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Embryonic Stem Cells Expressing Both Platelet Endothelial Cell Adhesion Molecule-1 and Stage-Specific Embryonic Antigen-1 Differentiate Predominantly into Epiblast Cells in a Chimeric Embryo¹

Development and Differentiation Laboratory,³ Developmental Biology Department, Insect and Animal Sciences Division, National Institute of Agrobiological Sciences, Ibaraki, 305-8602, Japan Reproductive Cell Biology Laboratory,⁴ Department of Animal Breeding and Reproduction, National Institute of Livestock and Grassland Science, Ibaraki, 305-0901, Japan

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We examined the expression of cell-surface markers on subpopulations of mouse embryonic stem (ES) cells to identify those that were associated with cells that had the highest pluripotency. Flow cytometry analysis revealed a wide variation in the expression of platelet endothelial cell adhesion molecule 1 (PECAM-1) and stage-specific embryonic antigen (SSEA)-1 in ES cells. Almost all SSEA-1⁺ cells expressed a high level of PECAM- 1, and reversible repopulation was observed between PECAM- 1⁺SSEA-1⁺ and PECAM-1⁺SSEA-1^– cells. The ES cells carrying the lacZ gene were sorted into three subpopulations: PECAM- 1^–SSEA-1^–, PECAM-1⁺SSEA-1^–, and PECAM-1⁺SSEA-1⁺. Quantitative reverse transcription–polymerase chain reaction revealed a low level of Oct3/4 mRNA expression and an elevation in differentiation maker gene expression in PECAM-1^– cells. To compare the pluripotency of these three subpopulations, a single cell from each was injected into eight-cell embryo and ES cells identified at later stages by X-gal staining. At the blastocyst stage, PECAM-1⁺ SSEA-1^+/– cells were found to have differentiated into epiblast cells in high numbers. In contrast, PECAM- 1^– cell derivatives localized in the primitive endoderm or trophectoderm. At 6.0–7.0 days post coitum, many PECAM-1⁺SSEA- 1⁺ cells were found in the epiblast, but few ß-gal⁺ cells were detected in any regions of embryos that were injected with cells from the other two populations. These results showed that the expression levels of PECAM-1 and SSEA-1 in ES cells correlated closely with their pluripotency and/or their ability to incorporate into the epiblast of chimeric embryos.

early development, embryo, developmental biology

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Embryonic stem (ES) cells maintained in culture for long periods of time retain the ability to differentiate into both somatic and germ cell lineages. However, the cells that comprise ES cells are not homogeneous, and some populations of these cells tend to differentiate spontaneously, even under adequate conditions. Having a source of undifferentiated, pluripotent ES cells would be useful for generating chimeric animals as well as for the further elucidation of the mechanism of ES cell differentiation. We searched surface markers, which expressed on a particular population of ES cells, and found widely variable expression of platelet endothelial cell adhesion molecule 1 (PECAM-1) and stage-specific embryonic antigen (SSEA)-1. Although these antigens are known markers of undifferentiated ES cells [1–4], we showed that the expression of these antigens correlated with each other and that ES cells expressing both PECAM-1 and SSEA-1 differentiated predominantly into epiblast cells in a chimeric embryo.

	MATERIALS AND METHODS

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Cell Cultures

Three ES, one embryonic germ (EG), and one embryonic carcinoma (EC) cell line were used in this study. ROSA cells, established from a ROSA26 (C57BL/6 background) x CBA F1 embryo, and TT2 cells [5] were maintained on an STO cell feeder layer in Dulbecco modified Eagle (DME) medium containing 17.5% knockout serum replacement (Invitrogen, Carlsbad, CA), 10^–4 M 2-mercaptoethanol, 1 mM nonessential amino acids (Invitrogen), and human leukemia inhibitory factor (LIF, 20 ng/ml). ZHBTc4 cells [6] were maintained on gelatin-coated dishes in DME medium containing 20% fetal calf serum (FCS) as described. The EG cell line TM1 was established by culturing primordial germ cells obtained from an 8.5-day post coitum C57BL/6 mouse fetus [7] and maintained under the same conditions described above for ROSA and TT2 cells. The EC cell line F9 was cultured on gelatin-coated dishes in DME medium containing 10% FCS and 10^–4 M 2-mercaptoethanol.

