Animals

C57BL/6N (B6), DBA/2, and (B6 × DBA/2) F(1) (BDF1) strains of mice were purchased from Japan SLC Inc. (Shizuoka, Japan), and ICR strain mice were purchased from CLEA Japan Inc. (Tokyo, Japan). Experiments were approved by the Animal Experimentation Committees of the RIKEN Tsukuba Institute and were performed in accordance with the committees' guiding principles.

Cell Lines

Most cell culture experiments in this study were performed with TSC lines established by the conventional method developed by Tanaka et al. [2] (“conventional TSC lines” hereafter). Additional experiments were performed using a TSC line established with a chemically defined culture condition (“defined TSC line” hereafter) [6]. The cell lines used are listed in Table 1, together with their genetic background. Prior to these experiments, we introduced the gene encoding enhanced green fluorescent protein (EGFP) into a TSC line, B6TS4, using a lentiviral vector as described previously [7]. Conventional TSCs were cultured as described previously [2]. In brief, cells were cultured on mitomycin-C-treated (Sigma-Aldrich, St. Louis, MO) primary MEFs in RPMI1640 medium (11875-093; Thermo Fisher Scientific, San Jose, CA) with 20% fetal bovine serum (Thermo Fisher Scientific), 25 ng/ml human recombinant FGF4 (062-04341; Wako, Osaka, Japan), 1 μg/ml heparin (H3149; Sigma-Aldrich), 100 μM 2-mercaptoethanol (Sigma-Aldrich), 1% Glutamax (Thermo Fisher Scientific), and 1 mM sodium pyruvate (Thermo Fisher Scientific). Trophoblast stem cells established with chemically defined medium were cultured under feeder-free, serum-free conditions as described previously [6].

TABLE 1

List of TSC lines and samples used for microarray analysis.

a

 ExE, extraembryonic ectoderm; EPC, ectoplacental cone.

b

 Referred to as “CD1-TS” in the text.

Immunofluorescence

Trophoblast stem cells were fixed in 4% paraformaldehyde in PBS for 30 min and were permeabilized with 0.5% Triton X-100 in PBS for 10 min, followed by blocking with 1% bovine serum albumin in PBS for 1 h. They were treated with a primary antibody overnight at 4°C and with a secondary antibody for 1 h at room temperature. The primary antibodies used were a rabbit monoclonal anti-CDX2 (1:500; ab76541; Abcam, Cambridge, U.K.) and a rat monoclonal anti-mouse CDH1 (1:100; ECCD2; M108; TaKaRa Bio Inc., Otsu, Japan). The secondary antibodies were goat anti-rabbit immunoglobulin G (IgG) conjugated to Alexa Fluor 555 (1:500) and goat anti-rat IgG conjugated to Alexa Fluor 546 (1:500; Thermo Fisher Scientific). Samples were also stained with 4′,6-diamidino-2-phenylindole (DAPI) for localizing the nuclei. Images were acquired using a confocal scanning laser microscope (CV1000; Yokogawa Electric, Kanazawa, Japan) or a fluorescence microscope (BZ-9000; Keyence, Osaka, Japan) equipped with a Plan-Fluor ELWD 20×/0.45 objective lens (Nikon, Tokyo, Japan).

Differential Interference Contrast Imaging

Trophoblast stem cells were dissociated with a mixture of TrypLE Select (1×; 12563-011; Thermo Fisher Scientific) and 0.1 mg/ml polyvinyl alcohol (P-8136; Sigma-Aldrich) at 37°C under 5% CO₂ in humidified air for 2 min, and were subsequently inactivated with an equal volume of fetal bovine serum. A drop of TSC suspension was placed on a glass slide. Differential interference contrast images were acquired using an Olympus BX53 microscope (Olympus, Tokyo, Japan) equipped with a 60×/0.90 UPlanSApo objective lens (Olympus) and a NEX-5T camera (Sony, Tokyo, Japan).

