CN110218696A - 一种用于化学诱导多能性干细胞生成的培养体系以及使用该培养体系的化学重编程方法 - Google Patents
一种用于化学诱导多能性干细胞生成的培养体系以及使用该培养体系的化学重编程方法 Download PDFInfo
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Abstract
本文公开了用于化学诱导多能性干细胞生成的培养体系,其包括基础培养基和促进化学重编程的组合物,其中,所述促进化学重编程的组合物包含胸腺嘧啶类似物,cAMP激活剂,TGF‑β受体抑制剂,骨形成蛋白,RA受体激活剂,GSK3抑制剂和碱性成纤维细胞生长因子,并且,其中,所述培养体系不包含血清。在通过本文提供的方法对体细胞进行化学重编程的过程中,无需对细胞进行频繁的分盘,这相对于目前已有的培养方法而言,简化了操作步骤,降低了在分盘过程中所产生的细胞损失,并且,采用本文提供的培养体系不需要使用血清,这进一步简化了后续的多能性干细胞的收集和分子机理分析,更便于后续建立诱导多能干细胞无动物源性培养体系。
Description
技术领域
本发明涉及一种用于化学诱导多能性干细胞生成的培养体系以及使用该培养体系的化学重编程方法。
背景技术
从体细胞诱导生成多能性干细胞是生物领域和医学领域的革命性发现。尤其对于医学领域而言,从体细胞诱导生成多能性干细胞这一概念能够指导研制再生药物作为患者的特异性干细胞,用于治疗诸如帕金森疾病和阿尔兹海默疾病之类的退行性疾病。在生物领域中,目前已对使用Yamanaka因子(Oct4,Sox2,Klf4和Myc)驱动的体细胞重编程进行了详细研究,例如,目前已详细研究了控制细胞命运及其转化的分子和细胞机理。对细胞命运的这种详细研究进一步揭示了从体细胞诱导生成多能性干细胞在癌症治疗、神经系统疾病的治疗和心血管疾病的治疗方面的应用。
本领域已提出采用化学小分子来代替Yamanaka因子诱导体细胞重编程生成多能性干细胞。北京大学的邓宏魁研究组已报道了采用小分子Forskolin来代替Yamanaka因子中的Oct4而生成化学诱导的多能性干细胞(Hou,P.等人,Pluripotent Stem CellsInduced From Mouse Somatic Cells by Small-molecule Compounds,Science 341,651-654),但是他们报道的培养体系无法有效诱导生成多能性干细胞并且在培养过程中需要对细胞进行频繁分盘,该培养体系仍然需要进行改进(Zhao Y.等人,A XEN-like StateBridges Somatic Cells to Pluripotency During Chemical Reprogramming.Cell,163,1678-1691)。
因此,还需要在采用化学小分子替代Yamanaka因子诱导生成多能性干细胞方面进行进一步深入研究。
发明内容
一方面,本文提供用于化学诱导多能性干细胞生成的培养体系,其包括基础培养基和促进化学重编程的组合物,其中,所述促进化学重编程的组合物包含胸腺嘧啶类似物,cAMP激活剂,TGF-β受体抑制剂,骨形成蛋白,RA受体激活剂,GSK3抑制剂和碱性成纤维细胞生长因子,并且,其中,所述培养体系不包含血清。
在一种实施方式中,所述胸腺嘧啶类似物的浓度为0.01-10μM。在一种实施方式中,所述cAMP激活剂的浓度为0.01-10μM。在一种实施方式中,所述TGF-β受体抑制剂的浓度为0.01-5μM。在一种实施方式中,所述骨形成蛋白的浓度为0.01-10ng/ml。在一种实施方式中,所述碱性成纤维细胞生长因子的浓度为0.01-10ng/ml。在一种实施方式中,所述RA受体激活剂的浓度为0.01-0.05μM。在一种实施方式中,所述GSK3抑制剂的浓度为0.01-3μM。在一种实施方式中,所述胸腺嘧啶类似物是Brdu。在一种实施方式中,所述cAMP激活剂是FSK。在一种实施方式中,所述TGF-β受体抑制剂是RepSox。在一种实施方式中,所述骨形成蛋白是BMP4。在一种实施方式中,所述碱性成纤维细胞生长因子是FGF2。在一种实施方式中,所述RA受体激活剂是AM580。在一种实施方式中,所述GSK3抑制剂是CHIR99021。
所述促进化学编程的组合物还包含维生素C,DOT1L抑制剂,组蛋白去乙酰化酶抑制剂和/或DZNep。
在一种实施方式中,所述维生素C的浓度为0.01-50μg/ml。在一种实施方式中,所述DOT1L抑制剂的浓度为0.01-5μM。在一种实施方式中,所述组蛋白去乙酰化酶抑制剂的浓度为0.01-0.5mM。在一种实施方式中,所述DZNep的浓度为0.001-0.05μM。
在一种实施方式中,所述DOT1L抑制剂是EPZ5676和/或SGC0946;所述组蛋白去乙酰化酶抑制剂是丙戊酸。
所述促进化学重编程的组合物还包含EZH2抑制剂和/或cMet抑制剂。在一种实施方式中,所述EZH2抑制剂的浓度为0.01μM-10μM。在一种实施方式中,所述cMet抑制剂的浓度为0.01μM-10μM。在一种实施方式中,所述EZH2抑制剂为EPZ6438。在一种实施方式中,所述cMet抑制剂为Capmatinib。
在本发明的一种示例性的实施方式中,所述用于化学诱导多能性干细胞生成的培养体系包含诸如iCD1之类的基础培养基以及含有下列成分的促进化学重编程的组合物:
10ng/ml BMP4;
50μg/ml维生素C;
5μM EPZ5676;
10μM Brdu;
0.05μM AM580;
3μM CHIR99021;
10ng/ml FGF2;
10μM Forsklin;
5μM RepSox;
0.1mM丙戊酸;
0.05μM DZNep;和/或
5μM SGC0946。
在本发明的另一示例性的实施方式中,所述用于化学诱导多能性干细胞生成的培养体系包含诸如iCD1之类的基础培养基以及含有下列成分的促进化学重编程的组合物:
10ng/ml BMP4;
50μg/ml维生素C;
5μM EPZ5676;
10μM Brdu;
0.05μM AM580;
3μM CHIR99021;
10ng/mlFGF2;
10μMForsklin;
5μMRepSox;
0.1mM丙戊酸;
0.05μM DZNep;
