Pluripotential stem cells derived from migrating primordial germ cells

Pluripotent stem cells termed embryonic germ cells (EGCs) have earlier been derived from pre- and post-migrating mouse primordial germ cells (PGCs). We have recently obtained four EGC lines from migrating PGCs of 9.5 days post coitum (dpc) embryos. All lines were male with normal karyotype and showed properties that are similar to previously established EGC lines, including colony morphology, expression of alkaline phosphatase (AP), and expression of SSEA-1 antigen. The developmental potency of two of these lines was tested in vivo. They contributed to a range of tissues in fetal chimeras including heart, lung, kidney, intestine, muscle, brain and skin. We also examined the methylation status of the imprinted genes: Igf2r, p57Kip2, Lit1, H19 and Igf2. Igf2r, p57Kip2 and Lit1 were unmethylated in all analysed EGC lines, whereas H19 and Igf2 showed significant hypo-methylation in the 9.5 dpc EGC-1 line when compared to previously derived 11.5 dpc male EGC lines. This suggests that imprint erasure in the male germ line occurs prior to 9.5 dpc for all imprinted genes examined.     

Impaired meiotic competence in putative primordial germ cells produced from mouse embryonic stem cells

There are still several unanswered questions and problems about the recently claimed possibility of producing functional germ cells in vitro from pluripotent embryonic stem cells (ESCs). In the present paper, we compared by single-cell analysis the capability of putative primordial germ cells (PGCs), produced in vitro from ESCs, and that of endogenous PGCs isolated from embryos, to enter and progress through meiotic prophase I. Using a protocol previously reported to be suitable to produce female germ cells from mouse ESC monolayers, we first identified putative PGCs by analysing the expression pattern of several markers such as SSEA1, APase, OCT4, NANOG, MVH and SCP3 of pre- and post migratory PGCs. Next, after isolation of such cells from culture, we tested their meiotic capability. The evaluation at 2-5 days of culture of the number of cells showing meiotic nuclear SCP3 staining in cytospreads showed that it remained nearly constant in the putative PGCs, whereas it increased markedly in endogenous PGCs. Moreover, we observed that in putative PGCs, the nuclear distribution or expression of SCP3 and other meiotic markers such as DMC1, gH2AX and SCP1 were always highly abnormal in comparison to that observed in endogenous cultured PGCs. We conclude that although the formation of cells showing characteristics of PGCs can occur efficiently from ESCs in vitro, these cells possess impaired capability to enter and progress through meiotic prophase I.     

Metastable primordial germ cell-like state induced from mouse embryonic stem cells by Akt activation

Specification to primordial germ cells (PGCs) is mediated by mesoderm-induction signals during gastrulation. We found that Akt activation during in vitro mesodermal differentiation of embryonic stem cells (ESCs) generated self-renewing spheres with differentiation states between those of ESCs and PGCs. Essential regulators for PGC specification and their downstream germ cell-specific genes were expressed in the spheres, indicating that the sphere cells had commenced differentiation to the germ lineage. However, the spheres did not proceed to spermatogenesis after transplantation into testes. Sphere cell transfer to the original feeder-free ESC cultures resulted in chaotic differentiation. In contrast, when the spheres were cultured on mouse embryonic fibroblasts or in the presence of ERK-cascade and GSK3 inhibitors, reversion to the ESC-like state was observed. These results indicate that Akt signaling promotes a novel metastable and pluripotent state that is intermediate to those of ESCs and PGCs.     

Chondrocytes derived from mouse embryonic stem cells

Our knowledge of cellular differentiation processes during chondro- and osteogenesis, in particular the complex interaction of differentiation factors, is still limited. We used the model system of embryonic stem (ES) cell differentiation in vitro via cellular aggregates, so called embryoid bodies (EBs), to analyze chondrogenic and osteogenic differentiation. ES cells differentiated into chondrocytes and osteocytes throughout a series of developmental stages resembling cellular differentiation events during skeletal development in vivo. A lineage from pluripotent ES cells via mesenchymal, prechondrogenic cells, chondrocytes and hypertrophicchondrocytes up to osteogenic cells was characterized. Furthermore, we found evidence for another osteogenic lineage, bypassing the chondrogenic stage. Together our results suggest that this in vitro system will be helpful to answer so far unacknowledged questions regarding chondrogenic and osteogenic differentiation. For example, we isolated an as yet unknown cDNA fragment from ES cell-derived chondrocytes, which showed a developmentally regulated expression pattern during EB differentiation. Considering ES cell differentiation as an alternative approach for cellular therapy, we used two different methods to obtain pure chondrocyte cultures from the heterogenous EBs. First, members of the transforming growth factor (TGF)-β family were applied and found to modulate chondrogenic differentiation but were not effective enough to produce sufficient amounts of chondrocytes. Second, chondrocytes were isolated from EBs by micro-manipulation. These cells initially showed dedifferentiation into fiboblastoid cells in culture, but later redifferentiated into mature chondrocytes. However, a small amount of chondrocytes isolated from EBs transdifferentiated into other mesenchymal cell types, indicating that chondrocytes derived from ES cells posses a distinct differentiation plasticity. This revised version was published online in July 2006 with corrections to the Cover Date.     

