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BOR - Papers in Press, published online ahead of print August 6, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.017384
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BIOLOGY OF REPRODUCTION 69, 1863–1871 (2003)
DOI: 10.1095/biolreprod.103.017384
© 2003 by the Society for the Study of Reproduction, Inc.

Embryo

Identification of a Biological Activity That Supports Maintenance and Proliferation of Pluripotent Cells from the Primitive Ectoderm of the Mouse1

Joy Rathjen, Jennifer M. Washington3, Michael D. Bettess4, and Peter D. Rathjen2

School of Molecular and Biomedical Sciences, and Australian Research Council Special Research Centre for Molecular Genetics of Development, University of Adelaide, Adelaide, South Australia 5005, Australia


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pluripotent cell development in the mammalian embryo results in the sequential formation of several developmentally distinct populations, inner cell mass, primitive ectoderm, and the primordial germ lineage. Factors within medium conditioned by HepG2 cells (MEDII) have been implicated in the formation and maintenance of primitive ectoderm from inner cell mass cells both in vitro and in vivo. Here we demonstrate that MEDII, but not LIF, is able to support the maintenance and proliferation in culture of pluripotent cells derived from primitive ectoderm formed in vitro or during embryonic development. This distinguishes primitive ectoderm and inner cell mass (ICM) on the basis of cytokine responsiveness and validates the biological activity proposed for factors within MEDII in primitive ectoderm establishment and maintenance. Further, it potentially provides an alternative technology for the isolation of pluripotent cells from the mammalian embryo.

developmental biology, early development, gene regulation, growth factors


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
By 3.5 days postcoitus (d.p.c.) of mammalian embryogenesis, the pluripotent cell population is compartmentalized within the inner cell mass (ICM), located at one pole of the blastocyst. As development progresses, outer ICM cells, in contact with the blastocoelic cavity, differentiate to form the primitive endoderm, an extraembryonic lineage that gives rise to both the visceral and parietal endodermal lineages. Internal cells retain pluripotence and give rise to a second pluripotent cell population, the primitive ectoderm, distinguished by morphology, differentiation potential [1–6], and gene expression [7–12]. Importantly, primitive ectoderm does not differentiate to extraembryonic endoderm but gives rise, at gastrulation, to the three primary germ layers of the embryo and the progenitor cells of the germ lineage.

Isolation and maintenance of pluripotent cells from the ICM of 129 mouse blastocysts was initially demonstrated by Martin [13] and Evans and Kaufman [14]. These cells, embryonic stem (ES) cells, can be maintained indefinitely in culture in the presence of members of the IL-6 family of cytokines, which signal through a common receptor subunit, gp130 [15–22]. Indeed, it has been shown that LIF is sufficient for establishment and maintenance of pluripotent cells from the 129 mouse blastocyst [17, 18]. A more complex mixture of growth factors and cytokines, including LIF, has enabled the derivation of pluripotent cell lines, termed embryonic germ (EG) cells, from the primordial germ lineage of the embryo [23, 24]. The derivation of pluripotent cell lines with apparently similar properties from developmentally distinct pluripotent cell populations in the ICM and primordial germ lineage led Rossant [25] to suggest that ES-like cells could be generated from all pluripotent cell populations of the developing embryo. However, this potential has not yet been realized for isolation of pluripotent cells from the primitive ectoderm, presumably reflecting limitations in existing procedures and culture conditions, particularly knowledge of the factors involved in primitive ectoderm establishment and maintenance.

We have described a conditioned medium, MEDII, that supports formation and maintenance of early primitive ectoderm-like (EPL) cells from ES cells in vitro [26]. Characterization of EPL cells has indicated equivalence with the pluripotent cells of the primitive ectoderm, with a shared gene expression profile, developmental potential in vitro and in vivo and similar responsiveness to cytokines [12, 26, 27]. MEDII or factors contained within MEDII are required for both the formation and maintenance of the EPL cell phenotype, with withdrawal of MEDII resulting in either spontaneous differentiation or, when the cytokine LIF is present, reversion to an ES cell phenotype [26]. Accumulating evidence suggests that the formation of primitive ectoderm in vivo requires signaling from the visceral endoderm (reviewed in Rodda et al. [28]). Consistent with this, the bioactive components of MEDII have been identified as equivalent to signals associated with the pregastrulation embryo or expressed by visceral endoderm-like cell lines [unpublished results, 28].