Time-Lapse Live Imaging

Trophoblast stem cell colonies in culture were monitored using an Axio Observer Z1 fluorescence microscope equipped with a 10×/0.30 Plan-Neofluar Ph1 objective lens, a Colibri LED light source, and an AxioCam MRm camera (all from Carl Zeiss Microimaging GmbH, Jena, Germany). The culturing environment was maintained with a Heating Insert P S and the incubation system S (Carl Zeiss) at 37°C under 5% CO₂ in air with 100% humidity. The fields of view were acquired every 30 min for 48 h to monitor any conversion of type 1 colonies. Images were acquired, merged, and processed using AxioVision 4.8 software (Carl Zeiss).

Quantification of Single Cell Sizes

The EGFP fluorescent signals were detected and analyzed quantitatively using a fluorescence microscope (BZ-9000; Keyence) equipped with a Plan-Fluor ELWD 40×/0.60 objective lens (Nikon). Images were recorded with the BZ-II viewer software, and the diameters of single cells were measured using BZ-II analyzer software (Keyence). The nuclear shape was observed after staining with Hoechst 33342.

RNA Isolation, Amplification, and Hybridization

Separation of EPC and ExE lines was performed as described previously [8]. Each colony of TSCs was picked up with a pulled-glass capillary pipette. Total RNA was extracted with TRIzol reagent (15596018; Thermo Fisher Scientific) from 30–50 TSC colonies or from the extraembryonic tissues of Embryonic Day 6.5 (E 6.5). RNA samples (500 pg) were amplified with a two-round RNA amplification procedure performed with the TargetAmp 2-Round Aminoallyl-aRNA Amplification Kit 1.0 (TAA2R4924; Epicenter Biotechnologies, Madison, WI) according to the manufacturer's instructions. Amplified RNA was labeled with Cy3 dye (GE Healthcare, Little Chalfont, U.K.) and hybridized to a Whole Mouse Genome oligo DNA microarray (8 × 60K; G4858A; Agilent Technologies, Palo Alto, CA) for 16 h at 65°C according to the manufacturer's instructions.

Microarray Analysis

Hybridized microarray slides were exposed to a DNA microarray scanner (Agilent Technologies) at 3-μm resolution. Fluorescence intensities on scanned images were quantified, corrected for background, and normalized using Feature Extraction software (Agilent Technologies). The raw signals were imported into Gene Spring GX 12.5 software (Agilent Technologies) and normalized using the default settings. Data sets for each array were filtered by expression levels to remove unreliable raw intensity values that were less than 100 (27 874 probes). The normalized and filtered data were analyzed to characterize the global relationships of individual samples by two-way hierarchical clustering and principal component analysis (PCA). To extract differentially expressed genes, one-way ANOVA and Tukey post hoc statistical analysis at a 5% significance level were performed using the Benjamini and Hochberg false-discovery rate for correcting multiple tests. Additionally, 2-fold change selection was performed to identify significantly differentially expressed genes compared with type 4 colonies. Gene Ontology (GO) analysis was performed with FuncAssociate 2.1 ( http://llama.mshri.on.ca/funcassociate/)[9]. Statistical analysis was done using a modified Fisher exact test at a 5% significance level for enrichment terms and resampling-based multiple testing.

