Extraembryonic Cell Lineages Research Articles

Naïve pluripotent-like characteristics of non-tumorigenic Muse cells isolated from human amniotic membrane

Multilineage-differentiating stress-enduring (Muse) cells are non-tumorigenic pluripotent-like stem cells that exhibit triploblastic differentiation and self-renewability at the single-cell level, and are collectable as pluripotent surface marker SSEA-3(+) from the bone marrow (BM), peripheral blood, and organ connective tissues. SSEA-3(+) cells from human amniotic membrane mesenchymal stem cells (hAMSCs) were compared with hBM-Muse cells. Similar to hBM-Muse cells, hAMSC-SSEA-3(+) cells expressed pluripotency genes (OCT3/4, NANOG, and SOX2), differentiated into triploblastic cells from a single cell, self-renewed, and exhibited non-tumorigenicity. Notably, however, they exhibited unique characteristics not seen in hBM-Muse cells, including higher expression of genes related to germline- and extraembryonic cell-lineages compared with those in hBM-Muse cells in single-cell RNA-sequencing; and enhanced expression of markers relevant to germline- (PRDM14, TFAP2C, and NANOS3) and extraembryonic cell- (CDX2, GCM1, and ID2) lineages when induced by cytokine subsets, suggesting a broader differentiation potential similar to naïve pluripotent stem cells. t-SNE dimensionality reduction and Gene ontology analysis visualized hAMSC-SSEA-3(+) cells comprised a large undifferentiated subpopulation between epithelial- and mesenchymal-cell states and a small mesenchymal subpopulation expressing genes relevant to the placental formation. The AM is easily accessible by noninvasive approaches. These unique cells are a potentially interesting target naïve pluripotent stem cell-like resource without tumorigenicity.

Eomes function is conserved between zebrafish and mouse and controls left-right organiser progenitor gene expression via interlocking feedforward loops.

The T-box family transcription factor Eomesodermin (Eomes) is present in all vertebrates, with many key roles in the developing mammalian embryo and immune system. Homozygous Eomes mutant mouse embryos exhibit early lethality due to defects in both the embryonic mesendoderm and the extraembryonic trophoblast cell lineage. In contrast, zebrafish lacking the predominant Eomes homologue A (Eomesa) do not suffer complete lethality and can be maintained. This suggests fundamental differences in either the molecular function of Eomes orthologues or the molecular configuration of processes in which they participate. To explore these hypotheses we initially analysed the expression of distinct Eomes isoforms in various mouse cell types. Next we compared the functional capabilities of these murine isoforms to zebrafish Eomesa. These experiments provided no evidence for functional divergence. Next we examined the functions of zebrafish Eomesa and other T-box family members expressed in early development, as well as its paralogue Eomesb. Though Eomes is a member of the Tbr1 subfamily we found evidence for functional redundancy with the Tbx6 subfamily member Tbx16, known to be absent from eutherians. However, Tbx16 does not appear to synergise with Eomesa cofactors Mixl1 and Gata5. Finally, we analysed the ability of Eomesa and other T-box factors to induce zebrafish left-right organiser progenitors (known as dorsal forerunner cells) known to be positively regulated by vgll4l, a gene we had previously shown to be repressed by Eomesa. Here we demonstrate that Eomesa indirectly upregulates vgll4l expression via interlocking feedforward loops, suggesting a role in establishment of left-right asymmetry. Conversely, other T-box factors could not similarly induce left-right organiser progenitors. Overall these findings demonstrate conservation of Eomes molecular function and participation in similar processes, but differential requirements across evolution due to additional co-expressed T-box factors in teleosts, albeit with markedly different molecular capabilities. Our analyses also provide insights into the role of Eomesa in left-right organiser formation in zebrafish.

Borcs6 is required for endo-lysosomal degradation during early development.

Early development and differentiation require precise control of cellular functions. Lysosomal degradation is a critical component of normal cellular homeostasis, allowing for degradation of signaling molecules, proteins, and other macromolecules for cellular remodeling and signaling. Little is known about the role of lysosomal function in mammalian embryos before gastrulation. Borcs6 is a protein involved in lysosomal trafficking as well as endo-lysosomal and autophagosome fusion. Here, we show that Borcs6 is necessary for efficient endo-lysosomal degradation in the early embryo. Although embryos lacking Borcs6 are developmentally comparable to control littermates at E5.5, they are characterized by large cells containing increased levels of late endosomes and abnormal nuclei. Furthermore, these embryos display a skewed ratio of extraembryonic and embryonic cell lineages, are delayed by E6.5, and do not undergo normal gastrulation. These results demonstrate the essential functions of lysosomal positioning and fusion with endosomes during early embryonic development and indicate that the early lethality of BORCS6 mutant embryos is primarily due to defects in the HOPS-related function of BORC rather than lysosomal positioning.

