Generation and characterization of pluripotent stem cells from cloned bovine embryos

Bovine embryonic stem (ES) cell lines reported to date vary in morphology and marker expression (e.g., alkaline phosphatase [ALPL], stage-specific embryonic antigen 4 [SSEA4], and OCT4) that normally are associated with the undifferentiated, pluripotent state. These observations suggest that the proper experimental conditions for consistently producing bovine ES cells have not been identified. Here, we report three bovine ES cell lines, one from in vitro-fertilized and two from nuclear transfer embryos. These bovine ES cells grew in large, multicellular colonies resembling the mouse ES and embryonic germ (EG) cells and human EG cells. Throughout the culture period, most of the cells within the colonies stained positive for ALPL and the cell surface markers SSEA4 and OCT4. The staining patterns of nuclear transfer ES cells were identical to those of the blastocysts generated in vitro yet different from most previously reported bovine ES cell lines, which were either negative or not detected. After undifferentiated culture for more than 1 yr, these cells maintained the ability to differentiate into embryoid bodies and derivatives of all three EG layers, thus demonstrating their pluripotency. However, unlike the mouse and human ES cells, following treatment with trypsin, type IV collagenase, or protease E, our bovine ES cells failed to self-renew and became spontaneously differentiated. Presumably, this resulted from an interruption of the self-renewal pathway. In summary, we generated pluripotent bovine ES cells with morphology similar to those of established ES cells in humans and mice as well as marker-staining patterns identical to those of the bovine blastocysts.     

Analysis of long-term culture properties and pluripotent character of two sibling human embryonic stem cell lines derived from discarded embryos

We had earlier reported the derivation and characterization of two new sibling human embryonic stem cell lines BJNhem19 and BJNhem20, from discarded grade III embryos of Indian origin. We report here the characteristics of the two sibling cell lines after long-term continuous culture for over 2 yr during which they have been passaged over 200 times. We show that both cell lines adapt well to culture on various mouse and human feeders as well as in feeder-free conditions. The cells show normal diploid karyotype and continue to express all pluripotency markers. Both cell lines differentiate to derivatives of all three germ layers in vitro. However as reported earlier, BJNhem19 is unable to generate teratomas in nude or SCID mice or differentiate to beating cardiomyocytes when tested over several passages during long-term stable culture. On the other hand, the cardiac differentiation capacity of BJNhem20 is greatly increased, and it can generate beating cardiomyocytes that proliferate when isolated and cultured further. In conclusion, the two cell lines have maintained a stable phenotype for over 2 yr and are indeed immortal. Their derivation from grade III embryos does not seem to have any adverse effect on their long-term phenotype. The cells can be obtained for research purposes from the UK Stem Cell Bank and from the authors.     

Derivation, propagation and differentiation of human embryonic stem cells

Embryonic stem (ES) cells are in vitro cultivated pluripotent cells derived from the inner cell mass (ICM) of the embryonic blastocyst. Attesting to their pluripotency, ES cells can be differentiated into representative derivatives of all three embryonic germ layers (endoderm, ectoderm and mesoderm) both in vitro and in vivo. Although mouse ES cells have been studied for many years, human ES cells have only more recently been derived and successfully propagated. Many biochemical differences and culture requirements between mouse and human ES cells have been described, yet despite these differences the study of murine ES cells has provided important insights into methodologies aimed at generating a greater and more in depth understanding of human ES cell biology. One common feature of both mouse and human ES cells is their capacity to undergo controlled differentiation into spheroid structures termed embryoid bodies (EBs). EBs recapitulate several aspects of early development, displaying regional-specific differentiation programs into derivatives of all three embryonic germ layers. For this reason, EB formation has been utilised as an initial step in a wide range of studies aimed at differentiating both mouse and human ES cells into a specific and desired cell type. Recent reports utilising specific growth factor combinations and cell-cell induction systems have provided alternative strategies for the directed differentiation of cells into a desired lineage. According to each one of these strategies, however, a relatively high cell lineage heterogeneity remains, necessitating subsequent purification steps including mechanical dissection, selective media or fluorescent or magnetic activated cell sorting (FACS and MACS, respectively). In the future, the ability to specifically direct differentiation of human ES cells at 100% efficiency into a desired lineage will allow us to fully explore the potential of these cells in the analysis of early human development, drug discovery, drug testing and repair of damaged or diseased tissues via transplantation.     