5μM SGC0946;
5μM EPZ6438;和/或
5μM Capmatinib。
另一方面,本文提供用于诱导体细胞进行化学重编程的方法,所述方法包括使体细胞在上述培养体系中培养持续足以使体细胞重编程至多能状态的时间段。所述方法还可包括在体细胞重编程至多能状态之后,将培养基更换为补充有下述成分的DMEM培养基并在该培养基中培养持续足以维持胚胎干细胞的原始状态的时间段,其中,所述成分包含:PD0325901;非必须氨基酸;GlutaMaxTM;人白细胞抗原B27;CHIR99021;白血病抑制因子,和/或N2添加剂。在一种实施方式中,PD0325901的浓度为1μM。在一种实施方式中,非必须氨基酸的浓度为培养基总体积的1%。在一种实施方式中,GlutaMaxTM的浓度为培养基总体积的1%。在一种实施方式中,人白细胞抗原B27的浓度为培养基总体积的2%。在一种实施方式中,CHIR99021的浓度为3μM。在一种实施方式中,N2添加剂的浓度为培养基总体积的1%。在一种实施方式中,白血病抑制因子的浓度为1U-1000U。
在本文提供的方法中,所述足以使体细胞重编程至多能状态的时间段可通过在培养过程中观察细胞的形态变化以及克隆的形成来确定,在一种实施方式中,所述所述足以使体细胞重编程至多能状态的时间段可为8天至22天。在本文提供的方法中,所述足以维持胚胎干细胞的原始状态的时间段可通过在培养过程中观察细胞的形态变化以及克隆的形成来确定,在一种实施方式中,所述足以维持胚胎干细胞的原始状态的时间段可为1天至18天。
采用本文提供的方法进行重编程的体细胞可选自:成纤维细胞、骨髓衍生的单核细胞、骨骼肌细胞、脂肪细胞、外周血单核细胞、巨噬细胞、肝细胞、角质细胞、口腔角质细胞、头发毛囊真皮细胞、胃上皮细胞、肺上皮细胞、滑膜细胞、肾细胞、皮肤上皮细胞、成骨细胞、神经干细胞和真皮细胞,但不限于此。
再一方面,本文提供用于化学诱导多能性干细胞生成的试剂盒,其中包括上文所述的培养体系。
在通过本文提供的方法对体细胞进行化学重编程的过程中,无需对细胞进行频繁的分盘,这相对于目前已有的培养方法而言,简化了操作步骤,降低了在分盘过程中所产生的细胞损失,并且,采用本文提供的培养体系不需要使用血清,这进一步简化了后续的多能性干细胞的收集和分子机理分析,更便于后续建立诱导多能干细胞无动物源性培养体系。
附图说明
图1是根据本发明的一种实施方式的化学诱导体细胞重编程的两阶段方法的示意图。
图2显示了小鼠胚胎成纤维细胞、化学诱导的多能性干细胞以及小鼠胚胎干细胞中的内源性多能性基因Oct4,Nanog,Sox2,Esrb,Rex1Dappa5,Sall4和Cdh1的表达水平,其中,CiPS-1,CiPS-2和CiPS-3表示来源于不同克隆株的化学诱导的多能性干细胞。
图3显示了化学诱导多能性干细胞、小鼠胚胎干细胞和小鼠成纤维细胞的的转录组学特性。
图4显示了多能性基因Oct4,Nanog,Sox2,Esrb和Rex1的在化学诱导的多能性干细胞中的蛋白质表达水平。
图5显示了化学诱导的多能性干细胞在小鼠体内形成畸胎瘤并分化形成三个胚层的细胞。
图6显示了化学诱导的多能性干细胞在传代过程中保持核型正常。
图7显示了将化学诱导的多能性干细胞注入假孕小鼠体内得到的嵌合子代小鼠。
具体实施方式
除非另有定义,本文所使用的科技术语的含义与本领域普通技术人员所理解的含义相同。
释义
本文使用的术语“多能的”或“多能性的”指细胞能够通过其后代产生成体动物中可见的数种不同细胞类型。
本文使用的术语“分化”是指如下过程:通过该过程使较不特化的细胞变成更特化的细胞以形成至少一种新细胞类型的后代。“去分化”是指如下细胞过程:其中部分分化细胞或终末分化细胞回复至较早的发育阶段,例如多潜能性或多能性。“转分化”是将一种分化细胞类型转化成另一种分化细胞类型的过程。在某些条件下,具有新细胞类型的特征的后代的比例可以为至少约1%、5%、25%或更大。
本文中使用的术语“体细胞”可选自小鼠体细胞和人体细胞,优选地,所述小鼠体细胞和所述人体细胞选自:成纤维细胞、骨髓衍生的单核细胞、骨骼肌细胞、脂肪细胞、外周血单核细胞、巨噬细胞、神经干细胞、肝细胞、角质细胞、口腔角质细胞、头发毛囊真皮细胞、胃上皮细胞、肺上皮细胞、滑膜细胞、肾细胞、皮肤上皮细胞、成骨细胞、神经干细胞和真皮细胞。
本文中使用的术语“分离的”是指以机械方式或化学方式分离细胞。分离的细胞的实例是发育中的细胞团、细胞培养物和细胞系。
本文使用的表达活性的术语“抑制剂”或“激活剂”分别是指通过对靶蛋白(或编码多核苷酸)的表达或活性进行体外和体内检测所鉴定的抑制分子或活化分子,例如,配体、激动剂、拮抗剂、及其同系物和模拟物。抑制剂是指如下试剂:例如,抑制表达或结合,部分或全部阻断活化或蛋白酶抑制剂活性,降低,防止,延迟活化,失活,去稳定化,或下调所述靶蛋白活性的试剂,例如拮抗剂。激活剂是例如,诱导或活化所述靶蛋白表达或结合,刺激、增加、开放、活化、促进、增强活化或蛋白酶抑制剂活性,致敏或上调所述靶蛋白(或编码多核苷酸)的活性,例如,激动剂。所述关于抑制剂和激活剂的测定包括,例如,将特定的调节剂化合物应用到表达所述靶蛋白的细胞,然后确定对所述靶蛋白活性的功能性作用,如上所述。将用潜在的激活剂或抑制剂处理包括所述靶蛋白的样品或测定与无抑制剂或激活剂的对照样品进行比较,以检验作用程度。对照样品(未用调节剂处理)被赋予100%的相对活性值。当活性值相对于对照为约80%,50%,25%,10%,5%或1%时,实现所述靶蛋白的抑制。当活性值相对于对照为约110%,150%,200%,300%,400%,500%,或1000%-3000%或更高时,实现所述靶蛋白的活化。
用于化学诱导多能件干细胞生成的培养体系
一方面,本发明提供用于化学诱导多能性干细胞生成的培养体系,其包括基础培养基和促进化学重编程的组合物,其中,所述促进化学重编程的组合物包含:胸腺嘧啶类似物,cAMP激活剂,TGF-β受体抑制剂,骨形成蛋白,RA受体激活剂,GSK3抑制剂和碱性成纤维细胞生长因子,并且,所述培养体系不包含血清。所述促进化学编程的组合物还可包含维生素C,DOT1L抑制剂,组蛋白去乙酰化酶抑制剂和/或DZNep,并且,还可包含EZH2抑制剂和/或cMet抑制剂。本发明的培养体系中的各个成分在下文中详细描述。