RT-Quantitative PCR

Total RNA for RT-quantitative PCR (RT-qPCR) was extracted with RNeasy Micro kits (74004; Qiagen, Venlo, the Netherlands) from 60–80 TSC colonies. Following extraction, the first-strand cDNA was synthesized with a SuperScript III reverse transcriptase reagent set (18080-044; Thermo Fisher Scientific). Gene expression was assessed by qPCR on a StepOnePlus instrument (Thermo Fisher Scientific) using Quantitect SYBR Green PCR kits (204145; Qiagen) according to the manufacturer's instructions. The Actb gene in each sample was used as an endogenous reference [10]. Data analysis was performed using StepOne software v. 2.1 (Thermo Fisher Scientific). The primer sets used for quantification were as follows: Cdx2, 5′-GCAGTCCCTAGGAAGCCAAG-3′ and 5′-GCAGCCAGCTCACTTTTCCT-3′; Hand1, 5′-AGCAAGCGGAAAAGGGAGT-3′ and 5′-GTGCGCCCTTTAATCCTCTT-3′; Elf5, 5′-GTGGCATCCTGGAATGGGAA-3′ and 5′-CACTAACCTCCGGTCAACCC-3′; and Actb, 5′-CTGTCGAGTCGCGTCCA-3′ and 5′-ACCCATTCCCACCATCACAC-3′.

Aggregation Chimeras

BDF1 strain female mice were induced to superovulate by injections of 7.5 IU of pregnant mare serum gonadotropin (Sankyo, Tokyo, Japan), followed by an injection of human chorionic gonadotropin (Sankyo) 48–50 h later. Mature metaphase II oocytes were collected from oviducts and ovarian follicles, and epididymal spermatozoa were collected from male C57BL/6N (B6) mice. In vitro fertilization was performed as described previously [11]. Aggregation of eight-cell-stage embryos with TSCs was used as the protocol for generating embryonic stem cells (ESCs) [12]. Clumps of 10–15 TSCs after dissociation were chosen and transferred into drops containing zona-free eight-cell embryos. However, type 4 colonies (see below) have low adhesion, so the TSCs of such colonies were attached to the surfaces of an eight-cell embryo. Aggregates were cultured for 40–48 h at 37°C under 5% CO₂ in humidified air until the blastocyst stage. Images were acquired with an Olympus IX-70 inverted microscope equipped with both Hoffman modulation and fluorescence optics. Chimeric blastocysts were stained with 5 μg/ml Hoechst 33342 (Dojindo, Kumamoto, Japan) for 10 min at 37°C under 5% CO₂ in humidified air and were subsequently observed using an HS all-in-one fluorescence microscope (BZ-9000) equipped with a Plan-Fluor ELWD 20×/0.45 objective lens (Nikon). In some experiments, chimeric blastocysts were transferred into the uteri of E 2.5 pseudopregnant females, which had been mated with vasectomized males. The placentas were removed from the uteri at E 8.5, E 9.5, E 12.5, or E13.5 for determining the presence of TSC-derived placental cells. The primer set used for the detection of EGFP transgene was: 5′-GGCAAGCTGACCCTGAAGTT-3′ and 5′-TCAGCTCGATGCGGTTCAC-3′.

Statistical Analysis

For comparing gene expression levels between colony types within the TSC lines, a multiple comparison test (Shirley-Williams test) was performed and P < 0.05 was considered statistically significant.

TSC Colonies Are Heterogeneous in Shape and Contain Two Different Cell Types

Trophoblast stem cell colonies could be classified into four major types by their morphology (Fig. 1A): type 1 was small, compact, and dome shaped; type 2 was compact and flattened; type 3 was similar to type 2, but the colonies had loose and multilayered cell clusters in their centers; and type 4 was similar to type 3 but with an extensive multilayered area. There was also an additional type 5, with a sparse monolayered appearance that was observed only rarely but expanded rapidly once it appeared. Immunostaining revealed that all colony types were positive for CDX2, a marker for undifferentiated trophoblastic cells, but the intensity of the CDX2-positive cells was lower in type 5 colonies than in the others (Fig. 1A). Therefore, we did not perform further detailed analyses for type 5 in this study. Besides these TSC colonies, TGCs appeared in the vicinity of types 2 and 3 colonies. Trophoblast giant cells were negative for CDX2 (Fig. 1A). The defined TSC line, CD1-TS, formed type 1-like and type 2-like colonies of different sizes, but not type 3-like or type 4-like colonies (Fig. 1B). Unlike conventional TSC colonies, the defined TSC line showed a relatively homogeneous colony transition; most type 1-like colonies transformed synchronously into large type 2-like colonies within 4 days (Fig. 1B).

