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CN1602351B - 源自人胚胎干细胞的胰岛细胞 - Google Patents

源自人胚胎干细胞的胰岛细胞 Download PDF

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Publication number
CN1602351B
CN1602351B CN028243676A CN02824367A CN1602351B CN 1602351 B CN1602351 B CN 1602351B CN 028243676 A CN028243676 A CN 028243676A CN 02824367 A CN02824367 A CN 02824367A CN 1602351 B CN1602351 B CN 1602351B
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cells
cell
islet
differentiation
insulin
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CN1602351A (zh
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G·J·菲斯克
M·S·伊库玛
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Asterias Biotherapeutics Inc
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Geron Corp
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Abstract

本发明提供由胚胎干细胞生产胰岛细胞的系统。用促进胰岛前体细胞和成熟胰岛素分泌细胞产生的药物将分化引向并定向于内胚层细胞。由此可产业规模地生产大量胰岛细胞以用于研究、药物筛选或再生药物。

Description

源自人胚胎干细胞的胰岛细胞 
                            技术领域
本发明涉及细胞生物学，胚胎干细胞和细胞分化。更具体地说，本发明提供具有胰腺内分泌功能的分化细胞。 
                            相关申请
本发明要求2001年12月7日的美国临时申请60/338,885的优先权。 
                            背景技术
美国糖尿病协会估计，目前全美有5百万糖尿病患者，1千万高危人群。 
该病及其后遗症对美国经济的损耗巨大。每年因糖尿病的医疗支出总计980亿美元，约占全美医疗保健支出的七分之一。每年新增24,000例糖尿病致盲病例。糖尿病是肾衰竭的主要原因，在新增透析患者中占40％。而且，糖尿病还是下肢截肢最常见的原因，每年约56,000人因此丧失下肢。糖尿病人每年的人均医疗支出为10,071美元，非糖尿病人则仅为2,699美元。 
重症I型糖尿病(胰岛素依赖型糖尿病)占糖尿病总数的5-10％。其病理是：患者胰腺内分泌胰岛素的β细胞被自身免疫反应清除。目前的治疗手段包括定期注射胰岛素，长期的饮食控制，以及持续监测胰岛素水平以调整胰岛素剂量。据估计，到2005年，对重组胰岛素的市场需求将达到40亿美元。显然，胰岛素的可得性对于I型糖尿病人来说是性命攸关的。但是，糖尿病人必需遵守的每日治疗无疑是繁琐的，而且并非普遍有效。 
因此，目前正在进行数项给糖尿病人移植分离自供体胰岛的胰岛细胞的临床试验。此类试验的可行性是基于胰岛细胞分离和培养方面的最新进展。美国专利4,797,213描述了Langerhans胰岛的分离。美国专利4,439,521报道了一种生产自我复制性胰岛样结构物的方法。美国专利5,919,703报道了胰岛的制备和储存方法。美国专利5,888,816报道了用下丘脑和脑垂体的提取物培养胰岛细胞的方法。WO00/72885报道了在非胰岛组织内诱导胰腺激素有调产生的方法。WO00/78929报道了制备胰岛细胞的方法。Kim等(Genes Dev.，15：111，2001)论述了调节胰腺发育及其功能的胞内信号。Yamaoka等(Int.J.Mol.Med.，3：247，1999)论述了胰岛的发育，并推测了Sonic hedgehog和活化素等因子，PDX1和Isl1等转录因子，EGF和HGF等生长因子，胰岛素和生长素等激素以及N-CAM和钙粘素等细胞粘附分子的推定作用。 
Peck等(Ann.Med.，33：186，2001)提出将胰干细胞作为构建单元用于构建更好的代胰岛继而用于治疗I型糖尿病。WO00/47721报道了诱导胰岛素阳性祖细胞的方法。WO01/39784报道了分离自胰岛细胞的nestin阳性胰干细胞。WO01/77300报道了人胰上皮祖细胞能够分化为胰泡细胞、胰管(ductal)细胞和胰岛细胞。Deutsch等(Development，128：871，2001)描述了胚胎内胚层中胰肝双能前体细胞群。Zulewski等(Diabetes，50：521，2001)描述了分离自成人胰岛的nestin阳性多能干细胞，它们可分化为内分泌、外分泌和肝表型。美国专利6,326,201(Curis Inc.)报道了自胰管中解离然后细胞培养而制得的胰祖细胞。Bretzel等(Exp.Clin.Endocrinol.Diabetes，190(Suppl.2)：S384，2001)和Oberholzer等(Ann.N.Y.Acad.Sci.，875：189，1999)论述了胰岛细胞移植方面的现有临床经验。目前的临床试验通常涉及将来自至少两个胰供体的细胞融合。既使这种方法被证明有效，目前来源仍无法提供足够的材料来满足所有I型糖尿病人治疗所需。 
已有多家研究机构开始致力于研究和开发胚胎多能干细胞可分化为其它类型细胞的能力。据报道，已从鼠胚胎细胞获得了具有胰岛细胞系特征的细胞。Lumelsky等(Science，292：1389，2001)报道了鼠胚胎细胞可分化为类似胰岛的胰岛素分泌结构物。Soria等(Diabetes 49：157，2000)报道衍生自鼠胚胎干细胞的胰岛素分泌细胞令得链脲霉素诱导的糖尿病小鼠的高血糖恢复正常。 
遗憾的是，胚胎干细胞发育的小鼠模型只是特例，未能获得适用其它物种的分化策略。事实上，目前只能从很少的其它哺乳动物中重复分离得到多能干细胞。直到最近，Thomson等才从人胚泡中分离获得了胚胎干细胞(Science，282：114，1998)。同时，Gearhart及其同事由胎性腺组织获得了人胚生殖(hEG)细胞系(Shamblott等，Proc.Natal.Acad.Sci.USA，95：13726，1998)。与只要用白血病抑制因子(LIF)即可阻止分化的鼠胚胎干细胞不同，人胚胎干细胞必需在非常特殊的条件下才能维持(美国专利6,200,806；WO99/20741；WO01/51616)。所以，必需开发全新的模型以使人多能细胞分化为具有完全功能的不同细胞类型。 
Jacobson等(Transplant.Proc.，33：674，2001)报道了恒河猴胚胎干细胞向小肠和胰腺内胚层的分化。Assady等(Diabetes，50：1691，2001)通过对培养基的免疫组织化学和酶联免疫试验鉴定到人胚胎干细胞分化为胚状体后的胰岛素产生。当然，胚状体包含了多种不同的细胞类型(WO01/51616)，而Assady没有试图分离分泌胰岛素的细胞，也没有试图确定可富集细胞群的分化条件。 
要使胚胎干细胞衍生的胰岛细胞满足产业应用的要求，仍需要开发新的方法以获得高纯度的胰岛细胞群。 
                            发明内容
本发明提供一种生产灵长动物细胞的系统，所述细胞是由多能细胞分化得到的胰岛细胞系细胞。本发明获得了具有相当富集程度的胰岛祖细胞群。然后，胰岛祖细胞进一步分化为分别分泌胰岛素、胰高血糖素、抑生长素或以上三者的细胞集落。 
所以，本发明实施方式之一是一种令灵长动物多能干细胞(pPS)(例如胚胎干细胞)分化而获得的细胞群，其中至少5％的细胞分泌以下内源基因编码的4种胰岛细胞蛋白中的至少一种：胰岛素，胰高血糖素，抑生长素和看似惰性的被称为胰多肽的产物。这些细胞可以呈细胞簇，其中包含分泌以上三种激素中任一种的细胞，并可以经加工而尽可能去除其它细胞类型。本发明的细胞可根据后文所述的表型标记来鉴定。功能和效果可根据给予高血糖症患者后空腹血糖水平的改善来确认。 
