鸡胚胎生殖细胞多向分化调控的研究
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摘要
与哺乳动物相比,鸡胚具有易于获取和操作、结合病毒转染和电转移可进行转基因操作等优势,因而常被作为发育生物学和比较医学研究的模型动物。尤其是近年来有关鸡胚各种组织器官发育分子机理的研究进展较快,这为鸡胚更好的作为模型动物奠定了一定的基础。为了更深层次揭示细胞发育和分化的机理,在内源性调节的基础上力图通过外源性调控,在体外获得干细胞来源、可用于组织功能修复的组织和器官,本实验以艾维因鸡胚为材料,分离了胚胎期原始生殖细胞(primordial germ cell, PGC)并通过传代培养获得了大量胚胎生殖(embryonic germ cell, EG)细胞,建立了EG细胞体内形成畸胎瘤及体外形成类胚体(embryoid body, EB)的模型,探讨了多种因素对EG细胞体外多向分化的调控,研究了EG细胞体外向成熟神经细胞、脂肪细胞、肝细胞和心肌细胞分化的潜能。
1.鸡胚胎生殖细胞分离和培养模型的建立
在体视显微镜下用玻璃细针分离3.5-4日胚龄鸡胚生殖嵴部位,用胰蛋白酶-EDTA消化分散组织,然后将分散的细胞进行培养以建立PGC的原代培养模型。所用的高糖DMEM培养液中添加5%胎牛血清(FCS)、10ng/ml白血病抑制因子(LIF)、10ng/ml碱性成纤维生长因子(bFGF)、10ng/ml干细胞生长因子(SCF), 0.1mmol/L MEM非必需氨基酸、0.1mmol/Lβ-巯基乙醇、2mmol/L L-谷氨酰胺(Gln)、100IU/ml青霉素和100μg/ml链霉素(完全培养液)。与体细胞共培养5d后,利用RT-PCR方法检测PGC特异性基因Cvh、Dazl和DEH在原代培养形成的PGC集落中的表达。将表达PGC特异基因的集落进行传代培养,然后用过碘酸雪夫氏反应(PAS)、SSEA-1和SSEA-3及SSEA-4免疫组化、和干细胞多能性相关基因PouV、Nanog和Sox2 RT-PCR检测均证实传代后形成的集落为EG阳性克隆。上述结果表明,PGC-体细胞原代及传代共培养并添加多种因子的条件下,PGC在体外可转变为EG细胞,这为研究生殖干细胞的分化机制提供了细胞来源。
2.鸡胚胎生殖细胞体内形成畸胎瘤
通过灌饲具有免疫抑制作用的药物环孢素A (CsA,5mg/kg)制作免疫抑制雏鸡,将含有1.0×106个鸡EG细胞的PBS注射到受到免疫抑制的雏鸡皮下,观察其分化情况。5周后取皮下形成的组织瘤进行切片和HE染色及免疫组化染色鉴定。结果表明,EG细胞在免疫缺陷的雏鸡皮下可自发分化为含有神经细胞、软骨细胞、脂肪细胞、血管、骨细胞、平滑肌、横纹肌和腺上皮细胞等三个胚层来源的多种细胞类型的畸胎瘤。而注射同样数量EG细胞到未饲喂环孢素A组(模型对照组)和注射不含有EG细胞的免疫抑制雏鸡组(细胞对照组)均未观察到组织瘤的形成。上述结果表明通过饲喂免疫抑制作用的药物环孢素A可制作免疫抑制雏鸡模型,本实验利用此模型证明了鸡EG细胞在体内具有多向分化为含有三个胚层来源细胞畸胎瘤的潜能,这为开展生殖干细胞体内分化潜能的研究提供了实验平台。
3.鸡胚胎生殖细胞体外形成EB模型的建立与优化
将EG克隆与体细胞进行胰酶消化,并利用差速贴壁方法富集EG细胞。然后无饲养层、无LIF和β-巯基乙醇条件下将鸡EG细胞接种到涂有1%琼脂的培养板中悬浮培养形成EB。形成的EB进行丫啶橙(AO)染色后在荧光共聚焦显微镜下观察其内部结构。对形成7d和14d的类胚体分别进行切片和HE组织学染色,并通过RT-PCR检测内胚层标志基因AFP、外胚层标志基因Sox3、中胚层标志基因GATA6和滋养层标志基因Cdx2在其中的表达。结果表明：将富集的鸡EG单细胞采用不含LIF的培养液进行悬浮培养4-7d可形成简单EB,继续悬浮培养则其内部形态也发生改变,最终形成囊状EB。不同时间点的EB切片显示随着时间的变化,EB中出现含有神经细胞、脂肪细胞和胰腺细胞等三个胚层的细胞类型。此外,在体外悬浮培养时比较了FCS和Gln对EB形成的影响。统计显示,随着悬浮培养时间的延长,EB的面积不断增大,FCS浓度和Gln添加量对EB的形成产生影响显著,其中以含15%FCS、2 mmol/L Gln的培养液中EB的形成数量和状态较好。同时,在EG细胞体外分化形成EB的第0d、3d、5d和10d分别检测多能性相关基因转录调控因子如PouV、Nanog和Sox2 mRNA的表达。结果显示：在EB体外分化过程中,调控干细胞多能性和自我更新的三个重要转录调控因子PouV、Nanog和Sox2表达量均呈明显下调趋势,提示PouV、Nanog和Sox2在维持鸡EG细胞多潜能性中起着重要作用。上述结果表明,鸡胚EG细胞通过悬浮培养方法可制备具有向三个胚层分化能力的EB,此结果为研究EG细胞体外定向诱导分化为特定的细胞类型奠定了实验基础。
4.鸡胚EG细胞体外多向分化调控的研究
在贴壁培养条件下,利用含10-6 mol/L RA的ITS诱导液可将EG细胞定向诱导分化为表达成熟神经元、星型胶质细胞和小胶质细胞特异蛋白及基因的神经细胞。部分神经元经免疫组化及RT-PCR方法鉴定,表明其具有合成多巴胺和胆碱能神经递质的功能。利用10μg/ml胰岛素、1μmol/L地塞米松和0.2 mmol/L吲哚美辛联合处理,可诱导EG细胞定向分化为含有大量脂滴并表达脂肪细胞特异性基因PPARγ, GLUT1和LPL的成熟脂肪细胞。利用与肝细胞条件培养联合培养的方式可定向诱导EG细胞分化为肝细胞,利用PAS染色、免疫细胞化学、RT-PCR方法鉴定EG诱导来源的肝细胞,提示EG细胞具有分化为含有糖原并分泌白蛋白功能的成熟的肝细胞。利用添加去甲基化试剂5-aza的培养液悬浮培养,可将EG细胞诱导分化为心肌细胞,表明5-aza具有显著提高形成规律性收缩EB比例的作用,其中以添加5μmol／L 5-aza组形成的比例最高。形成的EB经贴壁分化培养后,采用透射电镜观察、荧光免疫组化及RT-PCR方法鉴定EG细胞来源的心肌细胞,结果显示5-aza可诱导EG细胞分化为含有肌小结和缝隙连接、表达心肌细胞特异性蛋白和标志基因的心肌细胞。
以上实验结果表明：从鸡胚生殖嵴分离的PGC与体细胞原代共培养并经传代所建立的培养模型可用于EG细胞制备和分化调控的研究,经PAS组化、SSEA-1和SSEA-3及SSEA-4免疫细胞化学染色、干细胞多能性相关基因转录调控因子PouV、Nanog和Sox2的RT-PCR法检测、在免疫抑制雏鸡体内形成含有三个胚层来源细胞的畸胎瘤等多种方法均证实了EG细胞的干细胞特性。将富集的鸡EG细胞悬浮培养形成简单EB和囊状EB,培养液中添加15%FCS和2mmol／L Gln可促进EB的形成。在EB体外分化过程中,PouV Nanog和Sox2表达量均呈明显下调趋势,而三个胚层的标志基因AFP.Sox3和GATA6及滋养层标志基因Cdx2出现表达,表明EG细胞通过悬浮培养方法可制备具有向三个胚层分化能力的EB。贴壁培养的EB经不同的诱导剂处理后,经细胞特异性的标志基因或蛋白的表达鉴定,EB可定向分化成有功能的神经细胞、脂肪细胞、肝细胞及心肌细胞。以上结果提示利用所建立的EG／EB体外制备和定向诱导分化模型可用于生殖干细胞发育调控机制的研究,EG细胞可诱导分化成三个胚层的细胞,这为禽类干细胞发育生物学和比较医学的研究提供了实验平台和理论基础。
Although the chick embryo has long been used as a model for developmental biology, its potential use as an experiment model for the repair and regeneration of adult tissues is often overlooked. This model has several advantages over mammalian systems for in vivo studies, as it is cost-effective, easily manipulated, and can be used for transgenesis in conjunction with viral vectors or electroporation. The molecular mechanisms underlying the development of nearly all major organ systems have been elucidated in the chick, making data interpretation more complete within this knowledge landscape. In this study, embryonic germ (EG) cells were derived from primordial germ cells (PGCs) of genital ridges of 3.5-4-days-old chicken embryos. These cells satisfied the criteria previously used for defining chicken EG cells by using the expression of markers characteristic to ES cells. When injected subcutaneously, chicken EG cells could form teratoma that enable differentiation into a wide range of tissue types of all three primary cell lineages including neural cells, cartilage, forming bone, adipocytes, blood vessels, smooth muscle, striated muscle and secretory epithelia in recipient. Furthermore, cells in embryoid bodies (EBs) expressed lineage-specific markers of three germ layers and could be induced to differentiate into more advanced stages of various committed cell types, including dopamine and cholinergic neurons, astrocytes, oligodendrocytes, adipocytes, cardiomyocytes and hepatocytes, that were demonstrated by immunocytochemical staining or RT-PCR analysis. These findings support the multiple differentiation capability of chicken pluripotent EG cells, thus confirming the presumption that chicken embryos may used as a potential model for better understanding the mechanisms of tissue-specific differentiation and regeneration, that will help to devise strategies based on the transplantation of stem cell-derived tissues for restoring function to damaged or diseased tissues.
1. Preparation of of chicken EG cells and establishment of the culture models
Genital ridges were collected by dissection of chicken embryos at 3.5-4 days with a fine glass needle under a microsurgery scope. For primary culture, cell suspension containing both PGCs and somatic cells was seeded onto gelatin-treated 35 mm culture plates at a density of 1×106/well in DMEM supplemented with 5% fetal calf serum (FCS), 10ng/ml leukemia inhibitory factor (LIF), 10ng/ml human basic fibroblast growth factor (bFGF), 0.1mmol/L MEM nonessential amino acids, O.lmmol/L 2-mercaptoethanol,2mmol/L L-glutamine (Gln), 100U/ml penicillin and 100μg/ml streptomycin. The seeded cells were then maintained at 38.5℃in 5% CO2/95% air with 60%-70% relative humidity until the PGCs colonized as a primary culture. To trace the origin of the colonies, the primary formed colonies were picked up with a fine glass needle, dissociated with 0.25% trypsin-EDTA 5 days after plating and then subjected to RT-PCR analysis for expression of PGC-specific markers. For further subculture, colonies that were positive for PGC markers were picked up and treated with 0.25% trypsin-EDTA to achieve single cell suspension and reseeded onto 6-well dishes. After three passages, staining of periodic acid-Schiff regent (PAS), stage-specific embryonic antigens (SSEA-1, SSEA-3 and SSEA-4) immunocytochemistry, and the expression of the pluripotency-associated genes cPouV, cNanog and Sox2 analysis all confirmed the characteristics of cultured EG cells. The above results indicated that the primary and subculture models of PGCs could be used for obtaining EG cells which represents a source of EG cells for studies about differentiation of germline stem cells (GSC).
2. Teratoma formation of chicken EG cells in vivo
Seven-day-old chicks were obtained from a commercial hatchery and fed with cyclosporine (5 mg/kg BW) known as an immunosuppressive agent to dampen down the body's immune reaction. EG colonies were collected and treated with 0.25% trypsin-EDTA to achieve single cell suspension. About 1×106 cells in 200μl PBS were injected subcutaneously into axilla region of the chicks. After 5 weeks, chicks were sacrificed and emerging tissue materials were dissected. Tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Sections (5μm) were placed on slides for HE and irnmunohistochemical staining. Results showed that chicken EG cells were capable of forming teratomas after subcutaneous injection into the immunosuppressed chicks. The amount of differentiated tissues varied among individual teratomas. Each teratoma contained a broad variety of tissues, including neural cells, cartilage, forming bone, adipocytes, blood vessels, smooth muscle, striated muscle and secretory epithelia. There was no teratoma formed in chicks without immunosuppression or no EG cells injection. The above results indicated that the immunodeficiency chick model could be made by fed with immunosuppressive agents cyclosporine, and the present study using this chick model showed that the chicken EG cells could form teratomas containing a variety of differentiated tissues and cell types, as well as some patches of undifferentiated cells in vivo.
3. Formation of EBs from chicken EG cells in vitro
EG colonies in culture were picked up and dissociated into single cells, transferred onto gelatin-coated plates for a 30 min period to remove contaminating somatic cells and then transferred onto Ultra Low Attachment plates to allow their aggregation and prevent adherence to the plate. Usually about 106 EG cells were incubated in each 35 mm plate accompanied by the withdrawal of LIF and 2-mercaptoethanol from the medium. During this period, the cells grew into compact aggregates and became simple EBs within 2-5 days, especially more easily accompanied by 15% FCS and 2 mmol/L Gln, then turned to cystic EBs with the formation of a central cavity. Inner structure of EBs were observed by acridine orange staining or HE staining of the EBs sections on Day 7 and Day 17 of suspension culture respectively. HE staining analysis of EB sections showed heterogeneous structure with the ability to differentiate into multiple cell types, including neural tube like structure, vascular structures, connective tissues, and glandular-like cells. Furthermore, expression of the lineage-specific markers of three germ layers, including endoderm specific gene characterized by AFP, mesoblast specific gene characterized by Gata6, ectoderm specific gene characterized by Sox3 and the trophectoderm specific gene characterized by Cdx2 was detected in EBs, suggesting their differentiation potential in vitro. In addition, we demonstrated the expression profiles of pluripotency-associated markers, including chicken Oct4 homologue PouV (cPouV), chicken Nanog (cNanog) and Sox2 genes during the formation of EBs, which were required for the maintenance of pluripotency in ES cells. All of these genes were expressed in undifferentiated EG cells, and down regulated during the formation of EBs, suggesting their presumable effects in the inhibition of chicken EG cells differentiation. The above results demonstrate the formation potential of EG cells into EBs, which will facilitate the establishment of the differentiation model of EG cells in vitro.