FIG. 1

Colony types in conventional TSC lines. A) Morphology of TSC colonies in the B6TS4 line. They were immunostained for CDX2 (red); nuclei are stained with DAPI (blue). Most cells in the type 5 colonies were negative for CDX2 (see merged image). Trophoblastic giant cells were also CDX2 negative (arrowheads). Bar = 100 μm. B) Colonies in defined TSCs. They are similar to types 1 and 2 colonies in conventional TSCs. During culture, most type 1 colonies were transformed synchronously into large type 2 colonies, maintaining a relatively homogeneous colony appearance (see the transition from 40 to 80 h). Bar = 100 μm. C) Differential interference contrast microscope images of single cells from types 1–2, and type 4 colonies in the B6TS4 line. Cells in type 1 colonies were small and had cytoplasmic protrusions (pseudopods; arrowheads). Cells in type 4 colonies were large and had a clear, round nucleus containing a few large nucleoli (arrows). Type 2 colonies had a mixture of cells of both types. Bar = 20 μm.

We next observed the morphology of individual TSCs by dissociating colonies of different types into single-cell suspensions. The cell types were roughly divided into two categories (Fig. 1C and Supplemental Fig. S1A; Supplemental Data are available online at  www.biolreprod.org): a smaller cell type 6–18 μm in diameter with an unclear nuclear membrane, and having an irregular contour because of small cytoplasmic protrusions (pseudopods) on the surface; and a larger round cell type 10–26 μm in diameter with a clear, round nucleus containing a few large nucleoli. The ratios of these two cell types varied according to the colony type and the TSC line. In the B6TS4 line, type 1 colonies were predominantly composed of the smaller cells (70%–80%), whereas types 2 and 3 colonies had a mixture of cells of both types, and type 4 colonies contained only the larger cells (Fig. 1B). This caused differences in the cell size populations among the colony types. As expected, types 1 and 2 colonies contained significantly smaller cells than types 3 and 4 (Supplemental Fig. S1B and Supplemental Table S1). In the EGFP-TS_3.5 and XGFP lines derived from ICR cells (see below), type 1 colonies contained smaller populations of the smaller cells (20%–40%), and types 2, 3, and 4 colonies were composed of only the larger cells. Notably, in the defined TSC line, only the larger cells were observed (data not shown).

Type 1 Is the Primary Colony Category

To evaluate how this heterogeneity of TSC colonies was established in culture, we undertook a time-lapse live-imaging analysis after passaging a TSC line. Trophoblast stem cells from the B6TS4 line were dissociated into single-cell suspensions and then seeded onto a feeder cell layer. Among the single cells traced, the smaller cells typically found in type 1 colonies moved actively on the feeder cell layer and occasionally collided, forming small cell masses (Fig. 2A and Supplemental Movie S1). These cell masses increased with time and formed small type 1 colonies within 24 h after seeding (Fig. 2A and Supplemental Movie S1). Thus, type 1 was the category that first appeared after passage. However, TSC colonies were never formed from single cells in low-density cultures. Trophoblast stem cells from type 1 or type 4 colonies could not divide under single-cell culture conditions and finally underwent apoptosis (Fig. 3 and Supplemental Movies S2 and S3). Next, we traced the transitions of a single type 1 colony, which reached its maximal size at 48 h after seeding (the end of Day 2; Fig. 4A). The results are summarized in a dendrogram with the numbers of each colony type every 12 h (Fig. 4B). All colony types were derived from type 1, and these conversions were mostly irreversible (Fig. 4B); only one case among all 38 conversions was a reversion into type 1 from type 2 at 24 h. The proportion of type 1 colonies decreased with time, but it remained at about 20% at 48 h (Fig. 4C). Type 4 was the colony category that emerged from type 1, 2, or 3 at the final stage of culture (48 h). Only one type 5 colony was found in this experiment. These results indicate that type 1 is the primary colony category that can give rise to all types of TSC colonies in conventional TSC lines.

