CN104334719A - 人胚胎干细胞向胰腺内分泌细胞的分化 - Google Patents
人胚胎干细胞向胰腺内分泌细胞的分化 Download PDFInfo
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Abstract
本发明提供促进胰腺内胚层细胞分化成胰腺内分泌富集聚簇并增强激素表达细胞中的胰岛素表达的方法。
Description
相关申请的交叉引用
本申请要求于2012年6月8日提交的美国临时专利申请序列号61/657,160的权益,其全文以引用方式并入本文以用于任何目的。
技术领域
本发明在细胞分化领域中。更具体地,本发明公开了肝配蛋白配体和鞘氨醇-1-磷酸作为多能干细胞向内分泌细胞分化的调节因子的用途。
背景技术
用于I型糖尿病的细胞替代疗法的进展以及可移植胰岛的缺乏已使得注意力集中在开发适于植入的胰岛素生成细胞或β细胞的来源上。一种方法是从多能干细胞诸如例如胚胎干细胞产生功能性β细胞。
在脊椎动物的胚胎发育中,多能细胞可在称为原肠胚形成的过程中产生包括三个胚层(外胚层、中胚层和内胚层)的细胞群体。组织(诸如甲状腺、胸腺、胰腺、肠和肝脏)将从内胚层经由中间阶段发育而来。该过程中的中间阶段是定形内胚层的形成。定形内胚层细胞表达多种标记物,诸如HNF3β、GATA4、MIXL1、CXCR4和SOX17。
到原肠胚形成为止,内胚层划分成可通过一组因子的表达来识别的前-后域,所述因子唯一地标记内胚层的前、中和后区。例如,Hhex和Sox2鉴定内胚层的前区,而Cdx1、2和4鉴定内胚层的后半部分。
内胚层组织的迁移使内胚层与不同的中胚层组织紧密接近,这帮助肠管的区域化。这通过多种分泌因子诸如FGF、Wnt、TGF-B、视黄酸(RA)和BMP配体及其拮抗剂来完成。例如,FGF4和BMP促进预定的后肠内胚层中的Cdx2表达并阻遏前基因Hhex和SOX2的表达(2000 Development,127:1563–1567)。WNT信号传导也已显示与FGF信号传导平行工作,以促进后肠发育并抑制前肠命运(2007 Development,134:2207–2217)。最后,由间充质分泌的视黄酸调节前肠-后肠边界(2002 Curr Biol,12:1215–1220)。
特异性转录因子的表达水平可用于指定组织的身份。在定形内胚层转化成原肠管的过程中,肠管变得区域化成广泛域,所述广泛域可通过限制性基因表达模式在分子水平上进行观察。例如,肠管中区域化的胰腺域显示PDX-1的极高表达以及CDX2和SOX2的极低表达。相似地,高水平的Foxe1的存在指示食道组织;在肺组织中高度表达的是NKX2.1;SOX2/Odd1(OSR1)在胃组织中高度表达;PROX1/Hhex/AFP的表达在肝组织中较高;SOX17在胆道结构组织中高度表达;PDX1、NKX6.1/PTf1a和NKX2.2在胰腺组织中高度表达;并且CDX2的表达在肠组织中高。上文概括摘自Dev Dyn 2009,238:29–42和Annu Rev Cell Dev Biol 2009,25:221-251。
胰腺的形成起于定形内胚层分化成胰腺内胚层(2009 Annu Rev Cell DevBiol,25:221-251;2009 Dev Dyn,238:29-42)。背侧和腹侧胰腺域产生自前肠上皮。前肠也产生食道、气管、肺、甲状腺、胃、肝、胰腺和胆管系统。
胰腺内胚层的细胞表达胰-十二指肠同源盒基因PDX1。在不存在Pdx1时,胰腺形成腹胰芽和背胰芽后不再发育。因而,Pdx1表达标志着胰腺器官发生中的一个关键步骤。除了其他细胞类型,成熟的胰腺还包括外分泌组织和内分泌组织。外分泌和内分泌组织产生自胰腺内胚层的分化。
D’Amour等人描述了在高浓度激活素和低血清的存在下,产生人胚胎干细胞(ES)衍生出的定形内胚层的富集培养物(Nature Biotechnol 2005,23:1534-1541;美国专利7,704,738)。将这些细胞移植在小鼠的肾包膜下导致分化成具有内胚层组织特征的更成熟的细胞(美国专利7,704,738)。在添加FGF-10和视黄酸后,人胚胎干细胞衍生出的定形内胚层细胞还可进一步分化成PDX1阳性细胞(美国专利公开2005/0266554A1)。这些胰腺前体细胞在免疫缺陷小鼠的脂肪垫中的后续移植导致在3-4个月成熟期后形成功能胰腺内分泌细胞(美国专利7,993,920和美国专利7,534,608)。
Fisk等人报道用于由人胚胎干细胞产生胰岛细胞的系统(美国专利7,033,831)。在这种情况下,分化途径分成三个阶段。人胚胎干细胞首先使用丁酸钠和激活素A的组合而分化成内胚层(美国专利7,326,572)。然后将细胞和与EGF或β细胞素(betacellulin)结合的BMP拮抗剂诸如头蛋白(Noggin)一起培养,以生成PDX1阳性细胞。通过烟酰胺诱导终末分化。
小分子抑制剂也已用于诱导胰腺内分泌前体细胞。例如,TGF-B受体和BMP受体的小分子抑制剂(Development 2011,138:861-871;Diabetes2011,60:239-247)已用于显著提高胰腺内分泌细胞的数目。另外,小分子活化剂也已用于生成定形内胚层细胞或胰腺前体细胞(Curr Opin Cell Biol 2009,21:727-732;Nature Chem Biol 2009,5:258-265)。
虽然已在由人多能干细胞产生胰腺细胞的方案方面取得重大进步,但仍然需要建立产生功能性内分泌细胞且尤其是β细胞的方案。在此,我们证实一类肝配蛋白配体和鞘氨醇-1-磷酸或鞘氨醇受体的激动剂增强了内分泌细胞的产生并加速内分泌激素和内分泌前体细胞的聚簇。
发明内容
在一个实施例中,本发明涉及通过在包含肝配蛋白A4或肝配蛋白A3的培养基中培养胰腺内胚层细胞群来增强胰岛素和NKX6.1的表达的方法。在一些实施例中,胰腺内胚层细胞群基本上不表达CDX2或SOX2。在一些实施例中,胰腺内胚层细胞群通过多能细胞的逐步分化获得。在一些实施例中,多能细胞是人胚胎多能细胞。
