Generation of cloned calves and transgenic chimeric embryos from bovine embryonic stem-like cells

Bovine embryonic stem-like cells (ES-like cells) appear to maintain a normal diploid karyotype indefinitely during culture in vitro and to express marker proteins that are characteristic of ES cells from mice, namely, alkaline phosphatase (AP), stage-specific embryonic antigen-1 (SSEA-1), STAT-3, and Oct 4. After proliferation of undifferentiated ES-like cells in vitro, some bovine ES-like cells differentiated to neural precursor cells, which were cultured in the presence of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF). In addition, calves were successfully cloned using ES-like cells and the frequency of term pregnancies for blastocysts derived from ES-like cells was higher than those of early pregnancies and maintained pregnancies after nuclear transplantation (NT) with bovine somatic cells. Successful cloning from bovine ES-like cells should allow the introduction into cattle of specific genetic characteristics of biomedical and/or agricultural importance.     

Derivation and induction of the differentiation of animal ES cells as well as human pluripotent stem cells derived from fetal membrane

We succeeded in the derivation and maintenance of pluripotent embryonic stem (ES) cells from equine and bovine blastocysts. These cells expressed markers that are characteristics of mouse ES cells, namely, alkaline phosphatase, stage-specific embryonic antigen 1, STAT 3 and Oct 4. We confirmed the pluripotential ability of these cells, which were able to undergo somatic differentiation in vitro to neural progenitors and to endothelial or hematopoietic lineages. We were able to use bovine ES cells as a source of nuclei for nuclear transfer and we generated cloned cattle with a higher frequency of pregnancies to term than has been achieved with somatic cells. On the other hand, we established human fetal membrane derived stem cell lines by the colonial cloning techniques using MEMalpha culture medium containing 10 ng/ml of EGF, 10 ng/ml of LIF and 10% fetal bovine serum (FBS). These cells appeared to maintain normal karyotype in vitro and expressed markers characteristics of stem cells. Furthermore, these cells contributed to the formation of chimeric murine embryoid bodies and gave rise to all three germ layers in vitro. Results from animal ES cells and human fetal membrane derived stem cells clearly demonstrate that these cells might be used for providing different types of cells for regenerative medicine as well as used for targeted genetic manipulation of the genome.     

Incomplete reactivation of Oct4-related genes in mouse embryos cloned from somatic nuclei

The majority of cloned animals derived by nuclear transfer from somatic cell nuclei develop to the blastocyst stage but die after implantation. Mouse embryos that lack an Oct4 gene, which plays an essential role in control of developmental pluripotency, develop to the blastocyst stage and also die after implantation, because they lack pluripotent embryonic cells. Based on this similarity, we posited that cloned embryos derived from differentiated cell nuclei fail to establish a population of truly pluripotent embryonic cells because of faulty reactivation of key embryonic genes such as Oct4. To explore this hypothesis, we used an in silico approach to identify a set of Oct4-related genes whose developmental expression pattern is similar to that of Oct4. When expression of Oct4 and 10 Oct4-related genes was analyzed in individual cumulus cell-derived cloned blastocysts, only 62% correctly expressed all tested genes. In contrast to this incomplete reactivation of Oct4-related genes in somatic clones, ES cell-derived cloned blastocysts and normal control embryos expressed these genes normally. Notably, the contrast between expression patterns of the Oct4-related genes correlated with efficiency of embryonic development of somatic and ES cell-derived cloned blastocysts to term. These observations suggest that failure to reactivate the full spectrum of these Oct4-related genes may contribute to embryonic lethality in somatic-cell clones.     