Environmental Exposures around Conception: Developmental Pathways Leading to Lifetime Disease Risk.

Environment around conception can influence the developmental programme with lasting effects on gestational and postnatal phenotype and with consequences for adult health and disease risk. Peri-conception exposure comprises a crucial part of the ‘Developmental Origins of Health and Disease’ (DOHaD) concept. In this review, we consider the effects of maternal undernutrition experienced during the peri-conception period in select human models and in a mouse experimental model of protein restriction. Human datasets indicate that macronutrient deprivation around conception affect the epigenome, with enduring effects on cardiometabolic and neurological health. The mouse model, comprising maternal low protein diet exclusively during the peri-conception period, has revealed a stepwise progression in altered developmental programming following induction through maternal metabolite deficiency. This progression includes differential effects in extra-embryonic and embryonic cell lineages and tissues, leading to maladaptation in the growth trajectory and increased chronic disease comorbidities. The timeline embraces an array of mechanisms across nutrient sensing and signalling, cellular, metabolic, epigenetic and physiological processes with a coordinating role for mTORC1 signalling proposed. Early embryos appear active participants in environmental sensing to optimise the developmental programme for survival but with the trade-off of later disease. Similar adverse health outcomes may derive from other peri-conception environmental experiences, including maternal overnutrition, micronutrient availability, pollutant exposure and assisted reproductive treatments (ART) and support the need for preconception health before pregnancy.

Mouse totipotent stem cells captured and maintained through spliceosomal repression

Since establishment of the first embryonic stem cells (ESCs), invitro culture of totipotent cells functionally and molecularly comparable with invivo blastomeres with embryonic and extraembryonic developmental potential has been a challenge. Here we report that spliceosomal repression in mouse ESCs drives a pluripotent-to-totipotent state transition. Using the splicing inhibitor pladienolide B, we achieve stable invitro culture of totipotent ESCs comparable at molecular levels with 2- and 4-cell blastomeres, which we call totipotent blastomere-like cells (TBLCs). Mouse chimeric assays combined with single-cell RNA sequencing (scRNA-seq) demonstrate that TBLCs have a robust bidirectional developmental capability to generate multiple embryonic and extraembryonic cell lineages. Mechanically, spliceosomal repression causes widespread splicing inhibition of pluripotent genes, whereas totipotent genes, which contain few short introns, are efficiently spliced and transcriptionally activated. Our study provides a means for capturing and maintaining totipotent stem cells.

Establishment of bovine expanded potential stem cells

Embryonic stem cells (ESCs) and induced pluripotent stem cells have the potential to differentiate to all cell types of an adult individual and are useful for studying development and for translational research. However, extrapolation of mouse and human ESC knowledge to deriving stable ESC lines of domestic ungulates and large livestock species has been challenging. In contrast to ESCs that are usually established from the blastocyst, mouse expanded potential stem cells (EPSCs) are derived from four-cell and eight-cell embryos. We have recently used the EPSC approach and established stem cells from porcine and human preimplantation embryos. EPSCs are molecularly similar across species and have broader developmental potential to generate embryonic and extraembryonic cell lineages. We further explore the EPSC technology for mammalian species refractory to the standard ESC approaches and report here the successful establishment of bovine EPSCs (bEPSCs) from preimplantation embryos of both wild-type and somatic cell nuclear transfer. bEPSCs express high levels of pluripotency genes, propagate robustly in feeder-free culture, and are genetically stable in long-term culture. bEPSCs have enriched transcriptomic features of early preimplantation embryos and differentiate in vitro to cells of the three somatic germ layers and, in chimeras, contribute to both the embryonic (fetal) and extraembryonic cell lineages. Importantly, precise gene editing is efficiently achieved in bEPSCs, and genetically modified bEPSCs can be used as donors in somatic cell nuclear transfer. bEPSCs therefore hold the potential to substantially advance biotechnology and agriculture.

