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Influence of feeder layer type on the efficiency of isolation of porcine embryo-derived cell lines
THERIOGENOLOGY INFLUENCE OF FEEDER LAYER TYPE ON THE EFFICIENCY OF ISOLATION OF PORCINE EMBRYO-DERIVED CELL LINES
J.A. Piedrahitalpa , G.B. Andersonland R.H. BonDurant' 'Department of Animal Science and *Department of Reproduction, School of Veterinary Medicine, University of California, Davis, USA
Received for publication: May 83, 1990 Accepted: S~ptembw 14, 1990
ABSTRACT Experiments were conducted to determine the effects of feeder layers composed of different cell types on the efficiency of isolation and the behavior of porcine embryo-derived cell lines. Inner cell masses (ICM) isolated from 7- to I-d-old embryos were plated on feeder layers composed of Buffalo rat liver cells (BRL), a continuous cell line of murine embryonic fibroblasts (STO), ST0 combined with BRL at a 9:l and I:1 ratio, ST0 with BRL-conditioned medium (ST0 + CM), porcine embryonic fibroblasts (PEF), PEF combined with BRL at a 9:l and 1:l ratio, porcine uterine epithelial cells (PUE), murine embryonic fibroblasts (MEF), or an epithelial-like porcine embryo-derived cell line (PH3A). It was found that embryo-derived cell lines could be isolated only from the ST0 and the ST0 with BRL-conditioned medium treatments. The isolated cell lines were of epithelial-like and embryonic stem cell-like (ES-like) morphology. The feeders tested had an effect on the behavior of plated ICM. Some feeders, represented by PUE, BRL, STO:BRL (l:l), PEF:BRL (l:l), and PH3A, did not promote attachment of the ICM to the feeder layer; others, represented by ST0 and MEF, allowed attachment, differentiation and proliferation. On PEF feeders the ICM spread onto the feeder layer after attachment without apparent signs of proliferation or differentiation. None of the feeders tested increased the efficiency of isolation or the growth characteristics of embryo-derived (both ES-like and epitheliallike) cell lines over that of ST0 feeders. Key words:
embryonic stem cells, feeder cells, porcine
Acknowledgements This research was supported by a USDA Competitive Research Grant (GBA and RHB) and a Jastro-Shields Graduate Research Award (JAP). The authors thank Akiko Stevens, Kent Parker and Dan Sehnert for technical assistance and Kay Allen and Susan Donahue for help with manuscript preparation. aCurrent address: Department of Pathology, School of University of North Carolina, Chapel Hill, NC 27599-7525
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INTRODUCTION Murine embryonic stem (ES) cells have been isolated from whole embryos (1) and isolated ICM (2,3). The ES cells are capable of differentiating into several tissue types, including germ cells (4,5) and, therefore, are similar to cells from the early mouse embryo. Furthermore, ES cells can be maintained in culture in an undifferentiated state and, under appropriate culture conditions, can be induced to differentiate in a more or less organized fashion (6). Chimeras produced by combining genetically modified ES cells and normal embryos have been used to study the regulation of gene expression during development (7,8). In addition, ES cells have been used to study targeted modification of genes by homologous recombination (9-11) and to introduce the targeted modifications into the germ line (12-15). The combined abilities of isolating ES cells from early embryos, of genetically modifying ES cells in vitro, and of obtaining germ line transmission of the manipulated genome have led to the production of genetically modified individuals carrying mutations in specific sites (11,15). Such modified individuals can be used to study not only the basic aspects of gene regulation during growth and development but also ways in which traits of economic importance can be modified in agriculturally important species. Embryonic stem cells have been isolated from murine embryos using either mouse primary embryonic fibroblasts (3) or a continuous cell line of mouse embryonic fibroblasts (ST0;16) as feeder cells. Although cell lines with ES-like morphology can be isolated from porcine embryos plated on STO, their behavior and characteristics differ drastically from those of murine ES cell lines (17 and in unpublished observations). Recently it has been shown that ES cells can be isolated from hamster embryos using feeders composed of mouse primary embryonic fibroblasts (18). Smith and Hooper (19) reported the ability of conditioned medium from Buffalo rat liver (BRL) cells to support the growth of murine ES cells in the undifferentiated state in the absence of a feeder layer. Attempts at isolating ES cells from ovine species by culturing embryos on ovine skin fibroblasts or ovine embryonic fibroblasts in the presence or absence of BRL-conditioned media have been unsuccessful, however (20). The purpose of these experiments was to determine whether selected feeder layers have an effect on the growth and proliferation of porcine isolated ICM. We report here major differences in behavior of porcine ICM plated on different feeder layers and the isolation of epithelial-like and ES-like embryo-derived cell lines. The terms ES-like and epitheliallike are used to distinguish embryo-derived cell lines on the basis of Cells referred to as epithelial-like had a flattened, morphology. cuboidal shape and, when grown to confluence, tended to form structures reminiscent of epithelial sheets. Porcine epithelial-like cell lines express cytokeratin 18 (unpublished observation), a known marker for epithelial cells (21). Cells referred to as ES-like were small and rounded and had a large nucleus with one or two prominent nucleoli. The latter morphological characteristics have also been described for murine (2,3) and porcine (17) ES cells. The term ES-like is not used to imply that the cell lines had pluripotential capabilities.
