胚胎干细胞研究进展

胚胎干细胞研究进展郑瑞珍（中国科学院发育生物学研究所北京１０００８０）一、前言胚胎干细胞（ＥｍｂｒｙｏｎｉｃＳｔｅｍｃｅｌｌ,ＥＳ）是从早期胚胎的内细胞团分离来的,能在体外培养的一种高度未分化的细胞,在某种意义上也可以说它是一种具有发育全能性的细胞。英国的Ｅｖａｎｓ和Ｋａｕｆｍａｎ最早于１９８１年［１］用延缓着床的胚泡首次建立了小鼠胚胎干细胞。     

肺动脉内皮细胞与肺动脉平滑肌细胞增殖的相互调节

血管内皮细胞和血管平滑肌细胞在结构和功能上关系密切,二者的相互关系在血管舒缩和血管壁结构的调节中起重要作用。本文观察了培养的小牛肺动脉内皮细胞（ＰＡＥＣ）和肺动脉平滑肌细胞（ＰＡＳＭ）在细胞增殖方面的相互调节作用。混合培养的ＰＡＥＣ和ＰＡＳＭ细胞的３Ｈ－ＴｄＲ参入明显降低（Ｐ＜０．００１,与对照组相比）。无论向培养的ＰＡＥＣ和ＰＡＳＭ中分别加入ＰＡＳＭ和ＰＡＥＣ的条件培养基还是二者共培养时,均发现ＰＡＥＣ的３Ｈ－ＴｄＲ参入明显降低,而ＰＡＳＭ的３Ｈ－ＴｄＲ明显升高（Ｐ＜０．０５,与对照组相比）。流式细胞测定也发现共培养时ＰＡＥＣ的Ｇ１期细胞增多,Ｇ２／Ｍ期细胞减少;而ＰＡＳＭ的Ｇ１期细胞减少,Ｇ２／Ｍ期细胞增多。共培养的ＰＡＳＭ细胞内ｃＡＭＰ增加,ｃＧＭＰ含量降低;而ＰＡＥＣ细胞的ｃＡＭＰ和ｃＧＭＰ含量均降低（Ｐ＜０．０１,与对照组相比）。上述结果提示,ＰＡＥＣ和ＰＡＳＭ相互作用可能通过第二信使而调节它们本身的增殖     

小鼠原生殖细胞的研究进展

在动物的整个生命周期中，生殖细胞提供了各代间的连续性。对于多数动物来说，其生殖细胞不是在生殖腺中产生的，其前身原生殖细胞（ＰｒｉｍｏｒｄｉａｌＧｅｒｍＣｅｌ简称ＰＧＣ）是迁移到正在发育着的生殖腺中的。近年来，由于ＰＧＣ体外培养技术的不断完善，ＰＧＣ的...     

衰老细胞转录因子AP_1/CREB结合活性对EGF的可诱导性下降

转录因子与ＤＮＡ的结合是基因转录的关键环节．通过迁移位移法分析了转录因子ＡＰ１、ｃＡＭＰ反应元件结合蛋白（ＣＲＥＢ）、糖皮质激素反应元件（ＧＲＥ）结合蛋白在衰老细胞中的结合活性．结果表明,ＡＰ１与ＣＲＥＢ的结合活性在衰老细胞中对表皮生长因子（ＥＧＦ）的反应性低于年轻细胞;而ＧＲＥ结合蛋白的结合活性在衰老细胞中对ＥＧＦ的反应性无明显变化．这提示ＥＧＦ不能有效刺激衰老细胞增殖可能与其转录因子结合活性的改变有关     

表皮生长因子对鼠胚成纤维细胞细胞周期的调节作用

本文观察了表皮生长因子（ＥＧＦ）对小鼠胚胎成纤维细胞Ｃ３Ｈ／１０Ｔ１／２ＣＬ８（简称Ｃ３Ｈ／１０）细胞周期的影响。结果表明：ＥＧＦ使Ｓ期提前,细胞周期缩短。进一步探讨了ＥＧＦ对细胞周期影响的机制,发现ＥＧＦ可活化在细胞周期调节中起重要作用的Ｐ３４＾ｃｄｃ２激酶（简称ＣＤ２Ｋ）,使ＣＤ２Ｋ在周期中活性高峰出现的时间提前,提示ＥＧＦ对细胞周期的影响可能通过作用于ＣＤ２Ｋ实现的。     