Unexpected low expression of porcine endogenous retroviruses (PERVs) in porcine expanded potential stem cells (EPSCs)

Expanded potential stem cells (EPSCs) have been recently derived from porcine preimplantation embryos (Gao et al., 2019). These cells were shown to express key pluripotency genes, to be genetically stable and differentiate to derivatives of the three germ layers and additionally to trophoblast. Their molecular features and expanded potency to contribute to all embryonic and extra-embryonic cell lineages are generally not seen in the embryo-derived or induced pluripotent stem cells (iPSCs). Therefore porcine EPSCs represent a unique state of cellular potency. In the past it had been shown that human and murine embryonic stem cells (ESCs) show an increased expression of murine and human endogenous retroviruses, respectively, and retroviral expression patterns were used as markers of ESC pluripotency. An increased expression of porcine endogenous retroviruses (PERVs) was also detected in porcine iPSCs. Here we investigated 24 passages of five different clones of porcine EPSCs derived from German landrace pigs and show that they harbour PERV-A, PERV-B and PERV-C, but their expression was very low and did not change during cultivation. No recombinant PERV-A/Cs were found in these cells. The low expression despite the presence of spliced mRNA, and negative infection assay and electron microscopy results indicate that no PERV particles were released. Therefore, the absence of PERV expression seems to be a unique feature of porcine EPSCs. Most importantly, the copy number of PERV proviruses was much lower in EPSCs than in young and older pigs (29.1 copies compared with 35.8), indicating an increase in copy number during life time.

Establishment of porcine and human expanded potential stem cells.

We recently derived mouse expanded potential stem cells (EPSCs) from individual blastomeres by inhibiting the critical molecular pathways that predispose their differentiation. EPSCs had enriched molecular signatures of blastomeres and possessed developmental potency for all embryonic and extra-embryonic cell lineages. Here, we report the derivation of porcine EPSCs, which express key pluripotency genes, are genetically stable, permit genome editing, differentiate to derivatives of the three germ layers in chimeras and produce primordial germ cell-like cells in vitro. Under similar conditions, human embryonic stem cells and induced pluripotent stem cells can be converted, or somatic cells directly reprogrammed, to EPSCs that display the molecular and functional attributes reminiscent of porcine EPSCs. Importantly, trophoblast stem-cell-like cells can be generated from both human and porcine EPSCs. Our pathway-inhibition paradigm thus opens an avenue for generating mammalian pluripotent stem cells, and EPSCs present a unique cellular platform for translational research in biotechnology and regenerative medicine.

Maternal Eed knockout causes loss of H3K27me3 imprinting and random X inactivation in the extraembryonic cells.

Genomic imprinting is essential for mammalian development. Recent studies have revealed that maternal histone H3 Lys27 trimethylation (H3K27me3) can mediate DNA methylation-independent genomic imprinting. However, the regulatory mechanisms and functions of this new imprinting mechanism are largely unknown. Here we demonstrate that maternal Eed, an essential component of the Polycomb group complex 2 (PRC2), is required for establishing H3K27me3 imprinting. We found that all H3K27me3-imprinted genes, including Xist, lose their imprinted expression in Eed maternal knockout (matKO) embryos, resulting in male-biased lethality. Surprisingly, although maternal X-chromosome inactivation (XmCI) occurs in Eed matKO embryos at preimplantation due to loss of Xist imprinting, it is resolved at peri-implantation. Ultimately, both X chromosomes are reactivated in the embryonic cell lineage prior to random XCI, and only a single X chromosome undergoes random XCI in the extraembryonic cell lineage. Thus, our study not only demonstrates an essential role of Eed in H3K27me3 imprinting establishment but also reveals a unique XCI dynamic in the absence of Xist imprinting.

Maternal H3K27me3 controls DNA methylation-independent imprinting.

Mammalian sperm and oocytes have different epigenetic landscapes and are organized in different fashions. After fertilization, the initially distinct parental epigenomes become largely equalized with the exception of certain loci, including imprinting control regions. How parental chromatin becomes equalized and how imprinting control regions escape from this reprogramming is largely unknown. Here we profile parental allele-specific DNase I hypersensitive sites in mouse zygotes and morula embryos, and investigate the epigenetic mechanisms underlying these allelic sites. Integrated analyses of DNA methylome and tri-methylation at lysine 27 of histone H3 (H3K27me3) chromatin immunoprecipitation followed by sequencing identify 76 genes with paternal allele-specific DNase I hypersensitive sites that are devoid of DNA methylation but harbour maternal allele-specific H3K27me3. Interestingly, these genes are paternally expressed in preimplantation embryos, and ectopic removal of H3K27me3 induces maternal allele expression. H3K27me3-dependent imprinting is largely lost in the embryonic cell lineage, but at least five genes maintain their imprinted expression in the extra-embryonic cell lineage. The five genes include all paternally expressed autosomal imprinted genes previously demonstrated to be independent of oocyte DNA methylation. Thus, our study identifies maternal H3K27me3 as a DNA methylation-independent imprinting mechanism.