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THERIOGENOLOGY MATERIALS AND METHODS Embryo Collection and ICM Isolation Seven- to eight-day embryos (Day 0 - first day of estrus) were flushed from the uteri of slaughtered donors, using phosphate buffered saline (PBS) containing 2% fetal bovine serum (FBS; GIBCO, Grand Island, NY), penicillin (100 IU/ml), and streptomycin (100 ug/ml). The ICM were isolated by 15- to 25-min incubation in heat-inactivated rabbit antiporcine antiserum diluted l/8 in Dulbecco's modified Eagle's medium (DMEM), followed by a lo- to 20-min incubation in guinea pig serum (GIBCO) diluted l/10 in DMEM. Rabbit anti-porcine antiserum was prepared from blood collected from rabbits immunized with three weekly injections of 100 x 106porcine spleen cells (22). Preparation of Feeder Layers and BRL-Conditioned Media Embryonic fibroblasts were obtained from 30- to 40-d-old porcine embryos (PEF), and 14- to 16-d-old mouse fetuses (MEF). After removal of the head, liver, and heart, the embryonic fetal tissues were minced and incubated 45 min at 37'C in trypsin/EDTA (0.5% trypsin/0.2% EDTA; GIBCO). Large cell clumps were removed and the remaining cells plated on loo-mm tissue culture plates in DMEM containing 10% calf serum (GIBCO), 5% FBS, glutamine (2 mM), penicillin (100 IU/ml), and streptomycin (100 pg/ml). Cells were allowed to grow to confluence and used to prepare feeder layers up to and including the tenth passage. After the tenth passage, cells were discarded due to their slow growth rate and new fibroblast cultures were prepared. Porcine uterine epithelial cells (PUE) were prepared from explant cultures of porcine uterine endometrium obtained from 6- to 8-d pregnant sows. Using explant culture a mixture of epithelial and fibroblast cells was obtained. Only cultures that were visually determined to contain greater than 70% epithelial cells were used to prepare feeder layers. Porcine H3A (PH3A) cell line arose from a cultured porcine isolated ICM, had epithelial-like morphology, and showed the presence of cytokeratin 18 but not vimentin (unpublished observations). BRL cells were a gift from G.R. Martin (University of California, San Francisco). All cell lines, except the PH3A cell line, were maintained in DMEM (GIBCO) supplemented with 10% calf serum (GIBCO), 5% FBS (GIBCO), glutamine (2 mM), 2-mercaptoethanol (0.1 mM), penicillin (100 IU/ml) and streptomycin (100 pg/ml). For culturing the PH3A cell line, FBS (GIBCO) was increased to 10% and a different batch of calf serum used (Sigma, St. Louis, MO), as this was the medium in which the cell line had been isolated. Cells harvested by trypsinization were inactivated by xirradiation with 4500 or 6000 Rads and plated in 35-mm tissue culture dishes (Falcon, Becton Dickinson, Lincoln, NJ) coated with gelatin. Coating was accomplished by adding a solution of 0.1% gelatin to the dish and incubatin at room temperature for 20 min. Onto each 35-mm plate either 2 x 106gcells (PEF and PH3A cells) or 1X lo6 (all other cell types) were added. Feeders were used within a week of preparation. BRL-conditioned medium was prepared by a modification of the method of Smith and Hooper (19). Media from confluent cultures of BRL, plated on
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lOO-mm tissue culture plates, were collected every third day for up to 2 weeks, filtered through Millipore membrane (0.8 pm pore size) and frozen at -7OOC. Upon thawing, BRL-conditioned medium was diluted 1:l (v/v) with medium used to isolate embryonic stem cells. As a control, culture medium left in the incubator for 3 d was frozen and diluted under the same conditions as BRL-conditioned medium. To determine the influence of feeder layer type on the isolation of embryo-derived cell lines, embryos were plated on feeders composed of STO, STO:BRL at a 9:l ratio, STO:BRL at a 1:l ratio, ST0 with BRL-conditioned media (ST0 + CM), PEF, PEF:BRL at a 9:l ratio, PEF:BRL at a 1:l ratio, BRL, PUE, MEF, or PH3A. Survival profiles after repeated passage and the number and morphology of embryo-derived cell lines were recorded for each type of feeder cell. The effects of feeder cell types on the survival profiles of embryo-derived cell lines were compared by the Friedman twoway analysis of variance by rank (23). Multiple-comparisons to determine individual treatment differences were carried out using the formula IRjRj'l 2 z J(bk (k+l) + 6) where Rj and Rj' are the jth and j'th treatment rank totals, z-a+6, b - number of passages, and k - number of treatments (23). Isolation of Embryo-Derived Cell Lines With ES-Like Morphology Isolated ICM were plated onto feeder layers in DMEM containing 10% FBS (GIBCO), 10% calf serum (Sigma), L-glutamine (2 mM), 2-mercaptoethanol (0.1 mM), penicillin (100 IV/ml) and streptomycin (100 pg/ml). Batches of FBS and calf serum were selected on the basis of their ability to allow high growth and in vitro differentiation of PSA teratocarcinoma stem cells. Approximately 10 d after plating, growing ICM were dissociated by gentle trypsinization (3 to 5 min) and transferred to a fresh feeder layer with the aid of a mouth-operated micropipette. Then 7 to 10 d later, and at 7- to 10-d intervals thereafter, colonies composed of cells with embryonic stem cell-like morphology were selected for further passage. Colonies were subcultured into increasingly larger culture dishes (35 mm and 100 mm). Isolated cell lines were tested for mycoplasma contamination by DNA hybridization using a DNA probe (Gen-Probe) specific for mycoplasma and acholeplasma ribosomal RNA (24). In Vitro Differentiation of Isolated Cell Lines To induce in vitro differentiation, embryo-derived cell lines with ESlike morphology were seeded onto tissue culture plates in the absence of a feeder layer. After 7 to 10 d of culture, colonies were gently dislodged and placed in nonadhesive petri plates. Suspension cultures were monitored daily and media were changed every other day. At selected intervals, samples of floating colonies were obtained, rinsed in PBS, and fixed in 2.5% glutaraldehyde for 1 h at room temperature and 12 to 18 h at 4OC. After fixation, samples were stored in PBS containing 0.02% sodium azide and stored at 4OC for up to 2 wk. Samples to be analyzed by light microscopy were dehydrated with ethanol, embedded in paraffin, and 8-pm sections prepared with a JKB microtome. Sections were stained with eosin and hematoxylin.
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THERIOGENOLOGY RESULTS Isolation of Embryo-Derived Cell Lines With ES-Like Morphology The sequence of events leading to production of embryo-derived cell lines with ES-like morphology was different for the various feeder layers. When isolated ICM were plated on STO, attachment to the feeder layer occurred during the first 12 to 24 h in culture. After attachment, cells from the ICM began to spread onto the feeder layer. Thereafter, the ICM either continued to spread or degeneration progressed and the ICM slowly disappeared. In general, ICM that continued to spread produced epithelial-like rather than ES-like colonies. After 3 to 8 d of culture, the central portion of the colony either began a process of degeneration, or a new cell type (ES-like) could be seen migrating from the edge of the ICM. In many cases no ES-like cells were observed to originate from the ICM. When this occurred, the colony was passed after approximately 10 d. Commonly, ES-like cells appeared for the first time at the second, third, or even fourth passage. While ICM plated on ST0 proliferated, ICM plated on STO:BRL (9:1), STO:BRL (f:l), PEF, PEF:BRL (9:1), PEF:BRL (l:lf, BRL, PUE, MEF, and PH3A did not. In contrast to relatively early attachment of the ICM to ST0 feeders, attachment occurred 48 to 60 h and 60 to 72 h after plating for STO:BRL (9:l) and STO:BRL (1:l) feeders, respectively. Only ST0 and ST0 + CM supported the growth of embryo-derived colonies for longer than 10 passages (Table 1). The ICM survived plating and attached to PUE, BRL, and PH3A feeders only in approximately 10% of the cases (Table l), and attachment occurred 60 to 72 h after plating. The attached ICM showed no proliferative growth but appeared to quickly differentiate (1 to 2 d) into structures composed of fluid accumulating cells (FAG; 25; Figure 1). None of the differentiating ICM or their cell derivatives could be kept longer than three passages (Table 1). The remaining 90% of the ICM remained in suspension and either degenerated within 1 to 2 d or formed structures that resembled disorganized trophoblastic vesicles (Figure 1). When ICM were plated on PEF, PEF:BRL (l:l), or PEF:BRL f9:1), they attached to the feeder after 36 to 48 h (PEF and PEF:BRL [9:1]) but no resulting cell line could be maintained for longer than five passages (Table 1). Fewer than 10% of the ICM remained alive after the second passage. Although the rate of cell line survival after repeated passage on PEF-containing feeders, PUE, MEF and PH3A feeders resembled one another (Table l), the morphology of the ICM differed. The ICM plated on PEF, PEF:BRL (l:l), or PEF:BRL (9:l) remained as well-delineated colonies composed of cells with large nuclei and prominent nucleoli, which contrasted with the highly disorganized colony described above for PUE-, MEF- and PH3A-plated ICM (Figure 1). The ICM attached to MEF feeders 24 The morphology of ICM plated on PEF or PEFto 48 h after plating. containing feeders closely resembled that of murine embryonic stem cells (data not shown). The ICM plated on PEF and PEF:BRL (9:l) attached to the feeder layer 36 to 48 h after plating; attachment occurred after 48 to 60 h on PEF:BRL (1:l) feeders.