Flow Cytometry

Cells were dispersed by Accutase (Innovative Cell Technologies, La Jolla, CA) that was diluted 1:3 with PBS and then suspended in cold staining buffer (PBS containing 0.2% BSA). R-phycoerythrin-conjugated anti-mouse CD31/PECAM-1 antibody (40 ng/10⁶ cells; BD Biosciences, San Jose, CA) and anti-SSEA-1 antibody (200 ng/10⁶ cells, Kyowa Medex, Shizuoka, Japan) were added to the cell suspension; the cell suspension was then incubated for 30 min on ice. Subsequently, the cells were washed with staining buffer and resuspended in staining buffer containing fluorescein isothiocyanate-conjugated anti-mouse IgM (BD Biosciences). After a 30-min incubation on ice, the cells were resuspended in staining buffer and kept on ice prior to fluorescence-activated cell sorting.

Quantitative Reverse Transcription-Polymerase Chain Reaction

Total RNA was extracted using the RNeasy minikit (Qiagen, Hilden, Germany) with DNase treatment, according to the manufacturer's protocol. Total RNA (0.2 µg) was then subjected to oligo-dT-primed reverse transcription (RT) with Muloney murine leukemia virus-reverse transcriptase (Promega, Madison, WI). The single-strand cDNA products were purified using a polymerase chain reaction (PCR) purification kit (Qiagen) and 1/ 200 to 1/50 of the cDNA products were used for each PCR amplification. Quantitative RT-PCR was performed using LightCycler FastStart DNA Master SYBR Green I (Roche, Indianapolis, IN) on a LightCycler instrument (Roche). Gene-specific primers were designed based on published sequences (Table 1). The reaction conditions for each amplification were chosen as recommended by manufacturer.

Mapping of ES-Derived Cells and Generation of Chimeric Mice

Sorted ES cells were kept on ice until they were injected into host embryos within 2 h after sorting. A single, sorted ROSA cell was injected into 8-cell embryos [8] that were cultured in M16 media containing 10% FCS. After 18–36 h, embryos at Embryonic Day (E) 3.5–4.0 were fixed with 0.1% glutaraldehyde-PBS containing 0.2% Nonidet P-40 and stained with X-gal. For mapping at later developmental stages, ES cell-injected embryos that were cultured for 16–18 h in M16 media containing 10% FCS were transplanted into the uterine horn of pseudopregnant ICR recipient mice. After 4 days, embryos at the E6.0–7.0 stage were harvested from the uteri and were fixed for X-gal staining. ß-Gal-positive embryos were embedded in paraffin and sectioned for microscopic examinations. To generate chimeric mice, five sorted TT2 cells were injected into 8-cell ICR embryos that were then transplanted into pseudopregnant ICR recipient mice. Surgery was performed under barbiturate anesthesia, and all experiments were carried out under the Guidelines Concerning Animal Experiments at the National Institute of Agrobiological Sciences.

Statistical Analysis

Statistical significance between groups was determined using the chi- square or Fisher test.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Variation of PECAM-1 and SSEA-1 Expression in Mouse ES Cells

Flow cytometry analysis revealed a wide degree of variation in the expression of PECAM-1 and SSEA-1 in ROSA ES cells (Fig. 1A). Two additional independent ES cell lines (i.e., TT2 and ZHBTc4 [6]) showed similar variations in their expression of SSEA-1 and PECAM-1 (data not shown), which contrasted with a lesser degree of variation in the expression of these antigens in EG and EC cells. Approximately 80% of the above total cell populations expressed PECAM-1, whereas the expression of SSEA-1 was found only on cells expressing high levels of PECAM-1 (15.2% of the total population).

