Isolation and characterization of embryonic stem-like cells from canine blastocysts

Embryonic stem (ES) cells are pluripotent cells with the capacity to generate any type of cell. Here we describe the isolation of ES-like cells from canine blastocysts. Canine embryos were collected from beagle bitches at day 11-16 of first estrus. A total of 80 normal embryos were obtained from 15 dogs. Of the embryos, 13 were at the morulae stage, 39 at the blastocyst stage, and 28 at the hatched blastocyst stage. The inside of morulae or inner cell masses (ICMs) of blastocysts were isolated mechanically, and cultured onto mouse embryonic fibroblasts (MEF) as feeder layers. Primary cell colonies were formed in 0% (0/13) of morulae, 25.6% (10/39) of blastocysts, and 67.9% (19/28) of hatched blastocysts. These colonies were separated either by enzymatic dissociation or by mechanical disaggregation. Dissociation with collagenase resulted in immediate differentiation, but with mechanical disaggregation these cells remained undifferentiated, and two ES-like cell lines (cES1, cES2) continued to grow in culture after eight passages. These cells had typical stem cell-like morphology and expressed specific markers such as alkaline phosphatase activity, stage specific embryonic antigen-1 and Oct-4. These cells formed embryoid bodies (EBs) in a suspension culture; extended culture of EBs resulted in the formation of cystic EBs. When the simple EBs were cultured on tissue culture plates, they differentiated into several types of cells including neuron-like, epithelium-like, fibroblast-like, melanocyte-like, and myocardium-like cells. These observations indicate that we successfully isolated and characterized canine ES-like cells.     

Isolation, culture, and characterization of embryonic cell lines from vitrified sheep blastocysts

This study was conducted to isolate, to culture, and to characterize embryonic cell lines from in vitro produced vitrified sheep blastocysts. Embryos were produced and vitrified at the expanded blastocyst stage. Ten inner cell masses arising from day 6-7 blastocysts were isolated by immunosurgery, disaggregated, and cultured onto mitomocin-C-inactivated mouse STO fibroblasts (MIF). After 5 or 6 days of culture the primary cell colonies were disaggregated, seeded in a new MIF, and cultured for 3 or 4 days to form new colonies called Passage 1. These cells were then disaggregated and cultured for other two passages. The primary cell colonies and Passage 2 colonies expressed stage specific embryonic markers SSEA-1, SSEA-3, and SSEA-4, and were alkaline phosphatase positive. In the absence of feeder layer and human leukemia inhibitory factor (LIF), these cells differentiated into variety of cell types and formed embryoid bodies. When cultured for an extended period of time, embryoid bodies differentiated into derivatives of three embryonic germ (EG) layers. These were characterized by detection of specific markers for differentiation such early mesoderm (FE-C6), embryonic myosin (F1-652), neural precursor (FORSE-1), and endoderm (anti-cytokeratin 18). To our knowledge, this is the first time that embryonic cell lines from in vitro produced and vitrified ovine blastocysts have been isolated and examined for detection of SSEA markers, and embryoid bodies have been cultured and examined for specific cell surface markers for differentiation.     

Parameters influencing derivation of embryonic stem cells from murine embryos

The derivation of ES cells is poorly understood and varies in efficiency between different strains of mice. We have investigated potential differences between embryos of permissive and recalcitrant strains during diapause and ES cell derivation. We found that in diapause embryos of the recalcitrant C57BL/6 and CBA strains, the epiblast failed to expand during the primary explant phase of ES cell derivation, whereas in the permissive 129 strain, it expanded dramatically. Epiblasts from the recalcitrant strains could be expanded by reducing Erk activation. Isolation of 129 epiblasts facilitated very efficient derivation of ES cell lines in serum- and feeder-free conditions, but reduction of Erk activity was required for derivation of ES cells from isolated C57BL/6 or CBA epiblasts. The results suggest that the discrepancy in ES cell derivation efficiency is not attributable merely to variable prodifferentiative effects of the extra-embryonic lineages but also to an intrinsic variability within the epiblast to maintain pluripotency.     