FIG. 2

Time-lapse live imaging for tracing the formation of primary colonies after seeding single TSC cells. See also Supplemental Movies S1 and S4. A) B6TS4 line. Small cells with pseudopods (arrowheads) moved actively and aggregated to form small colonies. B) CD1-TS line. Round, larger TSCs were transformed into fibroblast-like cells upon attachment to the bottom of the dish, and started random movements. Then, adjacent cells formed small colonies while they continued cell division. Subsequently, groups of many small colonies aggregated into a smaller number of large ones. Bar = 100 μm.

FIG. 3

Time-lapse live imaging to trace the behavior of single B6TS4 cells after low-density culture. Typical TSCs from type 1 (A; small, amorphous cell) and type 4 (B; large, round cell) colonies did not divide, and they underwent apoptosis. See also Supplemental Movies S2 and S3. Bar = 20 μm.

FIG. 4

A representative pattern of the transition of colony types from a single type 1 colony in the B6TS4 line. A) A fully developed type 1 colony at 48 h (the end of Day 2) was selected and traced by time-lapse live imaging. B) A schematic diagram of the colony transition from a type 1 colony at every 12 h. All colony types appeared from a single type 1 colony and the conversion was irreversible, except for one case in which a type 1 colony was formed from a type 2 colony (arrowhead). C) A pie chart indicating the percentages of colony types counted every 12 h.

In the defined TSC line, which did not contain the smaller cell type, primary colonies were formed in a different way. Round cells of the larger type were transformed into fibroblast-like cells upon attachment to the bottom of the dish, and they started random movements. Then, adjacent cells formed small colonies while they continued cell division. Subsequently, groups of many small colonies aggregated into a smaller number of large ones (Fig. 2B and Supplemental Movie S4).

Global Gene Expression Profiles of TSC Colonies Are Line and Genotype Dependent

To characterize the colony types by their gene expression patterns, we performed microarray analysis using pooled colony samples from each cell line, together with in vivo-derived E 6.5 extraembryonic tissues. We analyzed types 1 and 2, the forms predominating in early days after passage, and type 4, the form that appeared in later days. The samples analyzed are listed in Table 1. To visualize the relationship between the samples, we analyzed the data by hierarchical clustering and PCA. Both analytical methods suggested that the samples could be grouped principally by their cell line rather than by their colony type (Fig. 5). Intriguingly, samples from the B6 cell line (B6TS4) proved closer to in vivo-derived samples (ExE and EPC) than other cell lines derived from ICR cells (EGFP-TS_3.5, XGFP, and CD1-TS). As a result, the defined TSC line (CD1-TS) was classified as one of the ICR-derived cell lines. These results indicate that the genetic background of the mice (B6 or ICR) was a primary determining factor that distinguished the TSC colonies in their gene expression profiles.

FIG. 5

Microarray analysis for global gene expression profiles in TSC colonies and in vivo-derived extraembryonic tissues. A) Hierarchical clustering analysis. Trophoblast stem cell samples were clustered by their genetic background (B6 vs. ICR strain) rather than by their colony type. B) Principal component analysis. The TSC samples were grouped by their genetic backgrounds. For the numbers of samples, see Table 1.

