CN111808812B - 一种用于多能干细胞向造血干细胞分化的材料和方法 - Google Patents
一种用于多能干细胞向造血干细胞分化的材料和方法 Download PDFInfo
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Abstract
本发明公开了一种用于多能干细胞向造血干细胞分化的材料和方法,特别是诱导多能干细胞向CD34+CD43+CD45+造血干细胞的定向分化的培养基及方法。利用上述材料(例如添加剂Ⅰ、添加剂Ⅱ和添加剂Ⅲ,或,培养基Ⅰ、培养基Ⅱ和培养基Ⅲ)和方法,可在常氧条件下实现诱导多能干细胞的定向分化,高效获得CD34+造血干细胞。本发明的材料化学成分确定,成本低,安全性高,质量稳定;本发明的方法具有耗时短、分化效率高等优点,可为组织工程、药物研发和细胞治疗等领域提供大量细胞来源,具有较佳的应用前景和商业价值。
Description
技术领域
本发明涉及生物医药技术领域,具体涉及一种用于多能干细胞向造血干细胞分化的材料和方法,特别是诱导多能干细胞向CD34+CD43+CD45+造血干细胞的定向分化的培养基及方法。
背景技术
近年来,造血干细胞(hematopoietic stem cell,HSC)越来越多地被应用到临床上来治疗血液系统恶性肿瘤、实体瘤、骨髓衰竭性疾病以及一些先天性疾病等,同时在儿科的血液病、遗传病、肿瘤的治疗中也表现出一定的应用发展前景。随着包括脐血和同源供体在内的干细胞移植替代来源的发展及新调理疗法的运用,极大地提高和改善了病人的预后。来源于脐血的造血干细胞因含量丰富且扩增能力强、免疫原性低、采集方便和对供体无害等优势,在多种血液系统疾病的临床治疗中具有广阔应用前景。但是目前仅通过供体无偿献血方式所提供的脐血的供应量远远满足不了临床大量需求,因单份脐带血体积小,难以满足成年患者的治疗需要,严重制约了脐带血造血干细胞的临床应用。加之临床用血存在着HIV、HCV等病原体的污染风险,以及受移植失败、移植物抗宿主病(GVHD)和造血构建延迟等不利因素的影响,导致50%以上的患者终生残疾或无法治愈。所以,即使用造血干细胞治疗,也面临着一个比较严峻的问题:如何能有足够量的造血干细胞用于移植。
诱导多能干细胞是获得造血干细胞的重要来源之一。诱导多能干细胞(iPS)是一类通过基因转染等细胞重编程技术人工诱导获得的多能干细胞,其首先由日本科学家Yamanaka在2006年运用逆转录病毒转导的方法,将Sox2、Oct3/4、c-Myc和Klf4四个转录因子转入小鼠胚胎成纤维(Mouse Embryonic Fibrobalst)细胞中,从而诱导出的重编程为一类具有类似于胚胎干细胞(ES)多能性分化潜力的干细胞。利用人类诱导多能干细胞能够在体外定向诱导分化成多种来自各个胚层的细胞,造血干细胞便是其中一种。通过使用人类诱导多能干细胞定向分化出大量的造血干细胞为临床提供安全、有效的细胞来源,成为治疗相关血液疾病有希望的新方法之一。
人类干细胞在Xeno-free的系统下分化成CD34+造血干细胞有重大的研究和临床应用价值。Uenishi, Gene, et al. "Tenascin C promotes hematoendothelialdevelopment and T lymphoid commitment from human pluripotent stem cells inchemically defined conditions." Stem cell reports 3.6 (2014): 1073-1084中公开了利用基础培养基IF9S结合10余种小分子在低氧条件下诱导人类干细胞分化成CD34+造血干细胞。但该分化方法过程较复杂,细节较多,人为误差太大,重复性差,诱导效率得不到保证,且分化培养需在低氧条件下进行,初期分化细胞死亡严重。
发明内容
本发明提供一种用于添加到干细胞培养基中起诱导分化作用的添加剂Ⅰ,其包含:BMP4、FGF2和VEGF,其中BMP4、FGF2和VEGF的质量比为1:0.1-10(如0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1、2、3、4、5、6、7、8、9、10):0.1-10(如0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1、2、3、4、5、6、7、8、9、10)。
