CN112980771B - 一种制备胰腺beta细胞的方法及其应用 - Google Patents
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Abstract
本发明涉及一种制备分化的胰腺beta细胞的方法及其应用,具体的,所述方法包括如下步骤:(1)多能干细胞的三维悬浮驯化培养;(2)诱导驯化后的细胞分化为胰腺beta细胞。本发明还涉及由所述的分化的胰腺beta细胞制备海藻酸钠‑聚赖氨酸‑海藻酸钠(APA)微囊化的人工胰岛的方法,所述方法为:使用海藻酸钠溶液制备微囊化的人工胰岛。本发明还涉及所述的胰腺beta细胞及人工胰岛的应用。
Description
技术领域
本发明属于医药生物技术领域,具体的,涉及一种制备胰腺beta细胞的方法及其应用。
背景技术
糖尿病是胰岛功能减退或衰竭而导致机体糖代谢紊乱的疾病。根据国际糖尿病联盟(International Diabetes Federation,IDF)的最新统计,2019年,全球超过4.63亿人患有糖尿病,到2045年将有7亿人患糖尿病;中国以1.16亿人糖尿病患者位居世界第一,到2045年预计将达到1.47亿人。2019年,全球约有420万人(20-79岁)死于糖尿病或其并发症,每6秒就有1人死于糖尿病,约占全球全死因死亡的11.3%。
1型糖尿病占糖尿病总数的5-10%,是由beta细胞数量和功能的完全丧失引起的。目前治疗手段主要是胰岛素长期注射治疗和胰岛移植,胰岛素注射在根本上无法治愈糖尿病及并发症;新型的胰岛素泵也有携带不方便、血糖控制不稳定和给病人带来很多痛苦的劣势。另外,胰岛移植策略中胰岛的提取与纯化过程非常复杂,器官资源的匮乏以及如何高效稳定地获得能满足临床移植要求的胰岛也是胰岛移植的主要挑战。针对以上问题,开发干细胞治疗技术,用以重新恢复体内原本缺失的胰腺beta细胞是根治糖尿病的更好选择(Pagliuca FW等,2013)。
目前有关干细胞分化为beta细胞的方法主要包括:(1)基因转染方法,是将控制胚胎向胰腺发育的多个转录因子PDX-1,NKX6.1,Ngn3,NeuroD,Pax4等转染至细胞中,使其诱导分化为胰岛素分泌细胞(Noguchi H等,2006)。然而,基因转染方法存在潜在致癌风险,它是利用病毒载体将外源基因***细胞基因组中,可能会使抑癌基因失活。除此之外,该方法获得的胰岛素分泌细胞并不成熟,不能够很好的对葡萄糖的刺激做出响应。(2)细胞因子诱导法,该方法是根据胚胎向胰腺的发育过程,在体外模拟胰腺发育中的关键步骤,将干细胞诱导分化为胰腺beta细胞或胰腺祖细胞(Pagliuca FW等,2014;Rezania A等,2014;KroonE等,2008)。然而该方法步骤复杂,分化时间较长;而且不成熟的分化方法容易带来分化批次稳定性较差,细胞得率较低,规模化生产受限等问题。但开发出成熟稳定的细胞因子诱导分化方法是能够很好的攻克以上问题的,这也可以在体外获得稳定的、规模化的、成熟的功能性胰腺beta细胞。
另外,移植成熟的胰腺beta细胞至异体环境中,往往会受到异体的免疫排斥,进而使得移植的beta细胞丧失原有的功能。目前有关异体移植过程中解决免疫排斥问题的主要策略是联合免疫抑制剂的使用,但免疫抑制效果往往不太理想且面临着终生服药的痛苦。最新的策略显示开发安全有效的免疫隔离工具成为异体移植过程中解决免疫排斥问题的更好选择(Omid Veiseh等,2015;Arturo J Vegas等,2016;Daniel G Anderson等,2016)。免疫隔离工具中所用包裹材料的选择是移植成功的关键,由于受不同包裹材料的影响,分化所得到的beta细胞体内功能评价效果差异也比较明显。目前常用的包裹材料包括:(1)天然材料。脂质、多糖和蛋白质;(2)半合成材料。纤维素类衍生物等;(3)合成材料。包括可降解性和非降解性材料。其中许多材料移植体内会出现严重的纤维化包裹、无免疫隔离功能等问题,严重影响到包裹细胞的活性和功能。然而,开发出一种理想的包裹材料便可以解决上述异种移植过程中存在的问题。
因此,多能干细胞衍生的胰腺beta细胞有望通过细胞替代策略治疗糖尿病,但如何更好地规模化稳定的在体外获得成熟的胰腺beta细胞并且解决异体来源胰腺beta细胞移植后出现的免疫排斥问题一直是研究热点。
发明内容
本发明涉及一种多能干细胞(PSC,人胚胎干细胞或人诱导多能干细胞)三维悬浮定向分化为成熟胰腺beta细胞的方法,所述方法包括如下步骤:
(1)多能干细胞的三维悬浮驯化培养;
(2)诱导驯化后的细胞分化为胰腺beta细胞;
步骤(1)所述的多能干细胞的三维悬浮驯化培养的步骤包括:
1)Y27632预处理,优选的,在悬浮培养前2小时将平面培养的多能干细胞进行换液,更换为含10μM浓度的Y27632的mTeSR1培养基;
2)使用DMEM/F12培养基清洗中和预处理细胞并对细胞进行计数;
3)在mTeSR1+Y27632培养基中搅拌培养所述多能干细胞,在mTeSR1+Y27632培养基中传代5-10次,每天更换一半培养基,传代时去除培养上清中的单细胞和聚集的大细胞团块;
优选的,所述的多能干细胞的接种密度为3-8*105/mL,使用一次性生物反应器(Disposable spinner flasks,Corning,3152/3153)进行三维搅拌悬浮培养,参数为:50-120rpm转速、恒温37℃、5%CO2、100%湿度,所述培养基为含10μM浓度的Y27632的mTeSR1培养基;
优选的,所述去除大细胞团块为使用400μm筛网过滤去除大于400μm的细胞团块;
4)检测驯化后的多能干细胞,细胞呈球状悬浮生长且Oct4+/SSEA4+双阳细胞比例在95%以上即为驯化合格;
步骤(2)所述的诱导驯化后的细胞分化和微囊化的步骤包括:
所使用的培养基为:
S1培养基:MCDB131基础培养基+1:10000-100000胰岛素-转铁蛋白-硒-乙醇胺(ITS-X)+1-5mM谷氨酰胺,必要的缓冲盐(NaHCO3)、抗生素、抗氧化剂(VC)、葡萄糖及血清白蛋白;
S2培养基:MCDB131基础培养基+ITS-X 1:10000-1:100000+1-5mM谷氨酰胺,必要的缓冲盐(NaHCO3)、抗生素、抗氧化剂(VC)、葡萄糖及血清白蛋白;
S3/S4培养基:MCDB131基础培养基+ITS-X 1:50-1:500+1-5mM谷氨酰胺,必要的缓冲盐(NaHCO3)、抗生素、抗氧化剂(VC)、葡萄糖及血清白蛋白;