Elf5 Might Be a Reliable Marker for Distinguishing the Status of Differentiation in TSC Colonies

We next observed the expression levels of marker genes for undifferentiated or differentiated trophoblastic cells using a microarray analysis. Among the undifferentiated state markers (Cdx2, Esrrb, Sox2, and Elf5), Elf5 was specifically decreased in type 4 colonies in both B6 (B6TS4) and ICR (EGFP-TS_3.5) TSC lines (Fig. 6). These four genes were downregulated in the EPC compared with the ExE, which is consistent with the fact that the EPC differentiates from the ExE (Fig. 6). A spongiotrophoblast cell marker, Ascl2, was expressed consistently in the B6ST4 and EGFP-TS_3.5 lines but was downregulated in type 1 colonies in the XGFP line. A TGC marker gene, Hand1, was consistently expressed in these three conventional TSC lines but was downregulated in the defined TSC line (CD1-TS). These findings suggest that Elf5 is a reliable marker for distinguishing the status of differentiation in TSC colonies, and that TSC lines established by a conventional method inherently contained differentiating TSCs, whereas the TSC line established by a chemically defined method was at a relatively undifferentiated state. Some of the other trophoblast marker genes—Eomes, Fgfr2, Ets2, Tfap2c, Prl3d1/Pl-1, and Krt7I [1314–15]—also showed expression levels in a colony type-dependent manner (Supplemental Fig. S2). Genes specific for endoderm, mesoderm, and ectoderm lineages showed no expressions, indicating that these TSCs did not undergo transdifferentiation during culture (Supplemental Fig. S3).

FIG. 6

Expression levels of TSC marker genes. Among the four undifferentiated markers (Cdx2, Esrrb, Sox2, and Elf5), only Efl5 showed a colony type-specific expression pattern in both B6 (B6TS4) and ICR (XGFP) lines. In the in vivo-derived samples, EPC consistently showed lower expression levels than ExE for these four genes. A spongiotrophoblast cell marker, Ascl2, and a TGC marker gene, Hand1, were consistently expressed in the three conventional lines, with slight variations, but Hand1 was downregulated in the defined line. Bars represent the means of each group. *P < 0.05; **P < 0.01; ***P < 0.001.

Type 4 Colonies Decrease the Expression of “Cell Junction” Categories of Genes

The results shown above indicate that type 4 might represent a differentiated colony type. Therefore, we sought to identify those genes with significantly different expression levels between type 4 and types 1 and 2 in different TSC lines. However, when the data from all TSC lines were analyzed together, very few common differentially expressed genes were extracted, probably because of variations in the gene expression levels among the TSC lines. Therefore, we used the data from B6TS4 and EGFP-TS_3.5 cells as representatives of the B6 and ICR lines, respectively, because the EGFP-TS_3.5 line has been used extensively elsewhere [3, 16]. We identified 71 commonly upregulated and 116 commonly downregulated genes in these two cell lines (Fig. 7A). The GO analysis revealed that the two major categories for upregulated genes in type 4 colonies were angiogenesis-related GO terms (Fig. 7B). This is in accordance with differentiation of the trophoblast lineage in the hemochorial placenta of rodents, because in such placental types trophoblastic cells make direct contact with the maternal blood to form vessels for the maternal blood supply [17, 18]. The upregulated angiogenesis-related genes included Nos3 (nitric oxide synthase 3), Robo4 (roundabout homolog 4), and Ramp2 (receptor [calcitonin] activity-modifying protein 2), which are known to be produced by trophoblastic lineage cells and have direct or indirect effects on vasodilatation and angiogenesis [1920–21]. The genes downregulated in type 4 colonies were enriched in cell junction-related GO terms. This might reflect the loosened cell-cell contacts in type 4 colonies. Of these GO terms, “tight junction” and “cell-cell junction” included Cdh1, the gene encoding E-cadherin (CDH1). The decreased expression levels of Cdh1 are shown in Figure 7C. CDH1 is known to be one of the key molecules necessary for establishing cell polarity and the structural integrity of several tissues [22]. We then investigated the localization of CDH1 in TSC colonies by immunostaining. Fluorescence microscopy images for CDH1 showed meshwork patterns in types 1 and 2 colonies, indicating that this protein was localized in entire cell-to-cell adjacent areas (Fig. 7D). By contrast, the localization of CDH1 was coarse and patchy in type 4 colonies, reflecting their weaker cell contacts (Fig. 7D).