具体地,添加剂Ⅰ中,BMP4、FGF2和VEGF的质量比可以为1:0.5-1:0.5-2;在本发明的一个实施例中,该比例为1:0.625:1。
本发明还提供一种添加剂Ⅱ,其包含:SCF和VEGF,其中SCF和VEGF的质量比为1:0.1-10(如0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1、2、3、4、5、6、7、8、9、10)。
具体地,添加剂Ⅱ中,SCF和VEGF的质量比可以为1:0.5-2;在本发明的一个实施例中,该比例为1:1。
本发明还提供一种添加剂Ⅲ,其包含:SCF、FLT3L和TPO,其中SCF、FLT3L和TPO的质量比为1:0.1-10(如0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1、2、3、4、5、6、7、8、9、10):0.1-10(如0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1、2、3、4、5、6、7、8、9、10)。
具体地,添加剂Ⅲ中,SCF、FLT3L和TPO的质量比可以为1:0.5-2:0.5-2;在本发明的一个实施例中,该比例为1:1:1。
本发明提供一种添加剂组合产品,其包含上述添加剂Ⅰ、添加剂Ⅱ、添加剂Ⅲ中的一种或多种(当该产品包含两种或三种添加剂时,各添加剂是分离状态的,而非混合的)。
在本发明的一个实施方式中,上述产品包含添加剂Ⅰ、添加剂Ⅱ和添加剂Ⅲ。
具体地,上述产品还包含干细胞培养基,如IF9S培养基,其与各添加剂是分离状态的,可在用于干细胞培养前临时混合。
在本发明的一个实施方式中,上述产品为试剂盒。
本发明还提供一种培养基Ⅰ,其包含补充有BMP4、FGF2和VEGF的干细胞培养基Ⅰ。
具体地,培养基Ⅰ中,BMP4的终浓度为30-100ng/ml(具体如30、35、40、45、50、55、60、70、75、76、77、78、79、80、81、82、83、84、85、90、95、100ng/ml),特别是50-100ng/ml。
具体地,培养基Ⅰ中,FGF2的终浓度为30-100ng/ml(具体如30、40、45、46、47、48、49、50、51、52、53、54、55、60、65、70、75、80、90、100ng/ml),特别是30-70ng/ml。
具体地,培养基Ⅰ中,VEGF的终浓度为30-100ng/ml(具体如30、35、40、45、50、55、60、70、75、76、77、78、79、80、81、82、83、84、85、90、95、100ng/ml),特别是50-100ng/ml。
在本发明的一个实施例中,培养基Ⅰ中的干细胞培养基Ⅰ为IF9S培养基。
在本发明的一个实施例中,培养基Ⅰ为补充有BMP4、FGF2和VEGF的IF9S培养基,其中BMP4、FGF2和VEGF的终浓度分别为80ng/ml、50ng/ml、80ng/ml。
本发明还提供一种培养基Ⅱ,其包含补充有SCF和VEGF的干细胞培养基Ⅱ。
具体地,培养基Ⅱ中,SCF的终浓度为30-100ng/ml(具体如30、35、40、45、50、55、60、70、75、76、77、78、79、80、81、82、83、84、85、90、95、100ng/ml),特别是50-100ng/ml。
具体地,培养基Ⅱ中,VEGF的终浓度为30-100ng/ml(具体如30、35、40、45、50、55、60、70、75、76、77、78、79、80、81、82、83、84、85、90、95、100ng/ml),特别是50-100ng/ml。
在本发明的一个实施例中,培养基Ⅱ中的干细胞培养基Ⅱ为IF9S培养基。
在本发明的一个实施例中,培养基Ⅱ为补充有SCF和VEGF的IF9S培养基,其中SCF和VEGF的终浓度分别为80ng/ml、80ng/ml。
本发明还提供一种培养基Ⅲ,其包含补充有SCF、FLT3L和TPO的干细胞培养基Ⅲ。
具体地,培养基Ⅲ中,SCF的终浓度为30-100ng/ml(具体如30、40、45、46、47、48、49、50、51、52、53、54、55、60、65、70、75、80、90、100ng/ml),特别是30-70ng/ml。