Nanog基因的功能与调控研究进展

胚胎干细胞的无限增殖能力和亚全能性决定了它在再生医学、新药开发及发育生物学基础研究中具有巨大的应用前景。探索维持胚胎干细胞亚全能性的因子及其网络的调控功能成为胚胎干细胞生物学研究的热点。已研究发现多个与维持胚胎干细胞亚全能性相关的基因如Oct4, Nanog, Sox2等,其中Nanog是2003年5月末发现的一个基因,它对维持胚胎干细胞亚全能性起关键性作用,能够独立于L1F/Stat3维持ICM和胚胎干细胞的亚全能性。几年来,Nanog的生物学功能及其与 Oct4, Sox2等亚全能性维持基因之间的相互作用关系已有较为深入的研究,并发现多个调控Nanog表达的转录因子,从而进一步明晰Nanog与已知调控胚胎发育的信号通路之间的关系。本文在综述Nanog基因的表达特征和功能的基础上、重点探讨Nanog基因表达调控以及Oct4, Sox2等亚全能性维持基因之间的相互作用关系,并对未来的研究趋势予以展望。     

Nanog overcomes reprogramming barriers and induces pluripotency in minimal conditions

Induced pluripotency requires the expression of defined factors and culture conditions that support the self-renewal of embryonic stem (ES) cells. Small molecule inhibition of MAP kinase (MEK) and glycogen synthase kinase 3 (GSK3) with LIF (2i/LIF) provides an optimal culture environment for mouse ES cells and promotes transition to naive pluripotency in partially reprogrammed (pre-iPS) cells. Here we show that 2i/LIF treatment in clonal lines of pre-iPS cells results in the activation of endogenous Nanog and rapid downregulation of retroviral Oct4 expression. Nanog enables somatic cell reprogramming in serum-free medium supplemented with LIF, a culture condition which does not support induced pluripotency or the self-renewal of ES cells, and is sufficient to reprogram epiblast-derived stem cells to naive pluripotency in serum-free medium alone. Nanog also enhances reprogramming in cooperation with kinase inhibition or 5-aza-cytidine, a small molecule inhibitor of DNA methylation. These results highlight the capacity of Nanog to overcome multiple barriers to reprogramming and reveal a synergy between Nanog and chemical inhibitors that promote reprogramming. We conclude that Nanog induces pluripotency in minimal conditions. This provides a strategy for imposing naive pluripotency in mammalian cells independently of species-specific culture requirements.     

Self-renewal and differentiation of mouse embryonic stem cells as measured by Oct4 expression: the role of the cAMP/PKA pathway

In this study we examined the role of the cAMP/protein kinase A (PKA) pathway in affecting IOUD2 ES cell self-renewal and differentiation, Oct4 expression, and cell proliferation. Forskolin, the adenylate cyclase agonist, alone had no effect on ES cell self-renewal. However, when cells were treated with the differentiation-inducing agent retinoic acid, forskolin significantly promoted ES cell self-renewal. Effectively, forskolin rescued cells from a pathway of differentiation. Culturing ES cells in the presence of the phosphodiesterase inhibitor IBMX had no effect on ES cell self-renewal but did increase cell proliferation. In the presence of 100 μM IBMX without LIF, 10 μM forskolin significantly increased ES cell self-renewal. The cell permeable cAMP analog 8-Br-cAMP (1 and 5 mM) promoted ES cell differentiation in the presence of LIF, while in the absence of LIF, it promoted ES cell self-renewal. The effect of the PKA specific inhibitors H89 and KT5720 on Oct4 expression was, again, LIF-dependent. In the presence of LIF, these inhibitors decreased Oct4 expression, while they increased Oct4 expression in the absence of LIF. In general, ES cells maintained on a self-renewal pathway through the presence of LIF show little effect from altered cAMP signaling except at higher levels. However, in strict contrast, when ES cell are on a differentiation pathway through exposure to retinoic acid or the removal of LIF, altering cAMP levels can rescue the self-renewal process promoting Oct4 expression. This study clearly shows that the cAMP/PKA pathway plays a role in ES cell self-renewal pathways. This work was partly funded by the Millennium Research Fund National University of Ireland Galway.     

