CN116355832A - 尿液上皮样细胞来源的iPSCs诱导分化制备人胰腺β细胞的方法和用途 - Google Patents
尿液上皮样细胞来源的iPSCs诱导分化制备人胰腺β细胞的方法和用途 Download PDFInfo
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Abstract
本发明属于生物技术领域,具体涉及一种尿液上皮样细胞来源的iPSCs诱导分化制备人胰腺β细胞的方法和用途。本发明提供了一种尿液上皮样细胞来源的iPSCs诱导分化制备人胰腺β细胞的方法。通过提取健康人尿液中的尿液上皮样细胞并进行培养扩增,再通过病毒感染的方式重编程,获得克隆的UiPSCs,再将克隆的UiPSCs在特定的培养基中进行诱导培养,通过诱导培养基和诱导培养方式的共同作用,在诱导14天后,得到能冻存和复苏的胰腺祖细胞。在诱导22天后,得到具有胰岛细胞类似功能的类β细胞,并能对葡萄糖刺激做出响应,体内移植不成瘤,安全性好。
Description
技术领域
本发明属于生物技术领域,具体涉及一种尿液上皮样细胞来源的iPSCs诱导分化制备人胰腺β细胞的方法和用途。
背景技术
I型糖尿病(Type 1 diabetes,T1D)又被称为胰岛素依赖型糖尿病(Insulin-dependent diabetes mellitus,IDDM),是一种慢性的多因素自身免疫疾病,其发病机制是免疫系统攻击自身胰岛,导致胰腺中分泌胰岛素的胰岛β细胞受损或死亡,最终导致高血糖症。
临床上对于T1D的治疗主要集中实体胰腺器官移植、胰岛移植和外源胰岛素注射三个方面。胰腺器官和胰岛移植面临的问题不仅仅是供体来源缺乏,费用昂贵,更主要是无法避免体内的免疫排斥反应。因此,供体组织的短缺和免疫抑制药物的使用(这些药物通常对β细胞有毒性)限制了人胰岛移植的广泛应用。重组胰岛素的合成和给药方式的进步从根本上降低了糖尿病相关疾病的发病率和死亡率,尽管该治疗方式能有效改善糖代谢,提高患者生存质量,但这种治疗不能模拟胰腺β细胞的实时胰岛素分泌模式,导致仍有部分患者遭受心血管疾病、糖尿病肾病、视网膜病变和神经病变等并发症,而不得不接受其他临床治疗。
因此,发展可再生的胰岛素分泌细胞来源以此重建血糖控制能力,对T1D的临床治疗具有重大意义。利用hESCs/iPSCs定向诱导分化获取功能性胰岛β细胞并进行体内细胞移植治疗是重要的发展方向,但大部分获取体细胞的方式都具有侵入性,同时面临着取材困难、费用昂贵等问题。但如何以非侵入性的方式获取iPSCs、如何进一步提高β细胞的诱导效率有待解决的重要科学和技术问题。
发明内容
本发明要解决的技术问题为:现有利用hESCs/iPSCs定向诱导分化获取功能性胰岛β细胞的方法取材困难、费用昂贵,并且有创、安全性不高的问题。
本发明解决上述技术问题的技术方案为:提供了一种尿液上皮样细胞来源的iPSCs诱导分化制备人胰腺β细胞的方法。该方法包括以下步骤:
a、尿液上皮样细胞的获取、培养、扩增;
提取尿液,离心,初始培养基重悬,并于30-40℃下培养2-5天,之后加入RE/MC扩增培养基培养3-15天,直到出现贴壁的克隆样的尿液上皮样细胞团,待细胞汇合度达90%时,胰蛋白酶消化,胰酶抑制剂终止,继续扩增培养及传代,得到P1代和P2代的尿液上皮样细胞;
b、重编程获取iPSCs;
获取小鼠胚胎成纤维细胞MEF,并进行辐照;
培养HEK-293T细胞,待密度达80-90%时,将感染复合物滴加入其中进行感染,感染后继续培养4-6小时,更换成含5%-20%的胎牛血清的DMEM培养基,继续培养12-36小时24小时;
将步骤a得到的尿液上皮样细胞消化后,按照3000个-10000个细胞/cm2的密度均匀接种至0.1%-0.5%明胶包被的板中;收集含各病毒颗粒的HEK-293T细胞培养上清,过滤,各上清等比例混合后,加入聚凝胺,将病毒混合液加入到含尿液上皮样细胞的六孔板中;重复上述步骤进行二次感染,感染12-36小时后将尿液上皮样细胞的培养基更换为RE/MC扩增培养基;
将辐照后的MEF细胞培养,待感染病毒后的尿液上皮样细胞达到80-90%时,用胰蛋白酶消化,并用ESC/dFBS培养基终止消化、重悬,接种至含MEF的板中,在ESC/dFBS培养基中加入转化生长因子β的I型受体抑制剂、糖原合成酶激酶-3抑制剂、新型的Rho相关激酶抑制剂、环状抑制剂-α氢溴酸盐和丁酸钠,于第2天、第4天、第6天及第8天对重编程后的细胞进行换液;采用mTeSR培养基于第10天、第12天、第14天及第16天进行换液;从第18天开始每天用mTeSR培养基进行换液,培养5-7天,得到克隆的UiPSCs;
c、3D悬浮诱导分化iPSCs为类β细胞;
消化重悬iPSCs细胞,将iPSCs单细胞进行接种,加入Y27632,诱导培养24小时,记为第0天,第1天采用诱导分化培养基A培养;第2-3天,采用诱导分化培养基B培养;第4-6天,采用诱导分化培养基C培养;第7-9天,采用诱导分化培养基D培养;第10天,采用诱导分化培养基E培养;第11-14天,采用诱导分化培养基F培养;第15-22天,采用诱导分化培养基G培养,直至培养得到类β细胞。
其中,上述尿液上皮样细胞来源的iPSCs诱导分化制备人胰腺β细胞的方法中,步骤a所述离心共进行3次,条件分别为:第1次室温400g,离心10分钟;第2、3次室温200g,离心10分钟。
