Establishment of Trophoblast Stem Cells under Defined Culture Conditions in Mice

The inner cell mass (ICM) and trophoblast cell lineages duet early embryonic development in mammals. After implantation, the ICM forms the embryo proper as well as some extraembryonic tissues, whereas the trophoectoderm (TE) exclusively forms the fetal portion of the placenta and the trophoblast giant cells. Although embryonic stem (ES) cells can be derived from ICM in cultures of mouse blastocysts in the presence of LIF and/or combinations of small-molecule chemical compounds, and the undifferentiated pluripotent state can be stably maintained without use of serum and feeder cells, defined culture conditions for derivation and maintenance of undifferentiated trophoblast stem (TS) cells have not been established. Here, we report that addition of FGF2, activin A, XAV939, and Y27632 are necessary and sufficient for derivation of TS cells from both of E3.5 blastocysts and E6.5 early postimplantation extraembryonic ectoderm. Moreover, the undifferentiated TS cell state can be stably maintained in chemically defined culture conditions. Cells derived in this manner expressed TS cell marker genes, including Eomes, Elf5, Cdx2, Klf5, Cdh1, Esrrb, Sox2, and Tcfap2c; differentiated into all trophoblast subtypes (trophoblast giant cells, spongiotrophoblast, and labyrinthine trophoblast) in vitro; and exclusively contributed to trophoblast lineages in chimeric animals. This delineation of minimal requirements for derivation and self-renewal provides a defined platform for precise description and dissection of the molecular state of TS cells.


Introduction
The placenta plays essential roles in mammalian embryonic development. This vital organ serves as a point of contact between the fetal and maternal circulation, allowing transfer of nutrients and oxygen to the fetus and metabolic waste back to the mother. In addition to its role as an interface between the developing embryo and its mother, the placenta also produces hormones that play a variety of roles during pregnancy and provides the fetus with immune protection. The primary components of the placenta are derived from the trophoblast lineage, a single layer of cells within the blastocyst that surrounds the inner cell mass (ICM), from which the embryo is ultimately derived [1]. Over the past decades, ICM-derived embryonic stem (ES) cells and trophoectoderm (TE)-derived trophoblast stem (TS) cells have substantially advanced our understanding of the molecular mechanisms of early embryonic and placental development. ES cells were first manipulated and characterized in the early 1980s [2,3]. The conditions for derivation and culture of ES cells in the laboratory, based on a large body of experimental work, include a wide variety of components, including non-dividing ''feeder cells'' that provide growth factors and extracellular matrix support; conditioned media from other cell types, which also provide soluble factors; serum; and individual empirically determined factors such as growth factors, cytokines, and hormones [2][3][4][5][6][7]. Currently, as a result of extensive trial and error, undifferentiated pluripotent stem cells can efficiently be derived and maintained under chemically defined culture conditions in the presence of leukemia inhibitory factor (LIF) and/or small-molecule chemical compounds (i.e., PD0325901, PD184352, PD173074, SU5402, CHIR99021, etc.) [8,9]. On the other hand, since TS cells were first described [10], they have been derived and maintained using various combinations of primary mouse embryonic fibroblast (MEF) cells, conditioned media, fetal bovine serum (FBS), fibroblast growth factor 4 (FGF4), and heparin. Recently, Kubaczka et al. reported derivation and maintenance of TS cells using a chemically defined medium containing FGF4, heparin, and TGFb1 on Matrigel [11]. Here we report that fibroblast growth factor 2 (FGF2), activin A, and the small molecules XAV939 and Y27632 in chemically defined medium (CDM/FAXY) on fibronectin are sufficient for derivation and maintenance of TS cells from both E3.5 TE and E6.5 early-postimplantation extraembryonic ectoderm (ExE). XAV939 is a canonical Wnt signaling inhibitor, which stimulates b-catenin degradation by stabilizing axin via inhibition of poly-ADP-ribosylating enzymes tankyrase 1 and tankyrase 2 [12], and Y27632 is an inhibitor of the Rho-associated protein kinase p160ROCK [13]. The TS cells that grew under these conditions satisfied all the criteria for undifferentiated TS cells: self-renewal capacity, marker gene expression, differentiation competence in vitro, and ability to contribute to the placenta in chimeric mice.
We then analyzed and compared expression levels of FGF ligands in TS and ES cells. The FGF family consists of at least 23 members in vertebrates [18]. Only FGF4 was expressed at high levels in ES cells (Fig. 2b). We also analyzed expression levels of FGF receptor genes in TS and ES cells. TS cells expressed Fgfr1 and Fgfr2, which is strongly expressed in diploid trophoblast [19], at high levels and Fgfr3 at low levels ( Fig. 2c). ES cells expressed only Fgfr1 at high levels. Next, we used microarray analysis to compare global gene expression between the new and conventional TS cells. In total, 3066 genes were differentially expressed by at least 2-fold. Among those 3066 genes, 1935 were overexpressed in the new TS cells, and 1131 genes were underexpressed (Fig. 2d, Table. S1). Both the new and conventional TS cells exhibited similar expression levels of trophoblast stem cell marker genes (Cdx2, Eomes, Sox2, Esrrb, Klf5, Elf5, and Tcfap2c) (Fig. 2e). Relative to conventional cells, the new TS cells expressed lower levels of Hand1 and PL-1 (giant cell marker genes) and Esx1 (a labyrinthine trophoblast marker gene) (Fig. 2e).