在一个实施例中,本发明涉及通过在包含激活素A或激活素C的培养基中培养胰腺内胚层细胞来增强生长抑素的表达同时抑制胰岛素、胰高血糖素和生长素释放肽的表达的方法。在一些实施例中,经激活素A或激活素C处理的胰腺内胚层细胞群表达的生长抑素比未经激活素A或激活素C处理的胰腺内胚层细胞群更多。在一些实施例中,与未经激活素A或激活素C处理的胰腺内胚层细胞群中胰岛素的表达相比,胰岛素的表达在经激活素A或激活素C处理的胰腺内胚层细胞群中受到抑制。在一些实施例中,与未经激活素A或激活素C处理的胰腺内胚层细胞群中胰高血糖素的表达相比,经激活素A或激活素C处理的胰腺内胚层细胞群中胰高血糖素的表达受到抑制。在一些实施例中,与未经激活素A或激活素C处理的胰腺内胚层细胞群中生长素释放肽的表达相比,生长素释放肽的表达在经激活素A或激活素C处理的胰腺内胚层细胞群中受到抑制。在一些实施例中,胰腺内胚层细胞基本上不表达CDX2或SOX2。在一些实施例中,经激活素A或激活素C处理的胰腺内胚层细胞通过多能细胞的逐步分化获得。在一些实施例中,衍生出胰腺内胚层细胞的多能细胞是人胚胎多能细胞。
在一个实施例中,本发明涉及通过在包含信号素3a或上皮细胞有丝分裂蛋白抗体(Epigen)的培养基中处理胰腺内胚层细胞来增强NKX6.1的表达的方法。在一些实施例中,与未经包含信号素3a或上皮细胞有丝分裂蛋白抗体的培养基处理的胰腺内胚层细胞相比,经包含信号素3a或上皮细胞有丝分裂蛋白抗体的培养基处理的胰腺内胚层细胞群表达增大的量的NKX6.1。在一些实施例中,与未经包含信号素3a或上皮细胞有丝分裂蛋白抗体的培养基处理的胰腺内胚层细胞相比,激素诸如胰岛素、胰高血糖素和生长素释放肽的表达水平在经包含信号素3a或上皮细胞有丝分裂蛋白抗体的培养基处理的胰腺内胚层细胞中未受到影响。在一些实施例中,胰腺内胚层细胞基本上不表达CDX2或SOX2。在一些实施例中,经包含信号素3a或上皮细胞有丝分裂蛋白抗体的培养基处理的胰腺内胚层细胞通过多能细胞的逐步分化获得。在一些实施例中,衍生出胰腺内胚层细胞的多能细胞是人胚胎多能细胞。
在一些实施例中,本发明涉及分化多能细胞的逐步方法,该方法包括将胰腺内胚层细胞培养于包含肝配蛋白A4、肝配蛋白A3、激活素A、激活素C、信号素3a或上皮细胞有丝分裂蛋白抗体的培养基中。在一些实施例中,胰腺内胚层细胞培养于包含肝配蛋白A4或肝配蛋白A3的培养基中。在一些实施例中,胰腺内胚层细胞培养于包含激活素A或激活素C的培养基中。在一些实施例中,胰腺内胚层细胞培养于包含信号素3a或上皮细胞有丝分裂蛋白抗体的培养基中。在一些实施例中,衍生出胰腺内胚层细胞的多能干细胞是人胚胎多能干细胞。
在一个实施例中,本发明涉及通过用鞘氨醇-1受体激动剂处理胰腺内分泌细胞来诱导内分泌聚簇的表达的方法。在一些实施例中,用于处理胰腺内分泌细胞的鞘氨醇-1受体激动剂是鞘氨醇-1-磷酸(S1P)。
还设想为本发明的实施例的是通过本发明的方法制备的细胞以及使用本发明的细胞的方法。
附图说明
图1A至图1G示出了实例1中所述的分化的人胚胎干细胞系H1的细胞中下列基因的表达的实时PCR分析的数据:胰岛素(图1A)、生长抑素(图1B)、生长素释放肽(图1C)、胰高血糖素(图1D)、PDX-1(图1E)、NKX6.1(图1F)和NGN3(图1G)。
图2A至图2C示出了对胰岛素免疫染色的细胞的图像。图2A,对照;图2B,经50ng/mL的肝配蛋白A3处理的细胞;并且图2C,经100ng/mL的肝配蛋白A3处理的细胞,如实例2中所述。
图3A至图3C示出了对胰岛素免疫染色的细胞的图像。图3A,对照;图3B,经50ng/mL的肝配蛋白A4处理的细胞;并且图3C,经100ng/mL的肝配蛋白A4处理的细胞,如实例2中所述。
图4A至图4D示出了经鞘氨醇-1-磷酸(S1P)处理并在第1天(图4A)、第7天(图4B)和在10天两种不同放大倍率(图4C和图4D)成像的细胞的S6培养物的相差图像。所述图像显示,在第7天,内分泌细胞有明显的聚簇迹象,并且在第10天,所述聚簇彼此被胰腺内胚层上皮薄层分开。
图5A至图5D示出了经S1P处理并对Hb9(图5A)和NKX6.1(图5B)免疫染色或对胰岛素(图5C)和Hb9(图5D)免疫染色的细胞的图像。
图6A和图6B示出了经10μM的S1P处理并在第6阶段开始后三天收获的细胞的不同放大倍率的相差图像。图6C和图6D示出了对NKX2.2免疫染色的细胞的图像。图6C,对照细胞;图6D,经S1P处理的细胞。
具体实施方式
为了以不受限制的方式清楚说明本公开,将本发明的具体实施方式分成下列描述或示出本发明某些特征、实施例或应用的小节。
定义
干细胞是通过其在单细胞水平上既自我更新又分化的能力来定义的未分化细胞。干细胞可产生子代细胞,包括自我更新祖细胞、非更新祖细胞和终末分化细胞。干细胞的特征还在于其在体外分化成来自多个胚层(内胚层、中胚层和外胚层)的多种细胞谱系的功能细胞的能力。干细胞还在移植后产生多种胚层的组织,并且在注射到胚泡内之后,促成基本上至大部分(如果不是所有的话)组织。
干细胞通过其发育潜能分类为:(1)全能的,意指能够产生所有胚胎和胚胎外细胞类型;(2)多能的,意指能够产生所有胚胎细胞类型;(3)多潜能的,意指能够产生细胞谱系子类,但全部在特定组织、器官或生理系统内(例如造血干细胞(HSC)可产生的后代包括HSC(自我更新)、局限于血细胞的寡能祖细胞、以及其为正常血液组分的所有细胞类型和成分(例如血小板));(4)寡能的,意指能够产生比多潜能干细胞更受限制的细胞谱系子类;以及(5)单能的,意指能够产生单细胞谱系(例如生精干细胞)。
分化是未特化的(“未定向的”)或特化不足的细胞获得特化细胞(诸如例如神经细胞或肌肉细胞)的特征的过程。分化的细胞或分化诱导的细胞是已在细胞谱系中占据更特化的(“定向的”)位置的细胞。当应用于分化的过程时,术语“定向的”指在分化途径中已进行到这样的点的细胞:其中在正常环境下,它继续分化成特定的细胞类型或细胞类型子类,并且在正常环境下,不能分化成不同细胞类型或回复到分化程度较低的细胞类型。“去分化”指细胞通过其回复到在细胞谱系内特化(或定向)程度较低的位置的过程。本文所用的“细胞的谱系”限定细胞的遗传性,即它来自哪些细胞以及它能产生什么细胞。细胞谱系将细胞定位于发育和分化的遗传计划内。谱系特异性标记物指与所关注谱系的细胞的表型明确地相关联的特征,可用来评估未定向细胞向所关注谱系的分化。
如本文所用,“标记物”是在所关注细胞中差异表达的核酸或多肽分子。在该语境中,差异表达意指与未分化细胞相比阳性标记物的水平升高并且阴性标记物的水平下降。标记物核酸或多肽的可检测水平,在所关注细胞中充分地高于或低于在其他细胞中,使得可使用多种本领域已知的方法中的任何一种将所关注细胞与其他细胞鉴别和区分开来。
如本文所用,当在细胞中检测到特定标记物时,细胞对于“特定标记物”是“阳性的”或是“阳性的”。相似地,当在细胞中未检测到特定标记物时,细胞对于“特定标记物”是“阴性的”或是“阴性的”。