本发明的另一实施方式是一种能够自我更新并能够产生成熟胰岛细胞后代的分化细胞群。这表示，此类细胞不再是多能的，但它们保留了通过增殖形成胰岛细胞的能力。该细胞群内中未分化多能细胞应尽可能少，既使残留也不应是通过进一步增殖可形成胰岛细胞的那些。可选的是，可通过提高端粒末端转移酶的活性来提高干细胞的复制能力。 
本发明的另一实施方式是一种获得分泌多肽的细胞的方法，其中，通过例如形成含内胚层的胚状体或其它形式引发pPS细胞的分化。然后，将细胞培养在多种分化因子的混合物中，所述分化因子例如TGF-β拮抗剂样的Noggin，活化素A，正丁酸盐，或后文其它因子。此外，或作为这一步骤的替代，可对细胞进行遗传改造使之表达某种胰转录因子，例如神经生成素3。 
本发明的另一实施方式是一种利用本发明细胞组合物来筛选能够调节胰岛细胞功能的化合物的方法。 
本发明的另一实施方式是一种通过培养本发明胰岛细胞来生产胰岛素、胰高血糖素或抑生长素的方法。本发明还包括包含本发明细胞的药物组合物和装置。本发明细胞、组合物和装置可用于在个体，尤其是但不限于I型糖尿病人中恢复胰岛细胞功能。 
以下是对本发明上述及其它实施方式的详细描述。 
                            附图说明
图1：分化和成熟过程中的hES衍生的肝细胞(10×，40×)。A行显示在含5mM正丁酸钠的培养基中培养4天后的细胞。培养物中80％以上的细胞含有大 细胞核和粒状细胞质。将这些细胞转移到特化肝细胞培养基中培养2-4天(B行和C行)。此时，多的是多核多边形细胞。ES衍生的肝细胞与新鲜分离的成人肝细胞(D行)和胎肝细胞(E行)具有相同的形态学特征。 
图2：表达胰岛素的hES衍生的细胞。分化策略基于假设肝细胞和胰细胞具有相同的早期祖细胞。早期分化与肝细胞相似，区别仅在于培养基中加有环杷明。接着，用胰岛分化因子活化素A、烟酰胺、环杷明和低浓度正丁酸盐培养细胞。最后，细胞在活化素A、β-动物纤维素、烟酰胺和IGF-1中培养11天以促进胰岛细胞长出。上图显示DAPI染色的细胞核。下图中的相应区域显示胰岛素的迷散性红色抗体染色。 
图3：沿胰岛细胞个体发育路径逐步引导hSE细胞分化的结果。在mRNA水平分析三种肠道内胚层标记的表达，并相对胰腺内的水平进行归一化。未分化细胞和非特异性分化细胞内的表达水平很低，但是正丁酸盐和活化素联用造成了向具有肠道内胚层特征的细胞的分化或选择。 
图4：hSE细胞先在过渡培养基中长时间聚集培养，然后在含有促分裂素和胰岛细胞分化因子Noggin的培养基中培养的结果。上图和中图分别为低倍放大和高倍放大的胰岛素c-肽染色情况，显示有陈述的胰腺β细胞簇。下图显示抑生长素，一种δ细胞标记的染色情况。 
图5：一例胰岛细胞分化试验中的细胞，以含转录调节剂基因神经生成素3(Neurogenin 3)的载体转染后9天。据抗体测定，这些细胞表现出高水平的胰高血糖素表达。 
图6(A)和(B)显示神经生成素3转染细胞内(实心柱)的mRNA表达水平，与阴性对照(阴影柱)相比。转染基因早在第2天起就表现出上调下游基因NeuroD1和Nkx2.2的表达，由此提高了胰岛素和胰高血糖素的表达。 
                            详细描述
本发明从多能干细胞进行分化解决了大量生产人胰岛细胞的问题。 
已发现，可以诱导干细胞沿胰岛细胞分化路径分化，即引发向内胚层细胞系的分化，并通过在促进胰岛细胞长出的因子存在下的培养实现分化过程的聚焦。本发明提供了一种生产富集多能胰岛细胞祖细胞的细胞群的方法，由此能够形成成熟的胰岛。根据需要，分化过程可继续，从而维持成熟的激素分泌细胞。 
为了以至于该分化过程的优化，本发明提供了一种将给分化路径解析为一系列后续阶段进行的策略。由此，可鉴定在分化路径各个阶段推动细胞分化的因子。 
在实施例5中，分化过程如下。1期，诱导来自无饲养细胞培养物的未分化 人胚胎干细胞的分化，从而在悬浮培养物中形成含内胚层细胞的混合细胞聚集体。用维甲酸作为初始分化剂，与之联用的是富集因子硒和T3。2期，在含有Noggin(200ng/ml)，EGF(20ng/ml)和bEGF(2ng/ml)的培养基中培养，诱导向胰祖细胞的分化。3期：清除Noggin、EGF和bEGF培养基代之以在10mM烟酰胺存在下培养，从而诱导向终期胰岛细胞分化。如图4所示，获得了合成胰岛素c-肽和抑生长素的细胞簇，产物达到可用抗体检测的水平。 
由pPS细胞生产胰岛干细胞的方法意义重大，因为pPS细胞可无限增殖。本发明提供了一种可用于产生无限量胰岛祖细胞及其后代定向发育为成熟胰岛细胞的系统。 
后文进一步描述本发明胰岛细胞的生产和鉴定，并且，广泛论述了这些细胞如何用于研究、药物研发和胰岛细胞功能紊乱相关病症的治疗。 
定义
就本发明而言，“胰岛细胞”指分化末期的分泌胰岛素的细胞，以及定向形成一般称为胰腺的后代的各种前体细胞。胰岛细胞表现出某些公认为胰岛细胞系所特有的形态特征和表型标记(见后文举例)。成熟α细胞分泌胰高血糖素；成熟β细胞分泌胰岛素；成熟δ细胞分泌抑生长素；PP细胞分泌胰多肽。 
“胰岛祖细胞”、“胰岛前体细胞”或“胰岛干细胞”指基本上不内分泌但保留了增殖能力并能够产生最终分化细胞的胰岛细胞，可能还具有自我更新能力。早期胰岛祖细胞具有多能性，即能够形成至少两种或全部四种成熟胰岛细胞类型。 
“胰祖细胞”、前体或肝细胞即能够形成胰内分泌细胞也能够形成胰外分泌细胞。 
讨论细胞的个体发育时，定语“分化”是相对而言的。“分化细胞”与对照细胞相比，已进行到了发育路径的较下游阶段。本发明假设：正常个体发育过程中的多能胚胎干细胞首先分化为能够形成胰细胞的内胚层细胞和其它内胚层细胞类型。进一步的分化进入胰细胞路径，其中，约98％的细胞成为外分泌管细胞或基质细胞，约2％成为内分泌细胞。早期内分泌细胞即胰岛祖细胞，可进一步分化为专职分泌胰岛素、胰高血糖素、抑生长素或胰多肽的功能性内分泌细胞。 
“分化剂”在本文中指用于本发明培养系统产生属于胰岛细胞系的分化细胞(包括前体细胞和最终分化细胞)的化合物。对此类化合物的作用方式没有限定。例如，所述分化剂辅助分化的机理可以是通过诱导或辅助表型改变、促进特定表型的细胞生长或抑制其它细胞的生长。分化剂还可以是其它因子的抑制剂，所述 其它因子存在于培养基中或由细胞群合成，如果不被抑制则可能将分化引向非期望的细胞类型。 
原型“灵长动物多能干细胞”(pPS)是衍生自受精后任何阶段的胚前、胚或胎组织的多能细胞，其特征在于能够在合适的条件下产生衍生自全部三个胚层(内胚层、中胚层、外胚层)的数种不同细胞类型的后代，这可根据标准试验例如在8-12周龄SCID小鼠内形成畸胎瘤的能力来确定。该术语包括已确立的各种类型的干细胞系和根据上述方式确认为多能的由原代(primary)组织获得的细胞。 
pPS细胞的定义包括各种类型的胚胎细胞，例如人胚胎干细胞(hES)，见Thomson等(Science 282：1145，1998)；其它灵长动物的胚胎干细胞，例如恒河猴干细胞(Thomson等，Proc.Natl.Acad.Sci.USA 92：7844，1995)，绒猴干细胞(Thomson等，Biol.Reprod.55：254，1996)和人胚胎生殖(hEG)细胞(Shamblott等，Proc.Natl.Acad.Sci.USA 95：13726，1998)以及其它类型的多能细胞。该术语包括源自所有三胚层、能够产生后代的灵长动物细胞，不论它们是来自胚组织、胎组织或是其它来源。pPS细胞最好不是来自恶性瘤组织。通常期望但非必须，此类细胞核型正常。 
当细胞群中大部分干细胞及其后代表现出明显区别于胚胎或成人已分化细胞的未分化细胞形态特征时，就称pPS细胞培养物为“未分化的”。本领域技术人员不难识别未分化pPS细胞，在二维显微镜下，它们表现为高核/质比和具有明显核仁的细胞集落。一般认为，细胞群内未分化细胞集落通常被临近的已分化细胞所包围。 
“饲养细胞”指与另一种细胞共培养，用于为后者提供适宜的生长环境的细胞。某些pPS细胞可用小鼠原代胚胎成纤维细胞，无限增殖化的小鼠胚胎成纤维细胞或hES细胞分化而成的人成纤维细胞样细胞来支持。如果自分裂后细胞已培养了至少一个周期，期间没有加新鲜饲养细胞来支持pPS细胞生长，则称这样的pPS细胞群为“基本无”饲养细胞。 
“胚状体”与“聚集体”同义，指已分化和未分化细胞的聚集体，出现在pPS细胞在单层培养物中过度生长或悬浮培养时。胚状体是不同细胞类型(通常源自不同胚层)的混合物，可根据形态学标准和免疫组织化学可测细胞标记来鉴别。 
“生长环境”指适宜所需细胞增殖、分化或成熟的环境。此类环境的特征包括用于培养细胞的培养基，可能存在的生长因子或分化因子，以及可能存在的支持结构(例如固体表面上的基质)。 
