趙云程，陳博，周川，張秀華，黃俊成

1 新疆維吾爾自治區(qū)動(dòng)物生物技術(shù)重點(diǎn)開放實(shí)驗(yàn)室，烏魯木齊 830000

2 農(nóng)業(yè)部草食家畜繁育生物技術(shù)重點(diǎn)開放實(shí)驗(yàn)室，烏魯木齊 830000

干細(xì)胞專欄

家畜胚胎干細(xì)胞多能性候選信號(hào)通路及分子標(biāo)志

趙云程1,2，陳博1,2，周川1,2，張秀華1,2，黃俊成1,2

1 新疆維吾爾自治區(qū)動(dòng)物生物技術(shù)重點(diǎn)開放實(shí)驗(yàn)室，烏魯木齊 830000

2 農(nóng)業(yè)部草食家畜繁育生物技術(shù)重點(diǎn)開放實(shí)驗(yàn)室，烏魯木齊 830000

家畜胚胎干細(xì)胞具有重要的生物學(xué)意義和廣闊的應(yīng)用前景。以下對(duì)比了小鼠、人胚胎干細(xì)胞多能性調(diào)控信號(hào)通路的異同，闡述了小鼠、人胚胎干細(xì)胞與家畜胚胎干細(xì)胞在多能性分子標(biāo)志上的差異，并結(jié)合本實(shí)驗(yàn)室開展綿羊胚胎干細(xì)胞研究的實(shí)際經(jīng)驗(yàn)，對(duì)目前家畜胚胎干細(xì)胞建系中可能存在的多能性候選信號(hào)通路及分子標(biāo)志進(jìn)行了探討。

家畜，胚胎干細(xì)胞，多能性，信號(hào)通路，候選基因

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

胚胎干細(xì)胞 (Embryonic stem cell，ESC)，是由哺乳動(dòng)物著床前囊胚期內(nèi)細(xì)胞團(tuán) (Inner cell mass，ICM) 經(jīng)體外特定培養(yǎng)環(huán)境選擇、適應(yīng)后，獲得的具有無(wú)限增殖能力和多向分化潛能的細(xì)胞系，其特定的生物學(xué)特性能夠使其與ICM重新整合，并參與到胚胎發(fā)育的全部過(guò)程中。自1989年采用ESC技術(shù)獲得了第一個(gè)轉(zhuǎn)基因小鼠以來(lái)[1]，ESC技術(shù)在家畜遺傳育種中展現(xiàn)出巨大前景。采用ESC克隆技術(shù)，其整合效率遠(yuǎn)遠(yuǎn)高于傳統(tǒng)核移植技術(shù)，可在短期內(nèi)生產(chǎn)較多的具有遺傳同質(zhì)性的動(dòng)物，免去后裔測(cè)定，大幅度提高良種家畜的繁殖效率。

然而，自 1981年小鼠胚胎干細(xì)胞成功建系以來(lái)，僅有小鼠[2]、大鼠[3-4]獲得了具備生殖系傳遞能力的ES細(xì)胞，極大地制約了ESC技術(shù)在家畜遺傳育種中的研究與應(yīng)用。目前，國(guó)內(nèi)外在家畜ESC的研究中，普遍借助小鼠胚胎干細(xì)胞 (Mouse embryonic stem cell，mESC)、人胚胎干細(xì)胞 (Human embryonic stem cell，hESC) 的成功經(jīng)驗(yàn)，將影響mESC、hESC增殖、分化的因素，如飼養(yǎng)層細(xì)胞、條件培養(yǎng)基、細(xì)胞生長(zhǎng)因子、激素、胎牛血清和血清提取物等進(jìn)行有機(jī)組合，依照mESC、hESC建系標(biāo)準(zhǔn)，直接應(yīng)用到家畜ESC建系中，從而試圖篩選出適合家畜ESC多能性維持的培養(yǎng)條件，然而卻收效甚微。導(dǎo)致家畜ESC建系失敗的原因之一是因?yàn)閙ESC和hESC多能性維持機(jī)制的差別，使得優(yōu)化組合各種因素變得十分困難；比如，mESC分子調(diào)控網(wǎng)絡(luò)中 BMP4 (Bone morphogenetic proteins 4) 與LIF協(xié)同維持其多能性，而在hESC中，BMP4會(huì)誘導(dǎo)hESC發(fā)生分化[5-6]。另外，家畜ESC多能性標(biāo)志的匱乏，是導(dǎo)致建系失敗的另一原因；研究表明mESC、hESC的多能性標(biāo)志，如SSEA-1、SSEA-4、POU5F1、Nanog、堿性磷酸酶活性等，不僅在牛、豬、山羊囊胚的ICM中表達(dá)，同時(shí)在其囊胚的滋養(yǎng)層上有所表達(dá)[7-11]。針對(duì)上述原因，就使得我們不得不考慮家畜 ESC至今未能建系，是因?yàn)樗梃b的mESC、hESC添加因子受體本身就不處于家畜ESC的分子調(diào)控網(wǎng)中？還是這些因子在激活家畜ESC自我更新通路的同時(shí)，又激活了另一個(gè)與之分化有關(guān)的通路？或是因?yàn)槎嗄苄苑肿訕?biāo)志的模棱兩可性，使得我們一再與家畜 ESC“擦肩而過(guò)”？因此，目前許多研究者普遍達(dá)成共識(shí)：家畜ESC的研究必須從多能性標(biāo)志和信號(hào)通路入手[12-14]，借鑒 mESC、hESC多能性維持的分子機(jī)制，了解對(duì)比家畜 ESC與 mESC、hESC多能性調(diào)控機(jī)制的異同，對(duì)家畜ESC研究具有十分重要的作用。

