Ultrastructure of the embryonic stem cells of the 8-day pig blastocyst before and after in vitro manipulation: Development of junctional apparatus and the lethal effects of PBS mediated cell–cell dissociation
The source of mouse, monkey, and human embryonic stem (ES) cell lines has most often been the culture of epiblast cells dissociated from primary blastocyst or inner cell mass (ICM) cultures (Brooks and Gardner, 1997; Robertson, 1987; Thompson and Marshall, 1998; Thompson et al., 1998). Because ultrastructural features of blastocysts vary between species, the epiblast cells of various species may differ in how or to what extent they are connected to one another, thus making their dissociation from each other more or less difficult (Blerkom and Motta, 1979; Enders, 1971). It is possible that differences in the survival of epiblast cells after dissociation may contribute to the success or failure of establishing ES cell lines from different species and from different strains of mice (Kawase et al., 1994; Stewart, 1991). For example, only recently have strains of mice other than 129/Sv yielded ES cell cultures and this for the most part at lower efficiencies (Brooks and Gardner, 1997; Kawase et al., 1994). Monkey and human ES cell lines do not survive single cell dissociation, i.e., clumps of cells with intact intercellular adhesions must be preserved to successfully passage the cells (Gearhart, 1998; Shamblott al., 1998; Thompson and Marshall, 1998; Thompson et al., 1998).
The few previously published electron microscopy studies of the hatched pig blastocyst did not detail the development of the connective and cytoskeletal elements of the epiblast cells (Barends et al., 1989; Giesert et al., 1982; Hall et al., 1965; Stroband et al., 1984). There is no information on the effects of immunodissection and primary culture on epiblast cellular and structural components in any species. Therefore, we undertook an electron microscopic examination of the ontogeny of the connective elements within and between pig epiblast cells shortly after the hatching of the blastocyst from the zona pellucida. To try to better understand what is happening to the epiblast cells during attempts to establish pig ES cells, we examined the epiblast cells immediately after immunodissection of the inner cell mass (ICM), after 2 days of ICM culture, after 1–2 days of primary culture of the physically dissected epiblasts, and after exposure of the epiblast cultures to cell–cell dissociating conditions.
MATERIALS AND METHODS
Blastocyst Collection, Immunodissection, and Cell Culture
Hatched pig blastocysts were collected by flushing uterine tracks 8 days after sequential natural and artificial insemination as previously described (Talbot et al., 1993a). Immunodissection of blastocysts was performed to isolate ICMs as previously described (Talbot et al., 1993a). Physical dissection to isolate epiblasts and their culture was performed as previously reported (Talbot et al., 1993a).
Coculture of isolated ICMs and epiblasts on STO feeder cells was done as previously described (Talbot et al., 1993a) using tissue culture plastic ware from Nunc (Denmark) or Falcon, Becton/Dickson (Lincoln Park, NJ). Fetal bovine serum (FBS) was obtained from Gibco (Gaithersburg, MD) or from HyClone (Logan, UT). Other cell culture reagents including, Dulbecco's phosphate buffered saline (PBS) without Ca++ and Mg++, media, trypsin-EDTA (0.025% trypsin; 0.43 mM EDTA), antibiotics, nonessential amino acids, and L-glutamine were from Gibco. STO feeder cells (CRL 1503, American Type Culture Collection, Rockville, MD) were prepared as described by Robertson (1987). ICMs and epiblasts were cultured in 10% DMEM-199, a 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Medium 199 with added nucleosides, 2-mercaptoethanol, and 10% FBS as previously described (Talbot and Paape, 1996).
Transmission electron microscopy (TEM) sample preparation and photomicroscopy was done with the assistance of JFE Enterprises (Brookeville, MD) as previously described (Talbot et al., 1998, 2000). Ultrastructural analysis was performed on the following samples in the study. Five large spherical (approximately 1,000–2,000 μm in diameter) and two small spherical (less than 400 μm in diameter) hatched 8-day pig blastocysts were examined. Two pig ICMs were examined immediately after immunodissection from large blastocysts. Two pig ICMs were examined after 48 hr coculture on STO feeder cells. Two epiblasts were examined after physical dissection and 24–48 hr of primary coculture on STO feeder cells. Two epiblasts in primary culture for 24–48 hr were fixed for TEM analysis after being exposed to Ca++-Mg++-free PBS at 35–37°C for 5–10 min. Scanning electron microscopy (SEM) was performed as previously described (Wergin, 1979) on two 48–72 hr primary cocultured epiblasts, one of which had been exposed to Ca++-Mg++-free PBS at 35–37°C for 5–10 min.
