Long-term culture and characterization of goat primordial germ cells

ELSEVIER

LONG-TERM

CULTURE AND CHARACTERIZATION PRIMORDIAL GERM CELLS

OF GOAT

B. Kiihholzer,‘~2 A. Baguisi,’ E.W. Overstrom”2.3 ‘Department of Biomedical Sciences *Department of Anatomy & Cellular Biology 3Program in Cell Molecular & Developmental Biology Tufts University, School of Veterinary Medicine, North Grafton, MA 01536, IJSA Received for publication: March 2, 1999 Accepted: October 8, 1999 ABSTRACT While the culture and identification of primordial germ cells (PGCs) in mice is established, only limited investigations on PGCs in livestock have been reported. This study was performed to characterize goat PGCs after culture and cryopreservation. Goat PGCs were isolated from Day 32 fetuses and cultured on a continuous cell line of murine embryonal tibroblasts (STO) as feedercells in the presence of leukemia inhibitory factor (LIF). The PGCs proliferated slowly and showed colony formation in early passages. Frozen-thawed PGCs continued to proliferate when stem cell factor (SCF) was added to the culture medium. However, differentiation into epitheliallike polygonal cells or neuronal cells was observed after 1 or 2 passages. The PGCs of 1 female and 1 male cell line were characterized by immunocytochemistry. The PGCs showed positive staining for anti stage-specific embryonic antigen-l (SSEA-1) and FMA-1 (monoclonal antibody produced against a glycoprotein cell surface antigen of the embryonal carcinoma Nulli SCCl), whereas the reactivity to alkaline phosphatase (AP), an established marker for PGCs in mice, was inconsistent. After differentiation, PGCs lost their positive reaction to SSEA-1, EMA- and AI’. In conclusion, SSEA-1 and EMA- can be used as reliable markers for identifying goat PGCs in addition to morphological criteria. The results indicate that goat PGCs can be kept in long-term culture without losing their morphological characteristics and their positive reaction to SSEA-1 and EMA-1, thus providing a promising source of donor-karyoplasts for nuclear transfer procedures. 0 2000 by Elsevier

Science

Inc.

Keywords: goat, fetal germ cells, alkaline phosphatase, SSEA-1, EMAINTRODUCTION Recent advances in the development and application of nuclear transfer technologies to livestock offer the possibility to use a variety of differentiated cell lines as donor karyoplasts, but efficiency remaining relatively low (1, 2, 16, 17). However, the use of undifferentiated, pluripotent fetal germ cells may provide an alternative source of developmentally competent karyoplasts with improved cloning efficiency. The migration of primordial germ cells (PGCs) in mammals from the base of the allantois via the dorsal mesenterium into the gonadal anlage is well described (3). In mice, PGCs have been Theriogenology 8 2000 Elsevier

531071-l Science

079.2000 Inc.

0093-691X/00/$-see PII 50093-691

front matter X(00)00253-3

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characterized, cultured and have been used to produce germ line chimeras following blastocyst injection (5, 7). In domestic species, very little is known about the characterization of PGCs. Most work has been conducted in swine, including the identification of PGCs by alkaline phosphatase (AP) staining, anti stage-specific embryonic antigen-l (SSEA-l), EMA(monoclonal antibody produced against a glycoprotein cell surface antigen of the embryonal carcinoma Nulli SCCI) and lectin-binding (4, 15). Results of long-term culture (12) and production of live chimeric piglets (9, IO. 13) have been recently reported Porcine PGCs have been successfully transfected with a construct containing the ‘humanized’ green fluorescent protein under the control of the cytomegalovirus promoter (9) yet germ line transmission of the transgene has not been demonstrated. Recently, cattle have been produced by using primordial germ cells as donor karyoplasts for nuclear transfer (14, 18) but no detailed identification or characterization of these PGCs was verified In goats, the successfkl isolation of PGCs and their initial characterization based on morphology, AP staining and the ability to differentiate in vitro has been documented (6). The aim of this study was to conduct a detailed assessment and characterization of cultured, subsequently frozen-thawed goat PGCs by immunocytochemical localization of AP, SSEA-I and EMAcellular markers. Specific intermediate filament staining of fixed colonies showed a positive expression of vimentin. Karyotyping of PGCs after spontaneous differentiation was also performed, showing the normal diploid number of chromosomes MATERIALS Isolation and Culture of Primordial