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56 100% 36 64% 32 57% 29 52% 23 41% 18 32% 16 29% 14 25% 8 14% 5 9% 2 5%
d" First Second Third Fourth Fifth sixth Seventh Eighth Ninth Tenth
14 100% 2 14% 0 0 0 0 0 0 0 0 0
BRLd
21 100% 7 33% 3 14% 2 10% 2 10% 2 10% 2 10% 0 0 0 0
sTO:mL~ (l:lI 10 100% 5 5oa 4 40% 3 30% 3 30% 3 30% 2 20% 2 20% 2 20% 2 20% 1 10%
STO+BP.L= CH 26 100% 4 15% 2 8% 1 4% 0 0 0 0 0 0 0
PE@
11100% 6 558 1 9% 1 9% 0 0 0 0 0 0 0
PEF:BRL* (l:lf
used as feeder.
22 100% 3 14% 1 5% 1 5% 1 5% 0 0 0 0 0 0
PEF:BRLd f9:lf 18 100% 1 68 1 6% 0 0 0 0 0 0 x
Pm+
17 100% 10 59% 1 6% 1 6% 0 0 0 0 0 0 0
KEFd
15 100% 1 7% 0 0 0 0 0 0 0 0 0
W3Ad
survival profile
'R' Columns with different letter superscripts differ significantly (P~0.05).
b Values for 0 passage are numbers of embryos plated for the various feeder types. For subsequent passages, values are numbers of embryos that survived.
I.5100% 6 40% 6 40% 6 40% 4 27% 2 13% 7% : 7% 0 0 0
sTo:mL~ (9:l-J
“See text (Abstract) for abbreviations
SmC
Cell me
Comparative behavior of porcine isolated ICH plated on different types of feeder layers: of embryo-derived cell lines during plating and repeated passage.
Passage pumber
Table 1.
THERIOGENOLOGY
ST0
BRL
PEF:BRL tl:lI PEF:BRL (9:lj
Figure 1.
Morphology of porcine ICM 3 to 5 days after attachment to Morphology of plated ICM differed the feeder layer. depending on feeder layer. See text for abbreviations and descriptions of ICM morphology.
When ICM were plated on feeders containing a combination of ST0 and BRL, the rate of cell line survival after repeated passage tended to be improved as the proportion of BRL in the feeders decreased (Table 1). The changes in rates of survival after repeated passage seen with different feeders paralleled changes in colony morphology. Thus, as the proportion of BRL in the feeders increased, the cell lines tended to become more
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disorganized morphologically and the rate of cell proliferation, as evidenced by changes in colony size, reduced (Figure 1). As the proportion of ST0 in the feeder increased, the morphology of the cell lines became more ES-like and a higher growth rate was observed. The proliferation was mostly concentrated in epithelial-like cells, which migrated from the differentiating ICM onto the feeder layer.
In Vitro Differentiation of Embryo-Derived Cell Lines Of three embryo-derived cell lines maintained for longer than 10 passages, two had ES-like morphology and one had epithelial-like morphology. Examples of ES-like colonies are shown in Figure 2. When embryo-derived cell lines with ES-like and epithelial-like morphology were cultured under conditions known to induce in vitro differentiation of murine ES cells (2), the epithelial-like cell line differentiated into structures resembling cystic embryoid bodies (2) at the light microscope level (Figure 3), but the ES-like cell lines did not undergo any obvious morphological changes even after 30 d of suspension culture. Histological examination of differentiating epithelial-like structures at various times after the initiation of culture showed a rapid organization of cells into pseudostratified columnar epithelium surrounding a loosely-packed group of cells (Figure 4). With prolonged culture the embryoid body-like structures showed a three- to four-fold increase in luminal size. At high magnification the lumen appeared to be surrounded by a single layer of cells with epithelial-like morphology (Figure 4).
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Figure
3.
In vitro differentiation of porcine embryo-derived, epithelial-like cell lines after culture under conditions known to induce differentiation of murine ES cells. TWO days after culture (A) the cell aggregates had a thin epithelial-like border and small lumen. The epitheliallike border of the colony became more pronounced by day 4 (B); similarly, the lumen increased in size. By the tenth day of suspension culture (C), the colony had increased in size several-fold and resembled a murine embryoid body with its large luminal cavity.
* :+ j”
i
Figure
4.
100x
In vitro differentiation of porcine embryo-derived, epithelial-like cell lines: A histological evaluation. Forty-eight h after the initiation of suspension culture the cell aggregates were composed of loosely organized groups of cells (100x). In a few of the aggregates the cells had organized into columnar epithelium surrounding a small luminal cavity. As culture progressed, the lumen By the seventh day of culture the increased in size. structures were well organized vesicles with large lumens At higher surrounded by columnar epithelium (160x). magnification (400x) the epithelium appeared to be of pseudostratified type.
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DISCUSSION Although embryo-derived cell lines can be isolated from porcine blastocysts plated on STO, their growth and morphological characteristics differ from those of murine embryo-derived cell lines (unpublished observations). Such differences could be due to species differences, inappropriate culture conditions, or a combination of both. The effects of different feeder layers on the behavior of isolated porcine ICM were examined. None of the feeders increased the efficiency of isolation or the growth characteristics of embryo-derived (both ES-like and epitheliallike) cell lines over that of ST0 feeders, but type of feeders did affect behavior of plated ICM (Table 1). One group of feeders, represented by PUE, BRL, STO:BRL (l:l), PEF:BRL (l:l), and PH3A, did not promote attachment of the ICM to the feeder layer, perhaps because of a reduced or missing extracellular matrix. Alternatively, the feeders could have induced changes in the ICM that reduced its ability to bind to the extracellular matrix. The PUE feeder did not have a beneficial effect on the survival of embryo-derived cell lines, but equivalent feeders compared to ST0 feeders promoted the development and hatching of early preimplantation porcine embryos (data not shown). Therefore, either the intact preimplantation porcine embryo responds differently to growth signals than does the isolated ICM and its derivatives, or the importance of the feeder layer lies more in having the proper extracellular matrix for attachment than in increasing the proliferative rate of the ICM by way of diffusible substances. Medium conditioned by BRL has been shown to inhibit the differentiation of murine ES cells (19), rendering the use of feeder cells unnecessary. Similarly, Smith et al. (26) and Williams et al. (27) have reported that culture of ES cells with leukocyte inhibitory factor (LIF) in the absence of feeder cells inhibits in vitro differentiation. Such results suggest that a diffusible substance may contribute to maintaining ES cells in an undifferentiated state. In preliminary experiments addition of BRLconditioned medium to porcine ICM plated on ST0 caused no apparent increase in the proliferation rate (data not shown). In an attempt to reduce the rate of differentiation of porcine ICM while at the same time providing conditions that would allow proliferation, BRL cells were incorporated into the feeder layer to increase the level of BRL-related secretory substances. When mixtures of STO:BRL and PEF:BRL were used, no increase in the proliferation rate was observed. Such results contradict those of Ware and First (28) who reported that plating of bovine, porcine, and ovine embryos on a combination of BRL and mouse primary fetal fibroblasts (1:l) resulted in the development of fast-growing embryoderived cells. An interesting effect related to inhibition of differentiation was observed when ICM were plated on PEF feeders. On such feeders the ICM spread onto the feeder layer after attachment without apparent signs of differentiation. The cells present after spreading did not change in morphology after several days of incubation, nor did the colony increase in size. These results suggest that the extracellular matrix, or substances released that are particular to PEF, inhibited differentiation of the porcine ICM. Unfortunately, proliferation also appeared to be inhibited. It may be possible to use PEF feeders to maintain the cells
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THERIOGENOLOGY in an undifferentiated state and attempt to find other methods to increase the rate of proliferation. It is interesting to note that the cell morphology of PEF-plated, undifferentiated ICM resembled that of isolated porcine ES-like colonies (Figures 1 and 2, respectively). In contrast to PEF feeder layers, ST0 and MEF feeder layers both allowed ICM proliferation and differentiation. The ICM plated on MEF showed, after attachment, differentiation into a presumptive endodermal or trophoblastic cell layer and a small, contained, central colony. The central colony was presumed to be of primitive ectodermal origin. Growth of the colony was limited, suggesting that conditions were inappropriate for optimal proliferation. The ICM plated on ST0 feeder layers showed a comparatively high rate of proliferation as inferred from changes in colony size. This proliferation was mostly concentrated in epithelial-like cells, which migrated from the differentiating ICM onto the feeder layer. From the results obtained in these experiments it appears that the function of the feeder layer in isolation of embryo-derived cell lines includes inhibition of differentiation and stimulation of proliferation. None of the feeder layers used in this experiment provided optimal conditions for isolation of porcine ES cells. Although embryo-derived cell lines with ES-like morphology were obtained from ICM plated on ST0 cells and ST0 cells with added BRL-conditioned media, the rate of proliferation, as estimated by changes in colony number and size, and the ability to differentiate in vitro differed from that reported for murine ES cells. REFERENCES 1.
