影响小鼠孤雌胚胎干细胞的建系效率因素初探

目的检测孤雌胚胎干细胞系的建系效率与小鼠品系以及培养体系的关系。方法将小鼠MⅡ期卵子孤雌激活发育至囊胚,然后从囊胚内细胞团分离孤雌胚胎干细胞。结果杂交和近交系小鼠的建系效率没有显著差异,建系的培养体系中加入ERK抑制剂或者采用血清替代品KSR时,建系效率显著提高。结论小鼠孤雌胚胎干细胞的建系效率与小鼠的遗传背景并没有直接关系,而与分离内细胞团的培养体系密切相关。     

小鼠孤雌胚胎干细胞的建立及其向运动神经元分化的初探

文章采用小鼠的孤雌囊胚建立胚胎干细胞系,探究其向运动神经元分化的可能,为临床治疗以及研究基因组印记与神经分化的的关系提供理论基础。结果表明:卵母细胞孤雌激活率达到93.26%,成功建立了8个孤雌胚胎干细胞系,建系率达到23.53%。克隆表达多潜能标记Oct4及细胞表面标记SSEA-1,有高水平的碱性磷酸酶活性,在细胞第10代和第30代时核型分析检测显示为正常的40条染色体。体内、外均分化出三胚层来源的细胞。联合应用全反式维甲酸(RA)、音猬因子(Shh)及细胞外基质,小鼠孤雌胚胎干细胞可被诱导表达运动神经元的标志性标记HB9、Olig2。     

Xist敲降可以改善孤雄单倍体囊胚质量

单倍体胚胎干细胞研究一直以来吸引着研究者们的注意,它可以用作基因修饰的工具或是用于药物筛选。随着孤雄胚胎干细胞系的成功建立,更扩展了单倍体胚胎干细胞的应用前景。但单倍体孤雄胚胎发育率低,胚胎质量差,制约着孤雄单倍体胚胎干细胞的建系。为改善孤雄单倍体胚胎发育潜能及胚胎干细胞建系效率低的问题,我们检测了小鼠(Mus musculus domesticus)孤雄单倍体胚胎的体外发育过程和该过程中Xist基因表达情况。结果发现,孤雄单倍体胚胎囊胚发育率只有10%~14%,发育至囊胚所需时间差异较大,从3.5~5.5 d不等。通过核糖核酸荧光原位杂交实验(RNA-FISH)跟踪Xist基因表达情况,发现其在发育至囊胚阶段的胚胎中呈抑制状态,而在早期胚胎中多呈表达状态。通过si RNA扰低Xist表达,虽然没有改变孤雄单倍体胚胎发育到囊胚的比例,但显著提高了囊胚质量,并提高了接种胚胎内细胞团(ICM)后建立细胞系的效率。结果说明,Xist基因的表达可能是导致小鼠孤雄单倍体胚胎质量差、干细胞建系率低的原因之一。     

小鼠孤雌激活来源囊胚线粒体基因表达

为了探索孤雌胚胎和正常体外受精胚胎线粒体基因表达的差异,本研究用ICR小鼠孤雌和正常受精胚胎为研究对象。孤雌和正常受精的胚胎分别发育至2-细胞期和囊胚期的比率,用q PCR方法检测囊胚线粒体基因Cox2、tom40、tim23和cytochrome C,以及多能性囊胚质量相关基因Oct4、Sox2和nanog的表达水平。结果发现孤雌激活不影响小鼠卵母细胞的卵裂(95%vs 97.6%,p0.05),但影响胚胎发育到囊胚(39.4%vs 75%,p0.05),孤雌囊胚细胞线粒体Cyto C和Cox2显著高于体外受精组(p0.05);正常受精囊胚多能性基因Oct4和nanog表达水平显著高于孤雌组(p0.05),但Sox2表达水平显著低于孤雌组(p0.05)。本研究表明孤雌激活可影响胚胎线粒体和细胞基因表达水平。     

山羊胚胎干细胞的分离与培养

对关中奶山羊配种后6～7天的桑椹胚和囊胚,分别采用全胚培养法、酶消化法和免疫外科法进行处理.将处理后的胚胎培养于小鼠胎儿成纤维细胞(MEF)饲养层上,分离培养山羊胚胎干细胞(Embryonic stem cell,ESC).对分离传代的山羊ESCs分别进行免疫组化染色,RT-PCR检测和体外诱导分化试验.结果表明.全胚培养法易于胚胎贴壁形成原代集落,采用全胚培养法获得的ESCs有一株目前已传至18代.山羊ESCs Nanong、Oct4、SSEA-3免疫组化染色呈阳性,SSEA-1免疫组化染色呈弱阳性,SSEA-4免疫组化染色呈阴性,RT-PCR检测显示其表达Nanog、Oct4、端粒酶、CD117.山羊ESCs经DMSO体外诱导可以向心肌细胞分化.这些试验均表明该细胞具有ESCs的生物学特性.     