I.胸腺嘧啶类似物
本文中的胸腺嘧啶类似物是指与胸腺嘧啶的结构类似的化学化合物,其是5-溴脱氧尿嘧啶核苷(Brdu),Brdu的特点是胸腺嘧啶环上5位C连接的甲基被溴取代,在细胞增殖DNA合成时可以与内源性胸腺嘧啶核苷酸竞争掺入到新合成的DNA中。Brdu的分子量为307.1,化学结构如下:
在本文所述的用于化学诱导多能性干细胞生成的培养体系中,Brdu的浓度为0.01-10μM,或0.1-10μM,或1-10μM,或2-10μM,或3-10μM,或4-10μM,或5-10μM,或6-10μM,或7-10μM,或8-10μM,或9-10μM,优选地为3μM,5μM,8μM,或10μM。
II.cAMP激活剂
腺苷酸环化酶(cAMP)是膜整合蛋白,能够将ATP转变成cAMP,引起细胞的信号应答,是G蛋白偶联系统中的效应物。本文中的腺苷酸环化酶(cAMP)激活剂可以通过细胞激活腺苷酸环化酶,从而升高细胞内的cAMP水平。示例性的cAMP激活剂包括,但不限于:FSK,FSH,米力农(milrinone),西洛酰胺(cilostamide),咯利普兰(rolipram),dbcAMP,8-Br-cAMP,IBMX,PGE2,NKH477,sp-8-br-cAMPs。本发明中使用的cAMP激活剂优选为FSK。在本文所述的用于化学诱导多能性干细胞生成的培养体系中,FSK的浓度为0.01-10μM,或0.1-10μM,或1-10μM,或2-10μM,或3-10μM,或4-10μM,或5-10μM,或6-10μM,或7-10μM,或8-10μM,或9-10μM,优选地为3μM,5μM,8μM,或10μM。
III.TGF-β受体抑制剂
本文中的TGF-β受体抑制剂能够抑制TGF-β信号通路并激发细胞特定功能分化,TGF-β受体抑制剂的实例包括,但不限于:Repsox(E-616452),SB431542,A8301,GW788388,SD208,SB525334,LY364947,D4476,SB505124,GW6604,SU5416,CAT-152,CAT-192,SB431542,SD-208,SM16,NPC-30345,Ki26894,SB-203580,SD-093,Gleevec。在本发明的一种实施方式中,所述用于化学诱导多能性干细胞生成的培养体系中的TGF-β受体抑制剂优选为RepSox,RepSox的浓度为0.01-5μM,或0.1-5μM,或1-5μM,或2-5μM,或3-5μM,或4-5μM,优选地为1μM,3μM,5μM。
IV.骨形成蛋白(BMP)
BMP是低分子量(约30,000Da)、不含胶原的糖蛋白。成熟BMP分子是由一个依赖半胱氨酸二硫键固定的双链(每链含有400个氨基酸)多肽二聚体分子。BMP是以一个大的前体蛋白的形式合成的,包括信号肽部分、前结构域和羧基末端区,蛋白水解酶将羧基末端从前体蛋白切割下来后即形成二聚体。本文中的BMP4是一个关键的成骨细胞因子,参与异位骨化的早期病变。BMP4对I型胶原蛋白、碱性磷酸酶、骨钙素的表达有刺激作用。在本文所述的用于化学诱导多能性干细胞生成的培养体系中,BMP4的浓度为0.01-10ng/ml,或0.1-10ng/ml,或1-10ng/ml,或2-10ng/ml,或3-10ng/ml,或4-10ng/ml,或5-10ng/ml,或6-10ng/ml,或7-10ng/ml,或8-10ng/ml,或9-10ng/ml,优选地为1ng/ml,3ng/ml,5ng/ml,8ng/ml,或10ng/ml。
V.RA受体激活剂
维甲酸类化合物(Retinoicacids,RA)在调控细胞生长、分化、凋亡等生命活动中起重要作用。其在体内的生理活性代谢产物包括全反式维甲酸(ATRA)、13-顺维甲酸(13-cis-RA)和9-顺维甲酸(9-cis-RA),它们可通过核维甲酸受体(RA受体)结合于DNA应答元件调节靶基因的转录。示例性的RA受体激活剂包括,但不限于:TTNPB,Ch55,Retinol,AM580,ATRA,13-cis-RA,Retinoic。本文所述的用于化学诱导多能性干细胞生成的培养体系中的RA受体激活剂优选为AM580,其浓度为0.01-0.05μM,或0.02-0.05μM,或0.03-0.05μM,或0.04-0.05μM,优选地为0.01μM,0.03μM,或0.05μM。
VI.GSK3抑制剂
糖原合成酶激酶-3(GSK-3)普遍存在于哺乳动物真核细胞中,除去最早发现的调控糖原合成酶(GS)的活性外,GSK-3还能作用于众多信号蛋白、结构蛋白和转录因子,调节细胞的分化、增殖、存活和凋亡。GSK3抑制剂可以活化,例如,Wnt/β-catenin途径。许多β-catenin下游基因共调节多能性基因网。例如,GSK抑制剂活化cMyc表达以及增强其蛋白稳定性和转录活性。GSK3抑制剂的实例包括,但不限于:CHIR99021,CHIR98014,TD114-2,BIO,Kenpaullone,TWS119,CBM1078,SB216763,3F8(TOCRIS),AR-A014418,FRATide,Indirubin-3’monoxime,L803,CT99021,CT20026,SB415286。本文所述的用于化学诱导多能性干细胞生成的培养体系本中使用的GSK3抑制剂优选为CHIR99021,其浓度为0.01-3μM,或0.1-3μM,或0.5-3μM,或1-3μM,或1.5-3μM,或2-3μM,或2.5-3μM,优选为0.5μM,1μM,2μM,或3μM。
VII.碱性成纤维细胞生长因子
碱性成纤维细胞生长因子是能促进中胚层和神经外胚层细胞分裂的多肽,具有强烈的血管生成作用。在体外,碱性成纤维细胞生长因子能刺激细胞增殖、迁移,诱导纤溶酶原激活物及胶原酶活性,其是与肝素具有高亲和力的细胞促分裂原。本文所述的用于化学诱导多能性干细胞生成的培养体系本中使用的碱性成纤维细胞生长因子的浓度为0.01-10ng/mL,或0.1-10ng/mL,或0.5-10ng/mL,或1-10ng/mL,或2-10ng/mL,或3-10ng/mL,或4-10ng/mL,或5-10ng/mL,或6-10ng/mL,或7-10ng/mL,或8-10ng/mL,或9-10ng/mL,优选地为1ng/ml,3ng/ml,5ng/mL,8ng/ml,或10ng/mL。
