CN103937743B - 一种利用三维诱导系统获得造血干细胞的方法 - Google Patents
一种利用三维诱导系统获得造血干细胞的方法 Download PDFInfo
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Abstract
本发明提供一种利用三维诱导体系高效获得造血干细胞的方法,是利用三维细胞培养基质或细胞培养支架,如水凝胶、海藻等材料制成的三维细胞培养系统,和/或联合基质细胞如骨髓细胞、小鼠骨髓细胞系OP9、OP9DL1等,和/或联合多种因子包括中胚层诱导因子、造血生长因子等诱导多能干细胞分化为造血干细胞。本发明建立了一种新的获得造血干细胞的方法,首次建立了利用三维诱导体系和/或联合骨髓细胞等基质细胞和/或多种因子等高效诱导多能干细胞分化为造血干细胞的系统,为获得临床可用的造血干细胞提供了理论基础和技术平台,并为多能干细胞来源的造血细胞在疾病机理探索、药物筛选等领域的应用开拓了新的方法和新的思路。
Description
技术领域
本发明属生物技术领域,涉及一种利用三维诱导系统高效获得造血干细胞的方法。
背景技术
造血的发生发展是一个复杂的过程。在人类胚胎第2周末就已经开始。此时,位于卵黄囊壁上一群未分化的、具有自我更新能力的胚外中胚层细胞组成血岛,成为人类最初的造血中心。从胚胎第6周开始,卵黄囊血岛产生的造血干细胞(Hematopoietic StemCells,HSCs)随血流迁移到肝脏,并种植在肝脏中,开始肝脏造血期。在胚胎第3个月,长骨髓已开始造血。第8个月时,骨髓造血已高度发育,这时,肝、脾造血功能减退,骨髓成为造血中心。位于骨髓中的造血干细胞是一群具有自我更新和多向分化潜能的成体干细胞,是人体血液系统中所有造血细胞和免疫细胞的来源,对于人体血液和免疫系统正常机能的维持具有重要作用。骨髓中的造血干细胞含量极低。青春期后随着年龄的增长,人类骨髓开始衰竭,并逐渐被脂肪组织所取代,造血干细胞数量逐渐减少,造成人体多种机能包括免疫机能下降。许多与血液和免疫系统相关的疾病都与造血干细胞的功能是否正常密切相关。目前,造血干细胞已被广泛应用于治疗多种疾病,包括血液系统恶性或非恶性肿瘤,如急性髓细胞白血病、慢性粒细胞白血病、再生障碍性贫血、地中海贫血等;免疫系统疾病,如部分自身免疫性疾病、重症联合免疫缺陷症等;部分实体瘤等。未来年纪较大且循环或免疫能力低下的人也可能受益于造血干细胞移植。造血干细胞移植具有广阔的临床应用前景,并已经成为应用最为广泛、技术最为成熟的一种组织干细胞,是再生医学的成功典范。
造血干细胞移植需要主要组织相容性复合体(Major HistocompatibilityComplex, MHC)或者白细胞抗原(HLA )配型相合的造血干细胞来源。根据中华骨髓库资料表明(截至2013年11月30日),患者申请查询人数,总计 34450人,而捐献造血干细胞人数只有3863例,需要配型的病人往往在等待过程中失去了疾病治疗的最佳时期。异基因造血干细胞移植是目前主要的细胞移植手段,但由于免疫排斥,往往导致移植物抗宿主病等一系列致命的移植并发症。虽然脐血可以替代成体造血干细胞进行移植,但脐血细胞中含有的造血干细胞数量非常少,很难满足成人对造血干细胞移植时细胞数量的要求。造血干细胞作为一种含量极低的成体干细胞,如何获得大量的功能性造血干细胞用于临床治疗,是目前开展造血干细胞相关的临床治疗和研究的主要问题。