In this study, we extend our observations on the bioactivity within MEDII and demonstrate the maintenance and proliferation of pluripotent cells from the primitive ectoderm, generated in vitro or during embryogenesis, in response to activities within the conditioned medium. LIF could not functionally replace MEDII in this assay, allowing functional discrimination between the pluripotent cell populations of the ICM and primitive ectoderm on the basis of cytokine responsiveness. This validates the biological activity within MEDII as a determinant of primitive ectoderm identity and maintenance. The use of MEDII extends the window of mammalian embryonic development from which pluripotent cells can be cultured to include cell populations present after cavitation of the epiblast, and potentially provides an alternative target population for derivation of pluripotent cell lines from the mammalian embryo.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture Conditions

ES cell lines E14 [29] and D3 [30] were used in this study. Routine culture of ES and EPL cells and production of MEDII- and sfMEDII-conditioned medium were as described in Rathjen et al. [26]. Briefly, ES cells were cultured in Dulbecco modified Eagle medium (DMEM; Invitrogen Corporation, Grand Island, NY) supplemented with 10% fetal calf serum (FCS; Commonwealth Serum Laboratories, Parkville, VIC, Australia), 40 mg/ml gentamycin, 1 mM L-glutamine, 0.1 mM ß-mercaptoethanol (ß-ME), and 1000 units of LIF (DMEM + LIF). For MEDII-containing medium, MEDII was mixed 1:1 with DMEM containing 10% FCS, and supplemented with 40 mg/ml gentamycin, 1 mM L-glutamine and 0.1 mM ß-ME (DMEM + 50% MEDII); 1000 units of LIF was added to DMEM + 50% MEDII where indicated (DMEM + 50% MEDII + LIF). Embryoid bodies (EBs) were formed and cultured as described in Lake et al. [27].

Formation and culture of single-cell suspensions from EBs were performed as follows. For the data presented in Figure 1B, equivalent plates of EBs were collected on Days 1 to 6 of development and trypsinized to a single-cell suspension. Total cell number was determined and 500 (Day 1), 1000 (Days 2 and 3), or 4000 (Days 4–6) cells were seeded into six 2-ml wells containing DMEM + LIF. For the data presented in Figure 1C, individual EBs, formed from either D3 or E14 ES cell lines and cultured for 5–8 days in culture, were washed with 0.5 mM EGTA in 1x PBS for 5 min before trypsinization to a single-cell suspension. Each single-cell suspension was divided equally between two gelatinized 2-ml tissue-culture wells containing either DMEM + LIF or DMEM + 50% MEDII + LIF.



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  		FIG. 1. MEDII supports the maintenance and proliferation of pluripotent cells from the primitive ectoderm of EBs. A) Northern blot analysis of 20 µg RNA from D3 ES cells and EBs on Days 1–6, probed with radioactively labeled cDNA probes to Rex1, Fgf5, Oct4, and mGAP. Transcript sizes are Rex1, 1.9 kb [9]; Fgf5, 2.7 kb [8]; Oct4, 1.55 kb [34]; and mGAP, 1.5 kb [47]. B) EB populations from (A) were trypsinized to a single-cell suspension, counted to give a total cell count (open circles), and plated into wells containing DMEM + LIF. After 5 days, wells were fixed and stained for alkaline phosphatase and positive colonies counted and expressed as cells/1000 seeded (closed squares). The data points represent data collected from six duplicate wells; error bars representing the standard error of the mean are shown. C) EBs formed from D3 or E14 ES cells were cultured for 5, 6, 7, and 8 days, as indicated, before being trypsinized to a single-cell suspension and seeded equally into wells containing DMEM + LIF (gray) or DMEM + 50% MEDII + LIF (black). After 5 days, wells were fixed and stained for alkaline phosphatase and positive colonies counted. The data points represented data collected from 12 EBs; error bars representing the standard error of the mean are shown. D) Northern blot analysis of 20 µg RNA from ES cells, EPL cells cultured for 2 and 8 days in MEDII with passaging every 2 days, and EPLB cells from either E14 EBs or D3 EBs isolated and maintained in culture in DMEM + 50% MEDII + LIF. Filters were probed with radioactively labeled cDNA probes to Fgf5, Oct4, and mGAP. Transcript sizes are as for A. E and F) EPLB cells cultured for 2 days in DMEM + 50% MEDII + LIF (E) or DMEM + LIF (F). Images were taken using phase-contrast illumination and a x20 objective

Primitive ectoderm explants and derivative cell populations were cultured in embryo culture medium (DMEM; Invitrogen Corporation), pH 7.4, containing high glucose, and supplemented with 15% FCS, 40 µg/ml gentamycin, 1 mM L-glutamine, and 0.1 mM ß-ME, supplemented with 1000 units of LIF under 10% CO2 in a humidified incubator. Inactivated feeder layers were prepared from either STO cells (ATCC CRL-1503) or primary mouse embryonic fibroblasts [31]. Inactivation of feeder cells was achieved by exposing cells to 30 gray of ionizing radiation. Feeder cells were seeded at least 6 h before use at a density of 1 x 105 cells/cm2 in DMEM onto tissue-culture plastic treated with human placental collagen IV (Sigma-Aldrich, St. Louis, MO) as per manufacturer's instructions. Feeder layers were washed once with DMEM before the addition of culture medium and embryonic explants.