Endothelial cells regulate cardiomyocyte development from embryonic stem cells

The molecules and environment that direct pluripotent stem cell differentiation into cardiomyocytes are largely unknown. Here, we determined a critical role of receptor tyrosine kinase, EphB4, in regulating cardiomyocyte generation from embryonic stem (ES) cells through endothelial cells. The number of spontaneous contracting cardiomyocytes, and the expression of cardiac‐specific genes, including α‐MHC and MLC‐2V, was significantly decreased in EphB4‐null ES cells. EphB4 was expressed in endothelial cells underneath contracting cardiomyocytes, but not in cardiomyocytes. Angiogenic inhibitors, including endostatin and angiostatin, inhibited endothelial cell differentiation and diminished cardiomyogenesis in ES cells. Generation of functional cardiomyocytes and the expression of cardiac‐specific genes were significantly enhanced by co‐culture of ES cells with human endothelial cells. Furthermore, the defects of cardiomyocyte differentiation in EphB4‐deficient ES cells were rescued by human endothelial cells. For the first time, our study demonstrated that endothelial cells play an essential role in facilitating cardiomyocyte differentiation from pluripotent stem cells. EphB4 signaling is a critical component of the endothelial niche to regulate regeneration of cardiomyocytes. J. Cell. Biochem. 111: 29–39, 2010. © 2010 Wiley‐Liss, Inc.     

Pluripotential rabbit embryonic stem (ES) cells are capable of forming overt coat color chimeras following injection into blastocysts

The isolation of pluripotent embryonic stem (ES) cell lines from preimplantation rabbit embryos and their in vitro properties have been previously described. In the present investigation, these ES cell lines were further characterized and their capacity to contribute to formation of adult, fertile animals upon injection into recipient New Zealand White blastocysts demonstrated. The efficiency of chimera formation was low (5% of live born), but the degree of chimerism, as assessed by coat color contribution from the Dutch belted strain, was high (10–50%). Thus a significant step is taken toward the development of gene-targeting technology in the rabbit, an animal whose physiology and size lend itself to unique applications in biomedical research. © 1996 Wiley-Liss, Inc.     

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.     

A method for cultivating morphologically undifferentiated embryonic stem cells from porcine blastocysts

Variable conditions were tested to determine an in-vitro cultivation method for the formation of morphologically undifferentiated embryonic stem cells from the inner cell mass (ICM) derived outgrowth of porcine blastocysts. Although all 16 Day-9 embryos failed to form colonies, 14 such colonies were obtained from a total of 69 Day-10 embryos when they were co-cultivated with porcine uterine fibroblast (PUF) cells over a 6-day period. The best results were obtained in Dulbecco's modified Eagle medium (DMEM) with 10% fetal calf serum and 10% porcine serum supplemented with bovine insulin and beta-mercaptoethanol, in which six out of seven embryos formed adequate ICM-derived colonies. Since murine fibroblasts were not found to be suitable feeder cells in this procedure, an endocrine synergistic interaction, which promotes embryonic attachment and colony formation, between porcine blastocysts and PUF cells is hypothesized. Continued propagation of the ICM-derived cells was not dependent on these factors; a total of seven cell lines were obtained after three to five subsequent passages on murine feeder-layers that resembled morphologically undifferentiated embryonic cells.     

Isolation and characterization of permanent cell lines from inner cell mass cells of bovine blastocysts

Inner cell masses (ICM) from in vitro produced day 8 or 9 bovine blastocysts were isolated by immunosurgery and cultured under different conditions in order to establish which of two feeder cell types and culture media were most efficient in supporting attachment and outgrowth of the bovine ICM cells. The efficiency of attachment and outgrowth of the ICM cells could be markedly improved when STO feeder cells were used instead of bovine uterus epithelial cells, and by using charcoal-stripped serum instead of normal serum to supplement the culture medium. More than 20 stable cell lines were obtained. Some of these lines were examined by immunofluorescence for developmentally regulated markers. From these results we conclude that the cell lines resemble epithelial cells, rather than pluripotent ICM cells. The developmental potential of cells of one of the lines was tested in the nuclear transfer assay. The cell line could support the initial development of enucleated oocytes, but none of the reconstructed embryos passed the eight-cell block. © 1995 Wiley-Liss, Inc.     

