CN116083349A - 人或恒河猴的多能干细胞诱导分化骨骼肌前体细胞的方法 - Google Patents
人或恒河猴的多能干细胞诱导分化骨骼肌前体细胞的方法 Download PDFInfo
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Abstract
本发明公开了一种人或恒河猴的多能干细胞诱导分化骨骼肌前体细胞的方法,将人或恒河猴的多能干细胞置于含有胚胎成纤维饲养层细胞的小皿中培养至多能干细胞生长至20%~30%汇聚度;培养基1培养5天;培养基2中培养5天;在培养基2中加入10ng/ml的肝细胞生长因子HGF,培养10天;将细胞消化分离成单细胞后,培养基3中培养10天;将细胞消化分离成单细胞后,重新铺在孔板中,待细胞生长至60%~70%汇聚度时,培养基4中培养。本发明在有饲养层存在的条件下,利用小因子调节WNT和BMP信号通路来达到体外高效分化骨骼肌细胞目的,为人和恒河猴来源的多能干细胞提供一种可靠的体外骨骼肌分化方法。
Description
技术领域
本发明涉及干细胞培养分化领域,尤其涉及一种人或恒河猴的多能干细胞诱导分化骨骼肌前体细胞的方法。
背景技术
骨骼肌主要由负责收缩和运动的分化多核肌纤维组成。此外,肌肉组织含有静止的单核干细胞群,称为卫星细胞,位于肌纤维的基底层和肌膜之间。卫星细胞在稳态条件下保持静止状态,但在组织损伤后会被激活。一旦被激活,卫星细胞会产生被称为未定型成肌细胞,然后分化并相互融合或与残留的肌纤维融合以再生受损组织。因此它们被证明具有自我更新和分化潜能。
虽然可以使用生长因子或小分子培养和适度扩增卫星细胞和成肌细胞,但目前的体外培养方案大多会有神经细胞和成纤维细胞等大量杂细胞的生成。并且,目前已报道的大多数培养基只适用于人的多能干细胞,对于猴的多能干细胞培养报道几乎没有。人的多能干细胞几乎都可以在无饲养层(小鼠胚胎成纤维细胞)提供生长因子的情况下,在Matrigel等基质胶上以商业化的Stemcell公司生产的mTeSR1或E8等培养基进行培养和传代。猴的多能干细胞在这些培养基中由于缺乏某些不确定因子,导致生长缓慢,干细胞克隆生长不致密和空泡化等现象发生,无法稳定生长和传代培养。这些现状也是导致猴的干细胞分化成骨骼肌细胞最主要的困难。
成人肌细胞生成的不同阶段表达不同转录因子和表面标记。例如,静止卫星细胞表达转录因子Pax7和表面标记血管细胞粘附分子1(VCAM-1),但缺乏肌源性分化因子MYOD表达。相比之下,激活的卫星细胞共表达Pax7和MyoD,而除了MyoD之外,成肌细胞和肌管的分化还会上调其他生肌因子,例如生肌调节因子4(MRF4或Myf6)和肌细胞生成素(Myogenin)。Pax7表达可作为静止细胞和活化卫星细胞的共用标记,通常用于遗传标记或纯化这些未成熟细胞群。
发明内容
本发明要解决的技术问题是克服现有技术的不足,提供一种人或恒河猴的多能干细胞诱导分化骨骼肌前体细胞的方法,目前已有多种人类多能干细胞定向分化骨骼肌的方法被报道,但是基于恒河猴干细胞的相关分化方法却少之又少。我们知道人与猴在亲缘关系以及基因进化相似性上都高度相似,是研究人类疾病的最重要的动物模型之一。无论是人或恒河猴的多能干细胞均能在饲养层细胞上稳定生长及传代,保持良好的干细胞特性。我们根据这一特点,在有饲养层存在的条件下,利用小因子调节WNT和BMP信号通路来达到体外高效分化骨骼肌细胞的目的,为人和恒河猴来源的多能干细胞提供一种可靠的体外骨骼肌分化方法。
为了实现本发明的目的,本发明提供了一种人或恒河猴的多能干细胞诱导分化骨骼肌前体细胞的方法,包括以下步骤:
S1、将人或恒河猴的多能干细胞置于含有CF1小鼠胚胎成纤维饲养层细胞的小皿中培养,,待多能干细胞生长至20%~30%汇聚度;
