Primordial germ cells contain subpopulations that have greater ability to develop into pluripotential stem cells

Primordial germ cells (PGCs) are undifferentiated germ cells in embryos. We previously found that some mouse PGCs develop into pluripotential cells (EG cells) when cultured on a feeder layer expressing the membrane bound form of Steel factor with culture medium containing leukemia inhibitory factor and basic fibroblast growth factor. To understand the mechanisms of the conversion of PGCs into EG cells, we attempted to identify PGC subpopulations that have the ability to develop into EG cells. Using flow cytometry, we fractionated PGCs by the expression of the cell surface antigen integrin α6, as well as by the detection of side‐population (SP) cells in which stem cells are enriched in various tissues. PGCs with negative or low integrin α6 expression and with SP cell phenotype showed higher potential to convert to EG cells. Negative or low integrin α6 expression in PGCs was also correlated with lower expression of Ddx4, which is specifically expressed in PGCs after embryonic day 10.5. The results indicate that the primitive PGC population showing the SP cell phenotype among undifferentiated PGCs has a higher ability of being converted into EG cells. Thus, conversion of PGCs into pluripotential stem cells may be regulated by being influenced by the natural status of individual PGCs as well as the reprogramming process after starting culture.     

Establishment and differentiation of murine EG cell lines derived from primordial germ cells]

Primordial germ cells (PGC) were isolated from 8.5, 10.5, 12.5 days post coitum (dpc) embryos of F1 (Balb/c x ICR), C57BL/6J, 129/svJ, 129/sv-ter mice, and cultured on mitotically inactive MEF or STO feeder layer cells with addition of leukemia inhibitory factor, stem cell factor and basic fibroblast growth factor in cultures. PGCs formed densely packed and AKP positive colonies with pluripotential marker gene (oct-4) expression resembling undifferentiated ES cells in morphology and growth pattern. Five EG cell lines derived from PGCs were established: EG1(8.5 dpc, F1), EG2 and EG3 (8.5 dpc, C57BL/6J), EG4 (10.5 dpc, 129/svJ), EG5 (10.5 dpc, 129/sv-ter). No long term culture was obtained from 12.5 dpc PGCs of 29 embryos. All five EG cell lines cultured on feeder layer cells or in LIF containing medium still remain undifferentiated state at 15 th passage. Under appropriate conditions, EG cells formed embryoid bodies in suspension culture and multiple types of differentiated cells in monolayer culture. When these EG cells were injected in nude mice, they formed teratocacinomas containing differentiated cells such as cartilage, neural tissue and epithelium. These results show that EG1-5 cell lines derived from 8.5, 10.5 dpc embryos are pluripotential.     

Zebrafish Staufen1 and Staufen2 are required for the survival and migration of primordial germ cells

In sexually reproducing organisms, primordial germ cells (PGCs) give rise to the cells of the germ line, the gametes. In many animals, PGCs are set apart from somatic cells early during embryogenesis. Work in Drosophila, C. elegans, Xenopus, and zebrafish has shown that maternally provided localized cytoplasmic determinants specify the germ line in these organisms (Raz, E., 2003. Primordial germ-cell development: the zebrafish perspective. Nat. Rev., Genet. 4, 690--700; Santos, A.C., Lehmann, R., 2004. Germ cell specification and migration in Drosophila and beyond. Curr. Biol. 14, R578-R589). The Drosophila RNA-binding protein, Staufen is required for germ cell formation, and mutations in stau result in a maternal effect grandchild-less phenotype (Schupbach,T., Weischaus, E., 1989. Female sterile mutations on the second chromosome of Drosophila melanogaster:1. Maternal effect mutations. Genetics 121, 101-17). Here we describe the functions of two zebrafish Staufen-related proteins, Stau1 and Stau2. When Stau1 or Stau2 functions are compromised in embryos by injecting antisense morpholino modified oligonucleotides or dominant-negative Stau peptides, germ layer patterning is not affected. However, expression of the PGC marker vasa is not maintained. Furthermore, expression of a green fluorescent protein (GFP):nanos 3'UTR fusion protein in germ cells shows that PGC migration is aberrant, and the mis-migrating PGCs do not survive in Stau-compromised embryos. Stau2 is also required for survival of neurons in the central nervous system (CNS). These phenotypes are rescued by co-injection of Drosophila stau mRNA. Thus, staufen has an evolutionarily conserved function in germ cells. In addition, we have identified a function for Stau proteins in PGC migration.     

