CN108570447A - 一种筛选分化hPSCs向MSCs的方法 - Google Patents

一种筛选分化hPSCs向MSCs的方法 Download PDF

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CN108570447A
CN108570447A CN201810075894.9A CN201810075894A CN108570447A CN 108570447 A CN108570447 A CN 108570447A CN 201810075894 A CN201810075894 A CN 201810075894A CN 108570447 A CN108570447 A CN 108570447A
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王淋立
***
陈月花
关春燕
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Haolsheng Bio Pharmaceutical Co Ltd
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Abstract

本发明公开了一种筛选分化hPSCs向MSCs的方法,其步骤是:1)将未分化hPSCs细胞株转移到细胞外基质包被的培养板上培养;2)将细胞消化并转移到由明胶类细胞外基质包被的培养板中,进行细胞筛选培养;3)将细胞消化并转移到贴壁用培养板上继续培养,获得MSCs。该技术方案操作简便,易于实现,并且极大缩短了MSCs细胞的培养时间,有利于MSCs细胞的大规模培养。

Description

一种筛选分化hPSCs向MSCs的方法
技术领域
本发明涉及细胞培养领域,特别涉及一种筛选分化hPSCs向MSCs的方法。
背景技术
目前“干细胞与再生医学(疗)”已成为当今21世纪重大的核心研究项目!作为临床再生医学移植的细胞来源主要包括人多能干细胞(human pluripotent stem cells,hPSCs)即人胚胎干细胞(human embryonic stem cells,hESCs)和人诱导多能干细胞(human induced pluripotent stem cells,hiPSCs)等的统称以及成体干细胞这两大类,其中成体干细胞包括造血干细胞,间充质干细胞等。来源于中胚层的间充质干细胞最初是由1966年Friedenstein等从老鼠骨髓抽出物中发现成纤维细胞样的细胞存在而且分离鉴定出来的,1991年Caplan最初将这些细胞称为间充质干细胞(mesenchymal stem cells,MSCs)。2006年国际细胞治疗协会(International Society for Cellular Therapy,ISCT)提议把这些细胞正式命名为‘multipotent mesenchymal stromal cells’或‘mesenchymalstem cells’(MSCs);并且把MSCs定义为1)可以贴壁生长;2)能向成骨、软骨和脂肪细胞等中胚层谱系分化;3)高水平表达CD105、CD90、CD73等MSCs阳性标志;基本不表达CD45、CD34、CD14或CD11b、CD79α或CD19以及HLA-DR等MSCs阴性标志。MSCs除了自我更新、具有分化成中胚层谱系的肌肉、骨骼、软骨、脂肪、肌腱和韧带,甚至还可以跨胚层转分化为神经、胰岛细胞等多谱系分化和免疫调节的能力,且避免发生肿瘤。MSCs移植手术自20世纪60年代以来一直被用于治疗白血病和其他疾病,但由于其风险,只有具有生命危险的患者才会接受移植手术,随着移植技术的进步使其更加可行的用于没有生命垂危的疾病,例如2010年JamesAnkrum等报道了一些再生医疗机构已经将人间充质干细胞(hMSCs)应用于心肌梗塞、中风、移植物抗宿主病(GvHD)、红斑狼疮、关节炎、克罗恩病、急性肺损伤、慢性阻塞性肺病、肝硬化、多发性硬化症、肌萎缩的横向硬化(ALS)和糖尿病等各种疾病患者并且取得了一定的疗效。根据美国一家最大的临床试验注册中心ClinicalTrials.gov2016年临床试验数据库(拥有201个国家的约25万种试验记录)显示~500个临床试验是与MSCs有关,其中有许多临床试验评估了MSC对多种疾病的影响,包括骨关节炎,创面愈合,退行性疾病,自身免疫性疾病等。可见目前的细胞基础治疗重点是使用MSCs。