如本文所用,“细胞密度”和“接种密度”在本文可互换使用,并且是指每单位面积的固体或半固体平坦或弯曲培养基所接种的细胞的数量。
如本文所用,“第1阶段”和“S1”在本文中可互换使用,以鉴定表达定形内胚层(DE)特征性标记物的细胞。
如本文所用,“定形内胚层”指这样的细胞,其具有在原肠胚形成过程中起于上胚层的细胞的特征,并形成胃肠道及其衍生物。定形内胚层细胞表达下述标记物中的至少一种:HNF3β、GATA4、SOX17、CXCR4、Cerberus、OTX2、goosecoid、C-Kit、CD99和MIXL1。
如本文所用,“肠管”指来源于定形内胚层的细胞,所述细胞表达下述标记物中的至少一种:HNF3-β、HNF1-β或HNF4-α。肠管细胞可产生所有内胚层器官,例如肺、肝、胰腺、胃和肠。
在本文中可互换使用的是“第2阶段”和“S2”,其鉴定表达原肠管特征性标记物的细胞。
“前肠内胚层”指产生食道、肺、胃、肝、胰腺、胆囊和一部分十二指肠的内胚层细胞。
“后前肠”指可产生后胃、胰腺、肝和一部分十二指肠的内胚层细胞。
“中肠内胚层”指可产生肠、一部分十二指肠、阑尾和升结肠的内胚层细胞。
“后肠内胚层”指可产生横结肠远端三分之一、降结肠、乙状结肠和直肠的内胚层细胞。
“第3阶段”和“S3”可互换使用,以鉴定表达前肠内胚层特征性标记物的细胞。如本文所用,“表达前肠谱系特征性标记物的细胞”指表达下述标记物中的至少一种的细胞:PDX-1、FOXA2、CDX2、SOX2和HNF4α。
本文可互换使用的是“第4阶段”和“S4”,以鉴定表达胰腺前肠前体特征性标记物的细胞。如本文所用,“表达胰腺前肠前体谱系特征性标记物的细胞”指表达下述标记物中的至少一种的细胞:PDX-1、NKX6.1、HNF6、FOXA2、PTF1a、Prox1和HNF4α。
如本文所用,“第5阶段”和“S5”可互换使用,以鉴定表达胰腺内胚层和胰腺内分泌前体细胞的特征性标记物的细胞。如本文所用的,“表达胰腺内胚层谱系特征性标记物的细胞”指表达下述标记物中的至少一种的细胞:PDX1、NKX6.1、HNF1β、PTF1α、HNF6、HNF4α、SOX9、HB9或PROX1。表达胰腺内胚层谱系特征性标记物的细胞基本上不表达CDX2或SOX2。
“胰腺内分泌细胞”或“胰腺激素表达细胞”或“表达胰腺内分泌谱系特征性标记物的细胞”或“第6阶段细胞”或“S6细胞”在本文中可互换使用,并且是指能够表达下列激素中的至少一种的细胞:胰岛素、胰高血糖素、生长抑素、生长素释放肽和胰多肽。
“胰腺胰岛素阳性细胞”是指表达胰岛素、HB9、NKX2.2和NKX6.1的内分泌细胞群。
“胰腺内分泌前体细胞”或“胰腺内分泌祖细胞”指能够变成胰腺激素表达细胞的胰腺内胚层细胞。此类细胞可表达下列标记物中的至少一种:NGN3、NKX2.2、NeuroD、ISL-1、Pax4、Pax6或ARX。
在本文中可互换使用的是“d1”、“d 1”和“第1天”;“d2”、“d 2”和“第2天”;“d3”、“d 3”和“第3天”等等。这些数字字母组合是指在本专利申请的逐步分化方案过程中的不同阶段中温育的具体天数。
“葡萄糖”和“D-葡萄糖”在本文中可互换使用,并且指右旋糖,在自然界中通常发现的糖。
在本文中可互换使用的是“NeuroD”和“NeuroD1”,其鉴定在胰腺内分泌祖细胞中表达的蛋白质及其编码基因。
在本文中可互换使用的是“LDN”和“LDN-193189”,以指示可得自美国加利福尼亚州Stemgent公司(Stemgent,CA,USA)的BMP受体抑制剂。
多能干细胞的分离、扩增和培养
多能干细胞可表达阶段特异性胚胎抗原(SSEA)3和4以及可用称为Tra-1-60和Tra-1-81的抗体检测的标记物中的一种或多种(Thomson等人,1998,Science 282:1145-1147)。多能干细胞在体外的分化导致SSEA-4、Tra-1-60和Tra-1-81表达的丧失。未分化的多能干细胞通常具有碱性磷酸酶活性,所述碱性磷酸酶活性可通过用4%多聚甲醛固定细胞,然后用矢量红(Vector Red)作为底物显色来检测,如由制造商(美国加利福尼亚州的维克多实验室公司(Vector Laboratories,CA,USA))描述的。如通过RT-PCR检测的,未分化的多能干细胞通常也表达OCT4和TERT。
增殖多能干细胞的另一种期望表型是分化成所有三种胚层的细胞的潜能:内胚层、中胚层和外胚层组织。干细胞的多能性可例如通过下述加以证实:将细胞注射到SCID小鼠内,使用4%多聚甲醛固定所形成的畸胎瘤,然后就来自三个胚层的细胞类型的证据对它们进行组织学检查。另选地,可以通过产生胚状体并评估胚状体中三个胚层相关的标记物的存在来确定多能性。
可以使用标准G带技术并与已公布的相应灵长类物种的核型相比较来分析增殖的多能干细胞系的核型。希望获得具有“正常核型”的细胞,其意指细胞为整倍体,其中所有人染色体都存在并且没有明显的改变。多能细胞在培养物中可使用多种饲养层或通过使用基质蛋白质涂布的容器容易地扩增。另选地,与成分确定的培养基诸如mTeSR1培养基(加拿大温哥华的干细胞科技公司(StemCell Technologies,Vancouver,Canada))组合的化学确定的表面可用于常规细胞扩增。多能细胞可使用酶促、机械或使用多种钙螯合剂诸如EDTA(乙二胺四乙酸)容易地从培养皿中取出。另选地,多能细胞可在不存在任何基质蛋白质或饲养层的情况下悬浮扩增。
多能干细胞的来源
可使用的多能干细胞的类型包括来源于妊娠后形成的组织的建立的多能细胞系,包括在妊娠期间的任何时间取得的胚胎前组织(诸如例如胚泡)、胚胎组织或胎儿组织,所述时间通常但不一定是在约10至12周妊娠前。非限制性例子是建立的人胚胎干细胞(hESC)系或人胚胎干细胞,诸如例如人胚胎干细胞系H1、H7和H9(美国威斯康星州麦迪逊的WiCell研究所(WiCell Research Institute,Madison,WI,USA))。另外合适的是取自已在不存在饲养细胞的情况下培养的多能干细胞群体的细胞。另外合适的是诱导多能细胞(IPS)或重新编程的多能细胞,其可使用多种多能相关的转录因子的被迫表达而来源于成人体细胞,所述转录因子诸如OCT4、NANOG、Sox2、KLF4和ZFP42(Annu Rev Genomics Hum Genet 2011,12:165-185)。本发明的方法中使用的人胚胎干细胞也可按照Thomson等人(美国专利5,843,780;Science,1998,282:1145-1147;Curr Top Dev Biol1998,38:133-165;Proc Natl Acad Sci U.S.A.1995,92:7844-7848)所述的方式制备。
