Origin of primordial germ cells in the prestreak chick embryo

The temporal and spatial pattern of segregation of the avian germline from the formation of the area pellucida to the beginning of primitive streak formation (stages VII–XIV, EG&K) was investigated using the culture of whole embryos and central and peripheral embryo fragments on vilelline membranes at stages VII–IX, immunohistological analysis of whole mount embryos and sections with monoclonal antibodies MC-480 against stage-specific embryonic antigen-1 (SSEA-1) and EMA-1, and with the culture of dispersed blastoderms at stages IX–XIV with and without an STO feeder layer. Whole embryos at intrauterine stages developed up to the formation of the primitive streak despite the absence of area pellucida expansion. Primordial germ cells (PGCs) appeared in the cultures of whole embryos and only in central fragments containing a partially formed area pellucida at stages VII–IX. When individual stage IX–XIV embryos were dispersed and cultured without a feeder layer, 25–45 PGCs/embryo were detected only with stage X–XIV, but not with stage IX blastoderms. However, the culture of dispersed cells from the area pellucida of stages IX–XIII on STO feeder layers yielded about 150 PGCs/embryo. The carbohydrate epitopes recognized by anti-SSEA-1 and EMA-1 first appeared at stage X on cells in association with polyingressing cells on the ventral surface of the epiblast and later on the dorsal surface of the hypoblast. The SSEA-1-positive hypoblast cells gave rise to chicken PGCs when cultured on a feeder layer of quail blastodermal cells. From these observations, we propose that the segregation and development of avian germline is a gradual, epigenetic process associated with the translocation of SSEA-1/EMA-1-positive cells from the ventral surface of the area pellucida at stage X to the dorsal side of the hypoblast at stages XI–XIV. © 1996 Wiley-Liss, Inc.     

Establishment of goat embryonic stem cells from in vivo produced blastocyst-stage embryos

Embryonic stem (ES) cells with the capacity for germ line transmission have only been verified in mouse and rat. Methods for derivation, propagation, and differentiation of ES cells from domestic animals have not been fully established. Here, we describe derivation of ES cells from goat embryos. In vivo-derived embryos were cultured on goat fetal fibroblast feeders. Embryos either attached to the feeder layer or remained floating and expanded in culture. Embryos that attached showed a prominent inner cell mass (ICM) and those that remained floating formed structures resembling ICM disks surrounded by trophectodermal cells. ICM cells and embryonic disks were isolated mechanically, cultured on feeder cells in the presence of hLIF, and outgrown into ES-like colonies. Two cell lines were cultured for 25 passages and stained positive for alkaline phosphatase, POU5F1, NANOG, SOX2, SSEA-1, and SSEA-4. Embryoid bodies formed in suspension culture without hLIF. One cell line was cultured for 2 years (over 120 passages). This cell line differentiated in vitro into epithelia and neuronal cells, and could be stably transfected and selected for expression of a fluorescent marker. When cells were injected into SCID mice, teratomas were identified 5-6 weeks after transplantation. Expression of known ES cell markers, maintenance in vitro for 2 years in an undifferentiated state, differentiation in vitro, and formation of teratomas in immunodeficient mice provide evidence that the established cell line represents goat ES cells. This also is the first report of teratoma formation from large animal ES cells.     

Analysis of germ line development in the chick embryo using an anti-mouse EC cell antibody

We have found that EMA-1, a monoclonal antibody originally raised against mouse embryonal carcinoma (Nulli SCC1) cells (Hahnel & Eddy, 1982), also labels chick primordial germ cells (PGCs). We have used this antibody in immunohistological studies to follow the development of PGCs in the chick embryo from the time of their initial appearance beneath the epiblast, through their migratory phase and subsequent colonization of the germinal epithelium. During hypoblast formation, individual EMA-1-labelled cells appeared to separate from the basal surface of the epiblast and enter the blastocoel, coincident with the appearance of morphologically identifiable PGCs in this same area. EMA-1 continued to label germ cells until the initiation of gametogenesis in each sex; specifically, labelling was absent by 7-8 days of incubation in females and started to decrease at 11 days of incubation in males. There was a recurrence of the epitope on oogonia at 15 days of incubation, but not on spermatogonia during the remainder of development through hatching. These observations are consistent with an epiblast origin for the avian germ line, and are strikingly similar to those reported for the early mouse embryo using the same antibody (Hahnel & Eddy, 1986).     

Characterization of stage-specific embryonic antigen-1 (SSEA-1) expression during early development of the turkey embryo.