View larger version (53K): [in this window] [in a new window]   FIG. 1. Expression of PECAM-1 and SSEA-1 in ES, EC, and EG cells. A) Cells were separated from feeder cells and analyzed by flow cytometry. B) Sorted ROSA ES cells were cultured for 4 days, and the cells that repopulated the cultures were analyzed by flow cytometry

ROSA ES cells were sorted into three subpopulations (i.e., PECAM-1^–SSEA-1^–, PECAM-1⁺SSEA-1^–, and PECAM-1⁺SSEA-1⁺) and repopulation assays were performed. After 4 days, each sorted cell population gave rise to progeny cells representing the other two populations (Fig. 1B). However, cells lacking PECAM-1 expression proliferated very poorly, and almost all of them demonstrated adhesive, differentiated cell morphology. PECAM- 1⁺SSEA-1⁺ and PECAM-1⁺SSEA-1^– cells formed compact colonies that were typical of undifferentiated ES cells and showed complete, reciprocal repopulation within 4 days.

Quantitative RT-PCR was performed to determine the level of expression of differentiation marker genes in each cell subpopulation (Fig. 2). Relative to the other subpopulations, PECAM-1^– cells showed reduced Oct-3/4 expression. The transcripts of the Oct-3/4 target genes Rex-1 also decreased in this cell population. Nanog [9, 10], a newly reported homeobox transcription factor, was also expressed at low levels in PECAM-1^– cells. More significantly, we observed a dramatic activation in primitive endodermal (Gata4 and collagen type IV) and primitive ectodermal markers (activin, Fgf-5) and in early embryonic mesodermal markers (Brachyury) in cells lacking PECAM-1 expression. PECAM-1⁺SSEA-1^– and PECAM-1⁺SSEA-1⁺ cells showed nearly identical patterns of expression of these differentiation marker genes. Only the level of mRNA for 1, 3-fucosyltransferase IX (Fuc-TIX) in PECAM- 1⁺SSEA-1⁺ cells, which is required for the synthesis of SSEA-1, were higher (2.8-fold) than in PECAM-1⁺SSEA- 1^– cells. High 1, 3-galactosyltransferase (Gal-T) transcript expression was detected in PECAM-1^–SSEA-1^– cells but not in PECAM-1⁺SSEA-1^– cells; it is known that Gal-T conceals the antigenicity of SSEA-1 by masking the epitope with an -galactose residue.

View larger version (27K): [in this window] [in a new window]   FIG. 2. Quantitative RT-PCR analysis of gene expressions in PECAM-1+SSEA-1–, PECAM-1+SSEA-1–, and PECAM-1+SSEA-1+ cells. Values were calculated from the first amplified cycle numbers and amplification of the Gapdh gene was used to standardize the data. Data were normalized relative to the values of gene expressions in PECAM-1+SSEA-1– cells. Data are the average of three independent experiments

PECAM-1⁺ SSEA-1⁺ ES Cells Predominantly Differentiate into Epiblast

To compare the pluripotency of the three ES cell subpopulations, a single cell from each subpopulation was injected into an 8-cell embryo, and the fate of the ES-derived cells was mapped at later developmental stages by X-gal staining. At the blastocyst stage, cells derived from the cell lacking PECAM-1 expression localized to the primitive endoderm or trophectoderm (Table 2 and Fig. 3A). In contrast, cells derived from a PECAM-1-positive cell most often differentiated into epiblast cells (Table 2 and Fig. 3B and C). We did not detect significant differences in the contribution to embryos between the PECAM-1⁺SSEA-1^– and PECAM- 1⁺SSEA-1⁺ subpopulations (Table 2). At E6.0–7.0, ß-gal- positive cells were only detected in three embryos that were injected with both PECAM-1^–SSEA-1^– and PECAM- 1⁺SSEA-1^– cells (5.0% and 4.7%, respectively, Table 3). Only injected PECAM-1^–SSEA-1^– cells were found to differentiate into visceral endoderm (Fig. 4, A, E, B, and F). PECAM-1⁺SSEA-1^– cells differentiated into epiblast cells in three cases, although their contribution was lower than that seen for PECAM-1⁺SSEA-1⁺ cell derivatives (Fig. 4C). In contrast, significant numbers of PECAM-1⁺SSEA- 1⁺ cell derivatives existed in the epiblast region (15.7% of embryos; Table 3 and Fig. 4, D and G). No ß-gal–positive cells derived from any populations were detected in the region of the trophectoderm at this developmental stage.