3. Multipledifferentiation regulation of chicken EG cells in vitro
For the generation of the neural cells, EBs were plated onto gelatin coated tissue culture plates at Day 4. Neural progenitors were induced in serum-free medium containing 10-6 M RA,2 mmol/L glutamine,5 mmol/L HEPES,25μg/ml insulin,100μg/ml transferrin, and 30 nmol/L sodium selenite for 2 days. Results showed that, RA treatment together with serum free culture condition significantly increased levels of neural specific gene expression. After additional 7 days in culture, cells possessing typical neuronal morphology were fixed and proved to be NSE, TH, ChAT, GFAP and CNP positive by immunocytochemical staining. RT-PCR analysis also showed that after induction, nestin was down-regulated, whereas NSE,β-tubulinⅢ, ChAT, TH, GFAP and CNP became positive, which displayed mature neuron, astrocyte and oligodendrocyte differentiation. To induce mature neural cells, these progenitors were continually cultured for 7 days. Adipogenic differentiation was induced by culture 7-day-old EBs in medium supplemented with 10% FCS,10μg/ml insulin,1μmol/L dexamethasone,0.2 mmol/L indomethacin for 10 days. The accumulation of lipid vacuoles in the cells was first detected 5 days after the addition of adipogenic induction medium and determined by staining for Oil Red O 10 days after induction. As differentiation progressed, expression of PPARγ, GLUT1 were significantly increased. Mature adipocytes marker gene lipoprotein lipase (LPL) was detected 10 days after induction, thereby confirming the mature adiogenic differentiation. Fetal chicken liver-derived cells were prepared as adherent cells from 7-day-old chicken embryos, after which 14-day-old EBs were plated onto the gelatin-coated culture dishes and incubated in the liver cells-conditioned medium. The AFP mRNA expression was detected in 14-day-old EBs. However, the expression of cytochrome P450 7a1 (CYP7A1), hepatocyte nuclear factor la (HNFla) and hepatocyte nuclear factor 4a (HNF4a), possible markers for embryonic endoderm-derived mature hepatocytes, were only observed after a co-culture with fetal liver cell derived conditioned medium. Immunocytochemical staining together with histochemical results demonstrated a high ratio of ALB and PAS positive cells 10 days after induction of hepatocyte differentiation, thus showing that co-culture of EG cells with liver cells-derived conditioned medium guided the differentiation toward mature and functional hepatocytes. We also investigated the effect of 5-azacytidine (5-aza) on EG cells to differentiate into cardiomyocytes. EBs were formed after 10 days with 5-aza in suspension culture, beating cell clusters were observed. Results showed that 5-aza (0～10μmol/L) promoted the production of beating cell clusters of EG cells in a dose-dependent manner. In response to 5μmol/L of 5-aza, EG cells expressed cardiac markers such as GATA-6, Nkx2.5, cardiac troponin T, a-actinin, MHC6, Desmin, ANF, CASQ, SMHC and SMA were up-regulated in a time-dependent manner after induction. Immunocytochemistry revealed the expression of smooth muscle SMA, sarcomeric a-actinin, cardiac troponin T and connexin 43 respectively.
Through these studies we successfully isolated positive cells for chicken PGC-specific markers from genital ridges. The positive cells proliferated to form colonies on somatic cells during primary culture. Firmly packed colonies with ES cell-like morphology could be observed after subculture. These cells showed important features of pluripotent stem cells especially the expression of the pluripotency-associated genes cPouV, cNanog and Sox2 and form teratoma in vivo. After suspension culture, simple and cystic EBs were formed from single EG cells and 15% FCS and 2 mmol/L Gln could promote their formation. The expression of the lineage-specific markers of three germ layers and the expression profiles of cPouV, cNanog and Sox2 genes during the EBs development in vitro were also examined. Results showed that cPouV, cNanog and Sox2 were down-Tregulated during the formation of EBs, and the the lineage-specific markers were expressed in 14-day-old EBs, suggesting their presumable effects in their regulatory effects in maintaining the pluripotency during differentiation of chicken EG cells. The differentiation potency into mature neuron, glial cells, adipocytes, hepatocytes and cardiomyocytes of EBs fully provide evidence that, chicken PGC-derived EG cells possess the pluripotency to differentiate into precursor cell lineages of all three germ layers and then into mature cells after the directed induction methods in vitro, thus making them a potential experimented model for further study the cellular mechanisms of tissue-specific differentiation and regeneration, that will help to devise strategies for restoring function to damaged or diseased tissues in humans.
1.鸡胚胎生殖细胞分离和培养模型的建立
在体视显微镜下用玻璃细针分离3.5-4日胚龄鸡胚生殖嵴部位,用胰蛋白酶-EDTA消化分散组织,然后将分散的细胞进行培养以建立PGC的原代培养模型。所用的高糖DMEM培养液中添加5%胎牛血清(FCS)、10ng/ml白血病抑制因子(LIF)、10ng/ml碱性成纤维生长因子(bFGF)、10ng/ml干细胞生长因子(SCF), 0.1mmol/L MEM非必需氨基酸、0.1mmol/Lβ-巯基乙醇、2mmol/L L-谷氨酰胺(Gln)、100IU/ml青霉素和100μg/ml链霉素(完全培养液)。与体细胞共培养5d后,利用RT-PCR方法检测PGC特异性基因Cvh、Dazl和DEH在原代培养形成的PGC集落中的表达。将表达PGC特异基因的集落进行传代培养,然后用过碘酸雪夫氏反应(PAS)、SSEA-1和SSEA-3及SSEA-4免疫组化、和干细胞多能性相关基因PouV、Nanog和Sox2 RT-PCR检测均证实传代后形成的集落为EG阳性克隆。上述结果表明,PGC-体细胞原代及传代共培养并添加多种因子的条件下,PGC在体外可转变为EG细胞,这为研究生殖干细胞的分化机制提供了细胞来源。
2.鸡胚胎生殖细胞体内形成畸胎瘤
通过灌饲具有免疫抑制作用的药物环孢素A (CsA,5mg/kg)制作免疫抑制雏鸡,将含有1.0×106个鸡EG细胞的PBS注射到受到免疫抑制的雏鸡皮下,观察其分化情况。5周后取皮下形成的组织瘤进行切片和HE染色及免疫组化染色鉴定。结果表明,EG细胞在免疫缺陷的雏鸡皮下可自发分化为含有神经细胞、软骨细胞、脂肪细胞、血管、骨细胞、平滑肌、横纹肌和腺上皮细胞等三个胚层来源的多种细胞类型的畸胎瘤。而注射同样数量EG细胞到未饲喂环孢素A组(模型对照组)和注射不含有EG细胞的免疫抑制雏鸡组(细胞对照组)均未观察到组织瘤的形成。上述结果表明通过饲喂免疫抑制作用的药物环孢素A可制作免疫抑制雏鸡模型,本实验利用此模型证明了鸡EG细胞在体内具有多向分化为含有三个胚层来源细胞畸胎瘤的潜能,这为开展生殖干细胞体内分化潜能的研究提供了实验平台。
3.鸡胚胎生殖细胞体外形成EB模型的建立与优化
将EG克隆与体细胞进行胰酶消化,并利用差速贴壁方法富集EG细胞。然后无饲养层、无LIF和β-巯基乙醇条件下将鸡EG细胞接种到涂有1%琼脂的培养板中悬浮培养形成EB。形成的EB进行丫啶橙(AO)染色后在荧光共聚焦显微镜下观察其内部结构。对形成7d和14d的类胚体分别进行切片和HE组织学染色,并通过RT-PCR检测内胚层标志基因AFP、外胚层标志基因Sox3、中胚层标志基因GATA6和滋养层标志基因Cdx2在其中的表达。结果表明：将富集的鸡EG单细胞采用不含LIF的培养液进行悬浮培养4-7d可形成简单EB,继续悬浮培养则其内部形态也发生改变,最终形成囊状EB。不同时间点的EB切片显示随着时间的变化,EB中出现含有神经细胞、脂肪细胞和胰腺细胞等三个胚层的细胞类型。此外,在体外悬浮培养时比较了FCS和Gln对EB形成的影响。统计显示,随着悬浮培养时间的延长,EB的面积不断增大,FCS浓度和Gln添加量对EB的形成产生影响显著,其中以含15%FCS、2 mmol/L Gln的培养液中EB的形成数量和状态较好。同时,在EG细胞体外分化形成EB的第0d、3d、5d和10d分别检测多能性相关基因转录调控因子如PouV、Nanog和Sox2 mRNA的表达。结果显示：在EB体外分化过程中,调控干细胞多能性和自我更新的三个重要转录调控因子PouV、Nanog和Sox2表达量均呈明显下调趋势,提示PouV、Nanog和Sox2在维持鸡EG细胞多潜能性中起着重要作用。上述结果表明,鸡胚EG细胞通过悬浮培养方法可制备具有向三个胚层分化能力的EB,此结果为研究EG细胞体外定向诱导分化为特定的细胞类型奠定了实验基础。
4.鸡胚EG细胞体外多向分化调控的研究
在贴壁培养条件下,利用含10-6 mol/L RA的ITS诱导液可将EG细胞定向诱导分化为表达成熟神经元、星型胶质细胞和小胶质细胞特异蛋白及基因的神经细胞。部分神经元经免疫组化及RT-PCR方法鉴定,表明其具有合成多巴胺和胆碱能神经递质的功能。利用10μg/ml胰岛素、1μmol/L地塞米松和0.2 mmol/L吲哚美辛联合处理,可诱导EG细胞定向分化为含有大量脂滴并表达脂肪细胞特异性基因PPARγ, GLUT1和LPL的成熟脂肪细胞。利用与肝细胞条件培养联合培养的方式可定向诱导EG细胞分化为肝细胞,利用PAS染色、免疫细胞化学、RT-PCR方法鉴定EG诱导来源的肝细胞,提示EG细胞具有分化为含有糖原并分泌白蛋白功能的成熟的肝细胞。利用添加去甲基化试剂5-aza的培养液悬浮培养,可将EG细胞诱导分化为心肌细胞,表明5-aza具有显著提高形成规律性收缩EB比例的作用,其中以添加5μmol／L 5-aza组形成的比例最高。形成的EB经贴壁分化培养后,采用透射电镜观察、荧光免疫组化及RT-PCR方法鉴定EG细胞来源的心肌细胞,结果显示5-aza可诱导EG细胞分化为含有肌小结和缝隙连接、表达心肌细胞特异性蛋白和标志基因的心肌细胞。
以上实验结果表明：从鸡胚生殖嵴分离的PGC与体细胞原代共培养并经传代所建立的培养模型可用于EG细胞制备和分化调控的研究,经PAS组化、SSEA-1和SSEA-3及SSEA-4免疫细胞化学染色、干细胞多能性相关基因转录调控因子PouV、Nanog和Sox2的RT-PCR法检测、在免疫抑制雏鸡体内形成含有三个胚层来源细胞的畸胎瘤等多种方法均证实了EG细胞的干细胞特性。将富集的鸡EG细胞悬浮培养形成简单EB和囊状EB,培养液中添加15%FCS和2mmol／L Gln可促进EB的形成。在EB体外分化过程中,PouV Nanog和Sox2表达量均呈明显下调趋势,而三个胚层的标志基因AFP.Sox3和GATA6及滋养层标志基因Cdx2出现表达,表明EG细胞通过悬浮培养方法可制备具有向三个胚层分化能力的EB。贴壁培养的EB经不同的诱导剂处理后,经细胞特异性的标志基因或蛋白的表达鉴定,EB可定向分化成有功能的神经细胞、脂肪细胞、肝细胞及心肌细胞。以上结果提示利用所建立的EG／EB体外制备和定向诱导分化模型可用于生殖干细胞发育调控机制的研究,EG细胞可诱导分化成三个胚层的细胞,这为禽类干细胞发育生物学和比较医学的研究提供了实验平台和理论基础。
Although the chick embryo has long been used as a model for developmental biology, its potential use as an experiment model for the repair and regeneration of adult tissues is often overlooked. This model has several advantages over mammalian systems for in vivo studies, as it is cost-effective, easily manipulated, and can be used for transgenesis in conjunction with viral vectors or electroporation. The molecular mechanisms underlying the development of nearly all major organ systems have been elucidated in the chick, making data interpretation more complete within this knowledge landscape. In this study, embryonic germ (EG) cells were derived from primordial germ cells (PGCs) of genital ridges of 3.5-4-days-old chicken embryos. These cells satisfied the criteria previously used for defining chicken EG cells by using the expression of markers characteristic to ES cells. When injected subcutaneously, chicken EG cells could form teratoma that enable differentiation into a wide range of tissue types of all three primary cell lineages including neural cells, cartilage, forming bone, adipocytes, blood vessels, smooth muscle, striated muscle and secretory epithelia in recipient. Furthermore, cells in embryoid bodies (EBs) expressed lineage-specific markers of three germ layers and could be induced to differentiate into more advanced stages of various committed cell types, including dopamine and cholinergic neurons, astrocytes, oligodendrocytes, adipocytes, cardiomyocytes and hepatocytes, that were demonstrated by immunocytochemical staining or RT-PCR analysis. These findings support the multiple differentiation capability of chicken pluripotent EG cells, thus confirming the presumption that chicken embryos may used as a potential model for better understanding the mechanisms of tissue-specific differentiation and regeneration, that will help to devise strategies based on the transplantation of stem cell-derived tissues for restoring function to damaged or diseased tissues.
1. Preparation of of chicken EG cells and establishment of the culture models
Genital ridges were collected by dissection of chicken embryos at 3.5-4 days with a fine glass needle under a microsurgery scope. For primary culture, cell suspension containing both PGCs and somatic cells was seeded onto gelatin-treated 35 mm culture plates at a density of 1×106/well in DMEM supplemented with 5% fetal calf serum (FCS), 10ng/ml leukemia inhibitory factor (LIF), 10ng/ml human basic fibroblast growth factor (bFGF), 0.1mmol/L MEM nonessential amino acids, O.lmmol/L 2-mercaptoethanol,2mmol/L L-glutamine (Gln), 100U/ml penicillin and 100μg/ml streptomycin. The seeded cells were then maintained at 38.5℃in 5% CO2/95% air with 60%-70% relative humidity until the PGCs colonized as a primary culture. To trace the origin of the colonies, the primary formed colonies were picked up with a fine glass needle, dissociated with 0.25% trypsin-EDTA 5 days after plating and then subjected to RT-PCR analysis for expression of PGC-specific markers. For further subculture, colonies that were positive for PGC markers were picked up and treated with 0.25% trypsin-EDTA to achieve single cell suspension and reseeded onto 6-well dishes. After three passages, staining of periodic acid-Schiff regent (PAS), stage-specific embryonic antigens (SSEA-1, SSEA-3 and SSEA-4) immunocytochemistry, and the expression of the pluripotency-associated genes cPouV, cNanog and Sox2 analysis all confirmed the characteristics of cultured EG cells. The above results indicated that the primary and subculture models of PGCs could be used for obtaining EG cells which represents a source of EG cells for studies about differentiation of germline stem cells (GSC).
2. Teratoma formation of chicken EG cells in vivo
Seven-day-old chicks were obtained from a commercial hatchery and fed with cyclosporine (5 mg/kg BW) known as an immunosuppressive agent to dampen down the body's immune reaction. EG colonies were collected and treated with 0.25% trypsin-EDTA to achieve single cell suspension. About 1×106 cells in 200μl PBS were injected subcutaneously into axilla region of the chicks. After 5 weeks, chicks were sacrificed and emerging tissue materials were dissected. Tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Sections (5μm) were placed on slides for HE and irnmunohistochemical staining. Results showed that chicken EG cells were capable of forming teratomas after subcutaneous injection into the immunosuppressed chicks. The amount of differentiated tissues varied among individual teratomas. Each teratoma contained a broad variety of tissues, including neural cells, cartilage, forming bone, adipocytes, blood vessels, smooth muscle, striated muscle and secretory epithelia. There was no teratoma formed in chicks without immunosuppression or no EG cells injection. The above results indicated that the immunodeficiency chick model could be made by fed with immunosuppressive agents cyclosporine, and the present study using this chick model showed that the chicken EG cells could form teratomas containing a variety of differentiated tissues and cell types, as well as some patches of undifferentiated cells in vivo.
3. Formation of EBs from chicken EG cells in vitro
EG colonies in culture were picked up and dissociated into single cells, transferred onto gelatin-coated plates for a 30 min period to remove contaminating somatic cells and then transferred onto Ultra Low Attachment plates to allow their aggregation and prevent adherence to the plate. Usually about 106 EG cells were incubated in each 35 mm plate accompanied by the withdrawal of LIF and 2-mercaptoethanol from the medium. During this period, the cells grew into compact aggregates and became simple EBs within 2-5 days, especially more easily accompanied by 15% FCS and 2 mmol/L Gln, then turned to cystic EBs with the formation of a central cavity. Inner structure of EBs were observed by acridine orange staining or HE staining of the EBs sections on Day 7 and Day 17 of suspension culture respectively. HE staining analysis of EB sections showed heterogeneous structure with the ability to differentiate into multiple cell types, including neural tube like structure, vascular structures, connective tissues, and glandular-like cells. Furthermore, expression of the lineage-specific markers of three germ layers, including endoderm specific gene characterized by AFP, mesoblast specific gene characterized by Gata6, ectoderm specific gene characterized by Sox3 and the trophectoderm specific gene characterized by Cdx2 was detected in EBs, suggesting their differentiation potential in vitro. In addition, we demonstrated the expression profiles of pluripotency-associated markers, including chicken Oct4 homologue PouV (cPouV), chicken Nanog (cNanog) and Sox2 genes during the formation of EBs, which were required for the maintenance of pluripotency in ES cells. All of these genes were expressed in undifferentiated EG cells, and down regulated during the formation of EBs, suggesting their presumable effects in the inhibition of chicken EG cells differentiation. The above results demonstrate the formation potential of EG cells into EBs, which will facilitate the establishment of the differentiation model of EG cells in vitro.