Gelatin was prepared as a 0.2% solution in 1x PBS and autoclaved. Other extracellular matrix (ECM) components were obtained from Sigma-Aldrich and used as per the manufacturers' specifications. Agarose-plugged wells were prepared by adding 0.5 ml 0.5% low melting-point agarose in DMEM medium base to a 2-ml tissue-culture well. Agarose plugs were washed 3 x 30 min in embryo culture medium before being pre-equilibrated against embryo culture medium containing 50% MEDII for 30 min at 37°C in a humidified 10% CO2 incubator.

Mice

CBA/C57 F2 embryos were dissected from the uteri of time-mated CBA/C57 F1 female mice on the days specified. The time 0.5 days postcoitum (d.p.c.) was designated as noon on the day of plugging. Mated female mice were killed by cervical dislocation or CO2 asphyxiation and uteri were kept in DMEM supplemented with 10 mM HEPES, pH 7.4, at 37°C until dissection of embryos. Embryos were removed, using standard dissection techniques, into DMEM supplemented with 10 mM HEPES, pH 7.4. Briefly, uterine horns were dissected into individual implantation sites. The uterine wall of each implantation site was dissected along the mesometrial side and the site opened to reveal the deciduum. The deciduum was gently teased open and the embryo removed. Reichart's membrane was removed with sharpened watchmakers forceps. Removal of extraembryonic endoderm was achieved mechanically by pipetting embryos through a pasteur pipette that had been pulled in a flame to a bore diameter slightly smaller than the embryo. Research conformed to the stipulations of the University of Adelaide Animal Ethics Committee.

Blastocyst Injection

Blastocyst injections were performed as described in Rathjen et al. [26]. F2 blastocysts derived from a C57/B6 x DBA F1 cross were used for injections.

Analysis of Gene Expression

Alkaline phosphatase enzyme activity was detected using Sigma kit 86-R with modifications as described in Rathjen et al. [26]. RNA isolation, Northern blot analysis, and probe production were essentially as described in Rathjen et al. [26]. Whole-mount in situ hybridization and production of probes was as described in Rathjen et al. [26].

Statistical Tests

The significance of data presented in Figure 2 was determined by a one-tailed, paired Student t-test.



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  		FIG. 2. Stability of ES and EPL cells cultured on purified matrix components. Two-milliliter tissue-culture wells were treated with gelatin (0.2% in PBS), collagen IV (5.7 µg/cm2), fibronectin (2.85 µg/cm2), laminin (1 µg/cm2), or a mixed matrix of collagen IV, fibronectin, and laminin (mix), and seeded with 2.5 x 102 D3 ES cells in either DMEM + LIF (A) or DMEM + 50% MEDII (B). Cells were cultured for 5 days, stained for alkaline phosphatase activity, and colonies classified as undifferentiated (white); partially differentiated (black), in which some cells lacking alkaline phosphatase could be detected; or differentiated (grey), in which no cells expressed alkaline phosphatase. n = 4, fibronectin, laminin, and the mixed matrix; n = 12, gelatin and collagen IV. Error bars represent standard error of the mean (SEM). *, P < 0.005, **, P < 0.0005


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
LIF Supports the Maintenance of Pluripotent Cells from ICM but Not Primitive Ectoderm Populations Formed In Vitro

Differentiation of ES cells as cell aggregates (embryoid bodies; EBs) results in the progressive appearance of differentiated cell populations in a manner that recapitulates differentiation of the pluripotent ICM cells during early mouse embryogenesis (reviewed in [32]). Outer cells of the aggregate differentiate and establish the extraembryonic endoderm lineage, while inner cells give rise to primitive ectoderm before undergoing further differentiation to cell populations representative of the three primary germ layers of the embryo. The sequential differentiation of pluripotent cells within EBs can be monitored by alterations in gene expression. By Day 3 of EB development, the pluripotent ES cell population within the embryoid body had differentiated to form a population comprised largely or entirely of primitive ectoderm, as determined by expression of the pluripotent cell marker, Oct4 [33–35], and primitive ectoderm marker, Fgf5 [7, 8, 12], and down regulation of the ES/ICM cell marker, Rex1 [9] (Fig. 1A).

The ability of LIF to maintain pluripotent cell populations equivalent to ICM and primitive ectoderm from differentiating EBs was investigated. EBs were trypsinized to a single-cell suspension on Days 1 to 6 of development and seeded into DMEM + LIF. Cells were fixed and stained for the pluripotent cell marker alkaline phosphatase [18, 36] on Day 5, and the number of alkaline phosphatase+ cell colonies was determined (Fig. 1B). Alkaline phosphatase+ cell colonies were detected readily on Day 1, at reduced numbers on Day 2, and at low levels, if at all, on the subsequent days of EB development, despite continued expression of Oct4 within the EBs. This suggested that LIF could support pluripotent cells present early in EB development, when Rex1 is expressed, but not those found later in EB development, a population equivalent to primitive ectoderm as assessed by Fgf5 and Oct4 expression.