人多能干细胞,包括胚胎干细胞和诱导性多能干细胞,可以在体外适当的培养条件下长期大量扩增,并具有分化为人体所需要的所有细胞类型包括造血干细胞的潜能,是一种解决造血干细胞来源问题的方法。因此,通过人多能干细胞诱导分化获得造血干细胞,具有十分重要的临床应用价值。目前,拟胚体法、基质细胞共培养法、诱导因子法、基质细胞联合诱导因子法等是人们诱导多能干细胞分化为造血干细胞的常用方法。其中基质细胞一般为与造血环境密切相关的基质细胞,如鼠骨髓基质细胞OP9或S17、小鼠主动脉-性腺-中肾区基质细胞AGM、鼠卵黄囊内皮细胞系C166、人或鼠胎肝细胞、改造的基质细胞系OP9-DL1等,诱导因子一般为中胚层诱导因子BMP4、PD98059,造血和内皮细胞诱导因子有SCF、FLT3L、VEGF、IL-3、IL-6、VEGF等。经过一段时间(一般为2-3周左右)的共培养或者诱导,获得的功能性造血干细胞的比例却很小,表达早期造血干细胞表面标志CD34+CD38-的比例只有0.1%–2%。在功能上,这些细胞虽然能够形成部分造血集落,生成红系祖细胞、粒系祖细胞、巨噬细胞等,但是不能同时获得髓系和淋系细胞。如何高效诱导人多能干细胞向功能性造血干细胞分化是多能干细胞临床应用的主要瓶颈,对于体外研究人造血发育过程也具有极为重要的意义。
发明内容
本发明的一个目的是提供一种利用三维诱导系统获得造血干细胞的方法,通过以下技术方案实现:机械法收集多能干细胞,根据厂家说明书对各种水凝胶、海藻、纳米材料等三维细胞培养材料进行处理,和/或联合基质细胞(包括人或者小鼠骨髓细胞、胎盘细胞、小鼠骨髓细胞系OP9、OP9-DL1或其它相关诱导造血分化的基质细胞),和/或联合相关小分子物质(包括中胚层诱导因子骨形态建成蛋白4(BMP4),干细胞生长因子(SCF),FMS样酪氨酸激酶3配体(Flt3L),血管内皮生长因子(VEGF)、血小板生成素(TPO),前列腺素2(PGE2)等),经过10-14天的诱导和培养,形成造血干细胞的方法。
造血干细胞是由人诱导性多能干细胞包括人胚胎干细胞或诱导性多能干细胞分化获得的。
所述人诱导性多能干细胞是利用含有转录因子Oct4,Sox2,Klf4和/或c-Myc四种因子的逆转录病毒、慢病毒、腺病毒或仙台病毒等方式诱导人体细胞,包括成纤维细胞、骨髓间充质干细胞等,重编程获得的诱导性多能干细胞。所述人胚胎干细胞为可从商业途径获得的人胚胎干细胞系H1,H3。
所述细胞三维培养材料包括多种三维细胞培养基质或细胞培养支架,如各种水凝胶、海藻、纳米材料等制成的三维细胞培养材料。
进一步利用造血相关因子包括造血细胞因子如SCF,Flt3L、白介素-3(IL-3)、白介素-6(IL-6)、PGE2等进一步扩增获得的造血干细胞。在获得造血干细胞的基础上,再利用SCF、IL-3、IL-6、白介素-7(IL-7)、白介素-2(IL-2)、粒细胞-巨噬细胞集落刺激(GM-CSF)、促红细胞生成素(EPO)、TPO等诱导整个体系向多个血系分化。
流式细胞技术检测获得的造血干细胞的比例:在诱导和扩增的过程中,利用流式细胞技术检测系统中CD34造血干细胞表面标记的表达情况。
甲基纤维素半固体培养法检测造血干细胞生成造血集落(CFU)的潜能:将获得的造血干细胞分散成单个细胞,种植于含有SCF、GM-CSF、IL-3、IL-6、TPO、EPO等细胞因子的甲基纤维素半固体培养基中,培养皿需低粘附,继续培养2周左右。观察CFU形成情况,并计数。
本发明方法的特点是利用三维细胞培养系统,和/或联合基质细胞,和/或联合中胚层诱导因子、造血细胞因子等诱导多能干细胞分化为造血干细胞,建立了高效获得造血干细胞的方法。该方法为获得临床可用的造血干细胞提供可靠的细胞来源,并积极促进多能干细胞在临床上的应用。本发明建立了一种新的获得造血干细胞的方法,首次建立了利用三维诱导体系和/或联合骨髓细胞等基质细胞和/或多种因子等高效诱导多能干细胞分化为造血干细胞的系统,为获得临床可用的造血干细胞提供了理论基础和技术平台,并为多能干细胞来源的造血细胞在疾病机理探索、药物筛选等领域的应用开拓了新的方法和新的思路。