1 ESC多能性維持機(jī)制中的關(guān)鍵信號(hào)轉(zhuǎn)導(dǎo)通路

近年來(lái)，隨著對(duì)mESC、hESC研究的逐步深入，mESC、hESC多能性維持的分子調(diào)控機(jī)制已逐步揭示。在小鼠中，mESC多能性的維持需要通過(guò)LIF-LIFR/gp130-Jak-STAT3途徑和 BMP-Id協(xié)同來(lái)完成，與之不同的是，hESC多能性的維持則需要 Activin/Nodal和 FGF的共同參與。mESC與hESC二者在多能性維持的分子調(diào)控機(jī)制上存在顯著差異。

1.1 LIF-STAT3途徑

白血病抑制因子 (Leukemia inhibitor factor，LIF)屬于白介素6 (IL-6) 細(xì)胞因子家族中的一員，是一種多功能的細(xì)胞因子。目前研究證實(shí)，LIF-LIFR/gp130-Jak-STAT3途徑是 mESC自我更新的重要途徑。對(duì)mESC體外多潛能性的維持，保持未分化狀態(tài)發(fā)揮著重要作用[15-16]。研究發(fā)現(xiàn)，LIF與LIFR結(jié)合后，LIFR與gp130 (glycoprotein 130) 迅速聚合形成異源二聚體激活下游的 Janus 酪氨酸蛋白激酶(Janus-associated tyrosine kinase，JAK)、信號(hào)轉(zhuǎn)導(dǎo)子和轉(zhuǎn)錄激活子3 (Signal transducer and activation of transcription，STAT) 途徑。STAT3的激活是mESC細(xì)胞自我更新的關(guān)鍵[17-18]。缺少IL-6家族成員、撤去MEF，均會(huì)導(dǎo)致STAT3失活和mESC分化；外源LIF因子、血清，活化 STAT3，則足以維持 mESC的多能性[19]。大鼠胚胎干細(xì)胞 (rESC) 研究表明，外源hLIF能有效促進(jìn)rESC集落形成率，因此Buehr推測(cè)，LIF-STAT3應(yīng)該是“真正”意義上ESC的本質(zhì)特性[3]。

在hESC研究中，起初認(rèn)為L(zhǎng)IF對(duì)hESC沒(méi)有效果[20]，或是LIF有利于hESC多能性的維持[21]。隨著研究的進(jìn)一步深入，研究表明，外源hLIF因子能有效使hESC-STAT3的Tyr (705)、Ser (727) 發(fā)生磷酸化 (p-STAT3)，但 p-STAT3并不能維持 hESC多能性[6,22-23]。Daheron等研究發(fā)現(xiàn)，雖然 mLIF與hLIF同源程度較高 (氨基酸序列一致性為78%)，但 mLIF卻表現(xiàn)出種屬特異性，無(wú)法使 hESC的STAT3發(fā)生磷酸化[23]。LIF-STAT3信號(hào)通路在mESC與 hESC上的顯著差異，可能與其各自在胚胎發(fā)育中的不同發(fā)育階段有關(guān)[24]。