Formation and cultivation of medaka primordial germ cells

Primordial germ cell (PGC) formation is pivotal for fertility. Mammalian PGCs are epigenetically induced without the need for maternal factors and can also be derived in culture from pluripotent stem cells. In egg-laying animals such as Drosophila and zebrafish, PGCs are specified by maternal germ plasm factors without the need for inducing factors. In these organisms, PGC formation and cultivation in vitro from indeterminate embryonic cells have not been possible. Here, we report PGC formation and cultivation in vitro from blastomeres dissociated from midblastula embryos (MBEs) of the fish medaka (Oryzias latipes). PGCs were identified by using germ-cell-specific green fluorescent protein (GFP) expression from a transgene under the control of the vasa promoter. Embryo perturbation was exploited to study PGC formation in vivo, and dissociated MBE cells were cultivated under various conditions to study PGC formation in vitro. Perturbation of somatic development did not prevent PGC formation in live embryos. Dissociated MBE blastomeres formed PGCs in the absence of normal somatic structures and of known inducing factors. Most importantly, under culture conditions conducive to stem cell derivation, some dissociated MBE blastomeres produced GFP-positive PGC-like cells. These GFP-positive cells contained genuine PGCs, as they expressed PGC markers and migrated into the embryonic gonad to generate germline chimeras. Our data thus provide evidence for PGC preformation in medaka and demonstrate, for the first time, that PGC formation and derivation can be obtained in culture from early embryos of medaka as a lower vertebrate model.     

小鼠原生殖细胞体外培养及其应用研究

原生殖细胞（ｐｒｉｍｏｒｄｉａｌｇｅｒｍｃｅｌｌ,ＰＧＣ）是胚胎生殖谱系最原始形式的细胞,在体胚胎迁移期ＰＧＣ增殖极为旺盛。体外培养的小鼠迁移期ＰＧＣ在饲养层细胞和三种生长因子（干细胞生长因子、碱性成纤维细胞生长因子及白血病抑制因子）的共同作用下,可发展为长期增殖并维持不分化状态的胚胎性干细胞,即胚胎生殖细胞（ｅｍｂｒｙｏｎｉｃｇｅｒｍｃｅｌｌ,ＥＧ）,具全能性发育潜能。ＥＧ建系成功对于研究生殖细胞发育以及寻找新的转基因动物操作的有效载体具有重要价值。     

Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture.

Steel factor (SF) and LIF (leukemia inhibitory factor) synergistically promote the proliferation and survival of mouse primordial germ cells (PGCs), but only for a limited time period in culture. We show here that addition of bFGF to cultures in the presence of membrane-associated SF and LIF enhances the growth of PGCs and allows their continued proliferation beyond the time when they normally stop dividing in vivo. They form colonies of densely packed, alkaline phosphatase-positive, SSEA-1-positive cells resembling undifferentiated embryonic stem (ES) cells in morphology. These cultures can be maintained on feeder layers for at least 20 passages, and under appropriate conditions give rise to embryoid bodies and to multiple differentiated cell phenotypes in monolayer culture and in tumors in nude mice. PGC-derived ES cells can also contribute to chimeras when injected into host blastocysts. The long-term culture of PGCs and their reprogramming to pluripotential ES cells has important implications for germ cell biology and the induction of teratocarcinomas.     

小鼠孤雌胚胎干细胞集落的建立

ＥＳＴＡＢＬＩＳＨＭＥＮＴＯＦＳＴＥＭＣＥＬＬＣＯＬＯＮＩＥＳＦＲＯＭＰＡＲＴＨＥＮＯＧＥＮＥＴＩＣＡＬＬＹＤＥＲＩＶＥＤＢＬＡＳＴＯＣＹＳＴＳＯＦＭＯＵＳＥ小鼠孤雌胚胎干细胞集落的建立ＫｅｙｗｏｒｄｓＭｏｕｓｅ,Ｐａｒｔｈｅｎｏｇｅｎｅｔｉｃｅｍ...     