鸭胚胎干细胞分离与鉴定

采用药勺法分离仙湖3号肉鸭鸭胚的胚盘细胞,并以鸭胚成纤维细胞作为饲养层,培养鸭胚胎干细胞。在此基础上,通过对体外培养的鸭胚胎干细胞形态观察,碱性磷酸酶染色(AKP)以及胚胎阶段表面特异性抗原(SSEA-1)免疫组化等方法,分离与鉴定鸭胚胎干细胞。结果表明传至第3代的鸭胚胎干细胞经AKP和SSEA-1鉴定均为阳性,AKP染色为深蓝色,SSEA-1染色呈绿色荧光,表明培养至第3代的鸭胚胎干细胞仍保持干细胞未分化特性,具有胚胎干细胞的特征。结果提示本试验分离、培养的鸭胚盘细胞为鸭胚胎干细胞。     

4个品系小鼠胚胎干细胞系的建立

探索高效的不同品系的小鼠胚胎干细胞的建系方法。B6D2F1(C57BL/6×DBA/2)、129/SV×DBA/2、C57BL/6、BALB/C等4个不同品系小鼠,孕马血清促性腺激素(pregnant mare serum gonadotrophin,PMSG) 人绒毛膜促性腺激素(human chorionic gonadotropin,HCG)促排,3.5天交配后(days post coitus,dpc)冲洗子宫取囊胚,或者2.5dpc冲洗输卵管,卵裂球体外培养获取囊胚。囊胚种植到小鼠成纤维细胞饲养层上干细胞培养液培养,4～5天内细胞团扩增后玻璃毛细管挑出,种植到新的饲养层上过夜再行胰蛋白酶消化,3～4天传代一次。对所建立的小鼠ES细胞系进行形态学、染色体核型、AKP染色、体内外分化能力,干细胞分子标记物荧光免疫染色等鉴定。获得10株小鼠胚胎干细胞,具有典型的胚胎干细胞生长特性,符合ES细胞的鉴定标准。结果表明成功的建立了来自B6D2F1(C57BL/6×DBA/2)、129/SV×DBA/2、C57BL/6、BALB/C等4个不同品系小鼠的10株ES细胞系。内细胞团挑出过夜增殖后消化的培养方法可能有助于提高ES细胞的建系率。     

小鼠类胚胎干细胞分离培养及用于制作嵌合体小鼠的实验研究

目的探讨分离小鼠囊胚内细胞群类胚胎干细胞及用于制作嵌合体小鼠的方法及应用价值。方法分离3.5d小鼠囊胚内细胞群的类胚胎干细胞作为供体细胞,通过显微注射方法将分离的类胚胎干细胞注射到供体小鼠的囊胚腔中,再将注射后的囊胚移植到假孕雌鼠的子宫中制作嵌合体小鼠。结果分离36枚囊胚的内细胞群类胚胎干细胞,注射256只昆明小鼠囊胚中,移植32只假孕雌鼠子宫中,获产崽2窝,共12只,其中2只获毛色嵌合体小鼠。结论采用该技术分离所获得的类胚胎干细胞作为供体细胞制作嵌合体小鼠获得成功,该方法为ES细胞介导的转基因动物制作增添了一条新的途径,在同种不同品系的动物改良及遗传病基因治疗中有一定的应用价值,尤其是对未能建立ES细胞系的大动物的遗传工程操作具有一定意义。     