MEDII Contains a Bioactivity That Supports Maintenance of Primitive Ectoderm Populations Formed In Vitro

The ability of MEDII to support the maintenance of primitive ectoderm formed within EBs was assessed. Single-cell suspensions from individual EBs, cultured for 5 days in DMEM + LIF or DMEM + 50% MEDII + LIF, were stained for alkaline phosphatase (Fig. 1C). As expected, alkaline phosphatase-positive colonies were observed rarely (E14), if at all (D3), after culture in medium supplemented with LIF alone. In contrast, numerous alkaline phosphatase-positive colonies, with a morphology consistent with EPL cells but distinct from ES cells, were maintained in medium supplemented with 50% MEDII + LIF (Fig. 1C) or 50% MEDII (unpublished results).

Cells derived from individual EBs (EPLB cells) in the presence of MEDII were expanded to establish cell lines. Expression of both Oct4 and Fgf5 by the majority of these cell lines (Fig. 1D) indicated that they maintained a gene expression profile consistent with pluripotent cells originating from primitive ectoderm and with their morphological equivalents, EPL cells, but distinct from both the ICM and ES cells. These data indicate that MEDII + LIF, but not LIF alone, can support the maintenance and proliferation of pluripotent cells from the primitive ectoderm of EBs and that the pluripotent cell population within the EBs is responsive to factors in MEDII.

Consistent with the properties of EPL cells formed by differentiation of ES cells in adherent culture, EPLB cells could be reverted to ES cells, as assessed by morphology and gene expression, when cultured in the presence of LIF and the absence of MEDII (Fig. 1, E and F; data not shown). Also consistent with the properties of EPL cells, EPLB isolated from D3 ES cell-derived EB and reverted to ES cells contributed to chimeric mice. Of 28 blastocysts injected with reverted EPLB cells, 19 pups resulted, of which 10 were chimeric as assessed by coat color (see also [37]).

Determination of Conditions Permissive for the Culture of Primitive Ectoderm-Derived Pluripotent Cells in Culture

The ability of MEDII to support pluripotent cells derived from primitive ectoderm is consistent with the role of MEDII in establishment of primitive ectoderm in vitro [26] and the suggested biological relevance of MEDII signaling in vivo [28]. The responsiveness of embryonic primitive ectoderm to the bioactivity was investigated to establish a biological relevance for MEDII. Initial experiments suggested that MEDII could not maintain pluripotent cells from a single-cell suspension of embryonic primitive ectoderm (data not shown), so individual parameters were assessed to allow optimization of the culture conditions.

Purified Extracellular Matrix (ECM) Components Stabilize EPL Cells in Culture

Primitive ectoderm-like cells can be maintained in culture; however, previous reports indicate that these cells are inherently less stable than ES cells, showing comparatively higher levels of spontaneous differentiation in culture [26]. In these reports, EPL cells were cultured on tissue-culture plastic pretreated with 0.1% gelatin, a relatively crude preparation of ECM components extracted from porcine skin. The effect of highly purified extracellular matrix components on the stability of EPL/primitive ectoderm cells in culture was tested by seeding ES cells at low density (2.5 x 102 cells/cm2) in DMEM + LIF or DMEM + 50% MEDII onto tissue-culture plastic that had been pretreated with either gelatin, purified human plasma fibronectin, mouse laminin, mouse collagen IV, or a combination of plasma fibronectin, laminin, and collagen IV. These purified ECM components have been shown to be present in the extracellular matrix adjacent to the pluripotent cells of the early embryo [38–41]. Cells were cultured for 5 days and stained for alkaline phosphatase activity, and colonies were classified on the presence or absence of differentiated (alkaline phosphatase negative) cell types within the colony (Fig. 2).

Of the conditions tested, gelatin was the least favorable for EPL cell stability, with 77.9% of all EPL cell colonies containing at least some differentiated cells. The purified matrix components all resulted in significantly more undifferentiated EPL cell colonies when compared with gelatin, with collagen IV and the matrix mix containing collagen IV supporting the growth of 59% and 61.7% undifferentiated colonies, respectively. ES cells cultured in the absence of MEDII showed greater stability compared with EPL cells on all tested matrices, although significantly more (P < 0.002) undifferentiated colonies were observed in conditions containing collagen IV or plasma fibronectin (Fig. 2).

Biological Activity Within MEDII, but Not LIF, Maintains Pluripotent Cells Within Embryonic Primitive Ectoderm

