家畜胚胎干细胞多能性候选信号通路及分子标志

赵云程，陈博，周川，张秀华，黄俊成

1 新疆维吾尔自治区动物生物技术重点开放实验室，乌鲁木齐 830000

2 农业部草食家畜繁育生物技术重点开放实验室，乌鲁木齐 830000

干细胞专栏

家畜胚胎干细胞多能性候选信号通路及分子标志

赵云程1,2，陈博1,2，周川1,2，张秀华1,2，黄俊成1,2

1 新疆维吾尔自治区动物生物技术重点开放实验室，乌鲁木齐 830000

2 农业部草食家畜繁育生物技术重点开放实验室，乌鲁木齐 830000

家畜胚胎干细胞具有重要的生物学意义和广阔的应用前景。以下对比了小鼠、人胚胎干细胞多能性调控信号通路的异同，阐述了小鼠、人胚胎干细胞与家畜胚胎干细胞在多能性分子标志上的差异，并结合本实验室开展绵羊胚胎干细胞研究的实际经验，对目前家畜胚胎干细胞建系中可能存在的多能性候选信号通路及分子标志进行了探讨。

家畜，胚胎干细胞，多能性，信号通路，候选基因

Abstract:Domesticated ungulates embryonic stem (ES) cells have great significances in biology and wide application prospects.This review compared the key signaling pathways related with pluripotency between mouse and human ES cells, and the difference of transcription factors in mouse, human and domesticated ungulates ES cells were elaborated. Finally the pluripotency candidate signaling network and transcription factors related in the derivation of domesticated ungulates ES cell were discussed combined with practical experience of ovine embryonic stem cell derivation in our laboratory.

Keywords:domesticated ungulates, embryonic stem cells, pluripotency, signaling pathway, candidate genes

胚胎干细胞 (Embryonic stem cell，ESC)，是由哺乳动物着床前囊胚期内细胞团 (Inner cell mass，ICM) 经体外特定培养环境选择、适应后，获得的具有无限增殖能力和多向分化潜能的细胞系，其特定的生物学特性能够使其与ICM重新整合，并参与到胚胎发育的全部过程中。自1989年采用ESC技术获得了第一个转基因小鼠以来[1]，ESC技术在家畜遗传育种中展现出巨大前景。采用ESC克隆技术，其整合效率远远高于传统核移植技术，可在短期内生产较多的具有遗传同质性的动物，免去后裔测定，大幅度提高良种家畜的繁殖效率。

然而，自 1981年小鼠胚胎干细胞成功建系以来，仅有小鼠[2]、大鼠[3-4]获得了具备生殖系传递能力的ES细胞，极大地制约了ESC技术在家畜遗传育种中的研究与应用。目前，国内外在家畜ESC的研究中，普遍借助小鼠胚胎干细胞 (Mouse embryonic stem cell，mESC)、人胚胎干细胞 (Human embryonic stem cell，hESC) 的成功经验，将影响mESC、hESC增殖、分化的因素，如饲养层细胞、条件培养基、细胞生长因子、激素、胎牛血清和血清提取物等进行有机组合，依照mESC、hESC建系标准，直接应用到家畜ESC建系中，从而试图筛选出适合家畜ESC多能性维持的培养条件，然而却收效甚微。导致家畜ESC建系失败的原因之一是因为mESC和hESC多能性维持机制的差别，使得优化组合各种因素变得十分困难；比如，mESC分子调控网络中 BMP4 (Bone morphogenetic proteins 4) 与LIF协同维持其多能性，而在hESC中，BMP4会诱导hESC发生分化[5-6]。另外，家畜ESC多能性标志的匮乏，是导致建系失败的另一原因；研究表明mESC、hESC的多能性标志，如SSEA-1、SSEA-4、POU5F1、Nanog、碱性磷酸酶活性等，不仅在牛、猪、山羊囊胚的ICM中表达，同时在其囊胚的滋养层上有所表达[7-11]。针对上述原因，就使得我们不得不考虑家畜 ESC至今未能建系，是因为所借鉴的mESC、hESC添加因子受体本身就不处于家畜ESC的分子调控网中？还是这些因子在激活家畜ESC自我更新通路的同时，又激活了另一个与之分化有关的通路？或是因为多能性分子标志的模棱两可性，使得我们一再与家畜 ESC“擦肩而过”？因此，目前许多研究者普遍达成共识：家畜ESC的研究必须从多能性标志和信号通路入手[12-14]，借鉴 mESC、hESC多能性维持的分子机制，了解对比家畜 ESC与 mESC、hESC多能性调控机制的异同，对家畜ESC研究具有十分重要的作用。

1 ESC多能性维持机制中的关键信号转导通路

近年来，随着对mESC、hESC研究的逐步深入，mESC、hESC多能性维持的分子调控机制已逐步揭示。在小鼠中，mESC多能性的维持需要通过LIF-LIFR/gp130-Jak-STAT3途径和 BMP-Id协同来完成，与之不同的是，hESC多能性的维持则需要 Activin/Nodal和 FGF的共同参与。mESC与hESC二者在多能性维持的分子调控机制上存在显著差异。

1.1 LIF-STAT3途径

白血病抑制因子 (Leukemia inhibitor factor，LIF)属于白介素6 (IL-6) 细胞因子家族中的一员，是一种多功能的细胞因子。目前研究证实，LIF-LIFR/gp130-Jak-STAT3途径是 mESC自我更新的重要途径。对mESC体外多潜能性的维持，保持未分化状态发挥着重要作用[15-16]。研究发现，LIF与LIFR结合后，LIFR与gp130 (glycoprotein 130) 迅速聚合形成异源二聚体激活下游的 Janus 酪氨酸蛋白激酶(Janus-associated tyrosine kinase，JAK)、信号转导子和转录激活子3 (Signal transducer and activation of transcription，STAT) 途径。STAT3的激活是mESC细胞自我更新的关键[17-18]。缺少IL-6家族成员、撤去MEF，均会导致STAT3失活和mESC分化；外源LIF因子、血清，活化 STAT3，则足以维持 mESC的多能性[19]。大鼠胚胎干细胞 (rESC) 研究表明，外源hLIF能有效促进rESC集落形成率，因此Buehr推测，LIF-STAT3应该是“真正”意义上ESC的本质特性[3]。