Requirement for FGF2, Activin A, and XAV939
In order to determine which factors are required to maintain the tight stem cell-like colony morphology of the TS cells, we observed the morphological changes in TS cells resulting from removal of FGF2, activin A, or XAV939. For five passages prior to these experiments, the undifferentiated state of TS cells was maintained in CDM-FAXY (50 ng/ml FGF2). The removal of FGF2 or Activin A dramatically reduced the proliferation rate and induced differentiation, mainly into flat epithelial cells (Fig. 3a). The removal of XAV939 resulted in the consistent appearance of differentiated cells at the edges of colonies (Fig. 2a, Fig. S1).

Requirement for Y27632
To verify the requirement for Y27632, we removed only Y27632 from cultures and investigated the effects. At 24 hours after the removal of Y27632, in contrast to the removal of FGF2, activin A, or XAV939, ,60% of cells were poly-caspase-positive apoptotic cells, and very few cells survived (Fig. 4a, 4b). In addition, we screened for extracellular matrix that would allow TS cells could survive even after Y27632 removal. We found that the TS cells could be maintained on Matrigel-coated dishes for at least 20 passages in the absence of Y27632. These TS cells exhibited compact and dome-shaped colony morphology (Fig. 4c).

Ability to contribute to placenta in chimeric mice
To analyze the ability of TS cells to contribute to placenta, we injected these cells into C57BL/6 blastocyst embryos (n = 100). To permit visualization of donor TS cells, the injected cells were first labeled with CAG-EGFP by lentivirus infection [20]. Donor TS cells contributed to the fetal portion of the placenta only at E14.5 (6/69, 8.7%) (Fig. 5a). TS cells differentiated into cells of all trophoblast subtypes: trophoblast giant cells, spongiotrophoblast cells, and labyrinthine trophoblasts (Fig. 5b). There were no TEderived cells in the maternal decidua or extraembryonic mesodermal chorionic membrane (Fig. 5b).