当用合适的人工手段将一段聚核苷酸转移到某细胞内时，或该细胞是被改造 细胞的后代并继承了上述聚核苷酸时，则称这样的细胞为经“遗传改造”或“转染”的细胞。 
常规技术
分子遗传学和基因工程中的常规方法可参见“分子克隆：实验室手册”(Sambrook等，Cold Spring Harbor)；“哺乳动物细胞基因转移载体”(Miller & Calos编)和“现代分子生物学方法”(F.M.Ausubel等，Wiley & Sons)。细胞生物学、蛋白质化学和抗体技术可参见“现代蛋白质科学方法”(J.E.Colligan等，Wiley &Sons)；“现代细胞生物学方法”(J.S.Bonifacino等，Wiley & Sons)和“现代免疫学方法”(J.E.Colligan等，Wiley & Sons)。试剂、克隆载体和基因操作所用的试剂盒可向例如BioRad、Stratagene、Invitrogen、ClonTeck和Sigma-Aldrich Co.等厂商购买。 
细胞培养方法可参见最新版的“动物细胞培养：基本技术手册”(R.I.Freshney编，Wiley & Sons)；细胞培养常规技术(M.A.Harrison & I.F.Rae.，Cambridge Univ.Press)和“胚胎干细胞：方法和规范”(K.Turksen等，Humana Press)。组织培养材料和试剂可向Gibco/BRL、Nalgene-Nunc International，Sigma Chemical Co.和Biomedicals等厂商购买。 
与本发明相关的专门著作包括：“胰岛比较生理学”，J.E.Brinn，Springer-Verlag，1988；“胰岛细胞再生和发育”，E.J.Vinlk等，Kluwer，1992；“胰岛细胞的免疫建模”(“胰岛移植”，Vol.2)，R.P.Lanza等编，Springer Verlag，1994。 
干细胞来源
本发明可用各种干细胞来实施。其中，包括衍生自妊娠后形成的组织如胚泡的灵长动物多能干细胞(pPS)，或衍生自妊娠期间胎或胚组织的pPS细胞。非限定性例子是胚胎干细胞或胚胎生殖细胞的原代培养物或细胞系，详见后文。 
本发明也可直接用原代胚或胎组织来实施，即直接由能够产生胰岛细胞的原代细胞衍生获得胰岛细胞而不先建立未分化细胞系。某些情况下，本发明还可用来自脐带血或某些成人组织的多能细胞进行。 
pPS细胞未分化时的增殖
采用促进增殖但不促进分化的条件，可使pPS细胞在培养物中持续增殖。例 如采用含血清的ES培养基，其中含80％DMEM(例如敲除DMEM，Gibco)，20％胎牛血清(FBS，Hyclone)或代血清(WO98/30679)，1％非必需氨基酸，1mM L-谷氨酰胺和0.1mM β-巯基乙醇。临用前，加入4ng/ml人bFGF(WO99/20741，GeronCorp.)。通常将ES细胞培养在一层饲养细胞上，饲养细胞一般为衍生自胚组织或胎组织的成纤维细胞。 
Geron的科学家发现，既使不用饲养细胞也可将pPS细胞维持于未分化状态。无饲养细胞培养的条件包括合适的培养基质，尤其是胞外基质，例如 或层粘连蛋白。通常，在细胞完全分散前终止酶解(例如，IV胶原酶处理约5分钟)。然后将10-2000细胞左右的细胞块直接接种到基质上平板培养，不再进一步分散。 
用含有支持细胞增殖但不支持其分化的因子的营养培养基来支持无饲养细胞培养物。此类因子的引入可通过在培养基中培养分泌此类因子的细胞，例如辐照过的(约4000rad)原代小鼠胚胎成纤维细胞、调聚(telomerized)小鼠成纤维细胞或衍生自pPS细胞的成纤维细胞样细胞。可调整培养基，例如在无血清培养基(例如补充了20％代血清和4ng/ml bFGF的KO DMEM)中以5-6×104/cm2左右的密度平板培养饲养细胞。在如此调整1-2天的培养基中进一步补充bFGF，然后用于支持pPS细胞培养物1-2天。无饲养细胞培养法的其它细节可参见WO01/51616和Xu等，Nat.Biotechnol.19：971，2001。 
显微镜下，ES细胞呈高核/质比，核仁明显，菌落致密，几乎观察不到细胞接合腺。灵长动物ES细胞表达阶段特异性胚胎抗原(SSEA)3和4，以及可用抗体测知的标记Tra-1-60和Tra-1-81(Thomson等，Science 282：1145，1996)。小鼠ES细胞可用作SSEA-1的阳性对照和SSEA-4、Tra-1-60和Tra-1-81的阴性对照。SSEA-4也存在于人胚胎性癌(hEC)细胞上。pPS的体外分化不表达SSEA-4、Tra-1-60和Tra-1-81，但提高SSEA-1表达，该抗原也存在于未分化hEG细胞上。 
制备胰岛细胞及其衍生物的材料和方法
本发明的胰岛细胞通过在富集所需表型(培养出所需的细胞或抑制或杀死其它细胞类型)的特殊生长环境中培养、分化或增殖干细胞制得。这些方法适用于许多类型的干细胞，包括灵长动物多能干细胞(pPS)。 
分步骤分化 
本发明假设：形成能够形成胰细胞和其它细胞类型的早期多能祖细胞可促进自pPS细胞产生胰岛细胞并富集。一般说来，该策略需首先形成一个富集了相关定型前体细胞的细胞群，然后进一步分化为越来越向形成胰岛特化的更成熟的细胞。根据该策略，pPS细胞向成熟胰岛的分化细分为数个阶段进行。 
未分化pPS细胞与成熟胰岛之间的中间体之一是不成熟的内胚层细胞。个体发育早期，内胚层细胞能够形成消化道和呼吸系统和重要消化器官(肝和胰)的上皮细胞。胰岛细胞可用一种两阶段方法生成。1期，获得共同内胚层前体细胞群。2期，使内胚层前体细胞成熟为胰内分泌细胞。如实施例3所述，可用肝细胞分化剂正丁酸盐引发pPS细胞沿内胚层分化路径分化。关于肝细胞分化的详述可参见WO01/81549(Geron Corporation)。Sonic Hedgehog被认为参与了肝的特化，所以，在培养基中加入环杷明(一种Sonic Hedgehog抑制剂)被认为有助于驱使细胞向胰细胞系分化。然后，可用末期分化因子烟酰胺(在环杷明和活化素A存在下)进一步促进分化。 
对该方法进一步的解说中，该分化路径被分成3个阶段。pPS细胞首先分化为内胚层(1期)，然后分化为第二中间体(2期)，可能为定型胰前体细胞水平(可根据标记Pdx1鉴定)。如果需要获得成熟胰岛，可进行下一步分化(3期)。为了完成1期，可用正丁酸盐+活化素A使pPS细胞分化为具有消化道内胚层标记的细胞(实施例4)。或者，可在富集剂(硒和甲状腺素例如T3)存在下用维甲酸培养pPS细胞来制备包含内胚层细胞的混合细胞群(实施例5)。为了完成2期，可用例如Noggin的TGF-β拮抗剂+促分裂素(属于FGF家族，可与EGF或动物纤维素混合)培养细胞(实施例5)。用环杷明阻断Hedgehog信号传递也许有帮助。3期可用前述末期分化因子烟酰胺来完成(实施例5)。或者，可通过使得Pdx1阳性胰前体细胞进化到成熟胰岛细胞的直接操作活化转录因子(实施例6)。 
以上分步骤分化的方法只是一种指导，本发明并非局限于此。以上分化路径还可分解为更多的阶段，从而以阶进方式优化分步骤分化。例如，可能存在于胰前体细胞和成熟胰岛之间的一种中间体是定向于形成胰内分泌细胞的前体细胞。另一方面，根据条件，可同时将多种有效的分化剂联合起来作用于不同阶段的细胞，或者促进沿分化路径的级联效应。 
所需的末期细胞群部分取决于预期用途。例如，就治疗胰岛功能不全和体外 研究胰岛分化而言，特别具有价值的是定型胰岛祖细胞。更早期的祖细胞可能具有更强的自我更新能力。当需要立即产生激素时，则需要高水平合成胰岛素或其它内分泌激素的成熟细胞。分化过程可根据需要来制定，使之中止于达到所需成熟度的阶段。 
以下是关于促进分化的组织培养和转基因技术。 
引发分化 
有两种方法可使pPS细胞向内胚层细胞分化。其一是将细胞平板培养在新的基质上，或更换培养基以去除胞外基质或可溶性分化抑制因子。这种方法有时称为“直接分化法”，参见WO01/51616和美国专利公开20020019046。在直接分化法中，通常优选从无饲养细胞的pPS细胞培养物开始，从而避免残留饲养细胞在分化过程中造成的干扰。实施例4通过引入含有早期分化因子的培养基，用直接分化法生成了消化道内胚层。 
另一种方法是悬浮培养未分化pPS细胞，通常使它们形成胚状体或聚集体。例如，通过胶原酶简短消化收获pPS细胞，分散为细胞簇，在非粘附细胞培养平板上传代。每隔数天加一次聚集体，经适当时间，通常4-8天后收获细胞(实施例1和5)。有时，在培养基中加入其它因子来加强分化，例如维甲酸(实施例5)或二甲基亚砜(DMSO)。根据情况，聚集体通常先形成一个混合细胞群，其中主要为内胚层细胞。然后，可将这些胚状体分散，并再平板培养在诸如层粘连蛋白或纤维结合素等基质上，以便进入下一分化期；或用非粘附平板和合适的培养基进行悬浮培养传代。 