表皮生长因子对鼠胚成纤维细胞细胞周期的调节作用

本文观察了表皮生长因子（ＥＧＦ）对小鼠胚胎成纤维细胞Ｃ_３Ｈ／１０Ｔ_１／２ＣＬ_８（简称Ｃ_３Ｈ／１０）细胞周期的影响。结果表明：ＥＧＦ使Ｓ期提前,细胞周期缩短。进一步探讨了ＥＧＦ对细胞周期影响的机制,发现ＥＧＦ可活化在细胞周期调节中起重要作用的Ｐ３４~（ｃｄｃ２）激酶（简称ＣＤ２Ｋ）,使ＣＤ２Ｋ在周期中活性高峰出现的时间提前,提示ＥＧＦ对细胞周期的影响可能通过作用于ＣＤ２Ｋ实现的。     

MCF细胞中人干细胞因子基因新调控元件的鉴定

人干细胞因子（ｈｕｍａｎ ｓｔｅｍ ｃｅｌｌ ｆａｃｔｏｒ,ｈＳＣＦ）是一个多功能细胞生长因子,调节某些哺乳动物干细胞如原始胚细胞的增殖、分化和移居。为研究ｈＳＣＦ基因的转录调控机理,对ｈＳＣＦ基因５’旁侧序列不同长度片段进行亚克隆,构建系列重组ｐＧＬ２荧光素酶报告基因载体,转染人乳腺癌细胞株ＭＣＦ并检测ｌｕｃ基因一过性表达活性。然后应用电泳迁移率变动分析技术,鉴定活性片段与ＭＣＦ细胞核抽提蛋白质     

颗粒子宫腺细胞的分离、纯化与细胞化学研究

胰蛋白酶消化小鼠子宫腺细胞区,制成单细胞悬液,滴片后进行细胞学及细胞化学研究。结果：有多种类型细胞存在于悬液中,其中中等大小细胞（直径２０μｍ左右）大部分为颗粒子宫腺细胞（ＧｒａｎｕｌａｔｅｄＭｅｔｒｉａｌＧｌａｎｄＣｅｌｌｓ,ＧＭＧｃｅｌｌｓ）。ＧＭＧ细胞约占所有细胞的５０％。利用Ｐｅｒｃｏｌｌ非连续性密度梯度离心可以提高ＧＭＧ细胞的纯度,使之达８０％以上。细胞化学研究显示ＧＭＧ细胞含ＡＣＰ,ＮＳＥ,ＬＮＡｓｅ及糖蛋白成份。     

细胞周期、限制点、关卡

１细胞周期、限制点、关卡的概念细胞周期（ｃｅｌｌｃｙｃｌｅ）是指亲代细胞分裂结束到子细胞分裂结束之间的间隔时期。人们一般将细胞周期分为Ｇ１、Ｓ、Ｇ２和Ｍ期等四个分期。还有一种所谓的静止期细胞（Ｇ０期细胞）暂时不增殖,在某种情况下可离开Ｇ０期而重新进入增殖周期。在细胞周期时相转变过程中有两个限制点（ｒｅｓｔｒｉｃｔｉｏｎｐｏｉｎｔｓ）决定细胞是继续增殖还是进入静止状态。当在不合适条件下,如营养匮乏、缺乏血清生长因子和抑素（ｃｈａｌｏｈｅ）等等,可以使细胞不能通过限制点而阻滞在细胞增殖周期中。一个主…     