其中,上述尿液上皮样细胞来源的iPSCs诱导分化制备人胰腺β细胞的方法中,步骤a所述的初始培养基组成包括:DMEM/F12基础培养基,10%胎牛血清,2%的肾上皮细胞培养基和0.2%的原代细胞抗生素。采用上述初始培养基,可使尿液中的上皮样细胞能快速的生长和出现。
其中,上述尿液上皮样细胞来源的iPSCs诱导分化制备人胰腺β细胞的方法中,步骤a所述的RE/MC扩增培养基组成包括:REGM基础培养基与高糖DMEM培养基按体积比1:1的比例混合后,添加5%胎牛血清、0.5%谷氨酰胺、0.5%非必需氨基酸、100U/mL青霉素-链霉素、5%肾上皮细胞培养基和终浓度均为5ng/mL的碱性成纤维细胞生长因子、血小板衍生细胞生长因子AB、表皮生长因子。
其中,上述尿液上皮样细胞来源的iPSCs诱导分化制备人胰腺β细胞的方法中,步骤a所述的胰蛋白酶浓度为0.25%。
其中,上述尿液上皮样细胞来源的iPSCs诱导分化制备人胰腺β细胞的方法中,步骤a所述的扩增培养时每天使用RE/MC扩增培养基进行半量换液。
其中,上述尿液上皮样细胞来源的iPSCs诱导分化制备人胰腺β细胞的方法中,步骤b所述的感染采用的复合物的制备方法为:在150μL的Opti-MEM培养基中依次加入5μg包装质粒AMPHO和5μg目的质粒,在另外150μL的Opti-MEM培养基中加入30μL的PEI溶液,两者混合后,室温静置15-20分钟,得到感染复合物。
进一步的,所述的目的质粒包括:PMXs-Oct4,PMXs-Sox2,PMXs-Klf4和PMXs-cMyc。
进一步的,所述的PEI溶液的制备方法为:每10mg的PEI粉末,溶解于9mL水中,充分溶解后调节pH至6.9-7.1,定容至10mL。
其中,步骤b中所述的mTeSR培养基中在加入转化生长因子β的I型受体抑制剂、糖原合成酶激酶-3抑制剂、新型的Rho相关激酶抑制剂、环状抑制剂-α氢溴酸盐和丁酸钠的基础上,还加入了分裂原活化抑制剂的抑制剂。
其中,上述尿液上皮样细胞来源的iPSCs诱导分化制备人胰腺β细胞的方法中,步骤c所述诱导分化培养基A组成包括:RPIM1640基础培养基,含0.2%胎牛血清、100U/mL青霉素-链霉素、1%谷氨酰胺、0.02%胰岛素-转铁蛋白-硒-氨基乙醇、100ng/mL激活素A以及50ng/mL Wnt3A。
其中,上述尿液上皮样细胞来源的iPSCs诱导分化制备人胰腺β细胞的方法中,步骤c所述诱导分化培养基B组成包括:RPIM1640基础培养基,含0.2%胎牛血清、100U/mL青霉素-链霉素、1%谷氨酰胺、0.02%胰岛素-转铁蛋白-硒-氨基乙醇、100ng/mL激活素A。
其中,上述尿液上皮样细胞来源的iPSCs诱导分化制备人胰腺β细胞的方法中,步骤c所述诱导分化培养基C组成包括:DMEM/F12基础培养基,含2%胎牛血清、100U/mL青霉素-链霉素、0.1% ITS-X和50ng/mL角质细胞生长因子。
其中,上述尿液上皮样细胞来源的iPSCs诱导分化制备人胰腺β细胞的方法中,步骤c所述诱导分化培养基D组成包括:DMEM基础培养基,含1% B27添加剂、1%谷氨酰胺、100U/mL青霉素-链霉素、0.25μM环巴胺、2μM视黄酸及50ng/mL指头蛋白。
其中,上述尿液上皮样细胞来源的iPSCs诱导分化制备人胰腺β细胞的方法中,步骤c所述诱导分化培养基E组成包括:DMEM基础培养基,含1% B27添加剂、1%谷氨酰胺、100U/mL青霉素-链霉素、50ng/mL角质细胞生长因子、50ng/mL表皮细胞生长因子、50ng/mL指头蛋白和100nM视黄酸。
其中,上述尿液上皮样细胞来源的iPSCs诱导分化制备人胰腺β细胞的方法中,步骤c所述诱导分化培养基F组成包括:DMEM基础培养基,含1% B27添加剂、1%谷氨酰胺、100U/mL青霉素-链霉素、1μM间变性淋巴瘤激酶2型抑制剂、50nM苯并内酰胺衍生的可渗透细胞的蛋白激酶C活化物、100nM视黄酸和100ng/mL指头蛋白。
其中,上述尿液上皮样细胞来源的iPSCs诱导分化制备人胰腺β细胞的方法中,步骤c所述诱导分化培养基G组成包括:DMEM基础培养基,含1%非必需氨基酸、1% B27添加剂、1%谷氨酰胺、100U/mL青霉素-链霉素、10μM间变性淋巴瘤激酶2型抑制剂、500nM选择性骨形态发生蛋白I型受体抑制剂、1μMγ分泌酶抑制剂二苯并氮卓、1μM T3三碘甲状腺原氨酸、0.5mM维生素C、1mM乙酰半胱氨酸、10μM硫酸锌和10ug/mL硫酸类肝素。
进一步的,还发明还提供了一种由上述方法诱导分化得到的人胰腺β细胞。
本发明还提供了一种上述人胰腺β细胞在制备人工仿生胰岛中的用途。
本发明还提供了上述人胰腺β细胞、人工仿生胰岛在制备预防或治疗糖尿病的药物中的用途。
进一步的,上述用途中,所述的糖尿病为I型糖尿病。
本发明的有益效果为:
本发明提供了一种尿液上皮样细胞来源的iPSCs诱导分化制备人胰腺β细胞的方法,采用无创的方式获取人尿液来源的上皮样细胞,将其重编程为iPSCs,并首次通过体外3D悬浮诱导分化培养,模拟了体内胚胎期的胰腺发育模式,高效地获得能分泌人胰岛素的类β细胞。本发明诱导得到的胰腺祖细胞能冻存和复苏,类β细胞能对葡萄糖刺激做出响应,体内移植不成瘤,安全性好。
