CN116790480A - 一种培养基及其应用 - Google Patents
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Abstract
本发明涉及干细胞技术领域,尤其涉及一种培养基及其应用。本发明的培养基体系成份明确,无血清、无异源蛋白,适合培养产品的临床转化。本发明的培养基支持在常氧(21%O2)条件下,建立、培养和扩增Naive hPSCs。在常氧条件下,生长在本发明的培养基体系下的Naive hPSCs在细胞形态、多能性标志物表达、转录组、甲基化水平和分化潜能等多个方面都能长期维持Naive多能状态。
Description
技术领域
本发明涉及干细胞技术领域,尤其涉及一种培养基及其应用。
背景技术
从细胞来源划分,人多能干细胞(human pluripotent stem cells,hPSCs)包括人胚胎干细胞(human embryonic stem cells,hESCs)和人诱导多能干细胞(human inducedpluripotent stem cells,hiPSCs)。从多能性状态划分,hPSCs包括原始态(Naive)和始发态(Primed)两种hPSCs。Naive hPSCs对应着床前囊胚中的上胚层,Primed hPSCs对应着床后晚期胚胎中的上胚层,即Naive hPSCs在发育时间上对应人胚胎中最早期的上胚层。
Naive hPSCs表达TFCP2L1、KLF4和KLF17等核心转录因子,不表达SSEA4和CD24等Primrd hPSCs的多能性标志物,在体外具有自我更新和无限增殖能力。在体内,将其注射到免疫缺陷小鼠皮下,能够形成具有三胚层结构的畸胎瘤。在体外,可以分化产生原始内胚层、滋养外胚层和胚外中胚层等胚外组织;将其培养到Primed培养体系,Naive hPSCs能够转化为Primed hPSCs,并和Primed hPSCs具有相似的分化潜能;通过Naive hPSCs甚至还可以重构出与人着床前囊胚高度相似的类囊胚。综上,通过Naive hPSCs,可以在体内和体外分化为特定组织的细胞类型或自我组装生成特定的类胚胎和类器官,理论上,Naive hPSCs在体外具有分化为所有胚外和胚胎组织,以及所有成体细胞的潜能。因此,Naive hPSCs在发育学研究、疾病模拟、药物筛选、器官再生、组织修复和细胞替代治疗等方面具有广阔的应用前景。截至目前,已建立的Naive hPSCs的所有培养体系如下:
(1)2014年,Smith实验室建立了培养基(Takashima等,Cell,2014;doi:10.1016/j.cell.2014.08.029);2019年,其团队在/>培养基的基础上进行改进,推出了现在的PXGL培养基(Bredenkamp等,Stem Cell Reports,2019;doi:10.1016/j.stemcr.2019.10.009);
(2)2014年,Jaenisch实验室建立了5i/LA培养基(Theunissen等,Cell StemCell,2014;doi:10.1016/j.stem.2014.07.002);2016年,其团队在5i/LA培养基的基础上进行改进,推出了4i/LA培养基(Theunissen等,Cell Stem Cell,2016;doi:10.1016/j.stem.2016.06.011);
(3)2021年,Hanna实验室建立了HENSM培养基(Bayerl等,Cell Stem Cell,2021;doi:10.1016/j.stem.2021.04.001)。
然而,上述所有培养体系都需要借助生理氧条件(5%O2),才能支持Naive hPSCs的建立和培养。当前,在正常氧条件下,是否能够建立和培养Naive hPSCs依然是未解之谜。此外,由于创造和维持细胞生长的生理氧条件,需要源源不断地往细胞培养箱补充氮气(N2)来稀释空气氧,这给日常的细胞培养操作增加了大量的人力和物力成本,且不利于Naive hPSCs的规模化培养和扩增。