附图说明
本发明中所用附图以水凝胶联合小鼠骨髓、OP9DL1和细胞因子的三维诱导系统这组实验数据为代表,进行说明。
图1是多能干细胞来源的造血干细胞三维诱导分化方案。
图2 是多能干细胞来源的造血干细胞三维诱导分化系统联合细胞因子的方案。
图3是造血干细胞三维诱导分化体系中第1天的细胞形态(4X)。
图4是造血干细胞三维诱导分化系统中第10-14天的细胞形态(10X)。
图5是造血干细胞三维诱导分化系统中第10-14天出现的葡萄状细胞克隆(20X)。
图6是造血干细胞三维诱导分化体系中第28天的细胞形态的变化(4X)。
图7是造血干细胞三维诱导分化体系中第28天出现类T细胞(20X)。
图8是造血干细胞三维诱导分化体系中第28天中出现的类造血集落的细胞形态(10X)。
图9是造血干细胞三维诱导分化体系中第28天中出现的类造血集落的细胞形态(10X)。
图10是造血干细胞三维诱导分化体系中诱导培养10-14天后人的细胞总量(以抗体TRA-1-851检测)以及人CD34+细胞比例检测。
图11是不同造血干细胞三维诱导分化系统中诱导培养10-14天时各组获得的CD34+细胞的比例。
图12是利用CD34+细胞磁珠分选技术分选造血干细胞三维诱导分化系统第10天后的CD34+细胞(20X)。
图13是流式细胞仪技术检测三维诱导系统后CD34阳性细胞的比例。
图14是造血干细胞三维诱导分化体系中获得的人CD34+细胞用甲基纤维素培养获得造血集落粒系集落, 粒系/巨噬系(4X)。
图15是造血干细胞三维诱导分化体系中获得的人CD34+细胞用甲基纤维素培养获得造血集落粒系集落, 粒系/巨噬系(4X)。
图16是造血干细胞三维诱导分化体系中获得的人CD34+细胞用甲基纤维素培养获得造血集红系集落, 混合系包括红系/粒系/巨噬系/巨核系的集落(4X)。
具体实施方式
本发明结合附图和实施例作进一步的说明。
实施例1 三维诱导系统将多能干细胞诱导分化为血细胞的制备。
本发明提供了利用三维细胞培养材料,包括水凝胶、海藻、纳米材料等制成的三维细胞培养材料,和/或联合骨髓基质细胞,和/或联合小鼠基质细胞系OP9或OP9-DL1,和/或联合多种中胚层诱导因子和造血相关因子,诱导多能干细胞分化为造血干细胞的方法。具体方案参见图1和图2。
1.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。
2.多能干细胞的培养、扩增和传代
①滋养层MEF制备(购自上海斯丹赛生物技术有限公司):按常规方法进行。
②将冻存的胚胎干细胞和多能干细胞复苏,种植在丝裂霉素C处理过的滋养层上,用多能干细胞专用培养基培养,包括DMEM/F12, 20% 血清替代物(KnockOut SerumReplacement,KSR), 2mM L-谷氨酰胺的衍生物GlutaMAX, 0.1mM β-巯基乙醇(β-mercaptoethanol), 1%非必需氨基酸(nonessential amino acids, NEAA), 50 U/mL 青霉素, 50 mg/mL链霉素和4ng/mL碱性成纤维细胞生长因子(basic fibroblast growthfactor, bFGF),37°C, 5% CO2的培养箱中培养。每天换液。每天观察克隆的生长状态。克隆长至一定大小,四型胶原酶消化,传代,或者备用。