S5培养基:MCDB131基础培养基+ITS-X 1:50-1:500+1-5mM谷氨酰胺+2-20μg/mL肝素钠盐,必要的缓冲盐(NaHCO3)、抗生素、抗氧化剂(VC)、葡萄糖及血清白蛋白;
诱导分化方法具体为:
使用步骤(1)驯化的多能干细胞作为分化的起始种子细胞
1)每毫升mTeSR1培养基(培养基中添加10μM Y27632)中接种0.3-0.7*106个散开的三维悬浮驯化的多能干细胞,培养24-72小时后,更换首日培养基,换液时体积减少10-20%,
首日培养基为:S1培养基中添加:50-200ng/ml重组人激活素A(Activin A)、10-100ng/mL重组人Wnt3a蛋白;
2)第2日更换第2日培养基,换液时培养基体积不变;
第2日培养基为:S1培养基中添加:50-200ng/ml Activin A;
3)分别于第4、6日更换第4日培养基,换液时培养基体积不变;
第4日培养基为:S2培养基中添加:20-100ng/ml重组人角化细胞生长因子蛋白(KGF)、1-5μM转化生长因子-βRI激酶抑制剂IV(TGF-βRI Kinase Inhibitor IV);
4)分别于第7、8日更换第7日培养基,换液时培养基体积不变;
第7日培养基为:S3/S4培养基中添加:20-100ng/ml KGF、0.1-0.5μM Sant1、1-5μM视黄酸(RA)、100-500nM 1,1,4,4-四苯基-1,3-丁二烯(TPB)、5-20μM Y27632;
5)分别于第9、11、13日更换第9日培养基,换液时培养基体积不变;
第9日培养基为:S3/S4培养基中添加:20-100ng/ml KGF、0.1-0.5μM Sant1、50-200nM RA、10-100ng/mL EGF、10-100ng/mL重组人头蛋白(NOG)、5-20μM Y27632;
6)分别于第14、16日均更换第14日培养基,换液时培养基体积不变;
第14日培养基为:S5培养基中添加:0.1-0.5μM Sant1、50-200nM RA、0.5-2μMγ-分泌酶抑制剂XXI(γ-Secretase Inhibitor,XXI)、5-20μM RepSox、0.5-2μM三典甲状腺氨酸(L-3,3',5-Triiodothyronine,T3)、5-50ng/ml重组人β细胞素(Recombinant HumanBetacellulin Protein)、50-500nM LDN193189盐酸盐(LDN193189hydrochloride)、10μM硫酸锌;
7)分别于第18、20日更换第18日培养基,换液时培养基体积不变;
第18日培养基为:S5培养基中添加:10-50nM RA、0.5-2μM XXI、5-20μM RepSox、0.5-2μM T3、5-50ng/ml Betacellulin、50-500nM LDN193189、1mM N-cys(Fmoc-N-Me-Cys(Trt)-OH);
8)第21天时,分化细胞使用TrypLE Express消化为单细胞,然后按照0.5-2*106细胞/毫升密度重新接种于反应器中,50-120rpm转速进行培养,培养箱的参数设置为恒温37℃,5%CO2和100%湿度,21-35天时,每两天更换一次S3培养基,培养过程中,使用10-50μm可逆滤器将重聚集的正常细胞团收集,弃去未聚团的上清,即得分化的胰腺beta细胞。
可选的,在诱导分化方法中,还可以包括任一或任意组合的如下步骤:
9)在上述分化步骤3)之前,可进行分化的CD177阳性的群的富集和培养,具体方法是:
取分化阶段细胞团,消化为单细胞,然后分选出CD177阳性的细胞亚群,然后将分选出细胞以2-10×105/mL的密度接种在含有10μM Y27632的上述第4日培养基中继续进行分化;
10)在上述分化步骤6)之前,可进行GP2阳性的群的富集和培养,具体方法是:
取分化阶段细胞团,消化为单细胞,然后分选出GP2阳性的细胞亚群,然后将分选出细胞以5-10×105/mL的密度接种在上述第14日培养基中继续进行分化;
11)在上述分化步骤8)之前,可进行Procr阳性的群的富集和培养,具体方法是:
取分化阶段细胞团,消化为单细胞,然后分选出Procr阳性的细胞亚群,富集的Proc阳性细胞每7-14天进行一次传代,在每次传代过程中,消化的Procr阳性单细胞悬液均以新鲜的human HUVEC细胞补充,细胞数比为1:1;然后混合Procr阳性细胞和human HUVEC细胞两种细胞,以1:4-1:6的比例重新接种培养,可使得分化的细胞得率显著提高;
12)在上述分化步骤8)的培养基中,可添加100ng/mLWNT4,可驱动分化细胞葡萄糖刺激的胰岛素分泌所必需的代谢成熟。
本发明还涉及由所述的分化的胰腺beta细胞制备海藻酸钠-聚赖氨酸-海藻酸钠(APA)微囊化的人工胰岛的方法,所述方法包括:
(1)海藻酸钠溶液的配制:溶解后的海藻酸钠依次经过0.8μm、0.45μm和0.22μm的PES无菌过滤装置过滤;检测溶液粘度为50-200cP,4℃保存;
(2)按方法一或方法二制备微囊化人工胰岛:
方法一:微流控法:
将1%-3%的海藻酸钠溶液与所述胰腺beta细胞混合,1mL海藻酸钠溶液与1-10*106细胞混合;
混合好后加入其中一个管道,另外一个管道加入0.1-2g/L氯化钙溶液;
在经过1-10cm的收集管进行交联成为凝胶,随后钙化5-30分钟;
加入0.01-0.1%的聚赖氨酸进行反应10-30分钟;
再加入0.1%-0.3%的海藻酸钠溶液反应2-10分钟;
再加入10-100mM的柠檬酸钠反应2-10分钟即形成APA微胶囊;
方法二:高压静电法:
将1%-3%的海藻酸钠溶液与所述胰腺beta细胞混合,1mL海藻酸钠溶液与1-10*106细胞混合;
混合好后使用20-40G针头的注射器吸入,在5-20KV电压、1-10脉冲、50-200Hz、流速100-1000μL/min的条件下,使得其形成射流进入0.1-2g/L氯化钙溶液中交联成为凝胶,随后钙化5-30分钟;
加入0.01-0.1%的聚赖氨酸进行反应10-30分钟;
再加入0.1%-0.3%的海藻酸钠溶液反应2-10分钟;
再加入10-100mM的柠檬酸钠反应2-10分钟即形成APA微胶囊。
本发明还涉及所述的胰腺beta细胞或人工胰岛在制备药物中的应用,所述的药物为治疗糖尿病的药物,优选的,所述的糖尿病为Ⅰ型糖尿病。