VIII.DOT1L抑制剂
由DOT1L基因编码的蛋白质是使组蛋白H3的赖氨酸-79甲基化的组蛋白甲基转移酶,其针对游离核心组蛋白是非活性的并且针对核小体表现出显著的组蛋白甲基转移酶活性。本发明中的DOT1L抑制剂是EPZ5676和SGC0946,本文所述的用于化学诱导多能性干细胞生成的培养体系本中使用的EPZ5676的浓度为0.01-5μM,或0.1-5μM,或0.5-5μM,或1-5μM,或2-5μM,或3-5μM,或4-5μM,优选为0.5μM,1μM,3μM,或5μM,SGC0946的浓度为0.01-5μM,或0.1-5μM,或0.5-5μM,或1-5μM,或2-5μM,或3-5μM,或4-5μM,优选为0.5μM,1μM,3μM,或5μM。
IX.组蛋白去乙酰化酶抑制剂
组蛋白去乙酰化酶抑制剂是一类化学化合物,具有干扰组蛋白去乙酰化酶的功能。组蛋白去乙酰化酶抑制剂通常可分为两大类:NAD+-依赖性酶和Zn2+依赖性酶。Zn2+依赖性蛋白酶包括HDACsI、II、IV亚族;NAD+-依赖性酶主要是HDAC III亚族。组蛋白去乙酰化酶抑制剂通过增加细胞内组蛋白的乙酰化程度,提高p21等基因的表达水平等途径,抑制肿瘤细胞的增殖,诱导细胞分化和(或)凋亡。本发明的用于化学诱导多能性干细胞生成的培养体系中使用的组蛋白去乙酰化酶抑制剂是属于HDAC亚族的丙戊酸(VPA),其浓度为0.01mM-0.5mM,或0.05-0.5mM,或0.1-0.5mM,或0.2-0.5mM,或0.3-0.5mM,或0.4-0.5mM,优选为0.1mM,0.3mM或0.5mM。
X.DZNep
3-Deazanep1anocin A(DZNep)是一种腺苷类似物,是竞争性S-adenosylhomocysteine hydrolase的抑制剂,其化学结构如下:
在本文所述的用于化学诱导多能性干细胞生成的培养体系中,DZNep的浓度为0.001-0.05μM,或0.005-0.05μM,或0.01-0.05μM,或0.02-0.05μM,或0.03-0.05μM,或0.04-0.05μM,优选地为0.01μM,0.03μM,或0.05μM。
XI.EZH2抑制剂
EZH2是细胞内的一种组蛋白甲基转移酶,它可促进细胞内组蛋白H3的第27个氨基酸上三甲基(H3K27me3)的甲基化,并被证明是一种原癌基因,与多种肿瘤的生长和转移相关。EZH2可通过提高细胞中H3K27me3甲基化水平,抑制抑癌基因表达,从而导致肿瘤发生。EZH2抑制剂是一种小分子化合物,其可选自但不限于:GSK503,GSK343,EPZ005687,EPZ6438,其分子结构如下所示:
在本文所述的用于化学诱导多能性干细胞生成的培养体系中使用的EZH2抑制剂优选为具有上述式IV结构的化合物EPZ6438,其浓度为0.01-10μM,或0.1-10μM,或1-10μM,或2-10μM,或3-10μM,或4-10μM,或5-10μM,或6-10μM,或7-10μM,或8-10μM,或9-10μM,优选地为3μM,5μM,8μM,或10μM。
XII.cMet抑制剂
受体酪氨酸激酶(cMet)肝细胞生长因子HGF受体。HGF/c-Met信号通路在肿瘤形成、生长和转移过程中被频繁激活,c-Met抑制剂是一种小分子化合物,其包括但不限于:Cabozantinib(一种有效的VEGFR2抑制剂,Capmatinib(一种新型的,ATP竞争性c-MET抑制剂)。在本文所述的用于化学诱导多能性干细胞生成的培养体系中使用的cMet抑制剂优选为Capmatinib,其结构式如下:
在本文所述的用于化学诱导多能性干细胞生成的培养体系中,Capmatinib的浓度为0.01-10μM,或0.1-10μM,或1-10μM,或2-10μM,或3-10μM,或4-10μM,或5-10μM,或6-10μM,或7-10μM,或8-10μM,或9-10μM,优选地为3μM,5μM,8μM,或10μM。
XIII.维生素C
维生素C的结构类似葡萄糖,是一种多羟基化合物,其分子中第2及第3位上两个相邻的烯醇式羟基极易解离而释出H+,故具有酸的性质,又称抗坏血酸。维生素C的结构式如下:
在本文所述的用于化学诱导多能性干细胞生成的培养体系中,维生素C的浓度为0.01-50μg/ml,或0.1-50μg/ml,或1-50μg/ml,或5-50μg/ml,或10-50μg/ml,或20-50μg/ml,或30-50μg/ml,或40-50μg/ml;优选地为5μg/ml,10μg/ml,20μg/ml,30μg/ml,40μg/ml,或50μg/ml。
在本发明的一种实施方式中,所述用于化学诱导多能性干细胞生成的培养体系包含诸如iCD1之类的基础培养基以及含有下列成分的促进化学重编程的组合物:
10ng/ml BMP4;
50μg/ml维生素C;
5μM EPZ5676;
10μM Brdu;
0.05μM AM580;
3μM CHIR99021;
10ng/ml FGF2;
10μM Forsklin;
5μM RepSox;
0.1mM丙戊酸;
0.05μM DZNep;
5μMSGC0946。
在本发明的另一实施方式中,所述用于化学诱导多能性干细胞生成的培养体系包含诸如iCD1之类的基础培养基以及含有下列成分的促进化学重编程的组合物:
10ng/ml BMP4;
50μg/ml维生素C;
5μMEPZ5676;
10μM Brdu;
0.05μM AM580;
3μM CHIR99021;
10ng/ml FGF2;
10μM Forsklin;
5μM RepSox;
0.1mM丙戊酸;
0.05μM DZNep;
5μM SGC0946;
5μM EPZ6438;
5μM Capmatinib。
采用该培养体系,无需使用血清并且无需进行细胞分盘,能够有效诱导体细胞重编程为多能性干细胞。由于无需分盘,使细胞培养操作得到了简化,降低了在分盘过程中所产生的细胞损失,并且,由于本发明的培养体系无需使用血清,这进一步简化了后续的多能性干细胞的收集和分子机理分析,更便于后续建立诱导多能干细胞无动物源性培养体系。
用于诱导体细胞进行化学重编程的方法