AND METHODS

Germ Cells

Two donor goats were mated naturally and pregnancy was detected by transabdominal ultrasonography on Day 30 of gestation. On Day 32 goats were sacrificed and their reproductive organs were aseptically removed. Three fetuses were recovered (1 singleton, I pair of twins) and stored within the fetal membranes in sterile PBS (without Ca*&/Mg”. PBS-) containing lo3 units/ml penicillin and 0.1 mg/mL streptomycin on ice until PGCs were isolated Each fetus was washed 3 times in PBS Subsequently the gonads, located medial to the mesonephren. were isolated with watchmaker forceps from the surrounding tissue Each pair of gonads was incubated in 0.02% EDTA (Sigma, St. Louis, MO) for 15 min at room temperature. Afterwards each pair was transferred to culture DMEM medium (4500 mg Dglucose/L; Gibco, Grand Island, NY), supplemented with 15% fetal calf serum (FCS, Sigma), 2 mM L-glutamine (Sigma), I mM Na-pyruvate (Gibco), lo3 units/mL penicillin, 0 1 mgimL streptomycin, 0.01 mM nonessential amino acids (MEM, Gibco), 0 I mM fi-mercaptoethanol (Sigma), and 10 ng/mL leukemia inhibitory factor (LIF, human recombinant. Pepro Tech, Rocky Hill, NJ). The gonadal tissue was mechanically disrupted by puncturing with a 30gneedle and repeated pipetting. The suspension of the gonadal tissue of I fetus was seeded on gelatin-coated culture dishes. Suspended PGCs from the other 2 fetuses were seeded on mitomycin C-inactivated ST0 feeder ceils (ATCC, Rockville, MD). All cultures were maintained at 39°C and 59/o CO2 in humidified atmosphere (I pair of gonads per 35-mm well)

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Fibroblasts were obtained from the remaining tissue of the fetus after removal of the head and internal organs except the dorsal mesenterium. Cells were isolated by incubating tissues in 0.1% trypsin/0.02% EDTA for 5 min. The suspension was washed twice and seeded on loo-mm culture dishes. The medium for fetal tibroblast culture consisted of DMEM, 10% FCS, 2 mM Lglutamine, 1 mM Na-pyruvate, lo3 units/ml penicillin, 0.1 mg/mL streptomycin. Fetal tibroblasts were cultured under the same conditions as described for PGCs. The PGCs were frozen in FCS supplemented with 10% dimethyl sulfoxide (DMSO) at -80°C for 1 d before they were transferred into liquid nitrogen (11). Subsequent cultures were generated from frozen PGCs that were thawed in a water bath at 38°C washed in culture medium, and seeded on fresh STO-feeder cells in PGC-medium supplemented with 30 ng/mL stem cell factor (SCF, human recombinant, Pepro Tech). Differentiated PGCs, showing an epithelial-like polygonal morphology, were transferred to loo-mm culture dishes without feeder cells, and were cultured in medium used for tibroblasts Differentiated cells were frozen in fibroblast medium containing 10% DMSO Monoclonal