Axelrod, H.R. Embryonic stem cell lines derived from blastocysts (1984). by a simplified technique. Dev. Biol. =:225-228
2.
Martin, G.R. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned to teratocarcinoma stem cells. Proc. Natl. Acad. Sci. s:7634-7638 (1981).
3.
Wobus, A.M., Holzhausen, H., Jakel, P. and Schoneich, J. Characterization of a pluripotent stem cell line derived from a mouse embryo. Exp. Cell Res. =:212-219 (1984).
4.
Bradley, A., Evans, M., Kaufman, M.H. and Robertson, E. Formation of germ-line chimeras from embryo-derived teratocarcinoma cell lines. Nature m:255-256 (1984).
5.
The relationship between Rossant, J. and Papaioannou, V.E. embryonic, embryonal carcinoma and embryo-derived stem cells. Cell Differentiation =:155-161 (1984).
6.
Martin, G.R. and Lock, L.F. Pluripotent cell lines derived from early mouse embryos in medium conditioned by teratocarcinoma stem cells. In: Silver, L.M., Martin, G.R. and Strickland, S. (eds), Teratocarcinoma Stem Cells. Vol 10. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1983, pp. 635-646.
NOVEMBER lB9OVOL. 34 NO. 5
875
7.
Stewart, C.L., Vanek, M. and Wagner, E.G. Expression of foreign genes from retroviral vectors in mouse teratocarcinoma chimeras. EMBO J. &:3701-3709 (1985).
8.
Wagner, E.F., Keller, G., Gilboa, E., Ruther, U. and Stewart, C. Gene transfer into murine stem cells and mice using retroviral vectors. Cold Spring Harbor Symposia on Quantitative Biol. 2:691699 (1985).
9.
Doetschman, T., Gregg, R. B., Maeda, N., Hooper, M. L., Melton, D. W Thompson, S. and Smithies, 0. Targeted correction of a mutant (1987). HPRT gene in mouse embryonic stem cells. Nature m:576:578
10.
Thomas, K.R. and Capecchi, M.R. Site-directed mutagenesis by gene Cell 2:503-512 targeting in mouse embryo-derived stem cells. (1987).
11.
Inactivating the E2-microglobulin Koller, B.H. and Smithies, 0. locus in mouse embryonic stem cells by homologous recombination. Proc. Natl. Acad. Sci. USA &:8932-8935 (1989).
12.
Gossler, A., Doetschman, T., Korn, R., Serfling E. and Kemler, R. Transgenesis by means of blastocyst-derived embryonic stem cell lines. Proc. Natl. Acad. Sci. USA u:9065-9069 (1986).
13.
Germ line Robertson, E., Bradley, A., Kuehn, M. and Evans, M. transmission of genes introduced into cultured pluripotential cells by retroviral vector. Nature =:445-448 (1986).
14.
Koller, B.H., Hagemann, L.J., Doetschman, T., Hagaman, J.R., Huang, S ., Williams, P.J., First, N.L., Maeda, N. and Smithies, 0. Germline transmission of a planned alteration made in a hypoxanthine phosphoribosyltransferase gene by homologous recombination in Proc. Natl. Acad. Sci. USA &:8927-8931 embryonic stem cells. (1989).
15.
Thompson, S., Clarke, A.R., Pow, A.M., Hooper, M.L. and Melton, D.W. Germ line transmission and expression of a corrected HPRT gene produced by gene targeting in embryonic stem cells. Cell 56:313321 (1989).
16.
Ware, L.M. and Axelrad, A.A. Inherited resistance to N- and Btropic murine leukemia virus in vitro: Evidence that congenic mouse strains SIM and S1M.R differ at the Fv-1 locus. Virology %:339348 (1972).
17.
Evans, M.J., Notarianni, E., Laurie, S. and Moor, R.M. Derivation and preliminary characterization of pluripotent cell lines from porcine and bovine blastocysts. Theriogenology =:125-128 (1990).
18.
Doetschman, T., Williams, P. and Maeda, N. Establishment of hamster blastocyst-derived embryonic stem (ES) cells. Dev. Biol. m:224227 (1988).
676
NOVEMBER 1990 VOL. 34 NO. 5
THERIOGENOLOGY 19.
Smith, A.G. and Hooper, M.L. Buffalo rat liver cells produce a diffusible activity which inhibits the differentiation of murine embryonal carcinoma and embryonic stem cells. Dev. Biol. u:l-9 (1987).
20.
Handyside, A., Hooper, M.L., Kaufman, M.H. and Wilmut, I. Towards the isolation of embryonal stem cell lines from the sheep. Roux's Arch. Dev. Biol. m:185-190 (1987).
21.
Mall, R., Franke, W.W., Schiller, D.L., Geiger, B. and Krepler, R. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell u:ll-24 (1982).
22.
Butler, J.E., Anderson, G.B., BonDurant, R.H., Pashen, R.L. and Penedo, M.C.T. Production of ovine chimeras by inner cell mass transplantation. J. Anim. Sci. =:317-324 (1987).
23.
Daniel, W.W. Applied Nonparametric Statistics. co., Palo Alto, CA, 1978, pp. 231-233.
24.
McGarrity, G.J. and Kotani, H. Detection of cell mycoplasmas by a genetic probe. Exp. Cell Res. w:273-278
25.
Isolation and Surani, M.A.H., Torchiana, D. and Barton, S.C. development of the inner cell mass after exposure of mouse embryos to calcium ionophore A23187. J. Embryol. Exp. Morph. 411237-247 (1978).
26.
Smith, A.G., Heath, J.K., Donaldson, D.D., Wong, G.W., Moreau, J., Stahl, M. and Rogers, D. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature 336:688690 (1988).
27.
Williams, R.L., Hilton, D.J., Pease, S., Wilson, T.A., Steward, C.L., Gearing, D.P., Wagner, E.F., Metcalf, D., Nicola, N. and Gough, N.M. Myeloid leukemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature =:684687 (1988).
28.
Ware, C.B. and First, N.L. Development of embryonic stem cell lines from farm animals. Biol. Reprod. 38(Suppl. 1):129 abstr. (1988).
