CN108570448B - 一种高效的hPSCs向MSCs分化的方法 - Google Patents
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Abstract
本发明公开了一种高效的hPSCs向MSCs分化诱导的方法,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后;换用含有BMP‑SMAD1/5/8信号通路激活剂、TGF‑β1/Activin/Nodal‑SMAD2/3信号通路激活剂、Wnt激活剂、PI3K抑制剂的分化培养基培养;2)去除旧培养基,换含有TGF‑β1/Activin/Nodal‑SMAD2/3信号通路抑制剂分化培养基继续培养;3)将细胞消化并转移到新的培养板上,换含有TGF‑β1/Activin/Nodal‑SMAD2/3信号通路抑制剂分化培养基继续培养;4)将细胞消化并转移到贴壁用培养板上继续培养,获得MSCs。本发明在体外建立了完整的hPSC通过中段原条阶段或侧中胚层阶段定向分化为MSCs的方案方法。相较于常规方法,本技术步骤精简,操作简易,再现性高,仅仅用12天便可以获得表型成熟、高质量的MSCs。
Description
技术领域
本发明涉及细胞培养领域,特别涉及一种高效的hPSCs向MSCs分化的方法。
背景技术
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 pluripotentstem 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存在成纤维细胞样的细胞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 stemcells,MSCs)。2006年国际细胞治疗协会(International Society for CellularTherapy,ISCT)提议把这些细胞正式命名为‘multipotent mesenchymal stromal cells’或‘mesenchymal stem 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,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。根据美国NIH临床试验注册中心ClinicalTrials.gov,2016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。由此可见MSCs在细胞再生医疗的重要位置。虽然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,但从组织中分离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,同种异体的免疫排斥(脐带来源)等问题。所以从临床需求而言,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小分子化合物SB431542阻碍转化生长因子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,从而获得表型成熟、高质量的hPSC-MSCs。但难免会导致衍生出不需要的细胞谱系。然而干细胞分化到所需的细胞谱系是相当复杂而难以掌控的一个过程,尤其是人胚胎生物学(限于伦理道德),所以在体外要高度模拟、遵循人胚胎发育期间的生物学原理来研究干细胞定向分化是一份十分艰巨而又非常具有挑战性的科研工作。因此,到目前为止还没有出现过将hPSCs定向分化成高度模拟胚胎期MSCs完整的方案方法。
发明内容
本发明的目的在于一种高效的hPSCs向MSCs分化的方法。
本发明所采取的技术方案是:
一种高效的hPSCs向MSCs分化诱导的方法,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后;换用含有BMP-SMAD1/5/8信号通路激活剂、TGF-β1/Activin/Nodal-SMAD2/3信号通路激活剂、Wnt激活剂、PI3K抑制剂的分化培养基培养;2)去除旧培养基,换含有TGF-β1/Activin/Nodal-SMAD2/3信号通路抑制剂分化培养基继续培养;3)将细胞消化并转移到新的培养板上,换含有TGF-β1/Activin/Nodal-SMAD2/3信号通路抑制剂分化培养基继续培养;4)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3)中,新的培养板是用明胶类细胞外基质包被的,进行细胞筛选。