可用后文所述标记监测直接分化或聚集体内分化，看是否存在内胚层细胞。一旦获得了足量的内胚层细胞，就可以重新对细胞进行平板培养或其它操作从而启动II期。某些情况下，如果细胞成小簇(例如50-5,000个细胞)，可促进胰岛细胞的分化和维持，这样，α-、β-和δ-细胞可直接相互作用。 
用这种方法获得共同的祖细胞后，可如下所述用特异性分化因子培养和/或用胰岛特异性基因或促进剂进行诱导。 
用可溶性因子进一步驱使细胞向胰岛分化 
为了驱使培养物进入胰岛分化路径的下一阶段，可用多种胰岛分化因子的混合物培养pPS细胞或它们的已分化后代。不论单用或联用，这些因子都能提高向 所需细胞类型转化的几率，从而长出具有胰岛表型的细胞，抑制其它细胞类型，或以其它方式富集胰岛细胞。要实施本发明并没有必要理解这种胰岛细胞富集的机制。以下是可选分化因子的举例。 
                表1：促进pPS细胞向胰岛细胞分化的因子 
因子             化合物类型或家族          推定功能                 起效浓度 
环杷明           甾族生物碱                Hedgehog信号传递抑       10μM 
                                           制剂，也可作为胆固醇 
                                           生物合成抑制剂 
β-动物纤维素    EGF家族成员               促分裂素，β细胞分化     4nM 
                                           促进剂。这两项功能缘 
                                           于该蛋白的不同域。 
活化素A          TGF-β家族成员            分化因子，使管样细胞     4nM 
                                           系分化为内分泌胰细 
                                           胞 
Extendin-4       胰高血糖素样肽1激         GLP-1的更稳定形式        20nM 
                 动剂                      (见后文) 
胰高血糖素样     肽激素，胰高血糖素        诱导葡萄糖生产、胰岛     20nM 
肽1(GLP-1)       基因的蛋白质产物之        素分泌，诱导β细胞生 
                 一：G蛋白偶联受体         成，诱导β细胞增殖 
                 的配体 
肝细胞生长因     c-Met受体的配体           在HGF过表达的转基        10ng/ml 
子(HGF)                                    因动物中使β细胞增 
                                           大，诱导由管细胞形成 
                                           β细胞 
烟酰胺           B族维生素                 影响细胞的核糖基化       10mM 
                                           和/或氧化状态：促进β 
                                           细胞分化 
胰岛素样生长     肽激素                    促β细胞分裂素           10nM 
因子1(IGF-1) 
正丁酸盐                                   组蛋白脱乙酰酶抑制        0.5-2.5mM 
                                           剂(用于产生肝细胞) 
维甲酸           维甲酸类                  分化因子                  10μM 
(全反式) 
生长素(人垂体    生长激素/催乳素家         促β细胞分裂素            100ng/ml 
生长激素)        族 
胎盘催乳素       生长激素/催乳素家         在PL过表达的转基因        50ng/ml 
                 族                        动物中使β细胞增大， 
                                           孕期内的促β细胞分裂 
                                           素 
脉管内皮生长     Flt-1和Flk-1是受体。      可能是内皮胰岛前体        50ng/ml 
因子(VEGF)       PDGF/VEGF家族             的促分裂素 
胰岛素样生长     胰岛素家族                与IGF-1类似，是促分       10nM 
因子II(IGF-II)                             裂素，但不如对胰发育 
                                           或功能的作用那么强 
3-异丁基-1-甲    磷酸二酯酶抑制剂，        增强胰岛素分泌            100μM 
基黄嘌呤         提高cAMP水平 
(IBMX) 
渥曼青霉素       PI-3激酶抑制剂            增强胰岛素分泌            30nM 
胃泌素           肽激素                    促进β细胞再生            10ng/ml 
胆囊收缩素       消化道激素                协助胰岛的成熟            5μM 
神经生长因子        NGF家族               增强胰岛素分泌           50ng/ml 
(NGF) 
表皮生长因子        EGF家族(见β-动物     转基因过表达将导致β            4nM 
(EGF)               纤维素)               细胞增殖 
角质细胞生长        FGF家族成员           转基因过表达将导致β            10ng/ml 
因子(KGF)，                               细胞扩增 
aka FGF7 
血小板衍生的        PDGF家族(将VEGF)      可能是前体细胞的促       50ng/ml 
生长因子                                  分裂素 
(PDGF) 
再生基因(Reg)       Reg家族               参与胰受伤后的胰岛       100ng/ml 
                                          再生 
胰岛再生相关        Reg家族               可能参与胰岛再生         100ng/ml 
蛋白(INGAP) 
结合相同受体的其它配体或抗体可认为与本文中的任一受体之配体相当。 
通常，至少2种、3种或更多种上述因子组成分化混合物。优选人源蛋白，但也可采用异种类似物或变体。读者可用结合相同受体或引发同一信号传导路径的其它配体，例如受体特异性抗体，取代上述因子。此外，可在培养基中加入其它成分来抵消可能存在的其它因子驱使细胞沿其它路径分化达到作用。 
可用矩阵法来测定任一上述因子的效果，从而获得一种可促进分化沿胰岛细胞路径前进一到两步的组合。可用例如后文所述的表型标记来监测所需中间体的表型或末期细胞的表现来评价上述效果。例如，被认为诱导内分泌性胰细胞分化的因子可根据它们在标准培养条件下诱导Pdx1表达和胰岛素表达的能力来评价其效果。 