S2、换成培养基1培养5天;
S3、换成培养基2中培养5天;
S4、在所述培养基2中加入10ng/ml的肝细胞生长因子HGF,继续培养10天;
S5、将细胞消化分离成单细胞后,换成培养基3中培养10天;
S6、将细胞消化分离成单细胞后,重新铺在孔板中,待细胞生长至60%~70%汇聚度时,更换为培养基4中培养;
所述培养基1包括骨骼肌基础培养基、1μM~6μM的CHIR99021和0.1μM~1μM的LDN193189;
所述培养基2包括骨骼肌基础培养基、20ng/ml的bFGF;
所述培养基3包括骨骼肌基础培养基、5% FBS、0.1mMβ-巯基乙醇、0.1%鸡胚提取物、20nM RepSox和20nM Forskolin;
所述培养基4包括骨骼肌肌管样细胞培养基。
上述的方法,进一步的,所述S1具体为:
S1-1、使用MEF-M培养基将胚胎成纤维细胞在小皿中铺匀培养一天;
S1-2、将多能干细胞接种于所述小皿中,用PSC-M培养基培养5天。
上述的方法,进一步的,所述S5和S6中,将细胞消化分离成单细胞的过程具体为:以不含钙离子和镁离子的DPBS轻轻洗两遍培养皿里的细胞,再用1ml 0.05%的胰酶覆盖细胞,于37℃温箱中静置消化细胞5~10分钟;待细胞从皿底脱落后,加入含有胎牛血清FBS的骨骼肌基础培养基终止胰酶消化,离心收集单细胞。
上述的方法,进一步的,所述S6中,培养基4培养5天内,在低浓度马血清存在条件下,大部分成肌细胞会自发融合成多核肌管样细胞。
上述的方法,进一步的,所述培养基1包骨骼肌基础培养基、3.5μM的CHIR99021和0.5μM的LDN193189。
与现有技术相比,本发明的优点在于:
(1)本发明提供了一种人或恒河猴的多能干细胞诱导分化骨骼肌前体细胞的方法,能够成功在饲养层培养条件下将人和恒河猴多能干细胞成功分化成成肌细胞,这些细胞能够特异性表达Pax7和Myogenin。并且,在含有2%马血清的DMEM培养基中成功融合成具有多核样特征的肌管样细胞,所产生的多核样肌管细胞有肌球重链蛋白(MHC)表达。体外分化骨骼肌的分化周期与体内发育主要阶段基本一致,均是由多能干细胞先向轴旁中胚层定向分化,再进一步发育成骨骼肌细胞。本技术中所报道的分化周期与其他方法分化周期几乎一致,均需要大于30天的培养周期,不能缩短体外从干细胞到骨骼肌分化时长。为了更快分化出用于下游实验研究的骨骼肌,培养周期过长是将来我们需要改进和主要克服的难点。
附图说明
为使本发明实施例的目的、技术方案和优点更加清楚,下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整的描述。
图1为本发明实施例2中从干细胞到骨骼肌分化简图。
图2为本发明实施例2中从干细胞到骨骼肌分化关键时间天数时明场下细胞生长状态图。
图3为本发明实施例2中从第0天人的干细胞到第40天融合后肌管样细胞对应关键时间天数时特异蛋白标记细胞免疫染色图。
图4为本发明实施例2中从第0天猴的干细胞到第40天融合后肌管样细胞对应关键时间天数时特异蛋白标记细胞免疫染色图。
图5为LDN浓度为1μM时或CHIR浓度为1μM时细胞生长情况结果示意图。
具体实施方式
以下结合具体优选的实施例对本发明作进一步描述,但并不因此而限制本发明的保护范围。以下实施例中所采用的材料和仪器均为市售。
表1:实施例1中所使用的小分子的购买途径,以及使用浓度及终浓度
一种人或恒河猴的多能干细胞诱导分化骨骼肌前体细胞的方法,胚胎发育过程中,外胚层、中胚层和内胚层细胞将来会发育成不同的结构组织。其中,中胚层又分为侧板中胚层和轴旁中胚层,侧板中胚层会进一步发育成为体壁间充质和四肢长骨,轴旁中胚层才会发育成为骨骼肌细胞。我们建立了二维贴壁培养条件下体外诱导骨骼肌分化的培养基,通过调节小因子使用浓度及方案,使用WNT通路激活小分子CHIR99021,结合BMP信号通路选择性抑制剂LDN-193189共同调节干细胞定向轴旁中胚层分化的过程。最终在高浓度成纤维生长因子bFGF存在下分化并维持成肌细胞生长。