近期，Intawicha 報(bào)道，mLIF能有效激活兔類胚胎干細(xì)胞 (ES-like cells) 的LIF-Jak-STAT3途徑，并提高其自我更新能力[25]；而牛 ES樣細(xì)胞研究表明，牛囊胚ICM、及ICM原代培養(yǎng)過(guò)程中的確存在LIFR及gp130信號(hào)轉(zhuǎn)導(dǎo)，但LIF對(duì)牛ES樣細(xì)胞增殖并無(wú)明顯作用[26]。另外，抑制 LIF-STAT3途徑，對(duì)豬外胚層細(xì)胞多能性并無(wú)顯著影響[27]；但 Brevini等研究發(fā)現(xiàn)，豬ES樣細(xì)胞中雖然并不存在LIFR，但LIF卻能有效抑制豬ES樣細(xì)胞類胚體的形成[28-29]。

1.2 MAPK/ERK途徑

研究發(fā)現(xiàn)，LIFR與gp130形成異源二聚體后，gp130除了能激活STAT3自我更新途徑以外，還能激活MAPK/ERK級(jí)聯(lián)反應(yīng)[24]，而Erk-1/2級(jí)聯(lián)反應(yīng)對(duì)mESC的分化具有十分重要的調(diào)控作用[19]，研究表明，Erk-1/2在不同品系小鼠早期胚胎發(fā)育過(guò)程中表現(xiàn)出應(yīng)答水平的不一致性[30]。利用小分子化合物阻斷 ERK-1/2級(jí)聯(lián)反應(yīng)，129Sv/ter、C57BL/6品系mESC建系率分別提高至76.5%和47%[31]，并且可從更多小鼠品系中建立 mESC (如 CBA、MF1、SCID、NOD品系)[32-34]。我們研究發(fā)現(xiàn)，添加FGFR、MEK 特異性抑制劑 (SU5402、PD0325901) 阻斷ERK-1/2分化級(jí)聯(lián)反應(yīng)后，能有效獲得昆明鼠 ES(KM-ES) 細(xì)胞，建系率81.48% (22/27)，KM-ES集落生長(zhǎng)穩(wěn)定，堿性磷酸酶活性顯著提高，分化得到明顯抑制 (圖 1)，有效解決了采用飼養(yǎng)層、血清替代物等常規(guī)培養(yǎng)體系中KM-ES集落分化率高、生長(zhǎng)不穩(wěn)定的問(wèn)題。與之類似的是，BMP4途徑的引入，正是依靠BMP4通過(guò)SMAD 1/5或SMAD 8途徑誘導(dǎo)產(chǎn)生Id蛋白 (Inhibitor of differentiation)，Id蛋白進(jìn)而抑制MAPK級(jí)聯(lián)反應(yīng)，并與LIF/STAT3協(xié)同維持mESC的多能性[35-37]。Li等采用FGF受體酪氨酸激酶和ERK-1/2級(jí)聯(lián)反應(yīng)特異性抑制劑SU5402和PD0325901，阻斷MAPK/ERK途徑后，成功建立了rESC系[4]。STAT3與ERK-1/2途徑相互協(xié)調(diào)，對(duì)維持mESC、rESC細(xì)胞的自我更新和分化之間的平衡具有重要作用。

圖1 KM-ES堿性磷酸酶活性檢測(cè)Fig.1 Alkaline phosphatase staining for KM-ES.