维持胚胎干细胞多能性的分子机制

胚胎干细胞（ES细胞）来源于早期发育的胚胎,具有分化为任何细胞类型的多能性,因此具有巨大的基础研究及潜在的应用前景.目前认为ES细胞主要通过一些外源性信号分子的作用及某些重要的内源性转录因子的表达共同起作用来达到其维持多能性的目的.外源性信号分子LIF、BMP4以及Wnt等介导的信号传导通路与内源性转录因子Oct4、Nanog、Sox2、FoxD3等共同起作用来抑制那些促进ES细胞分化的基因表达和激活那些有助于维持ES细胞多能性维持的基因表达,进而形成一个相互调控和依存的基因调控网络共同维持ES细胞的多能性.     

Insulin receptor substrate (IRS)-1 regulates murine embryonic stem (mES) cells self-renewal

Mouse embryonic stem (mES) cells are pluripotent cells that can be propagated in vitro with leukemia inhibitory factor (LIF) and serum. Intracellular signaling by LIF is principally mediated by activation of STAT-3, although additional pathways for self-renewal have been described. Here, we identified a novel role for Insulin receptor substrate-1 (IRS-1) as a critical factor in mES cells self-renewal and differentiation. IRS-1 is expressed and tyrosyl phosphorylated during mES cells self-renewal. Differentiation of mES cells, by LIF withdrawal, is associated with a marked reduction in IRS-1 expression. Targeting of IRS-1 by si-IRS-1 results in a severe reduction of Oct-4 protein expression and alkaline phosphatase activity, markers of undifferentiated mES cells. IRS-1 targeting does not interfere with LIF-induced STAT-3 phosphorylation, but negatively affects protein kinase B (PKB/AKT) and glycogen synthase kinase-3 (GSK-3beta) phosphorylation, which are downstream effectors of the LIF-mediated PI3K signaling cascade. Targeting of IRS-1 also results in a marked down regulation of Id-1 and Id-2 proteins expression, which are important components for self-renewal of ES cells. Conversely, over expression of IRS-1 inhibits mES cell differentiation. Taken together, these results suggest that expression and activity of IRS-1 are critical to the maintenance of the self-renewal program in mES cells.     

Conserved roles for Oct4 homologues in maintaining multipotency during early vertebrate development

All vertebrate embryos have multipotent cells until gastrulation but, to date, derivation of embryonic stem (ES) cell lines has been achieved only for mouse and primates. ES cells are derived from mammalian inner cell mass (ICM) tissue that express the Class V POU domain (PouV) protein Oct4. Loss of Oct4 in mice results in a failure to maintain ICM and consequently an inability to derive ES cells. Here, we show that Oct4 homologues also function in early amphibian development where they act as suppressors of commitment during germ layer specification. Antisense morpholino mediated PouV knockdown in Xenopus embryos resulted in severe posterior truncations and anterior neural defects. Gastrulation stage embryos showed reduced expression of genes associated with uncommitted marginal zone cells, while the expression of markers associated with more mature cell states was expanded. Importantly, we have tested PouV proteins from a number of vertebrate species for the ability to substitute Oct4 in mouse ES cells. PouV domain proteins from both Xenopus and axolotl could support murine ES cell self-renewal but the only identified zebrafish protein in this family could not. Moreover, we found that PouV proteins regulated similar genes in ES cells and Xenopus embryos, and that PouV proteins capable of supporting ES cell self-renewal could also rescue the Xenopus PouV knockdown phenotype. We conclude that the unique ability of Oct4 to maintain ES cell pluripotency is derived from an ancestral function of this class of proteins to maintain multipotency.     

Potential of embryonic stem cells

Embryonic stem (ES) cells are pluripotent cell lines established from undifferentiated embryonic cells characterized by nearly unlimited self-renewal and differentiation capacity. During differentiation in vitro, ES cells were found to be able to develop into specialized somatic cells types and to recapitulate processes of early embryonic development. These properties allow to use ES cells as model system for studying early embryonic development by gain- or loss-of-function approaches, or to investigate the effects of drugs and environmental factors on differentiation and cell function in embryotoxicity and pharmacology. Now, ES cells derived of human blastocysts may be used for the generation of somatic precursor or differentiated cells in cell and tissue therapy. The review presents data of mouse ES cell differentiation and gives an outlook on future perspectives and problems of using human ES cells in regenerative medicine.     