Derivation of Induced Trophoblast Cell Lines in Cattle by Doxycycline-Inducible piggyBac Vectors.

Trophectoderm lineage specification is one of the earliest differentiation events in mammalian development. The trophoblast lineage, which is derived from the trophectoderm, mediates implantation and placental formation. However, the processes involved in trophoblastic differentiation and placental formation in cattle remain unclear due to interspecies differences when compared with other model systems and the small repertoire of available trophoblast cell lines. Here, we describe the generation of trophoblast cell lines (biTBCs) from bovine amnion-derived cells (bADCs) using an induced pluripotent stem cell technique. bADCs were introduced with piggyBac vectors containing doxycycline (Dox)-inducible transcription factors (Oct3⁄4(POU5F1), Sox2, Klf4, and c-Myc). Colonies that appeared showed a flattened epithelial-like morphology similar to cobblestones, had a more definite cell boundary between cells, and frequently formed balloon-like spheroids similar to trophoblastic vesicles (TVs). biTBCs were propagated for over 60 passages and expressed trophoblast-related (CDX2, ELF5, ERRβ, and IFN-τ) and pluripotency-related genes (endogenous OCT3/4, SOX2, KLF4, and c-MYC). Furthermore, when biTBCs were induced to differentiate by removing Dox from culture, they formed binucleate cells and began to express pregnancy-related genes (PL, PRP1, and PAG1). This is the first report demonstrating that the induction of pluripotency in bovine amniotic cells allows the generation of trophoblastic cell lines that possess trophoblast stem cell-like characteristics and have the potential to differentiate into the extra-embryonic cell lineage. These cell lines can be a new cell source as a model for studying trophoblast cell lineages and implantation processes in cattle.

Embryos, DOHaD and David Barker.

The early embryo and periconceptional period is a window during which environmental factors may cause permanent change in the pattern and characteristics of development leading to risk of adult onset disease. This has now been demonstrated across small and large animal models and also in the human. Most evidence of periconceptional 'programming' has emerged from maternal nutritional models but also other in vivo and in vitro conditions including assisted reproductive treatments, show consistent outcomes. This short review first reports on the range of environmental in vivo and in vitro periconceptional models and resulting long-term outcomes. Second, it uses the rodent maternal low protein diet model restricted to the preimplantation period and considers the stepwise maternal-embryonic dialogue that comprises the induction of programming. This dialogue leads to cellular and epigenetic responses by the embryo, mainly identified in the extra-embryonic cell lineages, and underpins an apparently permanent change in the growth trajectory during pregnancy and associates with increased cardiometabolic and behavioural disease in adulthood. We recognize the important advice of David Barker some years ago to investigate the sensitivity of the early embryo to developmental programming, an insight for which we are grateful.

BMP signalling regulates the pre-implantation development of extra-embryonic cell lineages in the mouse embryo.

Pre-implantation development requires the specification and organization of embryonic and extra-embryonic lineages. The separation of these lineages takes place when asymmetric divisions generate inside and outside cells that differ in polarity, position and fate. Here we assess the global transcriptional identities of these precursor cells to gain insight into the molecular mechanisms regulating lineage segregation. Unexpectedly, this reveals that complementary components of the BMP signalling pathway are already differentially expressed after the first wave of asymmetric divisions. We investigate the role of BMP signalling by expressing dominant negative forms of Smad4 and Bmpr2, by down-regulating the pathway using RNAi against BMP ligands and by applying three different BMP inhibitors at distinct stages. This reveals that BMP signalling regulates the correct development of both extra-embryonic lineages, primitive endoderm and trophectoderm, but not the embryonic lineage, prior to implantation. Together these findings indicate multiple roles of BMP signalling in the early mouse embryo.