由多能干细胞形成表达胰腺内胚层谱系特征性标记物的细胞
多能干细胞的特征是本领域技术人员众所周知的,并且多能干细胞的另外的特征不断被鉴定。多能干细胞标记物包括例如下述中的一种或多种的表达:ABCG2、cripto、FOXD3、CONNEXIN43、CONNEXIN45、OCT4、SOX2、NANOG、hTERT、UTF1、ZFP42、SSEA-3、SSEA-4、Tra1-60、Tra 1-81。
适用于本发明的多能干细胞包括例如人胚胎干细胞系H9(NIH代码:WA09)、人胚胎干细胞系H1(NIH代码:WA01)、人胚胎干细胞系H7(NIH代码:WA07)和人胚胎干细胞系SA002(Cellartis,Sweden)。还适用于本发明的是表达下述多能细胞特征性标记物中的至少一种的细胞:ABCG2、cripto、CD9、FOXD3、CONNEXIN43、CONNEXIN45、OCT4、SOX2、NANOG、hTERT、UTF1、ZFP42、SSEA-3、SSEA-4、Tra 1-60和Tra 1-81。
定形内胚层谱系特征性标记物选自SOX17、GATA4、HNF3β、GSC、CER1、Nodal、FGF8、Brachyury、Mix样同源盒蛋白、FGF4、CD48、脱中胚蛋白(EOMES)、DKK4、FGF17、GATA6、CXCR4、C-Kit、CD99和OTX2。适用于本发明的是表达定形内胚层谱系特征性标记物中的至少一种的细胞。在本发明的一个方面,表达定形内胚层谱系特征性标记物的细胞为原条前体细胞。在另一方面,表达定形内胚层谱系特征性标记物的细胞是中内胚层细胞。在另一方面,表达定形内胚层谱系特征性标记物的细胞是定形内胚层细胞。
胰腺内胚层谱系特征性标记物选自PDX1、NKX6.1、HNF1β、PTF1α、HNF6、HNF4α、SOX9、HB9和PROX1。适用于本发明的是表达胰腺内胚层谱系特征性标记物中的至少一种的细胞。在本发明的一个方面,表达胰腺内胚层谱系特征性标记物的细胞是胰腺内胚层细胞,其中PDX-1和NKX6.1的表达基本上高于CDX2和SOX2的表达。
胰腺内分泌谱系特征性标记物选自NGN3、NEUROD、ISL1、PDX1、NKX6.1、PAX4、ARX、NKX2.2和PAX6。在一个实施例中,胰腺内分泌细胞能够表达下述激素中的至少一种:胰岛素、胰高血糖素、生长抑素和胰多肽。适用于本发明的是表达胰腺内分泌谱系特征性标记物中的至少一种的细胞。在本发明的一个方面,表达胰内分泌谱系特征性标记物的细胞为胰内分泌细胞。胰腺内分泌细胞可以是表达胰腺激素的细胞。另选地,胰腺内分泌细胞可以是分泌胰腺激素的细胞。
本发明的胰腺内分泌细胞是表达细胞谱系特征性标记物的细胞。表达细胞谱系特征性标记物的细胞表达PDX1以及下列转录因子中的至少一种:NKX2.2、NKX6.1、NEUROD、ISL1、HNF3β、MAFA、PAX4和PAX6。在本发明的一个方面,表达细胞谱系特征性标记物的细胞是细胞。
在一个实施例中,本发明涉及通过在包含肝配蛋白A4或肝配蛋白A3的培养基中培养第5阶段细胞群来增强胰岛素和NKX6.1的表达的方法。在一些实施例中,胰岛素和NKX6.1在细胞群中的表达增强到胰岛素和NKX6.1在未经处理的细胞群中的表达的至少2倍。在一些实施例中,第5阶段细胞群基本上不表达CDX2或SOX2。在一些实施例中,第5阶段细胞群通过多能细胞的逐步分化获得。在一些实施例中,多能细胞是人胚胎多能细胞。
在一个实施例中,本发明涉及通过在包含激活素A或激活素C的培养基中培养第5阶段细胞来增强生长抑素的表达同时抑制胰岛素、胰高血糖素和生长素释放肽的表达的方法。在一些实施例中,经处理的细胞群表达的生长抑素是未经处理的培养物的至少两倍。在一些实施例中,胰岛素的表达被抑制到未经处理培养物中胰岛素的表达的约一半。在一些实施例中,胰高血糖素的表达被抑制到未经处理培养物中胰高血糖素的表达的约1/10。在一些实施例中,生长素释放肽的表达被抑制到未经处理培养物中生长素释放肽的表达的约1/3。在一些实施例中,第5阶段细胞基本上不表达CDX2或SOX2。在一些实施例中,第5阶段细胞通过多能细胞的逐步分化获得。在一些实施例中,多能细胞是人胚胎多能细胞。
在一个实施例中,本发明涉及通过在包含信号素3a或上皮细胞有丝分裂蛋白抗体的培养基中处理第5阶段细胞来增强NKX6.1的表达的方法。在一些实施例中,经处理的细胞群表达的NKX6.1是未经处理培养物的至少两倍。在一些实施例中,与未经处理培养物相比,激素的表达水平在经处理的培养物中未受到影响。在一些实施例中,第5阶段细胞基本上不表达CDX2或SOX2。在一些实施例中,第5阶段细胞通过多能细胞的逐步分化获得。在一些实施例中,多能细胞是人胚胎多能细胞。
在一些实施例中,本发明涉及分化多能细胞的逐步方法,该方法包括将第5阶段细胞培养于包含肝配蛋白A4、肝配蛋白A3、激活素A、激活素C、信号素3a或上皮细胞有丝分裂蛋白抗体的培养基中。在一些实施例中,第5阶段细胞培养于包含肝配蛋白A4或肝配蛋白A3的培养基中。在一些实施例中,第5阶段细胞培养于包含激活素A或激活素C的培养基中。在一些实施例中,第5阶段细胞培养于包含信号素3a或上皮细胞有丝分裂蛋白抗体的培养基中。在一些实施例中,多能干细胞是人胚胎多能干细胞。
在一个实施例中,本发明涉及诱导胰岛素表达的方法,所述方法包括将胰腺内胚层细胞与肝配蛋白配体共培养。在一些实施例中,肝配蛋白配体选自肝配蛋白A3和肝配蛋白A4。在一些实施例中,胰腺内胚层细胞与肝配蛋白配体共培养增强了胰岛素和NKX6.1的表达。在一些实施例中,胰腺内胚层细胞与肝配蛋白配体共培养使胰腺内胚层细胞中胰岛素和NKX6.1的表达增强到未经处理胰腺内胚层细胞中胰岛素和NKX6.1的表达的至少2倍。在一些实施例中,胰腺内胚层细胞基本上不表达CDX2或SOX2。在一些实施例中,胰腺内胚层细胞通过多能干细胞的逐步分化获得。在一些实施例中,本发明的方法中使用的多能干细胞是人胚胎多能干细胞。
在一个实施例中,本发明涉及通过本发明的方法制备的胰岛素和NKX6.1表达细胞。
在一个实施例中,本发明涉及用于诱导内分泌聚簇形成的方法,所述方法包括将胰腺内胚层细胞与鞘氨醇-1受体激动剂共培养。在一些实施例中,胰腺内胚层细胞通过多能干细胞的逐步分化获得。在一些实施例中,多能干细胞是人胚胎多能干细胞。