3. Multipledifferentiation regulation of chicken EG cells in vitro
For the generation of the neural cells, EBs were plated onto gelatin coated tissue culture plates at Day 4. Neural progenitors were induced in serum-free medium containing 10-6 M RA,2 mmol/L glutamine,5 mmol/L HEPES,25μg/ml insulin,100μg/ml transferrin, and 30 nmol/L sodium selenite for 2 days. Results showed that, RA treatment together with serum free culture condition significantly increased levels of neural specific gene expression. After additional 7 days in culture, cells possessing typical neuronal morphology were fixed and proved to be NSE, TH, ChAT, GFAP and CNP positive by immunocytochemical staining. RT-PCR analysis also showed that after induction, nestin was down-regulated, whereas NSE,β-tubulinⅢ, ChAT, TH, GFAP and CNP became positive, which displayed mature neuron, astrocyte and oligodendrocyte differentiation. To induce mature neural cells, these progenitors were continually cultured for 7 days. Adipogenic differentiation was induced by culture 7-day-old EBs in medium supplemented with 10% FCS,10μg/ml insulin,1μmol/L dexamethasone,0.2 mmol/L indomethacin for 10 days. The accumulation of lipid vacuoles in the cells was first detected 5 days after the addition of adipogenic induction medium and determined by staining for Oil Red O 10 days after induction. As differentiation progressed, expression of PPARγ, GLUT1 were significantly increased. Mature adipocytes marker gene lipoprotein lipase (LPL) was detected 10 days after induction, thereby confirming the mature adiogenic differentiation. Fetal chicken liver-derived cells were prepared as adherent cells from 7-day-old chicken embryos, after which 14-day-old EBs were plated onto the gelatin-coated culture dishes and incubated in the liver cells-conditioned medium. The AFP mRNA expression was detected in 14-day-old EBs. However, the expression of cytochrome P450 7a1 (CYP7A1), hepatocyte nuclear factor la (HNFla) and hepatocyte nuclear factor 4a (HNF4a), possible markers for embryonic endoderm-derived mature hepatocytes, were only observed after a co-culture with fetal liver cell derived conditioned medium. Immunocytochemical staining together with histochemical results demonstrated a high ratio of ALB and PAS positive cells 10 days after induction of hepatocyte differentiation, thus showing that co-culture of EG cells with liver cells-derived conditioned medium guided the differentiation toward mature and functional hepatocytes. We also investigated the effect of 5-azacytidine (5-aza) on EG cells to differentiate into cardiomyocytes. EBs were formed after 10 days with 5-aza in suspension culture, beating cell clusters were observed. Results showed that 5-aza (0～10μmol/L) promoted the production of beating cell clusters of EG cells in a dose-dependent manner. In response to 5μmol/L of 5-aza, EG cells expressed cardiac markers such as GATA-6, Nkx2.5, cardiac troponin T, a-actinin, MHC6, Desmin, ANF, CASQ, SMHC and SMA were up-regulated in a time-dependent manner after induction. Immunocytochemistry revealed the expression of smooth muscle SMA, sarcomeric a-actinin, cardiac troponin T and connexin 43 respectively.
Through these studies we successfully isolated positive cells for chicken PGC-specific markers from genital ridges. The positive cells proliferated to form colonies on somatic cells during primary culture. Firmly packed colonies with ES cell-like morphology could be observed after subculture. These cells showed important features of pluripotent stem cells especially the expression of the pluripotency-associated genes cPouV, cNanog and Sox2 and form teratoma in vivo. After suspension culture, simple and cystic EBs were formed from single EG cells and 15% FCS and 2 mmol/L Gln could promote their formation. The expression of the lineage-specific markers of three germ layers and the expression profiles of cPouV, cNanog and Sox2 genes during the EBs development in vitro were also examined. Results showed that cPouV, cNanog and Sox2 were down-Tregulated during the formation of EBs, and the the lineage-specific markers were expressed in 14-day-old EBs, suggesting their presumable effects in their regulatory effects in maintaining the pluripotency during differentiation of chicken EG cells. The differentiation potency into mature neuron, glial cells, adipocytes, hepatocytes and cardiomyocytes of EBs fully provide evidence that, chicken PGC-derived EG cells possess the pluripotency to differentiate into precursor cell lineages of all three germ layers and then into mature cells after the directed induction methods in vitro, thus making them a potential experimented model for further study the cellular mechanisms of tissue-specific differentiation and regeneration, that will help to devise strategies for restoring function to damaged or diseased tissues in humans.
引文
[]]Agarwal S, KL Holton and R Lanza. Efficient Differentiation of Functional Hepatocytes from Human Embryonic Stem Cells. Stem Cells 2008; 26:1117-1127.
[2]Aleckovic M, Simon C. Is teratoma formation in stem cell research a characterization tool or a window to developmental biology? Reproductive BioMedicine 2008; 17:274-284.
[3]Aramaki S, Sato F, Kato T, Soh T, Kato Y, Hattori MA. Molecular cloning and expression of dead end homologue in chicken primordial germ cells. Cell and Tissue Res 2007; 330:45-52.
[4]Armstrong L, Saretzki G, Peters H, Wappler I, Evans J, Hole N, von Zglinicki T, and Lako M. Overexpression of telomerase confers growth advantage, stress resistance, and enhanced differentiation of ESCs toward the hematopoietic lineage. Stem Cells 2005; 23:516-529.
[5]Arsenijevic Y, Weiss S, Schneider B, Aebischer P. Insulin-Like Growth Factor-I Is Necessary for Neural Stem Cell Proliferation and Demonstrates Distinct Actions of Epidermal Growth Factor and Fibroblast Growth Factor-2. J. Neurosci 2001; 21:7194-7202.
[6]Babaie Y, Herwig R, Greber B, Brink TC, Wruck W, Groth D, Lehrach H, Burdon T, Adjaye J. Analysis of Oct4-dependent transcriptional networks regulating self-renewal and pluripotency in human embryonic stem cells. Stem Cells 2007; 25:500-510.
[7]Bain G, Kitchens D, Yao M, Huettner JE, Gottlieb DI. Embryonic stem cells express neuronal properties in vitro. Dev Biol.1995; 168:342-357.
[8]Boilly B, Vercoutter-Edouart AS, Hondermarck H, Nurcombe V, Le Bourhis X, Fernig DG. FGF signals for cell proliferation and migration through different pathways. Cytokine Growth Fact Rev2000; 11:295-302.
[9]Bowles J, Knight D, Smith C, Wilhelm D, Richman J, Mamiya S, Yashiro K, Chawengsaksophak K, Wilson MJ, Rossant J, Hamada H, Koopman P. Science 2006; 312:596-600
[10]Boyer LA, Lee TI, Cole MF, Johnstone SE, Levine SS, Zucker JP, Guenther MG, Kumar RM, Murray HL, Jenner RG, Gifford DK, Melton DA, Jaenisch R, Young RA. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 2005; 122:947-956
[11]Buehr M, Mclaren A, Bartley A, Darling S. Proliferation and migration of primordial germ cells in We/We mouse embryos. Dev Dyn 1993; 198:182-189.
[12]Burlacu A, Rosca AM, Maniu H, Titorencu I, Dragan E, Jinga V, Simionescu M. Promoting effect of 5-azacytidine on the myogenic differentiation of bone marrow stromal cells. Eur J Cell Biol 2008; 87:173-84.
[13]Cameron CM, Harding F, Hu WS, Kaufman DS. Activation of Hypoxic Response in Human Embryonic Stem Cell-Derived Embryoid Bodies. Experimental Biology and Medicine 2008; 233:1044-1057.
[14]Cameron CM, Hu WS, Kaufman DS. Improved development of human embryonic stem cell-derived embryoid bodies by stirred vessel cultivation. Biotechnol Bioeng 2006; 94:938-948.
[15]Canon S, Herranz C, Manzanares M. Germ cell restricted expression of chick Nanog. Dev Dynamic.2006; 235:2889-2894.
[16]Carpenedo Rd L, Sargent CY, McDevitt TC. Rotary Suspension Culture Enhances the Efficiency, Yield, and Homogeneity of Embryoid Body Differentiation. Stem Cells 2007; 25:
2224-2234.
[17]Chambers I, Colby D, Robertson M, Nichols J, Lee S, Tweedie S, Smith A. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 2003; 113:643-655.
[18]Chan SR, Blackburn EH. Telomere and telomerase. Phil Trans R Soc B 2004; 359:109-121.
[19]Chang IK, Tajima A, Chikamune T, Ohno T. Proliferation of chick primordial germ cells on stroma cells from the germinal ridge. Cell Biol Int 1995; 19:143-149.
[20]Chang TH, Polakis SE. Differentiation of 3T3-L1 fibroblasts to adipocytes. Effect of insulin and indomethacin on the levels of insulin receptors. J. Biol. Chem 1978; 253:4693-4696.
[21]Chen HF, Kuo HC, Chien CL, Shun CT, Yao YL, Ip PL, Chuang CY, Wang CC, Yang YS, Ho HN. Derivation, characterization and differentiation of human embryonic stem cells: comparing serum-containing versus serum-free media and evidence of germ cell differentiation. Hum. Reprod 2007; 22:567-577.
[22]Cherny R A, Merei J. Evidence for pluripotency of bovine primordial germ cell derived cell lines initiated in long-term culture. Theriogenoiogy 1994; 4:175.
[23]Chew JL, Loh YH, Zhang W, Chen X, Tam WL, Yeap LS, Li P, Ang YS, Lim B, Robson P, Ng HH. Reciprocal Transcriptional Regulation of Pou5fl and Sox2 via the Oct4/Sox2 Complex in Embryonic Stem Cells. Mol. Cell. Biol 2005; 25:6031-6046.
[24]Chinzei R, Tanaka Y, Shimizu-Saito K, Hara Y, Kakinuma S, Watanabe M, Teramoto K, Arii S, Takase K, Sato C, Terada N, Teraoka H. Embryoid-body cells derived from a mouse embryonic stem cell line show differentiation into functional hepatocytes. Hepatology 2002; 36:22-29.
[25]Choi D, Lee H, Jee S, Jin S, Koo SK, Paik SS, Jung SC, H SY, Lee KS, Oh B. In Vitro Differentiation of Mouse Embryonic Stem Cells:Enrichment of Endodermal Cells in the Embryoid Body. Stem Cells 2005; 23:817-827.
[26]Claswon RC and Domm LV. Developmental changes in glycogen content of primordial germ cells in chick embryo. Proc of the Society For Experi Biol Med 1963; 112:533-537.
[27]Coleman CM. Chicken embryo as a model for regenerative medicine. Birth Defects Res (Part C) 2008; 84:245-256.
[28]Dang SM, Zandstra PW. Scalable production of embryonic stem cell-derived cells. Methods Mol Biol 2005; 290:353-364.
[29]Dani C. Embryonic stem cell-derived adipogenesis. Cells Tissues Organs 1999; 165:173-180.
[30]De Felici M, McLaren A. Isolation of mouse primordial germ cells. Exp Cell Res 1982; 142:476-482.
[31]Desbaillets I, Ziegler U, Groscurth P, Gassmann M. Embryoid bodies:an in vitro model of mouse embryogenesis. Exp Physiol 2000; 85:645-651.
[32]Doetschman TC, Eistetter H, Katz M, Schmidt W, Kemler R. The in vitro development of blastocyst-derived embryonic stem cell lines:formation of visceral yolk sac, blood islands and myocardium. J Embryol Exp Morphol 1985; 87:27-45.
[33]Dolci S, Williams DE, Ernst MK, Resnick JL, Brannan CI, Lock LF, Lyman SD, Boswell HS, Donovan PJ. Requirement for mast cell growth factor for primordial germ cell survival in culture. Nature 1991; 352:809-811.
[34]Donovan PJ, Gearhart J. The end of the beginning for pluripotent stem cells. Nature 2001; 414:92-97.
[35]Donovan PJ, de Miguel MP. Turning germ cells into stem cells. Curr Opin Genet Dev 2003; 13:463-71.
[36]Durcova-Hills G, Adams IR, Barton SC, Surani MA, McLaren A. The role of exogenous fibroblast growth factor-2 on the reprogramming of primordial germ cells into pluripotent stem cells. Stem Cells 2006; 24:1441-1449.
[37]Durcova-Hills G, Wianny F, Merriman J, Zernicka-Goetz M, McLaren A. Development fate of embryonic germ cells(EGCs), in vivo an in vitro. Differentiation 2003; 71:135-141.
[38]Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature 1981; 292:154-156.
[39]Eyal-Giladi H, Ginsburg M and Farbarov A. Avian primordial germ cells are of epiblast origin. J of Embryo and Experi Morpho 1981; 63:139-147.
[40]Fang D, Leishear K, Nguyen TK, Finko R, Cai K, Fukunaga M, Li L, Brafford PA, Kulp AN, Xu X, Smalley KS, Herlyn M. Defining the Conditions for the Generation of Melanocytes from Human Embryonic Stem Cells. Stem Cells 2006; 24:1668-1677.
[41]Felici MD, Scaldaferri ML, Lobascio M, Iona S, Nazzicone V, Klinger FG, Farini D. Experimental approaches to the study of primordial germ cell lineage and proliferation. Hum. Reprod 2004; 10:197-206.
[42]Fiegel HC, Lioznov MV, Cortes-Dericks L, Lange C, Kluth D, Fehse B, Zander AR. Liver-specific gene expression in cultured human hematopoietic stem cells. Stem Cells 2003; 21:98-104.
[43]Fijnvandraat AC, van Ginneken AC, Schumacher CA, Boheler KR, Lekanne Deprez RH, Christoffels VM, Moorman AF. Cardiomyocytes purified from differentiated embryonic stem cells exhibit characteristics of early chamber myocardium. J Mol Cell Cardiol 2003; 35:1461-1472.
[44]Fok EY, Zandstra PW. Shear-Controlled Single-Step Mouse Embryonic Stem Cell Expansion and Embryoid Body-Based Differentiation. Stem Cells 2005; 23:1333-1342.
[45]Fong H, Hohenstein KA, Donovan PJ. Regulation of Self-renewal and pluripotency by Sox2 in human embryonic stem cells. Stem Cells 2008; 26:1931-938.
[46]Fujimoto T, Kuwana T, Maeda-Suga H. Attraction of chick primordial germ cells by gonadal anlage in vitro. Anatomical Record 1986; 215:403-406.
[47]Fujimito T, Ukeshima A, and Kivofuji R. The origin, migration and morphology of the primordial germ cells in the chick embryos. Anatomical Record 1976; 185:139-154.
[48]Fukuda K. Development of regenerative cardiomyocytes from mesenchymal stem cells for cardiovascular tissue engineering. Artif Organs 2001; 25:187-193.
[49]Gassanov N, Er F, Zagidullin N, Jankowski M, Gutkowska J, Hoppe UC. Retinoid acid-induced effects on atrial and pacemaker cell differentiation and expression of cardiac ion channels. Differentiation 2008 76:971-980.
[50]Gearhart J. New potential for human embryonic stem cells. Science 1998; 282:1061-1065.
[51]Gimeno RE, Klaman LD. Adipose tissue as an active endocrine organ:recent advances. Curr Opin Pharmacol 2005; 5:122-128.
