CN107338220A - 诱导性多能干细胞向造血干细胞分化的方法及其培养基 - Google Patents
诱导性多能干细胞向造血干细胞分化的方法及其培养基 Download PDFInfo
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Abstract
本发明涉及一种诱导性多能干细胞向造血干细胞分化的方法及其培养基。所述方法包括:将经预处理后的iPS用添加了重组来源的细胞因子组合Ⅰ的改进拟胚体形成培养基悬浮后离心,将离心后聚集在一起的iPS进行培养,获得拟胚体;再用添加了重组来源的细胞因子组合Ⅱ的造血干细胞诱导培养基培养,获得造血干细胞。该方法需要的初始iPS细胞量少,拟胚体形态整齐、发育和分化阶段统一;整个过程耗时短,操作简单;不涉及动物源性细胞和成分,大大提高了iPS来源的造血干细胞的安全性,为利用患者自身的iPS大规模生产临床应用级别的造血干细胞提供了一种新的途径和方法。
Description
技术领域
本发明涉及细胞工程领域,尤其是涉及一种无血清无动物源性诱导性多能干细胞向造血干细胞分化的方法及其培养基。
背景技术
临床上普遍使用造血干细胞移植,用于白血病、非霍奇金淋巴瘤、地中海贫血等病的治疗。造血干细胞的主要来源是脐带血、骨髓。如果直接进行骨髓移植,则需要进行人体的白细胞抗原配型,否则发生免疫排异会危及患者生命。现有脐带血库储存的造血干细胞免疫原性弱,但数量供不应求,使其在疾病中的临床应用中受到限制。诱导性多能干细胞(induced pluripotent stem cells,iPS)的出现成功地绕开了免疫原性和伦理性两个最关键的问题,而且来源广泛,可以建立患者特定的iPS细胞,做到个体化治疗,将为干细胞移植大规模在临床应用提供了可能性(Sontag S,M et al.ModellingIRF8Deficient Human Hematopoiesis and Dendritic Cell Development withEngineered iPS Cells.Stem Cells.2017Apr;35(4):898-908.)。
目前,iPS定向分化为血液细胞公认的技术有两个:①与OP9细胞共培养,②通过拟胚体(embryoid body,EB)途径。利用OP9细胞与iPS共培养,通过模拟体内的造血微环境,支持早期的造血干细胞分化(Klump H,Teichweyde N et al.Development of patient-specific hematopoietic stem and progenitor cell grafts from pluripotent stemcells,in vitro.Curr Mol Med.2013Jun;13(5):815-20.Schrimpf C,Wrede C etal.Differentiation of induced pluripotent stem cell-derived neutrophilgranulocytes from common marmoset monkey(Callithrix jacchus).Transfusion.2017Jan;57(1):60-69.)。拟胚法是指在特定的诱导条件下,多能干细胞在悬浮培养环境中分化形成的由三个胚层细胞组成的立体结构。在拟胚体形成的过程中,添加特定的细胞因子可以促进多能干细胞向中胚层分化,提高相应造血干细胞的比例。