Properties of murine embryonic stem cells maintained on human foreskin fibroblasts without LIF

In embryonic stem (ES) cells, leukemia inhibitory factor (LIF)/STAT3, wnt and nodal/activin signaling are mainly active to control pluripotency during expansion. To maintain pluripotency, ES cells are typically cultured on feeder cells of varying origins. Murine ES cells are commonly cultured on murine embryonic fibroblasts (MEFs), which senesce early and must be frequently prepared. This process is laborious and leads to batch variation presenting a challenge for high-throughput ES cell expansion. Although some cell lines can be sustained by exogenous LIF, this method is costly. We present here a novel and inexpensive culture method for expanding murine ES cells on human foreskin fibroblast (HFF) feeders. After 20 passages on HFFs without LIF, ES cell lines showed normal expression levels of pluripotency markers, maintained a normal karyotype and retained the ability to contribute to the germline. As HFFs do not senesce for at least 62 passages, they present a vast supply of feeders.     

FGF2 secreting human fibroblast feeder cells: a novel culture system for human embryonic stem cells

Human embryonic stem cell (hESC) lines are traditionally derived and maintained on mouse embryonic fibroblasts (MEF) which are xenogeneic and enter senescence rapidly. In view of the clinical implications of hESCs, the use of human fibroblast as feeders has been suggested as a plausible alternative. However, use of fibroblast cells from varying sources leads to culture variations along with the need to add FGF2 in cultures to sustain ES cell pluripotency. In this study we report the derivation of FGF2 expressing germ layer derived fibroblast cells (GLDF) from hESC lines. These feeders could support the pluripotency, karyotypes and proliferation of hESCs with or without FGF2 in prolonged cultures as efficiently as that on MEF. GLDF cells were derived from embryoid bodies and characterized for expression of fibroblast markers by RT-PCR, Immunofluorescence and by flow cytometry for CD marker expression. The expression and secretion of FGF2 was confirmed by RT-PCR, Western blot, and ELISA. The hESC lines cultured on MEF and GLDF were analyzed for various stemness markers. These feeder cells with fibroblast cells like properties maintained the properties of hESCs in prolonged culture over 30 passages. Proliferation and pluripotency of hESCs on GLDF was comparable to that on mouse feeders. Further we discovered that these GLDF cells could secrete FGF2 and maintained pluripotency of hESC cultures even in the absence of supplemental FGF2. To our knowledge, this is the first study reporting a novel hESC culture system which does not warrant FGF2 supplementation, thereby reducing the cost of hESC cultures.     

Comparative analysis of the developmental competence of three human embryonic stem cell lines in vitro

One of the goals of stem cell technology is to control the differentiation of human embryonic stem cells (hESCs), thereby generating large numbers of specific cell types for many applications including cell replacement therapy. Although individual hESC lines resemble each other in expressing pluripotency markers and telomerase activity, it is not clear whether they are equivalent in their developmental potential in vitro. We compared the developmental competence of three hESC lines (HSF6, Miz-hES4, and Miz-hES6). All three generated the three embryonic germ layers, extraembryonic tissues, and primordial germ cells during embryoid body (EB) formation. However, HSF6 and Miz-hES6 readily formed neuroectoderm, whereas Miz-hES4 differentiated preferentially into mesoderm and endoderm. Upon terminal differentiation, HSF6 and Miz-hES6 produced mainly neuronal cells whereas Miz-hES4 mainly formed mesendodermal derivatives, including endothelial cells, leukocyte progenitors, hepatocytes, and pancreatic cells. Our observations suggest that independently-derived hESCs may differ in their developmental potential.     

Patient-specific stem cell lines derived from human parthenogenetic blastocysts

Parthenogenetic activation of human oocytes may be one way to produce histocompatible cells for cell-based therapy. We report the successful derivation of six pluripotent human embryonic stem cell (hESC) lines from blastocysts of parthenogenetic origin. The parthenogenetic human embryonic stem cells (phESC) demonstrate typical hESC morphology, express appropriate markers, and possess high levels of alkaline phosphatase and telomerase activity. The phESC lines have a normal 46, XX karyotype, except one cell line, and have been cultured from between 21 to 35 passages. The phESC lines form embryoid bodies in suspension culture and teratomas after injection to immunodeficient animals and give differentiated derivatives of all three embryonic germ layers. DNA profiling of all six phESC lines demonstrates that they are MHC matched with the oocyte donors. The study of imprinted genes demonstrated further evidence of the parthenogenetic origin of the phESC lines. Our research has resulted in a protocol for the production of human parthenogenetic embryos and the derivation of stem cell lines from them, which minimizes the presence of animal-derived components, making the derived phESC lines more suitable for potential clinical use.     