优选的,细胞筛选所得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为阴性的细胞。
优选的,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。
优选的,步骤1)中,用含有BMP-SMAD1/5/8信号通路激活剂、TGF-β1/Activin/Nodal-SMAD2/3信号通路激活剂、Wnt激活剂、PI3K抑制剂的分化培养基培养时间为12~72h。
优选的,步骤1)中,BMP-SMAD1/5/8信号通路激活剂BMP2、BMP4、BMP7中的至少一种。
优选的,步骤1)中,TGF-β1/Activin/Nodal-SMAD2/3信号通路激活剂为ActivinA、Activin B、TGF-β1、Nodal中的至少一种。
优选的,步骤1)中,Wnt激活剂为CHIR99021,BIO、WNT-3a、R-spondin-2中的至少一种。
优选的,步骤1)中,PI3K抑制剂为TG100713、PIK90、PI-103中的至少一种。
优选的,TGF-β1/Activin/Nodal-SMAD2/3信号通路抑制剂为SB431542、SB505124、A8301、RepSox中的至少一种。
优选的,步骤3)中,继续培养的时间为2~30天。
优选的,在步骤2)继续培养之前,先用含有BMP-SMAD1/5/8信号通路激活剂、TGF-β1/Activin/Nodal-SMAD2/3信号通路激活剂、Wnt抑制剂的分化培养基培养。
优选的,在步骤2)中,继续培养的时间为2~10天。
优选的,Wnt抑制剂为Wnt-C59、XAV-939中的至少一种。
优选的,步骤4)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,在步骤1)至步骤4)中,至少一种培养基中含有碱性成纤维细胞生长因子bFGF。
优选的,获得的MSCs是使用诱导hPSCs通过中段原条阶段或侧中胚层阶段向MSCs定向分化的方法来获得的。
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:1)本发明是世界上首次在体外建立了完整的hPSCs通过中段原条阶段或侧中胚层阶段向MSC定向分化方案方法。为研究来源于人多能干细胞不同细胞的株间差,以及体内外MSCs的差异性提供了可靠的技术资源。
2)本发明hPSC-MSCs定向分化方案方法,步骤精简,操作简易,再现性高,仅仅用12天便可以获得表型成熟、高质量的来源于中胚层谱系的MSCs。
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为分化经中段原条获得的MSCs前体细胞。
图4为分化经侧中胚层获得的MSCs前体细胞。
图5为添加bFGF分化经中段原条获得的MSCs前体细胞。
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、梭型形态的MSCs细胞。
图8为没有发现可以粘附生长的细胞。
具体实施方式
hPSCs向MSCs分化的培养方法:
实施例1:
1)将未分化hPSCs细胞(图1和图2)转移到基质胶(Matrigel,MG;细胞外基质的一种)包被30min的24孔贴壁用培养板上,用0.5ml多能干细胞分化培养基,37℃、5%CO2培养24小时后(Day 0),改为0.5ml不含TGF-β1的多能干细胞分化培养基培养。另外,培养基还含有40ng/ml BMP4和30ng/mlActivin A,6μM CHIR99021,100nMPIK90。其中,BMP4为骨形态发生蛋白4,CHIR99021为6-[2-[4-(2,4-二氯苯基)-5-(4-甲基-1H-咪唑-2-基)嘧啶-2-基氨基]乙基氨基]吡啶-3-甲腈(CAS:252917-06-9),Activin A为激活素A,PIK90为N-(2,3-二氢-7,8-二甲氧基咪唑并[1,2-C]喹唑啉-5-基)-3-吡啶甲酰胺(CAS:677338-12-4)。培养24小时。检测MIXL1、BRACHYURY、HAND1等中段原条标记物的表达量。发现三个中段原条标记物均获得高表达,可以断定此时细胞处于中段原条的阶段(表1)。
2)去掉旧液,改为含有10uM/ml SB431542的0.5ml不含TGF-β1的多能干细胞分化培养基连续培养5天,隔天换液,期间不传代。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)。在经SB431542处理后,多能性标记Oct3/4、Nanog、Sox2等表达逐渐衰减(表2),BRACHYURY急减至基本不表达,侧中胚层标记物Nkx2.5、HAND1、FOXF1等持续高表达,可以断定此时细胞处于侧中胚层的阶段(表1)。