The growth factor environment defines distinct pluripotent ground states in novel blastocyst-derived stem cells

Pluripotent stem cell lines can be derived from blastocyst embryos, which yield embryonic stem cell lines (ES cells), as well as the postimplantation epiblast, which gives rise to epiblast stem cell lines (EpiSCs). Remarkably, ES cells and EpiSCs display profound differences in the combination of growth factors that maintain their pluripotent state. Molecular and functional differences between these two stem cell types demonstrate that the tissue of origin and/or the growth factor milieu may be important determinants of the stem cell identity. We explored how developmental stage of the tissue of origin and culture growth factor conditions affect the stem cell pluripotent state. Our findings indicate that novel stem cell lines, with unique functional and molecular properties, can be generated from murine blastocyst embryos. We demonstrate that the culture growth factor environment and cell-cell interaction play a critical role in defining several unique and stable stem cell ground states.     

Reactive oxygen species signaling in primordial germ cell development in Drosophila embryos

REDOX mechanisms that induce biosynthesis of the reactive oxygen species (ROS) have attracted considerable attention due to both the deleterious and beneficial responses elicited by the reactive radical. In several organisms including Drosophila melanogaster, modulation of ROS activity is thought to be crucial for the maintenance of cell fates in developmental contexts. Interestingly, REDOX mechanisms have been shown to be involved in maintaining progenitor fate of stem cells as well as their proliferation and differentiation. Here, we have explored the possible functions of ROS during proper specification and developmental progression of embryonic primordial germ cells (PGCs). Indicating its potential involvement in these processes, ROS can be detected in the embryonic PGCs and the surrounding somatic cells from very early stages of embryogenesis. Using both “loss” and “gain” of function mutations in two different components of the REDOX pathway, we show that ROS levels are likely to be critical in maintaining germ cell behavior, including their directed migration. Altering the activity of a putative regulator of ROS also adversely influences the ability of PGCs to adhere to one another in cellular blastoderm embryos, suggesting potential involvement of this pathway in orchestrating different phases of germ cell migration.     

Mouse embryonic stem cells and preimplantation embryos require signaling through the phosphatidylinositol 3-kinase pathway to suppress apoptosis

Whereas most mammalian cells require extracellular signals to suppress apoptosis, preimplantation embryos can survive and develop to the blastocyst stage in defined medium without added serum or growth factors. Since cells of these embryos are capable of undergoing apoptosis, it has been suggested that their lack of dependence upon exogenous growth factors results from the production of endogenous growth factors that suppress apoptosis by an autocrine signaling mechanism. In the present study, we have examined the growth factor requirements and intracellular signaling pathways that suppress apoptosis in both mouse preimplantation embryos and embryonic stem (ES) cells, which are derived from the blastocyst inner cell mass. Cultured ES cells, in contrast to intact embryos, required serum growth factors to prevent apoptosis. Suppression of ES cell apoptosis by serum growth factors required the phosphatidylinositol 3-kinase (PI 3-kinase) signaling pathway, since apoptosis was rapidly induced by inhibition of PI 3-kinase with LY294002. In contrast, inhibition of MEK/ERK signaling with U0126 or of mTOR with rapamycin had no detectable effect on ES cell survival. Thus, like most mammalian cells, the survival of ES cells is mediated by growth factor stimulation of PI 3-kinase signaling. Treatment with LY294002 (but not with U0126 or rapamycin) similarly induced apoptosis of mouse blastocysts in serum-free medium, indicating that intact preimplantation embryos are also dependent upon PI 3-kinase signaling for survival. These results demonstrate that PI 3-kinase signaling is required to suppress apoptosis of both ES cells and intact preimplantation embryos, consistent with the hypothesis that survival of preimplantation embryos is maintained by endogenous growth factors that stimulate the PI 3-kinase pathway.     

Cadherin-mediated cell interaction regulates germ cell determination in mice

The germ cell lineage segregates from the somatic cell lineages in early embryos. Germ cell determination in mice is not regulated by maternally inherited germplasm, but is initiated within the embryo during gastrulation. However, the mechanisms of germ cell specification in mice remain unknown. We located precursors to primordial germ cells (PGCs) within early embryos, and show here that cell-cell interaction among these precursors is required for germ cell specification. We found that the expression of a calcium-dependent cell adhesion molecule, E-cadherin, is restricted to the proximal region of extra-embryonic mesoderm that contains PGC precursors, and that blocking the functions of E-cadherin with an antibody inhibits PGC formation in vitro. These results showed that E-cadherin-mediated cell-cell interaction among cells containing PGC precursors is essential to directing such cells to the germ cell fate.     