Establishment of pluripotent cell lines from porcine preimplantation embryos

Embryonic stem (ES) cells are pluripotent cells isolated from in vitro culture of preimplantation embryos. Experiments were undertaken to identify preimplantation embryonic stages and culture conditions under which pluripotent, porcine embryo-derived cell lines could be isolated. Cell lines were established from in vitro culture of intact, porcine early hatched blastocysts and isolated inner cell masses (ICM) from intermediate and late hatched blastocysts on feeder layers prepared from permanent mouse embryonic fibroblasts (STO). The cells of these porcine embryo-derived cell lines had a morphology similar to that of murine ES cells, but colony morphology was more epithelial-like. The cell lines retained a normal diploid karyotype, consistently expressed alkaline phosphatase activity, and survived cryopreservation. When subjected to in vitro differentiation, either spontaneous or induced, the embryo-derived cell lines differentiated extensively into a wide range of cell types representing the 3 embryonic germ layers. In vivo pluripotency of the cells was demonstrated by birth of a chimeric piglet, documented by pigmentation and DNA markers, and the ability to direct the development of nuclear-transfer embryos to the blastocyst stage. Such pluripotent embryo-derived cells provide a potential route for porcine genetic manipulation.     

Pluripotency of bovine embryonic cell line derived from precompacting embryos

We report herein the establishment of three bovine pluripotent embryonic cell lines derived from 8-16-cell precompacting embryos. Two cell lines were cultured for 10 passages and underwent spontaneous differentiation. One cell line (Z2) has been cultured continuously for over 3 years and has remained undifferentiated. These cells express cell surface markers that have been used routinely to characterize embryonic stem (ES) and embryonic germ (EG) cells in other species such as stage-specific embryonic antigens SSEA-1, SSEA-3, and SSEA-4, and c-Kit receptor. In the absence of a feeder layer, these cells differentiated into a variety of cell types and formed embryoid bodies (EBs). When cultured for an extended period of time, EBs differentiated into derivatives of three EG layers - mesoderm, ectoderm, and endoderm - which were characterized by detection of specific cell surface markers. Our results indicate that the Z2 cell line is pluripotent and resembles an ES cell line. To our knowledge, this is the first bovine embryonic cell line that has remained pluripotent in culture for more than 150 passages.     

Derivation of stable zebrafish ES-like cells in feeder-free culture

Zebrafish offers an excellent opportunity to combine embryological, genetic and molecular analyses of vertebrate development in vivo. Embryonic stem (ES) cells have enormous potential to study developmental potency and differentiation in vitro and thus to complement in vivo approaches. Zebrafish ES-like cells have been produced on a feeder cell layer. Here, we report the derivation of Z428, a zebrafish ES-like cell line, from blastula embryos in feeder-free culture. Fetal bovine serum, fish serum, fish embryo extract, basic fibroblast growth factor, non-essential amino acids and 2-mercaptoethanol were found to be important for Z428 growth. After more than 120 passages and many freezing/thawing cycles over a period of 20 years, Z428 exhibits stable growth and manifests many ES cell features including an ES cell phenotype, high alkaline phosphatase activity and spontaneous differentiation in culture. Most importantly, Z428 was transplantable to blastula hosts and capable of contributing to embryonic tissues and organ systems of the three germ layers. Therefore, Z428 is a stable cell line and contains ES-like cells with pluripotency in vitro and in vivo, and a feeder layer is dispensable for ES-like cell derivation in zebrafish. The derivation and easy maintenance of zebrafish ES-like cells under feeder-free conditions provide a useful extension of the present toolbox for studying development and differentiation in the zebrafish model.     

家畜胚胎干细胞多能性候选信号通路及分子标志

家畜胚胎干细胞具有重要的生物学意义和广阔的应用前景。以下对比了小鼠、人胚胎干细胞多能性调控信号通路的异同,阐述了小鼠、人胚胎干细胞与家畜胚胎干细胞在多能性分子标志上的差异,并结合本实验室开展绵羊胚胎干细胞研究的实际经验,对目前家畜胚胎干细胞建系中可能存在的多能性候选信号通路及分子标志进行了探讨。     

Nuclear reprogramming and pluripotency of embryonic cells: Application to the isolation of embryonic stem cells in farm animals

Despite their biological and biotechnological interest, pluripotent embryonic stem cell lines (ES cells) have been isolated from cultured embryos only in a very limited number of mammalian species. Here we review the main molecular mechanisms that have been shown in mouse or primates to regulate the maintenance of pluripotency in vitro. We describe the main signaling pathways that participate in the self-renewal of ES cells and provide an outlook on the epigenetic associated mechanisms. We also propose a practical approach to stem cell differentiation that examines the relationships between the genotype of embryos and their culture conditions and consider nuclear reprogramming as a valuable approach in ES cell derivation in farm animals.     