表1
表2
3)去掉旧液,加2ml 0.05%Trypsin-EDTA将SB处理5天后的细胞消化5min,加4mlDMEM/F12(含10%FBS)终止,回收到15ml离心管,200g、5min离心。
4)除掉上清,加含有10uM/ml SB431542的1ml不含TGF-β1的多能干细胞分化培养基轻轻将细胞悬浮,移到经明胶(Gelatin,GT)包被30min、加含有10uM/ml SB431542的9ml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48h,得到具有贴壁性能、梭型形态特征的细胞(图3)。37℃、5%CO2继续培养3天,隔日换液,此时即为MSCs前体细胞阶段。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。
5)经GT阶段细胞汇合度可以到达~90%,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。
6)除掉上清,加1ml含有10uM/ml SB431542且不含TGF-β1抑制剂的多能干细胞分化培养基轻轻将细胞悬浮,移到(1:5传)没有任何包被的加有9ml含有10uM/ml SB431542且不含TGF-β1抑制剂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,流式鉴定结果如表3所示。经过FACS鉴定,细胞能高水平表达CD105、CD90、CD73等MSC阳性Markers;基本不表达CD45、CD34、CD14or CD11b、CD79α、CD19以及HLA-DR等MSC阴性Markers。另外,细胞是具有典型的梭型形态,可以认定细胞为MSCs细胞(图6和图7)。
表3
另外,将BMP4用BMP2、BMP7替换;Activin A用ActivinB、TGF-β1、Nodal替换,CHIR99021用BIO(CAS:667463-62-9)、WNT-3a(WNT信号蛋白3a)、R-spondin-2(R-spondin信号蛋白2)替换;SB431542用SB505124(CAS:694433-59-5)、A8301(CAS:909910-43-6)、RepSox(CAS:446859-33-2)PI-103替换;PIK90用TG100713(CAS:925705-73-3)、PI-103(CAS:371935-74-9)替换也可以产生类似的效果。
实施例2:
按照实施例1的方法进行分化培养,做如下改进:步骤2)换含有20ng/ml BMP4(BMP-SMAD1/5/8信号通路激活剂)、3uM/ml SB505124(TGF-β1/Activin/Nodal-SMAD2/3信号通路激活剂)、1uM/ml Wnt-C59(Wnt抑制剂)的分化培养基继续培养24小时,此时侧中胚层标记物Nkx2.5、HAND1、FOXF1等高表达(表4),可以断定此时细胞处于侧中胚层的阶段,然后换含有10uM/ml SB431542的0.5ml不含TGF-β1的多能干细胞分化培养基继续培养,隔天换液,期间不传代。SB505124是2-[4-(1,3-苯并二唑-5-基)-2-(1,1-二甲基乙基)-1H-咪唑-5-基]-6-甲基吡啶(CAS:694433-59-5),Wnt-C59是4-(2-甲基-4-吡啶基)-N-[4-(3-吡啶基)苯基]苯乙酰胺(CAS:1243243-89-1)。在步骤4)的时候会获得典型的梭型形态特征的MSCs前体细胞,如图4所示。其余步骤不变,最后经过FACS鉴定,细胞能高水平表达CD105、CD90、CD73等MSC阳性Markers;基本不表达CD45、CD34、CD14or CD11b、CD79αor CD19以及HLA-DR等MSC阴性Markers,如表5所示。
表4
表5
实施例3:
按照实施例1的方法进行分化培养,做如下改进:步骤1)开始在分化培养基基础上额外加上碱性成纤维细胞生长因子(Basic fibroblast growth factor,bFGF)后,在步骤4)的时候会获得更多、更典型的梭型形态特征的MSCs前体细胞,如图5所示。其余步骤不变,最后经过FACS鉴定,细胞能高水平表达CD105、CD90、CD73等MSC阳性Markers;基本不表达CD45、CD34、CD14、CD11b、CD79α、CD19以及HLA-DR等MSC阴性Markers,如表6所示。
表6
对比例