與mESC不同的是，hESC未分化狀態(tài)下則保持了較高的ERK活性，MAPK/ERK途徑對(duì)hESC的凋亡、增殖和分化過(guò)程具有一定調(diào)節(jié)作用[38]。研究表明，堿性成纖維細(xì)胞生長(zhǎng)因子 (Basic fibroblast growth factor，bFGF) 是hESC自我更新機(jī)制中的核心調(diào)控因子[39-40]，bFGF與細(xì)胞表面受體結(jié)合后，激活胞內(nèi)MAPK/ERK級(jí)聯(lián)反應(yīng)，對(duì)hESC多能性維持起到十分重要的調(diào)控作用[41-43]。目前研究發(fā)現(xiàn)，在未分化的 hESC中均能檢測(cè)到 FGFR-1、FGFR-2、FGFR-3、FGFR-4的表達(dá)，而以 FGFR-1的表達(dá)量最高[44-47]。采用 FGFR特異性抑制劑SU5402研究發(fā)現(xiàn)，F(xiàn)GFR受到抑制的條件下，hESC將會(huì)迅速發(fā)生分化[41]。hESC多能性的維持表現(xiàn)出對(duì)FGF的量性需求：當(dāng)bFGF添加量在4 ng/mL時(shí)，hESC的維持需要有滋養(yǎng)層細(xì)胞[20]；當(dāng)bFGF添加量達(dá)到8～40 ng/mL時(shí)，培養(yǎng)系統(tǒng)中不再需要飼養(yǎng)層細(xì)胞，而需要Noggin的參與 (Noggin為BMP4的拮抗蛋白)[41,48-49]；當(dāng)bFGF達(dá)到100 ng/mL時(shí)，hESC可處于不分化狀態(tài)[50]。采用MEK/ERK信號(hào)級(jí)聯(lián)反應(yīng)抑制劑PD98059、U0126，會(huì)迅速導(dǎo)致hESC發(fā)生分化[51]，這與mESC、rESC多能性調(diào)控機(jī)制存在顯著差別。

目前研究發(fā)現(xiàn)，家畜ES樣細(xì)胞與hESC更為相似[14]，bFGF能有效促進(jìn)豬ES樣細(xì)胞的原代集落形成率及自我更新[52-53]，并對(duì)兔ES樣細(xì)胞多能性的維持具有一定作用[54]。

1.3 Wnt途徑

近年來(lái)，研究表明Wnt途徑在mESC與hESC多能性維持中發(fā)揮著十分重要的作用，對(duì)ESC向皮膚、神經(jīng)系統(tǒng)、血液系統(tǒng)的分化起到調(diào)控作用[55]，與 LIF-STAT3、BMP4途徑不同的是，Wnt途徑在mESC與 hESC的自我更新中作用機(jī)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，解除對(duì)β連環(huán)蛋白的磷酸化，維持ESC的自我更新[57]。mESC、hESC本身可以自發(fā)激活Wnt途徑，而當(dāng)分化通路啟動(dòng)后，Wnt的表達(dá)量呈現(xiàn)下調(diào)趨勢(shì)[58]。采用GSK-3的特異性抑制劑 6-bromoindirubin-3′-oxime (BIO)，可以特異性抑制GSK-3的磷酸化活性，導(dǎo)致核內(nèi)β連環(huán)蛋白含量增高，從而激活 Wnt的下游信號(hào)轉(zhuǎn)導(dǎo)途徑，最終使得 mESC、hESC的多能性狀態(tài)得到維持[59]。Wnt的激活能夠調(diào)高c-Myc的水平，而c-Myc則是STAT3的靶位基因，表明Wnt途徑與LIF-STAT3協(xié)同作用于c-Myc，共同維持mESC的多能性[60-62]。

雖然，Wnt途徑在mESC、hESC多能性維持過(guò)程中的作用基本一致，但家畜ESC研究發(fā)現(xiàn)，采用Wnt途徑激活劑 (BIO、Wnt3a) 并不能阻止外胚層細(xì)胞的分化[63]。