在hESC研究中，起初认为LIF对hESC没有效果[20]，或是LIF有利于hESC多能性的维持[21]。随着研究的进一步深入，研究表明，外源hLIF因子能有效使hESC-STAT3的Tyr (705)、Ser (727) 发生磷酸化 (p-STAT3)，但 p-STAT3并不能维持 hESC多能性[6,22-23]。Daheron等研究发现，虽然 mLIF与hLIF同源程度较高 (氨基酸序列一致性为78%)，但 mLIF却表现出种属特异性，无法使 hESC的STAT3发生磷酸化[23]。LIF-STAT3信号通路在mESC与 hESC上的显著差异，可能与其各自在胚胎发育中的不同发育阶段有关[24]。

近期，Intawicha 报道，mLIF能有效激活兔类胚胎干细胞 (ES-like cells) 的LIF-Jak-STAT3途径，并提高其自我更新能力[25]；而牛 ES样细胞研究表明，牛囊胚ICM、及ICM原代培养过程中的确存在LIFR及gp130信号转导，但LIF对牛ES样细胞增殖并无明显作用[26]。另外，抑制 LIF-STAT3途径，对猪外胚层细胞多能性并无显著影响[27]；但 Brevini等研究发现，猪ES样细胞中虽然并不存在LIFR，但LIF却能有效抑制猪ES样细胞类胚体的形成[28-29]。

1.2 MAPK/ERK途径

研究发现，LIFR与gp130形成异源二聚体后，gp130除了能激活STAT3自我更新途径以外，还能激活MAPK/ERK级联反应[24]，而Erk-1/2级联反应对mESC的分化具有十分重要的调控作用[19]，研究表明，Erk-1/2在不同品系小鼠早期胚胎发育过程中表现出应答水平的不一致性[30]。利用小分子化合物阻断 ERK-1/2级联反应，129Sv/ter、C57BL/6品系mESC建系率分别提高至76.5%和47%[31]，并且可从更多小鼠品系中建立 mESC (如 CBA、MF1、SCID、NOD品系)[32-34]。我们研究发现，添加FGFR、MEK 特异性抑制剂 (SU5402、PD0325901) 阻断ERK-1/2分化级联反应后，能有效获得昆明鼠 ES(KM-ES) 细胞，建系率81.48% (22/27)，KM-ES集落生长稳定，碱性磷酸酶活性显著提高，分化得到明显抑制 (图 1)，有效解决了采用饲养层、血清替代物等常规培养体系中KM-ES集落分化率高、生长不稳定的问题。与之类似的是，BMP4途径的引入，正是依靠BMP4通过SMAD 1/5或SMAD 8途径诱导产生Id蛋白 (Inhibitor of differentiation)，Id蛋白进而抑制MAPK级联反应，并与LIF/STAT3协同维持mESC的多能性[35-37]。Li等采用FGF受体酪氨酸激酶和ERK-1/2级联反应特异性抑制剂SU5402和PD0325901，阻断MAPK/ERK途径后，成功建立了rESC系[4]。STAT3与ERK-1/2途径相互协调，对维持mESC、rESC细胞的自我更新和分化之间的平衡具有重要作用。

图1 KM-ES碱性磷酸酶活性检测Fig.1 Alkaline phosphatase staining for KM-ES.

与mESC不同的是，hESC未分化状态下则保持了较高的ERK活性，MAPK/ERK途径对hESC的凋亡、增殖和分化过程具有一定调节作用[38]。研究表明，碱性成纤维细胞生长因子 (Basic fibroblast growth factor，bFGF) 是hESC自我更新机制中的核心调控因子[39-40]，bFGF与细胞表面受体结合后，激活胞内MAPK/ERK级联反应，对hESC多能性维持起到十分重要的调控作用[41-43]。目前研究发现，在未分化的 hESC中均能检测到 FGFR-1、FGFR-2、FGFR-3、FGFR-4的表达，而以 FGFR-1的表达量最高[44-47]。采用 FGFR特异性抑制剂SU5402研究发现，FGFR受到抑制的条件下，hESC将会迅速发生分化[41]。hESC多能性的维持表现出对FGF的量性需求：当bFGF添加量在4 ng/mL时，hESC的维持需要有滋养层细胞[20]；当bFGF添加量达到8～40 ng/mL时，培养系统中不再需要饲养层细胞，而需要Noggin的参与 (Noggin为BMP4的拮抗蛋白)[41,48-49]；当bFGF达到100 ng/mL时，hESC可处于不分化状态[50]。采用MEK/ERK信号级联反应抑制剂PD98059、U0126，会迅速导致hESC发生分化[51]，这与mESC、rESC多能性调控机制存在显著差别。

目前研究发现，家畜ES样细胞与hESC更为相似[14]，bFGF能有效促进猪ES样细胞的原代集落形成率及自我更新[52-53]，并对兔ES样细胞多能性的维持具有一定作用[54]。

1.3 Wnt途径

近年来，研究表明Wnt途径在mESC与hESC多能性维持中发挥着十分重要的作用，对ESC向皮肤、神经系统、血液系统的分化起到调控作用[55]，与 LIF-STAT3、BMP4途径不同的是，Wnt途径在mESC与 hESC的自我更新中作用机理基本是一致的[56]，都能抑制糖原合成激酶3 (Glycogen synthase kinase 3，GSK-3) 的活性，解除对β连环蛋白的磷酸化，维持ESC的自我更新[57]。mESC、hESC本身可以自发激活Wnt途径，而当分化通路启动后，Wnt的表达量呈现下调趋势[58]。采用GSK-3的特异性抑制剂 6-bromoindirubin-3′-oxime (BIO)，可以特异性抑制GSK-3的磷酸化活性，导致核内β连环蛋白含量增高，从而激活 Wnt的下游信号转导途径，最终使得 mESC、hESC的多能性状态得到维持[59]。Wnt的激活能够调高c-Myc的水平，而c-Myc则是STAT3的靶位基因，表明Wnt途径与LIF-STAT3协同作用于c-Myc，共同维持mESC的多能性[60-62]。