The ability of mouse nuclear transfer embryonic stem cells to differentiate into primordial germ cells

Nuclear transfer embryonic stem cells (ntESCs) show stem cell characteristics such as pluripotency but cause no immunological disorders. Although ntESCs are able to differentiate into somatic cells, the ability of ntESCs to differentiate into primordial germ cells (PGCs) has not been examined. In this work, we examined the capacity of mouse ntESCs to differentiate into PGCs in vitro. ntESCs aggregated to form embryoid bodies (EB) in EB culture medium supplemented with bone morphogenetic protein 4(BMP4) as the differentiation factor. The expression level of specific PGC genes was compared at days 4 and 8 using real time PCR. Flow cytometry and immunocytochemical staining were used to detect Mvh as a specific PGC marker. ntESCs expressed particular genes related to different stages of PGC development. Flow cytometry and immunocytochemical staining confirmed the presence of Mvh protein in a small number of cells. There were significant differences between cells that differentiated into PGCs in the group treated with Bmp4 compared to non-treated cells. These findings indicate that ntESCs can differentiate into putative PGCs. Improvement of ntESC differentiation into PGCs may be a reliable means of producing mature germ cells.     

In vitro differentiation of germ cells from stem cells: a comparison between primordial germ cells and in vitro derived primordial germ cell-like cells

Stem cells are unique cell types capable to proliferate, some of them indefinitely, while maintaining the ability to differentiate into a few or any cell lineages. In 2003, a group headed by Hans R. Schöler reported that oocyte-like cells could be produced from mouse embryonic stem (ES) cells in vitro. After more than 10 years, where have these researches reached? Which are the major successes achieved and the problems still remaining to be solved? Although during the last years, many reviews have been published about these topics, in the present work, we will focus on an aspect that has been little considered so far, namely a strict comparison between the in vitro and in vivo developmental capabilities of the primordial germ cells (PGCs) isolated from the embryo and the PGC-like cells (PGC-LCs) produced in vitro from different types of stem cells in the mouse, the species in which most investigation has been carried out. Actually, the formation and differentiation of PGCs are crucial for both male and female gametogenesis, and the faithful production of PGCs in vitro represents the basis for obtaining functional germ cells.     

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.     

AKT signaling promotes derivation of embryonic germ cells from primordial germ cells

Primordial germ cells (PGCs) are embryonic germ cell precursors. Although the developmental potency of PGCs is restricted to the germ lineage, PGCs can acquire pluripotency, as verified by the in vitro establishment of embryonic germ (EG) cells and the in vivo production of testicular teratomas. PGC-specific inactivation of PTEN, which is a lipid phosphatase antagonizing phosphoinositide-3 kinase (PI3K), enhances both EG cell production and testicular teratoma formation. Here, we analyzed the effect of the serine/threonine kinase AKT, one of the major downstream effectors of PI3K, on the developmental potency of PGCs. We used transgenic mice that expressed an AKT-MER fusion protein, the kinase activity of which could be regulated by the ligand of modified estrogen receptor (MER), 4-hydroxytamoxifen. We found that hyperactivation of AKT signaling in PGCs at the proliferative phase dramatically augmented the efficiency of EG cell establishment. Furthermore, AKT signaling activation substituted to some extent for the effects of bFGF, an essential growth factor for EG cell establishment. By contrast, AKT activation had no effect on germ cells that were in mitotic arrest or that began meiosis at a later embryonic stage. In the transgenic PGCs, AKT activation induced phosphorylation of GSK3, which inhibits its kinase activity; enhanced the stability and nuclear localization of MDM2; and suppressed p53 phosphorylation, which is required for its activation. The p53 deficiency, but not GSK3 inhibition, recapitulated the effects of AKT hyperactivation on EG cell derivation, suggesting that p53 is one of the crucial downstream targets of the PI3K/AKT signal and that GSK3 is not.     

Hematopoietic development of primordial germ cell-derived mouse embryonic germ cells in culture.