1.4 TGFβ/Activin/Nodal途徑

TGFβ是一個(gè)大的超家族，超過(guò)40個(gè)成員，包括 TGFβ、Activin、Nodal和 BMP 等[56,64]。BMPs是TGFβ超家族中最大的成員，它在促進(jìn)ESC自我更新中作用不明顯，但是可以通過(guò)SMAD途徑激活I(lǐng)d的表達(dá)，而Id蛋白則能夠抑制神經(jīng)發(fā)生轉(zhuǎn)錄因子bHLH的表達(dá)，從而最終抑制向神經(jīng)系統(tǒng)的分化[65-66]；外源性Id蛋白將會(huì)模擬BMP4的生理學(xué)特性，維持mESC的多能性[65]。但是，BMP4在維持mESC細(xì)胞多能性的作用，需要在 LIF的存在下才能實(shí)現(xiàn)，這是因?yàn)锽MP4能誘導(dǎo)ESC細(xì)胞向內(nèi)胚層和中胚層分化，而LIF通過(guò)LIF-STAT3途徑抑制了BMP4的這一促分化作用，但同時(shí)對(duì)BMP4誘導(dǎo)的Id蛋白表達(dá)沒(méi)有作用，從而與LIF-STAT3共同維持mESC細(xì)胞的多能性[65]。與mESC自我更新維持機(jī)制截然相反，BMP4并不能維持hESC的自我更新，相反會(huì)使得 hESC向滋養(yǎng)層細(xì)胞和原始內(nèi)胚層細(xì)胞分化[5]，hESC多能性需要 BMP4的拮抗物 Noggin與bFGF配合才能得以維持[49]。此外，TGFβ超家族成員Activin A、Nodal在未分化的hESC中表達(dá)量較高[67]。hESC研究表明，Smad 2/3參與了 TGFβ/Activin/Nodal的信號(hào)轉(zhuǎn)導(dǎo)，在未分化過(guò)程中 Smad 2/3被激活，而Smad 1/5處于抑制狀態(tài)，隨著分化發(fā)生，Smad 2/3信號(hào)減弱，而Smad 1/5信號(hào)增強(qiáng)[68-69]。特異性小分子抑制劑BIO，可以模擬Wnt途徑從而保持hESC的未分化狀態(tài)，而此時(shí)Smad 2/3磷酸化水平仍然處于較高水平[69]。

TGFβ家族成員對(duì)mESC、hESC多能性的維持具有十分重要的作用，但其對(duì)家畜ESC的調(diào)控作用卻并不十分清楚。Pant等研究發(fā)現(xiàn)，添加Noggin抑制BMP4途徑后，牛ICM原代集落Nanog表達(dá)量顯著上調(diào)[70]。同時(shí)，Alberio等研究發(fā)現(xiàn)，添加BMP4將會(huì)導(dǎo)致豬外胚層細(xì)胞向滋養(yǎng)層細(xì)胞與生殖細(xì)胞方向分化；抑制Activin/Nodal途徑，豬外胚層細(xì)胞迅速分化為神經(jīng)細(xì)胞，因此提出Activin/Nodal信號(hào)途徑是在哺乳動(dòng)物細(xì)胞多能性維持過(guò)程中起調(diào)控作用的保守途徑[27]。而另有研究表明，添加 Activin及Noggin并不能阻止豬、馬外胚層細(xì)胞的分化，激活A(yù)ctivin途徑或抑制BMP4活性，對(duì)家畜ESC多能性的維持并無(wú)明顯作用[63]。

1.5 PI3K/AKT途徑

磷脂酰肌醇3激酶 (PI3K) 是一種脂質(zhì)激酶，對(duì)細(xì)胞的增殖、生長(zhǎng)、發(fā)育、遷移及細(xì)胞周期等生理活動(dòng)具有十分重要的調(diào)控作用[71-72]。PI3K/AKT途徑處于LIF、bFGF信號(hào)下游，在mESC中，當(dāng)PI3K的負(fù)調(diào)控基因PTEN缺失后，將會(huì)促進(jìn)細(xì)胞周期由G1期向S期的轉(zhuǎn)變，從而使得mESC增殖加速[72]。添加 PI3K特異性抑制劑，將會(huì)激活 LIF介導(dǎo)的MAPK/ERK途徑，降低LIF對(duì)mESC自我更新的調(diào)控作用，使得mESC增殖減慢[73-74]，同時(shí)編程性死亡發(fā)生率升高[75]。而hESC研究表明，PI3K/AKT與MAPK/ERK途徑，并不存在交匯作用，二者對(duì)hESC自我更新的維持存在疊加效應(yīng)[51]；阻斷 PI3K/AKT途徑，將會(huì)影響 hESC增殖，使編程性死亡發(fā)生率升高[51]。此外，研究發(fā)現(xiàn)采用PI3K特異性抑制劑，阻斷PI3K途徑后，將會(huì)增加早期附植胚胎編程性死亡的發(fā)生率[75]。