Effect of mitotic inducers and retinoic acid blocker on expression of pluripotent genes in ES cells derived from early stage in vitro-produced embryos in buffalo

So far, it has been difficult to generate embryonic stem (ES) cell from early stage preimplantation embryos of buffalo. These ES cells will be more helpful for efficient embryo cloning and generation of body cells as they are more primitive than inner cell mass (ICM)-derived ES cells. The present study was conducted to find the effect of lipopolysaccharide (LPS), melatonin (N-acetyl-5-methoxytryptamine, a pineal gland product), and citral (3,7-dimethyl-2,6-octadienal and a retinoic acid synthesis blocker) on establishment of primary ES cell colonies, the comparative size of the ES cell colonies, and expression of pluripotent genes during extended period of culture in buffalo. Zona-free eight-cell stage in vitro fertilization (IVF) embryos were cultured in ES cell medium supplemented with none (media I as control), LPS (media II), citral melatonin (media III), or melatonin (media IV). The multiplication of blastomere leading to ES cell colony formation and expression of pluripotent genes were assessed up to day 20 of culture. The primary colony formation, the comparative size of the ES cell colonies, and expression of pluripotent genes in these colonies were better in the medium supplemented with melatonin in all days of culture. Within melatonin supplementation, the colony size was comparatively larger on day 8 and day 12 of culture. Further, with this supplementation, the Oct-4 and Nanog expression was comparatively higher on all days of culture. The results indicated that supplementation of melatonin helped in the formation of better primary ES cell colony as well as in the maintenance of pluripotency. The results also indicated that primary colonies developed on day 8 to day 12 of culture may be better for passaging them for establishment of ES cell line from early stage preimplantation IVF embryos of in buffalo.     

dead end,a novel vertebrate germ plasm component,is required for zebrafish primordial germ cell migration and survival

In most animals, primordial germ cell (PGC) specification and development depend on maternally provided cytoplasmic determinants that constitute the so-called germ plasm. Little is known about the role of germ plasm in vertebrate germ cell development, and its molecular mode of action remains elusive. While PGC specification in mammals occurs via different mechanisms, several germ plasm components required for early PGC development in lower organisms are expressed in mammalian germ cells after their migration to the gonad and are involved in gametogenesis. Here we show that the RNA of dead end, encoding a novel putative RNA binding protein, is a component of the germ plasm in zebrafish and is specifically expressed in PGCs throughout embryogenesis; Dead End protein is localized to perinuclear germ granules within PGCs. Knockdown of dead end blocks confinement of PGCs to the deep blastoderm shortly after their specification and results in failure of PGCs to exhibit motile behavior and to actively migrate thereafter. PGCs subsequently die, while somatic development is not effected. We have identified dead end orthologs in other vertebrates including Xenopus, mouse, and chick, where they are expressed in germ plasm and germ-line cells, suggesting a role in germ-line development in these organisms as well.     

Developmentally regulated expression of mil-1 and mil-2, mouse interferon-induced transmembrane protein like genes,during formation and differentiation of primordial germ cells

In all multicellular organisms, germ cells originating from a fertilized egg have the highly specialized role of transmitting genetic information to the next generation. In many animal species, the establishment of the germ cell lineage is regulated by the maternally inherited germplasm. In mammals, however, germline determination is not based on the unequal distribution of maternal determinants. In the processes of mammalian germ cell formation and subsequent differentiation, the molecular basis of the acquisition of germ cell status is not well understood. Since migrating primordial germ cells (PGCs) are lineage-restricted to the germline, they have already acquired a germ cell specific fate distinct from that of pluri/multi-potent stem cells. However, there have been no molecules known to be expressed in migrating PGCs but not in the inner cell mass of blastocysts. Such molecules should be involved in early germ cell development, and they should make good markers for following the process of PGC formation. To identify such molecules, we performed a subtracted cDNA screening with migrating PGCs and blastocysts in mice, and isolated 11 clones preferentially expressed in PGCs. Here, we report the identification of two genes with similarity to human interferon-induced transmembrane protein (Ifitm) genes, and expression patterns of these genes in forming and in differentiating PGCs. During germ cell formation, mouse Ifitm like (mil)-1 was expressed in putative PGC ancestors in embryos at 6.5-7.5 days post coitum. In migrating PGCs, mil-1 expression was continuously observed and mil-2 expression was first detected during germ cell differentiation.