虽然，Wnt途径在mESC、hESC多能性维持过程中的作用基本一致，但家畜ESC研究发现，采用Wnt途径激活剂 (BIO、Wnt3a) 并不能阻止外胚层细胞的分化[63]。

1.4 TGFβ/Activin/Nodal途径

TGFβ是一个大的超家族，超过40个成员，包括 TGFβ、Activin、Nodal和 BMP 等[56,64]。BMPs是TGFβ超家族中最大的成员，它在促进ESC自我更新中作用不明显，但是可以通过SMAD途径激活Id的表达，而Id蛋白则能够抑制神经发生转录因子bHLH的表达，从而最终抑制向神经系统的分化[65-66]；外源性Id蛋白将会模拟BMP4的生理学特性，维持mESC的多能性[65]。但是，BMP4在维持mESC细胞多能性的作用，需要在 LIF的存在下才能实现，这是因为BMP4能诱导ESC细胞向内胚层和中胚层分化，而LIF通过LIF-STAT3途径抑制了BMP4的这一促分化作用，但同时对BMP4诱导的Id蛋白表达没有作用，从而与LIF-STAT3共同维持mESC细胞的多能性[65]。与mESC自我更新维持机制截然相反，BMP4并不能维持hESC的自我更新，相反会使得 hESC向滋养层细胞和原始内胚层细胞分化[5]，hESC多能性需要 BMP4的拮抗物 Noggin与bFGF配合才能得以维持[49]。此外，TGFβ超家族成员Activin A、Nodal在未分化的hESC中表达量较高[67]。hESC研究表明，Smad 2/3参与了 TGFβ/Activin/Nodal的信号转导，在未分化过程中 Smad 2/3被激活，而Smad 1/5处于抑制状态，随着分化发生，Smad 2/3信号减弱，而Smad 1/5信号增强[68-69]。特异性小分子抑制剂BIO，可以模拟Wnt途径从而保持hESC的未分化状态，而此时Smad 2/3磷酸化水平仍然处于较高水平[69]。

TGFβ家族成员对mESC、hESC多能性的维持具有十分重要的作用，但其对家畜ESC的调控作用却并不十分清楚。Pant等研究发现，添加Noggin抑制BMP4途径后，牛ICM原代集落Nanog表达量显著上调[70]。同时，Alberio等研究发现，添加BMP4将会导致猪外胚层细胞向滋养层细胞与生殖细胞方向分化；抑制Activin/Nodal途径，猪外胚层细胞迅速分化为神经细胞，因此提出Activin/Nodal信号途径是在哺乳动物细胞多能性维持过程中起调控作用的保守途径[27]。而另有研究表明，添加 Activin及Noggin并不能阻止猪、马外胚层细胞的分化，激活Activin途径或抑制BMP4活性，对家畜ESC多能性的维持并无明显作用[63]。

1.5 PI3K/AKT途径

磷脂酰肌醇3激酶 (PI3K) 是一种脂质激酶，对细胞的增殖、生长、发育、迁移及细胞周期等生理活动具有十分重要的调控作用[71-72]。PI3K/AKT途径处于LIF、bFGF信号下游，在mESC中，当PI3K的负调控基因PTEN缺失后，将会促进细胞周期由G1期向S期的转变，从而使得mESC增殖加速[72]。添加 PI3K特异性抑制剂，将会激活 LIF介导的MAPK/ERK途径，降低LIF对mESC自我更新的调控作用，使得mESC增殖减慢[73-74]，同时编程性死亡发生率升高[75]。而hESC研究表明，PI3K/AKT与MAPK/ERK途径，并不存在交汇作用，二者对hESC自我更新的维持存在叠加效应[51]；阻断 PI3K/AKT途径，将会影响 hESC增殖，使编程性死亡发生率升高[51]。此外，研究发现采用PI3K特异性抑制剂，阻断PI3K途径后，将会增加早期附植胚胎编程性死亡的发生率[75]。

同时研究表明，PI3K/AKT途径对ESC多能性的维持作用显著。在添加LIF及饲养层细胞条件下，抑制PI3K活性将会导致mESC、hESC发生分化，表明PI3K/AKT信号途径对ESC (如：小鼠、猴、人)多能性状态的维持具有十分重要的调控作用[73-74,76-78]。Storm等研究发现，抑制PI3K/AKT途径将会导致包括Nanog和Zscan4家族在内的646种基因的表达量发生改变，而其中Zscan4家族基因并未与Nanog基因关联[79-81]；此外研究表明，二细胞早期胚胎及ESC具备较高水平的Zscan4，下调Zscan4基因将会导致囊胚无法附植[82]，因此 PI3K/AKT途径可能通过Nanog与Zscan4两种方式调控mESC的多能性[79]。

目前研究表明，PI3K/AKT途径对兔ES样细胞多能性的维持具有十分重要的调控作用，与 hESC类似，兔ES样细胞自我更新过程中PI3K/AKT途径与 MAPK/ERK 途径并未存在交汇作用[83]；Brevini等研究发现，猪ES样细胞虽然不存在LIFR，但LIF可能通过PI3K/AKT途径，抑制猪ES样细胞类胚体的形成，从而维持猪ES样细胞的多能性[28-29,84]。

针对上述与mESC、hESC多能性密切相关的信号通路，我们在绵羊ESC研究过程中，通过对6～8 d囊胚ICM原代集落机械法传代后，添加LIF-STAT3、Wnt与 Noggin信号途径的有效激活因子 (mLIF、hLIF、Wnt3a、Noggin)，但结果表明绵羊ES样集落周边细胞呈弥散式生长，碱性磷酸酶活性降低，集落逐步呈平铺式生长，界限模糊 (图 2)，初步表明上述因子并不能有效阻止绵羊ES样细胞发生分化。

图2 mLIF、hLIF、Wnt3a、Noggin并不足以维持绵羊类胚胎干细胞的自我更新Fig.2 mLIF, hLIF, Wnt3a and Noggin fail to maintain self-renewal of ovine ES-like cells. (A) The 10 ng/mL mLIF and hLIF fail to maintain self-renewal of ovine ES-like cells(Alkaline phosphatase staining). (B) The 100 ng/mL Wnt3a fail to maintain self-renewal of ovine ES-like cells. (C) The 100 ng/mL noggin fail to inhibition of differentiation of ovine ES-like cells. Scale bars=50 μm.