The effect of MEDII on primitive ectoderm maintenance was assessed by placing 5.5 d.p.c. CBA/C57 F2 embryos individually into 2-ml tissue-culture wells pretreated with collagen IV, in embryo culture medium + LIF, or embryo culture medium + 50% MEDII + LIF, maintained at 37°C, 10% CO2. CBA/C57 F2 embryos were chosen as a source of embryos because they represented a strain of mice nonpermissive for pluripotent cell isolation. After 5 days in culture, explants were fixed with 4% paraformaldehyde and the presence of pluripotent Oct4-positive cells was assessed by in situ hybridization (Fig. 3). Of surviving embryos cultured in the presence of LIF alone (23.5%), none maintained a primitive ectoderm layer or contained any Oct4-positive cells after 5 days (Fig. 3A). In contrast, 42% of embryos cultured in the presence of 50% MEDII + LIF survived, and of these approaching 50% retained an embryonic organization characterized by a clearly identifiable cellular epithelium (Fig. 3B). Fifty-four percent of surviving explants cultured in the presence of 50% MEDII + LIF contained Oct4-positive cells after 5 days (Fig. 3, C and D), indicating that MEDII maintains pluripotent cells derived from the primitive ectoderm in culture. Comparative analysis of multiple experiments indicated that fresh MEDII, as opposed to MEDII that had been frozen, was more effective for maintenance in vitro of pluripotent cells derived from embryonic primitive ectoderm (data not shown).



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  		FIG. 3. MEDII promotes the survival of embryonic primitive ectoderm in vitro. A) A representative surviving 5.5 d.p.c. embryo explant cultured in DMEM + LIF. B) A representative surviving 5.5 d.p.c. embryo explant cultured in DMEM + LIF + MEDII. C) A representative surviving 5.5 d.p.c. embryo explant cultured in DMEM + LIF + MEDII stained by whole-mount in situ hybridization for the presence of Oct4 transcripts. D) A higher magnification of C showing the Oct4+ cells. A and D) Taken at x40 magnification, (B) at x20 magnification, and (C) at x10 magnification

Primitive Ectoderm from 5.5 d.p.c. Embryos, but Not Later Embryos, Responds to MEDII

The 5.5 d.p.c., 6.5 d.p.c., and 7.5 d.p.c. embryos were removed from time-mated females, dissected free of Reichart membrane, and placed individually into 2-ml tissue-culture wells pretreated with 0.1% gelatin for 30 min, in 1 ml embryo culture medium supplemented with 50% MEDII + LIF. After 3 days in culture at 37°C, 10% CO2, embryonic explants were stained for alkaline phosphatase expression, and the presence and abundance of alkaline phosphatase-positive cells was assessed. The number of embryo explants containing alkaline phosphatase-positive cells was greatest in explants of day 5.5 d.p.c. embryos (varying between 25 and 40%) and decreased significantly with increasing age of the embryo to less than 5% in explants of day 7.5 d.p.c. embryos. The 5.5 d.p.c. embryos were used as the source of primitive ectoderm in subsequent experiments.

MEDII Supports Maintenance of Pluripotent Cells from the Primitive Ectoderm of 5.5 d.p.c. Mouse Embryos

The above experiments established the primitive ectoderm of the 5.5 d.p.c. embryo as responsive to bioactive factors within MEDII and identified a requirement for improved matrix conditions in primitive ectoderm culture. Additional considerations for culture of the primitive ectoderm were suggested by observation of the behavior of embryonic explants in culture:

CBA/C57 F2 embryos were dissected from the uteri of time-mated female mice between 1000 and 1100 h on the morning of the sixth day of gestation (approximately 5.4 d.p.c.). Each embryo was dissected to remove Reichart membrane, the extraplacental cone, and the visceral endoderm (Fig. 4A), and was dissected at the embryonic/extraembryonic border to give a cup-shaped structure comprising primitive ectoderm in the absence of contaminating cell types. All tissues were removed from the embryos mechanically. Primitive ectoderm explants were placed individually into 2-ml wells plugged with agarose and cultured in 1 ml of embryo culture medium supplemented with LIF + 50% MEDII. Embryos were cultured at 37°C in a 10% CO2 humidified incubator. On the second or third day of culture, explants that comprised an epithelial sheet of cells reminiscent of primitive ectoderm with or without associated differentiation (Fig. 4, B and C), were redissected to remove any overtly differentiated cells, determined by morphological criteria, and placed back into 2-ml wells prepared as described above.



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  		FIG. 4. In vitro culture of primitive ectoderm explants. A) Day 0; 5.5 d.p.c. embryos were dissected from the uterus, the extraembryonic endoderm associated with the primitive ectoderm was removed mechanically and the embryo was dissected (indicated by the line) to give a portion containing the pluripotent primitive ectoderm cell layer. B and C) Day 2; representative embryo explants after 2 days in culture in embryo culture medium + 50% MEDII without (B) or with (C) associated differentiated cell populations (indicated by arrows). D and E) Day 7; representative explants maintained in suspension culture for 5 days in embryo culture medium + 50% MEDII were seeded onto collagen IV-treated tissue-culture plastic in embryo culture medium + 50% MEDII. Layers of prospective primitive ectoderm are indicated by arrows. F) Day 9; representative explants grown as for Day 7 were cultured for a further 2 days in embryo culture medium without MEDII, resulting in a loss of the cell layers and adoption of an ES cell-like colony morphology. G) Representative embryonic explant as for F fixed and probed by whole-mount in situ hybridization with a DIG-labeled antisense Oct4 probe. C) Photographed at x40 magnification, (A, B, E, and F) at x20 magnification, (D and G) at x10 magnification, (A through F) were visualized using phase-contrast illumination, (G) was visualized using brightfield illumination. Pictures were taken from a number of separate experiments and do not represent a single continuum. Days in culture denote the time elapsed from the day of original embryo dissection