另一方面,本发明提供一种用于诱导体细胞进行化学重编程的方法,所述方法包括使体细胞在上文所述的培养体系中培养持续足以使体细胞重编程至多能状态的时间段。本发明的方法还包括在体细胞重编程至多能状态之后,将培养基更换为第二培养基并在该培养基中培养持续足以维持胚胎干细胞的原始状态的时间段。所述第二培养基为补充有下述成分的DMEM培养基,其中,所述成分包含:PD0325901;非必须氨基酸;GlutaMaxTM;人白细胞抗原B27;CHIR99021;白血病抑制因子和/或N2添加剂。
在一种实施方式中,所述第二培养基中包含浓度为1μM的PD0325901;浓度为培养基总体积的1%的非必须氨基酸;浓度为培养基总体积的1%的GlutaMaxTM;浓度为培养基总体积的2%的人白细胞抗原B27;浓度为3μ.M的CHIR99021;浓度为培养基总体积的1%的N2添加剂;浓度为1U-1000U的白血病抑制因子。
在本发明的一种实施方式中,用于诱导体细胞进行化学重编程的方法包括使体细胞在上文所述的培养体系中培养持续8天至22天,优选为22天,随后将培养基更换为上文所述的第二培养基并继续培养1天至18天,优选为18天。
试剂盒
再一方面,本发明提供用于化学诱导体细胞进行重编程的试剂盒,其包括上文所述的促进化学重编程的组合物和基础培养基,以及任选地,培养体系使用说明书、细胞培养所需的培养平板。该试剂盒还可包括从供体收集细胞样本或组织样本所需的工具、保存所述细胞样本或组织样本的培养瓶等等。所述试剂盒中的说明书可向使用者提供关于培养体系的使用及其相关信息。该说明书可为任何合适的形式,包括,但不限于,印刷物、录像带、电脑可读盘或光盘。
实施例
本实施例以小鼠胚胎成纤维细胞、小鼠新生成纤维细胞、小鼠肺成纤维细胞,小鼠尾尖成纤维细胞和小鼠神经干细胞以及小鼠肝细胞为例来说明本发明的用于化学诱导多能性干细胞生成的培养体系。本实施例中使用的小鼠胚胎成纤维细胞来源于OG2小鼠E13.5胚胎;小鼠新生成纤维细胞来源于OG2小鼠出生后第1-3天真皮层;小鼠尾尖成纤维细胞来源于6至8周OG2小鼠尾部结缔组织;小鼠肺成纤维细胞来源于6至8周OG2小鼠肺部组织,这些成纤维细胞均保持在含有10%FBS,GlutMax(100x)和NEAA(100x)的DMEM培养基中培养。本实施例中使用的小鼠神经干细胞来源于OG2小鼠E13.5胚胎大脑组织,保持在含有1%N2,2%B27,bFGF(10ng/ml),EGF(10ng/ml),GlutMax(100x)和NEAA(100x)的DMEM/F12培养基中培养。作为对照使用的小鼠干细胞来源于6至8周OG2小鼠肝组织,保持在HCM培养基中培养。
实施例1.在用于化学诱导多能性干细胞的生成的培养体系中生成多能性干细胞
将上述小鼠胚胎成纤维细胞,小鼠新生成纤维细胞,小鼠肺成纤维细胞,小鼠尾尖成纤维细胞和小鼠神经干细胞以20,000个细胞/孔(12个孔)的密度或50,000个细胞/孔(6个孔)的密度,小鼠肝细胞以500,000万/孔(6孔板)接种于如下用于化学诱导生成多能性干细胞的化学重编程培养基。该培养基包含作为基础培养基的iCD1培养基和10ng/ml BMP4,10μM Brdu,5μM RepSox,10μM Forsklin,0.1mM VPA,0.05μM AM580,5μM EPZ5676,0.05μMDZNep,5μMSGC0946,50μg/ml维生素C,3μM CHIR99021和10ng/ml碱性成纤维细胞生长因子。每天更换培养基,培养22天之后,将上述化学重编程培养基更换为下述含有N2(100X),B27(50X),GlutMax(100X),NEAA(100X),3μM的CHIR99021,1μM的PD0325901以及LIF(1000U)的DMEM培养基,并继续培养持续14天至18天(如图1所示)。随后收集化学诱导的多能性干细胞(ciPSC)进行分析。
实施例2.化学诱导的多能性干细胞的表征
通过定量RT-PCR技术监测ciPSC中的内源性多能性基因Oct4,Nanog,Sox2,Esrb,Rex1Dappa5,Sall4和Cdh1的表达水平。如图2所示,这些内源性多能性基因的表达水平与在小鼠胚胎干细胞中所检测到的内源性多能性基因Oct4,Nanog,Sox2,Esrb,Rex1Dappa5,Sall4和Cdh1的表达水平相同。
通过RNA-seq分析来检测ciPSC的转录组学特性。如图3所示,ciPSC的转录组学特性与小鼠胚胎干细胞相同。
进一步地,如图4所示,通过免疫荧光确认了多能性基因Oct4,Nanog,Sox2,Esrb和Rex1的在化学诱导的多能性干细胞中的蛋白质表达水平。
实施例3.化学诱导的多能性干细胞的小鼠体内分化特性
将实施例1得到的ciPSC皮下注射至NOD-SCID小鼠体内。如图5所示,ciPSC在小鼠体内形成畸胎瘤并分化形成三个胚层的细胞(软骨:中胚层;肌肉:中胚层;神经:外胚层;消化道样上皮:内胚层)。如图6所示,ciPSC在传代过程中保持核型正常。将ciPSC注入小鼠囊胚中,如图7所示,子代小鼠为嵌合小鼠。
通过上述实施例2和实施例3的表征结果可以看出,根据本发明的方法,在本发明的培养体系中培养得到的化学诱导的多能性干细胞具有完全多能性。由此可见,本发明的培养体系在无需采用血清并且无需进行细胞分盘的条件下,通过两阶段培养能够有效地将体细胞重编程而生成多能性干细胞。并且,本发明的方法无需对细胞进行分盘使得培养操作得到简化,培养体系无需采用血清,使得后续的多能性细胞的收集和分子机理分析得到简化,更加便于后续建立诱导多能干细胞无动物源性培养体系。
检测方法
定量RT-PCR分析和RNA-seq分析:采用TRIzol从细胞中分离总RNA并采用ReverTraAce(Toyobo)和寡-dT(Takara)将总RNA转化为cDNA,随后采用Premix Ex Taq(Takara)通过qPCR进行分析。TruSeq RNA样本试剂盒(RS-122-2011,Illumina)用于文库构建。对于RNA-seq而言,采用Miseq Reagent试剂盒(MS-102-2011,Illumina)完成测序。测序过程中使用的引物列于下表1。