这两种常用的技术虽然能够在一定程度上诱导iPS细胞向造血干细胞分化,但是这两种诱导技术也存在着一定的缺点,主要表现在:①造血干细胞诱导体系中存在非人源成分,给诱导分化后的造血干细胞临床应用带来安全隐患。比如OP9细胞为小鼠骨髓基质细胞,如果用于人类造血干细胞诱导体系,存在鼠源性污染细胞的可能性。目前的人类造血干细胞分化技术涉及到胎牛血清(fetal bovine serum,FBS)或者牛血清白蛋白(bovineserum albumin,BSA)等均为动物源性成分,加之FBS的成分的不确定性,都极大的限制了iPS来源的造血干细胞在临床应用。②用拟胚体途径诱导分化造血干细胞的技术,存在消耗iPS细胞量大,拟胚体分化阶段不整齐等问题,造成造血干细胞分化效率较低(Féraud O,Valogne Y,et al.Donor Dependent Variations in Hematopoietic Differentiationamong Embryonic and Induced Pluripotent Stem Cell Lines.PLoS One.2016Mar 3;11(3):e0149291.),不利于iPS向造血干细胞定向诱导分化在临床应用上的大规模开展。
发明内容
针对现有技术中存在的缺陷,本发明的目的在于提供一种诱导性多能干细胞向造血干细胞分化的方法及其培养基。本发明不涉及动物源性细胞和成分,大大提高了iPS细胞来源的造血干细胞的安全性,为利用患者自身的iPS细胞大规模生产临床应用级别的造血干细胞提供了一种新的途径和方法。
为达到以上目的,本发明采取的技术方案是:
诱导性多能干细胞向造血干细胞分化的方法,包括如下步骤:
S1.将诱导性多能干细胞(具体可为人源、皮肤成纤维细胞)进行预处理;预处理的目的是提高细胞的活性和分化能力;
S2.收集经步骤S1预处理后的诱导性多能干细胞,用添加了重组来源的细胞因子组合Ⅰ的改进拟胚体形成培养基悬浮后离心,将离心后聚集在一起的诱导性多能干细胞在所述添加了重组来源的细胞因子组合Ⅰ的改进拟胚体形成培养基中培养,获得拟胚体;
S3.收集步骤S2获得的拟胚体,用添加了重组来源的细胞因子组合Ⅱ的造血干细胞诱导培养基培养,获得造血干细胞。
在上述诱导性多能干细胞向造血干细胞分化的方法中,步骤S1中所述预处理具体包括如下步骤:将诱导性多能干细胞先用DPBS洗(具体为洗两遍,每次1min),再用0.5mMEDTA消化(具体为37℃下处理5min),最后用完全培养基(如E8完全培养基)终止消化。
在上述诱导性多能干细胞向造血干细胞分化的方法中,步骤S2中,所述改进拟胚体形成培养基为将常规的拟胚体形成培养基中动物源性细胞和成分(如:胎牛血清和/或牛血清白蛋白)替换为人血清白蛋白。
在上述诱导性多能干细胞向造血干细胞分化的方法中,所述改进拟胚体形成培养基中所述人血清白蛋白的终浓度为1-5mg/ml,优选为2.5mg/ml。
在上述诱导性多能干细胞向造血干细胞分化的方法中,所述改进拟胚体形成培养基包含如下组分:
IMDM液体培养基30-50%(体积百分含量),优选为42.8%;
含L-丙氨酰-L-谷氨酰胺的F12营养培养基30-50%(体积百分含量),优选为42.8%;
人血清白蛋白1-5mg/ml,优选为2.5mg/ml;
聚乙烯醇5%(体积百分含量);
无蛋白杂交瘤细胞培养基II 5%(体积百分含量);
L-丙氨酰-L-谷氨酰胺(为含L-丙氨酰-L-谷氨酰胺的F12营养培养基之外另添加的)2mM;
胰岛素-转铁蛋白-硒10ug/ml。
在上述诱导性多能干细胞向造血干细胞分化的方法中,所述重组来源的细胞因子组合Ⅰ为干细胞因子、人血管内皮细胞生长因子和人骨形成蛋白4,其添加于所述改进拟胚体形成培养基中的终浓度(即使用浓度)分别为:30-50ng/ml、15-25ng/ml和15-25ng/ml,优选为:40ng/ml、20ng/ml和20ng/ml。
在上述诱导性多能干细胞向造血干细胞分化的方法中,步骤S2中,所述离心的条件为:1400-1600转/分离心4-6min;
和/或,所述培养的条件为:温度35-38℃,湿度为饱和湿度,二氧化碳浓度为4-6%;
和/或,所述培养的时间为:7-10天;
和/或,所述培养还包括在所述培养的第3-5天和第6-10天用所述添加了重组来源的细胞因子组合Ⅰ的改进拟胚体形成培养基半换液的步骤;
