鱼类的胚胎干细胞

胚胎干细胞(ES)是未分化的细胞培养物，来自动物的早期胚胎。它们能成为稳定的细胞系和长期冻存。在适当的条件下，ES细胞能分化成各种细胞类型，包括生殖细胞。这样，ES细胞就提供了一个有效的纽带，将动物基因组的体外和体内遗传操作连系起来。ES细胞的魅力就由其在产生和分析基因敲除老鼠中显现出来。目前，ES细胞技术仅见之老鼠，因其它脊椎动物的ES细胞的培养和建系难获成功。在鱼类，人们已做了大量的尝试。我们以青鳉(Oryzias latipes)作为建立鱼类ES细胞技术的模式，通过建立并应用无滋养层细胞的培养条件，获得了来自中期囊胚的ES细胞系。青鳉的ES细胞和老鼠的ES细胞有很多共同特征，如二倍体核型、分化潜力和形成嵌合体。因此，在鱼类建立和应用ES细胞技术是可能的。青鳉ES细胞的培养条件已成功地应用到其它鱼类如斑马鱼甚至海水鱼。本文旨在以青鳉为模式，综述获得和应用模式鱼和经济鱼ES细胞的主要进展和前景。     

Efficiency of cell culture derivation from blastula embryos and of chimera formation in the medaka (Oryzias latipes) depends on donor genotype and passage number

 Embryonic stem (ES) cells from early vertebrate embryos only rarely retain their full developmental potential under in vitro culture conditions, but undergo differentiation and lose their ability for chimeric embryogenesis. This is reflected by the fact that the ES cell technology to date could only be fully developed in mice. In the fish Oryzias latipes, the medaka, one ES-like cell line, MES1, has been established which gives rise to a high frequency of somatic chimeras but a low degree of chimerism. Here we have tested the effect of donor genotype and cultivation time on the efficiency of cell culture derivation and on chimera formation. The HB12A, HB32C and HNI strains of medaka most efficiently and reproducibly give rise to blastula-derived cell cultures that produce pigmented chimeras in albino hosts. Seven chimeras grew to male or female adults with normal fertility, although none of them showed obvious donor germline contribution. During prolonged in vitro propagation the frequency of chimeras and the degree of chimerism dropped to a value retained in the long-term cultured MES1 cells. Obviously, genetic factors in host/donor compatibility and physiological changes during prolonged in vitro culture may compromise, but do not abolish, the developmental potential of medaka ES-like cells. Thus, elucidation of conditions that will expand the developmental potential of medaka blastula cell cultures should lead to a further improvement towards establishment of the ES cell technology in medaka. Received: 5 June 1998 / Accepted: 6 July 1998     

Medaka cleavage embryos are capable of generating ES-like cell cultures

Mammalian embryos at the blastocyst stage have three major lineages, which in culture can give rise to embryonic stem (ES) cells from the inner cell mass or epiblast, trophoblast stem cells from the trophectoderm, and primitive endoderm stem cells. None of these stem cells is totipotent, because they show gene expression profiles characteristic of their sources and usually contribute only to the lineages of their origins in chimeric embryos. It is unknown whether embryos prior to the blastocyst stage can be cultivated towards totipotent stem cell cultures. Medaka is an excellent model for stem cell research. This laboratory fish has generated diploid and even haploid ES cells from the midblastula embryo with ~2000 cells. Here we report in medaka that dispersed cells from earlier embryos can survive, proliferate and attach in culture. We show that even 32-cells embryos can be dissociated into individual cells capable of producing continuously growing ES-like cultures. Our data point to the possibility to derive stable cell culture from cleavage embryos in this organism.     

Medaka fish stem cells and their applications

Stem cells are present in developing embryos and adult tissues of multicellular organisms. Owing to their unique features, stem cells provide excellent opportunities for experimental analyses of basic developmental processes such as pluripotency control and cell fate decision and for regenerative medicine by stem cell-based therapy. Stem cell cultures have been best studied in 3 vertebrate organisms. These are the mouse, human and a small laboratory fish called medaka. Specifically, medaka has given rise to...     