在整篇文档中引用的出版物据此全文以引用方式并入。本发明通过下述实例进一步举例说明,但并不受其限制。
实例
实例1
将肝配蛋白A4鉴定为胰岛素表达的强诱导物
进行该实例以理解各种蛋白质对由人ES细胞分化产生胰腺内胚层/内分泌培养物的作用。
将人胚胎干细胞系H1(hESC H1,第40代)的细胞作为单细胞以1×105细胞/cm2接种在MATRIGELTM(1:30稀释度;新泽西州的BD生物科学公司(BD Biosciences,NJ))涂布的培养皿上的1培养基(加拿大温哥华的干细胞科技公司(StemCell Technologies,Vancouver,Canada))中,所述培养基补充有10μM的Y27632(Rock抑制剂,目录号Y0503,密苏里州圣路易斯的西格玛奥德里奇公司(SigmaAldrich,St.Louis,MO))。接种后四十八小时,将培养物在不完全PBS(不含Mg或Ca的磷酸盐缓冲盐水溶液)中洗涤。按照如下方法使培养物分化成胰腺内胚层/内分泌谱系:
a)第1阶段(定形内胚层(DE)-3天):细胞在下述第1阶段培养基中培养一天:MCDB-131培养基(目录号10372-019,加利福尼亚州卡尔斯巴德的Invitrogen公司(Invitrogen,Carlsbad,CA)),其补充有0.1%的无脂肪酸BSA(目录号68700,爱荷华州安克尼的Proliant公司(Proliant,Ankeny,IA))、0.0012g/mL的碳酸氢钠(目录号S3187,密苏里州圣路易斯的西格玛奥德里奇公司(SigmaAldrich,St.Louis,MO))、1X的GlutaMaxTM(Invitrogen公司目录号35050-079)、4.5mM的D-葡萄糖(西格玛奥德里奇公司目录号G8769)、100ng/mL的GDF8(明尼苏达州明尼阿波利斯的研发体系公司(R&D Systems,Minneapolis,MN))和1μM的MCX化合物(GSK3B抑制剂,14-丙-2-烯-1-基-3,5,7,14,17,23,27-七氮杂四环[19.3.1.1~2,6~.1~8,12~]二十七-1(25),2(27),3,5,8(26),9,11,21,23-壬烯-16-酮,美国专利申请公开2010-0015711;该专利申请全文以引用的方式并入本文)。细胞随后在MCDB-131培养基中再培养一天,所述MCDB-131培养基补充有0.1%的无脂肪酸BSA、0.0012g/mL的碳酸氢钠、1X的GlutaMaxTM、4.5mM的D-葡萄糖、100ng/mL的GDF8和0.1μM的MCX化合物。细胞随后在MCDB-131培养基中再培养一天,所述MCDB-131培养基补充有0.1%的无脂肪酸BSA、0.0012g/mL的碳酸氢钠、1X的GlutaMaxTM、4.5mM的D-葡萄糖和100ng/mL的GDF8,接下来是
b)第2阶段(原肠管-2天):用MCDB-131培养基处理细胞两天,所述MCDB-131培养基补充有0.1%的无脂肪酸BSA;0.0012g/mL的碳酸氢钠;1X的GlutaMaxTM;4.5mM的D-葡萄糖;0.25mM的抗坏血酸(密苏里州圣路易斯的西格玛公司(Sigma,St.Louis,MO))和25ng/mL的FGF7(明尼苏达州明尼阿波利斯的研发体系公司(RD Systems,Minneapolis,MN)),接下来是
c)第3阶段(前肠-2天):用MCDB-131培养基处理细胞1天,所述MCDB-131培养基补充有1:200稀释的ITS-X(Invitrogen公司);4.5mM的葡萄糖;1X的GlutaMaxTM;0.0017g/mL的碳酸氢钠;2%的无脂肪酸BSA;0.25μM的SANT-1(Sigma,St.Louis,MO);10ng/mL的激活素A(RD Systems);1μM的视黄酸(RA;Sigma);25ng/mL的FGF7;0.25mM的抗坏血酸;200nM的TPB(PKC活化剂;目录号565740;新泽西州吉布斯顿的EMD化学品公司(EMD Chemicals,Gibstown,NJ));10μM的佛司可林(FSK,Sigma)和100nM的LDN(BMP受体抑制剂;目录号04-0019;加利福尼亚州圣迭戈的Stemgent公司(Stemgent;San Diego,CA))。第2天用MCDB-131培养基处理细胞,所述MCDB-131培养基补充有1:200稀释的ITS-X;4.5mM的葡萄糖;1X的GlutaMaxTM;0.0017g/mL的碳酸氢钠;2%的无脂肪酸BSA;0.25μM的SANT-1;10ng/mL的激活素A;1μM的RA;25ng/mL的FGF7;0.25mM的抗坏血酸、200nM的TPB、10μM的佛司可林和10nM的LDN,接下来是
d)第4阶段(胰腺前肠前体-2天):用MCDB-131培养基处理细胞两天,所述MCDB-131培养基补充有1:200稀释的ITS-X;4.5mM的葡萄糖;1X的GlutaMaxTM;0.0015g/mL的碳酸氢钠;2%的无脂肪酸BSA;0.25μM的SANT-1;50nM的RA;50nM的LDN-193189;10μM的佛司可林;0.25mM的抗坏血酸;和100nM的TPB,接下来是
e)第5阶段(胰腺内胚层/内分泌-3天):用MCDB-131培养基处理第4阶段细胞三天,所述MCDB-131培养基补充有1:200稀释的ITS-X;20mM的葡萄糖;1X的GlutaMaxTM;0.0015g/mL的碳酸氢钠;2%的无脂肪酸BSA;0.25μM的SANT-1;50nM的RA;10μM的佛司可林;0.25mM的抗坏血酸,且添加100nM的ALk5抑制剂SD-208(公开于Molecular Pharmacology 2007,72:152-161)仅持续2至3天。
在第5阶段的第1天,将下表I中所列的因子添加到培养基中,并在S5完成时(第5阶段的第3天)收集mRNA进行相关胰腺内胚层/内分泌基因的PCR分析。作为对照,将细胞仅用上面列出的S5培养基处理。根据制造商说明书,用RNeasy微型试剂盒(加利福尼亚州瓦伦西亚的凯杰公司(Qiagen;Valencia,CA))提取总RNA并使用高容量cDNA逆转录试剂盒(加利福尼亚州福斯特城的应用生物系统公司(Applied Biosystems,FosterCity,CA))逆转录。使用预上样于定制Taqman阵列(应用生物系统公司)上的Taqman Universal Master Mix和Taqman Gene Expression Assays扩增cDNA。使用序列检测软件(应用生物系统公司(Applied Biosystems))分析数据,并使用ΔΔCt方法归一化为未分化的人胚胎干细胞(hES)。所有引物均购自应用生物系统公司。