Experimental embryology of mammals at the Jastrzebiec Institute of Genetics and Animal Breeding

Our Department of Experimental Embryology originated from The Laboratory of Embryo Biotechnology, which was organized and directed by Dr. Maria Czlonkowska until her premature death in 1991. Proving successful international transfer of frozen equine embryos and generation of an embryonic sheep-goat chimaera surviving ten years were outstanding achievements of her term. In the 1990s, we produced advanced fetuses of mice after reconstructing enucleated oocytes with embryonic stem (ES) cells, as well as mice originating entirely from ES cells by substitution of the inner cell mass with ES cells. Attempts at obtaining ES cells in sheep resulted in the establishment of embryo-derived epithelioid cell lines from Polish Heatherhead and Polish Merino breeds, producing overt chimaeras upon blastocyst injection. Successful re-cloning was achieved from 8-cell rabbit embryos, and healthy animals were born from the third generation of cloned embryos. Recently mice were born after transfer of 8-cell embryonic nuclei into selectively enucleated zygotes, and mouse blastocysts were produced from selectively enucleated germinal vesicle oocytes surrounded by follicular cells, upon their reconstruction with 2-cell nuclei and subsequent activation. Embryonic-somatic chimaeras were born after transfer of foetal fibroblasts into 8-cell embryos (mouse) and into morulae and blastocysts (sheep). We also regularly perform the following applications: in vitro production of bovine embryos from slaughterhouse oocytes or those recovered by ovum pick up; cryopreservation of oocytes and embryos (freezing: mouse, rabbit, sheep, goat; vitrification: rabbit, cow); and banking of somatic cells from endangered wild mammalian species (mainly Cervidae).     

Isolation of pluripotent embryonic stem cells from reprogrammed adult mouse somatic cell nuclei

Pluripotent human stem cells isolated from early embryos represent a potentially unlimited source of many different cell types for cell-based gene and tissue therapies [1-3]. Nevertheless, if the full potential of cell lines derived from donor embryos is to be realised, the problem of donor-recipient tissue matching needs to be overcome. One approach, which avoids the problem of transplant rejection, would be to establish stem cell lines from the patient's own cells through therapeutic cloning [3,4]. Recent studies have shown that it is possible to transfer the nucleus from an adult somatic cell to an unfertilised oocyte that is devoid of maternal chromosomes, and achieve embryonic development under the control of the transferred nucleus [5-7]. Stem cells isolated from such a cloned embryo would be genetically identical to the patient and pose no risk of immune rejection. Here, we report the isolation of pluripotent murine stem cells from reprogrammed adult somatic cell nuclei. Embryos were generated by direct injection of mechanically isolated cumulus cell nuclei into mature oocytes. Embryonic stem (ES) cells isolated from cumulus-cell-derived blastocysts displayed the characteristic morphology and marker expression of conventional ES cells and underwent extensive differentiation into all three embryonic germ layers (endoderm, mesoderm and ectoderm) in tumours and in chimaeric foetuses and pups. The ES cells were also shown to differentiate readily into neurons and muscle in culture. This study shows that pluripotent stem cells can be derived from nuclei of terminally differentiated adult somatic cells and offers a model system for the development of therapies that rely on autologous, human pluripotent stem cells.     