具体地,培养基Ⅲ中,FLT3L的终浓度为30-100ng/ml(具体如30、40、45、46、47、48、49、50、51、52、53、54、55、60、65、70、75、80、90、100ng/ml),特别是30-70ng/ml。
具体地,培养基Ⅲ中,TPO的终浓度为30-100ng/ml(具体如30、40、45、46、47、48、49、50、51、52、53、54、55、60、65、70、75、80、90、100ng/ml),特别是30-70ng/ml。
在本发明的一个实施例中,培养基Ⅲ中的干细胞培养基Ⅲ为IF9S培养基。
在本发明的一个实施例中,培养基Ⅲ为补充有SCF、FLT3L和TPO的IF9S培养基,其中SCF、FLT3L和TPO的终浓度分别为50ng/ml、50ng/ml、50ng/ml。
本发明还提供一种培养基组合产品,其包含本发明上述培养基Ⅰ、培养基Ⅱ、培养基Ⅲ中的一种或多种(当该产品包含两种或三种培养基时,各培养基是分离状态的,而非混合的)。
在本发明的一个实施例中,上述产品包含培养基Ⅰ、培养基Ⅱ和培养基Ⅲ。
在本发明的一个实施方式中,上述产品为试剂盒。
本发明中所涉及干细胞培养基为现有技术已知的任何合适的干细胞培养基,其可采用市售可得商品,也可根据现有技术已知制备方法进行制备。例如IF9S培养基,其可参见“Uenishi, Gene, et al. "Tenascin C promotes hematoendothelial development andT lymphoid commitment from human pluripotent stem cells in chemically definedconditions." Stem cell reports 3.6 (2014): 1073-1084”中表S1所示组成进行制备。
本发明还提供一种用于多能干细胞分化、制备造血干细胞的方法,其包括如下步骤:
(1)将多能干细胞接种于本发明上述培养基Ⅰ中,进行培养;
(2)将步骤(1)的培养体系中的培养基更换为本发明上述培养基Ⅱ,进行培养;
(3)将步骤(2)的培养体系中的培养基更换为本发明上述培养基Ⅲ,进行培养。
具体地,上述方法还包括步骤(4):向将步骤(3)的培养体系中添加本发明上述培养基Ⅲ,进行培养。
具体地,步骤(1)中接种密度为5×103-2×104cell/ml。
具体地,上述方法的步骤均在正常氧气浓度下的气体环境下进行。
具体地,上述气体环境中包括95%空气、5%二氧化碳。
具体地,上述方法的步骤均在37℃的温度下进行。
具体地,上述步骤(1)-(4)中培养时间各自独立地为2-3天。
具体地,步骤(1)中的多能干细胞可以为诱导多能干细胞、间充质干细胞、胚胎干细胞等;在本发明的一个实施例中,步骤(1)中的多能干细胞为诱导多能干细胞。
具体地,步骤(1)中的多能干细胞为人类多能干细胞。
具体地,步骤(1)中的多能干细胞为单细胞或小细胞团。
在本发明的一个实施方式中,上述方法包括如下步骤:
(1)第0天,将多能干细胞接种于本发明上述培养基Ⅰ中,进行培养;
(2)第3天,将步骤(1)的培养体系中的培养基更换为本发明上述培养基Ⅱ,进行培养;
(3)第6天,将步骤(2)的培养体系中的培养基更换为本发明上述培养基Ⅲ,进行培养;
(4)第9天,向将步骤(3)的培养体系中添加本发明上述培养基Ⅲ,培养2-3天。
具体地,上述方法还可在步骤(1)前还包括预处理的步骤。
具体地,上述预处理步骤包括将多能干细胞系消化为单细胞或小细胞团,然后终止消化的步骤。
具体地,上述预处理步骤还包括将多能干细胞计数的步骤。
在本发明的一个实施方式中,上述预处理步骤包括:利用EDTA消化液消化iPSC细胞系为单细胞或者小细胞团,然后用E8培养液终止消化,离心去上清,利用E8培养基重悬计数。
本发明还提供一种上述方法得到的造血干细胞。
具体地,上述造血干细胞为CD34+造血干细胞。
具体地,上述造血干细胞为CD34+CD43+CD45+造血干细胞。
本发明还提供一种药物组合物,其包含本发明上述造血干细胞,以及药学上可接受的辅料。
本发明还提供上述添加剂Ⅰ、添加剂Ⅱ、添加剂Ⅲ,包含其的添加剂组合产品(如试剂盒)在多能干细胞的定向分化、造血干细胞的制备中的应用。