分数因数设计可用来高效地筛选化合物。每一种因子被指定两个水平：例如，可在培养基质中加入第一水平的纤连蛋白，加入第二水平的层粘连蛋白。在64种因子组合(64个实验)中，可以确定约15-20种因子中哪些能够以统计学显著性影响分化。适合用这种方法分析的组合包括环杷明，TGF家族成员(TGF-α、活化素A、活化素B、TGF-β1，TGF-β3)，Extendin-4，烟酰胺，正丁酸盐，DMSO，全反式维甲酸，GLF-1，骨成形蛋白(BMP-2、BMP-5、BMP-6、BMP-7)，胰岛素样生长因子(IGF-I、IGF-II)，成纤维细胞生成因子(FGF7、FGF10、bFGF、FGF4)，其它生长因子(EGF、β-动物纤维素，生长素，HGF)，其它激素(催乳素、胆囊收缩素、胃泌素1、胎盘催乳素)，TGF-β族拮抗剂(Noggin，follistatin，chordin)，IBMX，渥曼青霉素，地塞米松，Reg，INGAP，cAMP或cAMP活化剂(毛喉萜)和胞外基质组分(层粘连蛋白、纤连蛋白)。产生的细胞群根据表型标记来评价，通过分析表达方式来确定哪些因子对分化路径具有促进作用，哪些具有反作用。 
用基因修饰过的细胞引导或监测分化 
有时，使用以载体进行过基因改造的细胞有助于某一分化或分离方法的优化，所述载体具有驱动某报道基因表达的组织特异性启动子。 
合适的胰岛祖细胞特异性启动子包括驱动Pdx1(NT_009799)，神经生成素3(NT_008583)，NeurolD1(NT_005265)，Nestin(NT_004858)和Ptf1a-p48(NT_008705)表达的那些启动子。合适的成熟胰岛细胞特异性启动子是驱动胰岛素(GenBank登录号NT_009308)，胰高血糖素(NT_022154)，抑生长素(NT_005962)或胰多肽(NT_010755)表达的那些。选定启动子的最小有效序列(约0.5至5kB上游序列)以PCR扩增，然后接入标准质粒，腺病毒、慢病毒或逆病毒载体中，接入位置使其可驱动合适的报道基因的表达。合适的报道基因编码荧光分子(例如绿荧光蛋白或荧光素酶)，编码可测酶(例如碱性磷酸酶)，或者产生可用抗体或通过血凝素接合作用检测的细胞表面抗原(各种异源蛋白或糖)。 
以组织特异性启动子转染的细胞可用于优化前述分化方法。例如，可对经转化的未分化pPS细胞或分化早期阶段细胞实施各种分化方案，然后分析表达胰岛特异性启动子所驱动报道基因的细胞所占百分比。这就能够迅速找出有效的分化剂、培养条件和时间安排。然后可将优化过程用于非转染细胞来产生高质量的含有天然基因型的胰岛细胞系的细胞群。 
以组织特异性启动子转染的细胞还可用于机械分选。例如，启动子驱动某抗药性表型的表达，例如新霉素抗性基因(赋予对G418的抗性)或博来霉素抗性基因。此时，细胞分化后立即用相应药物培养以促进所需细胞类型的长出。或者，报道基因编码一荧光分子或表达一可测的表面抗原。此时，根据直接或间接荧光或根据免疫吸附来分选细胞，由此从分化细胞中选出所需细胞。非复制非整合载体(例如腺病毒载体)可用于分选步骤的临时转染，然后随着细胞的增殖而淡出，不改变细胞的天然基因型。 
还可以以直接驱动细胞进一步向胰岛细胞或其祖细胞分化为目的对细胞进行基因改造。本发明假设，人为上调胰岛细胞内某基因的表达可使分化程度较低的细胞采用适合更成熟胰岛细胞的基因表达模式。合适的基因包括编码影响下游基因表达的转录调节剂的基因。用于促进胰个体发育的候选基因包括(大致按照它们在分化路径中的位置)：Sox17(GenBank登录号NM_022454)、Hlx9(NM_05515)、Pdx1(NM_000209)、神经生成素3(NM_020999)、Pax4(NM_006193)或NeuroD1(NM_0002500)、IsIl(NM_002202)、Nkx2.2(NM_002509)和Nkx6.1(NM_006168)。 
该方法的说明详见实施例6。在胰岛细胞分化中通过细胞转导诱导的神经生 成素3表达增强了下游调节基因和编码胰岛激素的基因的表达。神经生成素是一族neuroD相关bHLH转录因子：活跃于神经系统发育的神经元决定基因。 
已分化细胞的特征
细胞可根据表型标准来鉴定，例如显微观察其形态特征，检测或定量细胞标记的表达，可体外测知的功能指标，以及输入宿主动物后的表现。 
表型标记 
本发明细胞可根据它们是否表达各种胰岛细胞的特征性表型标记来鉴定。可用的标记见表2。 
                表2：用于细胞鉴定的表型标记 
SSEA-4                       胚胎干细胞和胚胎生殖细胞 
Oct-4                        未分化胚胎多能细胞 
端粒末端转移酶逆转录         能够无限复制的细胞(例如未分化pPS细胞) 
酶 
Pdx1                         胰芽形成前在十二指肠将产生胰的区域内表达。也在成熟β细 
                             胞内表达。 
神经生成素3(Ngn3)            胰岛前体细胞的标记 
胰高血糖素                   α细胞 
Nkx61                        β细胞标记 
葡糖激酶                     β细胞标记 
胰岛素或前胰岛素             成熟β细胞 
抑生长素                     δ细胞 
胰多肽                       PP细胞 
胰岛淀粉样多肽(IAPP)         胰岛标记 
胰岛-1                       胰岛标记，也在神经系统中表达 
β-2/NeuroD                  泛胰岛标记 
HNF3b                        内胚层标记 
HNF4a                        内胚层标记 
Sox17                        明确的内胚层标记 
淀粉酶                       外分泌细胞 
HES                          外分泌胰腺标记 
Nestin                       潜在前体细胞标记 
Sonic Hedgehog               信号分子，胰形成要求其不存在 
CK19                         胰管标记(可能是胰的前体) 
Glut-2                       葡萄糖转运体 
补丁类似物                   Hedgehog受体 
(Patched homologue) 
平滑类似物                   Hedgehog受体 
(Smoothened homologue) 
组织特异性标记可用各种适当的免疫学技术来检测，例如检测细胞表面标记的流式免疫细胞化学，检测胞内或细胞表面标记的免疫组织化学(例如对固定细胞或组织切片)。有关流式细胞计数发，详见Gallacher等，“血液”96：1740，2000。如果在标准免疫细胞化学或流式细胞计数分析中，检测到明显的抗体与抗原接 合，则认定为该细胞表面抗原表达阳性，所述分析可在细胞固定后进行，可采用经标记的第二抗体或其它偶联物来放大标记效果。目前特异性抗体来源包括：胰岛素，Sigma Aldrich(12018)，胰高血糖素，Sigma Aldrich(G2654)；抑生长素，Santa Cruz Biotech(sc-7820)；神经生成素3，Chemicon(AB5684)；nestin，BDTransduction Labs(N17220)；α-淀粉酶，Sigma Aldrich(A8273)；Glut-2，AlphaDiagnostics(GT-22A)。 
组织特异性基因产物的表达还可以通过Northern印染分析、斑点杂交分析或采用序列特异性引物按照标准扩增方法进行逆转录酶引发的聚合酶链反应(RT-PCR)进行mRNA水平的检测。详见美国专利5,843,780。本文中具体标记的序列可由公开的数据库例如GenBank获取。 
最初的评价可采用标记组合，从而获知宽泛的个体发育情况：例如，Pdx1指示早期胰细胞；Ngn3指示早期胰内分泌细胞；胰岛素指示成熟β细胞。定期(例如每周)从前述分化过程中收获细胞，测定分化动力学特征。然后，经测定呈上述标记阳性的细胞进一步分析是否表达其它标记，例如IAPP和Nkx6.1。明确细胞特征后，即可优化每一步骤的分化因子组合和时间设定。 
本发明的部分实施例涉及这样的细胞群：其中，至少2％、5％、10％或更多细胞带有一个或多个前述表面标记。内分泌功能对许多研究和治疗性用途来说意义重大，这些用途中，特别需要至少5％的细胞分泌胰岛素、胰高血糖素、抑生长素或胰多肽的细胞群或含有至少5％能够分化为此类内分泌激素分泌细胞的祖细胞的细胞群。本发明假设：α、β和δ细胞之间的相互作用对避免去分化和保持有效的内分泌特性非常重要。本发明还包括这样的细胞群或细胞簇(约50-5,000细胞)：含有两种或三种上述细胞类型，它们因自身产生的基质、培养基中含有的基质祖父，或因荚膜包裹而结合在一起。 