内皮素和bFGF对MC的促增殖作用及胰岛素的协同效应

采用３Ｈ－ＴｄＲ参入法,测定碱性成纤维细胞生长因子（ｂＦＧＦ）、胰岛素和内皮素－１（ＥＴ－１）对体外培养的大鼠肾小球系膜细胞（ＭＣ）增殖的影响,以及胰岛素与ｂＦＧＦ或ＥＴ－１促ＭＣ增殖的协同作用。结果表明,不同浓度的ｂＦＧＦ（５－２００ｎｇ／ｍｌ）和胰岛素（０．１－２．４Ｕ／ｍｌ）均显著升高ＭＣ的３Ｈ－ＴｄＲ参入值（ｃｐｍ值）（Ｐ＜０．０１）。ＥＴ－１对ＭＣ的ｃｐｍ值的影响依剂量不同呈现两种不同的效应,在１０－９－１０－７ｍｏｌ／Ｌ时,随着浓度的升高,ＭＣ的ｃｐｍ值明显升高（Ｐ＜０．０１）,并以１０－８ｍｏｌ／Ｌ作用最强;当升高到１０－６ｍｏｌ／Ｌ时,ＭＣ的ｃｐｍ值出现降低趋势。胰岛素与ｂＦＧＦ或低浓度ＥＴ－１（≤１０－８ｍｏｌ／Ｌ）共同作用于ＭＣ时,ＭＣ的ｃｐｍ值明显高于二者单独作用之和（Ｐ＜０．０１）,与高浓度ＥＴ－１（＞１０－７ｍｏｌ／Ｌ）共同作用于ＭＣ时,ＭＣ的ｃｐｍ值小于二者单独作用之和（Ｐ＞０．０５）。上述结果说明,胰岛素、ｂＦＧＦ和ＥＴ－１均能显著促进ＭＣ增殖;胰岛素与ｂＦＧＦ或低浓度的ＥＴ－１促ＭＣ增殖具有正协同作用,与高浓度ＥＴ－１呈现负协同作用。     

Primordial germ cells contain subpopulations that have greater ability to develop into pluripotential stem cells

Primordial germ cells (PGCs) are undifferentiated germ cells in embryos. We previously found that some mouse PGCs develop into pluripotential cells (EG cells) when cultured on a feeder layer expressing the membrane bound form of Steel factor with culture medium containing leukemia inhibitory factor and basic fibroblast growth factor. To understand the mechanisms of the conversion of PGCs into EG cells, we attempted to identify PGC subpopulations that have the ability to develop into EG cells. Using flow cytometry, we fractionated PGCs by the expression of the cell surface antigen integrin α6, as well as by the detection of side‐population (SP) cells in which stem cells are enriched in various tissues. PGCs with negative or low integrin α6 expression and with SP cell phenotype showed higher potential to convert to EG cells. Negative or low integrin α6 expression in PGCs was also correlated with lower expression of Ddx4, which is specifically expressed in PGCs after embryonic day 10.5. The results indicate that the primitive PGC population showing the SP cell phenotype among undifferentiated PGCs has a higher ability of being converted into EG cells. Thus, conversion of PGCs into pluripotential stem cells may be regulated by being influenced by the natural status of individual PGCs as well as the reprogramming process after starting culture.     

Derivation and characterization of pluripotent embryonic germ cells in chicken

Embryonic germ (EG) cell lines established from primordial germ cells (PGCs) are undifferentiated and pluripotent stem cells. To date, EG cells with proven germ-line transmission have been completely established only in the mouse with embryonic stem (ES) cells. We isolated PGCs from 5.5-day-old (stage 28) chicken embryonic gonads and established a putative chicken EG cell line with EG culture medium supplemented with stem cell factor (SCF), leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), interleukin-11 (IL-11), and insulin-like growth factor-I (IGF-I). These cells grew continuously for ten passages (4 months) on a feeder layer of mitotically active chicken embryonic fibroblasts. After several passages, these cells were characterized by screening with the periodic acid-Schiff reaction, anti-SSEA-1 antibody, and a proliferation assay. The chicken EG cells maintained characteristics of gonadal PGCs and undifferentiated stem cells. When cultured in suspension, the chicken EG cells successfully formed an embryoid body and differentiated into a variety of cell types. The chicken EG cells were injected into stage X blastodermal layer and produced chimeric chickens with various differentiated tissues derived from the EG cells. Chicken EG cells will be useful for the production of transgenic chickens and for studies of germ cell differentiation and genomic imprinting.     

Mass isolation of primordial germ cells from transgenic rainbow trout carrying the green fluorescent protein gene driven by the vasa gene promoter

Mass isolation of live primordial germ cells (PGCs) was demonstrated for the first time in ectothermal vertebrates. To establish a stem cell-mediated gene transfer system in fish, a stem cell line that retains the ability to develop into gametes is necessary. PGCs are well suited for use as the initial material for such a stem cell line. We established transgenic rainbow trout (Oncorhynchus mykiss) strains carrying the green fluorescent protein (GFP) gene driven by a rainbow trout vasa-like gene (RtVLG) promoter/enhancer. Because GFP expression was specific to the PGCs, PGCs were successfully visualized in all developmental stages examined. Isolated genital ridges containing GFP-labeled PGCs were enzymatically dissociated. To isolate PGCs from the complex pools of dissociated genital ridges, GFP-labeled cells were sorted by flow cytometry. The sorted GFP-positive cells were large and round with a large nucleus, typical characters of PGC morphology. The expression of RtVLG was detected only in the GFP-positive cell population, confirming that these cells were PGCs. This simple and efficient technique to purify a large number of viable PGCs opens the way for establishing a stem cell line, which can differentiate into the germline. The purified PGCs would also be a novel tool for cellular and molecular study of vertebrate germline stem cells.     