3.基质细胞制备
①小鼠骨髓细胞的制备:购买小鼠(种类、年龄和性别不限),断颈处死。75%酒精浸泡5-8分钟,带入无菌室。解剖台上固定,用高温灭菌过的手术器械,如剪刀、手术镊子等,剪开腿部毛皮,将整个腿骨取下,放入无菌生理盐水中。带入超净台,去除腿骨上携带的肌肉等。将骨两端剪开,用1ml注射器吸取生理盐水,冲洗骨髓腔。离心收集骨髓,红细胞裂解液裂解红细胞5-7分钟。生理盐水洗涤2-3次,收集细胞,备用。
②人骨髓细胞的制备。经志愿者的同意,签署知情同意书,髂骨取1-2ml骨髓。带入无菌室,红细胞裂解液裂解红细胞5-7分钟。生理盐水洗涤2-3次,收集细胞,备用。
③胎盘细胞的制备。经家属的同意,签署知情同意书,取4cm×4cm胎盘组织,放入含有抗生素(抗青霉素和抗链霉素)的无菌生理盐水中。带入无菌室,生理盐水冲洗5-8次,无菌剪刀剪碎成操作1mm×1mm的组织碎块,加入0.25%的胰酶/乙二胺四乙酸中,37度消化15-20分钟,消化过程中,每隔4-5分钟震荡一次。用含有胎牛血清的培养基终止消化,过200目网筛,收集细胞,备用。
④OP9、OP9-DL1的复苏、培养和传代。
将冻存的OP9、OP9-DL1从液氮中取出,2-3分钟快速解冻,放入培养皿中培养,培养基为含有20%优质胎牛血清(Ausgenex)的α-MEM培养基。每3天换液一次。待细胞长至80-90%融合状态,0.25%的胰酶/乙二胺四乙酸消化4-6分钟,待细胞变形时,轻轻震荡,细胞从皿中脱落。用含有胎牛血清的α-MEM培养基终止消化,离心,收集细胞,1:3传代培养,或者待用。
4.三维细胞培养基质或细胞培养支架的制备
根据厂家提供的说明书,将三维细胞培养材料,包括水凝胶、海藻、纳米材料等进行处理。以Beaver Nano™ 3D细胞培养水凝胶为代表,阐述水凝胶三维材料的制备。
BeaverNano™水凝胶主要成份是多肽类生物纳米材料。在PH=7.0左右的条件下,可自组装成网状纳米的立体支架,其孔径为50-200nm,形态类似天然的细胞外基质(ECM),可以为细胞的粘附、分化或增殖提供更接近体内的外环境。该水凝胶具有良好的生物相容性,降解产物为天然氨基酸,不含潜在污染物或致病因子。具体使用步骤如下:
①用超声波水浴锅(或涡旋震荡仪)处理水凝胶30分钟以降低水凝胶原液的粘性。
②将1%(重量/体积:w/v)水凝胶原溶液和20%(w/v)的无菌蔗糖原溶液配制成2×水凝胶工作液。备用。
5.利用三维细胞培养基质或细胞培养支架建立高效诱导多能干细胞分化为造血干细胞的系统
①四型胶原酶消化多能干细胞。在多能干细胞联合基质细胞的实验组中,胰蛋白酶消化OP9、OP9-DL1等目的细胞,离心,弃上清。将多能干细胞与基质细胞混合,用10%(w/v)的无菌蔗糖重悬目的细胞。离心,收集细胞。
②取适量细胞重新悬浮在20%的无菌蔗糖工作溶液中,调整细胞密度为4×106个细胞/ml。配制成2×工作浓度的细胞悬浮液。
③等体积混合2×水凝胶工作液和2×细胞悬浮液。
④沿着培养孔边缘轻轻将含有15%胎牛血清(Hyclone)的IMDM(Iscove’sModified Dulbecco’s Mediums)培养基细胞培养液注入到凝胶上部,细胞培养液中的盐离子会促使水凝胶自组装成纳米纤维结构的水凝胶。
⑤将培养板放于37℃培养箱中静置30~60分钟待其凝固。
⑥水凝胶凝固期间,小心地用200μL移液枪移去2/3~3/4体积的培养液,随后再次更换培养液,在30分钟内更换2次。