MSCs的来源广泛获取方法众多,临床应用一般是从骨髓、脐带及脂肪组织中分离而获得、虽然间充质干细胞分离、大规模培养及储存技术在不断提高,然而从组织中分离MSCs不但受到手术侵入性的痛苦,而且收率非常低,只占抽出骨髓的0.001%–0.01%或脂肪抽吸物的0.05%,至于具有细胞治疗优势的脐带来源MSCs也要经过漫长的1个多月才能扩增到大概8亿个的细胞,而临床一次治疗量所用的MSCs一般是100万~10亿。同时在体外培养和扩增的过程中,随着传代次数(P≤16)的增加,其增殖和分化能力会逐渐衰减。尽管脐带在分娩后被认为是一种医疗废物,但它的使用和ESCs一样必须考虑到伦理问题,此外2014年James A.A等在Nature Biotechnology杂志上表明同种异体的MSCs也不能完全的逃脱免疫排斥。由此可见从组织中分离MSCs都会面临低收率、存在混杂细胞以及纯化细胞、获得一个更成熟的表型所需时间较长(约1个多月),容易造成细胞衰老。所以从临床治疗量需求而言,其供体来源短缺依然是必须解决的问题。相比之下,从自体来源或免疫配型匹配并且无限增殖的hiPSCs定向分化衍生而来的MSCs,比从脂肪、脐血、骨髓以及ESCs来源的MSCs能更好地解决这些临床细胞治疗的问题。
目前,由hPSCs(hiPSCs和hESCs)分化衍生出MSCs(hPSC-MSCs)最经典的方法是通过加入转化生长因子β(TGF-β)抑制剂(小分子化合物SB431542,4-[4-(1,3-苯并二唑-5-基)-5-(2-吡啶基)-1H-咪唑-2-基]-苯酰胺水合物,CAS:301836-41-9;下略:SB)阻碍TGF-β(Transforming Growth Factor)信号通道中ALK5,4,7激酶,达到抑制SMAD2/3信号的作用。从而降解hiPSCs或hESCs的多能性,诱发上皮向间质转化EMT(Epithelial-to-MesenchymalTransition)的发生,促进hPSC-MSCs。参考最近报道hPSC-MSCs的诱导分化方案,根据分化过程可以将其归纳为两大类:
一)分化期间经过EB(embryoid body)球形成(即hPSC-EB-MSCs)
2010年Amer Mahmood等使用小分子SB对EB球连续刺激10天后,移到用纤维粘连蛋白(Fibronectin)包被,经CDM(chemically defined medium)培养4代后,再用10%FBS的α-MEM培养20天,约1个半月时间从hESCs得到了具有成熟表型的高质量“间叶祖细胞(Mesenchymal Progenitors)”。
二)分化期间不经过EB球形成(即hPSC-MSCs)
2012年YEN SHUN CHEN等在干细胞转化医学杂志(STEM CELLS TRANSLATIONALMEDICINE)报道的方法,其实就是Amer Mahmood的hPSC-EB-MSCs分化方法的简化版,不经过EB球形成从hPSCs分化成MSCs,其中也不经过CDM培养,直接是hPSCs在含有KOSR和SB的培养基刺激10天后,立即移到10%FCS(不经灭活)的DMEM-HG中培养,约1个月时间从hPSCs得到MSCs,然而MSCs的表型和质量的评价数据在此文中没有明确表明。
2014年Qingguo Zhao等在PNAS杂志报道了hiPSC-MSCs分化的又一个方案,也是在Amer Mahmood方法的基础上进行了较大的改动,其方案是把hPSCs播种在Matrigel包被的培养板上,经过mTeSR1培养基,在SB培养刺激25天后,转移到没有任何包被的培养板上,用ESC-MSC培养基+SB继续培养20天,其特点是整个hiPSC-MSCs分化过程都添加了SB,而且整个培养过程都在7.5%CO2条件下培养。经过45天从hiPSCs得到表型成熟的MSCs。
发明内容
本发明的目的在于公开一种筛选分化hPSCs向MSCs编程的方法。
本发明所采取的技术方案是:
一种筛选分化hPSCs向MSCs的方法,其步骤是:
1)将未分化hPSCs细胞株转移到细胞外基质包被的培养板上培养;
2)将细胞消化并转移到由明胶类细胞外基质包被的培养板中,进行细胞筛选培养;
3)将细胞消化并转移到贴壁用培养板上继续培养,获得MSCs。
优选的,步骤1)中,培养的方式是:用含有TGF-β1/Activin/Nodal-SMAD2/3信号通路抑制剂的分化培养基培养。
优选的,步骤2)的方法是:先将细胞消化悬浮,移到经明胶类细胞外基质包被的贴壁用培养板上,用含有TGF-β1/Activin/Nodal-SMAD2/3信号通路抑制剂的分化培养基继续培养。