2 家畜ESC多能性候选基因

目前，制约家畜ESC研究的一个核心关键问题是缺乏家畜ESC的多能性标志，这也直接导致研究过程中无法及时有效地筛选出“真正”意义上的家畜ESC。近年来，研究表明POU5F1(POU domain 5 transcript factor 1)、Sox2(SRY-box containing gene 2)和Nanog等转录因子对mESC、hESC的自我更新和分化具有十分重要的作用，当它们表达时，ESC的自我更新途径被激活，分化途径受到抑制[85-87]，三者彼此调控，同时对上述影响ESC自我更新和分化的外源信号分子作出应答，严格控制着ESC的自我更新与分化进程，最终形成了ESC多能性机制的调控中枢——POU5F1、Sox2、Nanog[88-89]。

POU5F1又称为OCT-4[90]，是植入前胚胎发育的重要调节因子，特异性地表达于多种多能性细胞：卵母细胞、原始生殖细胞、早期植入前胚胎、原始外胚层、ICM和ESC细胞[91-92]，是mESC和hESC细胞的多能性分子标志[91-93]。目前，多种POU5F1的靶位基因已经得到确认，包括Fgf4、Utfl、Opn、Rexl/Zfp42、Fbxl5和Sox2。其中，Sox2在小鼠早期胚胎发育过程中起到十分重要的作用，不仅在小鼠早期胚胎中表达，同时会在胚外外胚层的多能性细胞中表达，Sox2表达量降低将导致mESC向滋养外胚层分化和多倍体细胞的出现[94]。Sox2-/-突变使小鼠早期胚胎不能形成外胚层并引起胚胎死亡[95]，表明Sox2是维持小鼠早期胚胎发育所必需的转录因子。Sox2与POU5F1协同作用，共同阻止ESC向滋养外胚层分化，同时阻止染色体异常[88]。Nanog基因是ESC研究中发现的另一个多能性主导基因，它不仅对胚胎发育过程中ICM多能性的调控起关键作用，同时还可维持外胚层细胞多能性和阻止其向原始内胚层的分化[96]，可在ESC、EG (Embryonic germ)和EC (Embryonic carcinoma) 等多能性细胞中检测到[96-97]，是维持mESC、hESC多能性的关键转录因子[98]。研究表明，虽然mESC与hESC在形态学、表面标志和生长因子方面存在显著差别，但是它们的Nanog基因都十分保守[97]。

此外，诱导多能干细胞 (Induced pluripotent stem cells，iPS) 的出现及后期“Yamanaka因子”(POU5F1、Sox2、c-Myc、Klf4) 功能的逐渐明朗，为家畜ESC多能性候选基因的选择提供了新的参考依据。2006年，Yamanaka采用逆转录病毒将POU5F1、Sox2、c-Myc、Klf4导入小鼠胚胎成纤维细胞或成年小鼠尾部皮肤成纤维细胞中，建立了与mESC非常相似的iPS细胞[99]。之后，Yamanaka将上述 4个转录因子导入到人皮肤成纤维细胞中，也成功获得了iPS细胞[100]。与此同时，Thomson研究小组也报道了成功诱导胎儿成纤维细胞转化为具有hESC基本特征的人iPS细胞，所不同的是他们使用慢病毒作为载体，选择了POU5F1、Sox2、Nanog、Lin28等4个基因[101]。Park等发现POU5F1和Sox2在诱导重构为 iPS细胞过程中是必需的，正是这 2个转录因子维持了人类iPS细胞的多潜能性，而Klf4和c-Myc的作用是改变染色质的结构，利于POU5F1和Sox2的结合，以提高诱导效率[102]，从而进一步明确了iPS多潜能性诱导过程中POU5F1、Sox2的重要作用。Huangfu等采用组蛋白脱乙酰基酶抑制剂，将POU5F1和Sox2基因导入到人类皮肤成纤维细胞中，也成功获得了iPS细胞[103]。Kim等将OCT4与Sox2或OCT4转录因子导入到小鼠神经干细胞中，成功获得了iPS细胞[104-105]。iPS相关技术在家畜iPS研究中的应用，为家畜ESC多能性候选基因的选择提供了参考依据。研究表明，采用iPS技术，导入包括POU5F1、Sox2、Nanog在内的多个基因，可以获得与 hESC各项生物学特性极为相似的猪多能性干细胞[106-107]，并已有通过iPS细胞获得嵌合体猪的报道[108]。

目前的家畜ESC研究表明，mESC、hESC多能性分子标志POU5F1、NANOG、SOX2在家畜多能性鉴定中应当被谨慎使用[12]。比如，POU5F1、NANOG、SOX2基因除在牛、猪、山羊囊胚的ICM表达外，在滋养层细胞、内胚层细胞中同样表达[7-8,12,109-110]。值得注意的是，上述多能性分子标志在ICM与滋养层细胞中的定位存在差别，Pant等研究发现：NANOG与POU5F1在牛ICM与滋养层细胞的核仁中表达，而NANOG除在ICM核仁表达外，在ICM核质中也有所表达[70]。近期研究表明POU5F1、NANOG的 mRNA及其编码的蛋白在山羊、绵羊ICM表达，而在囊胚滋养层细胞中mRNA表达量显著降低[9,111]；Hall等通过对11 d猪胚胎外胚层与滋养层细胞对比研究，发现POU5F1、Nanog、SOX2、FGFR1在外胚层细胞中的表达是特异的[112]；且牛、猪 ICM与外胚层细胞发生明显分化前，POU5F1、NANOG、SOX2的表达量发生明显变化[70,113-114]。上述相关研究成果为家畜ESC多能性标识的选择提供了一定依据。此外，值得注意的是，家畜胚胎POU5F1、NANOG、SOX2的基因表达量还受到外源环境的调控，比如Chio等研究发现，与体外胚胎相比，马体内胚胎 ICM 中POU5F1、SOX2、NANOG的表达量显著高于滋养层细胞[115]；另外，胚胎体外培养过程中，培养液的选择 (KSOM 与 SOF) 同样会对囊胚中上述相关候选基因的表达量产生一定调控作用[116]。因此，上述候选基因虽然能够作为家畜ESC多能性分子标识使用，但仍需进一步加以确认，因此与小鼠Nanog相区别，家畜ESC的NANOG基因则用大写斜体字母表示。