Epiblast Fluorescent Phalloidin Labeling and Analysis
Pig epiblasts, cultured without STO feeder cells for 24–36 hr, were fixed in 4% paraformaldehyde, and the actin cytoskeleton was stained with Texas Red-x phalloidin. In brief, primary cultures of epiblasts were permeabilised with 1% Triton X-100, labeled with 2.5 U/ml (82.5 nM) Texas Red-x phalloidin (Molecular Probes, Eugene, OR) then mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA). The actin cytoskeleton was imaged using a Zeiss LSM 410 laser scanning confocal microscope (Carl Zeiss Inc., Thornwood, NY). Texas Red-x was excited with the 568 nm line of a mixed gas Argon/Krypton laser and the emitted light was passed through a 590 nm long-pass filter. Images were obtained using a C-apo 40× water immersion objective (1.2 NA) at a zoom factor of 1.5. Individual optical sections were digitally recombined into a single composite image using LSM software (Carl Zeiss Inc., Thornwood, NY).
Ultrastructure of Epiblasts of Intact 8-Day Pig Blastocysts
Hatched 8-day pig blastocysts varied in size, within and between animals, from less than 400 μm in diameter (“small”) to greater than 1 mm in diameter (“large”). The size differences probably reflected variation in developmental age, i.e., small blastocysts, composed of fewer cells, were earlier in development than larger blastocyst composed of more cells. This assumption was also made by Stroband et al. (1984), who also experienced wide variation in blastocyst size between sows despite obtaining the blastocysts at similar chronological ages post-fertilization. Small and large hatched blastocysts consisted of three cell types, the outermost trophectoderm cells, the epiblast cells that comprised the bulk of the ICM and the endoderm cells that lined the under side of the epiblast mass and the trophectoderm cells (Fig. 1).
The epiblast of the 8-day pig blastocyst consisted of a well defined group of approximately cuboidal cells with no obvious polarized character. For both larger and smaller blastocysts the epiblast cell's cytoplasm had abundant free ribosomes/polysomes, occasional and usually short pieces of rough endoplasmic reticulum (RER), and numerous mitochondria. The cristae of the mitochondria appeared to be both tubular and lamellar in structure but were nearly always enlarged, angular, and translucent, thus giving the intracristal spaces a swollen appearance (Figs. 1 and 2B). The translucence of the intracristal spaces was made more prominent by its juxtaposition with the mitochondrial matrix that was often more electron-dense than the surrounding cytoplasm and particularly regular in its granularity. A few pinocytotic vesicles and associated caveolae were observed at all areas of the peripheral cytoplasm and cell membrane. Epiblast cells from “small” 8-day blastocysts were characterized by having numerous and often very large lipid droplets (Fig. 1A), whereas epiblast cells from the larger 8-day blastocysts were devoid of large and very large lipid droplets (Fig. 1B).
Epiblast cell unions differed between small and large blastocysts. Few and incompletely formed desmosomes were observed and no tight junctions (TJ) were present between the epiblast cells of the small blastocysts (Fig. 2A). Desmosomal connections between trophectoderm and epiblast, however, were apparent. In contrast to the “small” 8-day blastocysts, the “large” 8-day blastocyst's epiblast cells were joined by well developed desmosomes and what appeared to be extensive TJ complexes (Fig. 2B). Desmosomal connections were again present between trophectoderm cells and epiblasts in the larger 8-day blastocysts. The desmosomes had associated microfilaments and occasional microfilaments were observed running through the cytoplasm, but in no particular order. Microtubules were often prominent in the epiblast cells and may have been conspicuous as a result of the epiblast cell's cytoplasm having few other cytoplasmic features.
Ultrastructure of Immunodissected and 48-Hr Cocultured Pig ICMs
Pig ICMs from large 8-day blastocysts were fixed immediately after immunosurgery for TEM analysis. The endoderm cells that were adjacent (underneath) to the trophectoderm cells had markedly disrupted cell membranes and cytoplasm, whereas the epiblast cells and their attendant endoderm cells appeared to be undamaged (Fig. 3A). The RER and the mitochondrial cristae were swollen in the disrupted endoderm cells. Such ICMs have been judged as undamaged by light microscopic examination and have been routinely cultured for subsequent epiblast isolation (Talbot et al., 1993a). Much of the cell damage is apparently repaired or the undamaged endoderm cells undergo vigorous cell division and growth on the STO feeder cells because a radially expanding monolayer of endoderm cells extending out from the epiblast is observed by 48 hr in culture (Talbot et al., 1993a).