SSEA-1 is a carbohydrate epitope associated with cell adhesion, migration and differentiation. In the present study, SSEA-1 expression was characterized during turkey embryogenesis with an emphasis on its role in primordial germ cell development. During hypoblast formation, SSEA-1 positive cells were identified in the blastocoel and hypoblast and later in the germinal crescent. Based on location and morphology, these cells were identified, as PGCs. Germ cells circulating through embryonic blood vessels were also SSEA-1 positive. During the active phase of migration, PGCs in the dorsal mesentery and gonad could no longer be identified using the SSEA-1 antibody. The presence of PGCs at corresponding stages was verified using periodic acid Schiff stain. Pretreatment of PGCs with trypsin, alpha-galactosidase and neuraminidase did not restore immunoreactivity to SSEA-1. In general, expression was not limited to the germ cell lineage. SSEA-1 was also detected on the ectoderm, yolk sac endoderm, gut and mesonephric tubules. During neural tube closure, SSEA-1 was expressed by the neural epithelium of the fusing neural folds. Later SSEA-1 was detected in regions of the developing spinal cord. Enzyme pretreatment unmasked the epitope on some neural crest cells and cells in the sympathetic ganglion. The temporal and spatial distribution of SSEA-1 in the turkey embryo suggests a role in early germ cell and neural cell development. The absence of SSEA-1 on turkey gonadal germ cells was different from that observed for the chick. Therefore, while features of avian germ cell development appear to be conserved, expression of SSEA-1 can vary with the species.     

Isolation,culture, and characterization of primordial germ cells in Mongolian sheep

This study was performed to culture and preliminarily identify the primordial germ cells (PGCs) isolated from the genital ridge of the Mongolian sheep fetus. The growth characteristics of the sheep PGCs were detected in different culture systems such as culture media, resources, and state and passages of feeder cells. The obtained embryonic germ (EG) cells were identified by morphology, enzymology, and immunofluorescence. The results showed that the sheep EG cell colonies were ridgy, typically nest like, and compact, and had regular edges. Alkaline phosphatase staining reaction was weakly positive. EG cells expressed Kit, Rex-1, Nanog, and Oct-4. Immunofluorescence detection was weakly positive for Oct3/4, whereas positive for SSEA-1, SSEA-3, SSEA-4, TRA-1-61, and TRA-1-80.     

Directed neural differentiation of duck embryonic germ cells

Although the avian primordial germ cells (PGCs) have been used to produce transgenic birds, their characteristics largely remain unknown. The isolation, culture, biological characterization, and directed neural differentiation of duck EG cells were assayed in this study. The Results showed that the EG cells were got by isolating embryonic gonad and surrounding tissue from 7-day-old duck embryo. The PGCs co-cultured with their gonadal somatic cells were well grown. After passaging, the EG cells were incubated in medium with cytokines and Mitomycin C on inactivated duck embryonic fibroblasts (DEFs) feeder layers. After several passages, alkaline phosphatase (ALP) and periodic acid-Schiff (PAS) resulted positive, cellular markers detection positive for SSEA-1, SSEA-4, TRA-1-60, and TRA-1-81. Karyotype analysis showed the EG cells kept diploid condition and the hereditary feature was stable in accordance with varietal characteristics of duck. These cells grew continuously for 11 passages on DEFs. Under induction of medium with BME, RA, and IBMX, the EG cells lost undifferentiated state, large amount of neural cells appeared with the formation of neural cells networks. Special Nissl body was found by toluidine blue stain after induced for 7 days. Immunofluorescence staining results indicated that differentiated EG cells expressed Nestin, NSE, and GFAP positive. The expression of Nestin, NSE, and GFAP mRNA were positive by RT-PCR. The results revealed that RA can obviously promote the directed differentiation of duck EG cells into neural lineage. The duck EG cells will be useful for the production of transgenic birds, for cell replacement therapy and for studies of germ cell differentiation.     

Serum-free culture of murine primordial germ cells and embryonic germ cells

Fetal calf serum (FCS) has usually been used for culture of embryonic stem (ES) cell as a component of the culture medium. However, FCS contains undefined factors, which promote cell proliferation and occasionally stimulate differentiation of ES cells. Recently, a chemically-defined serum replacement, Knockout Serum Replacement (KSR), was developed to maintain ES cells in an undifferentiated state. In this experiment, we examined the effects of KSR on the growth and differentiation of primordial germ cells (PGCs) and embryonic germ (EG) cells. PGCs were collected 8.5 days postcoitum (dpc) from B6D2F1 (C57BL/6JxDBA/2J) female mice mated with B6D2F1 males. Most of the PGCs that were cultured in FCS-supplemented medium (FCS medium) had alkaline phosphatase (AP) activity and acquired a fibroblast cell shape. In contrast, PGCs in KSR-supplemented medium (KSR medium) proliferated, maintaining round and stem cell-like morphology. In addition, EG cells were established more easily from PGCs cultured in KSR medium than from PGCs cultured in FCS medium. The percentage of undifferentiated colonies of EG cells was significantly higher in KSR medium than in FCS medium. The germ line chimera was also produced from EG cells established in KSR medium. These results suggest that KSR can be used for sustaining an undifferentiated state of PGCs and EG cells in vitro.     