优选的,TGF-β1/Activin/Nodal-SMAD2/3信号通路抑制剂为SB431542、A8301、SB505124或RepSox中的至少一种。
优选的,步骤1)中,含有TGF-β1/Activin/Nodal-SMAD2/3信号通路抑制剂的分化培养基培养时间为5~30天。
优选的,步骤2)中,培养时间为5~30天。
优选的,步骤2)中,细胞筛选培养得到的细胞为具有梭型形态特征,表面标志CD90、CD73、CD105为阳性,CD34、CD45、CD14或CD11b、CD79α或CD19以及HLA-DR为阴性。
优选的,步骤3)中继续培养的方法是:用含有间充质干细胞培养基培养。
优选的,步骤2)中,明胶类细胞外基质为:细胞培养用明胶或I型、III型、IV型、V型胶原蛋白和弹性蛋白以及层粘连蛋白。
一种MSCs细胞,该细胞由上述分化方法制备而成。
本发明的有益效果是:本技术方案,操作简便,易于实现,并且极大缩短了MSCs细胞的培养时间,有利于MSCs细胞的大规模培养。
附图说明
图1为hiPSCs未分化的细胞。
图2为hESCs未分化的细胞。
图3为采用明胶筛选培养得到的细胞。
图4为采用基质胶筛选培养得到的细胞,箭头所示为异质性细胞。
图5为采用纤维粘连蛋白筛选培养得到的细胞,箭头所示为异质性细胞。
图6为从hiPSCs分化成表型成、梭型形态的MSCs细胞。
图7为从hESCs分化成表型成熟、梭型形态的MSCs细胞。
具体实施方式
实施例
hPSCs(hiPSCs和hESCs)向MSCs分化的具体方法
1)将未分化hPSCs细胞株转移到基质胶(Matrigel,MG;细胞外基质的一种)包被30min的10cm贴壁用培养板(P100)上用10ml多能干细胞分化培养基,37℃、7.5%CO2培养24小时后(Day 0,图1和图2),改为10ml含有10uM/ml SB431542且不含TGF-β1的多能干细胞分化培养基连续培养8天,隔日换液,期间不传代(即MG阶段)。
2)去掉旧液,加2ml 0.05%Trypsin-EDTA将SB处理8天后的细胞消化5min,加4mlDMEM/F12(含10%胎牛血清FBS)终止,回收到15ml离心管,200g、5min离心。
3)除掉上清,加入1ml不含TGF-β1的多能干细胞分化培养基(培养基中含有10uM/ml SB431542)轻轻将细胞悬浮,分别移到经明胶(Gelatin,GT)、基质胶(MG)和纤维粘连蛋白(Fibronectin)包被30min、加有9ml含有10uM/ml SB431542且不含TGF-β1的多能干细胞分化培养基,48小时后观察,观察发现,培养在GT包被的培养板上,细胞是典型的梭型形态,细胞均质,没有明显异质性;而培养在MG和Fibronectin包被的培养板上,除了梭型形态的细胞,还存在大量的异质性细胞和未分化hPSCs细胞(图3~图5)。因此仅选择采用GT包被的培养板上的细胞,37℃、7.5%CO2继续培养6天,隔日换液,期间不传代(即GT阶段)。其中所述明胶为细胞培养用明胶或I型、III型、IV型、V型胶原蛋白和弹性蛋白以及层粘连蛋白。
4)经GT阶段细胞汇合度可以到达~85%,加2ml 0.05%Trypsin-EDTA将细胞消化3min,加4ml DMEM/F12(含10%FBS)终止,回收到15ml离心管,200g、5min离心。
5)除掉上清,加1ml含有10uM/ml SB431542且不含TGF-β1的多能干细胞分化培养基轻轻将细胞悬浮,移到没有任何包被的加有9ml含有10uM/ml SB431542且不含TGF-β1的多能干细胞分化培养基的P100贴壁用培养板上培养,24小时后换成10%FBS的α-MEM,37℃、5%CO2培养3天。
上述步骤中涉及的SB431542是4-[4-(1,3-苯并二唑-5-基)-5-(2-吡啶基)-1H-咪唑-2-基]-苯酰胺水合物(CAS:301836-41-9);也可以用A8301(CAS:909910-43-6)、RepSox(CAS:446859-33-2)、SB505124(CAS:694433-59-5)替换。
对细胞进行鉴定,结果表明细胞能够高水平表达CD105、CD90、CD73等MSCs阳性Markers;基本不表达CD45、CD34、CD14或CD11b、CD79α或CD19以及HLA-DR等MSCs阴性Markers;不表达TRA-1-60、TRA-1-81等多能性Markers(见下表)。另外细胞形态具有典型的梭型形态。可以判断细胞为MSCs(图6和图7)。