3 结语

虽然家畜ESC研究已开展了20多年，但目前尚无实质性突破。因此比对 mESC、hESC多能性分子调控网络的差异，借鉴mESC、rESC的成功经验，研究家畜ES样细胞生物学特性，对获得生殖系传递能力的家畜ESC具有十分重要的指导意义；此外，随着mESC研究的逐步深入，一系列与mESC生物学特性极为相似的细胞 (如FAB-SC、EpiSC细胞) 逐渐被发现，研究表明，这些类型的细胞可以通过简单的培养条件的转换从而具备mESC多能性的生物学特性[117-118]，因此对比家畜 ES样细胞与FAB-SC、EpiSC的生物学特性差异，将会为家畜ES样细胞向多能性方向的转变提供新的技术途径；另外，借助iPS研究成果，研究家畜iPS细胞生物学特性及多能性维持调控途径，将会对分离、培养、鉴定具备生殖系传递能力的家畜 ESC具有十分重要的指导意义。

REFERENCES

[1] Capecchi MR. The new mouse genetics: altering the genome by gene targeting.Trends Genet, 1989, 5(3):70−76.

[2] Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos.Nature, 1981,292(5819): 154−156.

[3] Buehr M, Meek S, Blair K,et al. Capture of authentic embryonic stem cells from rat blastocysts.Cell, 2008,135(7): 1287−1298.

[4] Li P, Tong C, Mehrian-Shai R,et al. Germline competent embryonic stem cells derived from rat blastocysts.Cell,2008, 135(7): 1299−1310.

[5] Xu RH, Chen X, Li DS,et al. BMP4 initiates human embryonic stem cell differentiation to trophoblast.Nat Biotechnol, 2002, 20(12): 1261−1264.

[6] Humphrey RK, Beattie GM, Lopez AD,et al.Maintenance of pluripotency in human embryonic stem cells is STAT3 independent.Stem Cells, 2004, 22(4):522−530.

[7] van Eijk MJT, van Rooijen MA, Modina S,et al.Molecular cloning, genetic mapping, and developmental expression of bovinePOU5F1.Biol Reprod, 1999, 60(5):1093−1103.

[8] Kirchhof N, Carnwath JW, Lemme E,et al. Expression pattern of Oct-4 in preimplantation embryos of different species.Biol Reprod, 2000, 63(6): 1698−1705.

[9] He SY, Pant D, Schiffmacher A,et al. Developmental expression of pluripotency determining factors in caprine embryos: novel pattern of NANOG protein localization in the nucleolus.Mol Reprod Dev, 2006, 73(12):1512−1522.

[10] Talbot NC, Powell AM, Rexroad CE Jr.In vitropluripotency of epiblasts derived from bovine blastocysts.Mol Reprod Dev, 1995, 42(1): 35−52.

[11] Vejlsted M, Avery B, Schmidt M,et al. Ultrastructural and immunohistochemical characterization of the bovine epiblast.Biol Reprod, 2005, 72(3): 678−686.

[12] Keefer CL, Pant D, Blomberg L,et al. Challenges and prospects for the establishment of embryonic stem cell lines of domesticated ungulates.Anim Reprod Sci, 2007,98(1/2): 147−168.

[13] Talbot NC, Blomberg le A. The pursuit of ES cell lines of domesticated ungulates.Stem Cell Rev, 2008, 4(3):235−254.

[14] Muñoz M, Díez C, Caamaño JN,et al. Embryonic stem cells in cattle.Reprod Domest Anim, 2008, 43(Suppl 4):32−37.

[15] Smith AG, Heath JK, Donaldson DD,et al. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides.Nature, 1988, 336(6200):688−690.

[16] Williams RL, Hilton DJ, Pease S,et al. Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells.Nature, 1988,336(6200): 684−687.

[17] Boeuf H, Hauss C, Graeve FD,et al. Leukemia inhibitory factor-dependent transcriptional activation in embryonic stem cells.J Cell Biol, 1997, 138(6):1207−1217.

[18] Niwa H, Burdon T, Chambers I,et al. Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3.Genes Dev, 1998, 12(13):2048−2060.

[19] Matsuda T, Nakamura T, Nakao K,et al. STAT3 activation is sufficient to maintain an undifferentiated state of mouse embryonic stem cells.EMBO J, 1999,18(15): 4261−4269.

[20] Thomson JA, Itskovitz-Eldor J, Shapiro SS,et al.Embryonic stem cell lines derived from human blastocysts.Science, 1998, 282(5391): 1145−1147.

[21] Schuldiner M, Yanuka O, Itskovitz-Eldor J,et al. Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells.Proc Natl Acad Sci USA, 2000, 97(21): 11307−11312.

[22] Sumi T, Fujimoto Y, Nakatsuji N,et al. STAT3 is dispensable for maintenance of self-renewal in nonhuman primate embryonic stem cells.Stem Cells,2004, 22(5): 861−872.

[23] Dahéron L, Opitz SL, Zaehres H,et al. LIF/STAT3 signaling fails to maintain self-renewal of human embryonic stem cells.Stem Cells, 2004, 22(5): 770−778.

[24] Liu N, Lu M, Tian XM,et al. Molecular mechanisms involved in self-renewal and pluripotency of embryonic stem cells.J Cell Physiol, 2007, 211(2): 279−286.

[25] Intawicha P, Ou YW, Lo NW,et al. Characterization of embryonic stem cell lines derived from New Zealand white rabbit embryos.Cloning Stem Cells, 2009, 11(1):27−38.

[26] Pant D, Keefer C. 4 gene expression in cultures of inner cell masses isolated fromin vitro-produced andin vivo-derived bovine blastocysts.Reprod Fertil Dev,2006, 18(2): 110−110.

[27] Alberio R, Croxall N, Allegrucci C. Pig epiblast stem cells depend on activin/nodal signaling for pluripotency and self-renewal.Stem Cells Dev, 2010, 19(10):1627−1636.