本发明还描述了一种未分化pPS细胞低残留的细胞群。较好的细胞群为：1％或0.2％以下SSEA-4+ve、Oct-4+ve或内源性端粒末端转移酶逆转录酶阳性。较好的细胞群中其它细胞类型(例如肝细胞(白蛋白阳性细胞)、骨骼肌细胞(myoD阳性)、平滑肌细胞(平滑肌肌动蛋白)、具有成纤维细胞形态的细胞或神经元(β-微管蛋白III或NCAM阳性细胞))的比例也较低(5％、1％或0.2％以下)。 
如果是衍生自己确立的pPS细胞系，本发明细胞群和分离细胞具有与其源细胞系相同的基因组。这表示，不论是否染色异常，如果胰岛细胞是由未分化细胞系通过正常有丝分裂获得的，即可推定pPS细胞与胰岛细胞之间的染色体DNA具有90％以上相同性。重组引入了某转基因或敲除了某内源基因的胰岛细胞仍被 认为具有与其源细胞系相同的基因组，因为所有未操作基因元素全被保留。 
动物模型实验 
胰岛细胞临床应用尤其关注的是细胞群重建宿主动物胰岛系统的能力。重建可用各种常用动物模型来检验。 
非肥胖糖尿病(NOD)小鼠模型带有遗传缺陷，导致数周龄时发生胰岛炎(Yoshida等，Rev.Immunogenet.2：140，2000)。20-30周时，已有60-90％雌性小鼠患上明显的糖尿病。这种免疫相关性病理学与人I型糖尿病类似。其它I型糖尿病模型是转基因小鼠和带有敲除突变的小鼠(Wong等，Immunol.Rev.169：93，1999)。最近，Lenzen等报道了一种自发性I型糖尿病的大鼠模型(Diabetologia 44：1189，2001)。也可通过一次腹膜内注射链脲霉素(Soria等，Diabetes 49：157，2000)或连续低剂量链脲霉素(Ito等，Environ.Toxicol.Phamacol.9：71，2001)在小鼠模型中诱导高血糖症。为了测定移植胰岛细胞的效果，可检测小鼠葡萄糖水平是否恢复正常(低于200mg/ml)。 
更大的动物是跟踪慢性高血糖症后遗症的良好模型。可通过胰切除(J.Endocrinol.158：49，2001)或半乳糖喂养(Kador等，Arch.Opthalmol.113：352，1995)使狗产生胰岛素依赖。还有一种建立在荷兰卷尾狮毛狗中的I型糖尿病遗传模型(Am.J.Pathol.105：194，1981)。用狗进行的早期工作(Banting等，Can.Med.Assoc.J.22：141，1922)导致数名加拿大人于1925年2月远涉重洋来到斯得哥尔摩。 
作为说明，可在以下动物中用pPS衍生的胰岛细胞进行前期试验：a)非糖尿病裸(T细胞缺乏)小鼠；b)链脲霉素致糖尿病的裸小鼠和c)部分胰切除后处于胰岛再生过程中的裸小鼠。移植细胞量相当于约1000-2000正常人胰岛，移植在肾囊下、肝内或胰内。对于非糖尿病小鼠，实验最后可评价移植物的存活(组织学检查)和通过生物化学分析、RIA、ELISA和免疫组织化学检测胰岛素产生。链脲霉素处理过和部分胰切除的动物可评价其存活、代谢控制(血糖)和体重。 
未分化细胞的基因修饰 
本发明胰岛前体细胞具有强大的增殖能力。如果需要，可通过提高细胞内端粒末端转移酶逆转录酶(TERT)水平，或提高内源基因的转录，或引入转基因来进一步增强这种复制能力。尤其适合的是人端粒末端转移酶(hTERT)的催化性组分，参见WO98/14592。关于人细胞内的端粒末端转移酶转染和表达参见Bodna等，科学279：249，1998和Jiang等，Nat.Genet.21：111，1999。可根据标准方法，评价遗传改造细胞的hTERT表达(RT-PCR)，端粒末端转移酶活性(TRAP试验)，hTERT的免疫细胞化学染色或复制能力。还可以考虑采用令细胞无限增殖化的其 它方法，例如用编码myc、SV40大T抗原或MOT-2的DNA转化细胞(美国专利5,869,243，WO97/32972和WO01/23555)。 
如果需要，可进一步体外去除本发明细胞群中未分化细胞，或确保不发生体内回复突变。从细胞群中清除未分化干细胞的方法之一是用以下载体转染细胞，所述载体带有引起效应基因在未分化细胞内优先表达的启动子，例如TERT启动子或OCT-4启动子。所述效应基因可以是用于细胞分选的报道基因，例如绿荧光蛋白。效应物可以直接裂解细胞，编码例如某毒素或介导凋亡，例如caspase(Shinoura等，Cancer Gene Ther.7：739，2000)。效应基因的作用可以是使细胞易受某外来试剂例如抗体或前药的毒性作用。实例之一是单纯性疱疹病毒胸苷激酶(tk)基因，其表达会使该细胞易受更昔洛韦攻击(USSN60/253,443)。或者，效应物可引起某种外源决定基的细胞表面表达，该决定基使得任何回复未分化表型的细胞易受体内天然抗体的攻击(USSN60/253,357)。 
胰岛细胞在研究和临床治疗中的用途
本发明提高了一种生产大量胰岛前体细胞和成熟胰岛细胞的方法。这些细胞群可用于多种重要的研究、开发和商业目的。 
本发明的细胞可用来制备cDNA文库，该文库将不含在其它细胞系细胞中优先表达的杂cDNA。本发明的已分化细胞还可用于通过标准方法制备具有胰岛前体或其衍生物的特异性单克隆或多克隆抗体。 
尤其值得关注的是本发明组合物用于药物开发和临床治疗。 
药物筛选 
本发明胰岛细胞可用来筛选影响胰岛前体细胞或其各种后代特征的因子(例如溶剂、小分子药物、肽、聚核苷酸)或环境条件。 
重要实例之一是用胰岛细胞簇或β细胞均一制备物检验小分子药物上调或下调胰岛素合成或分泌的能力。将细胞与待测化合物混合，然后通过例如RT-PCR或对培养基的免疫试验来监测表达或分泌速度变化。 
本发明的其它筛选方法涉及检验药物化合物对胰岛细胞生长、发育的影响或其毒性。这种筛选不仅是设计对胰岛细胞具有药理作用的化合物所必需的，而且是检测药物化合物胰岛相关性副作用所必需的。 
第三个例子中，(未分化或已分化的)pPS细胞被用于筛选促进胰岛细胞成熟、促进长期培养中胰岛细胞增殖和维持的因子。例如，将候选分化因子或成熟因子加给测试板不同格中的细胞，然后检测引起的表型变化，看是否符合进一步培养及使用这些细胞的标准。这样可获得改良的衍生和培养方法，这些方法不仅 适用于pPS衍生的胰岛细胞，而且适用于分离自胰腺的胰岛细胞及它们的祖细胞。 
本文读者可参考标准教科书“药物研究的标准体外方法”，Academic Press，1997和美国专利5,030,015。评价候选候选药物化合物的活性通常需将本发明的已分化细胞与候选化合物或该化合物与前体药物的组合物混合。研究者检测化合物引起的细胞的形态变化、标记表型或功能活性(与非处理细胞或接受惰性化合物处理的细胞比较)，然后将化合物作用与观察到的变化相关联。 
毒性首先可根据对细胞活度、存活率、形态以及某种标记或受体的表达来测定。药物作用于染色体DNA的效果可通过测定DNA合成或修复来测定。[2H]-胸苷或BrdU掺入，尤其是细胞周期意外时刻的掺入，或者高于细胞复制所需水平的掺入，都是药物效果的表征。副作用还可能包括根据分裂中期延长测知的姐妹染色单体交换速度异常。更多细节可参考A.Vickers(pp.375-410)“药物研究的标准体外方法”Academic Press，1997，375-410。 
胰岛功能的恢复 
本发明还提供了胰岛前体细胞或其衍生细胞用于患者恢复其胰岛功能的用途。任何与胰内分泌激素(胰岛素、胰高血糖素或抑生长素)产生不足或分泌失调相关的病症都可考虑用本发明细胞进行治疗。尤其值得关注的是I型(胰岛素依赖型)糖尿病的治疗。 
挑选确认长期依赖外源胰岛素并且危险不大的患者接受治疗。患者接受约10,000胰岛当量细胞/kg体重。为了克服同种异型错配，患者于手术前先接受抗排斥药物例如FK506和雷帕霉素(口服)和daclizumab(静脉注射)。门静脉导管输注胰岛细胞。然后给患者做腹部超声波检查和验血来检测肝功能。记录每日胰岛素需要量，如果需要可对患者进行再次移植。随访监测包括定期验血测定药物水平，免疫功能，总体健康状况和是否仍然依赖胰岛素。 
护理糖尿病人的通用方法可参见标准教科书，例如“内服药课本”第3版，W.N.Kelley编，Lippincott-Raven，1997；专业参考书籍，例如“糖尿病：基本和临床教程”第2版，D.Leroith，Lippincott Williams & Wilkins 2000；“糖尿病(临床内分泌导读Vol.2)”C.R.Kahn等，Blackwell Science 1999；和“I型糖尿病的医治”第3版，McGraw Hill 1998。用胰岛细胞治疗I型糖尿病的详细论述可参见“胰内的细胞间相互关系：胰岛移植所涉问题”，L.Rosenberg等，Chapman& Hall，1999；和“胎儿胰岛移植”，C.M.Peterson等，Kluwer 1995。 