TEM analysis of the 48 hr cultured ICMs in the vicinity of the epiblast mass showed that at least some of the endoderm cells were undergoing necrosis and a few apoptotic bodies were present in or around the epiblast cells (Fig. 3B). The cellular adhesion structures and cytoskeletal elements of the epiblast cells of the 48 hr ICM cultures were similar to those found in the intact “large” pig blastocyst except that microfilament bundles now appeared to be associated with some of the epiblast nuclei. The cellular organelles also looked similar except that the mitochondria of the cells, particularly the epiblast cells, were indistinct, i.e., had a similar electron density to that of the cytoplasm, had poorly defined cristae and had matrixes that were variable in density.
Ultrastructure of Cultured Pig Epiblasts
Pig epiblast cells cultured for 36 hr formed an epithelial sheet of approximately cuboidal cells (Figs. 4A and 8A). The cells were held together at their apical lateral surfaces by apparent TJ and adherens type junctions, with well organized bands of microfilaments running between them and parallel to the apical cell membranes (Fig. 4A,B). These were probably actin filaments because phalloidin staining of 24–48 hr epiblasts cultured without STO feeder cells demonstrated positive staining at all cell unions (Fig. 5). Desmosomes were also observed joining the lateral cell surfaces, and the apical microfilament bands were clearly connected to them when the desmosomes occurred near the cell's apex (Fig. 4B). Some of the pig epiblast cells also appeared to have points of union where a gross confluence of microfilaments and microtubules existed, possibly a midbody between daughter cells (Fig. 4A and 6B). The epiblast nuclei were now distinctly surrounded by microfilaments (Fig. 6A). Microvilli were present, although sparsely, on the apical surface indicating apical-basolateral polarization (Figs. 4A, 6A, and 8A). The epiblast cells also changed in comparison to those in vivo and in cultured ICM in appearing to have more and larger Golgi complexes (Fig. 7A), and most mitochondria now had better defined tubular/lamellar cristae with relatively undilated intracristal spaces and matrix regions of variable density (Fig. 7A). Finally, a solitary cilium was observed projecting from the apical surface of one pig epiblast cell (Fig. 7B).
Ultrastructure of Pig Epiblast Cells Exposed to a 5–10 Min Treatment With Ca++-Mg++-Free PBS
Pig epiblasts cocultured on STO feeder cells were exposed to Ca++-Mg++-free PBS for 5–10 min at 35–37°C to dissociate the cells from one another. The epiblast cells experienced various degrees of cytoplasmic blebbing after the PBS treatment, and in most cases severe damage to the cells was evident (Figs. 8B and 9). In some cells the blebbing appeared to be immediately catastrophic in that nuclei were observed to be badly distorted (Fig. 10A). Many cells, particularly those that were nearly free from their neighbors displayed a common characteristic of having their cytoplasmic organelles concentrated together and tucked in close to their nuclei (Fig. 10B). Unusually large and numerous bundles of microfilaments were also frequently observed within the concentrated mass of organelles (Fig. 10B). Mitochondria were usually normal in appearance, but in a few cells they appeared swollen. Most cells had also developed numerous round empty spaces that did not appear to be membrane bound. Junctional complexes at the apical portion of the cells, i.e., the zonula adherens, remained intact in most epiblast cells during the gross blebbing that ensued in the 5–10 min PBS treatment (Fig. 10A). Lateral desmosomes in contrast had come apart, or were coming apart, as a result of the treatment.
Several studies of the microanatomy of preimplantation pig embryos have been published although none have focused on the epiblast cells of the hatched 8-day blastocyst or its cultured ICM and epiblast. This study documented the formation of desmosomes and tight junctional complexes in the pig epiblast cells at the transition from small hatched blastocyst to large spherical hatched blastocyst (Figs. 1,2). Thus, the time at which desmosomes and TJ developed in the pig epiblast cells was approximately 48 hr after blastocyst hatching, and was coincident with blastocyst enlargement, but occurred before elongation and before loss of the overlaying trophectoderm cells, Rauber's layer (Anderson, 1978; Hunter, 1973; Stroband et al., 1984). Well organized microfilament bundles running between cell unions and parallel to the apical cytoplasmic membranes appeared in the epiblast cells shortly after their in vitro culture as a pure tissue (Figs. 4B and 7A). Coincident with this, microfilament bundles that were closely associated with the nucleus became a prominent feature of the epiblast cells. Perinuclear microfilament arrays were noted to be unusual to pig, armadillo, and Mexican funnel eared bat blastocysts, although their presence in pig epiblast cells was not previously shown (Enders,1971; Hall et al., 1965). As previously reported by others, the pig epiblast cells also lost their gross lipid (yolk) vacuoles and became less rounded and more columnar in shape as development of the blastocyst proceeded (Barends et al., 1989; Stroband et al., 1984). The results presented here, however, demonstrate that these changes, particularly the loss of the gross lipid vacuoles, occurred very quickly, probably within 48 hr after hatching, and ahead of embryonic disc formation and loss of Rauber's layer (Figs. 1 and 2). Finally, the TEM analysis made immediately after ICM isolation showed that care is warranted in immunodissection (i.e., careful titration of anti-serum and complement, and carefully timed lysis), especially for smaller blastocysts where the endoderm layer underneath the trophectoderm is not necessarily fully formed. ICMs that are apparently intact by light microscopic assessment could in fact have damaged epiblast cells.