Gene Transfer and Genome-Wide Insertional Mutagenesis by Retroviral Transduction in Fish Stem Cells

Retrovirus (RV) is efficient for gene transfer and integration in dividing cells of diverse organisms. RV provides a powerful tool for insertional mutagenesis (IM) to identify and functionally analyze genes essential for normal and pathological processes. Here we report RV-mediated gene transfer and genome-wide IM in fish stem cells from medaka and zebrafish. Three RVs were produced for fish cell transduction: rvLegfp and rvLcherry produce green fluorescent protein (GFP) and mCherry fluorescent protein respectively under control of human cytomegalovirus immediate early promoter upon any chromosomal integration, whereas rvGTgfp contains a splicing acceptor and expresses GFP only upon gene trapping (GT) via intronic in-frame integration and spliced to endogenous active genes. We show that rvLegfp and rvLcherry produce a transduction efficiency of 11~23% in medaka and zebrafish stem cell lines, which is as 30~67% efficient as the positive control in NIH/3T3. Upon co-infection with rvGTgfp and rvLcherry, GFP-positive cells were much fewer than Cherry-positive cells, consistent with rareness of productive gene trapping events versus random integration. Importantly, rvGTgfp infection in the medaka haploid embryonic stem (ES) cell line HX1 generated GTgfp insertion on all 24 chromosomes of the haploid genome. Similar to the mammalian haploid cells, these insertion events were presented predominantly in intergenic regions and introns but rarely in exons. RV-transduced HX1 retained the ES cell properties such as stable growth, embryoid body formation and pluripotency gene expression. Therefore, RV is proficient for gene transfer and IM in fish stem cells. Our results open new avenue for genome-wide IM in medaka haploid ES cells in culture.     

Fish germ cells

Fish, like many other animals, have two major cell lineages, namely the germline and soma. The germ-soma separation is one of the earliest events of embryonic development. Germ cells can be specifically labeled and isolated for culture and transplan-tation, providing tools for reproduction of endangered species in close relatives, such as surrogate production of trout in salmon. Haploid cell cultures, such as medaka haploid embryonic stem cells have recently been obtained, which are capable of mimicking spe...     

Fish <Emphasis Type="Italic">ES</Emphasis> Cells and Applications to Biotechnology

ES cells provide a promising tool for the generation of transgenic animals with site-directed mutations. When ES cells colonize germ cells in chimeras, transgenic animals with modified phenotypes are generated and used either for functional genomics studies or for improving productivity in commercial settings. Although the ES cell approach has been limited to mice, there is strong interest for developing the technology in fish. We describe the step-by-step procedure for developing ES cells in fish. Key aspects include avoiding cell differentiation, specific in vitro traits of pluripotency, and, most importantly, testing for production of chimeric animals as the main evidence of pluripotency. The entire process focuses on two model species, zebrafish and medaka, in which most work has been done. The achievements attained in these species, as well as their applicability to other commercial fish, are discussed. Because of the difficulties relating to germ line competence, mostly of long-term fish ES cells, alternative cell-based approaches such as primordial germ cells and nuclear transfer need to be considered. Although progress to date has been slow, there are promising achievements in homologous recombination and alternative avenues yet to be explored that can bring ES technology in fish to fruition.     