本发明还涉及所述的胰腺beta细胞或人工胰岛在制备细胞治疗术中的治疗活性成分中的应用,所述的细胞治疗术为针对糖尿病患者的细胞治疗术,优选的,所述的糖尿病为Ⅰ型糖尿病。
本发明还涉及内胚层祖细胞系的诱导方法,所述方法包括如下步骤:
(1)多能干细胞的三维悬浮驯化培养;
(2)内胚层祖细胞系的诱导;
步骤(1)所述的多能干细胞的三维悬浮驯化培养步骤与所述多能干细胞三维悬浮定向分化为成熟胰腺beta细胞的方法的相关步骤相同;
步骤(2)所述的内胚层祖细胞系的诱导的方法为:
使用步骤(1)驯化的多能干细胞作为分化的起始种子细胞
1)每毫升mTeSR1培养基(培养基中添加10μM Y27632)中接种0.3-0.7*106个散开的三维悬浮驯化的多能干细胞,培养24-72小时后,更换首日培养基,换液时体积减少10-20%,
首日培养基为:S1培养基中添加:50-200ng/ml重组人激活素A(Activin A)、10-100ng/mL重组人Wnt3a蛋白;
2)第2日更换第2日培养基,换液时培养基体积不变;
第2日培养基为:S1培养基中添加:50-200ng/ml Activin A;
3)分别于第4、6日更换第4日培养基,换液时培养基体积不变;
第4日培养基为:S2培养基中添加:20-100ng/ml重组人角化细胞生长因子蛋白(KGF)、1-5μM转化生长因子-βRI激酶抑制剂IV(TGF-βRI Kinase Inhibitor IV);
4)流式检测SOX17阳性细胞比例>90%时,取细胞团消化为单细胞,分选出CXCR4+/CD117+双阳性的细胞亚群(常见的,分化到第6日,即能够进行CXCR4+/CD117+双阳性的细胞亚群分选),此细胞亚群即为定向内胚层祖细胞;
5)将该群细胞接种在含有Matrigel的平板里,使用定向内胚层祖细胞培养和扩增培养基进行培养扩增;
所述定向内胚层祖细胞培养和扩增培养基的成分为:
S1培养基中添加:20-100ng/mL骨形态发生蛋白4(BMP4)、5-20ng/mL重组人碱性成纤维细胞生长因子蛋白(bFGF)、5-20ng/mL重组人血管内皮生长因子蛋白(VEGF)、5-20ng/mL重组人表皮细胞生长因子蛋白(EGF)。
本发明的有益效果在于:
本发明的目的在于针对现有分化的beta细胞批次稳定性及体内功能评价不佳的缺陷,提供了一种在体外获得成熟的、批次稳定的规模化三维悬浮定向分化beta细胞的方法。同时提供一种理想的包裹材料微囊化包裹分化的beta细胞的方法,使微囊化的beta细胞在动物体内很好的行使功能且无免疫排斥和纤维化包裹问题。具体包括(1)多能干细胞(包括诱导多能干细胞和胚胎干细胞)的三维规模化悬浮培养;(2)使用化合物组合体外三维悬浮动态定向分化为成熟胰腺beta细胞;(3)beta细胞的微囊化包裹;(4)微囊化beta细胞的体内外功能评价。
与现有技术相比,本发明提供的方法具有包括如下多方面的显著优势:本发明提供的PSC三维悬浮培养的方法可成功的维持PSC多能性基因的表达,细胞增殖很好,细胞核型稳定,特别是平面细胞在上3D反应器之前需更换为加Y27632新鲜培养基预处理细胞2-4h,合适的初始细胞接种密度、培养体系、转速等均显著增加PSC三维悬浮培养的成功率。三维培养过程中的细胞换液方式(半换液),上清单细胞处理方式、大于400μm大细胞团块处理方式以及正常细胞团获取方式既节约了成本也能很好的维持细胞的活率等。分化过程中各阶段使用的化合物组合方式能高效率的分化为该阶段的细胞类型,分化细胞比例显著提高。特别是分化前培养体系变更,组合物的配制方法和添加时间,分化过程中换液方式、分化上清单细胞处理方式等均很好的促进分化的效果,显著增加分化各批次间的稳定性且分化的beta细胞更加成熟,功能更强。改良的海藻酸钠微囊化包裹分化的beta细胞移植动物体内可短时间内(24h)逆转糖尿病小鼠的高血糖症,且具有很好的免疫隔离功能,纤维化包裹较少,能在免疫完全小鼠体内维持血糖正常较长时间。
附图说明
图1、平面培养过程中的干细胞多能性检测指标及结果,1A、镜检照片;1B,流式检测结果。
图2、hPSCs细胞3D培养后的境检结果照片。
图3、驯化过程中的细胞增殖情况,增殖倍数在2-4倍之间(图3A)、Oct4/SSEA4双阳细胞比例在99%以上(图3B)。
图4、海藻酸钠-聚赖氨酸-海藻酸钠(APA)包裹后的细胞团形态。
图5、微囊化的胰腺beta细胞移植至STZ诱导的C57BL/6小鼠腹腔进行体内功能评价结果。
图6、经典方法分化胰腺beta细胞过程中各阶段的qPCR检测结果及相关标志物在转录水平上的表达趋势。
图7、本发明优化后的分化方法获得胰腺beta细胞过程中各阶段的qPCR检测结果及相关标志物在转录水平上的表达趋势。
图8、本发明优化后的分化方法的各个分化阶段细胞集群的性质检测结果。
图9、分化过程中各阶段的检测指标示意图。
具体实施方式
实施例1、hPSCs细胞的平面培养、体外3D培养及驯化
一、hPSCs细胞平面培养
所需试剂配制方法:
mTeSR1多能干细胞培养基(STEMCELL,85850);
Accutase消化酶(STEMCELL,07920);
Rho激酶抑制剂Y27632(Abcam,ab120129):用DMSO配成10mM的储存溶液,使用时终浓度为10μM,即当1000X用;
mTeSR1+Y27632培养基:15mL mTeSR1需添加15μL 10mM的Y27632,混匀,不用时置于4℃保存,配好后两周内用完最佳。
hPSCs细胞(临床级人胚胎干细胞或人诱导多能干细胞,来源北京干细胞库)。
1、hPSCs复苏和培养
1.1、Matrigel包被培养板:取出6孔板,每孔内加入0.5-2mL基质胶(Matrigel,Corning,354277),轻轻晃动6孔板使Matrigel完全覆盖皿底,置于37℃培养箱中孵育1h~2h,实验前拿出并置于超净工作台/生物安全柜中室温下平衡10-40min。如果暂时不用,可用Parafilm封口后2-8℃储存,并于1-2周内使用。
一支冻存的干细胞的数量在1×106cells/mL左右,对应接种6孔板1孔;
1.2、先将2~3mL的mTeSR1培养基加入15mL离心管中备用。
1.3、解冻:将从液氮中取出的冻存管快速浸入37℃温水中,快速摇动,使其在1~2min内快速解冻;