Discussion
Mouse ES cells can contribute to all types of embryonic cells in chimeras (including the germline) by transplantation into early embryos, and can generate almost entirely ES cell-derived individuals by tetraploid chimera formation [21]. However, the generation of individuals from ES cells without the use of oocytes or early embryos has not yet been reported. A reasonable explanation for this phenomenon is that ES cells cannot generate the trophoblast lineage. To date, however, aggregation of ES and TS cells has also failed to solve this problem. In order to move toward reconstruction of the early embryo from ES and TS cells, it is necessary to develop a defined platform for precise description and dissection of the molecular state of TS cells and ES-TS cell interactions.
In this study, we established new TS cell lines in the CDM/ FAXY culture condition. In previous work, conventional TS cells have been derived and maintained on a feeder layer of primary mouse embryonic fibroblast (MEF) cells in the presence of FGF4, heparin, serum, and conditioned medium. This method allows the passage of colonies with a tight epithelial morphology. Removal of any one of the critical components of this system (FGF4, heparin, or the MEF feeder layer) quickly and significantly reduces the rate of cell division; after the cells cease proliferating, they differentiate into a state resembling giant cells [10]. Indeed, even when all components of the system are present, a small fraction of TS cells cultured in this manner differentiate into giant cells at the borders of colonies following passage [10]. Furthermore, the requirements for TS cell proliferation and maintenance are not precisely defined: because conditioned media from fibroblasts is necessary for long-term culture of TS cells in this system, it is likely that FGF4 is not the only factor required for trophoblast growth [10]. Recently, Kubaczka et al. reported derivation and maintenance of TS cells with chemically defined medium containing FGF4, heparin, and TGFb1 on Matrigel [11]. However, this was not strictly a chemically defined culture condition, because Matrigel contains various undefined factors. On the other hand, new TS cells could be established and maintained on fibronectin-coated dishes in the absence of FGF4 in media containing FGF2, activin A, XAV939, and Y27632. This condition allowed the passage of colonies with typical TS cell-like epithelial morphology and stemness. Unexpectedly, CDM containing 50 ng/ml FGF4, 20 ng/ml activin A, 10 nM XAV939, and 5 nM Y27632 could not give rise to any TS cell lines (Table 1).
Over the past decades, molecular and genetic experiments in mice have shown that the main signaling pathway involved in the interaction between the trophoblast and ICM is the FGF pathway. The FGF family consists of at least 23 members in vertebrates, but only Fgf4 was highly expressed in ES cells, and no Fgfs were  highly expressed in the TS cells (Fig. 2b). Before implantation, Fgf4 is widely expressed throughout the embryo. In the blastocyst, however, expression of Fgf4 is limited to the ICM, consistent with a model in which FGF4 secreted by embryonic cells stimulates proliferation of the trophoblasts that will ultimately form the main structural components of the placenta [22]. In Fgf4 mutant mice, embryos die at implantation, and both the ICM and trophoblast lineages exhibit developmental deficiencies [23]. Embryonic lethality in Fgf4 mutants might result primarily from the inability of trophoblasts to develop into a functional placenta, as Fgf4 2/2 ES cells themselves are viable [24]. We compared expression levels of FGF receptors in TS and ES cells. TS cells expressed Fgfr1 and Fgfr2 at high levels, but barely expressed Fgfr3 (Fig. 2c), whereas ES cells expressed only Fgfr1 at high levels (Fig. 2c). Fgfr2 expression levels in the diploid trophoblast are high in the blastocyst stage and immediately after implantation [19], consistent with the idea that FGFR2 is the critical receptor molecule in TS cells. As with Fgf4 mutations, deficiencies in Fgfr2 cause failures in placental development and embryonic lethality [25,26]. These results suggest that although FGF4 and Fgfr2 are an ICMderived ligand and receptor that support trophoblast proliferation in vivo, FGF4 cannot function as active ligand in vitro in the absence of a MEF feeder layer, FBS, and conditioned medium. FGF2, however, could act as an active FGF ligand, allowing derivation and maintenance of the TS cells in the absence of the undefined factors.
When we derived the TS cell lines in CDM/FAXY, epiblast stem cell (EpiSC) [15,16]-like non-TS cells often appeared in early passages of explants (Table. 1). The CDM-FAXY condition resembles a modified EpiSC culture condition [27], which can support the undifferentiated state of the EpiSCs closely other than their basal media. Therefore, if epiblast-like cells emerge from ICM-derived outgrowths and epiblast cells adjacent to the ExE contaminate the primary cultures used for TS cell derivation, CDM-FAXY may be able to give rise to EpiSCs.
The new and conventional TS cells exhibited similar expression levels of trophoblast stem cell marker genes (Cdx2, Eomes, Sox2, Esrrb, Klf5, Elf5, and Tcfap2c) (Fig. 2e). Relative to conventional cells, new TS cells expressed lower levels of giant cell marker genes, Hand1 and PL-1, and a labyrinthine trophoblast marker gene, Esx1 (Fig. 2e). The removal of FGF4 from conventional TS culture conditions (i.e., containing undefined serum factors and feeder cells) results in subsequent differentiation into cells with a giant cell-like phenotype [10]. By contrast, removal of FGF2 or activin A from CDM/FAXY resulted in a rapid decline in proliferation, with subsequent differentiation into cells of all trophoblast cell lineages: trophoblast giant cells, spongiotrophoblast cells, and labyrinthine trophoblasts (Fig. 3a, b, c, e, f). This unbiased differentiation potential suggests that the new TS cells retain their undifferentiated state in vitro. The removal of XAV939 resulted in gradual differentiation, down-regulation of some TS cell marker genes, and alterations in the expression levels of differentiated trophoblast lineage marker genes (Fig. 3a, 3d, 3g). These results suggested that XAV939 is necessary to stably maintain the undifferentiated state of TS cells, and that this compound acts via a pathway distinct from those FGF2 and activin A. The requirement of Y27632 for TS cell survival could be circumvented by culturing the cells on Matrigel-coated dishes, although the resultant cells had colony morphologies distinct from those of typical TS cells (Fig. 4c). This result suggests two possibilities: either Matrigel provides extracellular matrix that allows TS cells to survive independent of Y27632, or Matrigel contains undefined factors that satisfy the Y27632 dependency.
Interference with several signaling pathways promotes the pluripotency of ES cells: suppression of Fgf signaling by inhibition of Fgfr, mitogen-activated protein kinase kinase (Mek), and/or mitogen-activated protein kinase (Mapk); inhibition of Tgf-b/ activin signaling by the inhibition of their type I receptors, activin receptor-like kinase 4 (Alk4), Alk5, and Alk7; and activation of Wnt signaling by the inhibition of glycogen synthase kinase 3 (Gsk3) [8,9,28]. By contrast, our results show that activation of Fgf signaling by FGF2, activation of activin signaling by activin A, and suppression of Wnt signaling by XAV939 allow derivation and maintenance of TS cells in chemically defined condition. Thus, ES cells and TS cells, which respectively reflect the early embryonic properties of ICM and TE, are derived and maintained under dissimilar signaling conditions.
The TS cells we derived using CDM/FAXY could contribute to all trophoblast subtypes: trophoblast giant cells (gi), spongiotrophoblast cells (sp), and labyrinthine trophoblasts (la) (Fig. 5a, 5b). This unbiased differentiation potential, together with the trophoblast lineage-specific contribution in vivo in chimeras, suggests that TS cells retain their undifferentiated state.
Our results show that the TS cells satisfied all the criteria for undifferentiated trophoblast stem cells: self-renewal capacity, marker gene expression, differentiation competence into various trophoblast lineage cells in vitro, and the ability to contribute to the placenta in chimeras. Defining the signaling pathways that maintain TS cells may provide key insights into early trophoblast development, elucidate the molecular interactions between TE and ICM, and facilitate the development of technologies using TS and ES cells.