[52]Ginsburg M, Eyal-Giladi H. Primordial germ cells of the young chick blastoderm originate from the central zone of the area pellucida irrespective of the embryo-forming process. Development 1987; 101:209-219.
[53]Ginsburg M. Primordial germ cell formation in birds. In:J Marsh and J Goode. Germline development, Ciba Foundation Symposium 182. London.1994; 52-61.
[54]Ginsburg M and Eyal-Giladi H. Temporal and spatial aspects of the gradual migration of primordial germ cells from epiblast into the germinal crescent in the avian embryo. Embryo Experi Morpho 1986; 95:53-71.
[55]Glaser T, Opitz T, Kischlat T, Konang R, Sasse P, Fleischmann BK, Engel W, Nayernia K, Briistle O. Adult Germ Line Stem Cells as a Source of Functional Neurons and Glia. StemCells 2008; 26:2434-2443.
[56]Godin I, Deed R, Cooke J, Zsebo K, Dexter M, Wylie CC. Effects of the steel gene product on mouse primordial germ cells in culture. Nature 1991; 352:807-809.
[57]Godin I, Wylie CC. TGF R inhibits proliferation and has a chemotropic effect on mouse primordial germ cells in culture. Development 1991; 113:1451-1457.
[58]Godoy MC, Tarelli R, Ferrari CC, Sarchi MI, Pitossi FJ. Central and systemicIL-1 exacerbates neurodegeneration and motor symptoms in a model of Parkinson's disease. Brain 2008; 131:1880-1894.
[59]Goldfarb M. Signaling by fibroblast growth factors:the inside story. Science STKE 2001; 2001:PE37.
[60]Gu Y, Runyan Ch, Shoemaker A, Surani A, Wylie Chris. Steel factor controls primordial germ cell survival and motility from the time of their specification in the allantois, and provides a continuous niche throughout their migration. Development 2009; 136:1295-1303.
[61]Guo Y, Costa R, Ramsey H, Starnes T, Vance G, Robertson K, Kelley M, Reinbold R, Scholer H, Hromas R. The embryonic stem cell transcription factor Oct-4 and FoxD3 interact to regulate endodermal-specific promoter expression. Proc. Natl. Acad. Sci.U.S.A 2002; 99: 3663-3667.
[62]Haegele L, Ingold B, Naumann H, Tabatabai G, Ledermann B, Brandner S. Wnt signaling inhibits neural differentiation of embryonic stem cells by controlling bone morphogenetic protein expression. Mol Cell Neurosci 2003; 24:696-708.
[63]Hamazaki T, Iiboshi Y, Oka M, Papst PJ, Meacham AM, Zon LI, Terada N. Hepatic maturation in differentiating embryonic stem cells in vitro. FEBS Lett 2001; 497:15-19.
[64]Han JY. Germ cells and transgenesis in chickens. Comp Immunol Microbiol Infect Dis 2009; 32:61-80.
[65]Han JY, Park TS, Hong JH, Jeong DK, Kim JN, Kim KD, Lim JM. Production of germline chimeras by transfer of chicken gonadal primordial germ cells maintained in vitro for an extended period. Theriogenology 2002; 58:1531-1539.
[66]Hart AH, Hartley L, Ibrahim M, Robb L. Identification, cloning and expression analysis of the pluripotency promoting Nanog genes in mouse and human[J]. Dev Dyn 2004; 230:187-198.
[67]Hay DC, Sutherland L, Clark J, Burdon T. Oct-4 knockdown induces similar patterns of endoderm and trophoblast differentiation markers in human and mouse embryonic stem cells. Stem Cells 2004; 22:225-235.
[68]He JQ, Ma Y, Lee Y, Thomson JA, Kamp TJ. Human embryonicstem cells develop into multiple types of cardiac myocytes:action potential characterization. Circ Res 2003; 93:32-39.
[69]Hedlund E, Pruszak J, Ferree A, Vinuela A, Hong S, Isacson O, Kim KS. Selection of embryonic stem cell-derived enhanced green fluorescent protein-positive dopamine neurons using the tyrosine hydroxylase promoter is confounded by reporter gene expression in immature cell populations. Stem Cells 2007; 25:1126-1135.
[70]Heins N, Englund MC, Sjoblom C, Dahl U, Tonning A, Bergh C, Lindahl A, Hanson C, Semb H. Derivation, characterization, and differentiation of human embryonic stem cells. Stem Cells 2004; 22:367-376.
[71]Henderson JK, Draper JS, Baillie HS, Fishel S, Thomson JA, Moore H, Andrews PW. Preimplantation human embryos and embryonic stem cells show comparable expression of stage-specific embryonic antigens. Stem Cells 2002; 20:329-337.
[72]Hillel AT, Varghese S, Petsche J, Shamblott MJ, Elisseeff JH. Embryonic germ cells are capable of adipogenic differentiation in vitro and in vivo. Tissue Eng PartA 2009; 15:479-486.
[73]Hopfl G, Gassmann M, Desbaillets I. Differentiating embryonic stem cells into embryoid bodies. Methods Mol Biol 2004; 254:79-98
[74]Horiuchi H, Tategaki A, Yamashita Y, Hisamatsu H, Ogawa M, Noguchi T, Aosasa M, Kawashima T, Akita S, Nishimichi N, Mitsui N, Furusawa S, Matsuda H.Chicken leukemia inhibitory factor maintains chicken embryonic stem cells in the undifferentiated state. J Biol Chem 2004; 279:24514-24520.
[75]Hromas R, Ye H, Spinella M, Dmitrovsky E, Xu D, Costa RH. Genesisi, a winged helix transcriptional repressor, has embryonic expression limited to neural crest, and stimulates proliferation in vitro in a neural development model. Cell Tissue Res 1999; 297:371-382.
[76]Horiuchi H, Tategaki A, Yamashita Y, Hisamatsu H, Ogawa M, Noguchi T, Aosasa M, Kawashima T, Akita S, Nishimichi N, Mitsui N, Furusawa S, Matsuda H. Chicken leukemia inhibitory factor maintains chicken embryonic stem cells in the undifferentiated state. J Biol Chem 2004; 279:24514-24520.
[77]Hubner K, Fuhrmann G, Christenson LK, Kehler J, Reinbold R, De La Fuente R, Wood J, Strauss JF 3rd, Boiani M, Scholer HR. Derivation of oocytes from mouse embryonic stem cells. Science 2003; 300:1251-1256.
[78]Imamura O, Satoh Y, Endo S, Takishima K. Analysis of Extracellular Signal-Regulated Kinase 2 Function in Neural Stem/Progenitor Cells via Nervous System-Specific Gene Disruption. Stem Cells 2008; 26:3247-3256.
[79]Ishizaka S, Shiroi A, Kanda S, Yoshikawa M, Tsujinoue H, Kuriyama S, Hasuma T, Nakatani K, Takahashi K. Development of hepatocytes from ES cells after transfection with the HNF-3beta gene. FASEB J 2002; 16:1444-14446.
[80]Itskovitz-Eldor J, Schuldiner M, Karsenti D, Eden A, Yanuka O, Amit M, Soreq H, Benvenisty N. Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol Med 2000; 6:88-95.
[81]Ji J, Vijayaragavan K, Bosse M, Menendez P, Weisel K, Bhatia M. OP9 Stroma Augments Survival of Hematopoietic Precursors and Progenitors During Hematopoietic Differentiation from Human Embryonic Stem Cells. Stem Cells 2008; 26:2485-2495.
[82]Jiang Y, Jahaglrdar BN, Reinhardt RL. Plurlpotency of mesenchymal stem cell derived from adult marrow. Nature 2002; 418:41-49.
[83]Kanatsu-Shinohara M, Inoue K, Lee J, Yoshimoto M, Ogonuki N, Miki H, Baba S, Kato T, Kazuki Y, Toyokuni S, Toyoshima M, Niwa O, Oshimura M, Heike T, Nakahata T, Ishino F, Ogura A, Shinohara T. Generation of pluripotent stem cells from neonatal mouse testis. Cell 2004; 119:1001-1012.
[84]Kanatsu-Shinohara M, Inoue K, Ogonuki N, Miki H, Yoshida S, Toyokuni S, Lee J, Ogura A,
Shinohara T. Leukemia inhibitory factor enhances formation of germ cell colonies in neonatal mouse testis culture. Biol Reprod,2007; 76:55-62.
[85]Karagenc L, Petitte JN. Soluble factors and the emergence of chick primordial germ cells in vitro. Poult Sci 2000; 79:80-85.
[86]Kerr DA, Llado J, Shamblott MJ, Maragakis NJ, Irani DN, Crawford TO, Krishnan C, Dike S, Gearhart JD, Rothstein JD. Human Embryonic Germ Cell Derivatives Facilitate Motor Recovery of Rats with Diffuse Motor Neuron Injury. J Neurosci 2003; 23:5131-5140.
[87]Khoo ML, McQuade LR, Smith MS, Lees JG, Sidhu KS, Tuch BE. Growth and Differentiation of Embryoid Bodies Derived from Human Embryonic Stem Cells:Effect of Glucose and Basic Fibroblast Growth Factor. Biol Reprod 2005; 73:1147-1156.
[88]Khoo ML, Shen B, Tao H, Ma DD. Long-term serial passage and neuronal differentiation capability of human bone marrow mesenchymal stem cells. Stem Cells Dev 2008; 17:883-896.
[89]Kim JH, Auerbach JM, Rodriguez-Gomez JA, Velasco I, Gavin D, Lumelsky N, Lee SH, Nguyen J, Sanchez-Pernaute R, Bankiewicz K, McKay R. Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson's disease. Nature 2002; 418:50-56.
[90]Kim MS, Hwang NS, Lee J, Kim TK, Leong K, Shamblott MJ, Gearhart J, Elisseeff J. Musculoskeletal Differentiation of Cells Derived from Human Embryonic Germ Cells. Stem Cells 2005; 23:113-123.
[91]Kim YJ, Cho SY, Yun CH, Moon YS, Lee TR, Kim SH. Transcriptional activation of Cidec by PPARgamma2 in adipocyte. Biochem Biophys Res Commun 2008; 377:297-302
[92]Kleinsmith LJ, Pierce GB. Multipotentiality of single embryonal carcinoma cells. Cancer Res 1964; 24:1544-1551.
[93]Klug MG, Soonpaa MH, Koh GY, Field LJ. Genetically selected cardiomyocytes from differentiating embronic stem cells form stable intracardiac grafts. J Clin Invest 1996; 98:216-224.
[94]Koshimizu U, Taga T, Watanabe M, Saito M, Shirayoshi Y, Kishimoto T, Nakatsuji N. Functional requirement of gp130-mediated signaling for growth and survival of mouse primordialgerm cells in vitro and derivation of embryonic germ (EG) cells. Development 1996; 122:1235-1242.
[95]Kuhholzer B, Baguisi A, Overstrom EW. Long-term culture and characterization of goat primordial germ cells.2000; 53:1071-1079.
[96]Kuwana T, Fujimoto T. locomotion and scanning electron microscopic observation of primordial germ cells from embryonic chick blood in vitro. Anat Rec 1984; 209:337-343.
[97]Kuwana T. and Rogulska T. Migratory mechanism of chick primordial germ cells toward gonadal anlarge. Cellular and Mol Biol 1999; 45:725-736.
[98]Labosky PA, Barlow DP, Brigid LM. Mouse embryonic germ (EG) cell lines:transmission through the germline and differences in the methylation imprint of insulin-like growth factor 2 receptor (Igf2r) gene compared with embryonic stem (ES) cell lines. Development 1994; 120:3197-3204.
[99]Labosky PA, Kaestner KH. The winged helix transcriptional factor Hfh2 is expressed in neural crest and spinal cord during mouse development. Mech Dev 1998; 76:185-190.
[100]Ladewig J, Koch P, Endl E, Meiners B, Opitz T, Couillard-Despres S, Aigner L, Brustle O. Lineage selection of functional and cryopreservable human embryonic stem cell-derived neurons. Stem Cells 2008; 26:1705-1712.
[101]Lai E, Rosen OM, Rubin CS. Dexamethasone regulates the beta-adrenergic receptor subtype expressed by 3T3 L1 preadipocytes and adipocytes. J. Biol. Chem 1982; 257:6691-6696.
[102]Lavial F, Acloque H, Bertocchini F, Macleod DJ, Boast S, Bachelard E, Montillet G, Thenot S, Sang HM, Stem CD, Samarut J, Pain B. The Oct4 homologue PouV and Nanog regulate pluripotency in chicken embryonic stem cells. Development 2007; 134:3549-3563.
[103]Lazzari L, Henschler R, Lecchi L, Rebulla P, Mertelsmann R, Sirchia G. Interleukin-6 andinterleukin-11 act synergistically with thrombopoietin and stem cell factor to modulate ex vivo expansion of human CD41+and CD61+megakaryocytic cells. Haematologica 2000; 85:25-30.
[104]Lee HS, Huang GT, Chiang H, Chiou LL, Chen MH, Hsieh CH, Jiang CC. Multipotential Mesenchymal Stem Cells from Femoral Bone Marrow Near the Site of Osteonecrosis. Stem Cells 2003; 21:190-199.
[105]Liao W, Nguyen MT, Yoshizaki T, Favelyukis S, Patsouris D, Imamura T, Verma IM, Olefsky JM. Suppression of PPAR-y attenuates insulin-stimulated glucose up take by affecting both GLUT1 and GLUT4 in 3T3-L1 adipocytes. Am J Physiol Endocrinol Metab 2007; 293:E219-E227.
[106]Liew CG, Draper JS, Walsh J, Moore H, Andrews PW. Transient and stable transgene expression in human embryonic stem cells. Stem Cells 2007; 25:1521-1528.
[107]Liu H, Kim JM, Aoki F. Regulation of histone H3 lysine 9methylation in oocytes and early pre-implantation embryos. Development 2004; 131:2269-2280.
[108]Lopez-Carballo G, Moreno L, Masia S, Perez P, Barettino D. Activation of the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway by Retinoic Acid Is Required for Neural Differentiation of SH-SY5Y Human Neuroblastoma Cells. J. Biol. Chem 2002; 277:25297-25304.
[109]Marion RM, Strati K, Li H, Tejera A, Schoeftner S, Ortega S, Serrano M, Blasco MA. Telomeres acquire embryonic stem cell characteristics in induced pluripotent stem cells. Cell Stem Cell 2009; 4:141-154.
[110]Matsubara Y, K Sato, H Ishii and Y Akiba. Changes in mRNA expression of regulatory factors involved in adipocyte differentiation during fatty acid induced adipogenesis in chicken. Comp Biochem Physiol (Part A) 2005; 141:108-115.
[111]Matsuda T, Nakamura T, Nakao K, Arai T, Katsuki M, Heike T, Yokota T. STAT3 activation is sufficient to maintain an undifferentiated state of mouse embryonic stem cells. EMBO J 1999; 18:4261-4269.
[112]Matsui Y, Toksoz D, Nishikawa S, Williams D, Zsebo K, Hogan BL. Effect of steel factor and leukaemia inhibitory factor on murine primordial germ cells in culture. Nature 1991, 353:750-752.
[113]Matsui Y, Zsebo K, Hogan BL. Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture. Cell 1992; 70:841-847.
[114]Medvedev SP, Shevchenko AI, Mazurok NA, Zakiian SM. OCT4 and NANOG are the key genes in the system of pluripotency maintenance in mammalian cells. Genetika 2008; 44:1589-1608.
[115]Mika G. Primordial germ cell development in avian. Poultry Science 1997; 76:91-95.
[116]Mikkola HK, Lowry WE, Plath K, Clark AT. Derivation of Primordial Germ Cells from Human Embryonic and Induced Pluripotent Stem Cells Is Significantly Improved by Coculture with Human Fetal Gonadal Cells. Stem Cells 2009; 27:783-795.