Antibodies

Monoclonal antibodies against stage-specific embryonic antigen-l (anti-SSEA-1) and EMA-1, developed by D. Solter and M. Eddy, were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the IJniversity of Iowa, Department of Biological Sciences (Iowa City, IA 52242) Secondary antibodies conjugated to Cy3 (goat anti mouse IgG or IgM) were obtained from Accurate Chemical & Scientific Corporation (San Diego, CA). Primary antibodies (monoclonal) against a-vimentin, ctpancytokeratin and the AP-staining kit were purchased from Sigma. Immunocytochemistry Immunocytochemistry was performed on frozen-thawed cells, that were lsultured on gelatin coated glass cover slips for 2 d, and subsequently fixed in methanol/acetone (7:3) for 6 min at -2O“C. All antibodies, SSEA-1 (1: 1), EMA(1:2), cc-vimentin (1.200) cl-pancytokeratin (1:300) and Cy3-labeled goat-anti-mouse IgG and IgM (1.500) were diluted in PBS containing 0.1% polyvinyl-pyrrolidone (PVP; Sigma). To block nonspecific background staining, fixed PGCs were pre-incubated in blocking solution (PBS- containing 1% bovine serum albumin (BSA), 0.2% powdered milk, 2% goat serum, 0. I M glycine; Sigma) for 30 min at room temperature Primary antibodies were then applied to the cover slips and incubated for 90 min at room temperature. After incubation in blocking solution for another 30 min, cover slips were incubated with the secondary antibody over night at 4”C, followed by another 30 min blocking period before they were mounted (PBS containing 50% glycerol) on slides and sealed with nail polish. Immunostaining was evaluated and photo-documented by fluorescence microscopy at x 100 to 600 magnification using an IM-35 inverted microscope equipped with epifluorescence attachment (Cy3 has an absorbtion wavelength of 552 and an emission wavelength of 565nm, rhodamine filter; Zeiss, Oberkochen, Germany). Preparations were also stained for AP-activity before incubation with the primary antibody

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Karyotypic

Theriogenology Analysis

Chromosome counts were determined afier spontaneous differentiation of PGCs at Passage 6 and Passage 7 Cells were arrested at metaphase by culture in the presence of 0 02 pg/mL colcemid (Sigma) for 6 h After trypsinization. the resulting pellet was resuspended in hypotonic solution (75 mM KCL in H20) and incubated at 37°C for 20 min. Cells were then fixed by a 5 min incubation in ice-cold acetic acid-methanol (1.3) followed by three repeated washes in fixative before drops of the cell suspension were put on prewashed cover slips Afier air drying at room temperature, chromosomes w,ere stained with Giemsa solution (0.40/b, Sigma) for 10 min Twenty metaphase spreads were counted for each cell line under x 1000 magnification with oil immersion RESULTS Isolation, Culture and Cryopreservation

of Goat PGCs

PGCs were successfully isolated from gonadal tissues of all 3 fetuses. They were identified on morphological criteria as round (-15 to 20 pm) cells with a large nucleus and granulated cytoplasm. Isolated PGCs showed cytoplasmic blebbing the first d after isolation, and some cells were still blebbing after 48 h Cells were identified morphologically as PGCs when they showed the appearance of pseudopodia, a characteristic of migrating cells (Figure la). PGCs of I fetus (No 654-l) were cultured on gelatin without STO-feeder. PGCs proliferated slowly the first 5 d in culture, and subsequently differentiated into epithelial-like polygonal cells (Figure lc) Cells 593-3) were cultured on cells showed

suspensions derived from the gonads of the remaining 2 fetuses (Nos. 593-l and cultured on inactivated ST0 cells. Similar to PGCs cultured on gelatin alone, PGCs ST0 monolayer started to differentiate after 4 to 5 d Following differentiation, the extensive proliferation