同時(shí)研究表明，PI3K/AKT途徑對(duì)ESC多能性的維持作用顯著。在添加LIF及飼養(yǎng)層細(xì)胞條件下，抑制PI3K活性將會(huì)導(dǎo)致mESC、hESC發(fā)生分化，表明PI3K/AKT信號(hào)途徑對(duì)ESC (如：小鼠、猴、人)多能性狀態(tài)的維持具有十分重要的調(diào)控作用[73-74,76-78]。Storm等研究發(fā)現(xiàn)，抑制PI3K/AKT途徑將會(huì)導(dǎo)致包括Nanog和Zscan4家族在內(nèi)的646種基因的表達(dá)量發(fā)生改變，而其中Zscan4家族基因并未與Nanog基因關(guān)聯(lián)[79-81]；此外研究表明，二細(xì)胞早期胚胎及ESC具備較高水平的Zscan4，下調(diào)Zscan4基因?qū)?huì)導(dǎo)致囊胚無(wú)法附植[82]，因此 PI3K/AKT途徑可能通過(guò)Nanog與Zscan4兩種方式調(diào)控mESC的多能性[79]。

目前研究表明，PI3K/AKT途徑對(duì)兔ES樣細(xì)胞多能性的維持具有十分重要的調(diào)控作用，與 hESC類似，兔ES樣細(xì)胞自我更新過(guò)程中PI3K/AKT途徑與 MAPK/ERK 途徑并未存在交匯作用[83]；Brevini等研究發(fā)現(xiàn)，豬ES樣細(xì)胞雖然不存在LIFR，但LIF可能通過(guò)PI3K/AKT途徑，抑制豬ES樣細(xì)胞類胚體的形成，從而維持豬ES樣細(xì)胞的多能性[28-29,84]。

針對(duì)上述與mESC、hESC多能性密切相關(guān)的信號(hào)通路，我們?cè)诰d羊ESC研究過(guò)程中，通過(guò)對(duì)6～8 d囊胚ICM原代集落機(jī)械法傳代后，添加LIF-STAT3、Wnt與 Noggin信號(hào)途徑的有效激活因子 (mLIF、hLIF、Wnt3a、Noggin)，但結(jié)果表明綿羊ES樣集落周邊細(xì)胞呈彌散式生長(zhǎng)，堿性磷酸酶活性降低，集落逐步呈平鋪式生長(zhǎng)，界限模糊 (圖 2)，初步表明上述因子并不能有效阻止綿羊ES樣細(xì)胞發(fā)生分化。

圖2 mLIF、hLIF、Wnt3a、Noggin并不足以維持綿羊類胚胎干細(xì)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多能性候選基因

目前，制約家畜ESC研究的一個(gè)核心關(guān)鍵問(wèn)題是缺乏家畜ESC的多能性標(biāo)志，這也直接導(dǎo)致研究過(guò)程中無(wú)法及時(shí)有效地篩選出“真正”意義上的家畜ESC。近年來(lái)，研究表明POU5F1(POU domain 5 transcript factor 1)、Sox2(SRY-box containing gene 2)和Nanog等轉(zhuǎn)錄因子對(duì)mESC、hESC的自我更新和分化具有十分重要的作用，當(dāng)它們表達(dá)時(shí)，ESC的自我更新途徑被激活，分化途徑受到抑制[85-87]，三者彼此調(diào)控，同時(shí)對(duì)上述影響ESC自我更新和分化的外源信號(hào)分子作出應(yīng)答，嚴(yán)格控制著ESC的自我更新與分化進(jìn)程，最終形成了ESC多能性機(jī)制的調(diào)控中樞——POU5F1、Sox2、Nanog[88-89]。

POU5F1又稱為OCT-4[90]，是植入前胚胎發(fā)育的重要調(diào)節(jié)因子，特異性地表達(dá)于多種多能性細(xì)胞：卵母細(xì)胞、原始生殖細(xì)胞、早期植入前胚胎、原始外胚層、ICM和ESC細(xì)胞[91-92]，是mESC和hESC細(xì)胞的多能性分子標(biāo)志[91-93]。目前，多種POU5F1的靶位基因已經(jīng)得到確認(rèn)，包括Fgf4、Utfl、Opn、Rexl/Zfp42、Fbxl5和Sox2。其中，Sox2在小鼠早期胚胎發(fā)育過(guò)程中起到十分重要的作用，不僅在小鼠早期胚胎中表達(dá)，同時(shí)會(huì)在胚外外胚層的多能性細(xì)胞中表達(dá)，Sox2表達(dá)量降低將導(dǎo)致mESC向滋養(yǎng)外胚層分化和多倍體細(xì)胞的出現(xiàn)[94]。Sox2-/-突變使小鼠早期胚胎不能形成外胚層并引起胚胎死亡[95]，表明Sox2是維持小鼠早期胚胎發(fā)育所必需的轉(zhuǎn)錄因子。Sox2與POU5F1協(xié)同作用，共同阻止ESC向滋養(yǎng)外胚層分化，同時(shí)阻止染色體異常[88]。Nanog基因是ESC研究中發(fā)現(xiàn)的另一個(gè)多能性主導(dǎo)基因，它不僅對(duì)胚胎發(fā)育過(guò)程中ICM多能性的調(diào)控起關(guān)鍵作用，同時(shí)還可維持外胚層細(xì)胞多能性和阻止其向原始內(nèi)胚層的分化[96]，可在ESC、EG (Embryonic germ)和EC (Embryonic carcinoma) 等多能性細(xì)胞中檢測(cè)到[96-97]，是維持mESC、hESC多能性的關(guān)鍵轉(zhuǎn)錄因子[98]。研究表明，雖然mESC與hESC在形態(tài)學(xué)、表面標(biāo)志和生長(zhǎng)因子方面存在顯著差別，但是它們的Nanog基因都十分保守[97]。