发明内容
为解决上述难题,本发明提供了一种培养基,该培养基能够克服当前其他NaivehPSCs培养基依赖生理氧(5%O2)条件的缺陷,从而简化Naive hPSCs的培养条件,降低人力和物力成本,为Naive hPSCs的规模化培养和扩增奠定基础。
首先,本发明提供了一种培养基,包括:
mN2B27培养基、以及2-20ng/ml的重组人活化素A(Activin-A)、1-5μM的IWP2(WNT信号通路的抑制剂)、0.2-1μM的CHIR99021(WNT信号通路的激动剂)、0.5-2μM的PD0325901(MEK信号通路的抑制剂)、0.5-5μM的(PKC信号通路的抑制剂)和2-20ng/ml的重组人白血病抑制因子(LIF);
所述mN2B27培养基包括:
基础培养基、以及0.25-1%N2、0.5-2%B27、0.1-1%GlutaMAX、0.1-1%非必需氨基酸(NEAA)、0.01-0.1mMβ-巯基乙醇(β-ME)、1-100μg/ml左旋抗坏血酸-2-磷酸镁盐(Vc)、0.05-0.2%化学脂质浓缩液、1-20μg/ml胰岛素、0.01-0.05μg/ml黄体酮;
所述基础培养基为DMEM/F12培养基与Neurobasal培养基的混合培养基。本发明中的“%”均表示体积百分比。
优选地,所述培养基,包括:
mN2B27培养基、以及5-15ng/ml的重组人活化素A(Activin-A)、1-5μM的IWP2(WNT信号通路的抑制剂)、0.2-1μM的CHIR99021(WNT信号通路的激动剂)、0.5-2μM的PD0325901(MEK信号通路的抑制剂)、0.5-2μM的(PKC信号通路的抑制剂)和5-15ng/ml的重组人白血病抑制因子(LIF);
所述mN2B27培养基包括:
基础培养基、以及0.5-1%N2、1-2%B27、0.5-1%GlutaMAX、0.5-1%非必需氨基酸(NEAA)、0.05-0.1mMβ-巯基乙醇(β-ME)、25-75μg/ml左旋抗坏血酸-2-磷酸镁盐(Vc)、0.05-0.2%化学脂质浓缩液、5-15μg/ml胰岛素、0.01-0.05μg/ml黄体酮;
所述基础培养基为DMEM/F12培养基与Neurobasal培养基的混合培养基。
优选地,所述基础培养基中,DMEM/F12培养基与Neurobasal培养基的体积比为1:0.8~1.2。
更优选地,所述基础培养基中,DMEM/F12培养基与Neurobasal培养基的体积比为1:1。
优选地,DMEM/F12培养基中,DMEM培养基与F12培养基的体积比为1:0.8~1.2,更优选为1:1。
优选地,所述培养基中不含有血清。
优选地,所述培养基中不含有异源动物成分。
进一步,本发明提供了一种试剂或试剂盒,其中含有上述任一方案中的培养基。
进一步,本发明提供了一种生物材料,其中含有上述任一方案中的培养基。
进一步,本发明提供了上述任一方案中的培养基、试剂或试剂盒、或生物材料在建立、培养或扩增人原始态多能干细胞中的应用。
此外,本发明提供了一种在常氧下建立人原始态多能干细胞系的方法,包括:在建系和培养过程中采用上述任一方案中的培养基。
在一些实施方案中,通过囊胚建系或通过人始发态多能干细胞转换建立人原始态多能干细胞系。
本发明的培养基支持在常氧条件下,通过人囊胚高效(>67%效率)建立NaivehPSCs。
本发明的培养基支持在常氧条件下,通过Primed hPSCs转化,高效(100%的转化效率)建立Naive hPSCs。
本发明的培养基支持在常氧条件下,长期培养和扩增Naive hPSCs后,NaivehPSCs能够稳定维持Naive多能性状态。
本发明的常氧条件为氧气含量在18%~24%,优选为20%~22%,最优选为21%。
与现有技术相比,本发明的有益效果在于:
本发明的培养基成份明确,无血清、无异源蛋白,适合培养产品的临床转化。本发明的培养基支持在常氧条件下,建立、培养和扩增Naive hPSCs。在常氧条件下,生长在本发明的培养基里的Naive hPSCs在细胞形态、多能性标志物表达、转录组、甲基化水平和分化潜能等多个方面都能长期维持Naive多能状态。
附图说明
图1是建立AIC-N培养体系的示意图。
图2是在AIC-N体系中,常氧条件下通过囊胚建系或Primed hPSCs转化建立的Naive hPSCs的相差图。
图3是常氧条件下,在AIC-N体系中建立和培养的Naive hPSCs的免疫荧光染色图。
图4是生长在不同培养基条件下的hPSCs的基因表达谱的PCA分析图。
图5是不同hPSCs的全基因组CpG甲基化水平检测图。
图6是X染色体上的CpG甲基化水平检测图。
图7是AIC-N体系下建立的Naive hPSCs来源的类囊胚的相差和免疫荧光染色图。
图8是同时包含三种细胞谱系的类囊胚的比例统计图。
图9是对Naive hPSCs进行多能性和下胚层标志物的免疫荧光染色图。
图10是标志物的流式细胞术分析图。
图11是G-带核型分析图。
图12是畸胎瘤实验结果图。
图13是在不同培养条件下的hPSCs中,代表性的Primed和基因热图。
图14是全基因组甲基化比较图。
图15是Naive hPSCs分化产生滋养外胚层谱系的示意图。
图16是Naive hPSCs分化产生滋养外胚层谱系的相差和染色图。
所有图中的标尺长度为100μm。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面将对本发明中的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例中未注明具体技术或条件者,均为常规方法或者按照本领域的文献所描述的技术或条件进行,或者按照产品说明书进行。所用试剂和仪器等未注明生产厂商者,均为可通过正规渠道商购买得到的常规产品。
N2购买Thermo Fisher Scientific,货号为17502-048;
B27购买自Thermo Fisher Scientific,货号为17504-044;
GlutaMAX购买自Thermo Fisher Scientific,货号为35050-061;
非必需氨基酸购买自Thermo Fisher Scientific,货号为11140-050;
化学脂质浓缩液购买自Thermo Fisher Scientific,货号为11905-031。
下述实施例的培养基中的“%”均表示体积百分比。
实施例1
本实施例在常氧(氧气含量21%)和AIC-N培养条件下,在Feeders上建立和培养Naive hPSCs。
AIC-N培养基配方:向mN2B27培养基中添加重组人活化素A(Activin-A,浓度为10ng/ml)、IWP2(一种WNT信号通路的抑制剂,浓度为2μM)、CHIR99021(一种WNT信号通路的激动剂,浓度为0.3μM)。PD0325901(一种MEK信号通路的抑制剂,浓度为1μM)、(一种PKC信号通路的抑制剂,浓度为2μM)和重组人白血病抑制因子(LIF,浓度为10ng/ml);
其中,mN2B27培养基配方为:基础培养基(DMEM/F12(体积比1:1)与Neurobasal等体积比例混合)添加1%N2、2%B27、1%GlutaMAX、1%非必需氨基酸(NEAA)、0.1mMβ-巯基乙醇(β-ME)、50μg/ml左旋抗坏血酸-2-磷酸镁盐(Vc)、0.1%化学脂质浓缩液、10μg/ml胰岛素、0.02μg/ml黄体酮。
具体步骤包括:
①通过囊胚建立Naive hPSCs。将人着床(植入)前囊胚接种在丝裂霉素C灭活的小鼠胚胎成纤维细胞(以下简称Feeder)上,48小时后,胚胎贴壁,部分胚胎中的hPSC-likeoutgrowth开始变得明显,用玻璃针将hESC-like outgrowth机械分离并转移至50%TrypLE液滴(如果hESC-like outgrowth不明显,直接用50%TrypLE处理整个胚胎),37℃培养箱孵育15分钟。然后将hESC-like outgrowth(或整个胚胎)再次转移到AIC-N培养基液滴,用玻璃针上下来回轻轻吹打,将hESC-like outgrowth(或整个胚胎)吹打为单细胞或小细胞团块(每个团块包含3-5个细胞)。将解离后的单细胞或小细胞团块接种至新Feeder上。3-5天后,显微镜下能观察到少量的hPSC集落,并用50%TrypLE进一步对其进行单细胞传代。自此,Naive hPSCs建系成功,后续培养过程中,每两天换液,每3-4天传代,传代比例为1:5-1:10。整个建系和培养过程,往AIC-N培养基中添加Y27632(一种选择性ROCK1和ROCK2抑制剂;10μM)。
②通过转换Primed hPSCs建立Naive hPSCs。在转换起始的前两周,AIC-N培养基中添加丙戊酸(VPA;1mM),以促进Primed hPSCs转换为Naive hPSCs。具体操作如下:用50%TrypLE消化Primed hPSCs 5-10分钟,移液枪轻轻将其吹打为单细胞,离心去上清,用添加了VPA的AIC-N培养基重悬细胞,以5×103cells/cm2的细胞密度接种到Feeder上。48小时后,换液新鲜培养基。转换过程中,每两天换液,每4天传代(50%TrypLE消化,单细胞传代,传代比例为1:5-1:10),传2-3代,hPSCs可以从Primed状态转换为Naive状态。转换成功后,不再需要往AIC-N培养基中添加VPA。整个转换和培养过程,往AIC-N培养基中添加Y27632(10μM)。
之后将Naive hPSCs冻存,冻存液配方为90%AIC-N+10%DMSO+10μM Y27632,现用现配,使用前在2-8度冰箱预冷10分钟。冻存管规格:2ml。将消化为单细胞的Naive hPSCs重悬到预冷的冻存液中,500μl/管细胞悬液加入冻存管,每管包含3×105-1×106个细胞。然后将含有细胞悬液的冻存管放入程序降温盒,﹣80度冰箱放置过夜后,转入液氮长期保存。
实施例2
本实施例用上述实施例中的Naive hPSCs分化产生滋养层干细胞。具体步骤包括:
用50%TrypLE消化Naive hPSCs 5-10分钟,移液枪轻轻将其吹打为单细胞,离心去上清,用添加了PD0325901(一种MEK信号通路的抑制剂,浓度为1μM)和A83-01(一种TGF-βtype I receptor抑制剂,浓度为1μM)的mN2B27培养基重悬细胞,以6×103cells/cm2的细胞密度接种到Feeder上,之后每两天换液一次。三天后,培养基被更换为滋养层干细胞培养基,并且在第五天对细胞进行1:3传代。自此,每两天换液,每四天1:3传代。一般2-3代后,能够建立纯的滋养层干细胞。滋养层干细胞培养基配方如下:基础培养基DMEM/F12、0.1mMβ-巯基乙醇(β-ME)、0.2%胎牛血清、0.3%牛血清白蛋白、1%胰岛素-转铁蛋白-硒-乙醇胺添加剂(ITS-X)、50μg/ml左旋抗坏血酸-2-磷酸镁盐(Vc)、50ng/ml表皮细胞生长因子(EGF)、2μM CHIR99021(一种WNT信号通路的激动剂)、0.5μM A83-01(一种TGF-βtype I receptor抑制剂)、1μM SB431542(一种ALK5抑制剂)、0.8mM VPA(一种组蛋白去乙酰化酶抑制剂)。在整个滋养层干细胞的分化和培养过程中,往所有培养基中添加5μM Y27632(一种选择性ROCK1和ROCK2抑制剂)。
实施例3
本实施例用上述实施例中的Naive hPSCs重构类囊胚,具体步骤包括:
用1mg/ml IV型胶原酶(Collagenase type IV)消化Naive hPSCs60-90分钟,使Naive hPSC集落从Feeder上脱壁飘起,收集,自然沉降或离心去上清,50%TrypLE消化Naive hPSC集落5-10分钟,移液枪轻轻将其吹打为单细胞,用20μm孔径的细胞筛过滤细胞,去除细胞团或杂质,然后离心去上清,用mN2B27培养基重悬细胞并计数,接种2ml细胞悬液/孔(含8×104cells)至AggreWellTM 400 24孔板。24小时后,用PALLY培养基替换mN2B27培养基。72小时后,进一步用含4μM油酰基-L-α-溶血磷脂酸钠盐(LPA)的mN2B27培养基替换PALLY培养基。在第五天,Naive hPSCs形成类囊胚。在类囊胚的制备过程中,每24小时换液一次,且所有培养基都添加10μΜ Y27632。PALLY培养基配方如下:mN2B27培养基作为基础培养基,添加1μM PD0325901,1μM A83-01,4μM LPA和10ng/ml重组人白血病抑制因子(LIF)。
试验例
对上述实施例建立和培养的Naive hPSCs、以及滋养层干细胞和类囊胚进行表征,表征结果如下。
图1是建立AIC-N培养体系的示意图;图2是在AIC-N体系中,常氧条件下通过囊胚建系或Primed hPSCs转化建立的Naive hPSCs的相差图;图3是常氧条件下,在AIC-N体系中建立和培养的Naive hPSCs的免疫荧光染色图;图4是生长在不同培养基条件下的hPSCs的基因表达谱的PCA分析;图5是不同hPSCs的全基因组CpG甲基化水平,其中,CC-hES1、CC-H9、CC-S4为培养在常氧下的Primed hPSCs,培养方法参照公开发表文献(DOI:10.1016/j.biomaterials.2020.120015);图6是不同hPSCs的X染色体上的CpG甲基化水平;图7是AIC-N体系下建立的Naive hPSCs来源的类囊胚的相差和免疫荧光染色图;图8是同时包含三种细胞谱系的类囊胚的比例;图9是对Naive hPSCs进行多能性和下胚层标志物的免疫荧光染色图;图10是标志物的流式细胞术分析;图11是AIC-N体系下建立的Naive hPSCs的G-带核型分析;图12是畸胎瘤实验结果;图13是在不同培养条件下的hPSCs中,代表性的Primed和基因热图;图14是AIC-N体系下建立的Naive hPSCs全基因组甲基化比较;图15是Naive hPSCs分化产生滋养外胚层谱系的示意图;
图16是Naive hPSCs分化产生滋养外胚层谱系的相差和染色图;其中,所有图中的标尺长度为100μm。
以上结果表明,常氧下,在AIC-N体系里建立和培养的Naive hPSCs,表达常规多能性标志物(OCT4,SOX2,NANOG,PRDM14,Tra-1-60,Tra-1-85,E-cadherin)和关键Naive多能性标志物(TFCP2L1,KLF4,KLF17,SUSD2,ALPPL2),不表达Primed多能性标志物(SSEA4,CD24)和其他分化标志物(GATA6,SOX17)。其基因表达谱、DNA甲基化、X染色体活化状态、染色体核型、体内三胚层分化潜能、类囊胚形成能力和滋养外胚层细胞谱系的分化潜能都与低氧条件下(5i/LA、HENSM及其/>和5i/LA的优化体系)建立和培养的NaivehPSCs高度相似,/>5i/LA、HENSM及其/>和5i/LA的优化体系在图4和图13中作为对照组。
实施例4
本实施例在常氧和AIC-N培养条件下,在Feeders上建立和培养Naive hPSCs,本实施例仅与实施例1不同的是培养基配方不同。
AIC-N培养基配方:向mN2B27培养基中添加重组人活化素A(Activin-A,浓度为5ng/ml)、IWP2(一种WNT信号通路的抑制剂,浓度为1μM)、CHIR99021(一种WNT信号通路的激动剂,浓度为0.6μM)。PD0325901(一种MEK信号通路的抑制剂,浓度为0.8μM)、(一种PKC信号通路的抑制剂,浓度为1μM)和重组人白血病抑制因子(LIF,浓度为20ng/ml);