NOVEMBER 1990 VOL. 34 NO. 5
Houghton Mifflin
culture (1986).
877
J.A. Piedrahitalpa , G.B. Andersonland R.H. BonDurant' 'Department of Animal Science and *Department of Reproduction, School of Veterinary Medicine, University of California, Davis, USA
Received for publication: May 83, 1990 Accepted: S~ptembw 14, 1990
ABSTRACT Experiments were conducted to determine the effects of feeder layers composed of different cell types on the efficiency of isolation and the behavior of porcine embryo-derived cell lines. Inner cell masses (ICM) isolated from 7- to I-d-old embryos were plated on feeder layers composed of Buffalo rat liver cells (BRL), a continuous cell line of murine embryonic fibroblasts (STO), ST0 combined with BRL at a 9:l and I:1 ratio, ST0 with BRL-conditioned medium (ST0 + CM), porcine embryonic fibroblasts (PEF), PEF combined with BRL at a 9:l and 1:l ratio, porcine uterine epithelial cells (PUE), murine embryonic fibroblasts (MEF), or an epithelial-like porcine embryo-derived cell line (PH3A). It was found that embryo-derived cell lines could be isolated only from the ST0 and the ST0 with BRL-conditioned medium treatments. The isolated cell lines were of epithelial-like and embryonic stem cell-like (ES-like) morphology. The feeders tested had an effect on the behavior of plated ICM. Some feeders, represented by PUE, BRL, STO:BRL (l:l), PEF:BRL (l:l), and PH3A, did not promote attachment of the ICM to the feeder layer; others, represented by ST0 and MEF, allowed attachment, differentiation and proliferation. On PEF feeders the ICM spread onto the feeder layer after attachment without apparent signs of proliferation or differentiation. None of the feeders tested increased the efficiency of isolation or the growth characteristics of embryo-derived (both ES-like and epitheliallike) cell lines over that of ST0 feeders. Key words:
embryonic stem cells, feeder cells, porcine
Acknowledgements This research was supported by a USDA Competitive Research Grant (GBA and RHB) and a Jastro-Shields Graduate Research Award (JAP). The authors thank Akiko Stevens, Kent Parker and Dan Sehnert for technical assistance and Kay Allen and Susan Donahue for help with manuscript preparation. aCurrent address: Department of Pathology, School of University of North Carolina, Chapel Hill, NC 27599-7525
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INTRODUCTION Murine embryonic stem (ES) cells have been isolated from whole embryos (1) and isolated ICM (2,3). The ES cells are capable of differentiating into several tissue types, including germ cells (4,5) and, therefore, are similar to cells from the early mouse embryo. Furthermore, ES cells can be maintained in culture in an undifferentiated state and, under appropriate culture conditions, can be induced to differentiate in a more or less organized fashion (6). Chimeras produced by combining genetically modified ES cells and normal embryos have been used to study the regulation of gene expression during development (7,8). In addition, ES cells have been used to study targeted modification of genes by homologous recombination (9-11) and to introduce the targeted modifications into the germ line (12-15). The combined abilities of isolating ES cells from early embryos, of genetically modifying ES cells in vitro, and of obtaining germ line transmission of the manipulated genome have led to the production of genetically modified individuals carrying mutations in specific sites (11,15). Such modified individuals can be used to study not only the basic aspects of gene regulation during growth and development but also ways in which traits of economic importance can be modified in agriculturally important species. Embryonic stem cells have been isolated from murine embryos using either mouse primary embryonic fibroblasts (3) or a continuous cell line of mouse embryonic fibroblasts (ST0;16) as feeder cells. Although cell lines with ES-like morphology can be isolated from porcine embryos plated on STO, their behavior and characteristics differ drastically from those of murine ES cell lines (17 and in unpublished observations). Recently it has been shown that ES cells can be isolated from hamster embryos using feeders composed of mouse primary embryonic fibroblasts (18). Smith and Hooper (19) reported the ability of conditioned medium from Buffalo rat liver (BRL) cells to support the growth of murine ES cells in the undifferentiated state in the absence of a feeder layer. Attempts at isolating ES cells from ovine species by culturing embryos on ovine skin fibroblasts or ovine embryonic fibroblasts in the presence or absence of BRL-conditioned media have been unsuccessful, however (20). The purpose of these experiments was to determine whether selected feeder layers have an effect on the growth and proliferation of porcine isolated ICM. We report here major differences in behavior of porcine ICM plated on different feeder layers and the isolation of epithelial-like and ES-like embryo-derived cell lines. The terms ES-like and epitheliallike are used to distinguish embryo-derived cell lines on the basis of Cells referred to as epithelial-like had a flattened, morphology. cuboidal shape and, when grown to confluence, tended to form structures reminiscent of epithelial sheets. Porcine epithelial-like cell lines express cytokeratin 18 (unpublished observation), a known marker for epithelial cells (21). Cells referred to as ES-like were small and rounded and had a large nucleus with one or two prominent nucleoli. The latter morphological characteristics have also been described for murine (2,3) and porcine (17) ES cells. The term ES-like is not used to imply that the cell lines had pluripotential capabilities.
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THERIOGENOLOGY MATERIALS AND METHODS Embryo Collection and ICM Isolation Seven- to eight-day embryos (Day 0 - first day of estrus) were flushed from the uteri of slaughtered donors, using phosphate buffered saline (PBS) containing 2% fetal bovine serum (FBS; GIBCO, Grand Island, NY), penicillin (100 IU/ml), and streptomycin (100 ug/ml). The ICM were isolated by 15- to 25-min incubation in heat-inactivated rabbit antiporcine antiserum diluted l/8 in Dulbecco's modified Eagle's medium (DMEM), followed by a lo- to 20-min incubation in guinea pig serum (GIBCO) diluted l/10 in DMEM. Rabbit anti-porcine antiserum was prepared from blood collected from rabbits immunized with three weekly injections of 100 x 106porcine spleen cells (22). Preparation of Feeder Layers and BRL-Conditioned Media Embryonic fibroblasts were obtained from 30- to 40-d-old porcine embryos (PEF), and 14- to 16-d-old mouse fetuses (MEF). After removal of the head, liver, and heart, the embryonic fetal tissues were minced and incubated 45 min at 37'C in trypsin/EDTA (0.5% trypsin/0.2% EDTA; GIBCO). Large cell clumps were removed and the remaining cells plated on loo-mm tissue culture plates in DMEM containing 10% calf serum (GIBCO), 5% FBS, glutamine (2 mM), penicillin (100 IU/ml), and streptomycin (100 pg/ml). Cells were allowed to grow to confluence and used to prepare feeder layers up to and including the tenth passage. After the tenth passage, cells were discarded due to their slow growth rate and new fibroblast cultures were prepared. Porcine uterine epithelial cells (PUE) were prepared from explant cultures of porcine uterine endometrium obtained from 6- to 8-d pregnant sows. Using explant culture a mixture of epithelial and fibroblast cells was obtained. Only cultures that were visually determined to contain greater than 70% epithelial cells were used to prepare feeder layers. Porcine H3A (PH3A) cell line arose from a cultured porcine