Quantitative PCR (qPCR) analysis
Total RNA was purified from cells using RNeasy Mini kits (Qiagen). For reverse transcription, ReverTra Ace (Toyobo) and oligo (dT) 20 primer were used. For qPCR PCR analyses, Power SYBR Green PCR Master Mix (Applied Biosystems) and a CFX384 Real-Time System (Bio-Rad) were used. Transcript levels were determined in triplicate reactions and normalized against the corresponding levels of Gapdh. Primer sequences are shown in Table S2.

Microarray analysis
Total RNA was purified by using RNeasy Mini kit (QIAGEN). Microarray targets from 200 ng total RNA were synthesized and labeled using the Low RNA Input Linear Amp Kit (Agilent) and hybridized to Mouse 4644K Ver.2.0 arrays. Arrays were scanned on an Agilent Technologies Microarray scanner, and signal intensities were calculated using the Agilent Feature Extraction 10.7.3.1 software. GEO accession number is GSE59107.

Apoptosis analysis
Apoptotic cells were detected using the FAM-FLICA Poly Caspase Assay Kit (ImmunoChemistry Technologies), and cells were counterstained with Hoechst 33342 (200 mg/ml, Immuno-Chemistry Technologies).

Imaging
Images were acquired with an IX71 inverted microscope (Olympus) equipped with a DP72 camera (Olympus) or with an SZX12 dissection microscope (Olympus) equipped with a DP71 camera (Olympus).

Lentivirus production and infection
293FT cells were plated at 4610 6 cells per 100 mm dish and incubated overnight. Cells were then transfected with the pLV-CAG1.1-EGFP, pMDL g/p, pREV, and pVSVG vectors [20] using Lipofectamine 2000 (Invitrogen). At 48 and 72 hours after transfection, medium from the transfectants was collected and filtered through a 0.45mm pore cellulose acetate filter (Millipore); the filtrate was used as the viral supernatant. One day before transduction, TS cells were seeded at 1610 5 cells per well in 6-well plates (Greiner Bio One). On the day of transduction, the medium was replaced with viral supernatant supplemented with 4 mg/ml Polybrene (Nacalai Tesque), and then incubated for 24 hours.

Blastocyst injection
To generate chimeric mice, three to five TSCs were injected into C57BL/6 blastocysts, which were then transferred into the uterine horns of CD-1 pseudopregnant mice.

Animal ethics statement
All animal experiments conformed to our guidelines for the care and use of laboratory animals and were approved by the institutional committee for laboratory animal experimentation (RIKEN Kobe Institute). CO 2 inhalation was used for euthanasia. To ensure death following CO 2 asphyxiation, cervical dislocation was performed. Figure S1 Morphological changes of TS cells upon removal of XAV939 (-X, right) at higher magnification.