附图说明
图1所示为尿液中上皮样细胞的分离、培养及扩增(比例尺:100μm)。
图2所示为绿色荧光蛋白(GFP)逆转录病毒感染尿液上皮样细胞的效果(比例尺:100μm)。
图3所示为尿液上皮样细胞感染Oct4、Sox2、Klf4及c-Myc逆转录病毒混合液后的形态变化及iPSCs的扩增(比例尺:100μm)。
图4所示为3D悬浮培养不同阶段细胞的形态(比例尺:200μm)。
图5所示为免疫荧光鉴定胰腺祖细胞中PDX1和NKX6.1的表达(比例尺:50μm)。
图6所示为胰腺祖细胞冻存及复苏后的细胞形态(比例尺:50μm)。
图7所示为胰腺祖细胞冻存及复苏后的活率统计(*P≤0.05)。
图8所示为流式检测类β细胞中C肽单阳性细胞、C肽和GCG双阳性细胞、NKX6.1单阳性细胞及C肽和NKX6.1双阳性细胞比例。
图9所示为透射电子显微镜观察胰岛素颗粒结构(比例尺:200nm)。
图10所示为类β细胞的静态GSIS实验,分别在不同葡萄糖浓度刺激下检测类β细胞的胰岛素分泌量(***P≤0.001)。
图11所示为类β细胞移植体内不成瘤。
具体实施方式
本发明提供了一种尿液上皮样细胞来源的iPSCs诱导分化制备人胰腺β细胞的方法。通过提取健康人尿液中的尿液上皮样细胞并进行培养扩增,再通过病毒感染的方式重编程,获得克隆的UiPSCs,再将克隆的UiPSCs在特定的培养基中进行诱导培养,通过诱导培养基和诱导培养方式的共同作用,在诱导22天后,得到具有胰岛细胞类似功能的类β细胞。
本发明模拟了体内胚胎期的胰腺发育模式,能够高效地获得能分泌人胰岛素的类β细胞,得到的类β细胞能对葡萄糖刺激做出响应,具有胰岛细胞相似的功能,能够应用在I型糖尿病的细胞治疗中。本发明的方法能够进行产业化生产,能够冻存和复苏,可以制备成细胞制剂使用,使用方便。并且体内移植不成瘤,安全性好。
本发明的关键在于通过病毒感染重编程,获得大量克隆的UiPSCs,这是将尿液上皮样细胞转化成类β细胞的重要一环。此外,本发明在诱导培养阶段,为了更好的模拟体内的转化环境,经过大量实验筛选,最终得到了先“加入Y27632,诱导培养24小时,记为第0天,第1天采用诱导分化培养基A培养;第2-3天,采用诱导分化培养基B培养;第4-6天,采用诱导分化培养基C培养;第7-9天,采用诱导分化培养基D培养;第10天,采用诱导分化培养基E培养;第11-14天,采用诱导分化培养基F培养;第15-22天,采用诱导分化培养基G培养,直至培养得到类β细胞”的技术方案,本发明能成功诱导得到类β细胞,离不开各诱导培养基的配合,其中每种培养基的组成,和其中添加的小分子物质的种类和剂量,均是发明人经过大量的实验筛选才最终得到。尤其是诱导分化培养基F和G,更是经过了长期的实验摸索才最终确定的效果最理想的培养基,将其更换为其他培养基时,将不能诱导得到最终的类β细胞。
下面将通过实施例对本发明的具体实施方式做进一步的解释说明,但不表示将本发明的保护范围限制在实施例所述范围内。
实施例所用到的仪器和试剂均为普通市售产品。本申请中尿液样本捐献者的纳入标准为:1)不携带乙肝、梅毒、艾滋、结核等传染性疾病;2)不具有肿瘤、结缔组织疾病、全身免疫性疾病;3)此前未接受过干细胞治疗。
实验已获四川大学华西医院医学伦理委员会批准。
实施例1尿液上皮样细胞的获取、培养及扩增
1.尿液上皮样细胞的获取
具体的操作步骤如下:
1)收集中段尿液100-200mL于一次性容器中,置于冰上,在4小时内进行处理;
2)将尿液转移至50mL的离心管中,室温400g,离心10分钟;
3)弃掉上清,使离心管中剩余约1mL左右的液体;
4)重悬剩余的1mL尿液样品,并将其转移至新的50mL离心管中;
5)向离心管中加入10mL洗涤缓冲液(含100U/mL青霉素-链霉素的磷酸盐缓冲液),室温200g,离心10分钟;
6)弃掉上清,再加入10mL洗涤缓冲液,室温200g,离心10分钟;
7)弃掉上清,使离心管中剩余约0.2mL左右液体,加入1mL初始培养基进行重悬;
初始培养基的组成为:在DMEM/F12基础培养基中加入10%胎牛血清,2%的1×REGMTM SinglequotsTM Supplement Pack(中文名称:肾上皮细胞培养基,品牌为Lonza,货号CC-3190以及0.2%primocin(原代细胞抗生素)。
8)将重悬后的液体转移至预先包被了0.2%明胶溶液的12孔板中,37℃培养24小时,此时记为第0天;
9)每天加入1mL初始培养基,连续加入3天;
10)第4天时,孔板中约有4mL液体,小心弃掉3mL,并加入1mL RE/MC扩增培养基;
RE/MC扩增培养基的组成为:将REGM基础培养基与高糖DMEM培养基按照1:1的比例混合后,添加5%胎牛血清、0.5%谷氨酰胺、0.5%非必需氨基酸、100U/mL青霉素-链霉素、5%1×REGMTM SinglequotsTM Supplement Pack以及终浓度均为5ng/mL的bFGF(碱性成纤维细胞生长因子)、PDGF-AB(血小板衍生细胞生长因子AB)和EGF(表皮生长因子),配制好的培养基于4℃避光保存,并在2周内使用。
11)接下来每天使用RE/MC扩增培养基进行半量换液,直到孔板中出现贴壁的克隆样的尿液上皮样细胞团。
2.尿液上皮样细胞的培养及扩增