免疫荧光染色:在培养皿中培养细胞并采用4%多聚甲醛在室温下固定30分钟。随后,用PBS洗涤细胞三次并用0.1%Triton X-100进行透化持续30分钟。随后,采用3%BSA的PBS溶液在室温下封闭1小时,在4℃下,用一抗孵育过夜。第二天,用PBS洗涤细胞三次并用合适的二抗孵育1小时。采用DAPI对细胞核进行染色。在共聚焦显微镜(Zeiss 710 NLO)下进行观察。一抗和二抗均在3%的BSA中稀释,其为抗-Oct4(SC-5279,1∶400),抗-Sox2(sc-17320,1∶200),抗-Nanog(BETHYL no.A300-397A,1∶200),抗-Rexl(SC-50668,1∶100)和抗-SSEA1(RA,MAB2155,1∶100)。
畸胎瘤形成和嵌合小鼠的产生:将1x106个化学诱导的多能性干细胞皮下注射至NOD SCID小鼠体内,在4至8周形成畸胎瘤。对于嵌合小鼠的产生而言,将化学诱导的多能性干细胞注射至ICR囊胚中并移植至假孕ICR雌性小鼠体内,随后得到的嵌合小鼠通过使F2小鼠与ICR小鼠交配而用于种系传递。
表1.测序过程中使用的引物序列
虽然本文描述并显示了本发明的优选的实施方式,但是对于本领域技术人员而言显而易见的是,这些实施方式仅仅是举例说明。本领域技术人员可对这些实施方式作出多种改变、修改和替换,而不背离本发明。通过所附的权利要求来限定本发明的范围,并且这些权利要求范围内的方法和结构及其等同物也被所附的权利要求涵盖。
序列表
<110> 中国科学院广州生物医药与健康研究院
<120> 一种用于化学诱导多能性干细胞生成的培养体系以及使用该培养体系的化学重编程方法
<160> 36
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213> Oct4启动子正义序列(Oct4)
<400> 1
cattgagaac cgtgtgag 18
<210> 2
<211> 18
<212> DNA
<213> Oct4启动子反义序列(Oct4)
<400> 2
tgagtgatct gctgtagg 18
<210> 3
<211> 20
<212> DNA
<213> Nanog启动子正义序列(Nanog)
<400> 3
ctcaagtcct gaggctgaca 20
<210> 4
<211> 20
<212> DNA
<213> Nanog启动子反义序列(Nanog)
<400> 4
tgaaacctgt ccttgagtgc 20
<210> 5
<211> 20
<212> DNA
<213> Sox2启动子正义序列(Sox2)
<400> 5
agggctggga gaaagaagag 20
<210> 6
<211> 20
<212> DNA
<213> Sox2启动子反义序列(Sox2)
<400> 6
ccgcgattgt tgtgattagt 20
<210> 7
<211> 20
<212> DNA
<213> Esrrb启动子正义序列(Esrrb)
<400> 7
tttctggaac ccatggagag 20
<210> 8
<211> 20
<212> DNA
<213> Esrrb启动子反义序列(Esrrb)
<400> 8
agccagcacc tccttctaca 20
<210> 9
<211> 18
<212> DNA
<213> Dppa5启动子正义序列(Dppa5)
<400> 9
ccgtgcgtgg tggataag 18
<210> 10
<211> 19
<212> DNA
<213> Dppa5启动子反义序列(Dppa5)
<400> 10
gcgactggac ctggaatac 19
<210> 11
<211> 20
<212> DNA
<213> Rex1启动子正义序列(Rex1)
<400> 11
cagccagacc accatctgtc 20
<210> 12
<211> 23
<212> DNA
<213> Rex1启动子反义序列(Rex1)
<400> 12
gtctccgatt tgcatatctc ctg 23
<210> 13
<211> 19
<212> DNA
<213> Sall4启动子正义序列(Sall4)
<400> 13
ctaaggagga agaggagag 19
<210> 14
<211> 18
<212> DNA
<213> Sall4启动子反义序列(Sall4)
<400> 14
caaggctatg gtcacaag 18
<210> 15
<211> 20
<212> DNA
<213> Sox17启动子正义序列(Sox17)
<400> 15
cagtatctgc cctttgtgta 20
<210> 16
<211> 19
<212> DNA
<213> Sox17启动子反义序列(Sox17)
<400> 16
gcaatagtag accgctgag 19
<210> 17
<211> 19
<212> DNA
<213> Foxa2启动子正义序列(Foxa2)
<400> 17
gcagacactt cctactacc 19
<210> 18
<211> 18
<212> DNA
<213> Foxa2启动子反义序列(Foxa2)
<400> 18
tccactcagc ctctcatt 18
<210> 19
<211> 18
<212> DNA
<213> Gata4启动子正义序列(Gata4)
<400> 19
cagcagcagt gaagagat 18
<210> 20
<211> 19
<212> DNA
<213> Gata4启动子反义序列(Gata4)
<400> 20
gtctgagtga caggagatg 19
<210> 21
<211> 23
<212> DNA
<213> Gata6启动子正义序列(Gata6)
<400> 21
ggtctctaca gcaagatgaa tgg 23
<210> 22
<211> 19
<212> DNA
<213> Gata6启动子反义序列(Gata6)
<400> 22
tggcacagga cagtccaag 19
<210> 23
<211> 21
<212> DNA
<213> Cdh1启动子正义序列(Cdh1)
<400> 23
cagccttctt ttcggaagac t 21
<210> 24
<211> 22
<212> DNA
<213> Cdh1启动子反义序列(Cdh1)