和/或,所述培养的起始细胞量为:每毫升所述拟胚体形成培养基含2.8×104-3.2×104个诱导性多能干细胞。
在上述诱导性多能干细胞向造血干细胞分化的方法中,步骤S3中,所述造血干细胞诱导培养基包含如下组分:
IMDM液体培养基90-95%(体积百分含量);
热灭活人AB血清4-5%(体积百分含量);
L-丙氨酰-L-谷氨酰胺2mM;
胰岛素-转铁蛋白-硒10ug/ml;
肝素钠2IU/ml;
铁离子饱和人转铁蛋白10ug/ml。
在上述诱导性多能干细胞向造血干细胞分化的方法中,所述重组来源的细胞因子组合Ⅱ为干细胞因子、促血小板生成素、FMS样酪氨酸激酶受体-3配体、白介素3、白介素6、重组人骨形成蛋白4、重组人血管内皮生长因子和红细胞生成素,其添加于所述造血干细胞诱导培养基中的终浓度(即使用浓度)分别为:80-120ng/ml、80-120ng/ml、80-120ng/ml、3-8ng/ml、3-8ng/ml、8-12ng/ml、3-8ng/ml、1-5U/ml。
在上述诱导性多能干细胞向造血干细胞分化的方法中,步骤S3中,所述培养的条件为:温度35-38℃,湿度为饱和湿度,二氧化碳浓度为4-6%;
和/或,所述培养的时间为:8-10天;
和/或,所述培养还包括在所述培养的第4天和第8天用所述添加了重组来源的细胞因子组合Ⅱ的造血干细胞诱导培养基半换液。
本发明保护上述任一诱导性多能干细胞向造血干细胞分化的方法中所述的改进拟胚体形成培养基,或所述的添加了重组来源的细胞因子组合Ⅰ的改进拟胚体形成培养基。
本发明保护上述任一诱导性多能干细胞向造血干细胞分化的方法中所述的造血干细胞诱导培养基或添加了重组来源的细胞因子组合Ⅱ的造血干细胞诱导培养基。
本发明的有益效果如下:
本发明改进了拟胚体的制作技术和工艺,利用离心的方式将iPS细胞通过离心力的作用聚集在一起进而形成拟胚体,其优势是需要的初始iPS细胞量少,形成形态整齐,发育和分化阶段统一的Spin EB(Spin EB为利用离心力使iPS细胞聚集最后形成的拟胚体,Spin EB在形态学或生物学上与拟胚体EB无差异),通过添加重组来源的细胞因子(如:干细胞因子(SCF),血管内皮细胞生长因子VEGF,骨形成蛋白4(BMP4),促血小板生成素(TPO),FMS样酪氨酸激酶受体-3配体(FLT3L),白介素3(IL-3),白介素6(IL-6),红细胞生成素(EPO))可高效诱导iPS向造血干细胞定向分化,整个过程耗时短,操作简单,使用改进的Spin EB形成培养基成分,不涉及动物源性细胞和成分,大大提高了iPS细胞来源的造血干细胞的安全性,为利用患者自身的iPS细胞大规模生产临床应用级别的造血干细胞提供了一种新的途径和方法。
附图说明
本发明有如下附图:
图1为iPS细胞的Spin EB形成过程。
图2为造血干细胞的诱导分化过程。
图3为CD34+细胞分化效率的流式细胞仪检测结果。
图4为克隆形成(CFU)检测结果。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1、诱导性多能干细胞向造血干细胞分化
1.Spin EB的形成
(1)T25瓶中培养的iPS(具体为人皮肤成纤维细胞重编程后获得的iPS)。弃培养基,DPBS洗两遍,每次1min;
(2)加入0.5mM EDTA消化液2ml;
(3)37℃孵育5min后,弃EDTA,加入E8完全培养基终止消化;
(4)收集细胞悬液,细胞计数;
(5)取7.2×105细胞,800转/分离心5min;
(6)加入Spin EB形成培养基24mL重悬细胞,用200μL枪头加入96孔板中,每孔0.3×104个细胞,每孔Spin EB形成培养基为100μL,即制作4个96孔板的240个孔的spin EB;
(7)将加入细胞的96孔板置于96孔板离心机中,1500转/分离心5min;
(8)放回培养箱培养(条件:37℃,饱和湿度,5%二氧化碳)8天。
(9)分别于步骤(8)培养的第4天和第7天用Spin EB形成培养基半换液。