An ES-like cell line from the marine fish Sparus aurata: Characterization and chimaera production

Embryonic stem (ES) cells provide a unique tool for cell-mediated gene transfer and targeted gene mutations due to the possibility of in vitro selection of desired genotypes. When selected cells contribute to the germ line in chimaeric embryos, transgenic animals may be generated with modified genetic traits. Though the ES cell approach has up to now been limited to mice, there is an increasing interest to develop this technology in both model and commercial fish species, with so far promising results in the medaka and zebrafish. In this study, we present evidence regarding a long-term stable cell line (SaBE-1c), derived from embryonic cells of the aquaculture marine fish Sparus aurata which has been characterized for (i) cell proliferation, (ii) chromosome complement, (iii) molecular markers, and (iv) in vitro tests of pluripotency by alkaline phosphatase (AP) staining, telomerase activity, and induced cell differentiation. These cells have proved their pluripotent capacities by in vitro tests. Furthermore, we have demonstrated their ability to produce chimaeras and to contribute to the formation of tissues from all three embryonic germ layers. These features suggest that SaBE-1c cells have the potential for multiple applications for the ES technology in fish, with the added value of originating from an economically important species.     

Germline stem cells are critical for sexual fate decision of germ cells

Egg or sperm? The mechanism of sexual fate decision in germ cells has been a long‐standing issue in biology. A recent analysis identified foxl3 as a gene that determines the sexual fate decision of germ cells in the teleost fish, medaka. foxl3/Foxl3 acts in female germline stem cells to repress commitment into male fate (spermatogenesis), indicating that the presence of mitotic germ cells in the female is critical for continuous sexual fate decision of germ cells in medaka gonads. Interestingly, foxl3 is found in most vertebrate genomes except for mammals. This provides the interesting possibility that the sexual fate of germ cells in mammals is determined in a different way compared to foxl3‐possessing vertebrates. Considering the fact that germline stem cells are the cells where foxl3 begins to express and sexual fate decision initiates and mammalian ovary does not have typical germline stem cells, the mechanism in mammals may have been co‐evolved with germline stem cell loss in mammalian ovary.

     

Progress towards cell-mediated gene transfer in zebrafish.

Although the zebrafish possesses several favourable characteristics that make it an ideal model for genetic studies of vertebrate development, one disadvantage of this model system is the absence of methods for the production of gene knockouts. The authors' laboratory, and others, are working to develop zebrafish pluripotent embryonic stem (ES) and primordial germ cell (PGC) cultures that can be used for cell-mediated gene transfer and the production of knockout mutant lines of fish. Progress has been made in developing short-term cell cultures that possess the ability to contribute to multiple tissues, including the germ line of a host embryo, and transgenic lines of zebrafish have been established using the embryo cell cultures. Work is in progress to extend the length of time that the embryo cells can be maintained in culture without losing their ability to generate germ-line chimeras.     

Culture of cells from zebrafish (Brachydanio rerio) embryo and adult tissues

The zebrafish is a popular model for studies of vertebrate development and toxicology. However, in vitro approaches with this organism have not been fully exploited because cell culture systems have been unavailable. We developed methods for the culture of cells from blastula-stage diploid and haploid zebrafish embryos, as well as cells from the caudal and pelvic fin, gill, liver, and viscera of adult fish. The haploid embryo-derived cells differentiated in culture to a pigmented phenotype and expressed, upon exposure to 2,3,7,8-tetrachlorodibenzo p-dioxin, a protein that was immunologically and functionally similar to rainbow trout cytochrome P450IA1 Zebrafish cultures were grown in a complex basal nutrient medium supplemented with insulin, trout embryo extract, and low concentrations of trout and fetal bovine serum; they could not be maintained in conventional culture medium containing a high concentration of mammalian serum. Using calcium phosphate-mediated transfection, a plasmid constructed for use in mammalian cells was introduced into zebrafish embryo cell cultures and expressed in a stable manner. These results indicated that the transfection procedures utilized in mammalian systems can also be applied to zebrafish cell cultures, providing a means for in vitro alteration of the genotype and phenotype of the cells.[/ p]Abbreviations TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin - EROD, 7-ethyoxyresorufin - HDPDS, 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid - EDTA, ethylanediaminetetraacetic acid - FBS, fetal bovine serum - LDF, limit dilution factor - DMSO, dimethyl sulfoxide - ES, embryonal stem - PAH, polycylic aromatic hydrocarbons - ZG, zebrafish gill - ZBF, zebrafish pelvic fin - ZV, Zebrafish viscera - ZCF, zebrafish caudal fin - ZEM, diploid blastula-derived     