⑦在含有诱导因子的实验组中,培养基中加入相应的诱导因子。第一阶段,诱导液为含有10ng/ml BMP4、100ng/ml SCF、100ng/ml FLT3L、20ng/ml TPO、1ng/ml VEGF、2μMPGE2、 15%胎牛血清的IMDM,诱导5-7天。然后换成第二阶段的培养液,含有100ng/ml SCF、100ng/ml FLT3L、20ng/ml IL-3、20ng/ml IL-6、20ng/ml TPO、1ng/ml VEGF、2μM PGE2的无血清造血干细胞扩增培养基SFEM(STEMCELL TECHNOLOGY)。12-14天后,换用第三阶段的培养液,为含有20ng/ml SCF、20ng/ml IL-3、20ng/ml IL-6、20ng/ml GM-CSF、20ng/ml TPO、20ng/ml EPO、10ng/ml IL-7、10ng/ml IL-2、10%胎牛血清的IMDM,继续培养至21-28天。在整个诱导过程中,每天观察诱导系统中细胞形态的变化。细胞形态学的变化参见图3-图9,分别展示了三维诱导系统中第一天、第十至十四天、第二十八天的细胞形态学的动态变化过程。
6.流式细胞仪技术检测三维诱导系统中人的细胞(以TRA-1-85为检测指标)、造血干细胞指标CD34+造血干细胞的表达情况。
具体方法如下:
①分阶段收集各组处理的三维培养细胞,用枪头将三维材料轻轻吹打,收集细胞,0.25%胰酶/乙二胺四乙酸消化4-5分钟,轻轻吹打,用含有胎牛血清的培养基终止消化,过200目网筛。
②用不含Ca2+ 和 Mg2+的PBS(加入1% FBS和1mM 乙二胺四乙酸,以供细胞营养并防止细胞粘连)洗涤2-3次,1000转,5分钟。收集细胞,制备单细胞悬液,密度为106-107细胞/ml。
③各个实验组中,取20μl 细胞悬液,加入5μl相应的鼠抗人抗体,包括TRA-1-85-PE、CD34-PE-Cy5,室温避光孵育30min,同型IgG作为对照;用不含Ca2+ 和 Mg2+的PBS(加入1% FBS和1mM 乙二胺四乙酸)洗涤3次,500μl PBS(加入1% FBS和1mM 乙二胺四乙酸)重悬细胞,BD FACScalibur instrument (Becton Dickinson)检测三维诱导系统中人的细胞以及多种血细胞包括造血干细胞表面抗原表达情况。
④获得的数据用FlowJo Version 7.2.5的软件进行分析,分析三批相关样本。
结果参见图10-图11,分别显示的是水凝胶三维细胞培养材料联合小鼠骨髓、OP9DL1和细胞因子系统中TRA-1-85+、CD34+细胞表达情况。
7.富集造血干细胞
三维诱导系统中造血干细胞的富集是使用加拿大干细胞技术有限公司(STEMCELLTechnologies)EasySep 人CD34分选试剂盒。由于前期流式细胞仪分选结果显示诱导系统中CD34%起始百分比>20%,所以分选步骤进行了优化,具体如下:
①收集诱导10-14天的三维培养系统中的细胞,用枪头轻轻吹打三维材料,1200转,6分钟,收集细胞;
②用含有乙二胺四乙酸0.25%的胰酶消化5-6分钟,轻轻吹打,用含有胎牛血清的培养基终止消化,过85-目网筛;
③1200转,6分钟,收集细胞;
④用不含Ca2+ 和 Mg2+的PBS(加入1% FBS和1mM 乙二胺四乙酸)洗涤2-3次;
⑤将细胞调至1x108 细胞/毫升;
⑥按照200uL/mL 细胞比例,加入EasySep CD34分选试剂盒中的抗体;
⑥室温下孵育15分钟;
⑦加入磁珠 100微升/毫升细胞;
⑧室温下孵育10分钟;
⑨孵育后将细胞用缓冲液稀释至2.5毫升,放在磁极中,先做1x10分钟的分选,然后做2个5分钟的过磁极的分选;
