Isolation and characterization of embryonic stem cell-like cells from in vitro-produced buffalo (Bubalus bubalis) embryos

This study was carried out to isolate and characterize buffalo embryonic stem (ES) cell-like cells from in vitro-produced embryos. Inner cell mass (ICM) cells were isolated either mechanically or by enzymatic digestion from 120 blastocysts whereas 28 morulae were used for the isolation of blastomeres mechanically. The ICM cells/ blastomeres were cultured on mitomycin-C-treated feeder layer. Primary cell colony formation was higher (P < 0.05) for hatched blastocysts (73.1%, 30/41) than that for early/expanded blastocysts (25.3%, 20/79). However, no primary cell colonies were formed when blastomeres obtained from morulae were cultured. Primary colonies were formed in 14.1% (12/85) of intact blastocyst culture, which was significantly lower (P < 0.05) than that of 41.6% for ICM culture. These colonies were separated by enzymatic or mechanical disaggregation. Using mechanical disaggregation method, the cells remained undifferentiated and two buffalo ES cell-like cell lines (bES1, bES2) continued to grow in culture up to eight passages. However, disassociation through enzymatic method resulted in differentiation. Undifferentiated cells exhibited stem cell morphological features, normal chromosomal morphology, and expressed specific markers such as alkaline phosphatase (AP) and Oct-4. Cells formed embryoid bodies (EBs) in suspension culture; extended culture of EBs resulted in formation of cystic EBs. Following prolonged in vitro culture, these cells differentiated into several types of cells including neuron-like and epithelium-like cells. Furthermore, the vitrified-thawed ES cell-like cells also exhibited typical stem cell characteristics. In conclusion, buffalo ES cell-like cells could be isolated from in vitro-produced blastocysts and maintained in vitro for prolonged periods of time.     

The specific binding of peptide ligands to cardiomyocytes derived from mouse embryonic stem cells

Purification of pluripotent stem cell (PSC)‐derived cardiomyocytes is critical for the application of cardiomyocytes both in clinical and basic research. Finding a specific cell marker is a promising method for purifying induced cells. The present study employed phage display technology to search for particular cell markers that could bind specifically to PSC‐derived cardiomyocytes. After three rounds of biopanning, several peptides were obtained. The ELISA results show the no. 3 sequence peptide (QPFTTSLTPPAR), and other four sequences having a consensus motif [SS(Q)PPQ(S)], no. 9, 11, 14, and 10, have relatively high affinity and specificity to cardiomyocytes. Immunofluorescence confirmed that the selected peptides could bind specifically to the PSC‐derived cardiomyocytes. Competition tests with chemically synthesized peptides revealed the binding ability was caused by the peptide itself. Western blot analysis proved the phages were both bound to two 17 kDa cardiomyocyte membrane proteins and the no. 9 sequence showed a 55 kDa protein that was not observed in the no. 3 sequence. These results suggest that the selected peptides specifically target receptors on PSC‐derived cardiomyocyte membranes. The results will pave the way for further studies of cell surface markers and their applications, such as labeling, purification, and as vehicles for drug delivery. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Anther Culture Response of Barley Genotypes to Cold Pretreatments and Culture Media

The embryoid formation and plant regeneration in anther cultures of three barley (Hordeum vulgare L.) cultivars (Niki, Karina, Thermi), one F₁ hybrid (Niki × Thermi), two F₂ populations (Niki × Thermi, Niki × Karina), and two F₃ populations (Niki × Thermi, Niki × Karina) were investigated in two solid induction media after cold pretreatment for 14 and 28 days at 4°C . The media used (N6 and FHG) differed in their composition and source of energy (maltose in FHG vs. sucrose in N6). Embryoid frequency and green plant regeneration depended on both the induction medium composition and cold pretreatment. The combination of the FHG induction medium with 28-day-long cold pretreatment was the most efficient in haploid embryoid formation and green plant production. In addition, the green plant production was genotype-dependent. Cv. Thermi and F₁ hybrid Niki × Thermi exhibited the highest frequency of green plant production. The parent with high or even moderate frequency of embryoid formation in anther culture could lead to the effective production of green plants from the F₁ hybrid or the F₂ generation for breeding purposes.     

沙冬青愈伤组织对培养基的特殊要求和球形胚状分化结构的诱导

通过对沙冬青未成熟子叶进行离体培养,获得了愈伤组织。基本培养基采用Ｂ５,再配合ＭＳ培养基的铁盐成分,对沙冬青未成熟子叶愈伤组织的诱导和生长效果最佳。愈伤组织的诱导和生长对培养基的附加成分要求十分严格,２,４－Ｄ浓度为０．５ｍｇ／Ｌ,６－ＢＡ浓度为０．５ｍｇ／Ｌ,蔗粮不宜超过２％。愈伤组织最初为乳白色透明松散状态,生长比较缓慢,极易褐化死亡。如果培养基中的蔗糖浓度低于２％,两周后未成熟子叶愈伤组织逐     

Cell and tissue requirements for the gene eed during mouse gastrulation and organogenesis

Summary: Mouse embryos homozygous for the allele eed^{l7Rn5‐3354SB} of the Polycomb Group gene embryonic ectoderm development (eed) display a gastrulation defect in which epiblast cells move through the streak and form extraembryonic mesoderm derivatives at the expense of development of the embryo proper. Here we demonstrate that homozygous mutant ES cells have the capacity to differentiate embryonic cell types both in vitro as embryoid bodies and in vivo as chimeric embryos. In chimeric embryos, eed mutant cells can respond to wild‐type signals and participate in normal gastrulation movements. These results indicate a non–cell‐autonomous function for eed. Evidence of mutant cell exclusion from the forebrain and segregation within somites, however, suggests that eed has cell‐autonomous roles in aspects of organogenesis. A requirement for eed in the epiblast during embryonic development is supported by the fact that high‐contribution chimeras could not be rescued by a wild‐type extraembryonic environment. genesis 31:142–146, 2001. © 2001 Wiley‐Liss, Inc.     

Change of Superoxide Dismutase Activities During Somatic Embryogenesis in Asparagus officinalis L.

The superoxide dismutase activities in callus and somatic embryoids at different developmental stages of Asparagus officinalis L. were determined by means light of induced oxidation-reduction of nitro blue tetrazolium. It was shown that the superoxide dismutase activity was higher in callus than in somatic embryoids at different developmental stages. The activity increased with growth, differentiation and maturation of somatic embryoids during somatic embryogenesis. The relations between superoxide dismutade activity and somatic embryogenesis were discussed.     

一种利用STO饲养层细胞制备拟胚体的新方法

建立了一种利用STO饲养层细胞制备拟胚体的新方法。该方法选用生长至80%饱和密度的STO细胞,经丝裂霉素C(10 mg/ml)处理4 h后以8×104 cm-2的密度接种培养12 h,制备饲养层,再将ES-D3细胞以1×104 cm-2的密度接种其上,首先用含mLIF的DMEM培养液培养24 h,再更换拟胚体诱导培养液,5～9天后获得了各成熟阶段的拟胚体。形态结构和分化潜能等研究表明,该方法制备的拟胚体结构典型,具有产生3个胚层谱系来源的功能细胞的潜能。与传统拟胚体制作方法如悬滴培养法相比,具有操作简便,拟胚体形成率高,重复性好等优点,是开展哺乳动物早期胚胎发育和干细胞分化研究的理想工具。