表I-实例1在S5时测试的因子的列表
蛋白 | 浓度 | 研发体系公司目录号 |
上皮细胞有丝分裂蛋白抗体 | 20ng/mL | 6629-EP-025 |
信号素3a | 50ng/ml | 1250-S3-025 |
导蛋白4 | 100ng/ml | 1254-N4-025 |
半乳糖凝集素-8 | 100ng/ml | 1305-GA-050 |
类胰蛋白酶-Y-1 | 20ng/mL | 1667-SE-010 |
β细胞素 | 20ng/mL | 261-CE-010 |
基膜聚糖 | 100ng/ml | 2846-LU-050 |
表皮形态发生素 | 50ng/ml | 2936-EP-025 |
间皮素 | 50ng/ml | 3265-MS-050 |
软骨基质蛋白-4 | 100ng/ml | 3380-MN-050 |
镍纹蛋白 | 50ng/ml | 3475-MN-025 |
肝配蛋白-A4 | 100ng/ml | 369-EA |
表I–续
IBSP | 100ng/ml | 4014-SP-050 |
EFG-L6 | 50ng/ml | 4329-EG-025 |
R-脊椎蛋白-1 | 100ng/ml | 4645-RS-025 |
肝配蛋白-B1 | 100ng/ml | 473-EB-200 |
跨膜蛋白酶丝氨酸(Hepsin) | 50ng/ml | 4776-SE-010 |
激活素A | 20ng/mL | 338-AC-010 |
EphA4 | 50ng/ml | 6827-A4-050 |
神经粘蛋白 | 100ng/ml | 6508-NC-050 |
DKK1 | 100ng/ml | 5439-DK-010 |
激肽释放酶-4 | 50ng/ml | 1719-SE-010 |
EGF | 20ng/mL | 236-EG-200 |
BDNF | 20ng/mL | 248-BD-005 |
脊骨蛋白 | 50ng/ml | 2495-SE-010 |
HGF | 20ng/mL | 294-HG-005 |
EphB4 | 50ng/ml | 3038-B4-100 |
松弛素1 | 50ng/ml | 3257-RN-025 |
激活素C | 20ng/mL | 4879-AC-010 |
BMP5 | 20ng/mL | 615-BMC-020 |
IGF-1 | 20ng/mL | 291-G1-200 |
图1A至图1G示出了如实例1所概述并在表I中所列因子的存在下分化为第5阶段的人胚胎干细胞系H1的细胞中的下列基因的表达的实时PCR分析的数据:胰岛素(图1A)、生长抑素(图1B)、生长素释放肽(图1C)、胰高血糖素(图1D)、PDX-1(图1E)、NKX6.1(图1F)和NGN3(图1G)。
如图1所示,与对照培养物相比,肝配蛋白A4增强了NKX6.1和胰岛素的mRNA表达(图1F)同时示出对PDX-1(图1E)和NGN3表达(图1G)的影响最小。因子诸如激活素A和激活素C显著增强了生长抑素的表达(图1B)同时抑制了胰岛素(图1A)、胰高血糖素(图1D)和生长素释放肽(图1C)的表达。此外,与未经处理培养物相比,因子诸如信号素3a和上皮细胞有丝分裂蛋白抗体增强了NKX6.1表达,同时不影响激素的表达。在图1中,对照培养物中不同标记物的平均表达水平在图表中以虚线示出。
实例2
验证在S5时肝配蛋白对胰岛素表达的作用
该实例描述了实例1中鉴别的活性化合物(hits)的验证。具体地讲,描述了下列方案中在S5时添加肝配蛋白A3或肝配蛋白A4的作用。
将人胚胎干细胞系H1(hESC H1,第40代)的细胞作为单细胞以1×105细胞/cm2接种在MATRIGELTM(1:30稀释度;新泽西州的BD生物科学公司(BD Biosciences,NJ))涂布的培养皿上的补充有10μM的Y27632的1培养基中。接种后四十八小时,将培养物在不完全PBS(不含Mg或Ca的磷酸盐缓冲盐水溶液)中洗涤。按照如下方法使培养物分化成胰腺内胚层/内分泌谱系:
a)第1阶段(定形内胚层(DE)-3天):将细胞在第1阶段培养基中培养一天(参见以上实例1)。细胞随后在MCDB-131培养基中再培养一天,所述MCDB-131培养基补充有0.1%的无脂肪酸BSA、0.0012g/mL的碳酸氢钠、1X的GlutaMaxTM、4.5mM的D-葡萄糖、100ng/mL的GDF8和0.1μM的MCX化合物。细胞随后在MCDB-131培养基中再培养一天,所述MCDB-131培养基补充有0.1%的无脂肪酸BSA、0.0012g/mL的碳酸氢钠、1X的GlutaMaxTM、4.5mM的D-葡萄糖和100ng/mL的GDF8,接下来是
b)第2阶段(原肠管-2天):用MCDB-131培养基处理细胞两天,所述MCDB-131培养基补充有0.1%的无脂肪酸BSA;0.0012g/mL的碳酸氢钠;1X的GlutaMaxTM;4.5mM的D-葡萄糖;0.25mM的抗坏血酸(密苏里州的西格玛公司(Sigma,MO))和25ng/mL的FGF7(明尼苏达州的研发体系公司(RD Systems,MN)),接下来是
c)第3阶段(前肠-2天):用MCDB-131培养基处理细胞1天,所述MCDB-131培养基补充有1:200稀释的ITS-X(加利福尼亚州的Invitrogen公司(Invitrogen,Ca));4.5mM的葡萄糖;1X的GlutaMaxTM;0.0017g/mL的碳酸氢钠;2%的无脂肪酸BSA;0.25μM的SANT-1(密苏里州的西格玛公司(Sigma,MO));10ng/mL的激活素A(明尼苏达州的研发体系公司(RD Systems,MN));1μM的RA(密苏里州的西格玛公司(Sigma,MO));25ng/mL的FGF7;0.25mM的抗坏血酸;200nM的TPB(PKC活化剂;目录号565740;新泽西州吉布斯顿的EMD化学品公司(EMD Chemicals,Gibstown,NJ));10μM的佛司可林和100nM的LDN(BMP受体抑制剂;目录号04-0019;Stemgent公司)。第2天用MCDB-131培养基处理细胞,所述MCDB-131培养基补充有1:200稀释的ITS-X;4.5mM的葡萄糖;1X的GlutaMaxTM;0.0017g/mL的碳酸氢钠;2%的无脂肪酸BSA;0.25μM的SANT-1;10ng/mL的激活素A;1μM的RA;25ng/mL的FGF7;0.25mM的抗坏血酸、200nM的TPB、10μM的佛司可林和10nM的LDN,接下来是