⑩按照步骤(4)进行流式细胞仪的检测,检查获得的细胞形态学以及其表达CD34的情况。检测CD34的抗体克隆号为8G12藻红蛋白(PE)标记。图12是分选后的细胞形态学检测;图13是流式细胞仪检测分选出的C造血干细胞的纯度。
8.三维诱导系统中获得的CD34细胞在甲基纤维素半固体培养成生成造血集落(CFU)的潜能检测
①将分选后的CD34细胞,按照 5000个细胞/ml 种植于含有9% 甲基纤维素,0.1mM2-巯基乙醇, 2mM GlutaMAX, 20ng/mL SCF, 20ng/mL IL-3, 20ng/mL IL-6, 20ng/mL G-CSF ,20ng/mL GM-CSF , 20ng/mL TPO, 3U/mL EPO的伊思柯夫改良培养液(Iscove'sModified Dubecco's Medium,IMDM)的低粘附的培养皿中,继续培养 12-14天。
②观察集落形成情况。诱导14-17天左右,培养系统中有明显的集落形成。根据集落的形状,细胞体积大小,可见的细胞密度程度,判断、检测和比较各个造血细胞集落形成情况,并计数。结果参见图14-图16。图14是三维诱导系统中分选CD34细胞在含有造血生长因子的甲基纤维素半固体培养基中,培养14-17天左右,产生的粒系、粒系/巨噬系造血集落。图15是三维诱导系统中分选CD34细胞在含有造血生长因子的甲基纤维素半固体培养基中,培养14-17天左右,产生的巨核系、瀑式红系造血集落。图16是三维诱导系统中分选CD34细胞在含有造血生长因子的甲基纤维素半固体培养基中,培养14-17天左右,产生的红系、粒系/红系/巨噬系/巨核系造血集落。
Claims (2)
1.一种利用三维诱导系统获得造血干细胞的方法,其特征在于,通过以下技术方案实现:
利用三维诱导系统,根据细胞因子功能的不同,分阶段高效诱导多能干细胞分化获得造血干细胞,经过10-14天的诱导和扩增,获得高于40%的造血干细胞,所述的多能干细胞包括人胚胎干细胞和诱导性多能干细胞, 所述的三维诱导系统选择多种三维细胞培养基质或细胞培养支架与诱导分化因子和生长因子,所述的多种三维细胞培养基质或细胞培养支架选择由水凝胶、海藻材料制成的三维细胞培养材料,其中在三维细胞培养基质或细胞培养支架中混合基质细胞,所述基质细胞选择人或者小鼠骨髓细胞、胎盘细胞、小鼠骨髓细胞系OP9、OP9-DL1;所述的人胚胎干细胞选择从商业途径获得的人胚胎干细胞系;
其中第一阶段选择的细胞因子为10ng/ml BMP4、100ng/ml SCF、100ng/ml Flt3L、1ng/ml VEGF、20ng/ml TPO、2μM PGE2;第二阶段选择的细胞因子为造血因子和促进造血干细胞增值的细胞因子100ng/ml SCF、100ng/ml Flt3L、20ng/ml IL-3、20ng/ml IL-6、20ng/ml TPO、1ng/ml VEGF、2μM PGE2;第三阶段选择的细胞因子为促进造血细胞扩增和分化的细胞因子20ng/ml SCF、20ng/ml IL-3、20ng/ml IL-6、10ng/ml IL-7、10ng/ml IL-2、20ng/ml GM-CSF、20ng/ml EPO、20ng/ml TPO。
2.根据权利要求1所述的一种利用三维诱导系统获得造血干细胞的方法,其特征在于,诱导性多能干细胞选择利用含有转录因子Oct4,ox2,Klf4和/或c-Myc四种因子的逆转录病毒、慢病毒、腺病毒或仙台病毒方式诱导人体细胞获得的诱导性多能干细胞。
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