[117]Mitani T, Takahashi N, Kawase E. Proliferation of alkaline phosphatase-positive cells from cultured primordial germ cells in rat. Theriogenology 1994; 41:336.
[118]Mitsui K, Tokuzawa Y, Itoh H, Segawa K, Murakami M, Takahashi K, Maruyama M, Maeda M, Yamanaka S. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 2003; 113:631-642.
[119]Miyazaki S, Yamato E, Miyazaki J. Regulated Expression of pdx-1 Promotes In Vitro Differentiation of Insulin-Producing Cells From Embryonic Stem Cells. Diabetes 2004; 53:1030-1037.
[120]Mozdziak PE, Angerman-Stewart J, Rushton B, Pardue SL, Petitte JN. Isolation of chicken primordial germ cells using fluorescence-activated cell sorting. Poult Sci 2005; 84:594-600.
[121]Nakamura M, Kuwana T, Myiayama Y, Fujimoto T. Extragonadal distribution primordial germ cells in the early chick embryo. Anat Rec 1988; 222:90-94.
[122]Nakamura Y, Yamamoto Y, Usui F, Mushika T, Ono T, Setioko AR, Takeda K, Nirasawa K, Kagami H, TagamiT. Migration and Proliferation of Primordial Germ Cells in the Early Chicken Embryo. Poult Sci 2007; 86:2182-2193.
[123]Naugler KM, Baer KA, Ropeleski MJ. Interleukin-11 antagonizes Fas ligand-mediated apoptosis in IEC-18 intestinal epithelial crypt cells:role of MEK and Akt-dependent signaling. Am J Physiol Gastrointest Liver Physiol, Mar 2008; 294:G728-G737.
[124]Nichols J, Davidson D, Taga T, Complementary tissue-specific expression of LIF and LIF-receptor mRNAs in early mouse embryogenesis. Mech Dev 1996; 57:123-131.
[125]Nishimoto M, Miyagi S, Yamagishi T, Sakaguchi T, Niwa H, Muramatsu M, Okuda A. Oct-3/4 maintains the proliferative embryonic stem cell state via specific binding to a variant octamer sequence in the regulatory region of the UTF1 locus. Mol Cell Biol 2005; 25:5084-5094.
[126]Nieuwkoop PD, Sutasurya LA. Primordial Germ Cells in the Chordates. Cambridge University Press; 1979.
[127]Niwa H, Burdon T, Chambers I, Smith A. Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes & Dev 1998; 12:2048.
[128]Niwa H, Miyazaki J, Smith A. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nature Genetics 2000; 24:372-376.
[129]O'Connor MD, Kardel MD, Iosfina I, Youssef D, Lu M, Li MM, Vercauteren S, Nagy A, Eaves CJ. Alkaline phosphatase-positive colony formation is a sensitive, specific, and quantitative indicator of undifferentiated human embryonic stem cells. Stem Cells 2008; 26:1109-1116.
[130]Ono T, Machida Y. Immunomagnetic purification of viable primordialgerm cells of Japanese quail (Coturnix japonica). Comp Biochem Physiol A Mol Integr Physiol 1999; 122:255-259.
[131]Okada Y, T Shimazakia, G Sobueb and H Okano. Retinoic-acid-concentration-dependent acquisition of neural cell identity during in vitro differentiation of mouse embryonic stem cells. Dev Biol 2004; 275:124-142.
[132]Pan GJ, Chang ZY, Scholer HR, Pei D. Stem cell pluripotency and transcription factor Oct4. Cell Res 2002; 12:321-319.
[133]Pan Y, Chen X, Wang S, Yang S, Bai X, Chi X, Li K, Liu B, Li L. In vitro neuronal differentiation of cultured human embryonic germ cells. Biochem Biophys Res Commun 2005; 327:548-556.
[134]Paquin J, Danalache BA, Jankowski M, McCann SM, Gutkowska J. Oxytocin induces differentiation of P19 embryonic stem cells to cardiomyocytes. Proc Natl Acad Sci U S A 2002; 99:9550-9555.
[135]Park JH, Kim SJ, Lee JB, Song JM, Kim CG, Roh S 2nd, Yoon HS. Establishment of a human embryonic germ cell line and comparison with mouse and human embryonic stem cells. Molecules and Cells 2004; 17:309-315.
[136]Park TS, Han JY. Derivation and characterization of pluripotent embryonic germ cells in chicken. Mol Reprod Dev 2000; 56:475-482.
[137]Park TS, Galic Z, Conway AE, Lindgren A, van Handel BJ, Magnusson M, Richter L, Teitell MA, Wei W, Qing T, Ye X, Liu H, Zhang D, Yang W, Deng H. Primordial germ cell specification from embryonic stem cells. PLoS ONE 2008; 3:e4013.
[138]Park TS, Hong YH, Kwon SC, Lim JM, Han JY. Birth of germline chimeras by transfer of chicken embryonic germ (EG) cells into recipient embryos. Mol Reprod Dev 2003; 65:389-395.
[139]Park TS, JY Han. Derivation and characterization of pluripotent embryonic germ cells in chicken. Mol Reprod Dev 2000; 56:475-482.
[140]Park TS, YH Hong, SC Kwon, JM Lim and JY Han. Birth of germline chimeras by transfer of chicken embryonic germ (EG) cells into recipient embryos. Mol Reprod Dev 2003; 65:389-395.
[141]Pearl EJ, Horb ME. Promoting ectopic pancreatic fates:pancreas development and future diabetes therapies. Clin Genet 2008;74:316-324.
[142]Phipps SM, Love WK, Mott TE, Andrews LG, Tollefsbol TO. Differential expression of epigenetic modulators during human embryonic stem cell differentiation. Mol Biotechnol 2009; 41:201-207.
[143]Prudhomme W, Daley GQ, Zandstra P, Lauffenburger DA. Multivariate proteomic analysis of murine embryonic stem cell self-renewal versus differentiation signaling. Proc Nail Acad Sci USA 2004; 101:2900-2905.
[144]Qi X, Li TG, Hao J, Hu J, Wang J, Simmons H, Miura S, Mishina Y, Zhao GQ. BMP4 supports self-renewal of embryonic stem cells by inhibiting mitogen activated protein kinase pathways. Proc Natl Acad Sci USA 2004; 101:6027-6032.
[]Rattis FM, Voermans C, Reya T. Wnt signaling in the stem cell niche. Curr Opin Hematol 2004; 11:88-94.
[145]Resnick JL, Bixler LS, Cheng L, Donovan PJ. Long-term proliferation of mouse primordial germ cells in culture. Nature 1992; 359:550-551.
[146]Resnick JL, Ortiz M, Keller JR, Donovan PJ. Role of fibroblast growth factors and their receptors in mouse primordial germ cell growth. Biol Reprod 1998; 59:1224-1229.
[147]Richards M, Fong CY, Tan S, Chan WK, Bongso A. An efficient and safe xeno-free cryopreservation method for the storage of human embryonic stem cells. Stem Cells 2004; 22:779-789.
[148]Rodda DJ, Chew JL, Lim LH, Loh YH, Wang B, Ng HH, Robson P. Transcriptional regulation of Nanog by OCT4 and SOX2. J. Biol. Chem 2005; 280:24731-24737.
[149]Rius M, Stresemann C, Keller D, Brom M, Schirrmacher E, Keppler D, Lyko F. Human concentrative nucleoside transporter 1-mediated uptake of 5-azacytidine enhances DNA demethylation. Mol Cancer Ther 2009; 8:225-231.
[150]Rosen ED, P Sarraf, AE Troy, G Bradwin, K Moore, DS Milstone, BM Spiegelman and RM Mortensen. PPAR gamma is required for the differentiation of adipose tissue in vivo and in vitro. Mol Cell 1999; 4:611-617.
[151]Saito S, Liu B, Yokoyama K. Animal embryonic stem (ES) cells:self-renewal, pluripotency, transgenesis and nuclear transfer. Hum Cell 2004; 17:107-115.
[152]Sang H. Prospects for transgenesis in the chick. Mechanisms of Development 2004; 121:1179-1186.
[153]Sato N, Meijer L, Skaltsounis L, Greengard P, Brivanlou AH. 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; 10:55-63.
[154]Soto-Gutierrez A, N Navarro-Alvarez, DB Zhao, JD Rivas-Carrillo, J Lebkowski, N Tanaka, IJ Fox and N Kobayashi. Differentiation of mouse embryonic stem cells to hepatocyte-like cells by co-culture with human liver nonparenchymal cell lines. Nature Protocols 2007; 2:347-356.
[155]Sekkai D, Gruel G, Herry M, Moucadel V, Constantinescu SN, Albagli O, Tronik-Le Roux D, Vainchenker W, Bennaceur-Griscelli A. Microarray analysis of LIF/Stat3 transcriptional targets in embryonic stem cells. Stem Cells 2005; 23:1634-1642.
[156]Shevinsky LH, Knowles BB, Damjanov I, Solter D. Monoclonal antibody to murine embryos defines a stage specific embryonic antigen expressed on mouse embryos and human teratocarcinoma cells. Cell 1982; 30:697-705.
[157]Shim H, Gutierrez-A dan A, Chen LR, BonDurant RH, Behboodi E, Anderson GB. Isolation of pluripotent stem cells from cultured porcine primordial germ cells. Biol Reprod 1997; 57:1089-1095.
[158]Shim SW, Han DW, Yang JH, Lee BY, Kim SB, Shim H, Lee HT. Derivation of embryonic germ cells from post migratory primordial germ cells, and methylation analysis of their imprinted genes by bisulfite genomic sequencing. Mol Cells 2008; 25:358-367.
[159]Smith AG, Nichols J, Robertson M, Rathjen PD. Differentiation inhibiting activity(DIA/LIF) and mouse development[J]. Dev Biol 1992; 151:339-315.
[160]Solter D, Knowles BB. Monoclonal antibody defining astage-specific mouse embryonic antigen(SSEA-1). Proc Natl Acad Sci USA 1978; 75:5565-5569.
[161]Soodeen-Karamath S, Gibbins AM. Apparent absence of oct3/4 from the chicken genome. Mol Reprod Dev 2001; 58:137-148.
[162]Soto-Gutierrez A, N Navarro-Alvarez, DB Zhao, JD Rivas-Carrillo, J Lebkowski, N Tanaka, IJ Fox and N Kobayashi. Differentiation of mouse embryonic stem cells to hepatocyte-like cells by co-culture with human liver nonparenchymal cell lines. Nature Protocols 2007; 2:347-356.
[163]Sroczynska P, Lancrin C, Pearson S, Kouskoff V, Lacaud G. In Vitro Differentiation of Mouse Embryonic Stem Cells as a Model of Early Hematopoietic Development. Methods Mol Biol 2009; 538:1-18.
[164]Stern CD. The chick:a great model system becomes even greater. Dev Cell 2005; 8:9-17.
[165]Stewart CL, Gadi L, Bhatt H. Stem cells from primordial germ cells can reeter the germ line. Dev.Biol 1994; 161:626-628.
[166]Sulston JE, Schierenberg E, White JG, Thomson JN. The embryonic cell lineage of the nematode Caenorhabditis elegans. Dev Biol 1983; 100:64-119.
[167]Sutasurya LA. Appearance of primordial germ cells in young chick blastoderm cultured in vitro. Development Growth and Differentiation 1983; 25:517-521.
[168]Suter DM, Cartier L, Bettiol E, Tirefort D, Jaconi ME, Dubois-Dauphin M, Krause KH. Rapid generation of stable transgenic embryonic stem cell lines using modular lentivectors. Stem Cells 2006; 24:615-623.
[169]Sutton J, Costa R, Klug M, Field L, Xu D, Largaespada DA, Fletcher CF, Jenkins NA, Copeland NG, Klemsz M, Hromas R. Genesisi, a winged helix transcriptional repressor with expression restricted to embryonic stem cells. J Biol Chem 1996; 271:23126-23133.
[170]Swift CH. Origin and early history of the primordial germ cells in chick. Am J of anat 1914; 15:483-516.
[171]Takahashi K, Yamanaka S Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006; 126:663-676.
[172]Takaki M, Nakayama S, Misawa H, Nakagawa T, Kuniyasu H. In vitro formation of enteric neural network structure in a gut-like organ differentiated from mouse embryonic stem cells. Stem Cells 2006; 24:1414-1422.
[173]Takashi M, Hisako M. Glycoconjugate antigens expressed on stem cells and early embryonic cells. Glycoconjugate 2004; 21:41-45.
[174]Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jone JM. Embryonic stem cell line derived from human blastcysts. Science 1998; 282:1145-1147.
[175]Tilg H, Moschen AR. Adipocytokines:mediators linking adipose tissue, inflammation and immunity. Nat Rev Immunol 2006; 6:772-783.
[176]Till JE, McCulloch EA. A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat Res 1961; 14:213-222.
[177]Tompers DM, Foreman RK, Wang Q, Kumanova M, Labosky PA. Foxd3 is required in the trophoblast progenitor cell lineage of the mouse embryo. Dev Biol 2005; 285:126-137.
[178]Torihashi S, Hattori T, Hasegawa H, Kurahashi M, Ogaeri T, Fujimoto T.The expression and crucial roles of BMP signaling in development of smooth muscle progenitor cells in the mouse embryonic gut. Differentiation 2009; 77:277-289.
[179]Tseng YH, KM Kriauciunas, E Kokkotou and CR Kahn. Differential roles of insulin receptor substrates in brown adipocyte differentiation. Mol Cell Biol 2004; 24:1918-1929.
[180]Tsunekawa N, Naito M, Sakai Y, Nishida T and Noce T. Isolation of chicken vasa homolog gene and tracing the origin of primordial germ cells. Development 2000; 127:2741-2750.
[181]Turnpenny L, Brickwood S, Spalluto CM, Piper K, Cameron IT, Wilson DI, Hanley NA. Derivation of human embryonic germ cells:an alternative source of pluripotent stem cells. Stem Cells 2003; 21:598-609.
[182]Urven LE, Abbott UK, Erickson CA. Distribution of extraceliular matrix in the migratory pathway of avian primordial germ cells. Anatomical Record 1989; 224:14-21.
[183]Valbuena D, Galan A, Sanchez E, Poo ME, Gomez E, Sanchez-Luengo S, Melguizo D, Garcia A, Ruiz V, Moreno R, Pellicer A, Simon C. Derivation and characterization of three new Spanish human embryonic stem cell lines (VAL-3-4-5) on human feeder and in serum-free conditions. Reproductive BioMedicine 2006; 13:875-886.
[184]Vallier L, Alexander M, Pedersen R. Conditional Gene Expression in Human Embryonic Stem Cells. Stem Cells 2007; 25:1490-1497.
[185]van de Lavoir MC, Diamond JH, Leighton PA et al. Germline transmission of genetically modified primordial germ cells. Nature 2006; 441:766-769.
[186]Viswanathan S, Benatar T, Rose-John S, Lauffenburger DA, Zandstra PW. Ligand/receptor signaling threshold (LIST) model accounts for gp130-mediated embryonic stem cell self-renewal responses to LIF and HIL-6. Stem Cells 2002; 20:119-138.
[187]Wang J, Jiao F, Pan XH, Xie SY, Li ZL, Niu XH, Du LX. Directed differentiation of chick embryonic germ cells into neural cells using retinoic acid induction in vitro. J Neurosci Methods 2009; 177:168-176.
[188]Wang R, Clark R, Bautch VL. Embryonic stem cell-derived cysticembryoid bodies form vascular channels:an in vitro model of blood vessel development. Development 1992; 114:303-316.
[189]Wakitani S,Takaoka K, Hattori T, Miyazawa N, Iwanaga T, Takeda S, Watanabe TK, Tanigami A. Embryonic stem cells injected into the mouse knee joint form teratomas and subsequently destroy the joint. Rheumatology 2003; 42:162-165.
[190]Wdziekonski B, Villageois P, Dani C. Differentiation of mouse embryonic stem cells and of human adult stem cells into adipocytes. Curr Protoc Cell Biol 2007; 23:23-24.