Derivation and characterization of putative pluripotential embryonic stem cells from preimplantation rabbit embryos

We have derived putative embryonic stem (ES) cell lines from preimplantation rabbit embryos and report here their initial characterization. Two principal cell types emerged following serial passage of explanted embryos, and each has subsequently given rise to immortalized cell lines. One cell type has morphology identical to primary outgrowths of trophectoderm, is strictly feeder-cell dependent, and spontaneously forms trophectodermal vesicles at high cell density. The second type appears to represent pluripotent ES cells derived from the inner cell mass as evidenced by (1) ability to grow in an undifferentiated state on feeder layers, (2) maintenance of a predominantly normal karyotype through serial passage (over 1 year), and (3) ability to form embryoid bodies, which form terminally differentiated cell types representative of ectoderm, mesoderm, and endoderm. These ES cells may ultimately be suitable for introduction of germline mutations (via homologous recombination). The rabbit's size, reproductive capability, and well-characterized physiology make it suitable for a wide range of investigations, particularly for development of large animal models of human disease. © 1993 Wiley-Liss, Inc.     

Regulation of p53 in embryonic stem cells

Despite an increasing interest in the role of the p53 tumour suppressor protein in embryonic stem cells, not much is known about its regulation in this cell type.We show that the relatively high amount of p53 protein correlates with a higher amount of p53 RNA in ES cells compared to differentiated cells. Moreover, p53 RNA is more stable in embryonic stem cells and the p53 protein is more often transcribed. This is at least partly due to decreased expression of miRNA-125a and 125b in embryonic stem cells. Despite its cytoplasmic localisation, p53 is degraded in 26S proteasomes in embryonic stem cells. This process is controlled by Mdm2, the deubiquitinating enzyme Hausp and Ubc13. In contrast, the E3 ligase PirH2 appears to be less important for the control of p53 in embryonic stem cells. During differentiation, p53 protein and RNA levels are decreased which corresponds to increased expression of miRNA-125a and miRNA-125b.     

牛胚胎生殖细胞的分离与培养

胚胎生殖细胞 (Embryonicgermcells,EG)是由生殖嵴原始生殖细胞 (Primordialgermcells,PGCs)中分离得到的一种未分化而多潜能的干细胞。牛EG细胞的研究在EG细胞核移植、转基因及建立生物反应器方面具有广阔的应用前景。本研究从 2 9- 70日龄牛胎儿PGCs分离得到EG细胞 ,经过抑制分化培养 ,其中一个细胞系传至 6代。所分离得到的EG细胞具有典型的EG细胞形态 ,AP及PAS染色呈阳性 ,核型正常 ,同时观察到这些细胞在体外进行自发性分化 ,可形成类胚体、成纤维样细胞及神经样细胞     

Multicolor karyotype analyses of mouse embryonic stem cells

Summary The manipulation of embryonic stem (ES) cells to introduce directional genetic changes into the genome of mice has become an important tool in biomedical research. Monitoring of cell morphology before and after DNA manipulation and special culture conditions are a prerequisite to preserve the pluripotent properties of ES cells and thus their ability to generate chimera and effective germline transmission (GLT). It has been reported that prolonged cell culturing may affect the diploid chromosomal composition of cells and therefore the percentage of chimerism and GLT. Herein, we report multicolor-fluorescence in situ hybridization (M-FISH) analysis of four different ES cell lines/clones. Although the morphology of all four ES cell lines/clones appeared normal and all four expressed the early markers Oct-3/4 and Nanog, two cell lines presented consistent numerical and structural chromosome aberrations. We demonstrate that M-FISH is a sensitive and accurate method for a comprehensive karyotype analysis of ES cells and may minimize time, costs, and disappointment due to inadequate ES cell sources. Both authors contributed equally to this work.     