isolated ICM, had epithelial-like morphology, and showed the presence of cytokeratin 18 but not vimentin (unpublished observations). BRL cells were a gift from G.R. Martin (University of California, San Francisco). All cell lines, except the PH3A cell line, were maintained in DMEM (GIBCO) supplemented with 10% calf serum (GIBCO), 5% FBS (GIBCO), glutamine (2 mM), 2-mercaptoethanol (0.1 mM), penicillin (100 IU/ml) and streptomycin (100 pg/ml). For culturing the PH3A cell line, FBS (GIBCO) was increased to 10% and a different batch of calf serum used (Sigma, St. Louis, MO), as this was the medium in which the cell line had been isolated. Cells harvested by trypsinization were inactivated by xirradiation with 4500 or 6000 Rads and plated in 35-mm tissue culture dishes (Falcon, Becton Dickinson, Lincoln, NJ) coated with gelatin. Coating was accomplished by adding a solution of 0.1% gelatin to the dish and incubatin at room temperature for 20 min. Onto each 35-mm plate either 2 x 106gcells (PEF and PH3A cells) or 1X lo6 (all other cell types) were added. Feeders were used within a week of preparation. BRL-conditioned medium was prepared by a modification of the method of Smith and Hooper (19). Media from confluent cultures of BRL, plated on
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lOO-mm tissue culture plates, were collected every third day for up to 2 weeks, filtered through Millipore membrane (0.8 pm pore size) and frozen at -7OOC. Upon thawing, BRL-conditioned medium was diluted 1:l (v/v) with medium used to isolate embryonic stem cells. As a control, culture medium left in the incubator for 3 d was frozen and diluted under the same conditions as BRL-conditioned medium. To determine the influence of feeder layer type on the isolation of embryo-derived cell lines, embryos were plated on feeders composed of STO, STO:BRL at a 9:l ratio, STO:BRL at a 1:l ratio, ST0 with BRL-conditioned media (ST0 + CM), PEF, PEF:BRL at a 9:l ratio, PEF:BRL at a 1:l ratio, BRL, PUE, MEF, or PH3A. Survival profiles after repeated passage and the number and morphology of embryo-derived cell lines were recorded for each type of feeder cell. The effects of feeder cell types on the survival profiles of embryo-derived cell lines were compared by the Friedman twoway analysis of variance by rank (23). Multiple-comparisons to determine individual treatment differences were carried out using the formula IRjRj'l 2 z J(bk (k+l) + 6) where Rj and Rj' are the jth and j'th treatment rank totals, z-a+6, b - number of passages, and k - number of treatments (23). Isolation of Embryo-Derived Cell Lines With ES-Like Morphology Isolated ICM were plated onto feeder layers in DMEM containing 10% FBS (GIBCO), 10% calf serum (Sigma), L-glutamine (2 mM), 2-mercaptoethanol (0.1 mM), penicillin (100 IV/ml) and streptomycin (100 pg/ml). Batches of FBS and calf serum were selected on the basis of their ability to allow high growth and in vitro differentiation of PSA teratocarcinoma stem cells. Approximately 10 d after plating, growing ICM were dissociated by gentle trypsinization (3 to 5 min) and transferred to a fresh feeder layer with the aid of a mouth-operated micropipette. Then 7 to 10 d later, and at 7- to 10-d intervals thereafter, colonies composed of cells with embryonic stem cell-like morphology were selected for further passage. Colonies were subcultured into increasingly larger culture dishes (35 mm and 100 mm). Isolated cell lines were tested for mycoplasma contamination by DNA hybridization using a DNA probe (Gen-Probe) specific for mycoplasma and acholeplasma ribosomal RNA (24). In Vitro Differentiation of Isolated Cell Lines To induce in vitro differentiation, embryo-derived cell lines with ESlike morphology were seeded onto tissue culture plates in the absence of a feeder layer. After 7 to 10 d of culture, colonies were gently dislodged and placed in nonadhesive petri plates. Suspension cultures were monitored daily and media were changed every other day. At selected intervals, samples of floating colonies were obtained, rinsed in PBS, and fixed in 2.5% glutaraldehyde for 1 h at room temperature and 12 to 18 h at 4OC. After fixation, samples were stored in PBS containing 0.02% sodium azide and stored at 4OC for up to 2 wk. Samples to be analyzed by light microscopy were dehydrated with ethanol, embedded in paraffin, and 8-pm sections prepared with a JKB microtome. Sections were stained with eosin and hematoxylin.
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THERIOGENOLOGY RESULTS Isolation of Embryo-Derived Cell Lines With ES-Like Morphology The sequence of events leading to production of embryo-derived cell lines with ES-like morphology was different for the various feeder layers. When isolated ICM were plated on STO, attachment to the feeder layer occurred during the first 12 to 24 h in culture. After attachment, cells from the ICM began to spread onto the feeder layer. Thereafter, the ICM either continued to spread or degeneration progressed and the ICM slowly disappeared. In general, ICM that continued to spread produced epithelial-like rather than ES-like colonies. After 3 to 8 d of culture, the central portion of the colony either began a process of degeneration, or a new cell type (ES-like) could be seen migrating from the edge of the ICM. In many cases no ES-like cells were observed to originate from the ICM. When this occurred, the colony was passed after approximately 10 d. Commonly, ES-like cells appeared for the first time at the second, third, or even fourth passage. While ICM plated on ST0 proliferated, ICM plated on STO:BRL (9:1), STO:BRL (f:l), PEF, PEF:BRL (9:1), PEF:BRL (l:lf, BRL, PUE, MEF, and PH3A did not. In contrast to relatively early attachment of the ICM to ST0 feeders, attachment occurred 48 to 60 h and 60 to 72 h after plating for STO:BRL (9:l) and STO:BRL (1:l) feeders, respectively. Only ST0 and ST0 + CM supported the growth of embryo-derived colonies for longer than 10 passages (Table 1). The ICM survived plating and attached to PUE, BRL, and PH3A feeders only in approximately 10% of the cases (Table l), and attachment occurred 60 to 72 h after plating. The attached ICM showed no proliferative growth but appeared to quickly differentiate (1 to 2 d) into structures composed of fluid accumulating cells (FAG; 25; Figure 1). None of the differentiating ICM or their cell derivatives could be kept longer than three passages (Table 1). The remaining 90% of the ICM remained in suspension and either degenerated within 1 to 2 d or formed structures that resembled disorganized trophoblastic vesicles (Figure 1). When ICM were plated on PEF, PEF:BRL (l:l), or PEF:BRL f9:1), they attached to the feeder after 36 to 48 h (PEF and PEF:BRL [9:1]) but no resulting cell line could be maintained for longer than five passages (Table 1). Fewer than 10% of the ICM remained alive after the second passage. Although the rate of cell line survival after repeated passage on PEF-containing feeders, PUE, MEF and PH3A feeders resembled one another (Table l), the morphology of the ICM differed. The ICM plated on PEF, PEF:BRL (l:l), or PEF:BRL (9:l) remained as well-delineated colonies composed of cells with large nuclei and prominent nucleoli, which contrasted with the highly disorganized colony described above for PUE-, MEF- and PH3A-plated ICM (Figure 1). The ICM attached to MEF feeders 24 The morphology of ICM plated on PEF or PEFto 48 h after plating. containing feeders closely resembled that of murine embryonic stem cells (data not shown). The ICM plated on PEF and PEF:BRL (9:l) attached to the feeder layer 36 to 48 h after plating; attachment occurred after 48 to 60 h on PEF:BRL (1:l) feeders.