[]Wei H, Juhasz O, Li J, Tarasova YS, Boheler KR. Embryonic stem cells and cardiomyocyte differentiation:phenotypic and molecular analyses. J Cell Mol Med 2005; 9:804-817.
[191]Wentworth BC, Tsaihhallett JH, Ghikamune DS, Rajcic-Spasojevic. Manipulation of avian primordial germ cells and gondal differentiation. Poultry science 1989; 69:999-1010.
[192]Wichterle H, Lieberam I, Porter JA, Jessell TM. Directed differentiation of embryonic stem cells into motor neurons.Cell 2002; 110:385-397.
[193]Willson ML, McElnea C, Mariani J, Lohof AM, Sherrard RM. BDNF increases homotypic olivocerebellar reinnervation and associated fine motor and cognitive skill. Brain 2008; 131:1099-1112.
[194]Wilson EB. The Cell in Development and Inheritance.1896; Macmillan, New York.
[195]Yamamoto H, Quinn G, Asari A, Yamanokuchi H, Teratani T, Terada M, Ochiya T. Differentiation of embryonic stem cells into hepatocytes:Biological functions and therapeutic application. Hepatology 2003; 37:983-993.
[196]Yamamoto Y, Usui F, Nakamura Y, Ito Y, Tagami T, Nirasawa K, Matsubara Y, Ono T, Kagami H. A Novel Method to Isolate Primordial Germ Cells and Its Use for the Generation of Germline Chimeras in Chicken. Biol Reprod 2007; 77:115-119.
[197]Yasuda Y, Tajima A, Fujimoto T, Kuwana T. A method to obtain avian germ line chimeras using isolated primordial germ cells. J Reprod Fert 1992; 96:521-528.
[198]Ying QL, Nichols J, Chambers I, Smith A. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 2003;115:281-292.
[199]Yoon BS, Yoo SJ, Lee JE, You S, Lee HT, Yoon HS. Enhanced differentiation of human embryonic stem cells into cardiomyocytes by combining hanging drop culture and 5-azacytidine treatment. Differentiation 2006; 74:149-159.
[200]Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin Ⅱ, Thomson JA. Induced pluripotent stem cell lines derived from human somatic cells. Science 2007; 318:1917-1920.
[201]Zhang D, Jiang W, Liu M, Sui X, Yin X, Chen S, Shi Y, Deng H. Highly efficient differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells. Cell Res 2009; 19:429-438.
[202]李碧春,陈国宏,赵东伟,王克华,钱菊汾.鸡胚PGCs迁移与性腺发育关系的研究.扬州大学学报2002；23：18-26.
[203]李碧春,秦洁,肖小珺,陈国宏,吴信生,吴圣龙.鸡胚不同发育时期原始生殖细胞的分离方法.动物学报2003；49：835-842.
[2]Aleckovic M, Simon C. Is teratoma formation in stem cell research a characterization tool or a window to developmental biology? Reproductive BioMedicine 2008; 17:274-284.
[3]Aramaki S, Sato F, Kato T, Soh T, Kato Y, Hattori MA. Molecular cloning and expression of dead end homologue in chicken primordial germ cells. Cell and Tissue Res 2007; 330:45-52.
[4]Armstrong L, Saretzki G, Peters H, Wappler I, Evans J, Hole N, von Zglinicki T, and Lako M. Overexpression of telomerase confers growth advantage, stress resistance, and enhanced differentiation of ESCs toward the hematopoietic lineage. Stem Cells 2005; 23:516-529.
[5]Arsenijevic Y, Weiss S, Schneider B, Aebischer P. Insulin-Like Growth Factor-I Is Necessary for Neural Stem Cell Proliferation and Demonstrates Distinct Actions of Epidermal Growth Factor and Fibroblast Growth Factor-2. J. Neurosci 2001; 21:7194-7202.
[6]Babaie Y, Herwig R, Greber B, Brink TC, Wruck W, Groth D, Lehrach H, Burdon T, Adjaye J. Analysis of Oct4-dependent transcriptional networks regulating self-renewal and pluripotency in human embryonic stem cells. Stem Cells 2007; 25:500-510.
[7]Bain G, Kitchens D, Yao M, Huettner JE, Gottlieb DI. Embryonic stem cells express neuronal properties in vitro. Dev Biol.1995; 168:342-357.
[8]Boilly B, Vercoutter-Edouart AS, Hondermarck H, Nurcombe V, Le Bourhis X, Fernig DG. FGF signals for cell proliferation and migration through different pathways. Cytokine Growth Fact Rev2000; 11:295-302.
[9]Bowles J, Knight D, Smith C, Wilhelm D, Richman J, Mamiya S, Yashiro K, Chawengsaksophak K, Wilson MJ, Rossant J, Hamada H, Koopman P. Science 2006; 312:596-600
[10]Boyer LA, Lee TI, Cole MF, Johnstone SE, Levine SS, Zucker JP, Guenther MG, Kumar RM, Murray HL, Jenner RG, Gifford DK, Melton DA, Jaenisch R, Young RA. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 2005; 122:947-956
[11]Buehr M, Mclaren A, Bartley A, Darling S. Proliferation and migration of primordial germ cells in We/We mouse embryos. Dev Dyn 1993; 198:182-189.
[12]Burlacu A, Rosca AM, Maniu H, Titorencu I, Dragan E, Jinga V, Simionescu M. Promoting effect of 5-azacytidine on the myogenic differentiation of bone marrow stromal cells. Eur J Cell Biol 2008; 87:173-84.
[13]Cameron CM, Harding F, Hu WS, Kaufman DS. Activation of Hypoxic Response in Human Embryonic Stem Cell-Derived Embryoid Bodies. Experimental Biology and Medicine 2008; 233:1044-1057.
[14]Cameron CM, Hu WS, Kaufman DS. Improved development of human embryonic stem cell-derived embryoid bodies by stirred vessel cultivation. Biotechnol Bioeng 2006; 94:938-948.
[15]Canon S, Herranz C, Manzanares M. Germ cell restricted expression of chick Nanog. Dev Dynamic.2006; 235:2889-2894.
[16]Carpenedo Rd L, Sargent CY, McDevitt TC. Rotary Suspension Culture Enhances the Efficiency, Yield, and Homogeneity of Embryoid Body Differentiation. Stem Cells 2007; 25:
2224-2234.
[17]Chambers I, Colby D, Robertson M, Nichols J, Lee S, Tweedie S, Smith A. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 2003; 113:643-655.
[18]Chan SR, Blackburn EH. Telomere and telomerase. Phil Trans R Soc B 2004; 359:109-121.
[19]Chang IK, Tajima A, Chikamune T, Ohno T. Proliferation of chick primordial germ cells on stroma cells from the germinal ridge. Cell Biol Int 1995; 19:143-149.
[20]Chang TH, Polakis SE. Differentiation of 3T3-L1 fibroblasts to adipocytes. Effect of insulin and indomethacin on the levels of insulin receptors. J. Biol. Chem 1978; 253:4693-4696.
[21]Chen HF, Kuo HC, Chien CL, Shun CT, Yao YL, Ip PL, Chuang CY, Wang CC, Yang YS, Ho HN. Derivation, characterization and differentiation of human embryonic stem cells: comparing serum-containing versus serum-free media and evidence of germ cell differentiation. Hum. Reprod 2007; 22:567-577.
[22]Cherny R A, Merei J. Evidence for pluripotency of bovine primordial germ cell derived cell lines initiated in long-term culture. Theriogenoiogy 1994; 4:175.
[23]Chew JL, Loh YH, Zhang W, Chen X, Tam WL, Yeap LS, Li P, Ang YS, Lim B, Robson P, Ng HH. Reciprocal Transcriptional Regulation of Pou5fl and Sox2 via the Oct4/Sox2 Complex in Embryonic Stem Cells. Mol. Cell. Biol 2005; 25:6031-6046.
[24]Chinzei R, Tanaka Y, Shimizu-Saito K, Hara Y, Kakinuma S, Watanabe M, Teramoto K, Arii S, Takase K, Sato C, Terada N, Teraoka H. Embryoid-body cells derived from a mouse embryonic stem cell line show differentiation into functional hepatocytes. Hepatology 2002; 36:22-29.
[25]Choi D, Lee H, Jee S, Jin S, Koo SK, Paik SS, Jung SC, H SY, Lee KS, Oh B. In Vitro Differentiation of Mouse Embryonic Stem Cells:Enrichment of Endodermal Cells in the Embryoid Body. Stem Cells 2005; 23:817-827.
[26]Claswon RC and Domm LV. Developmental changes in glycogen content of primordial germ cells in chick embryo. Proc of the Society For Experi Biol Med 1963; 112:533-537.
[27]Coleman CM. Chicken embryo as a model for regenerative medicine. Birth Defects Res (Part C) 2008; 84:245-256.
[28]Dang SM, Zandstra PW. Scalable production of embryonic stem cell-derived cells. Methods Mol Biol 2005; 290:353-364.
[29]Dani C. Embryonic stem cell-derived adipogenesis. Cells Tissues Organs 1999; 165:173-180.
[30]De Felici M, McLaren A. Isolation of mouse primordial germ cells. Exp Cell Res 1982; 142:476-482.
[31]Desbaillets I, Ziegler U, Groscurth P, Gassmann M. Embryoid bodies:an in vitro model of mouse embryogenesis. Exp Physiol 2000; 85:645-651.
[32]Doetschman TC, Eistetter H, Katz M, Schmidt W, Kemler R. The in vitro development of blastocyst-derived embryonic stem cell lines:formation of visceral yolk sac, blood islands and myocardium. J Embryol Exp Morphol 1985; 87:27-45.
[33]Dolci S, Williams DE, Ernst MK, Resnick JL, Brannan CI, Lock LF, Lyman SD, Boswell HS, Donovan PJ. Requirement for mast cell growth factor for primordial germ cell survival in culture. Nature 1991; 352:809-811.
[34]Donovan PJ, Gearhart J. The end of the beginning for pluripotent stem cells. Nature 2001; 414:92-97.
[35]Donovan PJ, de Miguel MP. Turning germ cells into stem cells. Curr Opin Genet Dev 2003; 13:463-71.
[36]Durcova-Hills G, Adams IR, Barton SC, Surani MA, McLaren A. The role of exogenous fibroblast growth factor-2 on the reprogramming of primordial germ cells into pluripotent stem cells. Stem Cells 2006; 24:1441-1449.
[37]Durcova-Hills G, Wianny F, Merriman J, Zernicka-Goetz M, McLaren A. Development fate of embryonic germ cells(EGCs), in vivo an in vitro. Differentiation 2003; 71:135-141.
[38]Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature 1981; 292:154-156.
[39]Eyal-Giladi H, Ginsburg M and Farbarov A. Avian primordial germ cells are of epiblast origin. J of Embryo and Experi Morpho 1981; 63:139-147.
[40]Fang D, Leishear K, Nguyen TK, Finko R, Cai K, Fukunaga M, Li L, Brafford PA, Kulp AN, Xu X, Smalley KS, Herlyn M. Defining the Conditions for the Generation of Melanocytes from Human Embryonic Stem Cells. Stem Cells 2006; 24:1668-1677.
[41]Felici MD, Scaldaferri ML, Lobascio M, Iona S, Nazzicone V, Klinger FG, Farini D. Experimental approaches to the study of primordial germ cell lineage and proliferation. Hum. Reprod 2004; 10:197-206.
[42]Fiegel HC, Lioznov MV, Cortes-Dericks L, Lange C, Kluth D, Fehse B, Zander AR. Liver-specific gene expression in cultured human hematopoietic stem cells. Stem Cells 2003; 21:98-104.
[43]Fijnvandraat AC, van Ginneken AC, Schumacher CA, Boheler KR, Lekanne Deprez RH, Christoffels VM, Moorman AF. Cardiomyocytes purified from differentiated embryonic stem cells exhibit characteristics of early chamber myocardium. J Mol Cell Cardiol 2003; 35:1461-1472.
[44]Fok EY, Zandstra PW. Shear-Controlled Single-Step Mouse Embryonic Stem Cell Expansion and Embryoid Body-Based Differentiation. Stem Cells 2005; 23:1333-1342.
[45]Fong H, Hohenstein KA, Donovan PJ. Regulation of Self-renewal and pluripotency by Sox2 in human embryonic stem cells. Stem Cells 2008; 26:1931-938.
[46]Fujimoto T, Kuwana T, Maeda-Suga H. Attraction of chick primordial germ cells by gonadal anlage in vitro. Anatomical Record 1986; 215:403-406.
[47]Fujimito T, Ukeshima A, and Kivofuji R. The origin, migration and morphology of the primordial germ cells in the chick embryos. Anatomical Record 1976; 185:139-154.
[48]Fukuda K. Development of regenerative cardiomyocytes from mesenchymal stem cells for cardiovascular tissue engineering. Artif Organs 2001; 25:187-193.
[49]Gassanov N, Er F, Zagidullin N, Jankowski M, Gutkowska J, Hoppe UC. Retinoid acid-induced effects on atrial and pacemaker cell differentiation and expression of cardiac ion channels. Differentiation 2008 76:971-980.
[50]Gearhart J. New potential for human embryonic stem cells. Science 1998; 282:1061-1065.
[51]Gimeno RE, Klaman LD. Adipose tissue as an active endocrine organ:recent advances. Curr Opin Pharmacol 2005; 5:122-128.
[52]Ginsburg M, Eyal-Giladi H. Primordial germ cells of the young chick blastoderm originate from the central zone of the area pellucida irrespective of the embryo-forming process. Development 1987; 101:209-219.
[53]Ginsburg M. Primordial germ cell formation in birds. In:J Marsh and J Goode. Germline development, Ciba Foundation Symposium 182. London.1994; 52-61.
[54]Ginsburg M and Eyal-Giladi H. Temporal and spatial aspects of the gradual migration of primordial germ cells from epiblast into the germinal crescent in the avian embryo. Embryo Experi Morpho 1986; 95:53-71.
[55]Glaser T, Opitz T, Kischlat T, Konang R, Sasse P, Fleischmann BK, Engel W, Nayernia K, Briistle O. Adult Germ Line Stem Cells as a Source of Functional Neurons and Glia. StemCells 2008; 26:2434-2443.
[56]Godin I, Deed R, Cooke J, Zsebo K, Dexter M, Wylie CC. Effects of the steel gene product on mouse primordial germ cells in culture. Nature 1991; 352:807-809.
[57]Godin I, Wylie CC. TGF R inhibits proliferation and has a chemotropic effect on mouse primordial germ cells in culture. Development 1991; 113:1451-1457.
[58]Godoy MC, Tarelli R, Ferrari CC, Sarchi MI, Pitossi FJ. Central and systemicIL-1 exacerbates neurodegeneration and motor symptoms in a model of Parkinson's disease. Brain 2008; 131:1880-1894.
[59]Goldfarb M. Signaling by fibroblast growth factors:the inside story. Science STKE 2001; 2001:PE37.
[60]Gu Y, Runyan Ch, Shoemaker A, Surani A, Wylie Chris. Steel factor controls primordial germ cell survival and motility from the time of their specification in the allantois, and provides a continuous niche throughout their migration. Development 2009; 136:1295-1303.
[61]Guo Y, Costa R, Ramsey H, Starnes T, Vance G, Robertson K, Kelley M, Reinbold R, Scholer H, Hromas R. The embryonic stem cell transcription factor Oct-4 and FoxD3 interact to regulate endodermal-specific promoter expression. Proc. Natl. Acad. Sci.U.S.A 2002; 99: 3663-3667.
[62]Haegele L, Ingold B, Naumann H, Tabatabai G, Ledermann B, Brandner S. Wnt signaling inhibits neural differentiation of embryonic stem cells by controlling bone morphogenetic protein expression. Mol Cell Neurosci 2003; 24:696-708.
[63]Hamazaki T, Iiboshi Y, Oka M, Papst PJ, Meacham AM, Zon LI, Terada N. Hepatic maturation in differentiating embryonic stem cells in vitro. FEBS Lett 2001; 497:15-19.