Ketamine exposure in early development impairs specification of the primary germ cell layers

Preclinical and clinical evidence implicates N-methyl-d-aspartate receptor (NMDAr) signaling in early embryological development. However, the role of NMDAr signaling in early development has not been well studied. Here, we use a mouse embryonic stem cell model to perform a step-wise exploration of the effects of NMDAr signaling on early cell fate specification. We found that antagonism of the NMDAr impaired specification into the neuroectodermal and mesoendodermal cell lineages, with little or no effect on specification of the extraembryonic endoderm cell lineage. Consistent with these findings, exogenous NMDA promoted neuroectodermal differentiation. Finally, NMDAr antagonism modified expression of several key targets of TGF-β superfamily signaling, suggesting a mechanism for these findings. In summary, this study shows that NMDAr antagonism interferes with the normal developmental pathways of embryogenesis, and suggests that interference is most pronounced prior to neuroectodermal and mesoendodermal cell fate specification.

Epigenetic control of cell fate in mouse blastocysts: The role of covalent histone modifications and chromatin remodeling

The first cell-fate decision in mammalian preimplantation embryos is the segregation of the inner cell mass (ICM) and trophectoderm (TE) cell lineages. The ICM develops into the embryo proper, whereas the TE ensures embryo implantation and is the source of the extra-embryonic trophoblast cell lineages, which contribute to the functional components of the placenta. The development of a totipotent zygote into a multi-lineage blastocyst is associated with the generation of distinct transcriptional programs. Several key transcription factors participate in the ICM and TE-specific transcriptional networks, and recent studies indicate that post-translational histone modifications as well as ATP-dependent chromatin remodeling complexes converge with these transcriptional networks to regulate ICM and TE lineage specification. This review will discuss our current understanding and future perspectives related to transcriptional and epigenetic regulatory mechanisms that are implicated in the initial mammalian lineage commitment steps, with a focus on events in mice.

Contribution of Cells Derived from the Area Pellucida to Extraembryonic Mesodermal Cell Lineages in Heterospecific Quail Chick Blastodermal Chimeras

The current study has two main objectives: first, to determine if cells derived from the area pellucida are able to populate extraembryonic membranes, and second, to determine if donor cells have the potential to differentiate to endothelial (EC) and hematopoietic cells (HC) in the yolk sac and allantois, the two extraembryonic membranes functioning as hematopoietic organs in the avian embryo. To this end, quail chick chimeras were constructed by transferring dissociated cells from the areae pellucidae of the stage X–XII (EG&K) quail embryo into the subgerminal cavity of the unincubated chick blastoderm. The distribution of quail cells in the allantois, yolk sac, amnion, and chorion of resulting putative chimeras was examined using quail cell-specific antibody against a perinuclear antigen (QCPN) after 6 days of incubation. The presence of EC, HC, and smooth muscle cells among the QCPN⁺ donor cells was examined using QH-1, a quail-specific marker identifying HC and EC and an anti-α-smooth muscle actin antibody. Evidence gathered in the present study demonstrates that quail cells derived from the areae pellucidae are able to populate all of the extraembryonic membranes of resulting heterospecific quail chick chimeras and, most importantly, give rise to HC, EC, and smooth muscle cells, all of the three main mesodermal lineages derived from the posterior mesoderm both in the yolk sac and allantois.

The H19 lincRNA is a developmental reservoir of miR-675 that suppresses growth and Igf1r.

The H19 large intergenic noncoding RNA (lincRNA) is one of the most highly abundant and conserved transcripts in mammalian development, being expressed in both embryonic and extraembryonic cell lineages, yet its physiological function is unknown. Here we show that miR-675, a microRNA (miRNA) embedded within H19’s first exon, is expressed exclusively in the placenta from the gestational time point when placental growth normally ceases, and placentas that lack H19 continue to grow. Overexpression of miR-675 in a range of embryonic and extraembryonic cell lines results in their reduced proliferation; targets of the miRNA are upregulated in the H19 null placenta, including the growth promoting Insulin-like growth factor 1 receptor (Igf1r). Moreover, the excision of miR-675 from H19 is dynamically regulated by the stress response RNA binding protein HuR. These results suggest that H19’s main physiological role is in limiting growth of the placenta prior to birth, by regulated processing of miR-675. The controlled release of miR-675 from H19 may also allow rapid inhibition of cell proliferation in response to cellular stress or oncogenic signals.