<400> 24
ggtagacagc tccctatgac tg 22
<210> 25
<211> 20
<212> DNA
<213> Epcam启动子正义序列(Epcam)
<400> 25
cttgtgtctg cacgacctgt 20
<210> 26
<211> 22
<212> DNA
<213> Epcam启动子反义序列(Epcam)
<400> 26
ccaagcattt agacgccagt tt 22
<210> 27
<211> 19
<212> DNA
<213> Cdh2启动子正义序列(Cdh2)
<400> 27
ccatcatcgc tatccttct 19
<210> 28
<211> 20
<212> DNA
<213> Cdh2启动子反义序列(Cdh2)
<400> 28
cctccacctt cttcatcata 20
<210> 29
<211> 23
<212> DNA
<213> CD44启动子正义序列(CD44)
<400> 29
cgttaatgtt gatggctcct tac 23
<210> 30
<211> 18
<212> DNA
<213> CD44启动子反义序列(CD44)
<400> 30
gtcctggttc gcacttga 18
<210> 31
<211> 20
<212> DNA
<213> Twist2启动子正义序列(Twist2)
<400> 31
agatgaccag ctgcagctac 20
<210> 32
<211> 18
<212> DNA
<213> Twist2启动子反义序列(Twist2)
<400> 32
atgtgcaggt gggtcctg 18
<210> 33
<211> 20
<212> DNA
<213> Snail启动子正义序列(Snail)
<400> 33
ctcggatgtg aagagatacc 20
<210> 34
<211> 18
<212> DNA
<213> Snail启动子反义序列(Snail)
<400> 34
agactcttgg tgcttgtg 18
<210> 35
<211> 25
<212> DNA
<213> Gapdh启动子正义序列(Gapdh)
<400> 35
aactttggca ttgtggaagg gctca 25
<210> 36
<211> 25
<212> DNA
<213> Gapdh启动子反义序列(Gapdh)
<400> 36
ttggcagcac cagtggatgc aggga 25
Claims (19)
1.用于化学诱导多能性干细胞生成的培养体系,其包括基础培养基和促进化学重编程的组合物,其中,所述促进化学重编程的组合物包含胸腺嘧啶类似物,cAMP激活剂,TGF-β受体抑制剂,骨形成蛋白,RA受体激活剂,GSK3抑制剂和碱性成纤维细胞生长因子,并且,其中,所述培养体系不包含血清。
2.如权利要求1所述的培养体系,其中,
所述胸腺嘧啶类似物的浓度为0.01-10μM;
所述cAMP激活剂的浓度为0.01-10μM;
所述TGF-β受体抑制剂的浓度为0.01-5μM;
所述骨形成蛋白的浓度为0.01-10ng/ml;
所述碱性成纤维细胞生长因子的浓度为0.01-10ng/ml;
所述RA受体激活剂的浓度为0.01-0.05μM;和/或
所述GSK3抑制剂的浓度为0.01-3μM。
3.如权利要求2所述的培养体系,其中,
所述胸腺嘧啶类似物是Brdu;
所述cAMP激活剂是FSK;
所述TGF-β受体抑制剂是RepSox;
所述骨形成蛋白是BMP4;
所述碱性成纤维细胞生长因子是FGF2;
所述RA受体激活剂是AM580;和/或
所述GSK3抑制剂是CHIR99021。
4.如权利要求1至3中任一项所述的培养体系,其中,所述促进化学编程的组合物还包含维生素C,DOT1L抑制剂,组蛋白去乙酰化酶抑制剂和/或DZNep。
5.如权利要求4所述的培养体系,其中,
所述维生素C的浓度为0.01-50μg/ml;
所述DOT1L抑制剂的浓度为0.01-5μM;
所述组蛋白去乙酰化酶抑制剂的浓度为0.01-0.5mM;和/或
所述DZNep的浓度为0.001-0.05μM。
6.如权利要求5所述的培养体系,其中,
所述DOT1L抑制剂是EPZ5676和/或SGC0946;
所述组蛋白去乙酰化酶抑制剂是丙戊酸。
7.如权利要求1至3中任一项所述的培养体系,其中,所述促进化学重编程的组合物还包含EZH2抑制剂和/或cMet抑制剂。
8.如权利要求7所述的培养体系,其中,所述EZH2抑制剂的浓度为0.01μM-10μM,和/或所述cMet抑制剂的浓度为0.01μM-10μM。
9.如权利要求8所述的培养体系,其中,所述EZH2抑制剂为EPZ6438,所述cMet抑制剂为Capmatinib。
10.如权利要求6所述的培养体系,其中,所述促进化学重编程的组合物包含:
10ng/ml BMP4;
50μg/ml维生素C;
5μM EPZ5676;
10μM Brdu;
0.05μM AM580;
3μM CHIR99021;
10ng/ml FGF2;
10μM Forsklin;
5μM RepSox;
0.1mM丙戊酸;
0.05μM DZNep;和/或
5μM SGC0946。
11.如权利要求9所述的培养体系,其中,所述促进化学重编程的组合物包含:
10ng/ml BMP4;
50μg/ml维生素C;
5μM EPZ5676;
10μM Brdu;
0.05μM AM580;
3μM CHIR99021;
10ng/ml FGF2;
10μM Forsklin;
5μM RepSox;
0.1mM丙戊酸;
0.05μM DZNep;
5μM SGC0946;
5μM EPZ6438;和/或
5μM Capmatinib。
12.如权利要求1至11中任一项所述的培养体系,其中,所述基础培养基是iCD1培养基。
13.用于诱导体细胞进行化学重编程的方法,所述方法包括使体细胞在权利要求1至12中任一项所述的培养体系中培养持续足以使体细胞重编程至多能状态的时间段。