View larger version (52K): [in this window] [in a new window]   FIG. 3. Mapping of ES-derived cells at the blastocyst stage. A single sorted ROSA ES cell was injected into an eight-cell embryo and stained by X-gal at E3.5–4.0. PrimE, Primitive endoderm; TE, trophectoderm; EP, epiblast. Original magnification x200

View larger version (94K): [in this window] [in a new window]   FIG. 4. A–G) Mapping of ES-derived cells at E6.0–7.0. (A–D) A single sorted ROSA ES cell was injected into an 8-cell embryo that was then transplanted into recipient mice. After 4 days, the embryos were harvested from the uteruses and fixed for X-gal staining. E and G) ß-Gal-positive embryos were embedded in paraffin and sectioned for microscopic examination. Lines in two of the upper panels (A and D) show the direction of section. F) High-power view of B. EE, Extraembryonic ectoderm; PE, parietal endoderm; VE, visceral endoderm; PC, proamniotic cavity; EP, epiblast. Original magnifications x40 (A, D, E, and G) and x100 (B and C). H) Six chimeric mice were generated from PECAM-1+ SSEA-1+ ES cell-injected embryos. Original magnification x40 (A, D, E, and G) and x100 (B and C)

Finally, we compared the frequency of generation of chimeric mice between the nonenriched and enriched cells (PECAM-1⁺SSEA-1⁺ cells). Three of thirty mice (10%) derived from nonenriched parental ES cell-injected embryos exhibited chimerism, and one of three chimeras was confirmed as a germ-line chimera by mating (Table 4). In contrast, 8 of 23 pups (35%) from PECAM-1⁺SSEA-1⁺ cell- injected embryos exhibited chimerism (Table 4 and Fig. 4H). Unfortunately, two chimeras with high ES coat color contributions from PECAM-1⁺SSEA-1⁺ cells died before maturation, but at least two of six viable chimeras (33.3%) were confirmed as germ-line chimeras (Table 4).

	DISCUSSION

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During early development, PECAM-1 expression is first detected on cells of the inner cell mass (ICM) of the blastocyst [11, 12], which is also the source of ES cells. PECAM-1 plays an important role in vasculogenesis [13], angiogenesis [14–16], and leukocyte infiltration during the inflammatory response [17–20]. However, the role of PECAM-1 during early development prior to vasculogenesis is not clear. Though PECAM-1-deficient mice were shown to exhibit defects in leukocyte transbasement membrane migration [17–20], they were viable and their inheritance followed the expected Mendelian pattern [20]. Robson et al. [12] suggested the possibility that transhomophilic interaction of PECAM-1-expressing ICM cells prevented their attachment to the basement membrane, thereby maintaining their developmental pluripotency. ES cells also form tight clumps in culture, suggesting that PECAM-1 plays a similar role in cell-cell interactions and helps to maintain the pluripotency of ES cells. Furthermore, PECAM-1 was found to be directly associated with Src homology-2-containing adaptor molecules [21–23], ß-catenin [24], and signal transducer and activator of transcription (STAT)3/5 in epithelial cells [25]. Thus, PECAM-1 may modulate the Janus kinase-STAT3 signaling pathway via the LIF receptor in ES cells. The dramatic elevation in lineage marker genes seen in cells lacking PECAM-1 expression raised the possibility that PECAM-1 expression may be regulated by a key molecule(s) that ensures the pluripotency of ES cells. Studies are underway to more clearly define the mechanisms that control PECAM-1 expression in ES cells.