1.4、离心:冻存管中的冻存液解冻后,将其逐滴加入含有mTeSR1培养基的15mL离心管中,将15mL离心管置于低速离心机中配比平衡后,800-1800rpm离心3min;
1.5、重悬:离心后弃上清加0.5-2mL mTeSR1+Y27632细胞培养基对干细胞沉淀进行吹吸混匀,吹吸3~5次左右。
1.6接种:吹吸均匀后,将已经平衡好的Matrigel弃掉,将吹打均匀的干细胞悬液加入到已经包被好的6孔板中,并补全每孔2mL培养体系。
1.7培养:接种后的6孔板可以置于倒置相差显微镜下观察接种的干细胞密度,水平十字轻轻晃动6孔板使细胞均匀分布。并置于37℃,5%CO2的恒温培养箱培养,第2天观察细胞贴壁情况;
1.8换液:从复苏的时间开始每24-48h换液一次。
平面培养过程中,细胞形态正常,平面培养过程中的干细胞多能性检测指标及结果见图1;结果显示通过上述方法进行平面培养的hPSCs可以很好的维持干细胞克隆形态,干性相关基因表达很好;流式结果显示Oct4+/SSEA4+双阳细胞比例在99%以上,说明培养过程中干性维持很好;SSEA1细胞比例非常低,未出现细胞分化情况。
二、hPSCs传代进行3D培养
2.1加Y27632预处理:上3D反应器(Disposable spinner flasks,Corning,3152/3153)前2-4h需更换含Y27632的mTeSR1 mediμm预处理细胞;
2.2清洗:吸掉原有培养基,贴壁缓慢加入0.5-2mL DMEM/F12并轻轻晃动,然后沿培养皿边缘吸去DMEM/F12;消化:在6孔板中加入0.5-2mL/孔Accutase使之覆盖皿底,并置于37℃培养箱中2~5min;
2.3中和:加入1-4mL DMEM/F12中和,用移液枪扇形吹打培养皿底,轻柔吹打3~5次,使皿底干细胞集落脱落,并将其并转移到15mL离心管中;
2.4计数:800-1800rpm离心3-5min;弃上清,用干细胞培养基吹打细胞5-10次,取部分细胞悬液进行计数;
2.5接种:根据计数结果,将3-8*10^5/mL密度重悬于mTeSR1+Y27632培养基中,然后接种于Corning Spinner Flask中,40-300mL培养体系。并将Spinner flasks置于九位点磁力搅拌器上以50-120rpm转速进行培养,培养箱的参数设置为恒温37℃,5%CO2和100%湿度。
hPSCs细胞3D培养后的境检结果照片见图2
在细胞培养和分化的过程中,这种培养方式维持不变。若细胞接种密度不在该范围内,密度过小会导致细胞无法很好的成团,增殖受限;密度过大会导致细胞团易结成大团块,团与团之间粘连严重,细胞团粒径偏大会导致中部细胞乏氧,细胞活率降低且容易分化。若转速不在该范围内,转速过大会导致无法很好的成团,单细胞过多细胞活率降低,干性丢失;转速过小会导致细胞团与团之间粘连严重,细胞团粒径偏大会导致中部细胞乏氧,细胞活率降低且容易分化。
三、3D培养驯化的hPSCs细胞及功能检测
3.1在测定hPSCs细胞分化能力之前,在反应器中保证至少驯化5-10代。每天进行半换液,大于400μm大细胞团块经400μm筛网去除,培养上清中的单细胞加回至反应器中。传代时弃去培养上清中的单细胞,将正常团用37μm可逆滤器收集,用Accutase将细胞分散开,并在每毫升添加了5-20μM Y27632的mTeSR1培养基中接种0.3-0.7百万个散开的细胞。若不去除大于400μm大细胞团块,则会导致细胞团块粘连越来越严重,进而细胞团粒径偏大会导致中部细胞乏氧,细胞活率降低且容易分化。若培养上清中的单细胞不加回至反应器中,会导致细胞增殖受限,不利于细胞团的形成。
3.2培养48-72小时后进行分化,期间每天更换一半不含Y27632的mTeSR1培养基,分化前以保持细胞簇直径大小在300μm以内,若细胞簇直径大小大于300μm,会导致细胞团中部分细胞坏死或自发分化。具有不同倍增时间的细胞系可能需要不同的接种浓度或时间。
驯化过程中的细胞增殖情况见图3:可见,在驯化过程中,多能干细胞能够很好的增殖,增殖倍数在2-4倍之间(图3A)。在驯化的过程中hPSCs也能够很好的表达干性相关的基因,流式结果显示Oct4+/SSEA4+双阳细胞比例在99%以上(图3B),也说明驯化过程中3D多能干细胞的干性维持很好。
实施例2、hPSCs细胞分化为胰腺beta细胞
整个分化过程中所用到的培养基的名称和配方如下,
S1培养基:MCDB131基础培养基(Thermofisher,10372019)+2-20mM葡萄糖(Glucose,Sigma,G7528-250G)+1-5g/L碳酸氢钠(NaHCO3,Sigma,S5761-500G)+0.5-5%重组人血清白蛋白(Human serum albumin,HSA,禾元生物,HYC002M01)+1:10000-1:100000胰岛素-转铁蛋白-硒-乙醇胺(InsulinTransferrinSeleniumEthanolamine,ITS-X,Thermofisher,51500056)+1-5mM谷氨酰胺替代物(GlutaMAXTM Supplement,Glutamax,Thermofisher,35050061)+0.1-0.5mM维他命C(L-Ascorbic acid,Vitamin C,Sigma,A4544-25G)+1%青霉素/链霉素(Penicillin-Streptomycin,Pen/Strep,Thermofisher,15140122).
S2培养基:MCDB131+2-20mM Glucose+1-3g/L NaHCO3+0.5-5%HSA+ITS-X 1:10000-1:100000+1-5mM Glutamax+0.1-0.5mM Vitamin C+1%Pen/Strep.
S3/S4培养基:MCDB131+2-20mM Glucose+1-3g/L NaHCO3+0.5-5%HSA+ITS-X 1:50-1:500+1-5mM Glutamax+0.1-0.5mM Vitamin C+1%Pen/Strep.
S5培养基:MCDB131+5-50mM Glucose+1-3g/L NaHCO3+0.5-5%HSA+ITS-X 1:50-1:500+1-5mM Glutamax+0.1-0.5mM Vitamin C+1%Pen/Strep+2-20μg/mL肝素钠盐(Heparin sodium salt,Heparin,Sigma,H3149-500KU-9).