d)第4阶段(胰腺前肠前体-2天):用MCDB-131培养基处理细胞三天,所述MCDB-131培养基补充有1:200稀释的ITS-X;4.5mM的葡萄糖;1X的GlutaMaxTM;0.0015g/mL的碳酸氢钠;2%的无脂肪酸BSA;0.25μM的SANT-1;50nM的RA;50nM的LDN-193189;10μM的佛司可林;0.25mM的抗坏血酸;和100nM的TPB,接下来是
e)第5阶段(胰腺内胚层/内分泌-3天):用MCDB-131培养基处理第4阶段细胞三天,所述MCDB-131培养基补充有1:200稀释的ITS-X;4.5mM的葡萄糖;1X的GlutaMaxTM;0.0015g/mL的碳酸氢钠;2%的无脂肪酸BSA;0.25μM的SANT-1;50nM的RA;10μM的佛司可林;0.25mM的抗坏血酸;100nM的ALk5抑制剂(仅处理2-3天)(SD-208,公开于Molecular Pharmacology 2007,72:152-161)和+/-0-100ng/mL的肝配蛋白A3或肝配蛋白A4(明尼苏达州的研发体系公司)。
在第5阶段结束时,将对照和经肝配蛋白处理的培养物固定并对胰岛素蛋白表达进行染色(使用豚鼠抗胰岛素抗体,其得自马萨诸塞州剑桥的密理博公司(Millipore;Cambridge,MA))。图2示出了对胰岛素免疫染色的细胞的图像。图2A,对照细胞;图2B,经50ng/mL的肝配蛋白A3处理的细胞;图2C,经100ng/mL的肝配蛋白A3处理的细胞。图3示出了对胰岛素免疫染色的细胞的图像。图3A,对照细胞;图3B,经50ng/mL的肝配蛋白A4处理的细胞;图3C,经100ng/mL的肝配蛋白A4处理的细胞。这些数据表明,与实例1的数据相符,在第5阶段时添加肝配蛋白A3和肝配蛋白A4两者显著增强了胰岛素的蛋白质表达。
实例3
在S6时添加鞘氨醇-1-磷酸显著加速了包含内分泌激素的细胞聚簇的
形成
该实例描述了在第6阶段时内分泌聚簇形成的进展以及鞘氨醇-1-磷酸在加速内分泌富集聚簇形成方面的作用。
将人胚胎干细胞系H1(hESC H1,第40代)的细胞作为单细胞以1×105细胞/cm2接种在MATRIGELTM(1:30稀释度;新泽西州的BD生物科学公司(BD Biosciences,NJ))涂布的培养皿上补充有10μM的Y27632的1培养基(加拿大温哥华的干细胞科技公司(StemCell Technologies,Vancouver,Canada))中。接种后四十八小时,将培养物在不完全PBS(不含Mg或Ca的磷酸盐缓冲盐水溶液)中洗涤。按照如下方法使培养物分化成胰腺内胚层/内分泌谱系:
a)第1阶段(定形内胚层(DE)-3天):将细胞在第1阶段培养基中培养一天(参见以上实例1)。细胞随后在MCDB-131培养基中再培养一天,所述MCDB-131培养基补充有0.1%的无脂肪酸BSA、0.0012g/mL的碳酸氢钠、1X的GlutaMaxTM、4.5mM的D-葡萄糖、100ng/mL的GDF8和0.1μM的MCX化合物。细胞随后在MCDB-131培养基中再培养一天,所述MCDB-131培养基补充有0.1%的无脂肪酸BSA、0.0012g/mL的碳酸氢钠、1X的GlutaMaxTM、4.5mM的D-葡萄糖和100ng/mL的GDF8,接下来是
b)第2阶段(原肠管-2天):用MCDB-131培养基处理细胞两天,所述MCDB-131培养基补充有0.1%的无脂肪酸BSA;0.0012g/mL的碳酸氢钠;1X的GlutaMaxTM;4.5mM的D-葡萄糖;0.25mM的抗坏血酸(密苏里州的西格玛公司(Sigma,MO))和25ng/mL的FGF7(明尼苏达州的研发体系公司(RD Systems,MN)),接下来是
c)第3阶段(前肠-2天):用MCDB-131培养基处理细胞1天,所述MCDB-131培养基补充有1:200稀释的ITS-X(加利福尼亚州的Invitrogen公司(Invitrogen,Ca));4.5mM的葡萄糖;1X的GlutaMaxTM;0.0017g/mL的碳酸氢钠;2%的无脂肪酸BSA;0.25μM的SANT-1(密苏里州的西格玛公司(Sigma,MO));10ng/mL的激活素A(明尼苏达州的研发体系公司(RD Systems,MN));1μM的RA(密苏里州的西格玛公司(Sigma,MO));25ng/mL的FGF7;0.25mM的抗坏血酸;200nM的TPB(PKC活化剂;目录号565740;新泽西州吉布斯顿的EMD化学品公司(EMD Chemicals,Gibstown,NJ));10μM的佛司可林(FSK,密苏里州的西格玛公司(Sigma,MO))和100nM的LDN(BMP受体抑制剂;目录号04-0019;加利福尼亚州的Stemgent公司(Stemgent,CA))。第2天用MCDB-131培养基处理细胞,所述MCDB-131培养基补充有1:200稀释的ITS-X;4.5mM的葡萄糖;1X的GlutaMaxTM;0.0017g/mL的碳酸氢钠;2%的无脂肪酸BSA;0.25μM的SANT-1;10ng/mL的激活素A;1μM的RA;25ng/mL的FGF7;0.25mM的抗坏血酸、200nM的TPB和10nM的LDN,接下来是
d)第4阶段(胰腺前肠前体-2天):用MCDB-131培养基处理细胞两天,所述MCDB-131培养基补充有1:200稀释的ITS-X;4.5mM的葡萄糖;1X的GlutaMaxTM;0.0015g/mL的碳酸氢钠;2%的无脂肪酸BSA;0.25μM的SANT-1;50nM的RA;50nM的LDN-193189;10μM的佛司可林;0.25mM的抗坏血酸;2ng/mL的FGF7;1ng/mL的AA;和100nM的TPB,接下来是
e)第5阶段(胰腺内胚层/内分泌-3天):用MCDB-131培养基处理第4阶段细胞三天,所述MCDB-131培养基补充有1:200稀释的ITS-X;15mM的葡萄糖;1X的GlutaMaxTM;0.0015g/mL的碳酸氢钠;2%的无脂肪酸BSA;0.25μM的SANT-1;50nM的RA;10μM的佛司可林;0.25mM的抗坏血酸;和1ng/mL的FGF7;且仅在第2至3天添加100nM的ALK5抑制剂SD-208,接下来是