By Day 5 of culture, surviving explants, approximately 25% of starting explants placed into culture on Day 0 and usually containing identifiable epithelial sheets of Oct4-positive cells, were removed from suspension, redissected as before to remove differentiated or contaminating cells, and plated onto tissue-culture plastic pretreated with collagen IV in embryo culture medium + 50% MEDII. Explants were maintained in embryo culture medium + 50% MEDII for a further 2 days, by which time they usually comprised a convoluted epithelial sheet (Fig. 4, D and E).

EPL cells formed from culture of ES cells in medium containing MEDII [26, 27] or isolated from the primitive ectoderm of EBs (this work) can be reverted to ES cells by withdrawal of MEDII but supplementation of the medium with LIF. Reversion to an ES cell state is accompanied by alterations in gene expression and differentiation potential, suggesting that the pluripotent cells have adopted the more stable ES cell state. Withdrawal of MEDII from the explants in the continued presence of LIF resulted in flattening of the sheets and adoption of an ES cell colony-like appearance (Fig. 4F), suggestive of cell reversion. Analysis of these cells by in situ hybridization showed them to be pluripotent as assessed by Oct4 (Fig. 4G) and alkaline phosphatase expression.

On Day 9 or 10 of culture, the explants were disaggregated into single cells or small clumps of cells by treatment with 0.5 mM EGTA in PBS for 5 min before a brief trypsinization, then seeded into 2-ml wells pretreated with collagen IV or preseeded with inactivated STO fibroblasts. When proliferated, the cells derived by this method were equivalent in appearance to ES cells (Fig. 5, A and B), expressed the pluripotent cell markers Oct4 and alkaline phosphatase (Fig. 5, C through F), retained the ability to differentiate spontaneously as demonstrated by the appearance of overtly differentiated cells in cultures (Fig. 5, G and H), and could be passaged and proliferated in culture for at least 4 wk. ES-like cells were isolated from approximately 10% of embryos, an efficiency comparable with the isolation of ES cells from the ICM of 129 mouse blastocyst-stage embryos by standard techniques [6, 46].



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  		FIG. 5. MEDII supports the maintenance and proliferation of pluripotent cells derived from embryonic primitive ectoderm. A and B) Explants grown to the stage represented by Figure 4F were dissociated to small clumps and seeded onto either inactivated STO feeder layers (A) or collagen IV (B) and allowed to proliferate for 2 (Day 11; A) or 3 (Day 12; B) days in embryo culture medium. Cells were termed primitive ectoderm-derived cells. C and D) Primitive ectoderm-derived cells on collagen IV (C) or an inactivated STO feeder layer (D) were fixed and stained for the expression of alkaline phosphatase. Differentiated cells, negative for alkaline phosphatase activity, are denoted by the arrow. C) Primitive ectoderm-derived cells after three passages (Day 26). D) Primitive ectoderm-derived cells after three passages (Day 21). E and F) Primitive ectoderm-derived cells were fixed and probed by wholemount in situ hybridization with a DIG-labeled antisense Oct4 probe. E) Primitive ectoderm-derived cells after four passages (Day 27) in culture on inactivated STO feeders. F) Primitive ectoderm-derived cells after one passage (Day 14) in culture on inactivated STO feeders. G and H) Differentiated cell colonies arising during the passage of primitive ectoderm-derived cells. Both examples were taken after passage three (Day 15) in culture on collagen IV. A, B, and F through H) were taken at x40 magnification, (C through E) were taken at x20 magnification. All pictures were taken from a number of separate experiments. Days in culture denote the time elapsed from day of original embryo dissection


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
MEDII Contains Signals That Support the Maintenance of Pluripotent Primitive Ectoderm Cells

Pluripotent cells, or epiblast, of the embryo can be divided into two populations, ICM and primitive ectoderm, on the basis of morphology, gene expression, differentiation potential, and cytokine/growth factor responsiveness [1–12]. A detailed examination of gene expression within the epiblast [12] suggests that ICM (Oct4+, Rex1+, CRTR1+, Psc1+, PRCE+, Fgf5-) persists to 4.75 d.p.c. before undergoing a dynamic differentiation program that culminates in the establishment of primitive ectoderm (Oct4+, Rex1-, CRTR-, Psc1-, PRCE-, Fgf5+) between 5.25 and 5.5 d.p.c. This window of time, 4.75 to 5.25 d.p.c., is coincident with epiblast cavitation and morphological rearrangement of the pluripotent cells into a pseudostratified epithelial sheet. A population of cells analogous to primitive ectoderm is formed as ES cells differentiate in EBs, with the alterations of gene expression characteristic of primitive ectoderm formation (Oct4+, Rex1-, Fgf5+) occurring approximately 3 days after EB formation. LIF has been established as sufficient to support the isolation of ES cells from the blastocyst [17, 18]. Consistent with the interpretation of gene expression, the pluripotent cells within EBs could not be isolated in medium supplemented with LIF alone after Day 2, suggesting that they were no longer equivalent to ES cells/ICM.