14.如权利要求13所述的方法,所述方法还包括在体细胞重编程至多能状态之后,将培养基更换为补充有下述成分的DMEM培养基并在该培养基中培养持续足以维持胚胎干细胞的原始状态的时间段,其中,所述成分包含:PD0325901;非必须氨基酸;GlutaMaxTM;人白细胞抗原B27;CHIR99021;白血病抑制因子和/或N2添加剂。
15.如权利要求14所述的方法,其中,PD0325901的浓度为1μM;非必须氨基酸的浓度为培养基总体积的1%;GlutaMaxTM的浓度为培养基总体积的1%;人白细胞抗原B27的浓度为培养基总体积的2%;CHIR99021的浓度为3μM;N2添加剂的浓度为培养基总体积的1%;和/或白血病抑制因子的浓度为1U-1000U。
16.如权利要求13所述的方法,其中,所述足以使体细胞重编程至多能状态的时间段为8天至22天。
17.如权利要求14所述的方法,其中,所述足以维持胚胎干细胞的原始状态的时间段为1天至18天。
18.如权利要求9至13中任一项所述的方法,其中,所述体细胞选自:成纤维细胞、骨髓衍生的单核细胞、骨骼肌细胞、脂肪细胞、外周血单核细胞、巨噬细胞、肝细胞、角质细胞、口腔角质细胞、头发毛囊真皮细胞、胃上皮细胞、肺上皮细胞、滑膜细胞、肾细胞、皮肤上皮细胞、成骨细胞、神经干细胞和真皮细胞。
19.用于化学诱导多能性干细胞生成的试剂盒,其中包括权利要求1-12中任一项所述的培养体系。
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CN201810170553.XA CN110218696A (zh) | 2018-03-01 | 2018-03-01 | 一种用于化学诱导多能性干细胞生成的培养体系以及使用该培养体系的化学重编程方法 |
PCT/CN2019/076433 WO2019165988A1 (zh) | 2018-03-01 | 2019-02-28 | 一种用于化学诱导多能性干细胞生成的培养体系以及使用该培养体系的化学重编程方法 |
JP2020545485A JP2021514647A (ja) | 2018-03-01 | 2019-02-28 | 多能性幹細胞の生成を化学的に誘導するための培養系、および該培養系を用いる化学的リプログラミング方法 |
US16/977,403 US20210047624A1 (en) | 2018-03-01 | 2019-02-28 | Culture system for chemically inducing generation of pluripotent stem cells and chemical reprogramming method using same |
EP19760781.5A EP3760709A4 (en) | 2018-03-01 | 2019-02-28 | CULTURAL SYSTEM FOR CHEMICAL INDUCTION OF GENERATION OF PLURIPOTENT STEM CELLS AND METHOD OF CHEMICAL REPROGRAMMING USING THEM |
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CN110423721A (zh) * | 2018-05-01 | 2019-11-08 | 云南济慈再生医学研究院有限公司 | 一种年轻化的修复型成纤维细胞的制备方法及其应用 |
CN111304156A (zh) * | 2020-02-20 | 2020-06-19 | 南方医科大学南方医院 | 一种诱导型滋养层干细胞及其制备方法和应用 |
CN112481192A (zh) * | 2019-09-12 | 2021-03-12 | 海门雨霖细胞科技有限责任公司 | 体内外化学诱导成纤维细胞直接重编程为肝细胞的化学小分子组合物及方法 |
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CN114350608A (zh) * | 2022-01-27 | 2022-04-15 | 昭泰英基生物医药(香港)有限公司 | 一种诱导t细胞重编程为类nk细胞的组合物及其应用 |
CN115772505A (zh) * | 2023-02-13 | 2023-03-10 | 淇嘉科技(天津)有限公司 | 促进体细胞重编程为诱导多能干细胞的培养基及方法 |
WO2023217128A1 (zh) * | 2022-05-10 | 2023-11-16 | 上海赛立维生物科技有限公司 | 胃粘膜上皮前体样细胞及其制备方法和应用 |
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CN111304156A (zh) * | 2020-02-20 | 2020-06-19 | 南方医科大学南方医院 | 一种诱导型滋养层干细胞及其制备方法和应用 |
WO2022042527A1 (zh) * | 2020-08-24 | 2022-03-03 | 北京大学 | 治疗内耳及肠道上皮组织损伤相关疾病的小分子药物 |
CN114350608A (zh) * | 2022-01-27 | 2022-04-15 | 昭泰英基生物医药(香港)有限公司 | 一种诱导t细胞重编程为类nk细胞的组合物及其应用 |
CN114350608B (zh) * | 2022-01-27 | 2024-05-28 | 昭泰英基生物医药(香港)有限公司 | 一种诱导t细胞重编程为类nk细胞的组合物及其应用 |
WO2023217128A1 (zh) * | 2022-05-10 | 2023-11-16 | 上海赛立维生物科技有限公司 | 胃粘膜上皮前体样细胞及其制备方法和应用 |
CN115772505A (zh) * | 2023-02-13 | 2023-03-10 | 淇嘉科技(天津)有限公司 | 促进体细胞重编程为诱导多能干细胞的培养基及方法 |
CN117645970A (zh) * | 2023-11-17 | 2024-03-05 | 广州百康细胞生命科技有限公司 | 一种使用化学重编辑方法高效快速获得人多能干细胞 |
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