肌肉萎缩症中的肌肉重建;或腕神经麻痹等神经障碍伴随的肌肉萎缩的治疗等,但通过本发明得到的骨骼肌细胞的用途并不限定与上述例举的特定的用途。例如,可使用从患者自身提取的骨髓基质细胞通过本发明的方法制造骨骼肌细胞,对患者进行自身移植。另外,也可以通过同样的方法进行HLA适应化的他方移植。通过本发明的方法得到的骨骼肌细胞为含有干细胞的、基本上纯粹的骨骼肌细胞集落,通过移植,干细胞在生物体上附着生长,可针对重复性肌肉损伤进行再生,在临床应用中有着巨大的优点。
实施例
以下,通过实施例对本发明进行更深入具体的说明,但本发明的范围并不局限于下述实施例。
实施例1
根据特开2003-144155号公报中记载的方法,从骨骼肌经诱导的成体大鼠(Wister种)及人的骨髓中提取基质细胞(間質細胞)进行 培养。使用在Minimum Essential Medium(MEM)Eagle Modification(Sigma公司,M4526)中添加了15%胎牛血清(Biowhittaker公司,14-501F、Lot#61-1012)的产品作为培养基。经过4代传代培养,在达到80-90%的细胞密度的时间点时分散细胞将密度调节至1800~1900cell/cm2,次日在添加了5μM弗司扣林(Calbiochem,344273)、10ng/ml碱性成纤维细胞生长因子(Peprotech EC LTD,100-18B)、5ng/ml血小板来源生长因子-AA(Peprotech EC LTD,369-HB)、200ng/ml Heregulin(R&D Systems,396-HB)的培养基中交换培养3天。然后,按照特开2003-144155号公报实施例1中记载的方法在细胞中导入Notch细胞内结构域的基因。
在导入的第二天以200ng/ml的浓度添加G418硫酸盐(GibcoBRL,83-5027)进行10天的导入细胞的选择。细胞数恢复大致达到100%汇合以后,通过在含有10%FBS的MEM EagleModification培养基中添加5μg/ml Jagged1蛋白(recombinant RatJagged1/Fc Chimera,GT TECHNE公司,3599)培养5天进行融合诱导。在该培养中,在显微镜水平下观察到细胞在局部出现的融合的多核的骨骼肌细胞并呈经时性的增加,形成肌球蛋白重链及骨骼肌球蛋白呈阳性的多核骨骼肌细胞,在培养下显示自发的收缩运动。在这些细胞中确认了Myf6/MRF4等成熟标记的表达。骨骼肌细胞的诱导效率在融合诱导第14天约为40%。作为对照,采用特开2003-144155号公报中记载的经脱甲基化处理、共同培养、以及其后伴随弗司扣林的添加的方法进行骨骼肌细胞的诱导分化时,骨骼肌细胞的诱导率为20.8±3.5%。
实施例2
按照和上述1同样的方法进行骨骼肌细胞的分化诱导。其中,在诱导分化中使用无血清培养基。无血清培养基使用在含有10μg/ml胰岛素、5μg/ml铁传递蛋白、10nmol亚硒酸钠、1mg/ml BSA、60μg/ml卡那霉素的DMEM(该培养基称为“ITS-serum free medium”)中添加Jagged1蛋白(recombinant Rat Jagged1/Fc Chimera,GTTECHNE公司,3599)以使最终浓度为5μg/ml的产品作为培养基。结果,在培养第1天多核骨骼肌细胞的数目为每个35毫升的培养皿中16个,培养第5天达到了664。另一方面作为对照仅以上述无血清 培养基(未添加Jagged1蛋白)进行培养时,在培养第一天为13,培养第5天为487。该结果显示,通过在无血清培养基中添加Jagged1蛋白,可诱导分化为成熟的骨骼肌细胞。
产业实用性
通过本发明的方法,与特开2003-144155号公报中记载的骨骼肌细胞的诱导分化方法相比简化了整个步骤,可简便而有效的制备骨骼肌细胞。另外,在本发明的方法中,通过步骤(b)对做为骨骼肌细胞的前体细胞的成肌细胞进行诱导分化后,可对该成肌细胞阶段性的诱导分化为成熟骨骼肌细胞,可对分化阶段及细胞增殖等进行控制。另外,在本发明的方法中,未使用含有成肌细胞或骨骼肌细胞以外的细胞的混合体系,可有效的制备基本上纯粹的骨骼肌细胞集落,所得到的骨骼肌细胞在治疗肌肉疾病时可作为安全的医药品使用。
Claims (2)
1.一种从骨髓基质细胞诱导分化为骨骼肌细胞的方法,其包含下述步骤:
(a)在骨髓基质细胞的培养物中,添加
5μM弗司扣林、10ng/ml碱性成纤维细胞生长因子(bFGF)、5ng/ml血小板来源生长因子-AA(PDGF-AA)、200ng/ml神经调节蛋白(Neuregulin),其中,该骨髓基质细胞为未经过脱甲基化剂处理的细胞;
(b)在所述(a)步骤中得到的细胞中导入Notch细胞内结构域的基因(Intracellular Domain of Notch)进行培养以获得成肌细胞培养物的步骤,其中,所述培养不包含导入和未导入基因的细胞的共同培养;以及
(c)在所述步骤(b)中得到的成肌细胞培养物中添加1μg/ml到20μg/ml Jagged1蛋白进行培养的步骤。
2.如权利要求1所述的方法,所述步骤(c)在无血清培养基中进行。
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- 2005-12-22 CN CN2005800487149A patent/CN101128578B/zh not_active Expired - Fee Related
- 2005-12-22 DE DE602005027489T patent/DE602005027489D1/de active Active
- 2005-12-22 WO PCT/JP2005/023598 patent/WO2006068225A1/ja active Application Filing
- 2005-12-22 EP EP05820072A patent/EP1840208B1/en not_active Not-in-force
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2007
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Non-Patent Citations (3)
Title |
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M Higuchi et al.Differential expression of Notch1 and Notch2 in developing and adult mouse brain.Molecular Brain Research.1995,29(2),263-272. * |
M.Schuldiner et al.Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells.PNAS.2000,97(21),11307–11312. * |
WW Deng et al.In Vitro Differentiation of Human Marrow Stromal Cells into Early Progenitors of Neural Cells by Conditions That Increase Intracellular Cyclic AMP.Biochemical and Biophysical Research Communications.2001,282(1),148–152. * |
Also Published As
Publication number | Publication date |
---|---|
CN101128578A (zh) | 2008-02-20 |
JP4581085B2 (ja) | 2010-11-17 |
BRPI0518648A2 (pt) | 2008-12-02 |
IL184132A0 (en) | 2007-10-31 |
WO2006068225A1 (ja) | 2006-06-29 |
KR20070100944A (ko) | 2007-10-15 |
AU2005320101A1 (en) | 2006-06-29 |
AU2005320101B2 (en) | 2010-06-24 |
ES2362791T3 (es) | 2011-07-13 |
ATE505534T1 (de) | 2011-04-15 |
US20080113435A1 (en) | 2008-05-15 |
EP1840208A1 (en) | 2007-10-03 |
CA2592470A1 (en) | 2006-06-29 |
US7718429B2 (en) | 2010-05-18 |
EP1840208B1 (en) | 2011-04-13 |
EP1840208A4 (en) | 2008-09-17 |
DE602005027489D1 (de) | 2011-05-26 |
JP2006174784A (ja) | 2006-07-06 |
IL184132A (en) | 2012-05-31 |
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