此外，誘導(dǎo)多能干細(xì)胞 (Induced pluripotent stem cells，iPS) 的出現(xiàn)及后期“Yamanaka因子”(POU5F1、Sox2、c-Myc、Klf4) 功能的逐漸明朗，為家畜ESC多能性候選基因的選擇提供了新的參考依據(jù)。2006年，Yamanaka采用逆轉(zhuǎn)錄病毒將POU5F1、Sox2、c-Myc、Klf4導(dǎo)入小鼠胚胎成纖維細(xì)胞或成年小鼠尾部皮膚成纖維細(xì)胞中，建立了與mESC非常相似的iPS細(xì)胞[99]。之后，Yamanaka將上述 4個(gè)轉(zhuǎn)錄因子導(dǎo)入到人皮膚成纖維細(xì)胞中，也成功獲得了iPS細(xì)胞[100]。與此同時(shí)，Thomson研究小組也報(bào)道了成功誘導(dǎo)胎兒成纖維細(xì)胞轉(zhuǎn)化為具有hESC基本特征的人iPS細(xì)胞，所不同的是他們使用慢病毒作為載體，選擇了POU5F1、Sox2、Nanog、Lin28等4個(gè)基因[101]。Park等發(fā)現(xiàn)POU5F1和Sox2在誘導(dǎo)重構(gòu)為 iPS細(xì)胞過(guò)程中是必需的，正是這 2個(gè)轉(zhuǎn)錄因子維持了人類iPS細(xì)胞的多潛能性，而Klf4和c-Myc的作用是改變?nèi)旧|(zhì)的結(jié)構(gòu)，利于POU5F1和Sox2的結(jié)合，以提高誘導(dǎo)效率[102]，從而進(jìn)一步明確了iPS多潛能性誘導(dǎo)過(guò)程中POU5F1、Sox2的重要作用。Huangfu等采用組蛋白脫乙酰基酶抑制劑，將POU5F1和Sox2基因?qū)氲饺祟惼つw成纖維細(xì)胞中，也成功獲得了iPS細(xì)胞[103]。Kim等將OCT4與Sox2或OCT4轉(zhuǎn)錄因子導(dǎo)入到小鼠神經(jīng)干細(xì)胞中，成功獲得了iPS細(xì)胞[104-105]。iPS相關(guān)技術(shù)在家畜iPS研究中的應(yīng)用，為家畜ESC多能性候選基因的選擇提供了參考依據(jù)。研究表明，采用iPS技術(shù)，導(dǎo)入包括POU5F1、Sox2、Nanog在內(nèi)的多個(gè)基因，可以獲得與 hESC各項(xiàng)生物學(xué)特性極為相似的豬多能性干細(xì)胞[106-107]，并已有通過(guò)iPS細(xì)胞獲得嵌合體豬的報(bào)道[108]。