以上所有的培养基均要经过0.22μm bottle top filter Filter过滤除菌。
并且需要在培养基更换之前配置新鲜培养基(将小分子和生长因子在低光罩的安全柜中加入到基础培养基中)。
一、SC-β细胞(胰腺beta细胞)分化
1.经典方法胰腺beta细胞分化及功能检测
采用Melton DA.方法(该方法援引自:doi.org/10.1016/j.cell.2014.09.040),
在分化的每个阶段和最后阶段,分别取细胞用于分析进行体外功能评价。检测结果见下:
(1)S0阶段:hPSC,human pluripotent stem cells。流式检测Oct4+/SSEA4+双阳细胞比例正常,干性维持较好;
(2)S1阶段:DE,definitive endoderm cells。流式检测SOX17阳性细胞比例为82.9%,未到达90%以上,分化效率较低;
(3)S2阶段:PGT,primitive gut tube cells。流式检测HNF4a阳性细胞比例为47.9%,分化效率较低;
(4)S3阶段:PP1,early pancreatic progenitor cells。流式检测PDX1阳性细胞比例为40.6%,分化效率较低;
(5)S4阶段:PP2,later pancreatic progenitor cells。流式检测PDX1+/NKX6.1+双阳性细胞比例仅为8.62%,表达较低;
(6)S5阶段:EN,endocrine progenitor cells。流式检测NKX6.1+/C-peptide双阳性细胞比例仅为10.1%,且分化结果中C-peptide绝大多数为弱阳性表达;
(7)S6阶段:SC-βcells,stem cell-derivedβcells。流式检测NKX6.1+/C-peptide双阳性细胞比例仅为12.7%。内分泌细胞CHGA阳性细胞比例为47.71%,相对较低。连续三轮的葡萄糖刺激未有响应,且高糖刺激时Insulin的分泌量很低。
(8)使用该方法分化过程中各阶段的qPCR检测结果及相关Marker在转录水平上的表达趋势:
整个分化过程中取不同阶段的细胞进行RNA的提取,然后使用qPCR检测在整个分化过程中相关基因的表达趋势情况。由图6可见,该方法所得PDX1、NKX6.1和Insulin的相对表达量较低,只有初始细胞的几十倍。Oct4的表达直到Stage5阶段才降至较低水平,存在很大的安全性问题。
2.优化方法胰腺beta细胞分化及功能检测
使用在3D反应器中驯化5-20代(所述5-20代传数中含3D驯化过程的传代数)的多能干细胞作为分化的起始种子细胞。
2.1分化方法
开始进行SC-β细胞分化的时候,在每毫升添加了10μM Y27632的mTeSR1培养基中接种0.3-0.7百万个散开的hPSCs细胞。培养24-72小时后,更换Day 1media,此时是80-320mL培养体系转换为60-300mL分化体系(体积减少10-20%,初次换液时培养基用量相比未换液时减少10%-20%,之后每次换液维持培养体积不变),此时是分化的正式开始,以后依次按照以下时间进行培养基的更换(全换液,相同分化阶段的培养上清中的单细胞离心后加回反应器中,跨阶段时培养上清中的单细胞弃去)。若分化起始时体积不减少,则会导致分化后期细胞得率非常低。分化过程中使用的Vc、所有因子和小分子化合物均当日添加。若提前添加,则会大大降低分化的效率。并且在培养基更换之前需要新鲜配制相关培养基,并将小分子化合物和生长因子在低光罩的安全柜中加入到基础培养基中,换个过程全程避光。若不避光操作,则会导致部分小分子化合物因受到光源直射导致分解等,最后也会导致分化效率的降低。
首先在每毫升添加了10μM Y27632的mTeSR1培养基中接种0.3-0.7百万个散开的hPSCs细胞。培养24-72小时后,更换培养基并添加相应的细胞因子,根据培养进程,更换培养基及添加细胞因子的步骤如下:
Day 1:S1+50-200ng/ml重组人激活素A(Recombinant Human/Mouse/Rat ActivinAProtein,ActivinA,R&D systems,338-AC-050/CF)+10-100ng/mL重组人Wnt3a蛋白(Recombinant Human Wnt-3a Protein,Wnt3a,R&D systems,5036-WN-500).(初次换液时培养基用量相比未换液时减少10%-20%,之后每次换液维持培养体积不变)
Day 2:S1+50-200ng/ml ActivinA.
Days 4、6:S2+20-100ng/ml重组人角化细胞生长因子蛋白(Recombinant HumanKGF/FGF-7Protein,KGF,R&D systems,251-KG-050)+1-5μM转化生长因子-βRI激酶抑制剂IV(TGF-βRI Kinase Inhibitor IV,Sigma,616454-2MG).
Days 7、8:S3+20-100ng/ml KGF+0.1-0.5μM Sant1(Sigma,S4572-5MG)+1-5μM视黄酸(Retinoic acid,RA,Sigma,R2625-50MG)+100-500nM 1,1,4,4-四苯基-1,3-丁二烯(1,1,4,4-Tetraphenyl-1,3-butadiene,TPB,Sigma,185213-5G)+5-20μM Y27632.
Days 9、11、13:S3+20-100ng/ml KGF+0.1-0.5μM Sant1+50-200nM RA+10-100ng/mL重组人表皮细胞生长因子蛋白(Recombinant Human EGF protein,EGF,R&D systems,236-EG-200)+10-100ng/mL重组人头蛋白(Recombinant Human Noggin Protein,NOG,R&Dsystems,6057-NG-025)+5-20μM Y27632.
Days 14、16:S5+0.1-0.5μM Sant1+50-200nM RA+0.5-2μMγ-分泌酶抑制剂XXI(γ-Secretase Inhibitor XXI,Compound E,XXI,Millipore,565790-500UG)+5-20μMRepSox(Alk5i II,Sigma,R0158-5MG)+0.5-2μM三典甲状腺氨酸(L-3,3',5-Triiodothyronine,T3,Millipore,64245-250MG-M)+5-50ng/ml重组人β细胞素(Recombinant Human Betacellulin Protein,Betacellulin,R&D systems,261-CE-010)+50-500nM LDN193189盐酸盐(LDN193189hydrochloride,LDN193189,Sigma,SML0559-5MG)+10μM硫酸锌(ZnSO4,Sigma,Z0251).
Days 18、20:S5+10-50nM RA+0.5-2μM XXI+5-20μM RepSox+0.5-2μM T3+5-50ng/ml Betacellulin+50-500nM LDN193189+1mM N-cys(Fmoc-N-Me-Cys(Trt)-OH,Sigma,773069-1G).
Days 21-35(每两天更换一次培养基):S3培养基。
在分化的21天时,将分化细胞使用TrypLE Express消化为单细胞,然后按照0.5-2百万细胞/毫升密度重新接种于反应器中,50-120rpm转速进行培养,培养箱的参数设置为恒温37℃,5%CO2和100%湿度。