具体的操作步骤如下:
1)待12孔板中出现尿液上皮样细胞的克隆时,每天利用RE/MC扩增培养基进行换液;
2)当尿液上皮样细胞密度可传代时(细胞汇合度达90%以上),利用0.25%的胰蛋白酶进行消化,并使用胰酶抑制剂(DTI,购自Invitrogen,货号R-007-100)进行终止,将消化后的细胞铺板至新的孔板中进行扩增培养,此时的尿液上皮样细胞记为P1代;
3)继续利用RE/MC扩增培养基对P1代的尿液上皮样细胞进行扩增和传代,记为P2代,用于后续重编程实验,其余尿液上皮样细胞可利用CELLBANKER2细胞冻存液(购自ZENOAQ)进行保种。
我们收集了健康人尿液100-200mL,通过两次离心后使用初始培养基重悬沉淀的细胞团并将其接种到包被了0.2%明胶的12孔板中,记为第0天,此时视野中悬浮的细胞大多数为鳞状细胞及少量的血细胞。接下来我们用初始培养基继续培养3天后更换为扩增培养基,在接种第12天时孔板中出现了贴壁的细胞集落,该集落外观规则、具有边缘光滑的轮廓和鹅卵石状的细胞形态。每天更换扩增培养基,并对出现的细胞集落进行扩增培养到第20天,此时细胞密度达到90%以上的汇合度,整体细胞呈羽翼状或涡旋状排列(如图1所示)。上述实验结果共同说明了,本实验经扩增培养的尿液上皮样细胞状态良好。
实施例2重编程获取iPSCs
具体的操作步骤如下:
1、滋养层细胞的获取
1)分离怀孕13.5天的ICR小鼠子宫,在含有100U/mL青霉素-链霉素的PBS中漂洗3遍;
2)从子宫中将每个胚胎剥离开,去掉胚胎的头部、四肢、内脏组织及生殖腺;
3)将剩余的胚胎组织用预冷PBS漂洗3遍,进一步去除血渍及其他杂质;
4)用无菌剪刀将胚胎组织块剪碎;
5)按照每个小鼠胚胎1mL的量加入0.25%含EDTA的胰蛋白酶,在37℃培养箱中消化8分钟,期间每隔2分钟摇晃一次,直到消化液变浑浊;
6)加入两倍体积的含10%胎牛血清的DMEM培养基(购自Gibco,货号:11965092)终止消化;
7)用移液器上下多次缓慢吹吸消化产物,使组织块进一步分散成均一的悬液;
8)在4℃条件下,将上述悬液以300g的转速离心5分钟;
9)弃去上清,用含10%胎牛血清的DMEM培养基进行重悬;
10)将细胞计数后,将1×106个细胞(P0代)接种至0.2%明胶包被的10cm小平皿中,放在含5% CO2的37℃培养箱中培养;
11)当细胞密度达到90%以上汇合度时,按照22Gray的剂量对小鼠胚胎成纤维细胞(MEF)进行辐照;
12)将辐照后的细胞进行计数后铺板,继续培养后再进行传代及保种。
2、重编程获取iPSCs
1)复苏HEK-293T细胞(购自ATCC)至10cm的平皿中进行扩增培养,待细胞密度达到90%以上时进行消化传代,并将细胞铺板至6孔板中,摇晃均匀后置于含5% CO2的37℃培养箱中培养;
2)同时培养实施例1传代得到的P1-P3代尿液上皮样细胞,待用;
3)将HEK-293T细胞培养18-24小时后,观察细胞密度,待密度达到80-90%时开始准备感染复合物;
4)准备感染复合物前,将HEK-293T细胞的培养基更换为预热的不含血清的DMEM基础培养基;
5)准备1个1.5mL的EP管,加入150μL的Opti-MEM培养基(购自Gibco,货号31985070),依次加入5μg包装质粒AMPHO(购自淼灵质粒;货号P1257)和5μg目的质粒(购自淼灵质粒;如PMXs-Oct4,货号:P0937,PMXs-Sox2货号:P0936,PMXs-Klf4货号:P0938,PMXs-cMyc货号P0939);
6)再准备1个1.5mL的EP管,加入150μL的Opti-MEM培养基后加入30μL的PEI(聚乙烯亚胺)溶液;PEI溶液制备方法为:称取10mg PEI粉末,加入到9mL的超纯水中,将其在室温溶解5-10分钟后逐滴加入0.1N NaOH调节pH至6.9-7.1,用超纯水定量至10mL,最后用0.22μm滤器过滤除菌。
7)将两个1.5mL EP管中的溶液均匀混合,配制感染复合物,室温静置15分钟;
8)将上述感染复合物均匀滴加至HEK-293T细胞的培养皿中,混匀后将细胞放入培养箱,继续培养6小时;
9)6小时后,将HEK-293T细胞的培养基更换为预热的含10%胎牛血清的DMEM培养基,继续培养24小时;
10)将尿液上皮样细胞消化后,按照7000个细胞/cm2的密度均匀接种至0.2%明胶包被的6孔板中;
11)收集含病毒颗粒的HEK-293T细胞培养上清,用0.45μm的滤器进行过滤以除去细胞碎片;
12)将Oct4、Sox2、Klf4、c-Myc四种逆转录病毒上清等比例混合后,加入终浓度为8μg/mL的polybrene(聚凝胺),将病毒混合液按照8mL/孔的量加入到含尿液上皮样细胞的六孔板中;
13)继续添加预热的含10%胎牛血清的DMEM培养基于HEK-293T细胞中,培养24小时;
14)24小时后再次收取病毒上清,按照上述方法进行第二次感染;
15)第二次感染24小时后将尿液上皮样细胞的培养基更换为新鲜的RE/MC扩增培养基;
16)将辐照后的MEF(实施例2中,步骤1,滋养层细胞的获取步骤中得到的产物)作为滋养层细胞,并以18000个/cm2的密度进行铺板,置于含5% CO2的37℃培养箱中培养;