[64]Han JY. Germ cells and transgenesis in chickens. Comp Immunol Microbiol Infect Dis 2009; 32:61-80.
[65]Han JY, Park TS, Hong JH, Jeong DK, Kim JN, Kim KD, Lim JM. Production of germline chimeras by transfer of chicken gonadal primordial germ cells maintained in vitro for an extended period. Theriogenology 2002; 58:1531-1539.
[66]Hart AH, Hartley L, Ibrahim M, Robb L. Identification, cloning and expression analysis of the pluripotency promoting Nanog genes in mouse and human[J]. Dev Dyn 2004; 230:187-198.
[67]Hay DC, Sutherland L, Clark J, Burdon T. Oct-4 knockdown induces similar patterns of endoderm and trophoblast differentiation markers in human and mouse embryonic stem cells. Stem Cells 2004; 22:225-235.
[68]He JQ, Ma Y, Lee Y, Thomson JA, Kamp TJ. Human embryonicstem cells develop into multiple types of cardiac myocytes:action potential characterization. Circ Res 2003; 93:32-39.
[69]Hedlund E, Pruszak J, Ferree A, Vinuela A, Hong S, Isacson O, Kim KS. Selection of embryonic stem cell-derived enhanced green fluorescent protein-positive dopamine neurons using the tyrosine hydroxylase promoter is confounded by reporter gene expression in immature cell populations. Stem Cells 2007; 25:1126-1135.
[70]Heins N, Englund MC, Sjoblom C, Dahl U, Tonning A, Bergh C, Lindahl A, Hanson C, Semb H. Derivation, characterization, and differentiation of human embryonic stem cells. Stem Cells 2004; 22:367-376.
[71]Henderson JK, Draper JS, Baillie HS, Fishel S, Thomson JA, Moore H, Andrews PW. Preimplantation human embryos and embryonic stem cells show comparable expression of stage-specific embryonic antigens. Stem Cells 2002; 20:329-337.
[72]Hillel AT, Varghese S, Petsche J, Shamblott MJ, Elisseeff JH. Embryonic germ cells are capable of adipogenic differentiation in vitro and in vivo. Tissue Eng PartA 2009; 15:479-486.
[73]Hopfl G, Gassmann M, Desbaillets I. Differentiating embryonic stem cells into embryoid bodies. Methods Mol Biol 2004; 254:79-98
[74]Horiuchi H, Tategaki A, Yamashita Y, Hisamatsu H, Ogawa M, Noguchi T, Aosasa M, Kawashima T, Akita S, Nishimichi N, Mitsui N, Furusawa S, Matsuda H.Chicken leukemia inhibitory factor maintains chicken embryonic stem cells in the undifferentiated state. J Biol Chem 2004; 279:24514-24520.
[75]Hromas R, Ye H, Spinella M, Dmitrovsky E, Xu D, Costa RH. Genesisi, a winged helix transcriptional repressor, has embryonic expression limited to neural crest, and stimulates proliferation in vitro in a neural development model. Cell Tissue Res 1999; 297:371-382.
[76]Horiuchi H, Tategaki A, Yamashita Y, Hisamatsu H, Ogawa M, Noguchi T, Aosasa M, Kawashima T, Akita S, Nishimichi N, Mitsui N, Furusawa S, Matsuda H. Chicken leukemia inhibitory factor maintains chicken embryonic stem cells in the undifferentiated state. J Biol Chem 2004; 279:24514-24520.
[77]Hubner K, Fuhrmann G, Christenson LK, Kehler J, Reinbold R, De La Fuente R, Wood J, Strauss JF 3rd, Boiani M, Scholer HR. Derivation of oocytes from mouse embryonic stem cells. Science 2003; 300:1251-1256.
[78]Imamura O, Satoh Y, Endo S, Takishima K. Analysis of Extracellular Signal-Regulated Kinase 2 Function in Neural Stem/Progenitor Cells via Nervous System-Specific Gene Disruption. Stem Cells 2008; 26:3247-3256.
[79]Ishizaka S, Shiroi A, Kanda S, Yoshikawa M, Tsujinoue H, Kuriyama S, Hasuma T, Nakatani K, Takahashi K. Development of hepatocytes from ES cells after transfection with the HNF-3beta gene. FASEB J 2002; 16:1444-14446.
[80]Itskovitz-Eldor J, Schuldiner M, Karsenti D, Eden A, Yanuka O, Amit M, Soreq H, Benvenisty N. Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol Med 2000; 6:88-95.
[81]Ji J, Vijayaragavan K, Bosse M, Menendez P, Weisel K, Bhatia M. OP9 Stroma Augments Survival of Hematopoietic Precursors and Progenitors During Hematopoietic Differentiation from Human Embryonic Stem Cells. Stem Cells 2008; 26:2485-2495.
[82]Jiang Y, Jahaglrdar BN, Reinhardt RL. Plurlpotency of mesenchymal stem cell derived from adult marrow. Nature 2002; 418:41-49.
[83]Kanatsu-Shinohara M, Inoue K, Lee J, Yoshimoto M, Ogonuki N, Miki H, Baba S, Kato T, Kazuki Y, Toyokuni S, Toyoshima M, Niwa O, Oshimura M, Heike T, Nakahata T, Ishino F, Ogura A, Shinohara T. Generation of pluripotent stem cells from neonatal mouse testis. Cell 2004; 119:1001-1012.
[84]Kanatsu-Shinohara M, Inoue K, Ogonuki N, Miki H, Yoshida S, Toyokuni S, Lee J, Ogura A,
Shinohara T. Leukemia inhibitory factor enhances formation of germ cell colonies in neonatal mouse testis culture. Biol Reprod,2007; 76:55-62.
[85]Karagenc L, Petitte JN. Soluble factors and the emergence of chick primordial germ cells in vitro. Poult Sci 2000; 79:80-85.
[86]Kerr DA, Llado J, Shamblott MJ, Maragakis NJ, Irani DN, Crawford TO, Krishnan C, Dike S, Gearhart JD, Rothstein JD. Human Embryonic Germ Cell Derivatives Facilitate Motor Recovery of Rats with Diffuse Motor Neuron Injury. J Neurosci 2003; 23:5131-5140.
[87]Khoo ML, McQuade LR, Smith MS, Lees JG, Sidhu KS, Tuch BE. Growth and Differentiation of Embryoid Bodies Derived from Human Embryonic Stem Cells:Effect of Glucose and Basic Fibroblast Growth Factor. Biol Reprod 2005; 73:1147-1156.
[88]Khoo ML, Shen B, Tao H, Ma DD. Long-term serial passage and neuronal differentiation capability of human bone marrow mesenchymal stem cells. Stem Cells Dev 2008; 17:883-896.
[89]Kim JH, Auerbach JM, Rodriguez-Gomez JA, Velasco I, Gavin D, Lumelsky N, Lee SH, Nguyen J, Sanchez-Pernaute R, Bankiewicz K, McKay R. Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson's disease. Nature 2002; 418:50-56.
[90]Kim MS, Hwang NS, Lee J, Kim TK, Leong K, Shamblott MJ, Gearhart J, Elisseeff J. Musculoskeletal Differentiation of Cells Derived from Human Embryonic Germ Cells. Stem Cells 2005; 23:113-123.
[91]Kim YJ, Cho SY, Yun CH, Moon YS, Lee TR, Kim SH. Transcriptional activation of Cidec by PPARgamma2 in adipocyte. Biochem Biophys Res Commun 2008; 377:297-302
[92]Kleinsmith LJ, Pierce GB. Multipotentiality of single embryonal carcinoma cells. Cancer Res 1964; 24:1544-1551.
[93]Klug MG, Soonpaa MH, Koh GY, Field LJ. Genetically selected cardiomyocytes from differentiating embronic stem cells form stable intracardiac grafts. J Clin Invest 1996; 98:216-224.
[94]Koshimizu U, Taga T, Watanabe M, Saito M, Shirayoshi Y, Kishimoto T, Nakatsuji N. Functional requirement of gp130-mediated signaling for growth and survival of mouse primordialgerm cells in vitro and derivation of embryonic germ (EG) cells. Development 1996; 122:1235-1242.
[95]Kuhholzer B, Baguisi A, Overstrom EW. Long-term culture and characterization of goat primordial germ cells.2000; 53:1071-1079.
[96]Kuwana T, Fujimoto T. locomotion and scanning electron microscopic observation of primordial germ cells from embryonic chick blood in vitro. Anat Rec 1984; 209:337-343.
[97]Kuwana T. and Rogulska T. Migratory mechanism of chick primordial germ cells toward gonadal anlarge. Cellular and Mol Biol 1999; 45:725-736.
[98]Labosky PA, Barlow DP, Brigid LM. Mouse embryonic germ (EG) cell lines:transmission through the germline and differences in the methylation imprint of insulin-like growth factor 2 receptor (Igf2r) gene compared with embryonic stem (ES) cell lines. Development 1994; 120:3197-3204.
[99]Labosky PA, Kaestner KH. The winged helix transcriptional factor Hfh2 is expressed in neural crest and spinal cord during mouse development. Mech Dev 1998; 76:185-190.
[100]Ladewig J, Koch P, Endl E, Meiners B, Opitz T, Couillard-Despres S, Aigner L, Brustle O. Lineage selection of functional and cryopreservable human embryonic stem cell-derived neurons. Stem Cells 2008; 26:1705-1712.
[101]Lai E, Rosen OM, Rubin CS. Dexamethasone regulates the beta-adrenergic receptor subtype expressed by 3T3 L1 preadipocytes and adipocytes. J. Biol. Chem 1982; 257:6691-6696.
[102]Lavial F, Acloque H, Bertocchini F, Macleod DJ, Boast S, Bachelard E, Montillet G, Thenot S, Sang HM, Stem CD, Samarut J, Pain B. The Oct4 homologue PouV and Nanog regulate pluripotency in chicken embryonic stem cells. Development 2007; 134:3549-3563.
[103]Lazzari L, Henschler R, Lecchi L, Rebulla P, Mertelsmann R, Sirchia G. Interleukin-6 andinterleukin-11 act synergistically with thrombopoietin and stem cell factor to modulate ex vivo expansion of human CD41+and CD61+megakaryocytic cells. Haematologica 2000; 85:25-30.
[104]Lee HS, Huang GT, Chiang H, Chiou LL, Chen MH, Hsieh CH, Jiang CC. Multipotential Mesenchymal Stem Cells from Femoral Bone Marrow Near the Site of Osteonecrosis. Stem Cells 2003; 21:190-199.
[105]Liao W, Nguyen MT, Yoshizaki T, Favelyukis S, Patsouris D, Imamura T, Verma IM, Olefsky JM. Suppression of PPAR-y attenuates insulin-stimulated glucose up take by affecting both GLUT1 and GLUT4 in 3T3-L1 adipocytes. Am J Physiol Endocrinol Metab 2007; 293:E219-E227.
[106]Liew CG, Draper JS, Walsh J, Moore H, Andrews PW. Transient and stable transgene expression in human embryonic stem cells. Stem Cells 2007; 25:1521-1528.
[107]Liu H, Kim JM, Aoki F. Regulation of histone H3 lysine 9methylation in oocytes and early pre-implantation embryos. Development 2004; 131:2269-2280.
[108]Lopez-Carballo G, Moreno L, Masia S, Perez P, Barettino D. Activation of the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway by Retinoic Acid Is Required for Neural Differentiation of SH-SY5Y Human Neuroblastoma Cells. J. Biol. Chem 2002; 277:25297-25304.
[109]Marion RM, Strati K, Li H, Tejera A, Schoeftner S, Ortega S, Serrano M, Blasco MA. Telomeres acquire embryonic stem cell characteristics in induced pluripotent stem cells. Cell Stem Cell 2009; 4:141-154.
[110]Matsubara Y, K Sato, H Ishii and Y Akiba. Changes in mRNA expression of regulatory factors involved in adipocyte differentiation during fatty acid induced adipogenesis in chicken. Comp Biochem Physiol (Part A) 2005; 141:108-115.
[111]Matsuda T, Nakamura T, Nakao K, Arai T, Katsuki M, Heike T, Yokota T. STAT3 activation is sufficient to maintain an undifferentiated state of mouse embryonic stem cells. EMBO J 1999; 18:4261-4269.
[112]Matsui Y, Toksoz D, Nishikawa S, Williams D, Zsebo K, Hogan BL. Effect of steel factor and leukaemia inhibitory factor on murine primordial germ cells in culture. Nature 1991, 353:750-752.
[113]Matsui Y, Zsebo K, Hogan BL. Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture. Cell 1992; 70:841-847.
[114]Medvedev SP, Shevchenko AI, Mazurok NA, Zakiian SM. OCT4 and NANOG are the key genes in the system of pluripotency maintenance in mammalian cells. Genetika 2008; 44:1589-1608.
[115]Mika G. Primordial germ cell development in avian. Poultry Science 1997; 76:91-95.
[116]Mikkola HK, Lowry WE, Plath K, Clark AT. Derivation of Primordial Germ Cells from Human Embryonic and Induced Pluripotent Stem Cells Is Significantly Improved by Coculture with Human Fetal Gonadal Cells. Stem Cells 2009; 27:783-795.
[117]Mitani T, Takahashi N, Kawase E. Proliferation of alkaline phosphatase-positive cells from cultured primordial germ cells in rat. Theriogenology 1994; 41:336.
[118]Mitsui K, Tokuzawa Y, Itoh H, Segawa K, Murakami M, Takahashi K, Maruyama M, Maeda M, Yamanaka S. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 2003; 113:631-642.
[119]Miyazaki S, Yamato E, Miyazaki J. Regulated Expression of pdx-1 Promotes In Vitro Differentiation of Insulin-Producing Cells From Embryonic Stem Cells. Diabetes 2004; 53:1030-1037.
[120]Mozdziak PE, Angerman-Stewart J, Rushton B, Pardue SL, Petitte JN. Isolation of chicken primordial germ cells using fluorescence-activated cell sorting. Poult Sci 2005; 84:594-600.
[121]Nakamura M, Kuwana T, Myiayama Y, Fujimoto T. Extragonadal distribution primordial germ cells in the early chick embryo. Anat Rec 1988; 222:90-94.
[122]Nakamura Y, Yamamoto Y, Usui F, Mushika T, Ono T, Setioko AR, Takeda K, Nirasawa K, Kagami H, TagamiT. Migration and Proliferation of Primordial Germ Cells in the Early Chicken Embryo. Poult Sci 2007; 86:2182-2193.
[123]Naugler KM, Baer KA, Ropeleski MJ. Interleukin-11 antagonizes Fas ligand-mediated apoptosis in IEC-18 intestinal epithelial crypt cells:role of MEK and Akt-dependent signaling. Am J Physiol Gastrointest Liver Physiol, Mar 2008; 294:G728-G737.
[124]Nichols J, Davidson D, Taga T, Complementary tissue-specific expression of LIF and LIF-receptor mRNAs in early mouse embryogenesis. Mech Dev 1996; 57:123-131.
[125]Nishimoto M, Miyagi S, Yamagishi T, Sakaguchi T, Niwa H, Muramatsu M, Okuda A. Oct-3/4 maintains the proliferative embryonic stem cell state via specific binding to a variant octamer sequence in the regulatory region of the UTF1 locus. Mol Cell Biol 2005; 25:5084-5094.
[126]Nieuwkoop PD, Sutasurya LA. Primordial Germ Cells in the Chordates. Cambridge University Press; 1979.
[127]Niwa H, Burdon T, Chambers I, Smith A. Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes & Dev 1998; 12:2048.
[128]Niwa H, Miyazaki J, Smith A. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nature Genetics 2000; 24:372-376.
[129]O'Connor MD, Kardel MD, Iosfina I, Youssef D, Lu M, Li MM, Vercauteren S, Nagy A, Eaves CJ. Alkaline phosphatase-positive colony formation is a sensitive, specific, and quantitative indicator of undifferentiated human embryonic stem cells. Stem Cells 2008; 26:1109-1116.