本发明还提供上述培养基Ⅰ、培养基Ⅱ、培养基Ⅲ,包含其的培养基组合产品(如试剂盒)在多能干细胞的定向分化、造血干细胞的制备中的应用。
具体地,上述应用中,多能干细胞为诱导多能干细胞,特别是人类诱导多功能干细胞。
具体地,上述应用中,造血干细胞为CD34+造血干细胞,特别是CD34+CD43+CD45+造血干细胞。
本发明还提供一种上述造血干细胞、药物组合物在制备治疗疾病或病症的药物中的应用。
本发明还提供一种治疗有此需要的对象中的疾病或病症的方法,其包含向对象施用有效量的本发明上述造血干细胞、药物组合物的步骤。
在本发明的一个实施方式中,上述疾病或病症为血液系统疾病。
具体地,上述血液系统疾病可以为:血液系统恶性肿瘤(如白血病、淋巴瘤、多发性骨髓瘤、骨髓增生异常综合征等)、血液系统非恶性肿瘤疾病(如再生障碍性贫血、范可尼贫血、地中海贫血、镰状细胞贫血、骨髓纤维化、重型阵发性睡眠性血红蛋白尿症、无巨核细胞性血小板减少症、放射性疾病等)。
在本发明的一个实施方式中,上述疾病或病症为其它实体瘤,如乳腺癌、卵巢癌、睾丸癌、神经母细胞瘤、小细胞肺癌等。
在本发明的一个实施方式中,上述疾病或病症为免疫系统疾病,如重症联合免疫缺陷症、严重自身免疫性疾病。
在本发明的一个实施方式中,上述疾病或病症为再生医学/修复医学领域的疾病和损伤。
具体地,上述再生医学/修复医学领域的疾病和损伤选自:缺血性心脏病、糖尿病、脑出血、脑梗死、股骨头坏死、卵巢早衰、脑震荡、脑挫裂伤、大脑损伤、开放性颅脑损伤、肝硬化、纤维肝、脂肪肝、出血性坏死性胰腺炎、胰腺损伤、胰腺纤维化、各种肿瘤化疗后免疫功能减退综合症、放疗后免疫功能减退综合症的免疫功能重建、慢性肾上腺皮质功能减退症、腺垂体功能减退症、垂体前叶机能减退症、中枢性尿崩症、甲状腺切除术后甲状腺损伤、甲状腺功能减退症、甲状旁腺机能减退症和肾功能衰竭。
利用本发明公开的用于多能干细胞向造血干细胞分化的材料(例如添加剂Ⅰ、添加剂Ⅱ和添加剂Ⅲ,或,培养基Ⅰ、培养基Ⅱ和培养基Ⅲ)和方法,可在常氧条件下实现诱导多能干细胞的定向分化,可以高效获得CD34+造血干细胞,且细胞普遍停留在造血前体细胞或造血干细胞时期,其中部分细胞表达CD43和CD45,且表达峰值明显。本发明的材料化学成分确定,成本低,安全性高,质量稳定;本发明的方法具有耗时短、分化效率高等优点,可为组织工程、药物研发和细胞治疗等领域提供大量细胞来源,具有较佳的应用前景和商业价值。
附图说明
图1所示为本发明实施例1的空白对照的流式细胞检测结果。
图2所示为本发明实施例1的阳性对照的流式细胞检测结果。
图3所示为本发明实施例1的试验组的细胞分化形态图。
图4所示为本发明实施例1的试验组的流式细胞检测结果。
具体实施方式
除非另有定义,本发明中所使用的所有科学和技术术语具有与本发明涉及技术领域的技术人员通常理解的相同的含义。
本发明中部分术语与缩写对应如下:
诱导多能干细胞 iPSC(induced pluripotent stem cell)
胚胎干细胞 ESC(embryomic stem cell)
造血内皮祖细胞 HEP(hemogenic endothelial progenitor)
造血前体细胞 HP(hematopoietic progenitor)
造血干细胞 HSC(hematopoietic stem cell)
骨形态发生蛋白4 BMP4(bone morphogenetic protein 4)
成纤维生长因子2 FGF2(Fibroblast growth factor 2)
血管内皮生长因子 VEGF(vascular endothelial growth factor)
干细胞因子 SCF(stem cell factor)
FMS样酪氨酸激酶3配体 FLT3L(FMS like tyrosine kinase 3 ligand)
血小板生成素 TPO(thrombopoietin)
在本发明中,术语“分化”描述了非特化细胞通过其而获得特化细胞(例如皮肤、心脏、肌肉、造血细胞)的特征的过程;“定向分化”指操作干细胞培养条件来诱导分化成特定的细胞类型。