Turning germ cells into stem cells

Primordial germ cells (PGCs), the embryonic precursors of the gametes of the adult animal, can give rise to two types of pluripotent stem cells. In vivo, PGCs can give rise to embryonal carcinoma cells, the pluripotent stem cells of testicular tumors. Cultured PGCs exposed to a specific cocktail of growth factors give rise to embryonic germ cells, pluripotent stem cells that can contribute to all the lineages of chimeric embryos including the germline. The conversion of PGCs into pluripotent stem cells is a remarkably similar process to nuclear reprogramming in which a somatic nucleus is reprogrammed in the egg cytoplasm. Understanding the genetics of embryonal carcinoma cell formation and the growth factor signaling pathways controlling embryonic germ cell derivation could tell us much about the molecular controls on developmental potency in mammals.     

Interleukin-2 induces the proliferation of mouse primordial germ cells in vitro

Primordial germ cells (PGCs) are the stem cell precursors of the germ line. Several growth factors contribute to enlarging the PGC population by acting as mitogens, survival factors or both. Interleukin-2 (IL-2) has a growth-promoting activity for T and B-lymphocytes, but its role in PGCs had not yet been studied. Here, we show that PGCs isolated from 10.5, 11.5 and 12.5 day postcoitum (dpc) mouse embryos constitutively express the three subunits (alpha, beta and gamma) of the IL-2 receptor (IL-2R). In contrast, IL-2 mRNA was not detected in these cells. However, the addition of recombinant IL-2 to the culture medium increased the number of PGCs in vitro via a mitogenic effect, as indicated by bromodeoxyuridine incorporation assays. Neutralization of the IL-2 receptor using anti-IL-2R subunit antibodies inhibited this IL-2-mediated proliferative effect on PGCs from 11.5 dpc embryos. Together, these data are indicative of a paracrine effect of IL-2 on PGC proliferation. In this regard, we also compared the effect of IL-2 with other compounds such as basic fibroblast growth factor (bFGF), steel factor, leukemia inhibitory factor and forskolin, and found that the degree of proliferation induced by IL-2 was similar to that induced by bFGF and forskolin. These observations support the notion that similar patterns of molecular signaling may underlie the developmental pathways of hematopoietic and germ stem cell precursors.     

Long-term culture and characterization of goat primordial germ cells

While the culture and identification of primordial germ cells (PGCs) in mice is established, only limited investigations on PGCs in livestock have been reported. This study was performed to characterize goat PGCs after culture and cryopreservation. Goat PGCs were isolated from Day 32 fetuses and cultured on a continuous cell line of murine embryonal fibroblasts (STO) as feeder-cells in the presence of leukemia inhibitory factor (LIF). The PGCs proliferated slowly and showed colony formation in early passages. Frozen-thawed PGCs continued to proliferate when stem cell factor (SCF) was added to the culture medium. However, differentiation into epithelial-like polygonal cells or neuronal cells was observed after 1 or 2 passages. The PGCs of 1 female and 1 male cell line were characterized by immunocytochemistry. The PGCs showed positive staining for anti stage-specific embryonic antigen-1 (SSEA-1) and FMA-1 (monoclonal antibody produced against a glycoprotein cell surface antigen of the embryonal carcinoma Nulli SCC1), whereas the reactivity to alkaline phosphatase (AP), an established marker for PGCs in mice, was inconsistent. After differentiation, PGCs lost their positive reaction to SSEA-1, EMA-1 and AP. In conclusion, SSEA-1 and EMA-1 can be used as reliable markers for identifying goat PGCs in addition to morphological criteria. The results indicate that goat PGCs can be kept in long-term culture without losing their morphological characteristics and their positive reaction to SSEA-1 and EMA-1, thus providing a promising source of donor-karyoplasts for nuclear transfer procedures.