The apical junctional complexes with microfilament bundles running between them and parallel with the apical surface of the pig epiblast cells had the typical cortical organization of adhesion belts found in certain epithelium, such as intestinal epithelium or early embryonic neuroepithelium (Alberts et al., 1994). Microfilaments forming adhesion belts are composed of actin (Alberts et al., 1994) and this is presumably the case here as evidenced by positive phalloidin staining (Fig. 5), and by previous immunocytological studies that have demonstrated cortical arrangements of actin filaments in the cells of the pig blastocyst (Albertini et al., 1987; Reima et al., 1993; Wang et al., 1999). Similar cortical microfilament arrangements were observed in pig trophectoderm and endoderm cells in a much earlier EM study of preimplantation pig blastocysts (Hall et al., 1965). Our observations, therefore, extend this characteristic to pig epiblast cells, at least in those exposed to an in vitro culture environment. Other EM studies that examined pig epiblast cells out to the embryonic disc stage did not mention apical membrane microfilament arrays in the cells, so it is possible that this development is a response unique to in vitro culture, or a response that occurs later in the in vivo differentiation of the pig epiblast, i.e., primitive streak formation (Barends et al., 1989; Giesert et al., 1982; Stroband et al., 1984).
The development of the adhesion belts might indicate cell differentiation in response to culture, despite the fact that similarly cultured pig epiblast cells are highly positive for alkaline phosphatase expression, a marker of undifferentiated ES cells (Talbot et al., 1993b). The discovery of a cilium in one of the epiblast cells lends credence to this idea because this is a feature typical of notochordal cells, a very early differentiated cell type deriving from the nodal region of the primitive streak (Bellomo et al., 1996; Sulik et al., 1994). Similarly, cortical microfilament bundles running between apical junctional complexes are present in the epiblast cells of the chicken primitive-streak-stage blastoderm, a stage where gastrulation is rapidly ensuing (Vakaet and Vanroelen, 1982). Also, a study of differentiation markers showed an undifferentiated state of freshly isolated pig epiblasts taken from 7- and 11-day embryos, and then the differentiation of the epiblast cells within only a few days of culture (Wianny et al., 1997). Thus, it is possible that the appearance of microfilament bundles, cortical and perinuclear, within the cultured pig epiblast cells are differentiation events. In regard to establishing pig ES cell lines, such events may indicate an irreversible loss of epiblast cell totipotency, i.e., it may be past the time that ES cell lines can be established, or it may mean that, for in vitro manipulations, it is now difficult to separate the cells from each other without harming them.
Our previous attempts to establish porcine ES cell lines from primary cocultures of isolated epiblasts indicated that a critical problem might be cell lysis and death after dissociation of the cells from one another (unpublished observations). The electron microscopic results presented demonstrate for the first time the extensive and lethal cell blebbing and organelle derangement that occurs within minutes of exposure of primary cultured pig epiblast cells to Ca++ -Mg++-free conditions, i.e., Ca++- and Mg++-free PBS. Similarly, treatment of primary epiblast cultures for 5–10 min in Ca++-free medium or trypsin-EDTA solution resulted in lysis of individualized cells with complete disintegration of the cells within 30–60 min., as judged by light microscopy and subsequent assay of attachment and growth (unpublished observations). In contrast to the destructive effects shown here for pig epiblast cells, mouse epiblast cells appear to be more tolerant in that Brooks and Gardner (1997) reported high efficiency establishment of ES cells lines from various strains of mice after using an initial dissociation protocol that included a 30–40 min exposure to Ca++-free medium containing the Ca++ chelator ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA).