[28] Brevini TAL, Antonini S, Pennarossa G,et al. Recent progress in embryonic stem cell research and its application in domestic species.Reprod Domest Anim,2008, 43(Suppl 2): 193−199.

[29] Brevini TAL, Pennarossa G, Gandolfi F. No shortcuts to pig embryonic stem cells.Theriogenology, 2010, 74(4):544−550.

[30] Kunath T, Saba-El-Leil MK, Almousailleakh M,et al.FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embryonic stem cells from self-renewal to lineage commitment.Development,2007, 134(16): 2895−2902.

[31] Batlle-Morera L, Smith A, Nichols J. Parameters influencing derivation of embryonic stem cells from murine embryos.Genesis, 2008, 46(12): 758−767.

[32] Ying QL, Wray J, Nichols J,et al. The ground state of embryonic stem cell self-renewal.Nature, 2008,453(7194): 519−523.

[33] Nichols J, Jones K, Phillips JM,et al. Validated germline-competent embryonic stem cell lines from nonobese diabetic mice.Nat Med, 2009, 15(7): 814−818.

[34] Yang WF, Wei W, Shi C,et al. Pluripotin combined with leukemia inhibitory factor greatly promotes the derivation of embryonic stem cell lines from refractory strains.Stem Cells, 2009, 27(2): 383−389.

[35] Burdon T, Stracey C, Chambers I,et al. Suppression of SHP-2 and ERK signalling promotes self-renewal of mouse embryonic stem cells.Dev Biol, 1999, 210(1):30−43.

[36] Qi XX, Li TG, Hao J,et al. BMP4 supports self-renewal of embryonic stem cells by inhibiting mitogen-activated protein kinase pathways.Proc Natl Acad Sci USA, 2004,101(16): 6027−6032.

[37] Lodge P, McWhir J, Gallagher E,et al. Increased gp130signaling in combination with inhibition of the MEK/ERK pathway facilitates embryonic stem cell isolation from normally refractory murine CBA blastocysts.Cloning Stem Cells, 2005, 7(1): 2−7.

[38] Böttcher RT, Nichrs C. Fibroblast growth factor signaling during early vertebrate development.Endocr Rev, 2005, 26(1): 63−77.

[39] Amit M, Carpenter MK, Inokuma MS,et al. Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture.Dev Biol, 2000, 227(2): 271−278.

[40] Xu CH, Inokuma MS, Denham J,et al. Feeder-free growth of undifferentiated human embryonic stem cells.Nat Biotechnol, 2001, 19(10): 971−974.

[41] Dvorak P, Hampl A. Basic fibroblast growth factor and its receptors in human embryonic stem cells.Folia Histochem Cytobiol, 2005, 43(4): 203−208.

[42] Kang HB, Kim JS, Kwon HJ,et al. Basic fibroblast growth factor activates ERK and induces c-fos in human embryonic stem cell line MizhES1.Stem Cells Dev,2005, 14(4): 395−401.

[43] Levenstein ME, Ludwig TE, Xu RH,et al. Basic fibroblast growth factor support of human embryonic stem cell self-renewal.Stem Cells, 2006, 24(3): 568−574.

[44] Bhattacharya B, Miura T, Brandenberger R,et al. Gene expression in human embryonic stem cell lines: unique molecular signature.Blood, 2004, 103(8): 2956−2964.

[45] Brandenberger R, Wei H, Zhang S,et al. Transcriptome characterization elucidates signaling networks that control human ES cell growth and differentiation.Nat Biotechnol, 2004, 22(6): 707−716.

[46] Dvash T, Mayshar Y, Darr H,et al. Temporal gene expression during differentiation of human embryonic stem cells and embryoid bodies.Hum Reprod, 2004,19(12): 2875−2883.

[47] Ginis I, Luo YQ, Miura T,et al. Differences between human and mouse embryonic stem cells.Dev Biol, 2004,269(2): 360−380.

[48] Wang GW, Zhang H, Zhao Y,et al. Noggin and bFGF cooperate to maintain the pluripotency of human embryonic stem cells in the absence of feeder layers.Biochem Biophys Res Commun, 2005, 330(3): 934−942.

[49] Xu RH, Peck RM, Li DS,et al. Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells.Nat Methods, 2005,2(3): 185−190.

[50] Xu CH, Rosler E, Jiang JJ,et al. Basic fibroblast growth factor supports undifferentiated human embryonic stem cell growth without conditioned medium.Stem Cells,2005, 23(3): 315−323.

[51] Li J, Wang GW, Wang CY,et al. MEK/ERK signaling contributes to the maintenance of human embryonic stem cell self-renewal.Differentiation, 2007, 75(4): 299−307.

[52] Li M, Zhang D, Hou Y,et al. Isolation and culture of embryonic stem cells from porcine blastocysts.Mol Reprod Dev, 2003, 65(4): 429−434.

[53] Li M, Ma W, Hou Y,et al. Improved isolation and culture of embryonic stem cells from Chinese miniature pig.J Reprod Dev, 2004, 50(2): 237−244.

[54] Honda A, Hirose M, Ogura A. Basic FGF and Activin/Nodal but not LIF signaling sustain undifferentiated status of rabbit embryonic stem cells.Exp Cell Res, 2009, 315(12): 2033−2042.

[55] Cadigan KM, Nusse R. Wnt signaling: a common theme in animal development.Genes Dev, 1997, 11(24):3286−3305.

[56] Niwa H. Molecular mechanism to maintain stem cell renewal of ES cells.Cell Struct Funct, 2001, 26(3):137−148.

[57] Rattis FM, Voermans C, Reya T. Wnt signaling in thestem cell niche.Curr Opin Hematol, 2004, 11(2): 88−94.

[58] Sato N, Meijer L, Skaltsounis L,et al. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor.Nat Med,2004, 10(1): 55−63.

[59] Constantinescu S. Stemness, fusion and renewal of hematopoietic and embryonic stem cells.J Cell Mol Med, 2003, 7(2): 103−112.

[60] Hao J, Li TG, Qi XX,et al. WNT/β-catenin pathway up-regulatesStat3and converges on LIF to prevent differentiation of mouse embryonic stem cells.Dev Biol,2006, 290(1): 81−91.