[130]Ono T, Machida Y. Immunomagnetic purification of viable primordialgerm cells of Japanese quail (Coturnix japonica). Comp Biochem Physiol A Mol Integr Physiol 1999; 122:255-259.
[131]Okada Y, T Shimazakia, G Sobueb and H Okano. Retinoic-acid-concentration-dependent acquisition of neural cell identity during in vitro differentiation of mouse embryonic stem cells. Dev Biol 2004; 275:124-142.
[132]Pan GJ, Chang ZY, Scholer HR, Pei D. Stem cell pluripotency and transcription factor Oct4. Cell Res 2002; 12:321-319.
[133]Pan Y, Chen X, Wang S, Yang S, Bai X, Chi X, Li K, Liu B, Li L. In vitro neuronal differentiation of cultured human embryonic germ cells. Biochem Biophys Res Commun 2005; 327:548-556.
[134]Paquin J, Danalache BA, Jankowski M, McCann SM, Gutkowska J. Oxytocin induces differentiation of P19 embryonic stem cells to cardiomyocytes. Proc Natl Acad Sci U S A 2002; 99:9550-9555.
[135]Park JH, Kim SJ, Lee JB, Song JM, Kim CG, Roh S 2nd, Yoon HS. Establishment of a human embryonic germ cell line and comparison with mouse and human embryonic stem cells. Molecules and Cells 2004; 17:309-315.
[136]Park TS, Han JY. Derivation and characterization of pluripotent embryonic germ cells in chicken. Mol Reprod Dev 2000; 56:475-482.
[137]Park TS, Galic Z, Conway AE, Lindgren A, van Handel BJ, Magnusson M, Richter L, Teitell MA, Wei W, Qing T, Ye X, Liu H, Zhang D, Yang W, Deng H. Primordial germ cell specification from embryonic stem cells. PLoS ONE 2008; 3:e4013.
[138]Park TS, Hong YH, Kwon SC, Lim JM, Han JY. Birth of germline chimeras by transfer of chicken embryonic germ (EG) cells into recipient embryos. Mol Reprod Dev 2003; 65:389-395.
[139]Park TS, JY Han. Derivation and characterization of pluripotent embryonic germ cells in chicken. Mol Reprod Dev 2000; 56:475-482.
[140]Park TS, YH Hong, SC Kwon, JM Lim and JY Han. Birth of germline chimeras by transfer of chicken embryonic germ (EG) cells into recipient embryos. Mol Reprod Dev 2003; 65:389-395.
[141]Pearl EJ, Horb ME. Promoting ectopic pancreatic fates:pancreas development and future diabetes therapies. Clin Genet 2008;74:316-324.
[142]Phipps SM, Love WK, Mott TE, Andrews LG, Tollefsbol TO. Differential expression of epigenetic modulators during human embryonic stem cell differentiation. Mol Biotechnol 2009; 41:201-207.
[143]Prudhomme W, Daley GQ, Zandstra P, Lauffenburger DA. Multivariate proteomic analysis of murine embryonic stem cell self-renewal versus differentiation signaling. Proc Nail Acad Sci USA 2004; 101:2900-2905.
[144]Qi X, Li TG, Hao J, Hu J, Wang J, Simmons H, Miura S, Mishina Y, Zhao GQ. BMP4 supports self-renewal of embryonic stem cells by inhibiting mitogen activated protein kinase pathways. Proc Natl Acad Sci USA 2004; 101:6027-6032.
[]Rattis FM, Voermans C, Reya T. Wnt signaling in the stem cell niche. Curr Opin Hematol 2004; 11:88-94.
[145]Resnick JL, Bixler LS, Cheng L, Donovan PJ. Long-term proliferation of mouse primordial germ cells in culture. Nature 1992; 359:550-551.
[146]Resnick JL, Ortiz M, Keller JR, Donovan PJ. Role of fibroblast growth factors and their receptors in mouse primordial germ cell growth. Biol Reprod 1998; 59:1224-1229.
[147]Richards M, Fong CY, Tan S, Chan WK, Bongso A. An efficient and safe xeno-free cryopreservation method for the storage of human embryonic stem cells. Stem Cells 2004; 22:779-789.
[148]Rodda DJ, Chew JL, Lim LH, Loh YH, Wang B, Ng HH, Robson P. Transcriptional regulation of Nanog by OCT4 and SOX2. J. Biol. Chem 2005; 280:24731-24737.
[149]Rius M, Stresemann C, Keller D, Brom M, Schirrmacher E, Keppler D, Lyko F. Human concentrative nucleoside transporter 1-mediated uptake of 5-azacytidine enhances DNA demethylation. Mol Cancer Ther 2009; 8:225-231.
[150]Rosen ED, P Sarraf, AE Troy, G Bradwin, K Moore, DS Milstone, BM Spiegelman and RM Mortensen. PPAR gamma is required for the differentiation of adipose tissue in vivo and in vitro. Mol Cell 1999; 4:611-617.
[151]Saito S, Liu B, Yokoyama K. Animal embryonic stem (ES) cells:self-renewal, pluripotency, transgenesis and nuclear transfer. Hum Cell 2004; 17:107-115.
[152]Sang H. Prospects for transgenesis in the chick. Mechanisms of Development 2004; 121:1179-1186.
[153]Sato N, Meijer L, Skaltsounis L, Greengard P, Brivanlou AH. 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; 10:55-63.
[154]Soto-Gutierrez A, N Navarro-Alvarez, DB Zhao, JD Rivas-Carrillo, J Lebkowski, N Tanaka, IJ Fox and N Kobayashi. Differentiation of mouse embryonic stem cells to hepatocyte-like cells by co-culture with human liver nonparenchymal cell lines. Nature Protocols 2007; 2:347-356.
[155]Sekkai D, Gruel G, Herry M, Moucadel V, Constantinescu SN, Albagli O, Tronik-Le Roux D, Vainchenker W, Bennaceur-Griscelli A. Microarray analysis of LIF/Stat3 transcriptional targets in embryonic stem cells. Stem Cells 2005; 23:1634-1642.
[156]Shevinsky LH, Knowles BB, Damjanov I, Solter D. Monoclonal antibody to murine embryos defines a stage specific embryonic antigen expressed on mouse embryos and human teratocarcinoma cells. Cell 1982; 30:697-705.
[157]Shim H, Gutierrez-A dan A, Chen LR, BonDurant RH, Behboodi E, Anderson GB. Isolation of pluripotent stem cells from cultured porcine primordial germ cells. Biol Reprod 1997; 57:1089-1095.
[158]Shim SW, Han DW, Yang JH, Lee BY, Kim SB, Shim H, Lee HT. Derivation of embryonic germ cells from post migratory primordial germ cells, and methylation analysis of their imprinted genes by bisulfite genomic sequencing. Mol Cells 2008; 25:358-367.
[159]Smith AG, Nichols J, Robertson M, Rathjen PD. Differentiation inhibiting activity(DIA/LIF) and mouse development[J]. Dev Biol 1992; 151:339-315.
[160]Solter D, Knowles BB. Monoclonal antibody defining astage-specific mouse embryonic antigen(SSEA-1). Proc Natl Acad Sci USA 1978; 75:5565-5569.
[161]Soodeen-Karamath S, Gibbins AM. Apparent absence of oct3/4 from the chicken genome. Mol Reprod Dev 2001; 58:137-148.
[162]Soto-Gutierrez A, N Navarro-Alvarez, DB Zhao, JD Rivas-Carrillo, J Lebkowski, N Tanaka, IJ Fox and N Kobayashi. Differentiation of mouse embryonic stem cells to hepatocyte-like cells by co-culture with human liver nonparenchymal cell lines. Nature Protocols 2007; 2:347-356.
[163]Sroczynska P, Lancrin C, Pearson S, Kouskoff V, Lacaud G. In Vitro Differentiation of Mouse Embryonic Stem Cells as a Model of Early Hematopoietic Development. Methods Mol Biol 2009; 538:1-18.
[164]Stern CD. The chick:a great model system becomes even greater. Dev Cell 2005; 8:9-17.
[165]Stewart CL, Gadi L, Bhatt H. Stem cells from primordial germ cells can reeter the germ line. Dev.Biol 1994; 161:626-628.
[166]Sulston JE, Schierenberg E, White JG, Thomson JN. The embryonic cell lineage of the nematode Caenorhabditis elegans. Dev Biol 1983; 100:64-119.
[167]Sutasurya LA. Appearance of primordial germ cells in young chick blastoderm cultured in vitro. Development Growth and Differentiation 1983; 25:517-521.
[168]Suter DM, Cartier L, Bettiol E, Tirefort D, Jaconi ME, Dubois-Dauphin M, Krause KH. Rapid generation of stable transgenic embryonic stem cell lines using modular lentivectors. Stem Cells 2006; 24:615-623.
[169]Sutton J, Costa R, Klug M, Field L, Xu D, Largaespada DA, Fletcher CF, Jenkins NA, Copeland NG, Klemsz M, Hromas R. Genesisi, a winged helix transcriptional repressor with expression restricted to embryonic stem cells. J Biol Chem 1996; 271:23126-23133.
[170]Swift CH. Origin and early history of the primordial germ cells in chick. Am J of anat 1914; 15:483-516.
[171]Takahashi K, Yamanaka S Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006; 126:663-676.
[172]Takaki M, Nakayama S, Misawa H, Nakagawa T, Kuniyasu H. In vitro formation of enteric neural network structure in a gut-like organ differentiated from mouse embryonic stem cells. Stem Cells 2006; 24:1414-1422.
[173]Takashi M, Hisako M. Glycoconjugate antigens expressed on stem cells and early embryonic cells. Glycoconjugate 2004; 21:41-45.
[174]Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jone JM. Embryonic stem cell line derived from human blastcysts. Science 1998; 282:1145-1147.
[175]Tilg H, Moschen AR. Adipocytokines:mediators linking adipose tissue, inflammation and immunity. Nat Rev Immunol 2006; 6:772-783.
[176]Till JE, McCulloch EA. A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat Res 1961; 14:213-222.
[177]Tompers DM, Foreman RK, Wang Q, Kumanova M, Labosky PA. Foxd3 is required in the trophoblast progenitor cell lineage of the mouse embryo. Dev Biol 2005; 285:126-137.
[178]Torihashi S, Hattori T, Hasegawa H, Kurahashi M, Ogaeri T, Fujimoto T.The expression and crucial roles of BMP signaling in development of smooth muscle progenitor cells in the mouse embryonic gut. Differentiation 2009; 77:277-289.
[179]Tseng YH, KM Kriauciunas, E Kokkotou and CR Kahn. Differential roles of insulin receptor substrates in brown adipocyte differentiation. Mol Cell Biol 2004; 24:1918-1929.
[180]Tsunekawa N, Naito M, Sakai Y, Nishida T and Noce T. Isolation of chicken vasa homolog gene and tracing the origin of primordial germ cells. Development 2000; 127:2741-2750.
[181]Turnpenny L, Brickwood S, Spalluto CM, Piper K, Cameron IT, Wilson DI, Hanley NA. Derivation of human embryonic germ cells:an alternative source of pluripotent stem cells. Stem Cells 2003; 21:598-609.
[182]Urven LE, Abbott UK, Erickson CA. Distribution of extraceliular matrix in the migratory pathway of avian primordial germ cells. Anatomical Record 1989; 224:14-21.
[183]Valbuena D, Galan A, Sanchez E, Poo ME, Gomez E, Sanchez-Luengo S, Melguizo D, Garcia A, Ruiz V, Moreno R, Pellicer A, Simon C. Derivation and characterization of three new Spanish human embryonic stem cell lines (VAL-3-4-5) on human feeder and in serum-free conditions. Reproductive BioMedicine 2006; 13:875-886.
[184]Vallier L, Alexander M, Pedersen R. Conditional Gene Expression in Human Embryonic Stem Cells. Stem Cells 2007; 25:1490-1497.
[185]van de Lavoir MC, Diamond JH, Leighton PA et al. Germline transmission of genetically modified primordial germ cells. Nature 2006; 441:766-769.
[186]Viswanathan S, Benatar T, Rose-John S, Lauffenburger DA, Zandstra PW. Ligand/receptor signaling threshold (LIST) model accounts for gp130-mediated embryonic stem cell self-renewal responses to LIF and HIL-6. Stem Cells 2002; 20:119-138.
[187]Wang J, Jiao F, Pan XH, Xie SY, Li ZL, Niu XH, Du LX. Directed differentiation of chick embryonic germ cells into neural cells using retinoic acid induction in vitro. J Neurosci Methods 2009; 177:168-176.
[188]Wang R, Clark R, Bautch VL. Embryonic stem cell-derived cysticembryoid bodies form vascular channels:an in vitro model of blood vessel development. Development 1992; 114:303-316.
[189]Wakitani S,Takaoka K, Hattori T, Miyazawa N, Iwanaga T, Takeda S, Watanabe TK, Tanigami A. Embryonic stem cells injected into the mouse knee joint form teratomas and subsequently destroy the joint. Rheumatology 2003; 42:162-165.
[190]Wdziekonski B, Villageois P, Dani C. Differentiation of mouse embryonic stem cells and of human adult stem cells into adipocytes. Curr Protoc Cell Biol 2007; 23:23-24.
[]Wei H, Juhasz O, Li J, Tarasova YS, Boheler KR. Embryonic stem cells and cardiomyocyte differentiation:phenotypic and molecular analyses. J Cell Mol Med 2005; 9:804-817.
[191]Wentworth BC, Tsaihhallett JH, Ghikamune DS, Rajcic-Spasojevic. Manipulation of avian primordial germ cells and gondal differentiation. Poultry science 1989; 69:999-1010.
[192]Wichterle H, Lieberam I, Porter JA, Jessell TM. Directed differentiation of embryonic stem cells into motor neurons.Cell 2002; 110:385-397.
[193]Willson ML, McElnea C, Mariani J, Lohof AM, Sherrard RM. BDNF increases homotypic olivocerebellar reinnervation and associated fine motor and cognitive skill. Brain 2008; 131:1099-1112.
[194]Wilson EB. The Cell in Development and Inheritance.1896; Macmillan, New York.
[195]Yamamoto H, Quinn G, Asari A, Yamanokuchi H, Teratani T, Terada M, Ochiya T. Differentiation of embryonic stem cells into hepatocytes:Biological functions and therapeutic application. Hepatology 2003; 37:983-993.
[196]Yamamoto Y, Usui F, Nakamura Y, Ito Y, Tagami T, Nirasawa K, Matsubara Y, Ono T, Kagami H. A Novel Method to Isolate Primordial Germ Cells and Its Use for the Generation of Germline Chimeras in Chicken. Biol Reprod 2007; 77:115-119.
[197]Yasuda Y, Tajima A, Fujimoto T, Kuwana T. A method to obtain avian germ line chimeras using isolated primordial germ cells. J Reprod Fert 1992; 96:521-528.
[198]Ying QL, Nichols J, Chambers I, Smith A. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 2003;115:281-292.
[199]Yoon BS, Yoo SJ, Lee JE, You S, Lee HT, Yoon HS. Enhanced differentiation of human embryonic stem cells into cardiomyocytes by combining hanging drop culture and 5-azacytidine treatment. Differentiation 2006; 74:149-159.
[200]Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin Ⅱ, Thomson JA. Induced pluripotent stem cell lines derived from human somatic cells. Science 2007; 318:1917-1920.
[201]Zhang D, Jiang W, Liu M, Sui X, Yin X, Chen S, Shi Y, Deng H. Highly efficient differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells. Cell Res 2009; 19:429-438.
[202]李碧春,陈国宏,赵东伟,王克华,钱菊汾.鸡胚PGCs迁移与性腺发育关系的研究.扬州大学学报2002；23：18-26.
[203]李碧春,秦洁,肖小珺,陈国宏,吴信生,吴圣龙.鸡胚不同发育时期原始生殖细胞的分离方法.动物学报2003；49：835-842.