实施例1:
一种体外诱导骨骼肌分化的培养基,包括培养基1、培养基2,培养基3,培养基4。
培养基1包括骨骼肌基础培养基(MYO-M)、3.5μM的CHIR99021和0.5μM的LDN193189;
培养基2包MYO-M、20ng/ml的bFGF
培养基3包括MYO-M、5% FBS、0.1mMβ-巯基乙醇、0.1%鸡胚提取物、20nM RepSox和20nM佛司可林(Forskolin);
培养基4包括:骨骼肌肌管样细胞培养基(MYOTUBE-M)。
实施例2:
一种实施例1的培养基在人或恒河猴的多能干细胞诱导分化骨骼肌前体细胞中的应用,在干细胞培养阶段,为保证分化方法一致。我们采用能稳定维持恒河猴和人干细胞特性的小鼠胚胎成纤维饲养层细胞上培养,挑选生长状态较好的干细胞克隆进行传代并进行进一步分化。WNT信号的激活和BMP相关ALK信号通路的抑制是向轴旁中胚层分化最关键的步骤,我们采用稳定的WNT通路激活剂CHIR99021联合BMP信号抑制剂LDN193189定向轴旁中胚层细胞。经过小分子调控分化,此时干细胞中有大量的中胚层特异标记Brachyury表达。在培养后半阶段,我们在不同时间点加入bFGF和HGF,先用bFGF维持成肌细胞分化和生长,再加入HGF维持成肌细胞的增殖,维持成肌细胞的特性。整个培养过程连续,模拟体内三胚层分化后轴旁中胚层向骨骼肌分化的过程,这一发育过程在恒河猴和人类是一致的,因此,我们所采用的方法能够将这两种干细胞都成功分化出骨骼肌成肌细胞,最终在马血清的诱导下融合成多核样肌管细胞。
具体的应用方法参见图1,图1为从干细胞到骨骼肌分化简图。图中对应从干细胞PSC到骨骼肌Myotube不同细胞培养关键时间点表达的特异标记以及对应时间点加入培养基中的关键小因子。具体应用方法包括以下步骤:
(1)第-6天,准备一个35mm直径小皿,以3×105细胞量铺下CF1品系小鼠的经放射线处理或丝裂霉素处理过的胚胎成纤维细胞(该胚胎成纤维细胞能稳定维持恒河猴和人干细胞特性,饲养层细胞可从ATCC公司进行购买)。使用2ml饲养层细胞培养基MEF-M(表2)铺匀细胞。
表2:饲养层细胞培养基(MEF-M)的成分
名称 | 货号 | 体积 | 终浓度 |
DMEM | Gibco(11320) | 45ml | |
FBS | Gibco(26010074) | 5ml | 10%(vol/vol) |
NEAA | Gibco(111400) | 500ul | 1%(vol/vol) |
PS | Gibco(15070063) | 500ul | 1%(vol/vol) |
(2)第-5天,将生长状态良好,克隆致密且核质比高的干细胞(猴的多能干细胞由昆明理工大学灵长类转化医学研究院提供,人的多能干细胞H1购买于中国科学院细胞库)用玻璃拉针轻轻切割成1mm-2mm直径大小克隆,每个小皿接种50个左右的克隆即可。每日更换2ml新鲜的多能干细胞培养基PSC-M(表3)。
表3:多能干细胞培养基(PSC-M)成分
(3)第0天,此时,干细胞生长至20~30%汇聚度,更换成MYO-M(表4),在培养基中加入3.5uM的CHIR99021和0.5uM的LDN193189。每日换液,共计5天。在此过程中会有大量细胞的死亡,但仍有部分细胞贴壁生长,这部分细胞就是我们所需要的分化的中胚层细胞,应注意每日观察和换入新鲜培养基。此时干细胞由致密状态变得相对松散,并且特异性表达中胚层标记Brachyury。