[61] Cartwright P, McLean C, Sheppard A,et al. LIF/STAT3 controls ES cell self-renewal and pluripotency by a Myc-dependent mechanism.Development, 2005, 132(5):885−896.

[62] Kristensen DM, Kalisz M, Nielsen JH. Cytokine signalling in embryonic stem cells.APMIS, 2005,113(11/12): 756−772.

[63] Talbot NC, Blomberg Le A. The pursuit of ES Cell lines of domesticated ungulates.Stem Cell Rev, 2008, 4(3):235−254.

[64] Massagué J. TGF-β signal transduction.Annu Rev Biochem, 1998, 67: 753−791.

[65] Ying QL, Nichols J, Chambers I,et al. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3.Cell, 2003, 115(3): 281−292.

[66] Gerrard L, Rodgers L, Cui W. Differentiation of human embryonic stem cells to neural lineages in adherent culture by blocking bone morphogenetic protein signaling.Stem Cells, 2005, 23(9): 1234−1241.

[67] Sato N, Sanjuan IM, Heke M,et al. Molecular signature of human embryonic stem cells and its comparison with the mouse.Dev Biol, 2003, 260(2): 404−413.

[68] Beattie GM, Lopez AD, Bucay N,et al. Activin A maintains pluripotency of human embryonic stem cells in the absence of feeder layers.Stem Cells, 2005, 23(4):489−495.

[69] James D, Levine AJ, Besser D,et al. TGFβ/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells.Development, 2005,132(6): 1273−1282.

[70] Pant D, Keefer CL. Expression of pluripotency- related genes during bovine inner cell mass explant culture.Cloning Stem Cells, 2009, 11(3): 355−365.

[71] Cantley LC. The phosphoinositide 3-kinase pathway.Science, 2002, 296(5573): 1655−1657.

[72] Sun H, Lesche R, Li DM,et al. PTEN modulates cell cycle progression and cell survival by regulating phosphatidylinositol 3,4,5,-trisphosphate and Akt/protein kinase B signaling pathway.Proc Natl Acad Sci USA,1999, 96(11): 6199−6204.

[73] Jirmanova L, Afanassieff M, Gobert-Gosse S,et al.Differential contributions of ERK and PI3-kinase to the regulation of cyclin D1 expression and to the control of the G1/S transition in mouse embryonic stem cells.Oncogene, 2002, 21(36): 5515−5528.

[74] Paling NRD, Wheadon H, Bone HK,et al. Regulation of embryonic stem cell self-renewal by phosphoinositide 3-kinase-dependent signaling.J Biol Chem, 2004,279(46): 48063−48070.

[75] Gross VS, Hess M, Cooper GM. Mouse embryonic stem cells and preimplantation embryos require signaling through the phosphatidylinositol 3-kinase pathway to suppress apoptosis.Mol Reprod Dev, 2005, 70(3):324−332.

[76] Armstrong L, Hughes O, Yung S,et al. The role of PI3K/AKT, MAPK/ERK and NFκβ signalling in the maintenance of human embryonic stem cell pluripotency and viability highlighted by transcriptional profiling and functional analysis.Hum Mol Genet, 2006, 15(11):1894−1913.

[77] Watanabe S, Umehara H, Murayama K,et al. Activation of Akt signaling is sufficient to maintain pluripotency in mouse and primate embryonic stem cells Akt signalling in pluripotency of ES cells.Oncogene, 2006, 25(19):2697−2707.

[78] Kim SJ, Cheon SH, Yoo SJ,et al. Contribution of the PI3K/Akt/PKB signal pathway to maintenance of self-renewal in human embryonic stem cells.FEBS Lett,2005, 579(2): 534−540.

[79] Storm MP, Kumpfmueller B, Thompson B,et al.Characterization of the phosphoinositide 3-kinasedependent transcriptome in murine embryonic stem cells: identification of novel regulators of pluripotency.Stem Cells, 2009, 27(4): 764−775.

[80] Storm MP, Bone HK, Beck CG,et al. Regulation of Nanog expression by phosphoinositide 3-kinase-dependent signaling in murine embryonic stem cells.J Biol Chem,2007, 282(9): 6265−6273.

[81] Takahashi K, Mitsui K, Yamanaka S. Role of ERas in promoting tumour-like properties in mouse embryonic stem cells.Nature, 2003, 423(6939): 541−545.

[82] Falco G, Lee SL, Stanghellini I,et al.Zscan4: a novelgene expressed exclusively in late 2-cell embryos and embryonic stem cells.Dev Biol, 2007, 307(2): 539−550.

[83] Wang SF, Shen Y, Yuan XH,et al. Dissecting signaling pathways that govern self-renewal of rabbit embryonic stem cells.J Biological Chem, 2008, 283(51):35929−35940.

[84] Brevini TAL, Pennarossa G, Attanasio L,et al. Culture conditions and signalling networks promoting the establishment of cell lines from parthenogenetic and biparental pig embryos.Stem Cell Rev, 2010, 6(3):484−495.

[85] Pera MF, Reubinoff B, Trounson A. Human embryonic stem cells.J Cell Sci, 2000, 113(Pt 1): 5−10.

[86] Adewumi O, Aflatoonian B, Ahrlund-Richter L,et al.Characterization of human embryonic stem cell lines by the international stem cell initiative.Nat Biotechnol,2007, 25(7): 803−816.

[87] Boiani M, Schöler HR. Regulatory networks in embryo-derived pluripotent stem cells.Nat Rev Mol Cell Biol, 2005, 6(11): 872−884.

[88] Yamanaka S, Li JL, Kania G,et al. Pluripotency of embryonic stem cells.Cell Tissue Res, 2008, 331(1):5−22.

[89] Schoenhals M, Kassambara A, De Vos J,et al.Embryonic stem cell markers expression in cancers.Biochem Biophys Res Commun, 2009, 383(2): 157−162.

[90] Nichols J, Zevnik B, Anastassiadis K,et al. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4.Cell, 1998, 95(3):379−391.

[91] Pesce M, Gross MK, Schöler HR. In line with our ancestors: Oct-4 and the mammalian germ.Bioessays,1998, 20(9): 722−732.