1)将未分化hPSCs细胞转移到细胞外基质(MG)包被30min的24孔贴壁用培养板上,用0.5ml多能干细胞分化培养基,37℃、5%CO2培养24小时后(Day 0),换0.5ml不含TGF-β1的多能干细胞分化培养基,培养基中含有30ng/mlActivin A,6μM CHIR99021,100nM PIK90培养24小时。检测MIXL1、BRACHYURY、TBX6等前段原条标记物的表达量,发现均获得高表达,此时细胞处于前段原条阶段(表7)。
表7
2)去掉旧液,改为含有10uM/ml SB431542的0.5ml不含TGF-β1的多能干细胞分化培养基连续培养5天,隔天换液,期间不传代。
3)去掉旧液,加2ml 0.05%Trypsin-EDTA将SB处理5天后的细胞消化5min,加4mlDMEM/F12(含10%FBS)终止,回收到15ml离心管,200g、5min离心。
4)除掉上清,加含有10uM/ml SB431542的1ml不含TGF-β1的多能干细胞分化培养基轻轻将细胞悬浮,移到经明胶(GT)包被30min、加含有10uM/ml SB431542的9ml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48h,没有发现可以粘附生长的细胞(图8)。
Claims (17)
1.一种高效的hPSCs向MSCs分化诱导的方法,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后;换用含有BMP-SMAD1/5/8信号通路激活剂、TGF-β1/ Activin/ Nodal-SMAD2/3信号通路激活剂、Wnt激活剂和PI3K抑制剂的分化培养基培养;获得中段原条;
2)去除旧培养基,换含有TGF-β1/ Activin/ Nodal-SMAD2/3信号通路抑制剂分化培养基继续培养;
3)将细胞消化并转移到新的培养板上,换含有TGF-β1/ Activin/ Nodal-SMAD2/3信号通路抑制剂分化培养基继续培养;
4)将细胞消化并转移到贴壁用培养板上继续培养,获得MSCs;
上述获得的MSCs是使用诱导hPSCs通过中段原条阶段向MSCs定向分化的方法来获得的。
2.根据权利要求1所述的方法,其特征在于,步骤3)中,新的培养板是用明胶类细胞外基质包被的,进行细胞筛选。
3.根据权利要求2所述的方法,其特征在于,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。
4.根据权利要求1所述的方法,其特征在于,步骤1)中,用含有BMP-SMAD1/5/8信号通路激活剂、TGF-β1/ Activin/ Nodal-SMAD2/3 信号通路激活剂、Wnt激活剂和PI3K抑制剂的分化培养基培养时间为12~72h。
5.根据权利要求1所述的方法,其特征在于,步骤1)中,BMP-SMAD1/5/8信号通路激活剂BMP2、BMP4、BMP7中的至少一种。
6.根据权利要求1所述的方法,其特征在于,步骤1)中,TGF-β1/Activin/ Nodal-SMAD2/3 信号通路激活剂为Activin A、Activin B、TGF-β1、Nodal中的至少一种。
7.根据权利要求1所述的方法,其特征在于,步骤1)中, Wnt激活剂为CHIR99021,BIO、WNT-3a、R-spondin-2中的至少一种。
8.根据权利要求1所述的方法,其特征在于,步骤1)中, PI3K抑制剂为TG100713、PIK90、PI-103中的至少一种。
9.根据权利要求1所述的方法,其特征在于,TGF-β1/Activin/Nodal-SMAD2/3信号通路抑制剂为SB431542、SB505124、A8301、RepSox中的至少一种。
10.根据权利要求1所述的方法,其特征在于,步骤3)中,继续培养的时间为2~30天。
11.根据权利要求1所述的方法,其特征在于,在步骤2)继续培养之前,先用含有BMP-SMAD1/5/8信号通路激活剂、TGF-β1/ Activin/ Nodal-SMAD2/3信号通路激活剂和Wnt抑制剂的分化培养基培养。
12.根据权利要求1所述的方法,其特征在于,在步骤2)中,继续培养的时间为2~10天。
13.根据权利要求11所述的方法,其特征在于,Wnt抑制剂为Wnt-C59、XAV-939中的至少一种。
14.根据权利要求1所述的方法,其特征在于,步骤4)中继续培养的方法是:用含有间充质干细胞培养基培养。
15.根据权利要求1所述的方法,其特征在于,在步骤1)至步骤4)中,至少一种培养基中含有碱性成纤维细胞生长因子bFGF。
16.根据权利要求1所述的方法,其特征在于,获得的MSCs是使用诱导hPSCs通过侧中胚层阶段向MSCs定向分化的方法来获得的。
17.一种MSCs细胞,由权利要求1~16任意一项所述的方法制备而成。
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