表4:骨骼肌基础培养基MYO-M的成分
名称 | 货号 | 体积 | 终浓度 |
DMEM/F12 | Gibco(11320) | 49ml | |
IST-X | Gibco(51500056) | 500ul | 1%(vol/vol) |
NEAA | Gibco(111400) | 500ul | 1%(vol/vol) |
PS | Gibco(15070063) | 500ul | 1%(vol/vol) |
(4)第5天,撤掉CHIR和LDN,在MYO-M中加入20ng/ml的bFGF,每日换液,共计5天。
(5)第10天,除了bFGF外,在MYO-M中加入10ng/ml的肝细胞生长因子HGF,肝细胞生长因子被认为是非常重要的器官再生有丝分裂和维持机能重要生长因子,能促进骨骼肌前体细胞的增殖,并且能抑制骨骼肌前体细胞的分化,每日换液,继续维持至19天。
(6)第20天,此时细胞生长得非常致密,饲养层细胞由于营养因子的缺乏及自身老化,几乎快要全部死亡。大量的骨骼肌前体细胞尚未成熟,继续培养的骨骼肌细胞会变得难以消化分离成单细胞,在20天以后进行细胞消化是必要的过程。以DPBS(不含钙离子和镁离子)轻轻洗两遍培养皿里的细胞,再用1ml 0.05%的胰酶覆盖细胞,于37℃温箱中静置消化细胞5-10分钟。待细胞从皿底脱落后,加入含有胎牛血清FBS的骨骼肌基础培养基终止胰酶消化。用1ml移液枪适当的吹吸消化后的细胞团块能够促进细胞散开成单细胞。用40μm的细胞滤网将难以消化成单细胞的片状胶原纤维和死细胞团块滤过。把含有单细胞的细胞悬液在1000转/分钟的离心机中离心5分钟收集细胞,弃含有胰酶的上清,用手轻弹离心管壁,使细胞团块变得松散。
更换培养基为MYO-M,另加5% FBS、0.1mMβ-巯基乙醇、0.1%鸡胚提取物、20nMRepSox和20nM Forskolin。每日换液,至第30天。
(7)第31天,此时开始,大部分骨骼肌已经成熟,可以按照上一步骤消化细胞后,重新铺在12孔板或6孔板中,等细胞生长至60%~70%汇聚度时,更换为骨骼肌肌管样细胞培养基MYOTUBE-M(表5),通常在5天内,在低浓度马血清存在条件下,大部分成肌细胞会自发融合成多核肌管样细胞。融合后的骨骼肌肌管细胞能够在MYOTUBE-M中维持2个月以上。
表5:骨骼肌肌管样细胞培养基MYOTUBE-M的成分
名称 | 货号 | 体积 | 终浓度 |
DMEM | Gibco(11320) | 49ml | |
Horse Serum | Invitrogen(16050-130) | 1ml | 2%(vol/vol) |
PS | Gibco(15070063) | 500ul | 1%(vol/vol) |
上述培养基配好后一般在4℃冰箱里避光存放不超过2周。在使用前从4℃冰箱里去出平衡至室温即可,不要反复在37℃水浴锅中复温,影响培养基中小因子的活性。
图2为从干细胞到骨骼肌分化关键时间天数时明场下细胞生长状态图。干细胞克隆生长状态致密,核质比高且没有中心空泡化被认为是细胞生长状态良好的判断标准,可以开始进行肌细胞分化,定义为第0天,培养至第5天时,干细胞之间出现明显分离,且有部分细胞死亡。第10天时,细胞已经长得非常致密,几乎完全占满整个培养皿。当细胞继续培养至20天以上,细胞之间会更加紧密,如果继续培养,有可能会因为边缘卷起,而导致细胞卷缩成团,消化成单细胞变得非常困难,因此,很难收集到单个骨骼肌细胞。在20天左右进行细胞消化后,按照一定密度铺皿,并继续分化,至30天以上,就可以进行低浓度马血清诱导肌管融合成多核样细胞。