The reasons for the inability to separate the cultured pig epiblast cells from each other without rupture are speculative at this point. It may result from the development in the cells of adhesion belts and the extensive arrays of microfilaments that surround their nuclei as shown in the data presented here. This speculation is based partly on previous findings that showed that the epiblast cells of cultured mouse blastocysts did not have well defined bands of microfilaments joining their junctional complexes, nor did they have a visible network of microfilaments surrounding their nuclei (Solter et al., 1974). Similarly, undifferentiated mouse and human embryonal carcinoma (EC) cells and mouse ES cells do not have well developed junctional complexes (Andrews et al., 1987; Hogan et al., 1994; Saitou et al., 1998; Trevor and Steben, 1992). The actin-myosin filaments of the pig epiblast cell's adhesion belts may place continuous contractile tension on the cell's apical portion (Hagmann et al., 1999; Langanger et al, 1986; Odell et al., 1981). Purse string-like contraction of the epiblast cell adhesion belts is also a possibility in response to the removal of external calcium because this has been shown to occur in Madin-Darby canine kidney (MDCK) epithelial monolayers (Castillo et al., 1998; Lagunes et al., 1999; Ma et al., 2000). Such tension and contraction could exacerbate cell rupture at the epiblast cell's basolateral desmosomes as external Ca++ is withdrawn and Ca++-dependent cadherins, i.e., desmocollins and desmoglein, denature and dissociate (Collins and Fleming, 1995). Also, because the desmosome associated cytokeratin intermediate filaments are not well developed in undifferentiated epiblast cells (Lehtonen, 1987; Schwarz et al., 1995), the cells cytoskeletal network might be particularly fragile, and, therefore, sensitive to desmosome dissociation (Fuchs and Cleveland, 1998).
A number of other interacting cytoskeletal elements may be important. For example, because cell blebbing appears to result from a disassociation of the plasma membrane from the actin cytoskeleton (Torgerson and McNiven, 1998; Hagmann et al., 1999; Dai and Sheetz, 1999) the expression and function of cross-linker proteins such as plectin may be key to cell survival during cell–cell dissociation. Plectin, occurring in various forms as a result of differential transcript splicing, has been shown to interconnect not only with cytoskeletal elements (i.e., intermediate filaments, microtubules, actin and myosin), it also associates with cell organelles such as the nucleus and mitochondria (Wiche, 1998; Djabali, 1999; Guttman et al., 1999; Reipert et al., 1999). In any case, preliminary work indicates the importance of maintaining normal cytoskeletal architecture in that, even after extensive blebbing during cell–cell dissociation of cultured pig epiblast cells, a rapid reestablishment of substrate attachments by the cells fosters dramatic increases in their survival (unpublished observations).
If epiblast cells were dissociated before the development of adherens junctions, e.g., from the small 8-day blastocyst where epiblast TJ and desmosomes have not formed (Figs. 1 and 2), they might survive dissociation better and be less differentiated in phenotype so that they would be more likely to adopt a behavior in culture analogous to that of mouse, or monkey and human ES cell cultures. Our attempts to culture intact epiblasts from small 8-day pig blastocysts, however, have always resulted in no discernible growth and survival (unpublished observation). In contrast to our procedure of isolation and culture of pure epiblast tissue, a recent report using whole blastocyst culture found the success rate for the establishment of pig ES-like cell cultures was decidedly better (12 [21%] vs. none) from “early” hatched blastocysts than from “late” hatched blastocysts (Chen et al., 1999). The “early” hatched pig blastocysts corresponded in description to the small 8-day blastocysts examined here. Three of the resulting cultures were composed of alkaline phosphatase positive cells, a marker of the undifferentiated state, and one of these cultures displayed pluripotency in a chimeric offspring (Chen et al., 1999). As with rodent and primate ES cell culture establishment, Chen and coworkers cultured the entire blastocyst, for some time (days), before trypsin dissociation of the blastocyst outgrowth into small clumps of cells. Whether adherens type junctions with associated microfilament bundles developed in the epiblast cells under these conditions is unknown, and, thus, the possible relevance of the lack of such elements to the establishment of their pig ES-like cell cultures is also unknown. One aspect of our future work will be to compare the ultrastructure of pig epiblast cells from different stages of whole blastocyst culture to see if they remain relatively unconnected and without microfilament bundles as was shown here for the small 8-day blastocyst. If so, they would resemble cultures of undifferentiated mouse and human embryonal carcinoma (EC) cells (Andrews et al., 1987; Hogan et al., 1994; Trevor and Steben, 1992) and presumably mouse and primate ES cells (Saitou et al., 1998; Thomson and Marshall, 1998).
We wish to thank Drs. Sukanta K. Dutta, Vernon G. Pursel, and John M. Talbot for reading the manuscript and providing helpful scientific and editorial suggestions in its final preparation. We also thank Ms. Leah Schulman for pig blastocyst collection and Mr. Charles Murphy for SEM preparation and imaging.