Spin EB的形成如图1所示,离心前,iPS细胞在96孔板孔中呈弥散分布(图1中的A图,4×),离心后,iPS细胞聚集于96孔板孔的底部中心(图1中的B图,4×)。培养箱培养24小时后,spin EB形成,spin EB与周围单个细胞有明显边界(图1中的C图,4×)。培养73小时后,spin EB应为实心致密球体,边界明显且光滑(图1中的D图,4×)。
上述步骤1中所述spin EB形成培养基的配方如表1所示:
表1
注:表1中的“%”均为体积百分数,“终浓度”指在spin EB培养基中的浓度,“用量”指配制201mL spin EB培养基的加入量,spin EB培养基的溶剂为超纯水。
2.造血干细胞的诱导分化
(1)收集96孔板中的Spin EB于15mL离心管,静止3min,弃上清,添加造血干细胞诱导培养基,将Spin EB转移至6孔板中1个孔中;
(2)放回培养箱培养(37℃,饱和湿度,5%二氧化碳)9天;
(3)分别于步骤(2)培养的第4天和第8天用造血干细胞诱导培养基半换液。
上述步骤2中所述造血干细胞诱导培养基的配方如表2所示:
表2
注:表2中的“%”均为体积百分数,“终浓度”指在配制造血干细胞诱导培养基中的浓度,造血干细胞诱导培养基的溶剂为超纯水。
造血干细胞的诱导分化过程如图2所示:步骤1中的spin EB在96孔板中培养8天后,收集96孔板中的spin EB于6孔板1孔中,可见spin EB发育程度整齐,形态大小均一(图2中的A图,4×);经过步骤2造血干细胞诱导培养基培养4天后,实心的spin EB发育为有腔EB(图2中的B图,4×);经过步骤2造血干细胞诱导培养基培养9天后,可见大量CD34+细胞(图2中的C图,4×;图2中的D图,20×)。
3.CD34+细胞的收集
(1)收集经步骤2培养9天后6孔板中的Spin EB和从Spin EB分化脱落下来的单个细胞,800转/分离心5min后弃上清;
(2)加入5mL浓度为0.4U/mL的collagenase B(B型胶原酶)37℃消化30min后,800转/分离心5min后弃上清;
(3)加入10mL PBS 800转/分离心5min后弃上清;加入5mL cell dissociationbuffer(细胞解离缓冲液)37℃解离10min后,加入10mL所述造血干细胞诱导培养基800转/分离心5min后弃上清;
(4)加入5mL所述造血干细胞诱导培养基制备细胞悬液,过200目细胞筛,获得的单个细胞悬液用磁珠分选或者流式细胞分选,得到CD34+细胞。
4.造血干细胞分化效率的流式细胞仪检测
将步骤3获得的单个细胞悬液通过流式细胞仪鉴定CD34+细胞的数量,以未做诱导分化的未分化的iPS细胞为对照。
结果如图3所示,其中V1-L#和V1-R#是指流式细胞仪的检测通道。CON:指对照;CD348D:步骤3获得的单个细胞悬液;FL1-H:流式细胞仪单位图表的横坐标,对应的是FITC(绿色荧光)通道,即代表绿色荧光染料所染到的细胞数量。
图3的结果表明,iPS细胞向造血干细胞(CD34+细胞)的分化效率为16.5%(图3中的右图),显著高于对照的0.6%(图3中的左图)。
5.造血干细胞多能性的检测
将步骤3经过磁珠分选收集的CD34+细胞用于克隆形成(CFU)检测,结果表明,通过步骤1和2方法诱导iPS细胞分化而来的造血干细胞(CD34+细胞)能形成CFU-E(红细胞集落生成单位)(图4中的A图),CFU-G(粒系集落生成单位)(图4中的B图),CFU-GEMM(粒系红细胞巨核细胞巨噬细胞集落生成单位)(图4中的C图)和CFU-M(巨核细胞集落生成单位)(图4中的D图)。
本说明书中未作详细描述的内容属于本领域专业技术人员公知的现有技术。
Claims (10)
1.诱导性多能干细胞向造血干细胞分化的方法,包括如下步骤:
S1.将诱导性多能干细胞进行预处理;