目前的家畜ESC研究表明，mESC、hESC多能性分子標(biāo)志POU5F1、NANOG、SOX2在家畜多能性鑒定中應(yīng)當(dāng)被謹(jǐn)慎使用[12]。比如，POU5F1、NANOG、SOX2基因除在牛、豬、山羊囊胚的ICM表達(dá)外，在滋養(yǎng)層細(xì)胞、內(nèi)胚層細(xì)胞中同樣表達(dá)[7-8,12,109-110]。值得注意的是，上述多能性分子標(biāo)志在ICM與滋養(yǎng)層細(xì)胞中的定位存在差別，Pant等研究發(fā)現(xiàn)：NANOG與POU5F1在牛ICM與滋養(yǎng)層細(xì)胞的核仁中表達(dá)，而NANOG除在ICM核仁表達(dá)外，在ICM核質(zhì)中也有所表達(dá)[70]。近期研究表明POU5F1、NANOG的 mRNA及其編碼的蛋白在山羊、綿羊ICM表達(dá)，而在囊胚滋養(yǎng)層細(xì)胞中mRNA表達(dá)量顯著降低[9,111]；Hall等通過(guò)對(duì)11 d豬胚胎外胚層與滋養(yǎng)層細(xì)胞對(duì)比研究，發(fā)現(xiàn)POU5F1、Nanog、SOX2、FGFR1在外胚層細(xì)胞中的表達(dá)是特異的[112]；且牛、豬 ICM與外胚層細(xì)胞發(fā)生明顯分化前，POU5F1、NANOG、SOX2的表達(dá)量發(fā)生明顯變化[70,113-114]。上述相關(guān)研究成果為家畜ESC多能性標(biāo)識(shí)的選擇提供了一定依據(jù)。此外，值得注意的是，家畜胚胎POU5F1、NANOG、SOX2的基因表達(dá)量還受到外源環(huán)境的調(diào)控，比如Chio等研究發(fā)現(xiàn)，與體外胚胎相比，馬體內(nèi)胚胎 ICM 中POU5F1、SOX2、NANOG的表達(dá)量顯著高于滋養(yǎng)層細(xì)胞[115]；另外，胚胎體外培養(yǎng)過(guò)程中，培養(yǎng)液的選擇 (KSOM 與 SOF) 同樣會(huì)對(duì)囊胚中上述相關(guān)候選基因的表達(dá)量產(chǎn)生一定調(diào)控作用[116]。因此，上述候選基因雖然能夠作為家畜ESC多能性分子標(biāo)識(shí)使用，但仍需進(jìn)一步加以確認(rèn)，因此與小鼠Nanog相區(qū)別，家畜ESC的NANOG基因則用大寫斜體字母表示。

3 結(jié)語(yǔ)

雖然家畜ESC研究已開展了20多年，但目前尚無(wú)實(shí)質(zhì)性突破。因此比對(duì) mESC、hESC多能性分子調(diào)控網(wǎng)絡(luò)的差異，借鑒mESC、rESC的成功經(jīng)驗(yàn)，研究家畜ES樣細(xì)胞生物學(xué)特性，對(duì)獲得生殖系傳遞能力的家畜ESC具有十分重要的指導(dǎo)意義；此外，隨著mESC研究的逐步深入，一系列與mESC生物學(xué)特性極為相似的細(xì)胞 (如FAB-SC、EpiSC細(xì)胞) 逐漸被發(fā)現(xiàn)，研究表明，這些類型的細(xì)胞可以通過(guò)簡(jiǎn)單的培養(yǎng)條件的轉(zhuǎn)換從而具備mESC多能性的生物學(xué)特性[117-118]，因此對(duì)比家畜 ES樣細(xì)胞與FAB-SC、EpiSC的生物學(xué)特性差異，將會(huì)為家畜ES樣細(xì)胞向多能性方向的轉(zhuǎn)變提供新的技術(shù)途徑；另外，借助iPS研究成果，研究家畜iPS細(xì)胞生物學(xué)特性及多能性維持調(diào)控途徑，將會(huì)對(duì)分離、培養(yǎng)、鑒定具備生殖系傳遞能力的家畜 ESC具有十分重要的指導(dǎo)意義。

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] Mu?oz M, Díez C, Caama?o 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] B?ttcher 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, Sch?ler 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, Sch?ler HR. In line with our ancestors: Oct-4 and the mammalian germ.Bioessays,1998, 20(9): 722?732.

[92] Sch?ler 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

國(guó)家高技術(shù)研究發(fā)展計(jì)劃 (863計(jì)劃) (No. 2008aa101005)，新疆維吾爾自治區(qū)高技術(shù)研究發(fā)展計(jì)劃項(xiàng)目 (No. 200711104)，新疆畜牧科學(xué)院青年科研基金 (Nos. 2008QJ01, 2010QJ006) 資助。