In the repopulation assay, PECAM-1-negative cells proliferated poorly, and a minor population of this cell fraction seemed to repopulate the other two subpopulations. In contrast, PECAM-1⁺SSEA-1⁺ and PECAM-1⁺SSEA-1^– cells demonstrated entirely reciprocal repopulation. These results suggest that the immunoreactivity of SSEA-1 on PECAM- 1-positive cells may have temporarily changed and is not consistent with irreversible differentiation of ES cells. It has been reported that the change in immunoreactivity of SSEA-1 on F9 cells was due to the activity of Fuc-TIX and Gal-T [26]. We also observed the variation of mRNA expression levels of these carbohydrate enzymes among subpopulations. Gal-T mRNA levels were equivalent in PECAM-1⁺SSEA-1⁺ and PECAM-1⁺SSEA-1^– cells, but Fuc- TIX mRNA levels in PECAM-1⁺SSEA-1⁺ cells were higher (2.8-fold) than those seen in PECAM-1⁺SSEA-1^– cells, which might result in high SSEA-1 levels in PECAM- 1⁺SSEA-1⁺ cells. Although Fuc-TIX mRNA expression in PECAM-1^–SSEA-1^– cells showed the highest levels in three subpopulations, Gal-T mRNA expressed remarkably high levels in this subpopulation, which might lead the reduction of the antigenicity of SSEA-1 in this subpopulation. Taken together, these results suggest that the expression of Gal-T is stringently regulated by the factor(s) involved in the pluripotency of ES cells, but the change in Fuc-TIX is not. Fenderson et al. [27, 28] suggested that the changes in carbohydrate phenotype in the early embryo might play an important role in regulating the cell-cell interactions that are involved in lineage formation and morphogenesis. Similar changes might occur in ES cells, and fluctuations in the amount of SSEA-1 may be responsible for the differences in gene expression that influence their contributions to the epiblast in E6.0–7.0 embryos. Although it is unclear why PECAM-1⁺SSEA-1^–-derived cells that were observed in the blastocyst disappeared in later-stage embryos, we speculate that they might lack essential factor(s) or important epigenetic competence that would allow for organogenesis after the blastocyst stage.

It has been reported that mouse ES cells have the potential to differentiate into the trophectodermal lineage in vitro and in vivo [29]. We observed that the derivatives of ES cells that lacked PECAM-1 expression localized to the trophectoderm at the blastocyst stage but not at E6.0–7.0. Although PECAM-1-negative cells may have the potential to differentiate into the trophectodermal lineage, their rate of differentiation into functional extraembryonic tissues may be quite low. Interestingly, we have never observed the differentiation of an injected ES cell into multiple lineages in any population. If the injected cell derivatives had still kept multipotency or bipotency just after first cell division, we should observe the derivatives in multiple lineages (i.e., both primitive endoderm and the epiblast lineage). This result raised two possibilities: 1) the injected cell had already committed to a specific lineage or 2) the positional information imparted by the host embryo strongly affected the fate of the injected cell and/or its derivatives, which led them to the particular lineage commitment. To further investigate these possibilities, we are presently attempting to identify new surface markers that can be used to further divide PECAM-1-positive ES cells into more precise and definitive subpopulations and trace their fate in host embryos.

Enrichment of PECAM-1⁺SSEA-1⁺ cells was effective in generation of chimeric mice; however, the ES cell contributions in individual mice were equivalent to those in mice-derived nonenriched ES cells. The number of pups from the enriched ES cells was 11.3% less than those from nonenriched ES cells, and offspring with high ES coat color contributions died before maturation, suggesting that mortality of chimeric animals increases with the ES cell proportion. The reason is not clear; however, epigenetic alterations in imprinted genes may occur in some ES cell populations, and it may be associated with high mortality [30, 31].

This article describes the first evidence that the expression levels of PECAM-1 and SSEA-1 in ES cells correlated closely with their pluripotency and/or their ability to incorporate into the epiblast of chimeric embryos. Such studies should eventually enable us to produce homogeneous sources of ES cells that can be used to facilitate the development of effective methods for generating chimeric animals and better protocols for the differentiation of specific cell lineages.

	ACKNOWLEDGMENTS

 
We thank Dr. Hitoshi Niwa (CDB, Riken, Japan) and Dr. Takuro Horii (Kyoto University, Kyoto, Japan) for providing ZHBTc4 and ROSA ES cells, respectively; and Dr. Fumio Ike (BRC, Riken, Japan) for his technical advice regarding the cell sorting procedures.

	FOOTNOTES

 
¹ Supported by a grant from the Program for Promotion of Basic Research Activities for Innovative Biosciences.

² Correspondence: Tomoyuki Tokunaga, Development and Differentiation Laboratory, Developmental Biology Department, Insect and Animal Sciences Division, National Institute of Agrobiological Sciences, Ikenodai 2, Tsukuba, Ibaraki, 305-8602, Japan. FAX: 81 298 38 7383; tom;caaffrc.go.jp

Received: 14 October 2003.

First decision: 29 October 2003.

Accepted: 12 January 2004.

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