17)感染完病毒后的尿液上皮样细胞达到80-90%的汇合度时,用0.25%的胰蛋白酶进行消化,并用ESC/dFBS培养基终止消化,将细胞计数后按照200g转速离心5分钟,再用ESC/dFBS培养基进行重悬,按照1500个/cm2的密度接种至含MEF的6孔板中,记为第0天;
ESC/dFBS培养基组成为:将DMEM/F12基础培养基(厂家Gibco,货号10565042)与高糖DMEM基础培养基(厂家Gibco,货号11965092)按照1:1的比例混合,添加10%血清替代物(KSR)(厂家Gibco,货号10828010)、10%胎牛血清(厂家Gibco,货号10091)、1%非必须氨基酸(厂家Gibco,货号11140050)、1%谷氨酰胺(厂家Gibco,货号25030081)、100U/mL青霉素-链霉素(厂家Gibco,货号15070063)、100μMβ-巯基乙醇(厂家Sigma,货号M3148)及10ng/mLbFGF(碱性成纤维生长因子)。
18)在ESC/dFBS培养基中添加A 83-01(转化生长因子β的I型受体抑制剂,MCE,HY-10432)、CHIR99021,(糖原合成酶激酶-3抑制剂,Selleck,S1263
)、Thiazovivin(新型的Rho相关激酶抑制剂,Selleck,S1459)、CyclicPifithrin-αhydrobromide(环状抑制剂-α氢溴酸盐,Selleck,S5791)及Sodium butyrate(丁酸钠,Selleck,S1999),并在第2天、第4天、第6天及第8天对重编程后的细胞进行换液;
19)在mTeSR培养基(Stem Cell,货号85850)中添加A 83-01、CHIR99021、Thiazovivin、Cyclic Pifithrin-αhydrobromide、Sodium butyrate及PD0325901(分裂原活化抑制剂的抑制剂,Selleck,S1036)6种化合物,并在第10天、第12天、第14天及第16天进行换液;
20)从第18天开始每天用mTeSR培养基进行换液,直至UiPSCs克隆出现;
21)挑取边界清晰,克隆致密的细胞进行传代扩增。
我们将状态良好的尿液来源的上皮样细胞扩增到P1-P3代,并以7000个/cm2的密度接种到0.2%明胶包被的培养皿中。考虑到病毒质量对iPSCs的重编程至关重要,在制备逆转录病毒Oct4、Sox2、Klf4及c-Myc的同时,我们首先使用绿色荧光蛋白(GFP)逆转录病毒感染尿液上皮样细胞用于评价感染效果。在感染36小时后,在荧光显微镜下观察尿液上皮样细胞GFP的表达情况,结果显示逆转录病毒可以很好地感染尿液上皮样细胞(如图2所示)。
接下来,我们收集OSKM的逆转录病毒混合液并对尿液来源的上皮样细胞进行2次感染,当细胞汇合度达到90%时,将其以1500个/cm2的密度传代至MEF滋养层上,并用ESC/dFBS培养基培养2天,在ESC/dFBS培养基中添加A-83-01、CHIR99021、Thiazovivin、Cyclicpifithrin-a及Sodium butyrate继续培养8天,最后在添加了A-83-01、CHIR99021、Thiazovivin、Cyclic pifithrin-a、Sodium butyrate及PD0325901的mTeSR培养基中再培养8天以提高重编程的效率。当重编程完成时,与未感染病毒的尿液上皮样细胞相比,感染了O,S,K,M的病毒组中出现了致密的边界清晰的细胞克隆。随后,我们挑取了具有典型特征的细胞克隆并将其接种到经MEF铺板的培养皿中,记为iPSCs-P0代。在后续的研究中,我们将iPSCs接种到基质胶包被的培养皿中并进行了多次传代(iPSCs-P2-P7),所得到的iPSCs克隆致密、边界清晰、折光性强且核质比高,具有典型的胚胎干细胞(HES-H9)的克隆特征(如图3所示)。
实施例3 3D悬浮诱导分化iPSCs为类β细胞
具体的操作步骤如下:
1)使用10cm的培养皿对iPSCs进行扩大培养;
2)将汇合度在90%以上的iPSCs用Accutase细胞解离剂(Gibco,货号:A1110501)进行消化;
3)加入Accutase细胞解离剂3倍体积的mTeSR培养基终止消化,用移液枪轻轻吹打细胞为单细胞悬液,将单细胞悬液按照1500rpm的转速离心3分钟;
4)用mTeSR培养基重悬细胞沉淀后进行计数;
5)按照每毫升1×106个细胞的密度将iPSCs单细胞接种于低粘附6孔板中,6孔板每个孔的体系为5.5mL,并加入10μM Y27632(选择性ROCK1和ROCK2抑制剂,Selleck,S1049);
6)将低粘附6孔板置于细胞培养摇床上,摇床转速为95rpm,在5% CO2,37℃的培养箱中培养24小时,记为诱导第0天;
7)24小时后,低粘附6孔板中的单细胞悬液形成了大小均一的直径为100-200μm大小的球状体,收集球状体至50mL离心管中;
8)让球状体自然沉降至离心管底部;
9)配制第1天诱导分化培养基(RPIM1640含0.2%胎牛血清、100U/mL青霉素-链霉素、1%谷氨酰胺、0.02% ITS-X、100ng/mL Activin A以及50ng/mL Wnt3A);