Isolation of embryonic stem (ES) cells in media supplemented with recombinant leukemia inhibitory factor (LIF)

The isolation of pluripotent murine embryonic stem (ES) cells has previously been achieved by coculturing the ES cells with fibroblast feeder cells. In this report we demonstrate that ES cell lines can be isolated from murine 129/Sv He blastocysts in the absence of feeder cells in culture medium supplemented with recombinant leukemia inhibitory factor (LIF). Three of the ES cell lines (MBL-1, MBL-2, and MBL-3) were isolated by directly explanting blastocysts, whilst two ES cell lines (MBL-4 and MBL-5) were isolated from blastocysts pretreated by immunosurgery. Three of the ES cell lines contained the Y chromosome (MBL-1, MBL-2, and MBL-5) with a high proportion of the cells displaying a normal diploid karyotype with a modal chromosome number of 40. All of the ES cell lines tested expressed the stem cell markers ECMA-7 and alkaline phosphatase, which were lost on removal of LIF when the ES cells differentiated into a variety of cell types. The full developmental potential of the ES cells was determined by injecting cells from two of the independently derived ES cell lines, MBL-1 and MBL-5, into C57BL/6J blastocysts. A high proportion of the pups born were chimeric as judged by coat pigmentation. Subsequent breeding established that the ES cells had contributed to the germ line. These results demonstrate that feeder cells are not essential for the isolation of pluripotent ES cell lines.     

Normal specification of the extraembryonic lineage after somatic nuclear transfer

To examine the establishment and maintenance of trophectoderm (TE) lineage in somatic cloned blastocysts, the expression of Cdx2, a key molecule for specification of TE fate, was immunohistochemically examined simultaneously with Oct4 expression. Cloned mouse embryos were made by nuclear transfer using cumulus cells, tail-tip fibroblasts, and embryonic stem cells. After 96 h of culture, the rates of Oct4-expressing blastocysts were as low as 50% and 60% for cumulus and fibroblast clones, respectively. However, regardless of Oct4 expression, the majority of those cloned blastocysts (> 90%) normally expressed Cdx2. Thus, even though somatic cloned embryos have reduced potential to produce the inner cell mass lineage, the TE lineage can be established and maintained.     

Transcriptional activation by Oct4 is sufficient for the maintenance and induction of pluripotency

Oct4 is an essential regulator of pluripotency in vivo and in vitro in embryonic stem cells, as well as a key mediator of the reprogramming of somatic cells into induced pluripotent stem cells. It is not known whether activation and/or repression of specific genes by Oct4 is relevant to these functions. Here, we show that fusion proteins containing the coding sequence of Oct4 or Xlpou91 (the Xenopus homolog of Oct4) fused to activating regions, but not those fused to repressing regions, behave as Oct4, suppressing differentiation and promoting maintenance of undifferentiated phenotypes in vivo and in vitro. An Oct4 activation domain fusion supported embryonic stem cell self-renewal in vitro at lower concentrations than that required for Oct4 while alleviating the ordinary requirement for the cytokine LIF. At still lower levels of the fusion, LIF dependence was restored. We conclude that the necessary and sufficient function of Oct4 in promoting pluripotency is to activate specific target genes.     

Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will greatly facilitate the use of ES cells for developmental biology studies, disease modeling, drug discovery, and regenerative medicine (reviewed in ^1,2).To expedite the identification and characterization of novel regulators of ES cell maintenance and self-renewal, we developed a fluorescence reporter-based assay to quantitatively measure the self-renewal status in mouse ES cells using the Oct4GiP cells ³. The Oct4GiP cells express the green fluorescent protein (GFP) under the control of the Oct4 gene promoter region ^4,5. Oct4 is required for ES cell self-renewal, and is highly expressed in ES cells and quickly down-regulated during differentiation ^6,7. As a result, GFP expression and fluorescence in the reporter cells correlates faithfully with the ES cell identity ⁵, and fluorescence-activated cell sorting (FACS) analysis can be used to closely monitor the self-renewal status of the cells at the single cell level ^3,8.Coupled with RNAi, the Oct4GiP reporter assay can be used to quickly identify and study regulators of ES cell maintenance and self-renewal ^3,8. Compared to other methods for assaying self-renewal, it is more convenient, sensitive, quantitative, and of lower cost. It can be carried out in 96- or 384-well plates for large-scale studies such as high-throughput screens or genetic epistasis analysis. Finally, by using other lineage-specific reporter ES cell lines, the assay we describe here can also be modified to study fate specification during ES cell differentiation.