Alginate-chitosan coacervation in production of artificial seeds

Survival of secondary embryoids of winter oilseed rape (Brassica napus ssp. oleifera cv. Primor) has been used as an assay for the development of artificial seeds involving complex coacervation of alginate (polyanion) with chitosan (polycation). Germination frequency of 100% was achieved for encapsulated embryoids when alginate formed the inner matrix and chitosan the outer layer. When the matrix makeup was reversed, there was no germination of embryoids. The artificial seeds produced were hardened in dilute alkaline solutions of NaOH and Ca(OH)(2). An optimum setting time could be selected based on a quantitative measurement of resistance of hardened capsules to compression and the germination frequency of the encapsulated embryoids. (c) 1993 John Wiley & Sons, Inc.     

石刁柏体细胞胚胎发生过程中蛋白质及同工酶变化的研究

本文报道石刁柏胚性愈伤组织的可溶性蛋白质含量与组分、过氧化物酶和酯酶的活力及同工酶带均比其体细胞胚的要少。而在体细胞胚胎发生过程中,过氧化物酶和酯酶活力、可溶性蛋白质含量均以球形胚为最低,子叶分化期胚为最高而呈递增趋势;可溶性蛋白质组分以子叶分化期胚、成熟胚为最多,球形胚、香蕉形胚为最少;过氧化物酶同工酶带以梨形胚为最多,子叶分化期胚、成熟胚为最少;酯酶同工酶则以子叶分化期胚为最多,成熟胚为最少。胚性愈伤组织与体细胞胚均有其特异性可溶性蛋白质及同工酶带,可作为体细胞胚胎发生的分子标记。     

Serum replacement with albumin‐associated lipids prevents excess aggregation and enhances growth of induced pluripotent stem cells in suspension culture

Suspension culture systems are currently under investigation for the mass production of pluripotent stem (PS) cells for tissue engineering; however, the control of cell aggregation in suspension culture remains challenging. Existing methods to control aggregation such as microwell culture are difficult to scale up. To address this issue, in this study a novel method that incorporates the addition of KnockOut Serum Replacement (KSR) to the PS cell culture medium was described. The method regulated cellular aggregation and significantly improved cell growth (a 2‐ to 10‐fold increase) without any influence on pluripotency. In addition, albumin‐associated lipids as the major working ingredient of KSR responsible for this inhibition of aggregation were identified. This is one of the simplest methods described to date to control aggregation and requires only chemically synthesizable reagents. Thus, this method has the potential to simplify the mass production process of PS cells and thus lower their cost. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1009–1016, 2016     

Cryopreservation of mouse embryoid bodies

Cryopreservation of embryonic stem (ES) cells is essential to establish them as a resource for regenerative therapy. We evaluated survival adhesion rate, cell structure, gene expression, and multipotency of frozen and thawed embryoid bodies (EBs) derived from mouse ES cells. EBs were cryopreserved using the BICELL and the Program Freezer. After one week the EBs were thawed and cultured. EBs prepared in the Program Freezer had the highest survival adhesion (Program Freezer; 55-69%, BICELL; 30-38%). Though many cells in the thawed EBs were damaged, some were not, especially those prepared in the Program Freezer. RT-PCR analysis showed that genes characteristic of the three embryonic germ layers were expressed in thawed EBs cultured for one week. EBs transplanted into mice formed teratomas consisting of cells derived from the three germ layers. In conclusion, EBs frozen in the Program Freezer had higher survival adhesion rates compared to the BICELL and formed differentiated cells characteristic of the three embryonic germ layers.     

VEGF receptor 2/‐3 heterodimers detected in situ by proximity ligation on angiogenic sprouts

The vascular endothelial growth factors VEGFA and VEGFC are crucial regulators of vascular development. They exert their effects by dimerization and activation of the cognate receptors VEGFR2 and VEGFR3. Here, we have used in situ proximity ligation to detect receptor complexes in intact endothelial cells. We show that both VEGFA and VEGFC potently induce formation of VEGFR2/‐3 heterodimers. Receptor heterodimers were found in both developing blood vessels and immature lymphatic structures in embryoid bodies. We present evidence that heterodimers frequently localize to tip cell filopodia. Interestingly, in the presence of VEGFC, heterodimers were enriched in the leading tip cells as compared with trailing stalk cells of growing sprouts. Neutralization of VEGFR3 to prevent heterodimer formation in response to VEGFA decreased the extent of angiogenic sprouting. We conclude that VEGFR2/‐3 heterodimers on angiogenic sprouts induced by VEGFA or VEGFC may serve to positively regulate angiogenic sprouting.     

黄秋葵组培快繁的研究

黄秋葵 (ＨｉｂｉｓｃｕｓｅｓｃｕｌｅｎｔｕｓＬ .)为锦葵科一年生草本植物 ,别名羊角豆、秋葵。原产非洲 ,欧美及东南亚热带地区广泛栽培 (马传先 ,1999;李正应 ,1993;翁文 ,1997;雪珍等 ,1999)。黄秋葵是一种营养保健型蔬菜 ,其花、叶、芽、果均可食用 ,种子富含油脂、蛋白质及钾、钙、铁、锌、锰等矿物质 ,晒干后既可用于提取油脂和蛋白质 ,还可作为咖啡的添加剂或代用品。花、种子和根均可入药 ,对恶疮、痛疖有疗效。黄秋葵的愈伤组织在一定条件下比较容易产生体细胞胚并再生成完整植株 ,故黄秋葵的组培快繁研究也可为胚胎学研究和人…     

Generation of functional gut-like organ from mouse induced pluripotent stem cells

Induced pluripotent stem (iPS) cells have the pluripotency to differentiate into broad spectrum derivatives of all three embryonic germ layers. However, the in vitro organ differentiation potential of iPS cells to organize a complex and functional “organ” has not yet been demonstrated. Here, we demonstrate that mouse iPS cells have the ability to organize a gut-like organ with motor function in vitro by a hanging drop culture system. This “induced gut (iGut)” exhibited spontaneous contraction and highly coordinated peristalsis accompanied by a transportation of contents. Ultrastructural analysis identified that the iGut had large lumens surrounded by three distinct layers (epithelium, connective tissue and musculature). Immunoreactivity for c-Kit, a marker of interstitial cells of Cajal (ICCs, enteric pacemaker cells), was observed in the wall of the lumen and formed a distinct and dense network. The neurofilament immunoreactivity was identified to form large ganglion-like structures and dense neuronal networks. The iGut was composed of all the enteric components of three germ layers: epithelial cells (endoderm), smooth muscle cells (mesoderm), ICCs (mesoderm), and enteric neurons (ectoderm). This is the first report to demonstrate the in vitro differentiation potential of iPS cells into particular types of functional “organs.” This work not only contributes to understanding the mechanisms of incurable gut disease through disease-specific iPS cells, but also facilitates the clinical application of patient-specific iPS cells for novel therapeutic strategies such as patient-specific “organ” regenerative medicine in the future.