H3K27乙酰化模式在孤雌胚与正常胚中的差异及其对胚胎发育的影响

目的考察小鼠孤雌胚胎H3K27乙酰化模式与体内胚胎的差异,探究表观遗传模式对孤雌胚发育的影响。方法利用SrCl2激活卵母细胞,获得植入前各时期孤雌胚胎,并统计胚胎发育率;小鼠注射孕马血清激素(Pregnant Mare Serum Gonadotrophin,PMSG)和人绒毛膜促性腺激素(Human Chorionic Gonadotropin,hCG)超排后合笼,在不同发育时间采用体内冲胚的方法获得体内各时期胚胎;将获得的各期各类胚胎用H3K27乙酰化抗体与特异性位点结合,与连接有FITC荧光基团的二抗共同孵育,利用激光共聚焦显微镜检测荧光强度,获得小鼠植入前各时期孤雌胚和体内胚组蛋白H3K27乙酰化模式。结果用SrCl2激活成熟卵母细胞得到的孤雌胚的激活率和囊胚率分别为96.39%和69.54%,处于正常发育水平;孤雌胚H3K27乙酰化荧光强度从原核期相对较高的水平逐渐降低,2-细胞、4-细胞和8-细胞时期荧光强度都处于较低水平,到桑葚胚时期又突然升高,总体变化趋势和体内组先降低后升高的整体趋势一样,且原核期至8-细胞时期的荧光值孤雌胚高于体内胚,桑囊胚时期则相反;两组的H3K27乙酰化荧光强度值在原核期和桑葚胚时期差异不显著(P>0.05),在2-细胞、4-细胞、8-细胞和囊胚期差异显著(P<0.01)。结论本研究表明小鼠孤雌胚H3K27乙酰化模式与体内胚的模式存在差异,可能是影响孤雌胚发育能力的重要原因之一。进一步的深入研究将对纠正小鼠孤雌胚乙酰化模式和提高孤雌胚发育能力具有重要意义。     

一种新的人胚胎干细胞自身来源的滋养层支持其体外培养

摘要: 通过人胚胎干细胞(Human embryonic stem cells, hESCs)经体内分化获取间充质干细胞(Mesenchymal stem cells, MSCs)为人胚胎干细胞提供一种新的滋养层。将约5×106个hESCs注射入重症免疫联合缺陷小鼠形成畸胎瘤, 8周后再从畸胎瘤中分离MSCs并鉴定, 将MSCs作为hESCs的滋养层细胞, 并检测和观察hESCs的生长情况、细胞特性和分化能力。从畸胎瘤中获得了纯度较高的具有类似骨髓来源的MSC特性的细胞群, 其形态相似、表面抗原标志相似(CD34和CD45阴性, CD29、CD49b、CD105、CD73和CD90阳性), 经诱导可以向成骨细胞和成脂细胞分化。将hESCs在MSCs滋养层细胞上传代培养10代以上, hESCs依然具有正常的细胞形态, 反转录PCR证实其特异转录因子Oct4、Nanog的表达, 干细胞表面标记SSEA-1显示为阴性, SSEA-4、TRA-1-60、TRA-1-81显示为阳性, 碱性磷酸酶染色显示为阳性, 并且核型正常。体外EB形成和体内畸胎瘤形成证明了其全能性。因此来源于hESCs本身的MSCs可以被用来作为支持胚胎干细胞生长并维持其未分化状态的滋养层细胞。     

Human embryonic stem cell lines derived from the Chinese population

Six human embryonic stem cell lines were established from surplus blastocysts. The cell lines expressed alkaline phosphatase and molecules typical of primate embryonic stem cells, including Oct-4, Nanog, TDGF1, Sox2, EBAF, Thy-1, FGF4, Rex-1, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. Five of the six lines formed embryoid bodies that expressed markers of a variety of cell types; four of them formed teratomas with tissue types representative of all three embryonic germ layers. These human embryonic stem cells are capable of producing clones of undifferentiated morphology, and one of them was propagated to become a subline. Human embryonic stem cell lines from the Chinese population should facilitate stem cell research and may be valuable in studies of population genetics and ecology.     