S2.收集经步骤S1预处理后的诱导性多能干细胞,用添加了重组来源的细胞因子组合Ⅰ的改进拟胚体形成培养基悬浮后离心,将离心后聚集在一起的诱导性多能干细胞在所述添加了重组来源的细胞因子组合Ⅰ的改进拟胚体形成培养基中培养,获得拟胚体;
S3.收集步骤S2获得的拟胚体,用添加了重组来源的细胞因子组合Ⅱ的造血干细胞诱导培养基培养,获得造血干细胞。
2.如权利要求1所述的诱导性多能干细胞向造血干细胞分化的方法,其特征在于:步骤S1中所述预处理包括如下步骤:将诱导性多能干细胞先用DPBS洗,再用0.5mM EDTA消化,最后用完全培养基终止消化。
3.如权利要求1所述的诱导性多能干细胞向造血干细胞分化的方法,其特征在于:步骤S2中,所述改进拟胚体形成培养基为将常规的拟胚体形成培养基中动物源性细胞和成分替换为人血清白蛋白。
4.如权利要求3所述的诱导性多能干细胞向造血干细胞分化的方法,其特征在于:所述改进拟胚体形成培养基中所述人血清白蛋白的终浓度为1-5mg/ml。
5.如权利要求4所述的诱导性多能干细胞向造血干细胞分化的方法,其特征在于:所述改进拟胚体形成培养基包含如下组分:
IMDM液体培养基30-50%(体积百分含量);
含L-丙氨酰-L-谷氨酰胺的F12营养培养基30-50%(体积百分含量);
人血清白蛋白1-5mg/ml;
聚乙烯醇5%(体积百分含量);
无蛋白杂交瘤细胞培养基II 5%(体积百分含量);
L-丙氨酰-L-谷氨酰胺2mM;
胰岛素-转铁蛋白-硒10ug/ml;
和/或,所述重组来源的细胞因子组合Ⅰ为干细胞因子、人血管内皮细胞生长因子和人骨形成蛋白4,其添加于所述改进拟胚体形成培养基中的终浓度分别为:30-50ng/ml、15-25ng/ml和15-25ng/ml。
6.如权利要求1所述的诱导性多能干细胞向造血干细胞分化的方法,其特征在于:步骤S2中,
所述离心的条件为:1400-1600转/分离心4-6min;
和/或,所述培养的条件为:温度35-38℃,湿度为饱和湿度,二氧化碳浓度为4-6%;
和/或,所述培养的时间为:7-10天;
和/或,所述培养还包括在所述培养的第3-5天和第6-10天用所述添加了重组来源的细胞因子组合Ⅰ的改进拟胚体形成培养基半换液的步骤;
和/或,所述培养的起始细胞量为:每毫升所述拟胚体形成培养基含2.8×104-3.2×104个诱导性多能干细胞。
7.如权利要求1所述的诱导性多能干细胞向造血干细胞分化的方法,其特征在于:步骤S3中,
所述造血干细胞诱导培养基包含如下组分:
IMDM液体培养基90-95%(体积百分含量);
热灭活人AB血清4-5%(体积百分含量);
L-丙氨酰-L-谷氨酰胺2mM;
胰岛素-转铁蛋白-硒10ug/ml;
肝素钠2IU/ml;
铁离子饱和人转铁蛋白10ug/ml;
和/或,所述重组来源的细胞因子组合Ⅱ为干细胞因子、促血小板生成素、FMS样酪氨酸激酶受体3配体、白介素3、白介素6、重组人骨形成蛋白4、重组人血管内皮生长因子和红细胞生成素,其添加于所述造血干细胞诱导培养基中的终浓度分别为:80-120ng/ml、80-120ng/ml、80-120ng/ml、3-8ng/ml、3-8ng/ml、8-12ng/ml、3-8ng/ml、1-5U/ml。
8.如权利要求1所述的诱导性多能干细胞向造血干细胞分化的方法,其特征在于:步骤S3中,所述培养的条件为:温度35-38℃,湿度为饱和湿度,二氧化碳浓度为4-6%;
和/或,所述培养的时间为:8-10天;
和/或,所述培养还包括在所述培养的第4-5天和第8-10天用所述添加了重组来源的细胞因子组合Ⅱ的造血干细胞诱导培养基半换液。
9.权利要求3—5中任一所述的改进拟胚体形成培养基,或权利要求5中所述的添加了重组来源的细胞因子组合Ⅰ的改进拟胚体形成培养基。
10.权利要求7中所述的所述造血干细胞诱导培养基或添加了重组来源的细胞因子组合Ⅱ的造血干细胞诱导培养基。
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