Subtle differences were observed in the developmental progression of pluripotent cells in D3 and E14 EBs, evidenced by the persistence of LIF responsive cells in E14 EBs but not D3 EBs. The LIF responsive cell population relative to the MEDII responsive cells in E14 EBs declined with time but could still be detected 8 days after EB formation. The presence of this cell population suggests that the rate and extent of differentiation differs between the two cell lines and that the potential exists in some ES cell lines for the maintenance of pluripotent cells during differentiation. Maintenance of ES cells during differentiation has been noted previously [47] with the emergence of stem cell nests after prolonged culture of ES cells in the absence of LIF, a phenomenon attributed to the production of gp130 agonists by differentiated cells within the culture.

The molecular signals that regulate the formation of primitive ectoderm within EBs and in vivo have not been well characterized. MEDII was identified by its ability to induce and maintain primitive ectoderm cells from ES cells in vitro. Here, we strengthen the identification of this biological activity and expand the biological relevance of MEDII by demonstrating that MEDII was able to support the maintenance and proliferation of primitive ectoderm-derived pluripotent cells from post-Day 3 EBs and 5.5 d.p.c. embryos. This activity was specific to MEDII and could not be replaced by LIF alone or the presence of a feeder layer. The ability of MEDII to promote maintenance of embryonic primitive ectoderm in culture validates the role of factors within MEDII in primitive ectoderm formation and maintenance, deduced from in vitro experiments [26] and the sites of embryonic expression of active MEDII components [28].

The culture of blastocysts from mouse and rat is accompanied by the rapid loss of Oct4 expression [48], with the majority (96% of CBA blastocysts) of ICM outgrowths devoid of Oct4-expressing cells after 6 days in culture. This rapid loss of Oct4 was also observed in cultured 129 blastocysts, a strain of mouse permissive for the establishment of ES cell lines in culture, although the effect was less pronounced and small pockets of Oct4+ cells persisted in 44% of outgrowths. This is consistent with the rapid loss of Oct4+ cells in 5.5 d.p.c. CBA/C57 F2 embryos cultured in LIF and contrasts with the maintenance of Oct4+ cells in 5.5 d.p.c. CBA/C57 F2 embryos cultured in MEDII supplemented medium. Further, not only were Oct4-expressing cells maintained in epiblast derived from a nonpermissive mouse strain, but Oct4 expression was not restricted to small pockets of cells but observed broadly in both embryo outgrowths and explants. These data suggest that factors within MEDII act to maintain Oct4 expression in pluripotent cells. The potential application of factors within MEDII to the maintenance of pluripotence and phenotypic stability of nonmouse pluripotent cell lines has yet to be explored.

Primitive Ectoderm-Derived Cells Maintain Characteristics of Their Parental Population in Culture

Primitive ectoderm-derived cells from EB, termed EPLB cells, were maintained as a pluripotent cell population with a morphology, gene expression, and cytokine responsiveness equivalent to EPL cells and consistent with derivation from primitive ectoderm. Similarly, cell populations derived from the embryonic ectoderm and cultured in MEDII maintained characteristics of their parent population, including morphology and gene expression. This is in contrast with ES cells that retain a morphology and gene expression profile consistent with derivation from the ICM [26].

Establishment of pluripotent cell lines from the embryo has been achieved from ICM of blastocysts up to the 5th day of gestation (4.0–5.0 d.p.c.; [6]) by culture in LIF. The ability of MEDII, but not LIF, to support the maintenance of pluripotent cells from the 6th day of gestation (5.0–6.0 d.p.c.) suggests that the pluripotent cells of the embryo undergo an alteration in cytokine responsiveness between 5.0 d.p.c. and 6.0 d.p.c., correlating with the establishment of primitive ectoderm. The efficacy of MEDII was most pronounced with culture of early postimplantation embryos, between 5.25 and 5.5 d.p.c., after which epiblast could not be maintained efficiently in culture in MEDII. This window of opportunity defines a novel cell population characterized by an alternative responsiveness of the cells to extracellular signals, particularly to the biological activity in MEDII.

Reversion of Primitive Ectoderm-Derived Pluripotent Cells

EPLB, like EPL cells, were capable of reversion to an ES cell phenotype when cultured in medium supplemented with LIF alone. This is in contrast with the inability of LIF to support maintenance of primitive ectoderm derived directly from EBs. Similarly, after time in culture, embryonic primitive ectoderm could be reverted to an ES/ICM cell-like phenotype with removal of MEDII and maintenance of LIF signaling, as evidenced by loss of the stratified epithelial morphology and formation of three-dimensional domed colony morphology. The reversion of these cells to an ES cell-like state is consistent with the hypothesis that ES cells represent a base, or default, state for pluripotent cells in culture [25].