此后隔天换液,使用10-50μm可逆滤器将重聚集的正常团收集,弃去未聚团的上清。在分化的每个阶段和最后阶段,分别取细胞用于分析。
2.2Stage0-Stage6分化过程中各阶段的检测指标:
在2.1的方法的分化的每个阶段和最后阶段,分别取细胞用于分析进行体外功能评价。检测结果见下:
(1)S0阶段。hPSC,human pluripotent stem cells。检测结果见图8A;
流式检测Oct4+/SSEA4+双阳细胞比例大于95%,干性维持较好;
(2)S1阶段。DE,definitive endoderm cells。检测结果见图8B;
流式检测SOX17阳性细胞比例达到92.8%,说明绝大部分细胞分化为定向内胚层细胞。
另外,该阶段可进行流式分选,分选得到定向内胚层祖细胞系。主要方法是:
分化进行至此阶段时,取细胞团使用TryPLE消化为单细胞,然后将细胞标记上CXCR4和CD117,通过流式分选出CXCR4+/CD117+双阳性的群。
将该群细胞接种在含有Matrigel的平板里,使用定向内胚层祖细胞培养和扩增培养基进行培养。
其中定向内胚层祖细胞培养和扩增培养基的成分为:
S1 media+20-100ng/mL骨形态发生蛋白4(Recombinant Human BMP-4Protein,BMP4,R&D systems,314-BP-050)+5-20ng/mL重组人碱性成纤维细胞生长因子蛋白(Recombinant Human bFGF Protein,bFGF,R&D systems,233-FB-010)+5-20ng/mL重组人血管内皮生长因子蛋白(Recombinant Human VEGF 165Protein,VEGF,R&D systems,293-VE-010)+5-20ng/mL重组人表皮细胞生长因子蛋白(Recombinant Human EGF protein,EGF,R&D systems,236-EG-200)。
培养获得的定向内胚层祖细胞可作为beta细胞分化的起点,这可以进一步提高不同分化批次的稳定性和减少分化终末端beta细胞中干/祖细胞的残留,这也显著提高了移植体内的安全性。
(3)S2阶段。PGT,primitive gut tube cells。检测结果见图8C;
流式检测HNF4a阳性细胞比例达到98.4%,说明几乎所有细胞分化成PGT细胞,分化效率非常高。
(4)S3阶段。PP1,early pancreatic progenitor cells。检测结果见图8D;
流式检测PDX1阳性细胞比例达到57.4%,说明大部分细胞进入胰腺祖细胞早期阶段,分化效果很好。
(5)S4阶段。PP2,later pancreatic progenitor cells。检测结果见图8E;
流式检测PDX1+/NKX6.1+双阳性细胞比例为27%,说明此时进入下一分化阶段的细胞占一定比例,分化效果较好。
(6)S5阶段。EN,endocrine progenitor cells。检测结果见图8F;
流式检测NKX6.1+/C-peptide双阳性细胞比例达到24%,说明此时已经有超过20%的细胞分化为SC-beta细胞。
(7)S6阶段。SC-βcells,stem cell-derivedβcells。检测结果见图8G;
流式检测NKX6.1+/C-peptide双阳性细胞比例大于30%。有部分细胞表达GCG(胰岛α细胞)和SST(胰岛δ细胞),内分泌细胞CHGA阳性细胞比例大于90%。
有连续三轮的葡萄糖响应且刺激指数大于2,高糖刺激时Insulin的分泌量大于1.0-5.0μIU/1000cells。双硫腙染色呈现红色。
(8)分化过程中各阶段的qPCR检测结果及相关Marker在转录水平上的表达趋势:
整个分化过程中取不同阶段的细胞进行RNA的提取,然后使用qPCR检测在整个分化过程中相关基因的表达趋势情况。由图7可得,该方法所得PDX1、NKX6.1和Insulin的相对表达量较高,特别是Insulin的表达是初始的近20万倍。Oct4的表达到Stage2阶段便降至较低水平,安全性问题大大降低。
检测结论:
使用优化方法诱导hPSCs细胞分化为胰腺beta细胞的整个分化过程中需经历以下阶段,各个阶段的检测指标如下:
此数值为内部质控标准,当每个阶段达到以下数值后方可认为该批次分化正常,分化可顺利进行下去,最终得到的胰腺beta细胞的功能方可正常。
hPSCs细胞:八聚体结合转录因子4/阶段特异性胚胎抗原4(Octamer-bindingtranscription factor4/Stage-specific embryonic antigens 4,Oct4+/SSEA4+)双阳性细胞比例大于95%;
DE,定向内胚层:SOX转录因子家族成员17(Sex-determining region Y-box 17,Sox17+)阳性细胞比例大于90%;
PGT,原肠管:肝细胞核因子4a(Hepatocyte Nuclear Factor 4alpha,HNF4a+)阳性细胞比例大于80%;
PP1,胰腺祖细胞早期阶段:胰十二指肠同源框-1(Pancreatic and duodenalhomeobox 1,PDX1+)阳性细胞比例大于60%;
PP2,胰腺祖细胞晚期阶段:PDX1+/同源转录因子NKX6.1(homeoboxtranscription factor NK6 homeobox1,NKX6.1+)双阳性细胞比例大于25%;
EN,胰腺内分泌祖细胞:NKX6.1+/C-peptide+双阳性细胞比例大于10%;
SC-beta cluster,干细胞来源的胰腺beta细胞:NKX6.1+/C-peptide+双阳性细胞比例大于30%。
在不同阶段的指标达到上述质控标准后,能够很好地分化为功能性的beta细胞。
2.3在Stage0-Stage6整个分化过程中的某些阶段(具体阶段见上述9)-12)步骤)对细胞进行包括但不限于CD177,CD117,CXCR4,GP2,Procr等相关Marker的富集(这组富集是在分化的整个过程中穿插进行的,相当于分化过程中的优化,这些富集能够显著提高分化效率,是提高分化效率的一种选择方式)。
该富集可根据分化过程中细胞的形态来判断是否执行,若该批次分化细胞形态略有改变,可通过富集来进一步提高分化细胞的功能和得率:
(1)CXCR4+/CD117+双阳性的群的具体富集和培养方法是:
取准备进行富集的分化阶段细胞团使用TryPLE消化为单细胞,然后将细胞标记上CXCR4和CD117,通过流式分选出CXCR4+/CD117+双阳性的群。将该群细胞接种在含有Matrigel的平板里,使用定向内胚层祖细胞培养和扩增培养基进行培养。
其中内胚层祖细胞培养和扩增培养基的成分为:S1 media+BMP4(20-100ng/mL)+bFGF(5-20ng/mL)+VEGF(5-20ng/mL)+EGF(5-20ng/mL)。
培养获得的定向内胚层祖细胞进行分化可以进一步提高不同分化批次的稳定性和减少分化终末端beta细胞中干/祖细胞的残留,显著提高了安全性。
(2)CD177阳性的群的具体富集和培养方法是:
取分化阶段细胞团使用TryPLE消化为单细胞,然后将细胞标记上CD177,通过流式分选出CD177阳性的群。然后将分选出细胞以2-10×105/mL的密度接种在含有10μM Y27632的Stage1培养基中进行培养分化。
与未进行该富集的细胞相比,进行CD177+富集程序的细胞在体外可更均匀地分化为胰腺祖细胞,且最终会分化为功能更成熟的具有葡萄糖响应性beta细胞。
(3)GP2阳性的细胞群的富集和培养,具体方法是:
取分化阶段细胞团,消化为单细胞,然后分选出GP2阳性的细胞亚群,然后将分选出细胞以5-10×105/mL的密度接种在上述第14日培养基中继续进行分化;
该方法可使得分化的细胞PDX1+/NKX6.1+双阳细胞比例显著提高。
(4)Procr阳性的群的具体富集和培养方法是:
取分化阶段细胞团使用TryPLE消化为单细胞,然后将细胞标记上Procr,通过流式分选出Procr阳性的群。
富集的Proc+细胞可每7-14天进行一次传代,在每次传代过程中,消化的Procr+单细胞悬液均以新鲜的human HUVEC细胞补充,细胞数比为1:1。
然后混合Procr+单细胞和human HUVEC两种细胞以1:4-1:6的比例重新接种培养。该方法可使得分化的细胞得率显著提高。
(5)在分化的最后一个阶段可添加WNT4,可明显驱动代谢成熟,结果表明可以显著提高分化的beta细胞对葡萄糖的响应,且释放更多的Insulin。
实施例3、分化的胰腺beta细胞进行改良APA微囊化包裹
1、改良的超纯海藻酸钠溶液的配制
溶解后的海藻酸钠依次经过0.8μm、0.45μm和0.22μm的PES无菌过滤装置过滤;海藻酸钠的粘度在50-200cP;配好的无菌的海藻酸钠在4度冰箱中可以保存1-4周。
2、APA微囊化分化的beta细胞——微流控法或者高压静电法
2.1微流控法(微流控板由苏州汶颢微流控技术股份有限公司购买,WH-SP-01型号):
将1%-3%的海藻酸钠溶液与beta细胞混合,1mL海藻酸钠对应1-10*10^6细胞;
混合好后加入其中一个管道,另外一个管道加入0.1-2g/L氯化钙溶液;
在经过1-10cm的收集管进行交联成为凝胶,随后钙化5-30分钟;
加入0.01-0.1%的聚赖氨酸(Poly-L-lysine hydrobromide,Sigma,P7890-100MG)进行反应10-30分钟;
再加入0.1%-0.3%的海藻酸钠溶液反应2-10分钟;
再加入10-100mM的柠檬酸钠反应2-10分钟即形成APA微胶囊。
2.2高压静电法:
将1%-3%的海藻酸钠溶液与beta细胞混合,1mL海藻酸钠对应1-10*10^6细胞;
混合好后使用20-40G针头的注射器吸入,在5-20KV电压、1-10脉冲、50-200Hz、流速100-1000μL/min的条件下,使得其形成射流进入0.1-2g/L氯化钙溶液中交联成为凝胶,随后钙化5-30分钟;
加入0.01-0.1%的聚赖氨酸进行反应10-30分钟;
再加入0.1%-0.3%的海藻酸钠溶液反应2-10分钟;
再加入10-100mM的柠檬酸钠反应2-10分钟即形成APA微胶囊。
具体包裹后的细胞团形态见图4。较好的情况下是微囊的粒径控制在250-750μm。
实施例4、微囊化的胰腺beta细胞功能评价(单一移植功能评价)
目前已发现多种化合物可在动物模型诱发糖尿病。研究最多且常规使用的两种化合物分别是链脲佐菌素(Streptozotocin,STZ)和四氧嘧啶(Alloxan,ALX),其中又以STZ诱导最常用。STZ和ALX都是葡萄糖类似物,都通过β细胞中的葡萄糖转运蛋白GLUT2起作用,最终导致胰岛中β细胞几乎完全被破坏,致使小鼠严重缺乏胰岛素生成,表现出高血糖症和体重减轻,从而重现I型糖尿病的主要症状。本发明通过使用STZ进行药物诱导,致使C57BL/6小鼠产生I型糖尿病症状。
1、微囊化或者裸的胰腺beta细胞移植至STZ诱导的I型糖尿病C57BL/6小鼠皮下进行体内功能评价
1.1微囊化或者裸的胰腺beta细胞移植:
通过吸入2%至5%的异氟烷(Isoflurane USP,Clipper Distribution)麻醉接受移植的小鼠。然后进行皮下移植:在小腹上建立了一个小的皮肤切口(0.3-0.5cm),来形成一个左右下象限皮下“小口袋”,将微囊化的beta细胞或者裸的beta细胞与200-500μL的beta细胞活性基质胶混合后移植至小鼠的皮下“小口袋”中。其中beta细胞活性基质胶的组分包括:10X Media(M)199基础培养基(ThermoFisher,11825015)、L谷氨酰胺、胎牛血清、5-10%的碳酸氢钠和I型胶原蛋白。
1.2裸的beta细胞(未微囊化的beta细胞)移植还需联合免疫抑制剂使用,具体方案为:
维持免疫抑制由雷帕霉素(1-2mg/kg腹膜内,每天四次)组成,从移植当天开始,持续5-10d。
为了清除B细胞,使用10F4(由宾夕法尼亚大学病理学和实验室医学室M.Cancro提供),这是一种针对小鼠BLyS的单克隆抗体(移植前15-25d腹膜内注射50-200μg,分两次注射,间隔24小时)。从第10天开始,也逐渐给予10F4,剂量逐渐减少,从第2周的每周20-100μg降低到第8周的每周1-10μg。在第50-80天时,免疫抑制给药中止。
1.3移植后检测
检测小鼠血糖水平、糖化血红蛋白水平、体内葡萄糖刺激及人C肽情况等。取移植后的APA微囊化的beta细胞或者裸的beta细胞进行固定后做免疫荧光,进行相关Marker的鉴定。
另外,可切除胰岛移植部位,目的是为了确认胰岛移植物是维持血糖正常的唯一来源,对一群长期接受血糖正常血糖的胰岛受体行胰岛切除术(皮肤),这导致糖尿病在24-72小时内迅速复发。同样的结果显示,皮下移植的分化beta细胞与微囊化beta细胞腹腔移植后的功能相当(是指一种检测手段,即将移植的胰腺beta细胞去除后,动物又会复发糖尿病,这就说明是移植的胰腺beta细胞在行使功能)。
根据上述方法,每只小鼠移植量1-10*10^6细胞,使用留置针进行腹腔移植,移植后检测小鼠血糖水平、糖化血红蛋白水平、体内葡萄糖刺激及人C肽情况等。
结果显示:微囊化的beta细胞可以快速逆转糖尿病小鼠高血糖症状,此过程需要1-7天,随后维持正常血糖30天以上。在移植期间内随机检测移植小鼠血浆中人C肽的含量,结果显示,人C肽的含量远远高于对照组,在100-500pM之间。糖化血红蛋白HbA1C的百分含量也随之降低,达到6-12%。体内IPGTT实验显示微囊化的beta细胞能够快速感应血糖的上升,分泌足够量的Insulin维持血糖稳定。取移植后的APA微囊化的beta细胞进行固定后做免疫荧光,进行相关Marker的鉴定。微囊化的beta细胞可以很好地表达beta细胞相关Marker NKX6.1/C-peptide,双阳阳性率在大于30%。具体评价结果见图5中。
实施例5、微囊化的胰腺beta细胞功能评价(联合移植功能评价)
微囊化或者裸的beta细胞联合间充质干细胞(MSC)或内皮祖细胞(EPC)移植至STZ诱导的C57小鼠体内进行功能评价,实验步骤如下:
1、将Stage5-Stage6分化阶段的细胞团使用TryPLE消化酶消化为单细胞后,将间充质干细胞(MSC)或内皮祖细胞(EPC)的单细胞悬液按照1:1的比例与分化的单细胞悬液进行混合培养,混合培养所使用的培养基为S3分化基础培养基,在低吸附6孔板中置于轨道摇床上进行培养。
2、动物实验的细胞接种方法同实施例4,混合细胞接种密度为0.5-2*10^6/mL,培养体积为2-7mL/孔,轨道摇床转速为70-120rpm。单细胞在24-72h内重新聚集成团,经过7-14天进一步成熟后移植至STZ诱导的C57小鼠皮下或腹腔进行体内功能评价。将间充质干细胞或内皮祖细胞与胰岛细胞共同聚集培养,形成三维细胞团,在皮下或腹腔内注射单剂量的混合胰岛细胞团可以给机体提供长时间的血糖控制,Ⅰ型糖尿病小鼠在不使用抗免疫排斥药物或包封***的情况下,血糖能保持在正常范围以内。并且该过程中受体能免于使用抗免疫排斥药物或胰岛细胞包封***(微囊化)。