其中,RPIM1640购自Gibco,货号11875119;ITS-X:胰岛素-转铁蛋白-硒-氨基乙醇,购自Gibco,货号51500056;Activin A:激活素A,购自Peprotech,货号120-14P-50;Wnt3A:经典Wnt/β-catenin信号通路中的重要配体,购自R&D,货号5036-WN-010/CF。
10)吸走50mL离心管中大部分培养基,此时球状体聚集在离心管底部,并用适量含钙、镁的PBS将球状体重悬,轻微吹打球状体,使其重悬充分;
11)让球状体在含钙、镁的PBS中自然沉降;
12)尽可能吸走上清,加入5.5mL的第1天诱导分化培养基,将球状体重悬后转移至新的低粘附6孔板中,将低粘附6孔板置于摇床,继续按照95rpm的转速在5% CO2,37℃的培养箱中培养24小时;
13)收集球状体使其自然沉降,配制第2-3天的诱导分化培养基(RPIM1640含0.2%胎牛血清、100U/mL青霉素-链霉素、1%谷氨酰胺、0.02% ITS-X以及100ng/mL ActivinA);
14)吸掉离心管中约5mL左右的上清,并补充5mL新鲜配制的第2天诱导分化培养基将球状体进行重悬;
15)转移球状体悬液至低粘附6孔板中继续诱导分化培养,后续每天均按该方式进行换液;
16)配制第4-6天诱导分化培养基(DMEM/F12含2%胎牛血清、100U/mL青霉素-链霉素、0.1% ITS-X以及50ng/mL KGF(角质细胞生长因子),每天对球状体进行换液;
17)配制第7-9天诱导分化培养基(DMEM含1% B27添加剂细胞培养添加剂B27、1%谷氨酰胺、100U/mL青霉素-链霉素、0.25μM Cyclopamine-KAAD环巴胺、2μM视黄酸及50ng/mL Noggin(指头蛋白,骨形态发生蛋白的拮抗剂),调整摇床转速为105rpm,每天对细胞进行换液;上述诱导分化培养基能促使细胞从前肠向后前肠进行分化。
18)配制第10天诱导分化培养基(DMEM含1% B27添加剂、1%谷氨酰胺、100U/mL青霉素-链霉素、50ng/mL KGF、50ng/mL EGF、50ng/mL Noggin以及100nM视黄酸),对球状体进行换液;上述诱导分化培养基能促进位于后前肠阶段的细胞向胰腺祖细胞进行分化。
19)配制第11-14天诱导分化培养基(DMEM含1% B27添加剂、1%谷氨酰胺、100U/mL青霉素-链霉素、1μM ALKi II间变性淋巴瘤激酶2型抑制剂、50nM TPB苯并内酰胺衍生的可渗透细胞的蛋白激酶C活化物、100nM视黄酸及100ng/mL Noggin),调整摇床转速为95rpm,每天换液;上述诱导分化培养基能诱导分化出胰腺祖细胞。
20)配制第15-22天诱导分化培养基(DMEM含1%非必需氨基酸、1% B27添加剂、1%谷氨酰胺、100U/mL青霉素-链霉素、10μM ALKi II、500nM LDN193189选择性骨形态发生蛋白I型受体抑制剂、1μMγ分泌酶抑制剂XX二苯并氮卓、1μM T3三碘甲状腺原氨酸、0.5mMVitamin C维生素C、1mM N-acetyl Cysteine乙酰半胱氨酸、10μM Zinc sulfate硫酸锌、10ug/mL Herparin sulfate硫酸类肝素),调整摇床转速至100rpm,每天换液,以形成类β细胞。该培养基促使胰腺祖细胞向类β细胞进行分化。
对不同诱导阶段的细胞进行观察,在诱导24小时后,iPSCs可形成直径大小约100-200μm,边缘光滑的球状体结构。在诱导第4天时,球状体的边缘不再光滑,具有明显的出芽状结构;随后在向胰腺祖细胞形成的过程中,球状体中会出现明显的肠样褶皱,并且部分球状体大小不均一、形态不规则;但在最后诱导分化为类β细胞时,球状体的边缘又会再次变得光滑,大小较为均一且形态规则(如图4所示)。
我们对诱导到胰腺祖细胞阶段的球状体进行鉴定,结果发现胰腺祖细胞身份相关的标志性指标PDX1胰十二指肠同源框因子-1和NKX6.1同源盒蛋白NK-6同源物A的阳性率达到了70-80%左右(如图5所示)。说明了胰腺祖细胞(类β细胞的上一个阶段细胞)占70-80%以上,说明本发明的诱导效率高达70-80%。
实施例4诱导得到的胰腺祖细胞冻存与复苏实验
对实施例3步骤19得到的胰腺祖细胞进行冻存和复苏实验,以考察本发明的诱导方法是否可产业化实施。
胰腺祖细胞冻存的具体操作步骤如下:
1)收集胰腺祖细胞球状体,置于50ml离心管中,室温静置3分钟;
2)待胰腺祖细胞球状体因重力作用沉降到离心管底部时,吸走离心管中大部分的上清培养基(即第11-14天的诱导分化培养基);
3)用不含钙镁离子的磷酸盐缓冲液洗涤胰腺祖细胞球状体;
4)尽可能吸走残留的磷酸盐缓冲液;
5)向含有胰腺祖细胞球状体沉淀的离心管中加入CELLBANKER冻存液3mL,并轻轻重新重悬球状体;
6)将重悬后的球状体按照每管1ml的体系分装至冻存管中;
7)将冻存管放置在梯度降温盒中,并置于-80℃冰箱2天;
8)将冻存管转移至液氮罐中进行长期保存。
冻存后胰腺祖细胞复苏的具体操作如下:
1)从液氮罐中取出冻存管;
2)将冻存管快速置于37℃水浴锅中2分钟,使液体融化;
3)准备15ml离心管,并在离心管中加入3ml第11-14天的诱导分化培养基;
4)重悬冻存管中的细胞悬液,并缓慢滴加至步骤3)中的离心管中;