f)第6阶段(胰腺内分泌-3至10天):用MCDB-131培养基处理第5阶段细胞3至10天,所述MCDB-131培养基补充有1:200稀释的ITS-X;15mM的葡萄糖;1X的GlutaMaxTM;0.0015g/mL的碳酸氢钠;2%的无脂肪酸BSA;0.25μM的SANT-1;50nM的RA;0.25mM的抗坏血酸。在一些培养物中,添加10μM的鞘氨醇-1-磷酸(密苏里州的西格玛公司(Sigma,MO)),持续三天。
图4A至图4D示出了经鞘氨醇-1-磷酸(S1P)处理并在第1天(图4A)、第7天(图4B)和在10天以两种不同放大倍率(图4C和图4D)成像的细胞的S6培养物的相差图像。图像显示,在第7天,内分泌细胞有明显的聚簇迹象,并且在第10天,所述聚簇彼此被胰腺内胚层上皮薄层分开。
图5A至图5D示出了对Hb9(图5A)和NKX6.1(图5B)免疫染色或对胰岛素(图5C)和Hb9(图5D)免疫染色的细胞的图像。图5A和图5B示出了内分泌聚簇富含Hb9,而聚簇周围的胰腺上皮富含NKX6.1。在富含Hb9的聚簇中的细胞中的一些对于NKX6.1也呈阳性。如图5C和图5D中所示,聚簇富含胰岛素和Hb9。该形态变化非常类似于富含NKX6.1+PDX-1+的上皮产生内分泌聚簇的胰腺发育。在每种情况下,使用不同滤光片从相同视野的细胞获得该对图像。
图6A和图6B示出了经10μM的鞘氨醇-1-磷酸(S1P)处理并在第6阶段开始后三天收获的细胞的不同放大倍率的相差图像。这些图像示出,内分泌聚簇仅在第6阶段开始后3天出现。这比对照培养物中聚簇的形成要早约7天。
图6C和图6D示出了对NKX2.2免疫染色的对照细胞(图6C)和经S1P处理的细胞(图6D)的图像。在经S1P处理的培养物中,与NKX2.2+细胞均匀分布在整个培养物中的对照培养物(图6D)相比,内分泌聚簇也富含NKX2.2+细胞(图6C)。
Claims (27)
1. 一种在激素表达细胞中诱导胰岛素表达的方法,所述方法包括将胰腺内胚层细胞与肝配蛋白配体共培养。
2. 根据权利要求2所述的方法,其中所述胰腺内胚层细胞与所述肝配蛋白配体共培养也增强了NKX6.1的表达。
3. 根据权利要求2所述的方法,其中与未经处理的胰腺内胚层细胞中胰岛素和NKX6.1的表达相比,所述胰腺内胚层细胞与肝配蛋白配体共培养增强了所述胰腺内胚层细胞中胰岛素和NKX6.1的表达。
4. 根据权利要求3所述的方法,其中所述胰腺内胚层细胞基本上不表达CDX2或SOX2。
5. 根据权利要求4所述的方法,其中所述胰腺内胚层细胞表达约小于10%的CDX2或SOX2。
6. 根据权利要求1至5中任一项所述的方法,其中所述肝配蛋白配体是肝配蛋白A3或肝配蛋白A4。
7. 根据权利要求6所述的方法,其中所述胰腺内胚层细胞通过多能干细胞的逐步分化获得。
8. 根据权利要求7所述的方法,其中所述多能干细胞是人胚胎多能干细胞。
9. 通过权利要求1所述的方法制备的胰岛素和NKX6.1表达细胞。
10. 一种通过胰腺内胚层细胞与肝配蛋白配体共培养所制备的细胞群,其中与未经肝配蛋白配体处理的胰腺内胚层细胞相比,所述细胞群表达增强的胰岛素和NKX6.1。
11. 根据权利要求10所述的细胞群,其中所述肝配蛋白配体是肝配蛋白A3或肝配蛋白A4。
12. 一种增强激素表达细胞中生长抑素的表达的方法,所述方法包括将胰腺内胚层细胞培养于包含激活素A或激活素C的培养基中。
13. 根据权利要求12所述的方法,其中胰岛素、胰高血糖素和生长素释放肽的表达受到抑制。
14. 根据权利要求12所述的方法,其中经激活素A或激活素C处理的所述胰腺内胚层细胞群表达的生长抑素比未经激活素A或激活素C处理的胰腺内胚层细胞群更多。
15. 根据权利要求14所述的方法,其中与未经激活素A或激活素C处理的胰腺内胚层细胞群中胰岛素的表达相比,胰岛素的表达在经激活素A或激活素C处理的所述胰腺内胚层细胞群中受到抑制。
16. 根据权利要求12所述的方法,其中与未经激活素A或激活素C处理的胰腺内胚层细胞群中胰高血糖素的表达相比,经激活素A或激活素C处理的所述胰腺内胚层细胞群中胰高血糖素的表达受到抑制。
17. 根据权利要求12所述的方法,其中与未经激活素A或激活素C处理的胰腺内胚层细胞群中生长素释放肽的表达相比,所述生长素释放肽的表达在经激活素A或激活素C处理的所述胰腺内胚层细胞群中受到抑制。
18. 根据权利要求12至18中任一项所述的方法,其中所述胰腺内胚层细胞基本上不表达CDX2或SOX2。
19. 根据权利要求18所述的方法,其中所述经激活素A或激活素C处理的所述胰腺内胚层细胞通过多能细胞的逐步分化获得。
20. 根据权利要求19所述的方法,其中所述衍生出所述胰腺内胚层细胞的所述多能细胞是人胚胎多能细胞。
21. 一种通过在包含信号素3a或上皮细胞有丝分裂蛋白抗体的培养基中处理胰腺内胚层细胞来增强NKX6.1的表达的方法。
22. 根据权利要求21所述的方法,其中与未经包含信号素3a或上皮细胞有丝分裂蛋白抗体的培养基处理的胰腺内胚层细胞相比,经包含信号素3a或上皮细胞有丝分裂蛋白抗体的培养基处理的所述胰腺内胚层细胞群表达增大的量的NKX6.1。
23. 根据权利要求21所述的方法,其中与未经包含信号素3a或上皮细胞有丝分裂蛋白抗体的培养基处理的胰腺内胚层细胞相比,胰岛素、胰高血糖素和生长素释放肽的表达水平在经包含信号素3a或上皮细胞有丝分裂蛋白抗体的培养基处理的胰腺内胚层细胞中未受到影响。
24. 一种诱导内分泌聚簇的形成的方法,所述方法包括将胰腺内分泌细胞与鞘氨醇-1受体激动剂共培养。
25. 根据权利要求24所述的方法,其中用于处理胰腺内分泌细胞的所述鞘氨醇-1受体激动剂是鞘氨醇-1-磷酸(S1P)。
26. 根据权利要求24所述的方法,其中所述胰腺内分泌细胞通过多能干细胞的逐步分化获得。
27. 根据权利要求26所述的方法,其中所述多能干细胞是人胚胎多能干细胞。
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CN111094547B (zh) * | 2017-06-14 | 2024-02-09 | 德国亥姆霍兹慕尼黑中心健康与环境研究中心(有限公司) | 用于纯化源自人胚胎干细胞的内胚层和胰腺内胚层细胞的方法 |
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