Additionally, these data suggest that, with time in culture, primitive ectoderm-derived cells acquire the ability to respond to LIF. The possibility that culture conditions can alter the phenotype of stem cells has been proposed for some somatic stem cells that have been shown to acquire the ability to be capable of reversion to a more primitive phenotype, with acquisition of a less restricted differentiation potential in culture [49, 50]. Thus, the alterations in cytokine responsiveness observed in primitive ectoderm-derived cells in culture may be representative of a more general phenomenon.

LIF Is Not Sufficient for Prolonged In Vitro Culture of Pluripotent Cells Derived from Embryonic Primitive Ectoderm

Pluripotent cell cultures derived from primitive ectoderm in the presence of MEDII could be maintained for several weeks in culture following reversion in LIF, with continued expression of both Oct4 and alkaline phosphatase activity. However, unlike the establishment of EPLB lines from in vitro-derived primitive ectoderm, routine culture beyond 4 to 5 wk of the embryo-derived cells was not achieved. After time, embryonic primitive ectoderm-derived cells down regulated Oct4 expression and appeared differentiated, suggesting that molecules other than LIF are required for optimal growth of the population. A requirement for alternative molecules for the maintenance of pluripotent cells in culture has been suggested by the ability of LIF-/-, LIFR-/- and gp130-- mice to maintain and proliferate pluripotent cell populations during early embryogenesis [51–53]. Similarly, nonmouse pluripotent cell lines do not appear to require LIF in culture [54, 55].

A Requirement for Visceral Endoderm Signaling in Primitive Ectoderm Maintenance

Several lines of experimental evidence have suggested a requirement for extraembryonic endoderm signaling in the formation and maintenance of primitive ectoderm during embryogenesis and EB development. Ablation of genes expressed within the extraembryonic endoderm lineage perturbs both the maintenance and differentiation of primitive ectoderm during embryogenesis (reviewed in [56]). Furthermore, the primitive ectoderm defect in Hnf4-/- embryos can be corrected by recombination with Hnf4+/+ visceral endoderm in tetraploid chimeras, implicating signals from this tissue in pluripotent cell development [57]. Studies on EB differentiation implicate signals from the extraembryonic endoderm in formation of an amniotic cavity-like structure [42] and differentiation of ES cells to primitive ectoderm (reviewed in [28]).

Liver cells and cell lines, including HepG2 cells, share similarities in gene expression with extraembryonic visceral endoderm [58–60]. The established role of MEDII in the formation of EPL cells from ES cells and the ability of MEDII to influence the differentiation of EPL cells has been interpreted as evidence suggesting that MEDII provides a source of visceral endoderm-like signaling [26, 27, 45]. The role of MEDII in the maintenance of embryonic and EB primitive ectoderm is therefore likely to be attributable to the presence of signals analogous to visceral endoderm, and provides a model system for the molecular characterization of primitive ectoderm formation and maintenance.

Maintenance of pluripotent cells from embryonic primitive ectoderm in culture was compromised by the presence of visceral endoderm. Paradoxically, removal of visceral endoderm, but maintenance of MEDII signaling, improved the retention of alkaline phosphatase-expressing cells in the explants and reduced overt morphological indications of differentiation. Removal of the extraembryonic endoderm and differentiated cell populations, either mechanically or by use of selection agents, has also been shown to improve the efficiency of ES cell isolation from the blastocyst [6, 61, 62]. Apart from a role in formation and maintenance of primitive ectoderm, signaling from the visceral endoderm in pluripotent cell differentiation and cell fate selection has been well documented [42–45], and it is this signaling that is proposed to be deleterious during pluripotent cell isolation. The limited differentiation observed in pluripotent cells cultured in adherent culture in the presence of MEDII suggests that, although MEDII may functionally replace visceral endoderm signaling in the formation and maintenance of the primitive ectoderm and in subsequent differentiation to the neural lineage [63], other signaling activities involved in a more diverse range of developmental programs, such as differentiation and patterning, are not represented.


   ACKNOWLEDGMENTS
 
We would like to thank Joe Wrin for assistance with blastocyst injections, Drs. Austin Smith and Jennifer Nichols and the British Council for training in dissection of early mouse embryos, and Dr. Julie Lake for useful discussion.


   FOOTNOTES
 
1 This work was supported by the Australian Research Council and Bresagen Pty. Ltd. Back

3 Current address: School of Agriculture and Wine, Waite Campus, University of Adelaide, Urrbrae, South Australia 5034 Australia Back

4 Current address: Institute Suisse de Recherche Expérimentale sur le Cancer (ISREC), Chemin des Boveresses 155, CH-1066 Epalinges, Switzerland Back

2 Correspondence: FAX: 618 8303 4348; peter.rathjen{at}adelaide.edu.au Back

Received: 20 March 2003.

First decision: 6 April 2003.

Accepted: 4 August 2003.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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