Claims (10)

1.一种筛选分化hPSCs向MSCs的方法,其步骤是:
1)将未分化hPSCs细胞株转移到细胞外基质包被的培养板上培养;
2)将细胞消化并转移到由明胶类细胞外基质包被的培养板中,进行细胞筛选培养;
3)将细胞消化并转移到贴壁用培养板上继续培养,获得MSCs。
2.根据权利要求1所述的分化方法,其特征在于,步骤1)中,培养的方式是:用含有TGF-β1/Activin/Nodal-SMAD2/3信号通路抑制剂的分化培养基培养。
3.根据权利要求1所述的分化方法,其特征在于,步骤2)的方法是:先将细胞消化悬浮,移到经明胶类细胞外基质包被的贴壁用培养板上,用含有TGF-β1/Activin/Nodal-SMAD2/3信号通路抑制剂的分化培养基继续培养。
4.根据权利要求2或3所述的分化方法,其特征在于,TGF-β1/Activin/Nodal-SMAD2/3信号通路抑制剂为SB431542、A8301、SB505124或RepSox中的至少一种。
5.根据权利要求2所述的分化方法,其特征在于,步骤1)中,含有TGF-β1/Activin/Nodal-SMAD2/3信号通路抑制剂的分化培养基培养时间为5~30天。
6.根据权利要求1所述的分化方法,其特征在于,步骤2)中,培养时间为5~30天。
7.根据权利要求1所述的分化方法,其特征在于,步骤2)中,细胞筛选培养得到的细胞为具有梭型形态特征,表面标志CD90、CD73、CD105为阳性,CD34、CD45、CD14或CD11b、CD79α或CD19以及HLA-DR为阴性。
8.根据权利要求1所述的分化方法,其特征在于,步骤3)中继续培养的方法是:用含有间充质干细胞培养基培养。
9.根据权利要求1所述的分化方法,其特征在于,步骤2)中,明胶类细胞外基质为:细胞培养用明胶或I型、III型、IV型、V型胶原蛋白和弹性蛋白以及层粘连蛋白。
10.一种MSCs细胞,该细胞由权利要求1~9任意一项所述的分化方法制备而成。
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