通常，患者的挑选、给药方式和内分泌胰细胞的剂量最终都由主治医师负责。 
为了商业销售，本发明胰岛细胞一般以药物组合物的形式提供，其中包含等 渗赋形剂。组合物需在满足人用的足够严格的无菌条件下制备。本发明还包括生产、分发或使用过程中任何时候的成组细胞。这些细胞组包含两种或更多种本文所述细胞群的组合，例如但不限于已分化pPS细胞衍生的细胞(胰岛细胞及其前体、亚型等)与未分化pPS细胞或其它已分化细胞类型，有时它们具有相同的基因组。组内的各细胞类型可以包装在一起，或分装在同一装置的不同容器内，或放置在不同的位置，但都由同一实体或存在商务关系的不同实体掌控。 
关于细胞组合物的药用配制可参见“细胞治疗：干细胞移植，基因治疗和细胞免疫治疗”G.Morstyn & W.Sheridan，Cambridge University Press，1996。可将组合物装在合适的容器内，配以写明适宜用途例如治疗糖尿病的说明书。 
装置 
本发明细胞还可以用作用来生产一种或多种胰岛细胞内分泌多肽的装置的功能性组件。 
最简单的是，所述装置包含pPS衍生的胰岛细胞，将其置于一层半透膜之后，该半透膜可阻止细胞群透过从而将其保留在装置中，但允许细胞群所分泌的胰岛素、胰高血糖素或抑生长素透过。这包括微胶囊化的胰岛细胞，它们通常呈细胞簇的形式从而通过允许细胞间相互作用来抑制去分化。例如，美国专利4,391,909记载了一种胰岛细胞微胶囊，由大于3000mol.wt.的多糖交联聚合物构成的球状半透膜包裹，该半透膜允许胰岛素大小的蛋白质透过但不允许大于100,000mol.wt.的分子透过。美国专利6,023,009记载了包裹在琼脂糖和琼脂胶构成的半透膜中的胰岛细胞。这种微胶囊被制成适合输入糖尿病人体内的形式，且被认为有利于减少组织相容性问题或细菌感染。 
还可将本发明胰岛细胞用于更精细的装置，以用于植入体内或用于体外治疗。美国专利4,378,016记载了一种人造内分泌腺，包括一个体外部分，一个皮下部分和一个可替换包膜，包膜内包含了产激素的细胞。美国专利5,674,289记载了一种人造胰腺，具有一个胰岛腔，腔内由一张半透膜隔成一个或多个管状腔并与周围组织连通。可用的装置通常包括一个容纳胰岛细胞的腔室和一个由半透膜与胰岛细胞隔开的腔室，该腔室收集胰岛细胞分泌的蛋白质，并且还可能允许信号例如循环葡萄糖水平向胰岛细胞反馈。 
                               实施例
实施例1：胚胎干细胞的无饲养细胞增殖
将已建立的未分化人胚胎干细胞(hES)细胞系维持在基本无饲养细胞的培养条件下。 
事先用按照标准方法分离(WO01/51616)的原代小鼠胚胎成纤维细胞(mEF)制备条件培养基。 
将hES培养物移植到涂有Matrigel的平板上。接种后1周左右，培养物铺满平板并可以传代。培养物在这种条件下可维持180天以上，仍然呈现ES样形态。根据免疫组织化学检测，hES菌落表达SSEA-4、Tra-1-160、Tra-1-81和碱性磷酸酶，但位于菌落之间的已分化细胞则不表达这些标记。对这些细胞进行制造特定细胞类型的标准操作，从而确认它们的多能性。 
实施例2：hES细胞向肝细胞的衍生
在亚铺满培养物中加入DMSO引发全面分化，由此令未分化hES细胞分化为具有人肝细胞特征的细胞。然后，加入正丁酸钠，诱导细胞形成肝细胞样细胞。 
简而言之，分离后，将hES细胞在非分化培养条件下维持2-3天。此时，细胞铺满率约50-60％，将培养基换成含1％DMSO的非条件SR培养基(步骤II)。连续4天每天给培养物添加SR培养基，然后换成含1％DMSO+2.5％正丁酸钠的非条件SR培养基，连续6天每天添加(步骤III)。然后，将细胞接种到胶原蛋白平板上，培养在含亲肝细胞生长因子混合物的肝细胞成熟培养基中(步骤IV)。该过程可概括如表3所示。 
Figure G02824367619960328D000211
图1显示获得了肝细胞样细胞。左栏：10倍放大；右栏：40倍放大。加入正丁酸盐4天时，培养物中80％以上细胞直径较大，含有大细胞核和粒状细胞质(A行)。SR培养基中培养5天后，细胞改在HCM中培养。2天后，许多细胞形成多核并呈较大的多角形(B行)。HCM中培养4天后，多核多角细胞成为主体，且胞质溶胶颜色变深(C行)，据此标准，它们类似于新鲜分离的成人肝细胞(D行)或胎儿肝细胞(E行)。 
实施例3：由肝细胞路径中的早期细胞获得胰岛素分泌细胞
对以上肝细胞分化方案进行调整，从而由H9细胞系中的hES细胞衍生胰岛素分泌细胞。 
简而言之，步骤I，在最后一次传代后，在7-8天内，用实施例1所述采用 小鼠胚胎成纤维细胞的SR条件培养基将hES细胞培养至铺满。步骤II，换用含1％DMSO的非条件SE培养基并加入10μM环杷明，培养4天。步骤III，在加有B27，0.5mM正丁酸盐(肝细胞方案的五分之一)，10μM环杷明，4nM活化素A和10mM烟酰胺的RPMI 1640培养基中培养11天。然后，收获细胞，固定，对胰岛素表达进行染色(用1∶500稀释的Sigma小鼠多克隆抗胰岛素抗体)，并用DAPI反染色以检测细胞核。步骤IV，用补充了B27并可能含有胰岛分化因子的RPMI培养基培养11天以令细胞成熟。 
图2显示步骤IV中用4nM活化素A、4nMβ-动物纤维素、10nM IGF-1和10mM烟酰胺培养细胞的结果。上图中的圆圈代表DAPI染蓝的细胞核。集中在视野中间的迷散性染色是红色荧光，代表合成并固定在细胞内的胰岛素。据估计，测试格中约1％细胞表达胰岛素。同一载玻片上的其它细胞簇以及同种异型对照中都没有看到胰岛素染色。 
实施例4：通过优化消化道内胚层的形成来进入胰岛细胞路径
本实施例说明如何模拟子宫内的正常发育分步骤诱导pPS细胞分化为胰岛。 
用标准无饲养细胞条件将hES细胞的H7和H9细胞系培养至铺满，作为起始材料。为了引发分化，将培养基换成补充了B27(Invitrogen)和以下成分之一的RPMI 1640： 
无其它添加(培养基对照) 
4nM活化素A(R&D system) 
0.5mM正丁酸钠(Sigma) 
活化素A+正丁酸钠 
每日更换培养基(第一天除外)，共8天。 
结束时，去除培养基，用PBS洗涤细胞，加入400μl RTL裂解缓冲液(Qiagen)制备细胞裂解液。用Qiagen的“RNAeasy迷你试剂盒”按照其说明制备RNA。用DNAse处理RNA，用Invitrogen的“扩增前cDNA第一链试剂盒”按照其说明制备随机引发的cDNA第一链。用Sox17、HNF1-α和HNF3-β的相应异物-探针组对每份cDNA样品进行标准实时RT-PCR(Taqman)。为了将表达水平归一化，用cycophilin的探针和引物(Applied Biosystems)进行平行的Taqman试验。用deltadelta Ct法(Applied Biosystems)计算每份样品与胰标准品相比的相对丰度。 
图3显示各消化道内胚层标记的表达水平。所有结果相对人胎儿胰内的表达水平进行了标准化。同时还显示了未分化hES细胞(起始细胞系)内的表达水平以供比较。活化素A+正丁酸钠诱导这三种标记最有效。Sox17在未分化H9细胞内 高表达(2.3相对表达)，但用以上两种添加剂诱导时可达32相对表达(提高100倍以上)。H7细胞内，诱导效果更显著，从胎儿胰的0.007倍提高到79倍(提高10,000倍以上)。HNF3-β的表达由0.5以下相对表达提高至5.1或6.3(提高10倍以上)。HNF4-α表达由0.1以下相对表达提高至1.0以上(提高10倍以上)。 
这三种标记的强烈诱导证明这些hES衍生的细胞具有消化道内胚层的关键特征。然后对这些细胞进行继续培养，并用诱导胰形成的因子诱导，并监测关键性胰标记Pdx1的诱导。 
实施例5：在长期聚集体培养物中加入添加剂来获取胰岛细胞
将hES细胞的H7细胞系培养在实施例1所述mEF培养基中保持未分化形式。然后，在含有1∶1mEF条件培养基和1∶1DFB+的混合培养基中进行悬浮培养(形成胚状体)。DFB+即补充了B27(1x)、胰岛素(25μg/ml)、前列腺素(6.3ng/ml)、腐胺(10μg/ml)、砷(砷酸盐，100ng/ml)、转铁蛋白(50μg/ml)和甲状腺素受体的配体T3(40ng/ml)的DMEM/F12培养基。次日，加入10μM全反式(all-trans)维甲酸。第3天，将培养基换成含10μM全反式维甲酸的DFB+，然后隔日添加，持续7天(1期)。 
为了启动2期，去除维甲酸换以含Noggin(200ng/ml)、EGF(20ng/ml)和bEGF(2ng/ml)的培养基。隔日添加培养基，培养14天。 