5)将离心管按照300g的转速离心3分钟;
6)离心完成后,弃去上清,用第11-14天的诱导分化培养基再次重悬胰腺祖细胞沉淀;
7)将重悬后的胰腺祖细胞球状体悬液铺板至新的低粘附6孔板中继续培养。
我们发现3D悬浮培养的胰腺祖细胞可冻存与复苏(如图6所示),复苏后的胰腺祖细胞状态良好,细胞形态和结构与冻存前无显著差异。为了检测复苏后胰腺祖细胞的细胞活性,我们将冻存前后的胰腺祖细胞球消化成单细胞,进行台盼蓝染色和细胞活率统计,结果发现与冻存前的细胞活率(97.21%±1.463)相比,复苏后的胰腺祖细胞的活率有所下降,但细胞的活率仍然在80%以上(82.88%±4.836)(如图7所示)。这些结果证明3D悬浮培养的胰腺祖细胞可冻存和复苏,为未来细胞治疗临床转化应用奠定了基础。
实施例5类β细胞的诱导效率及功能鉴定
我们利用流式细胞术,检测了类β细胞的诱导效率。
操作步骤如下:
1)将类β细胞用Accutase细胞解离剂(Gibco,A1110501)消化8分钟后,用PBS重悬,制备成单细胞悬液,按照每管100μL的体系分装至流式管中;
2)向流式管中加入3μL流式抗体,每种抗体3μL(一管单独加C肽抗体、一管同时加C肽和同源盒蛋白NK-6同源物A抗体,一管同时加C肽和胰高血糖素抗体,一管单独加同源盒蛋白NK-6同源物A抗体),混匀后在冰上避光孵育30分钟;
3)用PBS重悬细胞,在4℃条件下,按照500g的转速离心3分钟,弃去上清,重复此步骤3次;
4)轻轻倒掉上清,用100μL PBS重悬细胞沉淀,在流式细胞仪上进行操作分析。
结果如图8所示:可见C肽阳性的细胞比例在80%以上。C肽是β细胞分泌的产物,与胰岛素具有共同的前体,实验中C肽的表达说明了我们诱导分化的类β细胞具有和人胰岛β细胞相似的功能,阳性细胞比例在80%以上,说明我们的诱导的效率高。
同时可见,诱导产物中几乎不表达GCG,其阳性比例不足1%。说明诱导分化的产物不含有α细胞和多激素细胞。
此外,我们利用透射电镜观察到了诱导产物具有典型的胰岛素颗粒结构(如图9所示),胰岛素颗粒颜色的深浅代表了不同的成熟度,颜色越深表征其成熟度越高。图9说明了诱导产物的成熟度很高。
为了进一步验证我们体外诱导的类β细胞是否具有葡萄糖响应的功能,我们收集诱导产物并在体外进行了葡萄糖刺激胰岛素分泌(Glucose stimulates insulinsecretion,GSIS)实验。
具体的步骤如下:
1)所有样品用Krebs-Ringer-HEPES(KRH)/BSA缓冲液漂洗三次;Krebs-Ringer-HEPES(KRH)/BSA:称量23.83mg乙烷磺酸、5.3676mg氯化钾、2.4648mg 7水硫酸镁、163.632mg氯化钠、3.3604mg碳酸氢钠、1.1998mg磷酸2氢钠以及7.644mg氯化钙粉末至18mL超纯水中,并加入0.1N氢氧化钠调节PH至7.4,再用超纯水定容至20mL后用0.22μm滤器过滤除菌,先制备出Krebs-Ringer-HEPES(KRH)缓冲液,随后称量20mg牛血清白蛋白溶解至10mLKrebs-Ringer-HEPES(KRH)溶液中,使其终浓度为0.2%w/v,制备出Krebs-Ringer-HEPES(KRH)/BSA缓冲液。
1)用Krebs-Ringer-HEPES(KRH)/BSA缓冲液覆盖样品,并置于培养箱中平衡30分钟;
2)移除缓冲液,用含2.8mM葡萄糖的缓冲液覆盖样品,并在培养箱中孵育1小时;
3)收集上清后,将样品移至含有20mM葡萄糖的缓冲液中,再在培养箱中孵育1小时,再次收集上清;
4)在-20℃的条件下,将样品置于-酸/乙醇溶液中过夜;
5)使用ELISA试剂盒(厂家Mercodia,货号10-1132-01)对收集的样本进行检测。
结果如图10所示,显示:在低糖浓度下(2mM),类β细胞所分泌的胰岛素浓度很低,而在高糖浓度刺激下(20mM),类β细胞能分泌高水平的胰岛素,且与低糖刺激下的胰岛素分泌量相比具有显著性的差异,证明诱导产生的类β细胞具有葡萄糖响应功能,本发明诱导得到的是具有胰岛细胞类似功能的功能型的类β细胞。
实施例6类β细胞的移植体内的安全性实验
为了进一步验证诱导不同阶段的细胞移植体内是否具有成瘤性。我们将iPSCs、胰腺祖细胞以及类β细胞按照5×106个/只的剂量移植到SCID小鼠的肾包膜中,进行为期3个月的成瘤性观察。结果发现:移植iPSCs组的4只小鼠中有2只因畸胎瘤形成过大导致小鼠在移植未满3个月时便死亡,畸胎瘤组织在体内与其他脏器相互粘连坏死无法获取,而剩余2只小鼠在移植完成3个月后其畸胎瘤直径均达到了2cm左右。移植胰腺祖细胞的小鼠中,发现有部分形成了畸胎瘤组织,但畸胎瘤的直径均在0.2-0.5cm左右。接受类β细胞移植的小鼠则未观察到任何畸胎瘤的形成(如图11所示)。上述实验结果共同说明了,通过本申请的方法诱导得到的类β细胞移植小鼠体内不成瘤,生物安全性高。
我们利用3D悬浮培养方法,首次将健康人尿液上皮样细胞来源的iPSCs按照定向内胚层、原始肠管、后前肠、胰腺祖细胞以及胰岛细胞的顺序,进行体外细胞定向诱导分化,获得了能冻存和复苏的胰腺祖细胞以及80% C肽阳性的类β细胞,类β细胞具有胰岛素颗粒的典型结构特征,能对葡萄糖刺激做出响应,体内移植不成瘤,安全性好。