Derivation of Putative Porcine Embryonic Germ Cells and Analysis of Their Multi-Lineage Differentiation Potential

Embryonic germ (EG) cells are cultured pluripotent stem cells derived from the primordial germ cells (PGCs) that migrate from the dorsal mesentery of the hindgut to the developing genital ridge. In this study, the morphology of the porcine genital ridge was assessed in embryos harvested on days 22–30 of pregnancy. PGCs from embryos at these stages were cultured to obtain porcine EG cell lines, and EG-like cells were derived from PGCs from embryos harvested on days 24–28 of pregnancy. The EG-like cells expressed Oct4, Sox2, Nanog, SSEA-3, SSEA-4 and alkaline phosphatase (AP). These cells were able to form embryoid bodies (EBs) in suspension culture and differentiate into cells representative of the three germ layers as verified by a-fetoprotein (AFP), α-smooth muscle actin (α-SMA), and Nestin expression. Spontaneous differentiation from the porcine EG-like cells of delayed passage in vitro showed that they could differentiate into epithelial-like cells, mesenchymal-like cells and neuron-like cells. In vitro directed differentiation generated osteocytes, adipocytes and a variety of neural lineage cells, as demonstrated by alizarin red staining, oil red O staining, and immunofluorescence for neuronal class Ⅲ β-tubulin (Tuj1), glial fibrillary protein (GFAP) and galactosylceramidase (GALC), respectively. These results indicate that porcine EG-like cells have the potential for multi-lineage differentiation and are useful for basic porcine stem cell research.     

免疫磁珠分选降低分化体系中胚胎干细胞的比例

研究目的:采用免疫磁珠分选系统（magnetic activated cell sorting, MACS）分离去除小鼠胚胎干细胞（murine embryonic stem cells, mES）向神经细胞分化时培养体系中的ES细胞,即对分化细胞进行纯化,以期减少移植致瘤性。方法：诱导mES细胞向神经细胞分化,取分化第四期的细胞,胰酶消化制成单细胞悬液,用mES特异性表面抗原SSEA-1（special stage embryonic antigen-1）单抗标记,间接免疫磁珠分选系统分离去除SSEA-1阳性细胞,流式细胞仪检测分选前后细胞中mES细胞的比例,台盼蓝染色检测分选前后细胞存活率。结果：经MACS分选后的阴性细胞中的SSEA-1阳性率可以由分选前的（7.19±1.36）%下降到（1.34±0.80）%,结果具有显著性差异;分选后的细胞存活率仍为92%左右,与分选前存活率无明显变化。结论 用SSEA-1作为表面标志,用MACS方法能有效地去除胚胎干细胞分化细胞中残存的胚胎干细胞,得到高纯度的分化细胞,并且细胞存活率不受影响,为下一步进行移植实验奠定基础。     

Identification of pig primordial germ cells by immunocytochemistry and lectin binding

Monoclonal antibodies anti-SSEA-1 and EMA-1, and the lectins DBA and LTA, bound to the surface of large, round cells randomly distributed in the 26-day pig genital ridge. Other antibodies, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81, did not react with any cells in the pig genital ridge. SSEA-1-positive cells displayed pseudopods and appeared to migrate from the dorsal mesentery of the hindgut (18-day) to the primordium of the gonad (day 23) and entered the genital ridge by 26 days. The number of SSEA-1-positive cells associated with the dorsal mesentery and genital ridge markedly increased from the 18-day to the 26-day pig embryo. It was concluded that the SSEA-1-positive cells were primordial germ cells (PGCs). Using these markers and alkaline phosphatase histochemistry, pig PGCs derived from the 26-day genital ridge showed no proliferation when grown in STO co-culture in the presence of human LIF, bFGF and SCF. Mol. Reprod. Dev. 46:567–580, 1997. Published 1997 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

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.     

Immunomagnetic isolation of primordial germ cells and the establishment of embryonic germ cell lines in the mouse

The stage-specific embryonic antigen 1 (SSEA-1) is a cell marker of primordial germ cells (PGCs). In the present study, it is shown that isolation and purification of PGCs from 8.5-11.5 days post coitum (dpc) embryos can be achieved by a immunomagnetic cell sorting method using SSEA-1 antibody-conjugated magnetic beads, and then the sorted PGCs can be used for long-term culture under strict culture conditions to derive embryonic germ (EG) cell lines. Five independent EG cell lines with male karyotypes have been established. They show both a strong alkaline phosphatase activity and expression of the SSEA-1 antigen, and are karyotypically stable with a modal number of chromosomes in more than 80% of the cells. One of the EG cell lines from 8.5-dpc embryos produced chimeras after injections of the cells into 8-cell host embryos. These procedures could provide a useful and simple method for isolation of undifferentiated cells from a heterogeneous cell population and for establishment of embryo-derived stem cell lines.     