图3为不同阶段人的干细胞培养明场拍摄图片及荧光拍摄图片。从第0天干细胞到第40天融合后肌管样细胞对应关键时间天数时特异蛋白标记细胞免疫染色图。从图中可以看出:第0天时干细胞标记Oct4、Sox2和Nanog标记均表达,第5天时WNT信号通路激活,中胚层标记Brachyury表达。第25天时,肌肉卫星细胞标记和肌肉标记Pax7和Myogenin表达。第40天时,在2%马血清培养基中,融合后的肌管样细胞表达MHC。
图4为不同阶段猴的干细胞培养明场拍摄图片及荧光拍摄图片。从图中可以看出,猴的干细胞培养情况与人的培养情况几乎一样。
实验一:考察LDN和CHIR浓度对细胞分化的影响。
在实施例2的步骤(3)分别加入梯度浓度的CHIR99021(1μM~6μM)和LDN193189(0.1μM~1μM),考察在分化过程中,细胞生长的情况。
图5为细胞生长情况结果示意图。左图显示LDN浓度为1μM时,第3天细胞几乎全部死亡。右图显示CHIR浓度为1μM时,肌细胞分化至40天,极少的肌管形成,并且细胞生长状态很差,分裂增殖能力很弱。证明,在分化过程中,当LDN浓度过高时,细胞几乎全部由于小因子毒性而死亡。当CHIR浓度浓度过低又会出现分化效率低的情况。
以上所述,仅是本发明的较佳实施例而已,并非对本发明作任何形式上的限制。虽然本发明已以较佳实施例揭示如上,然而并非用以限定本发明。任何熟悉本领域的技术人员,在不脱离本发明的精神实质和技术方案的情况下,都可利用上述揭示的方法和技术内容对本发明技术方案做出许多可能的变动和修饰,或修改为等同变化的等效实施例。因此,凡是未脱离本发明技术方案的内容,依据本发明的技术实质对以上实施例所做的任何简单修改、等同替换、等效变化及修饰,均仍属于本发明技术方案保护的范围内。
Claims (5)
1.一种人或恒河猴的多能干细胞诱导分化骨骼肌前体细胞的方法,其特征在于,包括以下步骤:
S1、将人或恒河猴的多能干细胞置于含有胚胎成纤维饲养层细胞的小皿中培养至多能干细胞生长至20%~30%汇聚度;
S2、换成培养基1培养5天;
S3、换成培养基2中培养5天;
S4、在所述培养基2中加入10ng/ml的肝细胞生长因子HGF,继续培养10天;
S5、将细胞消化分离成单细胞后,换成培养基3中培养10天;
S6、将细胞消化分离成单细胞后,重新铺在孔板中,待细胞生长至60%~70%汇聚度时,更换为培养基4中培养;
所述培养基1包括骨骼肌基础培养基、1μM~6μM的CHIR99021和0.1μM~1μM的LDN193189;
所述培养基2包括骨骼肌基础培养基、20ng/ml的bFGF;
所述培养基3包括骨骼肌基础培养基、5%FBS、0.1mMβ-巯基乙醇、0.1%鸡胚提取物、20nM RepSox和20nM Forskolin;
所述培养基4包括骨骼肌肌管样细胞培养基。
2.根据权利要求1所述的方法,其特征在于,所述S1具体为:
S1-1、使用MEF-M培养基将CF1小鼠胚胎成纤维细胞在小皿中铺匀培养一天;
S1-2、将多能干细胞接种于所述小皿中,用PSC-M培养基培养5天。
3.根据权利要求1所述的方法,其特征在于,所述S5和S6中,将细胞消化分离成单细胞的过程具体为:以不含钙离子和镁离子的DPBS轻轻洗两遍培养皿里的细胞,再用1ml0.05%的胰酶覆盖细胞,于37℃温箱中静置消化细胞5~10分钟;待细胞从皿底脱落后,加入含有胎牛血清FBS的骨骼肌基础培养基终止胰酶消化,离心收集单细胞。
4.根据权利要求1所述的方法,其特征在于,所述S6中,培养基4培养5天内,在低浓度马血清存在条件下,大部分成肌细胞会自发融合成多核肌管样细胞。
5.根据权利要求1至4中任一项所述的方法,其特征在于,所述培养基1包括骨骼肌基础培养基、3.5μM的CHIR99021和0.5μM的LDN193189。
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