Effect of ectopic expression of homeoprotein EGAM1C on the cell morphology, growth, and differentiation in a mouse embryonic stem cell line, MG1.19 cells

The homeoprotein EGAM1C was identified in preimplantation mouse embryos and embryonic stem (ES) cells. To explore the impact of EGAM1C on the hallmarks of mouse ES cells, MG1.19 cells stably expressing EGAM1C at levels similar to those in blastocysts were established using an episomal expression system. In the presence of leukemia inhibitory factor (+LIF), control transfectants with an empty vector formed flattened cell colonies, while Egam1c transfectants formed compacted colonies with increased E-CADHERIN expression. In Egam1c transfectants, the cellular contents of POU5F1 (OCT4), SOX2, TBX3, and NANOG increased. Cell growth was accelerated in an undifferentiated state sustained by LIF and in the course of differentiation. During clonal proliferation, EGAM1C stabilized the undifferentiated state. In adherent culture conditions, EGAM1C partly inhibited the progression of differentiation at least within a 4-day culture period in the presence of retinoic acid by preventing the downregulation of LIF signaling with a robust increase in TBX3 expression. Conversely, EGAM1C enhanced the expression of lineage marker genes Fgf5 (epiblast), T (mesoderm), Gata6 (primitive endoderm), and Cdx2 (trophectoderm) in -LIF conditions. In embryoid bodies expressing EGAM1C, the expression of marker genes for extraembryonic cell lineages, including Tpbpa (spongiotrophoblast) and Plat (parietal endoderm), increased. These results demonstrated that the ectopic expression of EGAM1C is capable of affecting the stabilization of an undifferentiated state and the progression of differentiation in MG1.19 ES cells, in addition to affecting cellular morphology and growth.

Choice of random rather than imprinted X inactivation in female embryonic stem cell-derived extra-embryonic cells

In female mammals, one of two X chromosomes is epigenetically inactivated for gene dosage compensation, known as X inactivation (Xi). Inactivation occurs randomly in either the paternal or maternal X chromosome in all embryonic cell lineages, designated as random Xi. By contrast, in extra-embryonic cell lineages, which are segregated from somatic cell lineages in pre-implantation development, the paternal X chromosome is selectively inactivated, known as imprinted Xi. Although it is speculated that erasure of the imprinted mark on either the maternal or paternal X chromosome in somatic cell lineages might change the mode of Xi from imprinted to random, it is not known when this event is completed in development. Here, we tested the mode of Xi during the differentiation of female mouse embryonic stem (ES) cells derived from the inner cell mass (ICM) of blastocyst-stage embryos toward trophectoderm (TE) and primitive endoderm (PrE) lineages induced by artificial activation of transcription factor genes Cdx2 and Gata6, respectively. We found that random Xi occurs in both TE and PrE cells. Moreover, cloned embryos generated by the transfer of nuclei from the female ES cells showed random Xi in TE, suggesting the complete erasure of all X imprints for imprinted Xi in ICM-derived ES cells.

Characterization of Trophoblast and Extraembryonic Endoderm Cell Lineages Derived from Rat Preimplantation Embryos

BackgroundPrevious attempts to isolate pluripotent cell lines from rat preimplantation embryo in mouse embryonic stem (ES) cell culture conditions (serum and LIF) were unsuccessful, however the resulting cells exhibited the expression of such traditional pluripotency markers as SSEA-1 and alkaline phosphatase. We addressed the question, which kind of cell lineages are produced from rat preimplantation embryo under “classical” mouse ES conditions.ResultsWe characterized two cell lines (C5 and B10) which were obtained from rat blastocysts in medium with serum and LIF. In the B10 cell line we found the expression of genes known to be expressed in trophoblast, Cdx-2, cytokeratin-7, and Hand-1. Also, B10 cells invaded the trophectodermal layer upon injection into rat blastocysts. In contrast to mouse Trophoblast Stem (TS) cells proliferation of B10 cells occurred independently of FGF4. Cells of the C5 line expressed traditional markers of extraembryonic-endoderm (XEN) cells, in particular, GATA-4, but also the pluripotency markers SSEA-1 and Oct-4. C5 cell proliferation exhibited dependence on LIF, which is not known to be required by mouse XEN cells.ConclusionsOur results confirm and extend previous findings about differences between blastocyst-derived cell lines of rat and mice. Our data show, that the B10 cell line represents a population of FGF4-independent rat TS-like cells. C5 cells show features that have recently become known as characteristic of rat XEN cells. Early passages of C5 and B10 cells contained both, TS and XEN cells. We speculate, that mechanisms maintaining self-renewal of cell lineages in rat preimplantation embryo and their in vitro counterparts, including ES, TS and XEN cells are different than in respective mouse lineages.