此方法中,在移植位置先进行间充质干细胞的移植,移植间充质干细胞的量为每只小鼠的移植部位移植量1-10*10^6细胞。主要目的是构建移植部位的血管生成环境,使得移植后的beta细胞能够的在血管化的环境中获得营养和氧气,更有利于移植的beta细胞的存活和功能。结果显示,预先移植的间充质干细胞能够使移植的部分产生血管化,产生血管时间在10-50天。随后移植的微囊化或者裸的beta细胞与微囊化beta细胞腹腔移植后的功能相当。
最后需要说明的是,以上实施例仅用于帮助本领域技术人员理解本发明的实质,不用于限定本发明的保护范围。
Claims (8)
1.一种多能干细胞三维悬浮定向分化为成熟胰腺beta细胞的方法,所述方法包括如下步骤:
(1)多能干细胞的三维悬浮驯化培养;驯化后检测多能干细胞,细胞呈球状悬浮生长且Oct4+/SSEA4+双阳细胞比例在95%以上即为驯化合格;所述的多能干细胞为人诱导多能干细胞;
(2)诱导驯化后的细胞分化为胰腺beta细胞;分化步骤为:
1)含10μM Y27632的mTeSR1培养基中接种0.3-0.7×106个散开的三维悬浮驯化的多能干细胞,培养24-72小时后,更换首日培养基,换液时体积减少10-20%,
首日培养基为:S1培养基中添加50-200ng/ml Activin A和10-100ng/mL Wnt3a;
2)第2日更换第2日培养基;
第2日培养基为:S1培养基中添加50-200ng/ml Activin A;
3)第4~6日更换第4日培养基;
第4日培养基为:S2培养基中添加20-100ng/ml KGF和1-5μM TGF-βRI KinaseInhibitor IV;
4)第7~8日更换第7日培养基;
第7日培养基为:S3/S4培养基中添加20-100ng/ml KGF、0.1-0.5μM Sant1、1-5μM视黄酸、100-500nM1,1,4,4-四苯基-1,3-丁二烯和5-20μM Y27632;
5)第9~13日更换第9日培养基;
第9日培养基为:S3/S4培养基中添加20-100ng/ml KGF、0.1-0.5μM Sant1、50-200nM视黄酸、10-100ng/mL EGF、10-100ng/mL重组人头蛋白和5-20μM Y27632;
6)第14~16日更换第14日培养基;
第14日培养基为:S5培养基中添加0.1-0.5μM Sant1、50-200nM视黄酸、0.5-2μMγ-分泌酶抑制剂XXI、5-20μM RepSox、0.5-2μM三典甲状腺氨酸、5-50ng/ml重组人β细胞素、50-500nM LDN193189盐酸盐和10μM硫酸锌;
7)第18~20日更换第18日培养基;
第18日培养基为:S5培养基中添加10-50nM视黄酸、0.5-2μMγ-分泌酶抑制剂XXI、5-20μM RepSox、0.5-2μM三典甲状腺氨酸、5-50ng/ml重组人β细胞素、50-500nM LDN193189和1mM Fmoc-N-Me-Cys(Trt)-OH;
8)第21天时,将分化细胞消化为单细胞并重新接种于反应器中再培养,培养过程中将重聚集的正常细胞团收集,弃去未聚团的上清,即得分化的胰腺beta细胞;
所述的各个培养基为:
S1培养基:MCDB131基础培养基+1:10000-100000胰岛素-转铁蛋白-硒-乙醇胺+1-5
mMGlutamax+1-5g/L碳酸氢钠+1%青霉素/链霉素+0.1-0.5mM维他命C+2-20mM葡萄糖+0.5-5%重组人血清白蛋白;
S2培养基:MCDB131基础培养基+1:10000-1:100000胰岛素-转铁蛋白-硒-乙醇胺+1-5mMGlutamax+1-3g/L碳酸氢钠+0.1-0.5mM维他命C+1%青霉素/链霉素+2-20mM葡萄糖+0.5-5%重组人血清白蛋白;
S3/S4培养基:MCDB131基础培养基+1:50-1:500胰岛素-转铁蛋白-硒-乙醇胺+1-5mMGlutamax+1-3g/L碳酸氢钠+0.1-0.5mM维他命C+1%青霉素/链霉素+2-20mM葡萄糖+0.5-5%重组人血清白蛋白;
S5培养基:MCDB131基础培养基+1:50-1:500胰岛素-转铁蛋白-硒-乙醇胺+1-5mMGlutamax+2-20μg/mL肝素钠盐+1-3g/L碳酸氢钠+0.1-0.5mM维他命C+1%青霉素/链霉素+5-50mM葡萄糖+0.5-5%重组人血清白蛋白。
2.根据权利要求1所述的方法,其特征在于,将分化细胞消化为单细胞并重新接种于反应器中再培养时,所述再培养的参数为:50-120rpm转速,恒温37℃,5%CO2和100%湿度,21-35天时,每两天更换一次S3培养基,使用10-50μm可逆滤器收集重聚集的正常细胞团。
3.根据权利要求1所述的方法,其特征在于,步骤(1)所述的多能干细胞的三维悬浮驯化培养的步骤包括:
1)预处理:在悬浮培养前2小时将平面培养的多能干细胞进行换液,更换为含10μM浓度的Y27632的mTeSR1培养基;
2)使用DMEM/F12培养基清洗中和预处理细胞;
3)在mTeSR1+Y27632培养基中传代5-10次,每天更换一半培养基,传代时去除培养上清中的单细胞和聚集的大细胞团块。
4.根据权利要求3所述的方法,其特征在于,
所述的多能干细胞的接种密度为3-8×105/mL,
三维搅拌培养的参数为:50-120rpm转速、恒温37℃、5%CO2、100%湿度,所述培养基为含10μM浓度的Y27632的mTeSR1培养基。
5.根据权利要求1所述的方法,其特征在于,在所述诱导驯化后的细胞分化为胰腺beta细胞步骤中,
步骤2)中,第2日更换第2日培养基,换液时培养基体积不变;
步骤3)中,分别于第4、6日更换第4日培养基,换液时培养基体积不变;
步骤4)中,分别于第7、8日更换第7日培养基,换液时培养基体积不变;
步骤5)中,分别于第9、11、13日更换第9日培养基,换液时培养基体积不变;
步骤6)中,分别于第14、16日均更换第14日培养基,换液时培养基体积不变;
步骤7)中,分别于第18、20日更换第18日培养基,换液时培养基体积不变。
6.根据权利要求1所述的方法,其特征在于,在所述诱导驯化后的细胞分化为胰腺beta细胞步骤中,还进一步包括以下任一步骤或任意步骤的组合:
9)在上述分化步骤3)之前,进行分化的CD177阳性的群的富集和培养,具体方法是:
取前一步骤分化的细胞团,消化为单细胞,然后分选出CD177阳性的细胞亚群,然后将分选出细胞以2-10×105/mL的密度接种在含有10μM Y27632的上述第4日培养基中继续进行分化;
10)在上述分化步骤6)之前,进行GP2阳性的群的富集和培养,具体方法是:
取前一步骤分化的细胞团,消化为单细胞,然后分选出GP2阳性的细胞亚群,然后将分选出细胞以5-10×105/mL的密度接种在上述第14日培养基中继续进行分化;
11)在上述分化步骤8)之前,进行Procr阳性细胞群的富集和培养,具体方法是:
取前一步骤分化的细胞团,消化为单细胞,然后分选出Procr阳性的细胞亚群,富集的Proc阳性细胞每7-14天进行一次传代,在每次传代过程中,消化的Procr阳性单细胞悬液均以新鲜的human HUVEC细胞补充,细胞数比为1:1;然后混合Procr阳性细胞和human HUVEC细胞两种细胞,以1:4-1:6的比例重新接种培养;
12)在上述分化步骤8)的培养基中,添加100ng/mL WNT4,驱动分化细胞葡萄糖刺激的胰岛素分泌所必需的代谢成熟。
7.权利要求1-6任一所述的方法在制备治疗糖尿病的药物中的应用。
8.根据权利要求7所述的应用,其特征在于,所述的糖尿病为Ⅰ型糖尿病。
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