Claims (10)
1.尿液上皮样细胞来源的iPSCs诱导分化制备人胰腺β细胞的方法,其特征在于,包括以下步骤:
a、尿液上皮样细胞的获取、培养、扩增;
提取尿液,离心,初始培养基重悬,并于30-40℃下培养2-5天,之后加入RE/MC扩增培养基培养3-15天,直到出现贴壁的克隆样的尿液上皮样细胞团,待细胞汇合度达90%时,胰蛋白酶消化,胰酶抑制剂终止,继续扩增培养及传代,得到P1代和P2代的尿液上皮样细胞;
b、重编程获取iPSCs;
获取小鼠胚胎成纤维细胞MEF,并进行辐照;
培养HEK-293T细胞,待密度达80-90%时,将感染复合物滴加入其中进行感染,感染后继续培养4-6小时,更换成含5%-20%的胎牛血清的DMEM培养基,继续培养12-36小时24小时;
将步骤a得到的尿液上皮样细胞消化后,按照3000个-10000个细胞/cm2的密度均匀接种至0.1%-0.5%明胶包被的板中;收集含各病毒颗粒的HEK-293T细胞培养上清,过滤,各上清等比例混合后,加入聚凝胺,将病毒混合液加入到含尿液上皮样细胞的六孔板中;重复上述步骤进行二次感染,感染12-36小时后将尿液上皮样细胞的培养基更换为RE/MC扩增培养基;
将辐照后的MEF细胞培养,待感染病毒后的尿液上皮样细胞达到80-90%时,用胰蛋白酶消化,并用ESC/dFBS培养基终止消化、重悬,接种至含MEF的板中,在ESC/dFBS培养基中加入转化生长因子β的I型受体抑制剂、糖原合成酶激酶-3抑制剂、新型的Rho相关激酶抑制剂、环状抑制剂-α氢溴酸盐和丁酸钠,于第2天、第4天、第6天及第8天对重编程后的细胞进行换液;采用mTeSR培养基于第10天、第12天、第14天及第16天进行换液;从第18天开始每天用mTeSR培养基进行换液,培养5-7天,得到克隆的UiPSCs;
c、3D悬浮诱导分化iPSCs为类β细胞;
消化重悬iPSCs细胞,将iPSCs单细胞进行接种,加入Y27632,诱导培养24小时,记为第0天,第1天采用诱导分化培养基A培养;第2-3天,采用诱导分化培养基B培养;第4-6天,采用诱导分化培养基C培养;第7-9天,采用诱导分化培养基D培养;第10天,采用诱导分化培养基E培养;第11-14天,采用诱导分化培养基F培养;第15-22天,采用诱导分化培养基G培养,直至培养得到类β细胞。
2.根据权利要求1所述的尿液上皮样细胞来源的iPSCs诱导分化制备人胰腺β细胞的方法,其特征在于:步骤a所述的RE/MC扩增培养基组成包括:REGM基础培养基与高糖DMEM培养基按体积比1:1的比例混合后,添加5%胎牛血清、0.5%谷氨酰胺、0.5%非必需氨基酸、100U/mL青霉素-链霉素、5%肾上皮细胞培养基和终浓度均为5ng/mL的碱性成纤维细胞生长因子、血小板衍生细胞生长因子AB、表皮生长因子。
3.根据权利要求1所述的尿液上皮样细胞来源的iPSCs诱导分化制备人胰腺β细胞的方法,其特征在于:步骤b所述的感染采用的复合物的制备方法为:在150μL的Opti-MEM培养基中依次加入5μg包装质粒AMPHO和5μg目的质粒,在另外150μL的Opti-MEM培养基中加入30μL的PEI溶液,两者混合后,室温静置15-20分钟,得到感染复合物。
4.根据权利要求3所述的尿液上皮样细胞来源的iPSCs诱导分化制备人胰腺β细胞的方法,其特征在于:所述的目的质粒包括:PMXs-Oct4,PMXs-Sox2,PMXs-Klf4和PMXs-cMyc;所述的PEI溶液的制备方法为:每10mg的PEI粉末,溶解于9mL水中,充分溶解后调节pH至6.9-7.1,定容至10mL。
5.根据权利要求1所述的尿液上皮样细胞来源的iPSCs诱导分化制备人胰腺β细胞的方法,其特征在于:步骤b中所述的mTeSR培养基中在加入转化生长因子β的I型受体抑制剂、糖原合成酶激酶-3抑制剂、新型的Rho相关激酶抑制剂、环状抑制剂-α氢溴酸盐和丁酸钠的基础上,还加入了分裂原活化抑制剂的抑制剂。
6.根据权利要求1所述的尿液上皮样细胞来源的iPSCs诱导分化制备人胰腺β细胞的方法,其特征在于:步骤c所述诱导分化培养基F组成包括:DMEM基础培养基,含1%B27添加剂、1%谷氨酰胺、100U/mL青霉素-链霉素、1μM间变性淋巴瘤激酶2型抑制剂、50nM苯并内酰胺衍生的可渗透细胞的蛋白激酶C活化物、100nM视黄酸和100ng/mL指头蛋白。
7.根据权利要求1所述的尿液上皮样细胞来源的iPSCs诱导分化制备人胰腺β细胞的方法,其特征在于:步骤c所述诱导分化培养基G组成包括:DMEM基础培养基,含1%非必需氨基酸、1%B27添加剂、1%谷氨酰胺、100U/mL青霉素-链霉素、10μM间变性淋巴瘤激酶2型抑制剂、500nM选择性骨形态发生蛋白I型受体抑制剂、1μMγ分泌酶抑制剂二苯并氮卓、1μM T3三碘甲状腺原氨酸、0.5mM维生素C、1mM乙酰半胱氨酸、10μM硫酸锌和10ug/mL硫酸类肝素。
8.权利要求1-7中任一项所述的方法诱导得到的人胰腺β细胞。
9.权利要求8所述的人胰腺β细胞在制备人工仿生胰岛中的用途。
10.权利要求8所述的人胰腺β细胞、权利要求9所述的人工仿生胰岛在制备预防或治疗糖尿病的药物中的用途。
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