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56 100% 36 64% 32 57% 29 52% 23 41% 18 32% 16 29% 14 25% 8 14% 5 9% 2 5%
d" First Second Third Fourth Fifth sixth Seventh Eighth Ninth Tenth
14 100% 2 14% 0 0 0 0 0 0 0 0 0
BRLd
21 100% 7 33% 3 14% 2 10% 2 10% 2 10% 2 10% 0 0 0 0
sTO:mL~ (l:lI 10 100% 5 5oa 4 40% 3 30% 3 30% 3 30% 2 20% 2 20% 2 20% 2 20% 1 10%
STO+BP.L= CH 26 100% 4 15% 2 8% 1 4% 0 0 0 0 0 0 0
PE@
11100% 6 558 1 9% 1 9% 0 0 0 0 0 0 0
PEF:BRL* (l:lf
used as feeder.
22 100% 3 14% 1 5% 1 5% 1 5% 0 0 0 0 0 0
PEF:BRLd f9:lf 18 100% 1 68 1 6% 0 0 0 0 0 0 x
Pm+
17 100% 10 59% 1 6% 1 6% 0 0 0 0 0 0 0
KEFd
15 100% 1 7% 0 0 0 0 0 0 0 0 0
W3Ad
survival profile
'R' Columns with different letter superscripts differ significantly (P~0.05).
b Values for 0 passage are numbers of embryos plated for the various feeder types. For subsequent passages, values are numbers of embryos that survived.
I.5100% 6 40% 6 40% 6 40% 4 27% 2 13% 7% : 7% 0 0 0
sTo:mL~ (9:l-J
“See text (Abstract) for abbreviations
SmC
Cell me
Comparative behavior of porcine isolated ICH plated on different types of feeder layers: of embryo-derived cell lines during plating and repeated passage.
Passage pumber
Table 1.
THERIOGENOLOGY
ST0
BRL
PEF:BRL tl:lI PEF:BRL (9:lj
Figure 1.
Morphology of porcine ICM 3 to 5 days after attachment to Morphology of plated ICM differed the feeder layer. depending on feeder layer. See text for abbreviations and descriptions of ICM morphology.
When ICM were plated on feeders containing a combination of ST0 and BRL, the rate of cell line survival after repeated passage tended to be improved as the proportion of BRL in the feeders decreased (Table 1). The changes in rates of survival after repeated passage seen with different feeders paralleled changes in colony morphology. Thus, as the proportion of BRL in the feeders increased, the cell lines tended to become more
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disorganized morphologically and the rate of cell proliferation, as evidenced by changes in colony size, reduced (Figure 1). As the proportion of ST0 in the feeder increased, the morphology of the cell lines became more ES-like and a higher growth rate was observed. The proliferation was mostly concentrated in epithelial-like cells, which migrated from the differentiating ICM onto the feeder layer.
In Vitro Differentiation of Embryo-Derived Cell Lines Of three embryo-derived cell lines maintained for longer than 10 passages, two had ES-like morphology and one had epithelial-like morphology. Examples of ES-like colonies are shown in Figure 2. When embryo-derived cell lines with ES-like and epithelial-like morphology were cultured under conditions known to induce in vitro differentiation of murine ES cells (2), the epithelial-like cell line differentiated into structures resembling cystic embryoid bodies (2) at the light microscope level (Figure 3), but the ES-like cell lines did not undergo any obvious morphological changes even after 30 d of suspension culture. Histological examination of differentiating epithelial-like structures at various times after the initiation of culture showed a rapid organization of cells into pseudostratified columnar epithelium surrounding a loosely-packed group of cells (Figure 4). With prolonged culture the embryoid body-like structures showed a three- to four-fold increase in luminal size. At high magnification the lumen appeared to be surrounded by a single layer of cells with epithelial-like morphology (Figure 4).
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Figure
3.
In vitro differentiation of porcine embryo-derived, epithelial-like cell lines after culture under conditions known to induce differentiation of murine ES cells. TWO days after culture (A) the cell aggregates had a thin epithelial-like border and small lumen. The epitheliallike border of the colony became more pronounced by day 4 (B); similarly, the lumen increased in size. By the tenth day of suspension culture (C), the colony had increased in size several-fold and resembled a murine embryoid body with its large luminal cavity.
* :+ j”
i
Figure
4.
100x
In vitro differentiation of porcine embryo-derived, epithelial-like cell lines: A histological evaluation. Forty-eight h after the initiation of suspension culture the cell aggregates were composed of loosely organized groups of cells (100x). In a few of the aggregates the cells had organized into columnar epithelium surrounding a small luminal cavity. As culture progressed, the lumen By the seventh day of culture the increased in size. structures were well organized vesicles with large lumens At higher surrounded by columnar epithelium (160x). magnification (400x) the epithelium appeared to be of pseudostratified type.
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DISCUSSION Although embryo-derived cell lines can be isolated from porcine blastocysts plated on STO, their growth and morphological characteristics differ from those of murine embryo-derived cell lines (unpublished observations). Such differences could be due to species differences, inappropriate culture conditions, or a combination of both. The effects of different feeder layers on the behavior of isolated porcine ICM were examined. None of the feeders increased the efficiency of isolation or the growth characteristics of embryo-derived (both ES-like and epitheliallike) cell lines over that of ST0 feeders, but type of feeders did affect behavior of plated ICM (Table 1). One group of feeders, represented by PUE, BRL, STO:BRL (l:l), PEF:BRL (l:l), and PH3A, did not promote attachment of the ICM to the feeder layer, perhaps because of a reduced or missing extracellular matrix. Alternatively, the feeders could have induced changes in the ICM that reduced its ability to bind to the extracellular matrix. The PUE feeder did not have a beneficial effect on the survival of embryo-derived cell lines, but equivalent feeders compared to ST0 feeders promoted the development and hatching of early preimplantation porcine embryos (data not shown). Therefore, either the intact preimplantation porcine embryo responds differently to growth signals than does the isolated ICM and its derivatives, or the importance of the feeder layer lies more in having the proper extracellular matrix for attachment than in increasing the proliferative rate of the ICM by way of diffusible substances. Medium conditioned by BRL has been shown to inhibit the differentiation of murine ES cells (19), rendering the use of feeder cells unnecessary. Similarly, Smith et al. (26) and Williams et al. (27) have reported that culture of ES cells with leukocyte inhibitory factor (LIF) in the absence of feeder cells inhibits in vitro differentiation. Such results suggest that a diffusible substance may contribute to maintaining ES cells in an undifferentiated state. In preliminary experiments addition of BRLconditioned medium to porcine ICM plated on ST0 caused no apparent increase in the proliferation rate (data not shown). In an attempt to reduce the rate of differentiation of porcine ICM while at the same time providing conditions that would allow proliferation, BRL cells were incorporated into the feeder layer to increase the level of BRL-related secretory substances. When mixtures of STO:BRL and PEF:BRL were used, no increase in the proliferation rate was observed. Such results contradict those of Ware and First (28) who reported that plating of bovine, porcine, and ovine embryos on a combination of BRL and mouse primary fetal fibroblasts (1:l) resulted in the development of fast-growing embryoderived cells. An interesting effect related to inhibition of differentiation was observed when ICM were plated on PEF feeders. On such feeders the ICM spread onto the feeder layer after attachment without apparent signs of differentiation. The cells present after spreading did not change in morphology after several days of incubation, nor did the colony increase in size. These results suggest that the extracellular matrix, or substances released that are particular to PEF, inhibited differentiation of the porcine ICM. Unfortunately, proliferation also appeared to be inhibited. It may be possible to use PEF feeders to maintain the cells
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THERIOGENOLOGY in an undifferentiated state and attempt to find other methods to increase the rate of proliferation. It is interesting to note that the cell morphology of PEF-plated, undifferentiated ICM resembled that of isolated porcine ES-like colonies (Figures 1 and 2, respectively). In contrast to PEF feeder layers, ST0 and MEF feeder layers both allowed ICM proliferation and differentiation. The ICM plated on MEF showed, after attachment, differentiation into a presumptive endodermal or trophoblastic cell layer and a small, contained, central colony. The central colony was presumed to be of primitive ectodermal origin. Growth of the colony was limited, suggesting that conditions were inappropriate for optimal proliferation. The ICM plated on ST0 feeder layers showed a comparatively high rate of proliferation as inferred from changes in colony size. This proliferation was mostly concentrated in epithelial-like cells, which migrated from the differentiating ICM onto the feeder layer. From the results obtained in these experiments it appears that the function of the feeder layer in isolation of embryo-derived cell lines includes inhibition of differentiation and stimulation of proliferation. None of the feeder layers used in this experiment provided optimal conditions for isolation of porcine ES cells. Although embryo-derived cell lines with ES-like morphology were obtained from ICM plated on ST0 cells and ST0 cells with added BRL-conditioned media, the rate of proliferation, as estimated by changes in colony number and size, and the ability to differentiate in vitro differed from that reported for murine ES cells. REFERENCES 1.