在本发明中,术语“多能干细胞”是指具有以下性质的细胞:(i)能够在未分化状态下在体外无限增殖;(ii)通过长期培养维持正常的核型;以及(iii)即使在长时间培养后,仍保持分化为所有三个胚胎胚层(内胚层、中胚层和外胚层)的衍生物的潜力。目前可获得的多能干细胞的非限制性实施例包括胚胎干细胞(ESC)和诱导多能干细胞(iPSC)。胚胎干细胞的非限制性实施例包括:可从ESI, Singapore获得的HES2(也称为ES02)细胞系和可从WiCell, Madison, WI获得的H1或H9(也称为WA01)细胞系。诱导多能干细胞是衍生自非多能细胞的人工衍生的干细胞(通常是成体体细胞),其通过诱导一种或多种干细胞特异性基因的表达而产生。
在本发明中,术语“药学上可接受的”,表示考虑到待治疗的疾病或病症以及相应的施用途径,所指出的材料不具有引起合理谨慎的医师避免将该材料施用于患者的性质。例如,通常要求这种材料基本上是无菌的。
在本发明中,术语“治疗”是指在伤害或干预之前、期间和/或之后预防、治愈、逆转、减弱、减轻、最小化、抑制、制止和/或停止疾病或病症的一种或多种临床症状。
在本发明中,术语“患者”或“对象”是指用药物组合物或根据本文所述的方法治疗的动物,包括哺乳动物,例如,鼠、犬、马、牛、猿或人类,特别是人类。
本文所引用的各种出版物、专利和公开的专利说明书,其公开内容通过引用整体并入本文。
下面将结合本发明实施例,对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例仅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1
细胞培养:人类BM-iPSC细胞系(Wicell)按照Wicell干细胞培养方法,培养在E8-Vitronectin系统,利用1mM EDTA(或加TrypLE)常规方法传代。分化前,传代1x105细胞到新的胶原预处理的6孔板(约104/cm2,添加终浓度10μM的Rocki增加存活率)。然后采用基础培养基(IF9S)加小分子3步法进行细胞分化:干细胞-中胚层-造血内皮祖细胞-CD34+CD43+CD45+造血干细胞。最后收取悬浮细胞,流式上机检测。
IF9S培养基成分参见“Uenishi, Gene, et al. "Tenascin C promoteshematoendothelial development and T lymphoid commitment from humanpluripotent stem cells in chemically defined conditions." Stem cell reports3.6 (2014): 1073-1084”中表S1所示。
试验组实验过程如下:
1、利用Wicell公司提供的E8-Vitronectin系统和方法常规培养人类诱导多功能干细胞系BM-iPSC细胞系,确定细胞生长良好,状态稳定;
2、分化实验开始前利用2ml 2.4μg/ml的胶原(Collagen IV)预处理包被6孔板,4℃包被过夜;
3、利用1ml 1mM EDTA(DPBS稀释)消化液消化BM-iPSC细胞系为单细胞或者小细胞团,9ml E8培养液终止消化,离心去上清,利用1 ml E8培养基重悬计数;
4、吸出预处理6孔板中的胶原,根据细胞计数接种1x105 BM-iPSC干细胞到6孔板中,E8培养基补足2ml;如果细胞死亡严重,需要加入终浓度为10μM的细胞凋亡抑制剂(Rocki,Y-27632);
5、转天更换细胞培养液为第一阶段细胞分化培养液2ml:基础培养基(IF9S)+表1所示成分,持续培养2~3天(第0~3天);
6、第3天更换细胞培养液为第二阶段细胞分化培养液2ml:基础培养基(IF9S)+表2所示成分,持续培养2~3天(第4~6天);
7、第6天更换细胞培养液为第三阶段细胞分化培养液2ml:基础培养基(IF9S)+表3所示成分,持续培养2~3天(第7~9天);
8、第9天添加第三阶段细胞分化培养液2ml:基础培养基(IF9S)+表3所示成分,持续培养2~3天(第10~12天);
9、细胞分化过程中的形态图见图3;之后常规细胞表面标记染色,流式检测见图4。
以上分化过程均在正常氧气浓度,5%二氧化碳37℃恒温培养箱中进行。
由图3可知,在常氧条件下细胞形态逐渐转变;由图4可知,通过流式检测,证明获得了CD34+细胞,且细胞普遍停留在造血前体细胞或造血干细胞时期,其中部分细胞表达CD43和CD45,且表达峰值明显。