Derivation of three new human embryonic stem cell lines

Human embryonic stem cells are pluripotent cells capable of extensive self-renewal and differentiation to all cells of the embryo proper. Here, we describe the derivation and characterization of three Sydney IVF human embryonic stem cell lines not already reported elsewhere, designated SIVF001, SIVF002, and SIVF014. The cell lines display typical compact colony morphology of embryonic stem cells, have stable growth rates over more than 40 passages and are cytogenetically normal. Furthermore, the cell lines express pluripotency markers including Nanog, Oct4, SSEA3 and Tra-1-81, and are capable of generating teratoma cells derived from each of the three germ layers in immunodeficient mice. These experiments show that the cell lines constitute pluripotent stem cell lines.     

Improved genetic manipulation of human embryonic stem cells

Low efficiency of transfection limits the ability to genetically manipulate human embryonic stem cells (hESCs), and differences in cell derivation and culture methods require optimization of transfection protocols. We transiently transferred multiple independent hESC lines with different growth requirements to standardized feeder-free culture, and optimized conditions for clonal growth and efficient gene transfer without loss of pluripotency. Stably transfected lines retained differentiation potential, and most lines displayed normal karyotypes.     

New approach for the establishment of mouse early embryonic stem cells and induction of their differentiation

Eleven early embryonic stem (EES) cell lines were established using a new novel method. Two cell stage embryos from the ddY mouse strain were cultured in alpha-MEM supplemented with 10% fetal calf serum (FCS) and embryotrophic factors (ETFs) and allowed to develop to the trilaminal germ disc embryonic stage. Only small round cells (EES cells) were isolated by the colony isolating technique and subsequently cultured in the same medium containing the ETFs and leukemia inhibitory factors (LIF-10 ng/ml). The newly established embryonic stem (ES) cells isolated from inner cell mass of blastocysts differentiated from two cell stage embryo in culture. The EES and ES cell lines were maintained in an undifferentiated state using Ham's F12 medium supplemented with 10% FCS and 1 ng/ml of LIF. The EES cells maintained their normal genetic and morphological features as well as their potential to differentiate into a broad spectrum of cell types as well as their ability to contribute to all cell lineages in chimeric mice. Moreover, these cell lines changed and differentiated into various kinds of cells by removing LIF and by the addition of ETFs to the vitro culture system. All 11 EES cell lines and 3 ES cell lines formed embryoid bodies; however, cell line EES-4 formed tube-like structures which extended, anastomosed with each other, and finally formed networks when the LIF were absent. Primitive germ organ-like structures composed of 3 germ layers were recognized in the cultures following the administration of ETFs. In conclusion, the new method devised by us is a novel, easy and reliable technique for establishing EES cell lines.     

Epigenetic stability of embryonic stem cells and developmental potential

Recent studies highlight the tremendous potential of human embryonic stem (ES) cells and their derivatives as therapeutic tools for degenerative diseases. However, derivation and culture of ES cells can induce epigenetic alterations, which can have long lasting effects on gene expression and phenotype. Research on human and mouse stem cells indicates that developmental, cancer-related genes, and genes regulated by genomic imprinting are particularly susceptible to changes in DNA methylation. Together with the occurrence of genetic alterations, epigenetic instability needs to be monitored when considering human stem cells for therapeutic and technological purposes. Here, we discuss the maintenance of epigenetic information in cultured stem cells and embryos and how this influences their developmental potential.     

Perspectives of pluripotent stem cells in livestock

The recent progress in derivation of pluripotent stem cells(PSCs)from farm animals opens new approaches not only for reproduction,genetic engineering,treatment and conservation of these species,but also for screening novel drugs for their efficacy and toxicity,and modelling of human diseases.Initial attempts to derive PSCs from the inner cell mass of blastocyst stages in farm animals were largely unsuccessful as either the cells survived for only a few passages,or lost their cellular potency;indicating that the protocols which allowed the derivation of murine or human embryonic stem(ES)cells were not sufficient to support the maintenance of ES cells from farm animals.This scenario changed by the innovation of induced pluripotency and by the development of the 3 inhibitor culture conditions to support na?ve pluripotency in ES cells from livestock species.However,the long-term culture of livestock PSCs while maintaining the full pluripotency is still challenging,and requires further refinements.Here,we review the current achievements in the derivation of PSCs from farm animals,and discuss the potential application areas.