Differentiation of embryonic stem cells into retinal neurons

Mouse embryonic stem (ES) cells are continuous cell lines derived from the inner mass of blastocysts. Neural progenitors derived from these cells serve as an excellent model for controlled neural differentiation and as such have tremendous potential to understand and treat neurodegenerative diseases. Here, we demonstrate that ES cell-derived neural progenitors express regulatory factors needed for retinal differentiation and that in response to epigenetic cues a subset of them differentiate along photoreceptor lineage. During the differentiation, they activate photoreceptor regulatory genes, suggesting that ES cell-derived neural progenitors recruit mechanisms normally used for photoreceptor differentiation in vivo. These observations suggest that ES cells can serve as an excellent model for understanding mechanisms that regulate specification of retinal neurons and as an unlimited source of neural progenitors for treating degenerative diseases of the retina by cell replacement.     

In vitro differentiation of rat embryonic stem cells into functional cardiomyocytes

The recent breakthrough in the generation of rat embryonic stem cells (rESCs) opens the door to application of gene targeting to create models for the study of human diseases. In addition, the in vitro differentiation system from rESCs into derivatives of three germ layers will serve as a powerful tool and resource for the investigation of mammalian development, cell function, tissue repair, and drug discovery. However, these uses have been limited by the difficulty of in vitro differentiation. The aims of this study were to establish an in vitro differentiation system from rESCs and to investigate whether rESCs are capable of forming terminal-differentiated cardiomyocytes. Using newly established rESCs, we found that embryoid body (EB)-based method used in mouse ESC (mESC) differentiation failed to work for the serum-free cultivated rESCs. We then developed a protocol by combination of three chemical inhibitors and feeder-conditioned medium. Under this condition, rESCs formed EBs, propagated and differentiated into three embryonic germ layers. Moreover, rESC-formed EBs could differentiate into spontaneously beating cardiomyocytes after plating. Analyses of molecular, structural, and functional properties revealed that rESC-derived cardiomyocytes were similar to those derived from fetal rat hearts and mESCs. In conclusion, we successfully developed an in vitro differentiation system for rESCs through which functional myocytes were generated and displayed phenotypes of rat fetal cardiomyocytes. This unique cellular system will provide a new approach to study the early development and cardiac function, and serve as an important tool in pharmacological testing and cell therapy.     

Isolation and culture of embryonic stem-like cells from pig nuclear transfer blastocysts of different days

This study was conducted to establish pig embryonic stem (ES)-like cell lines from nuclear transfer blastocysts. A green fluorescent protein (GFP)-expressing cell line was used as the source of donor cells injected into the enucleated oocytes. Blastocysts were collected at D5 (the fifth day), D7 (the seventh day) and D9 (the ninth day). Differential staining was used to assay the viability and development of blastocysts from the 3 days. The number of inner cell mass (ICM) cells increased from 1.83 ± 0.8 (D5) to 5.37 ± 1.2 (D7) to 7.56 ± 1.5 (D9). The expression profiles of embryonic stem (ES) cell factors (OCT4, SOX2, KLF4 and c-MYC) correlated best with the undifferentiated ES state and were identified by qPCR. The expression of the four factors was increased from D5 to D7, whereas the expression decreased from D7 to D9. We tried to isolate ES-like cells from these embryos. However, ES-like cells from the D7 blastocysts grew slowly and expressed alkaline phosphatase. The cells from the D9 blastocysts grew rapidly but did not express alkaline phosphatase. ES-like cells were not isolated from the D5 blastocysts. These results show that the cells from the D7 embryos are pluripotent but grow slowly. The cells from the D9 embryos grow rapidly but start to lose pluripotency.     

DNA羟甲基化在小鼠胚胎干细胞中的研究进展

简要总结DNA羟甲基化在小鼠胚胎干细胞(mouse embryonic stem cells,mESC)中的最新研究进展.DNA甲基化(DNAmethylation)影响染色质的结构与功能,在发育与疾病发生过程中具有重要作用.2009年Tahiliani等发现TET1可以催化甲基化胞嘧啶(5-methylcytosine,5mC)氧化为羟甲基化胞嘧啶(5-hydroxymethylcytosine,5hmC).DNA羟甲基化(DNAhydroxymethylation)被认为是调节DNA甲基化的一种重要方式,成为了表观遗传学的研究热点之一.