3期，去除Noggin、EGF和bFGF，将细胞培养在含烟酰胺(10mM)的DFB+培养基中。隔日更换培养基，持续5天。然后，将细胞铺在有涂层的分格载玻片上，放置1天，固定并用抗胰岛素c肽抗体或抗抑生长素抗体染色。C肽是前胰岛素的组分之一，分泌前被切除。该肽可用于区分细胞合成的胰岛素与存在于培养基中的胰岛素。 
图4显示了结果。上图和中图分别以低放大率和高放大率显示胰岛素c肽染色结果，该染色表征成熟胰β细胞。下图显示抑生长素染色，这是胰岛δ细胞的标记。Taqman实时RT-PCR在mRNA水平确认这些细胞表达胰岛素和胰高血糖素。 
实施例6：用神经生成素3基因表达驱动胰岛路径的末期
本实施例表明某胰岛相关基因过表达能够重新启用分化III期胰岛细胞系特有的基因表达模式。 
构建含有受组成型启动子调控的转录调节基因神经生成素3(Ngn3)的腺病毒载体。AdNgn3是一种复制缺陷型、E1-和E3-缺失的重组腺病毒5载体，用AdMAXTM载体构建试剂盒(Microbix Biosystems，Toronto)构建。将Ngn3 cDNA645bp(GenBank登录号NM_020999)克隆到pDC515穿梭载体的mCMV(鼠巨细胞 病毒极早期基因)启动子的下游。将紧接在ATG启动子上游的天然序列TAGAAAGG换成共有Kozak序列GCCACC。将含有Ngn3插入片段的穿梭质粒和Ad基因组质粒(AdMAX质粒)共转染到表达E1的293细胞内，以期发生同源重组。分离噬菌斑，扩增293细胞制备病毒原液，然后用CsCI密度梯度离心纯化。测定A280处的光密度以确定病毒颗粒浓度，用标准空斑试验测定感染效价。 
将人ES细胞的H7细胞悬浮培养在实施例5所述混合培养基中。第二天加入全反式维甲酸(10μM)。7天后，在Matrigel
Figure 10003_2
上将EB平板培养在加有EGF(20ng/ml)和bFGF(2ng/ml)的DFB+培养基中。5天后，用50MOI的AdNgn3载体感染细胞。用AdGFP(含绿荧光蛋白的相同载体，不会直接分化)作为阴性对照。细胞在6格培养板上于1ml加有EGF(20ng/ml)的DFB+培养基中培养，加入50Mol的AdNgn3或AdGFP病毒，培养4小时，然后每格添加2ml该培养基。此后隔日补充培养基。感染后第2天或第8天收获细胞，如实施例4所述用实时RT-PCR分离mRNA进行分析。将细胞铺在带格载玻片上以进行免疫组织化学染色，先用4％低聚甲醛固定，封闭，然后先用抗胰高血糖素抗体(1∶1000)接着用FITC偶联的山羊抗小鼠IgG(1∶500)染色。 
图5显示神经生成素3转导后9天转导细胞的免疫组织化学染色。平板上的胚状体显示明显的抗体可测水平的胰高血糖素表达。 
图6(A)和(B)显示与阴性对照(阴影柱)相比，神经生成素3转导细胞(实心柱)的mRNA表达水平。实时RT-PCR数据相对人胎儿胰内的表达水平进行了归一化。 
结果发现，在转导细胞内，数个位于神经生成素3下游的基因与对照相比被特异性地上调。NeuroD1和Nkx2.2在第2天显著上调；Nkx6.1在第8天上调。胰岛素和胰高血糖素表达也在第8天显著上调，表明基因转染显著促进了细胞的成熟，由此产生了具有重要临床意义的末期产物即胰岛细胞。 
对本领域技术人员来说显而易见的是，可对本发明进行常规优化，这并不背离本发明的主旨，也不超越本发明的范围。 
如果允许多项从属权利要求，则本发明的实施应包括以下内容： 
1.一种分离的细胞群，由分化灵长动物多能干细胞获得，其中至少5％细胞分泌一种或多种以下内源基因编码的蛋白质：胰岛素，胰高血糖素，抑生长素或胰多肽。 
2.一种分离的细胞簇，由分化灵长动物多能干细胞获得，包含分泌胰岛素的细胞，分泌胰高血糖素的细胞和分泌抑生长素的细胞。 
3.如前述权利要求中任一项所述的细胞群，其中至少5％细胞表达至少两种 以下标记：Pdx1，Ngn3，胰岛素，IAPP和Nkx6.1。 
4.如前述权利要求中任一项所述的细胞群，未分化灵长动物多能干细胞少于1％。 
5.如前述权利要求中任一项所述的细胞群，当其被移植到高血糖症NOD小鼠内时可使空腹葡萄糖水平降至200mg/L以下。 
6.如前述权利要求中任一项所述的细胞群，其经遗传改造而以高于正常的水平表达端粒末端转移酶逆转录酶。 
7.如前述权利要求中任一项所述的细胞群，与已建立的灵长动物胚胎干细胞的细胞系具有相同的基因组。 
8.两细胞细胞群，包含前述权利要求中任一项所述的已分化细胞群和作为其来源的未分化灵长动物多能干细胞。 
9.一种获得多肽分泌细胞的方法，包括在胰岛细胞分化因子混合物中培养灵长动物多能干细胞或其后代，由此获得一细胞群，其中至少5％细胞分泌一种或多种以下内源基因编码的蛋白质：胰岛素，胰高血糖素，抑生长素和胰多肽。 
10.如权利要求9所述的方法，所述灵长动物多能干细胞分化成胚状体或具有肝细胞或消化道内皮特征的细胞。 
11.如权利要求9或10所述的方法，所述胰岛细胞分化因子混合物包含至少一种以下物质：活化素A，烟酰胺，环杷明，β动物纤维素和IGF-1。 
12.如权利要求9或10所述的方法，所述胰岛细胞分化因子混合物包含TGF-β拮抗剂和一种或多种促分裂素。 
13.如权利要求9至12中任一项所述的方法，还包括改变细胞的基因使其表达胰转录因子，例如神经生成素3。 
14.如权利要求1-8中任一项所述的已分化细胞群，由权利要求9-13中任一项所述的方法制得。 
15.一种生产胰岛素、胰高血糖素或抑生长素的方法，包括： 
a)培养权利要求1-8中任一项所述的已分化细胞群或按照权利要求9-13中任一项所述方法制得的已分化细胞群，和 
b)收获培养细胞所分泌的胰岛素、胰高血糖素或抑生长素。 
16.一种生产胰岛素、胰高血糖素或抑生长素的装置，包含： 
a)权利要求1-8中任一项所述的已分化细胞群或按照权利要求9-13中任一项所述方法制得的已分化细胞群，和 
b)阻止所述细胞群通过但允许其分泌的胰岛素、胰高血糖素或抑生长素 通过的半透膜。 
17.一种药物组合物，包含权利要求1-8中任一项所述的已分化细胞群或按照权利要求9-13中任一项所述方法制得的已分化细胞群。 
18.一种筛选能够调节胰岛细胞功能的化合物的方法，包括：将化合物与如权利要求1-8中任一项所述或按照权利要求9-13任一项所述方法制得的已分化细胞群接触，测定接触化合物引起的表型或代谢改变，将这些改变与化合物调节胰岛素、胰高血糖素或抑生长素的能力相关联。 
19.一种恢复个体胰岛细胞功能的方法，包括给予如权利要求1-8中任一项所述或按照权利要求9-13任一项所述方法制得的已分化细胞群。 
20.一种治疗I型糖尿病的方法，包括给予如权利要求1-8中任一项所述或按照权利要求9-13任一项所述方法制得的已分化细胞群。 
21.如权利要求1-8中任一项所述或按照权利要求9-13任一项所述方法制得的已分化细胞群在制备治疗胰岛素、胰高血糖素或抑生长素缺乏相关性疾病的药物中的用途。 
22.如权利要求18所述多用途，所述疾病是I型糖尿病。 
23.前述权利要求中任一项所述的细胞群、装置、方法或用途，所述灵长动物多能细胞是由人胚泡获得的细胞的后代。 
24.前述权利要求中任一项所述的细胞群、装置、方法或用途，所述灵长动物多能细胞是人胚胎干细胞。 

Claims (13)

1.一种包含内胚层细胞的细胞培养物，所述内胚层细胞与已建立的灵长动物多能干细胞的细胞系具有相同的基因组。
2.如权利要求1所述的细胞培养物，所述内胚层细胞表达Sox17。
3.如权利要求1所述的细胞培养物，所述内胚层细胞表达HNF4α。
4.如权利要求1所述的细胞培养物，所述内胚层细胞表达HNF3β。
5.一种产生权利要求1中所述内胚层细胞的方法，包括将灵长动物多能干细胞培养在含活化素A的培养基中。
6.如权利要求5所述的方法，所述培养基还含有丁酸钠。
7.如权利要求5所述的方法，所述人多能干细胞在含有活化素A的培养基中培养8天。
8.如权利要求5所述的方法，在于含活化素A的培养基中进行培养之前由灵长类动物多能干细胞形成胚状体。
9.如权利要求5所述的方法，在于含活化素A的培养基中进行培养之前未由灵长类动物多能干细胞形成胚状体。
10.如权利要求5所述的方法，所述内胚层细胞的Sox17表达水平高于人多能干细胞。
11.如权利要求5所述的方法，所述内胚层细胞的HNF4α表达水平高于人多能干细胞。
12.如权利要求5所述的方法，所述内胚层细胞的HNF3β表达水平高于人多能干细胞。
13.如权利要求5至12中任一项所述的方法，所述人多能干细胞是人胚胎干细胞。
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