FIG. 7

Extraction of differentially expressed genes (DEGs) between type 4 and types 1 and 2 colonies and their GO analysis. A) A Venn diagram showing the overlaps of DEGs in two TSC lines. In these lines, 71 commonly upregulated and 116 commonly downregulated genes were identified. B) The GO analyses showing functional annotation of the overlapping genes extracted in the Venn diagram. C) Expression levels of Cdh1 showing lower expressions in type 4 colonies in both lines, although there was no statistical significance for the ICR line. *P < 0.05. D) Immunofluorescence staining for CDH1. Types 1 and 2 colonies showed intense meshwork patterns, whereas type 4 showed a patchy staining pattern. Bars = 100 μm.

Quantitative Gene Expression Analysis of Trophoblast Marker Genes

To confirm the results obtained by the microarray analysis, the expression levels of some marker genes were analyzed by RT-qPCR (Fig. 8). We used B6TS4 and EGFP-TS_3.5 cells, the representative B6- and ICR-derived conventional lines (see above). In both cell lines, Cdx2 was repressed in type 5 colonies, in agreement with the results from immunostaining experiments (Fig. 1A). Elf5 was repressed specifically in types 3 and 4 colonies. A marker for TGC, Hand1, did not show any colony type-dependent pattern. Thus, our RT-qPCR analysis further supported the findings obtained by the microarray analysis: Elf5 could be a better marker for the undifferentiated state than Cdx2, and Hand1 was consistently expressed in TSCs regardless of the undifferentiated/differentiated state of TSCs. Type 3 colonies, which were not analyzed by microarray, seemed to have characteristics intermediate to those of types 2 and 4.

FIG. 8

Analysis for TSC marker genes by RT-qPCR. Cdx2 was repressed in type 5 colonies in both TSC lines, consistent with the immunofluorescence analysis shown in Figure 1A. Elf5 showed different expression patterns according to the colony type. The expression levels of Hand1 were not significantly different among the samples. The relative expression levels were normalized against that of Actb. *P < 0.05.

TSCs from Types 1 and 2 Colonies, but Not from Type 4, Contribute to the TE in Chimeric Blastocysts

Finally, we examined the ability of TSCs from types 1, 2, and 4 colonies to contribute to polar TE formation after aggregation with normal embryos. The distributions of TSC-derived cells were identified from the EGFP expression levels of the original TSCs (B6TS4). At the blastocyst stage, TSCs from types 1, 2, and 4 colonies were localized to the polar TE of blastocysts in 27 of 35 (77%), 18 of 25 (72%), and 4 of 41 (9%), respectively (Fig. 9). Only TSCs from type 4 colonies contributed to the mural TE, but the proportion was low at 2 of 41 (5%). When the ability of TSCs to contribute to the placental tissues was examined by embryo transfer experiments, TSCs from types 1 and 2 were distributed to the placentas, but those from type 4 were not detected even with genomic PCR analysis (Supplemental Fig. S4A). We did not observe EGFP-specific fluorescence in these placentas, whereas those derived from chimeric blastocysts using TSCs from type 1 colonies of EGFP-TS_3.5 line did show it (Supplemental Fig. S4B). Although we do not know why B6TSC4-derived chimeric placentas showed no fluorescence, these findings indicate that not only TSCs in type 1 colonies but also those in type 2 colonies maintained their ability to contribute to a placenta. However, TSCs in type 4 colonies had lost this ability.

FIG. 9

Assessment of the chimera formation ability of TSCs by aggregation with eight-cell embryos. A) Images of chimeric blastocysts at 40–48 h after aggregation. Most EGFP-positive TSC-derived cells, if present, were distributed to the polar TE or ICM. Right side: representative images of blastocysts containing cells derived from type 1 colonies. B) The proportions of blastocysts classified by the distribution pattern of TSC-derived cells.