Stem cells and regenerative medicine

Stem cells have been defined as clonogenic cells that undergo both self-renewal and differentiation to more committed progenitors and functionally specialized mature cells. Of late years, stem cells have been identified in a variety of tissues of an adult body. Depending on the source, they have the potential to form one or more, or even all cell types of an organism. Stem cell research provided some outstanding contributions to our understanding of developmental biology and offered much hope for cell replacement therapies overcoming a variety of diseases. The establishment of human ES cell lines enabled us to generate all tissues we comprise. Recently, excitement has been evoked by the controversial evidence that adult stem cells have a much higher degree of developmental plasticity than previously imagined. More recently, the existence of multipotent somatic stem cells in bone marrow has been reported. Combined with these discoveries and achievements as well as the developing technologies, scientists are now trying to bring stem cell therapies to the clinic.     

Establishment and growth responses of Nile tilapia embryonic stem‐like cell lines under feeder‐free condition

Embryonic stem (ES) cells provide an invaluable tool for molecular analysis of vertebrate development and a bridge linking genomic manipulations in vitro and functional analysis of target genes in vivo. Work towards fish ES cells so far has focused on zebrafish (Danio renio) and medaka (Oryzias latipes). Here we describe the derivation, pluripotency, differentiation and growth responses of ES cell lines from Nile tilapia (Oreochromis niloticus), a world‐wide commercial farmed fish. These cell lines, designated as TES1‐3, were initiated from blastomeres of Nile tilapia middle blastula embryos (MBE). One representative line, TES1, showed stable growth and phenotypic characteristics of ES cells over 200 days of culture with more than 59 passages under feeder‐free conditions. They exhibited high alkaline phosphatase activity and expression of pluripotency genes including pou5f3 (the pou5f1/oct4 homologue), sox2, myc and klf4. In suspension culture together with retinoic acid treatment, TES1 cells formed embryoid bodies, which exhibited expression profile of differentiation genes characteristics of all three germ cell layers. Notably, PKH26‐labeled TES1 cells introduced into Nile tilapia MBE could contribute to body compartment development and led to hatched chimera formation with an efficacy of 13%. These results suggest that TES1 cells have pluripotency and differentiation potential in vitro and in vivo. In the conditioned DMEM, all of the supplements including the fetal bovine serum, fish embryonic extract, fish serum, basic fibroblast growth factor and non‐protein supplement combination 5N were mitogenic for TES1 cell growth. This study will promote ES‐based biotechnology in commercial fish.     

Ovarian germline stem cells in the teleost fish, medaka (Oryzias latipes)

In the mammalian testis germline stem cells keep producing many sperms, while there is no direct evidence for the presence of germline stem cells in the ovary. It is widely accepted in mammals that the mature oocytes are supplied from a pool of primordial follicles in the adult ovary. In other vertebrates, such as fish, however, there has been no investigation on the mechanism underlying the high egg-producing ability. In this review, we introduce the recently identified ovarian germline stem cells and the surrounding unique structure in teleost fish, medaka (Oryzias latipes) [Nakamura S et al. Science. 2010; 328: 1561-1563]. We also discuss about the expression and function of sox9 that characterizes this unique structure.     

Germline competence of mouse ES and iPS cell lines: Chimera technologies and genetic background