Isolation of multipotent stem cells in human periodontal ligament using stage-specific embryonic antigen-4

The periodontal ligament (PDL) comprises adult stem cells, which are responsible for periodontal tissue regeneration. In the present study, we investigated the specific profile of the stem cells in the human PDL. Microscopic analysis demonstrated that PDL cells showed a fibroblastic appearance, forming flat and loose aggregates. PDL cells expressed embryonic stem cell-associated antigens (SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, OCT4, NANOG, SOX2, and REX1, and alkaline phosphatase activity), as well as conventional mesenchymal stem cell markers. When PDL cells were cultured in the presence of all-trans-retinoic acid, the numbers of SSEA-3+ and SSEA-4+ PDL cells were significantly decreased, while that of SSEA-1+ was increased. SSEA-4+ PDL cells showed a greater telomere length and growth rate. SSEA-4+ PDL cells exhibited the potential to generate specialized cells derived from three embryonic germ layers: mesodermal (adipocytes, osteoblasts, and chondrocytes), ectodermal (neurons), and endodermal (hepatocytes) lineages. Our findings demonstrated that SSEA-4, a major antigen to distinguish human embryonic stem cells, could also be used to identify multipotent stem cells in the PDL. Hence, SSEA-4+ human PDL cells appear to be a promising source of stem cells for regenerative medicine.     

Efficient derivation of human embryonic stem cell lines from discarded embryos through increases in the concentration of basic fibroblast growth factor

We describe the derivation and characterization of three novel human embryonic stem (hES) cell lines (YT1, YT2, YT3). One hES line (YT1) was obtained from six discarded blastocysts in a culture medium supplemented with 12 ng/ml basic fibroblast growth factor (bFGF), and two lines (YT2,YT3)were obtained from three discarded blastocysts in the same medium but supplemented with 16 ng/ml bFGF. These cell lines were derived by partial or whole embryo culture followed by further expansion after manual dissection of the passaged cells. These cells were passaged continuously for more than 6 or 8 months and possessed all of the typical features of pluripotent hES cell lines, such as typical morphological characteristics and the expression of hES-specific markers (TRA-1-60, TRA-1-81, SSEA-4, SSEA-3, alkaline phosphatase, Oct4, Nanog) and pluripotency-related genes (Oct4, Nanog, TDGF1, Sox2, EBAF, Thy-1, FGF4, Rex1). The lines maintained normal karyotypes after long-term cultivation. The karyotype of YT1 and YT3 was 46,XX, and that of YT2 was 46, XY. Pluripotency was confirmed by in vitro and in vivo differentiation, and genetic identity was demonstrated by DNA fingerprinting.Our results indicate that higher concentrations of bFGF at the early culture stage support efficient the hES cell derivation.     

Derivation and characterization of human embryonic stem cell lines from the Chinese population

Human embryonic stem cells (hESCs) can self-renew indefinitely and differentiate into all cell types in the human body. Therefore, they are valuable in regenerative medicine, human developmental biology and drug discovery. A number of hESC lines have been derived from the Chinese population,but limited of them are available for research purposes. Here we report the derivation and characterization of two hESC lines derived from human blastocysts of Chinese origin. These hESCs express alkaline phosphatase and hESC-specific markers, including Oct4, Nanog, SSEA-3, SSEA-4,TRA-1-60 and TRA-1-81. They also have high levels of telomerase activity and normal karyotypes. These cells can form embryoid body in vitro and can be differentiated into all three germ layers in vivo by teratoma formation. The newly established hESCs will be distributed for research purposes.The availability of hESC lines from the Chinese population will facilitate studies on the differences in hESCs from different ethnic groups.     

Derivation and maintenance of human embryonic stem cell line on human adult skin fibroblast feeder cells in serum replacement medium

Human embryonic stem (hES) cells were originally isolated and maintained on mouse embryonic fibroblast (MEF) feeder layers in the presence of fetal bovine serum (FBS). However, if the hES cells are to be used for therapeutic applications, it is preferable to regulatory authorities that they be derived and cultured in animal-free conditions to prevent mouse antigen contamination that would exacerbate an immune response to foreign proteins, and the potential risk of transmission of retroviral and other zoonotic pathogens to humans. As a step towards this goal, we derived a new hES cell line (MISCES-01) on human adult skin fibroblasts as feeder cells using serum replacement (SR) medium. The MISCES-01 cells have a normal diploid karyotype (46XX), express markers of pluripotency (OCT4, GCTM-2, TRA-1-60, TRA-1-81, SSEA-3, SSEA-4, and alkaline phosphatase) and following in vitro and in vivo differentiation, give rise to derivatives of the three primary germ layers. This cell line can be obtained for research purposes from the Australian Stem Cell Centre (http://www.stemcellcentre.edu.au).