[92] Schöler HR, Dressler GR, Balling R,et al. Oct-4: a germline-specific transcription factor mapping to the mouse t-complex.EMBO J, 1990, 9(7): 2185−2195.

[93] Palmieri SL, Peter W, Hess H,et al. Oct-4 transcription factor is differentially expressed in the mouse embryo during establishment of the first two extraembryonic cell lineages involved in implantation.Dev Biol, 1994,166(1): 259−267.

[94] Li J, Pan GJ, Cui K,et al. A dominant-negative form of mouse SOX2 induces trophectoderm differentiation and progressive polyploidy in mouse embryonic stem cells.J Biol Chem, 2007, 282(27): 19481−19492.

[95] Avilion AA., Nicolis SK, Pevny LH,et al. Multipotent cell lineages in early mouse development depend on SOX2 function.Genes Dev, 2003, 17(1): 126−140.

[96] Chambers I, Colby D, Robertson M,et al. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells.Cell, 2003, 113(5):643−655.

[97] Pan GJ, Thomson JA. Nanog and transcriptional networks in embryonic stem cell pluripotency.Cell Res,2007, 17(1): 42−49.

[98] Darr H, Mayshar Y, Benvenisty N. Overexpression ofNANOGin human ES cells enables feeder-free growth while inducing primitive ectoderm features.Development, 2006, 133(6): 1193−1201.

[99] Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell, 2006, 126(4): 663−676.

[100] Takahashi K, Tanabe K, Ohnuki M,et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell, 2007, 131(5): 861−872.

[101] Yu JY, Vodyanik MA, Smuga-Otto K,et al. Induced pluripotent stem cell lines derived from human somatic cells.Science, 2007, 318(5858): 1917−1920.

[102] Park IH, Zhao R, West JA,et al. Reprogramming of human somatic cells to pluripotency with defined factors.Nature, 2008, 451(7175): 141−146.

[103] Huangfu D, Osafune K, Maehr R,et al. Induction of pluripotent stem cells from primary human fibroblasts with onlyOct4andSox2.Nat Biotechnol, 2008, 26(11):1269−1275.

[104] Kim JB, Sebastiano V, Wu GM,et al. Oct4-induced pluripotency in adult neural stem cells.Cell, 2009,136(3): 411−419.

[105] Kim JB, Zaehres H, Wu GM,et al. Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors.Nature, 2008, 454(7204): 646−650.

[106] Esteban MA, Xu JY, Yang JY,et al. Generation of induced pluripotent stem cell lines from Tibetan miniature pig.J Biol Chem, 2009, 284(26): 17634−17640.

[107] Wu Z, Chen JJ, Ren JT,et al. Generation of pig induced pluripotent stem cells with a drug-inducible system.J Mol Cell Biol, 2009, 1(1): 46−54.

[108] West FD, Terlouw SL, Kwon DJ,et al. Porcine induced pluripotent stem cells produce chimeric offspring.Stem Cells Dev, 2010, 19(8): 1211−1220.

[109] Wolf XA, Rasmussen MA, Schauser K,et al. OCT4 expression in outgrowth colonies derived from porcine inner cell masses and epiblasts.Reprod Domest Anim,2010, DOI: 10.1111/j.1439-053414.2010.01675.X.

[110] Pawar SS, Malakar D, De AK,et al. Stem cell-like outgrowths fromin vitrofertilized goat blastocysts.Indian J Exp Biol, 2009, 47(8): 635−642.

[111] Sanna D, Sanna A, Mara L,et al. Oct4 expression inin-vitro-produced sheep blastocysts and embryonicstem-like cells.Cell Biol Int, 2010, 34(1): 53−60.

[112] Hall VJ, Christensen J, Gao Y,et al. Porcine pluripotency cell signaling develops from the inner cell mass to the epiblast during early development.Dev Dyn,2009, 238(8): 2014−2024.

[113] Blomberg Le A, Schreier LL, Talbot NC. Expression analysis of pluripotency factors in the undifferentiated porcine inner cell mass and epiblast duringin vitroculture.Mol Reprod Dev, 2008, 75(3): 450−463.

[114] Oestrup E, Gjoerret J, Schauser K,et al. Characterisation of bovine epiblast-derived outgrowth colonies.Reprod Fertil Dev, 2010, 22(4): 625−633.

[115] Choi YH, Harding HD, Hartman DL,et al. The uterine environment modulates trophectodermal POU5F1 levels in equine blastocysts.Reproduction, 2009, 138(3):589−599.

[116] Purpera MN, Giraldo AM, Ballard CB,et al. Effects of culture medium and protein supplementation on mRNA expression ofin vitroproduced bovine embryos.Mol Reprod Dev, 2009, 76(8): 783−793.

[117] Chou YF, Chen HH, Eijpe M,et al. The growth factor environment defines distinct pluripotent ground states in novel blastocyst-derived stem cells.Cell, 2008, 135(3):449−461.

[118] Bao SQ, Tang FC, Li XH,et al. Epigenetic reversion of post-implantation epiblast to pluripotent embryonic stem cells.Nature, 2009, 461(7268): 1292−1295.

Pluripotency candidate signaling network and transcription factors in domesticated ungulates: a review

Yuncheng Zhao1,2, Bo Chen1,2, Chuan Zhou1,2, Xiuhua Zhang1,2, and Juncheng Huang1,2

1Key Laboratory of Animal Biotechnology of Xinjiang,Urumqi830000,China
2Key Laboratory of Livestock Reproduction & Biotechnology of MOA,Xinjiang Academy of Animal Science,Urumqi830000,China

Received:July 26, 2010;Accepted:October 20, 2010

Supported by:National High Technology Research and Development Program of China (863 Program) (No. 2008aa101005), High Technology Research and Development Program of Xinjiang Uighur Autonomous Region (No. 200711104), Youth Research Fund of Animal Science Academy in Xinjiang Uighur Autonomous Region (Nos. 2008QJ01, 2010QJ006).

Corresponding author:Juncheng Huang. E-mail: hjc@sina.com

国家高技术研究发展计划 (863计划) (No. 2008aa101005)，新疆维吾尔自治区高技术研究发展计划项目 (No. 200711104)，新疆畜牧科学院青年科研基金 (Nos. 2008QJ01, 2010QJ006) 资助。