Axelrod, H.R. Embryonic stem cell lines derived from blastocysts (1984). by a simplified technique. Dev. Biol. =:225-228
2.
Martin, G.R. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned to teratocarcinoma stem cells. Proc. Natl. Acad. Sci. s:7634-7638 (1981).
3.
Wobus, A.M., Holzhausen, H., Jakel, P. and Schoneich, J. Characterization of a pluripotent stem cell line derived from a mouse embryo. Exp. Cell Res. =:212-219 (1984).
4.
Bradley, A., Evans, M., Kaufman, M.H. and Robertson, E. Formation of germ-line chimeras from embryo-derived teratocarcinoma cell lines. Nature m:255-256 (1984).
5.
The relationship between Rossant, J. and Papaioannou, V.E. embryonic, embryonal carcinoma and embryo-derived stem cells. Cell Differentiation =:155-161 (1984).
6.
Martin, G.R. and Lock, L.F. Pluripotent cell lines derived from early mouse embryos in medium conditioned by teratocarcinoma stem cells. In: Silver, L.M., Martin, G.R. and Strickland, S. (eds), Teratocarcinoma Stem Cells. Vol 10. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1983, pp. 635-646.
NOVEMBER lB9OVOL. 34 NO. 5
875
7.
Stewart, C.L., Vanek, M. and Wagner, E.G. Expression of foreign genes from retroviral vectors in mouse teratocarcinoma chimeras. EMBO J. &:3701-3709 (1985).
8.
Wagner, E.F., Keller, G., Gilboa, E., Ruther, U. and Stewart, C. Gene transfer into murine stem cells and mice using retroviral vectors. Cold Spring Harbor Symposia on Quantitative Biol. 2:691699 (1985).
9.
Doetschman, T., Gregg, R. B., Maeda, N., Hooper, M. L., Melton, D. W Thompson, S. and Smithies, 0. Targeted correction of a mutant (1987). HPRT gene in mouse embryonic stem cells. Nature m:576:578
10.
Thomas, K.R. and Capecchi, M.R. Site-directed mutagenesis by gene Cell 2:503-512 targeting in mouse embryo-derived stem cells. (1987).
11.
Inactivating the E2-microglobulin Koller, B.H. and Smithies, 0. locus in mouse embryonic stem cells by homologous recombination. Proc. Natl. Acad. Sci. USA &:8932-8935 (1989).
12.
Gossler, A., Doetschman, T., Korn, R., Serfling E. and Kemler, R. Transgenesis by means of blastocyst-derived embryonic stem cell lines. Proc. Natl. Acad. Sci. USA u:9065-9069 (1986).
13.
Germ line Robertson, E., Bradley, A., Kuehn, M. and Evans, M. transmission of genes introduced into cultured pluripotential cells by retroviral vector. Nature =:445-448 (1986).
14.
Koller, B.H., Hagemann, L.J., Doetschman, T., Hagaman, J.R., Huang, S ., Williams, P.J., First, N.L., Maeda, N. and Smithies, 0. Germline transmission of a planned alteration made in a hypoxanthine phosphoribosyltransferase gene by homologous recombination in Proc. Natl. Acad. Sci. USA &:8927-8931 embryonic stem cells. (1989).
15.
Thompson, S., Clarke, A.R., Pow, A.M., Hooper, M.L. and Melton, D.W. Germ line transmission and expression of a corrected HPRT gene produced by gene targeting in embryonic stem cells. Cell 56:313321 (1989).
16.
Ware, L.M. and Axelrad, A.A. Inherited resistance to N- and Btropic murine leukemia virus in vitro: Evidence that congenic mouse strains SIM and S1M.R differ at the Fv-1 locus. Virology %:339348 (1972).
17.
Evans, M.J., Notarianni, E., Laurie, S. and Moor, R.M. Derivation and preliminary characterization of pluripotent cell lines from porcine and bovine blastocysts. Theriogenology =:125-128 (1990).
18.
Doetschman, T., Williams, P. and Maeda, N. Establishment of hamster blastocyst-derived embryonic stem (ES) cells. Dev. Biol. m:224227 (1988).
676
NOVEMBER 1990 VOL. 34 NO. 5
THERIOGENOLOGY 19.
Smith, A.G. and Hooper, M.L. Buffalo rat liver cells produce a diffusible activity which inhibits the differentiation of murine embryonal carcinoma and embryonic stem cells. Dev. Biol. u:l-9 (1987).
20.
Handyside, A., Hooper, M.L., Kaufman, M.H. and Wilmut, I. Towards the isolation of embryonal stem cell lines from the sheep. Roux's Arch. Dev. Biol. m:185-190 (1987).
21.
Mall, R., Franke, W.W., Schiller, D.L., Geiger, B. and Krepler, R. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell u:ll-24 (1982).
22.
Butler, J.E., Anderson, G.B., BonDurant, R.H., Pashen, R.L. and Penedo, M.C.T. Production of ovine chimeras by inner cell mass transplantation. J. Anim. Sci. =:317-324 (1987).
23.
Daniel, W.W. Applied Nonparametric Statistics. co., Palo Alto, CA, 1978, pp. 231-233.
24.
McGarrity, G.J. and Kotani, H. Detection of cell mycoplasmas by a genetic probe. Exp. Cell Res. w:273-278
25.
Isolation and Surani, M.A.H., Torchiana, D. and Barton, S.C. development of the inner cell mass after exposure of mouse embryos to calcium ionophore A23187. J. Embryol. Exp. Morph. 411237-247 (1978).
26.
Smith, A.G., Heath, J.K., Donaldson, D.D., Wong, G.W., Moreau, J., Stahl, M. and Rogers, D. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature 336:688690 (1988).
27.
Williams, R.L., Hilton, D.J., Pease, S., Wilson, T.A., Steward, C.L., Gearing, D.P., Wagner, E.F., Metcalf, D., Nicola, N. and Gough, N.M. Myeloid leukemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature =:684687 (1988).
28.
Ware, C.B. and First, N.L. Development of embryonic stem cell lines from farm animals. Biol. Reprod. 38(Suppl. 1):129 abstr. (1988).
NOVEMBER 1990 VOL. 34 NO. 5
Houghton Mifflin
culture (1986).
877