其中,mN2B27培养基配方为:基础培养基(DMEM/F12(体积比1:1)与Neurobasal等体积比例混合)添加0.5%N2、1%B27、0.5%GlutaMAX、0.5%非必需氨基酸(NEAA)、0.05mMβ-巯基乙醇(β-ME)、100μg/ml左旋抗坏血酸-2-磷酸镁盐(Vc)、0.05%化学脂质浓缩液、5μg/ml胰岛素、0.01μg/ml黄体酮。
经试验,在常氧条件下,在该AIC-N体系里建立和培养的Naive hPSCs,其在细胞形态、多能性标志物表达、转录组、甲基化水平和分化潜能等方面与实施例1的培养基配方接近。
实施例5
本实施例在常氧和AIC-N培养条件下,在Feeders上建立和培养Naive hPSCs,本实施例仅与实施例1不同的是培养基配方不同。
AIC-N培养基配方:向mN2B27培养基中添加重组人活化素A(Activin-A,浓度为20ng/ml)、IWP2(一种WNT信号通路的抑制剂,浓度为5μM)、CHIR99021(一种WNT信号通路的激动剂,浓度为1μM)。PD0325901(一种MEK信号通路的抑制剂,浓度为0.5μM)、(一种PKC信号通路的抑制剂,浓度为0.5μM)和重组人白血病抑制因子(LIF,浓度为2ng/ml);
其中,mN2B27培养基配方为:基础培养基(DMEM/F12(体积比1:1)与Neurobasal等体积比例混合)添加0.25%N2、0.5%B27、0.1%GlutaMAX、0.1%非必需氨基酸(NEAA)、0.01mMβ-巯基乙醇(β-ME)、5μg/ml左旋抗坏血酸-2-磷酸镁盐(Vc)、0.2%化学脂质浓缩液、2μg/ml胰岛素、0.05μg/ml黄体酮。
经试验,在常氧条件下,在该AIC-N体系里建立和培养的Naive hPSCs,其在细胞形态、多能性标志物表达、转录组、甲基化水平和分化潜能等方面与实施例1的培养基接近。
最后应说明的是:以上实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
Claims (10)
1.一种培养基,其特征在于,包括:
mN2B27培养基、以及2-20ng/ml的重组人活化素A、1-5μM的IWP2、0.2-1μM的CHIR99021、0.5-2μM的PD0325901、0.5-5μM的和2-20ng/ml的重组人白血病抑制因子;
所述mN2B27培养基包括:
基础培养基、以及0.25-1%N2、0.5-2%B27、0.1-1%GlutaMAX、0.1-1%非必需氨基酸、0.01-0.1mMβ-巯基乙醇、1-100μg/ml左旋抗坏血酸-2-磷酸镁盐、0.05-0.2%化学脂质浓缩液、1-20μg/ml胰岛素、0.01-0.05μg/ml黄体酮;
所述基础培养基为DMEM/F12培养基与Neurobasal培养基的混合培养基。
2.根据权利要求1所述的培养基,其特征在于,所述基础培养基中,DMEM/F12培养基与Neurobasal培养基的体积比为1:0.8~1.2。
3.根据权利要求2所述的培养基,其特征在于,DMEM/F12培养基中,DMEM培养基与F12培养基的体积比为1:0.8~1.2。
4.根据权利要求1~3中任一项所述的培养基,其特征在于,其中不含有血清。
5.根据权利要求1~3中任一项所述的培养基,其特征在于,其中不含有异源动物成分。
6.一种试剂或试剂盒,其特征在于,其中含有权利要求1~5中任一项所述的培养基。
7.一种生物材料,其特征在于,其中含有权利要求1~5中任一项所述的培养基。
8.权利要求1~5中任一项所述的培养基、或权利要求6所述的试剂或试剂盒、或权利要求7所述的生物材料在建立、培养或扩增人原始态多能干细胞中的应用。
9.一种在常氧下建立人原始态多能干细胞系的方法,其特征在于,包括:在建系和培养过程中采用权利要求1~5中任一项所述的培养基。
10.根据权利要求9所述的方法,其特征在于,通过囊胚建系或通过人始发态多能干细胞转换建立人原始态多能干细胞系。
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