In mice, gene targeting by homologous recombination continues to play an essential role in the understanding of functional genomics. This strategy allows precise location of the site of transgene integration and is most commonly used to ablate gene expression ("knock-out"), or to introduce mutant or modified alleles at the locus of interest ("knock-in"). The efficacy of producing live, transgenic mice challenges our understanding of this complex process, and of the factors which influence germline competence of embryonic stem cell lines. Increasingly, evidence indicates that culture conditions and in vitro manipulation can affect the germline-competence of Embryonic Stem cell (ES cell) lines by accumulation of chromosome abnormalities and/or epigenetic alterations of the ES cell genome. The effectiveness of ES cell derivation is greatly strain-dependent and it may also influence the germline transmission capability. Recent technical improvements in the production of germline chimeras have been focused on means of generating ES cells lines with a higher germline potential. There are a number of options for generating chimeras from ES cells (ES chimera mice); however, each method has its advantages and disadvantages. Recent developments in induced pluripotent stem (iPS) cell technology have opened new avenues for generation of animals from genetically modified somatic cells by means of chimera technologies. The aim of this review is to give a brief account of how the factors mentioned above are influencing the germline transmission capacity and the developmental potential of mouse pluripotent stem cell lines. The most recent methods for generating specifically ES and iPS chimera mice, including the advantages and disadvantages of each method are also discussed.     

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.     

Dorsomorphin Promotes Survival and Germline Competence of Zebrafish Spermatogonial Stem Cells in Culture

Zebrafish spermatogonial cell cultures were established from Tg(piwil1:neo);Tg(piwil1:DsRed) transgenic fish using a zebrafish ovarian feeder cell line (OFC3) that was engineered to express zebrafish Lif, Fgf2 and Gdnf. Primary cultures, initiated from testes, were treated with G418 to eliminate the somatic cells and select for the piwil1:neo expressing spermatogonia. Addition of dorsomorphin, a Bmp type I receptor inhibitor, prolonged spermatogonial stem cell (SSC) survival in culture and enhanced germline transmission of the SSCs following transplantation into recipient larvae. In contrast, dorsomorphin inhibited the growth and survival of zebrafish female germline stem cells (FGSCs) in culture. In the presence of dorsomorphin, the spermatogonia continued to express the germ-cell markers dazl, dnd, nanos3, vasa and piwil1 and the spermatogonial markers plzf and sox17 for at least six weeks in culture. Transplantation experiments revealed that 6 week-old spermatogonial cell cultures maintained in the presence of dorsomorphin were able to successfully colonize the gonad in 18% of recipient larvae and produce functional gametes in the resulting adult chimeric fish. Germline transmission was not successful when the spermatogonia were cultured 6 weeks in the absence of dorsomorphin before transplantation. The results indicate that Bmp signaling is detrimental to SSCs but required for the survival of zebrafish FGSCs in culture. Manipulation of Bmp signaling could provide a strategy to optimize culture conditions of germline stem cells from other species.     

Retention of the Developmental Pluripotency in Medaka Embryonic Stem Cells after Gene Transfer and Long-term Drug Selection for Gene Targeting in Fish

Embryonic stem (ES) cells provide a unique tool for introducing random or targeted genetic alterations, because it is possible that the desired, but extremely rare recombinant genotypes can be screened by drug selection. ES cell-mediated transgenesis has so far been limited to the mouse. In the fish medaka (Oryzias latipes) several ES cell lines have been made available. Here we report the optimized conditions for gene transfer and drug selection in the medaka ES cell line MES1 as a prelude for gene targeting in fish. MES1 cells gave rise to a moderate to high transfection efficiency by the calcium phosphate co-precipitation (5%), commercial reagents Fugene (11%), GeneJuice (21%) and electroporation (>30%). Transient gene transfer and CAT reporter assay revealed that several enhancers/promoters and their combinations including CMV, RSV and ST (the SV40 virus early gene enhancer linked to the thymidine kinase promoter) were suitable regulatory sequences to drive transgene expression in the MES1 cells. We show that neo, hyg or pac conferred resistance to G418, hygromycin or puromycin for positive selection, while the HSV-tk generated sensitivity to ganciclovir for negative selection. The positive-negative selection procedure that is widely used for gene targeting in mouse ES cells was found to be effective also in MES1 cells. Importantly, we demonstrate that MES1 cells after gene transfer and long-term drug selection retained the developmental pluripotency, as they were able to undergo induced differentiation in vitro and to contribute to various tissues and organs during chimeric embryogenesis.