Pluripotent differentiation in vitro of murine Es-D3 embryonic stem cells

Although the ES-D3 murine embryonic stem cell line was one of the first derived, little information exists on the in vitro differentiation potential of these cells. We have used immunocytochemical and flow cytometric methods to monitor ES-D3 embryoid body differentiation in vitro during a 21-d period. Spontaneous differentiation of embryoid body cells was induced by leukemia inhibitory factor withdrawal in the absence of feeder cells. The pluripotent stem cell markers Oct-3/4, SSEA-1, and EMA-1 were found to persist for at least 7 d, whereas the primitive endoderm marker cytokeratin endo-A was expressed at increasing levels from day 6. The localization of these antigens within the embryoid bodies suggested that embryonic ectoderm- and primitive endoderm-derived tissues were segregated. Localized expression of class III beta-tubulin and sarcomeric myosin also was detected, indicating that representatives of all three embryonic germ layers were present after induction of differentiation in vitro.     

人多潜能胚胎生殖细胞的分离和培养（简报）

To establish human pluripotent embryonic germ (EG) cell lines, human primordial germ cells (PGCs) of embryos aborted in 5-9 week were cultured on inactive mouse STO fibroblast feeder. The medium contained human leukemia inhibitory factor (hLIF), human basic fibroblast growth factor (hbFGF) and forskolin. The EG cells could be passaged continuously until 12 generations. Most cells were positive in alkaline phosphatase staining and expressed cell surface antigen SSEA-3 and pluripotent marker Oct-4. These EG cell populations that retained normal karyotype could form embryoid body in culture and differentiate further into neuron-like cells, mucous epithelial cells, epithelial cells and other types of the cells spontaneously. These results indicated the cell clones derived from human PGCs resemble pluripotent EG cells from mouse PGCs in appearance or nature.     

人多潜能胚胎生殖细胞的分离和培养

多潜能胚胎性干细胞来源有两条途经,从植入前的早期胚胎内细胞团(inner cell mass,ICM)分离出来的称胚胎干细胞(embryonic stem cells,ES);从原始生殖细胞(primordial germ cells,PGCs)分离得到的称胚胎生殖细胞(embryonic germ cells,EG)。这两种干细胞在小鼠嵌合体实验中,都证明具有参与生殖系传递的能力。这类干细胞在体外保持     

Human teratocarcinoma stem cells: glycolipid antigen expression and modulation during differentiation

Teratocarcinomas are germ cell tumors in which pluripotent stem cells, embryonal carcinoma (EC) cells, undergo differentiation along the pathways resembling those occurring during early embryogenesis. Human EC cell lines established in vitro provide a model for studying embryonic cellular differentiation in a way that is pertinent to early human development. The predominant glycolipid antigens expressed by EC cells of both humans and mice have globoseries core structures; in humans they are terminally modified to yield the monoclonal antibody-defined stage-specific embryonic antigens SSEA-3 and SSEA-4, and also globo-ABH antigens; in the mouse terminal modification yields the Forssman antigen rather than SSEA-3 and -4. These observations focus attention on the possible role of the P-blood group system, which regulates synthesis of globoseries oligosaccharides, in the behavior of cells in the early embryo and in teratocarcinomas. Marked changes in the core structures of the cell surface glycolipids occur as the EC cells differentiate; thus globoseries structures rapidly diminish and are replaced by lactoseries and then by ganglioseries glycolipids. During differentiation of the NTERA-2 line of pluripotent human EC cells into neurons and other cell types, the various subsets of differentiated cells that arise are distinguished by their differential expression of new glycolipid antigens, particularly ganglioside GT3 (recognized by antibody A2B5), and ganglioside 9-0-acetyl GD3 (recognized by antibody ME311). Neurons are found among the A2B5+/ME311- cells.     

Characterization of a pluripotent stem cell line derived from a mouse embryo

A pluripotent, karyotypically normal, male culture line ESC-BLC 1 of embryonal stem cells was established from delayed mouse blastocysts of strain 129/ter Sv. The cell line was isolated after cultivation of inner cell mass cells on X-irradiated feeder layer of mouse embryonal fibroblasts. The pluripotent status of the cell line was confirmed by in vivo and in vitro differentiation. For in vivo differentiation, cells were injected subcutaneously into syngeneic mice. The resulting tumors contained various tissues, derivatives of all three primary germ layers. In vitro cultivated pluripotent stem cells differentiated into endoderm-like, neuronal-like and tubular structures. Determination of alkaline phosphatase in cell line ESC-BLC 1 yielded a high specific activity; G-banding of metaphases revealed a normal, male karyotype.