空白对照组:以基础培养基(IF9S)在上述相同条件下培养细胞,作为空白对照,结果如图1所示。由图1可知,在基础培养基(IF9S)的条件下,几乎无CD34。
阳性对照组:以基础培养基(IF9S)+小分子:BMP4、FGF2、ActA、LiCl、VEGF、SCF、TPO、IL3、IL6、TGFb inhibitor SB(可参见Uenishi, Gene, et al. "Tenascin Cpromotes hematoendothelial development and T lymphoid commitment from humanpluripotent stem cells in chemically defined conditions." Stem cell reports3.6 (2014): 1073-1084.),在上述相同条件下培养细胞,作为阳性对照,结果如图2所示。由图2可知,在此条件下,可以获得CD34+细胞,但是细胞普遍停留在中胚层-造血内皮祖细胞分化时期,几乎不表达CD43和CD45。
表1 第一阶段细胞分化培养液添加成分
表2 第二阶段细胞分化培养液添加成分
表3 第三阶段细胞分化培养液添加成分
成分 | 终浓度 |
SCF | 50 ng/ml |
FLT3L | 50 ng/ml |
TPO | 50 ng/ml |
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换等,均应包含在本发明的保护范围之内。
本发明中描述的前述实施例和方法可以基于本领域技术人员的能力、经验和偏好而有所不同。
本发明中仅按一定顺序列出方法的步骤并不构成对方法步骤顺序的任何限制。
Claims (14)
1.一种用于多能干细胞分化的方法,其包括如下步骤:
(1)将多能干细胞接种于培养基Ⅰ中,进行培养;
(2)将步骤(1)的培养体系中的培养基更换为培养基Ⅱ,进行培养;
(3)将步骤(2)的培养体系中的培养基更换为培养基Ⅲ,进行培养;
(4)向将步骤(3)的培养体系中加入所述培养基Ⅲ,进行培养;
其中,
所述培养基Ⅰ包含补充有BMP4、FGF2和VEGF的干细胞培养基Ⅰ;
所述培养基Ⅱ包含补充有SCF和VEGF的干细胞培养基Ⅱ;
所述培养基Ⅲ包含补充有SCF、FLT3L和TPO的干细胞培养基Ⅲ;
所述培养基Ⅰ中,BMP4、FGF2和VEGF的终浓度分别为30-100ng/ml、30-100ng/ml、30-100ng/ml;
所述培养基Ⅱ中,SCF和VEGF的终浓度分别为30-100ng/ml、30-100ng/ml;
所述培养基Ⅲ中,SCF、FLT3L和TPO的终浓度分别为30-100ng/ml、30-100ng/ml、30-100ng/ml。
2.如权利要求1所述的方法,其特征在于,所述培养基Ⅰ中BMP4的终浓度为80ng/ml。
3.如权利要求1所述的方法,其特征在于,所述培养基Ⅰ中FGF2的终浓度为50ng/ml。
4.如权利要求1所述的方法,其特征在于,所述培养基Ⅰ中VEGF的终浓度为80ng/ml。
5.如权利要求1所述的方法,其特征在于,所述培养基Ⅱ中SCF的终浓度为80ng/ml。
6.如权利要求1所述的方法,其特征在于,所述培养基Ⅱ中VEGF的终浓度为80ng/ml。
7.如权利要求1所述的方法,其特征在于,所述培养基Ⅲ中SCF的终浓度分别为50ng/ml。
8.如权利要求1所述的方法,其特征在于,所述培养基Ⅲ中FLT3L的终浓度分别为50ng/ml。
9.如权利要求1所述的方法,其特征在于,所述培养基Ⅲ中TPO的终浓度分别为50ng/ml。
10.如权利要求1所述的方法,其特征在于,所述干细胞培养基Ⅰ、干细胞培养基Ⅱ、干细胞培养基Ⅲ均为IF9S培养基。
11.如权利要求1所述的方法,其特征在于,所述方法的步骤均在正常氧气浓度下进行。
12.如权利要求1所述的方法,其特征在于,所述多能干细胞为人类诱导多能干细胞。
13.如权利要求1所述的方法,其特征在于,所述方法的步骤均在37℃的温度下进行。
14.如权利要求1所述的方法,其特征在于,所述步骤(1)-(4)中培养时间各自独立地为2-3天。
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