Induction of hematopoiesis in an embryonic germ (EG) cell line derived from mouse primordial germ cells (PGCs) was examined. When single undifferentiated EG-1 cells were inoculated directly into the methylcellulose medium, both primitive and definitive erythropoiesis were seen in embryoid bodies derived from the EG cells as observed in ES cells, and production of myeloid cell lineages was stimulated by IL-3. These results indicate that EG cells acquired in vitro potency to differentiate toward hematopoietic cells, although they were derived from PGC and are distinct from inner cell mass-derived ES cells with regard to gene expression and patterns of DNA methylation corresponding to genomic imprinting. It turns out that they are useful for study of cell differentiation in the animals whose ES cells are not available.     

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.     

The germ cell determinant Blimp1 is not required for derivation of pluripotent stem cells

Blimp1 (Prdm1), the key determinant of primordial germ cells (PGCs), plays a combinatorial role with Prdm14 during PGC specification from postimplantation epiblast cells. They together initiate epigenetic reprogramming in early germ cells toward an underlying pluripotent state, which is equivalent to embryonic stem cells (ESCs). Whereas Prdm14 alone can?promote reprogramming and is important for the propagation of the pluripotent state, it is not known whether Blimp1 is similarly involved. By using a genetic approach, we demonstrate that Blimp1 is?dispensable for the derivation and maintenance of ESCs and postimplantation epiblast stem cells (epiSCs). Notably, Blimp1 is also dispensable for reprogramming epiSCs to ESCs. Thus, although Blimp1 is obligatory for PGC specification, it is not required for the reversion of epiSCs to ESCs and for their maintenance thereafter. This study suggests that reprogramming, including that of somatic cells to ESCs, may not entail an obligatory route through a Blimp1-positive PGC-like state.     

Bone morphogenetic protein 4 is an efficient inducer for mouse embryonic stem cell differentiation into primordial germ cell

Presence of specific growth factors and feeder layers are thought to be important for in vitro embryonic stem cell (ESCs) differentiation. In this study, the effect of bone morphogenetic protein 4 (BMP4) and mouse embryonic fibroblasts (MEFs) co-culture system on germ cell differentiation from mouse ESCs was evaluated. One-day-old embryoid body was cultured for 4?d in simple culture systems or on top of the MEFs, both in the presence or absence of BMP4. Data showed significant higher viability percent and proliferation rate in simple culture media compared to co-culture systems. Analysis of gene expression indicated that the germ cell-specific genes (VASA and Stra8) were expressed in a significant higher ratio in BMP4-treated cells in simple culture system. Also, the results of immunocytochemistry in simple culture systems showed that the mean percentage of immunostaining cells of VASA, the primordial germ cell (PGC) marker, was increased significantly in BMP4-treated cells compared with BMP4-free group. Meanwhile, CDH1, the late premiotic germ cell marker, showed no significant difference between these two groups. The results suggest that BMP4 is an efficient inducer in PGC derivation from mouse ESC. However, the employment of MEFs as feeder has no apparent effect on PGC derivation.     

Heterogeneity in imprinted methylation patterns of pluripotent embryonic germ cells derived from pre-migratory mouse germ cells

Pluripotent stem cells, termed embryonic germ (EG) cells, have been generated from both human and mouse primordial germ cells (PGCs). Like embryonic stem (ES) cells, EG cells have the potential to differentiate into all germ layer derivatives and may also be important for any future clinical applications. The development of PGCs in vivo is accompanied by major epigenetic changes including DNA demethylation and imprint erasure. We have investigated the DNA methylation pattern of several imprinted genes and repetitive elements in mouse EG cell lines before and after differentiation. Analysed cell lines were derived soon after PGC specification, “early”, in comparison with EG cells derived after PGC colonisation of the genital ridge, “late” and embryonic stem (ES) cell lines, derived from the inner cell mass (ICM). Early EG cell lines showed strikingly heterogeneous DNA methylation patterns, in contrast to the uniformity of methylation pattern seen in somatic cells (control), late EG cell and ES cell lines. We also observed that all analysed XX cell lines exhibited less methylation than XY. We suggest that this heterogeneity may reflect the changes in DNA methylation taking place in the germ cell lineage soon after specification.     

Isolation of mouse primordial germ cells

Primordial germ cells (PGCs) were obtained from fetal mouse gonads of both sexes days post coitum (dpc), either by collagenase treatment, or by mechanical procedures with or without prior EDTA treatment. With mechanical procedures alone, yield was relatively low and many of the cells released were dead. After EDTA treatment, both yield and viability were significantly improved. Collagenase treatment gave the best yield of cells, since the entire gonad was disaggregated, but contamination with somatic cells was substantial, and the adhesive properties of the germ cells were altered by the treatment. When cells released following EDTA treatment were fractionated on a simple Percoll gradient, several thousand viable PGCs per gonad could be obtained in 2–3 h, with not more than 10–20% somatic cell contamination.     

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.     

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.