After 4 d in culture fetal fibroblasts of 2 fetuses (Nos. 593-l and 593-2) showed round cells (PGCs) on the surface of the fibroblast monolayer Before passaging the fetal fibroblasts, those PGCs were disaggregated in 0.02% EDTA at room temperature while monitoring microscopically (x 50 magnification). The PGCs were isolated from the fibroblasts monolayer by carehI pipetting, washed and seeded on fresh STO-feeder layers. Further culture was performed on 35-mm dishes with PGC-culture medium PGCs continued to proliferate and after 3 d in culture colony formation was observed (see Figure lb) Some colonies were trypsinized after 6 d in culture (from P2) and passaged on fresh feeder cells in 23-well dishes to produce clonal lines. Some clonal lines differentiated into neuronal cells (see Figure Id) The remaining cells were passaged I:2 every 4 to 6 d depending on the extent of their proliferation. Remaining fibroblasts were proliferating rapidly as opposed to PGCs; therefore, the preplating technique was used to purify PGCs before transferring the cells onto fresh ST0 feeder From Passage 3 on. no further colony formation was observed The PGCs continued to proliferate, but only single cells or small groups of PGCs without any development to colonies were seen (Figure la) In this study, PGCs were kept in culture for up to 3 mo without

Theriogenology

Figure

1. a) Cultured goat PGCs on STO-feeder (x 200); b) Colony of PGCs (x 100); c, d) spontaneously differentiated PGCs: c) differentiated into polygonal cells (x 200) and d) differentiated into cells with neuronal morphology (arrow, x 200); e, f) Immunocytochemical analyzes of frozen-thawed PGCs: e) Two individual PGCs on STO-feeder: brightfield and f) same cells stained for SSEA-1 (x 600).

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differentiation. Frozen-thawed PGCs required 2 to 5 d before they continued to proliferate. Early passages showed colony formation similar to those prior to cryopreservation. However, most of the frozen-thawed samples started to differentiate into epithelial-like polygonal cells after 2 to 3 tirther passages. Immunocytochemistry All frozen-thawed cells, morphologically determined as PGCs, showed positive staining for SSEA-1 and EMA-I (see Figures le, f), whereas only a few of them stained positive for alkaline phosphatase activity. Some colonies from both lines failed to stain positive for SSEA-1 and EMA-1, indicating finther differentiation of these cells. However, all colonies that showed AP-staining also stained for SSEA- 1 or EMA- I. Results of immunocytochemistry for different cell lines are summarized in Table 1. Table 1. Immunocytochemistry

of frozen-thawed

PGCs before and after differentiationa

Cell line/ID No

AP

SSEA-1

EMA- 1

PGC, PGC, PGC, PGC, Diff,, Diff., ST0

+I+/+I+I-

+ +/+ +I-

+ +I+ +I-

593-l 593-1, colonies 593-2 593-2. colonies 593- 1 593-2 (control)

-

Vimentin ND + ND -1. + + +

pan-Cytokeratin ND ND

aAP, alkaline phosphatase; SSEA-I, stage-specific embryonic antigen-l ; EM A-l, monoclonal antibody against surface of embnjonal carcinoma cells (YJulli SCC I). Diff, differentiated polygonal cells. -, no apparent staining; I, positive staining, t-i-. inconsistent staining, ND. not determined. Control Experiments Nonspecific staining was excluded by incubating cells with secondary antibodies alone. No detectable fluorescence staining was observed after incubation with either of the secondary antibodies used in this study (goat anti-mouse IgG-Cy3 and goat anti-mouse IgM-Cy3, data not shown). Additionally ST0 cells and differentiated PGCs mere stained according to the same staining protocol described above Neither ST0 cells nor differentiated polygonal cells showed positive staining to AP, pan-cytokeratin, SSEA-I or EMA-I In contrast, both cell types stained positive for vimentin (see Table 1)

Karyotypic

Analysis

Metaphase spreads of both cell lines showed 60 chromosomes, indicating the normal diploid number of chromosomes in goats (8), line 593-l being female and line 593-2 male.

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DISCUSSION In this study PGCs were successfully isolated from gonads of goat fetuses on Day 32 of gestation, Additionally, a high percentage of PGCs proliferated when the dorsal mesenterium was included in the tissue used for preparation of fetal fibroblast lines. This indicates that a large number of PGCs is still present in the dorsal mesenterium early in the fifth week of gestation. When PGCs started to proliferate on the primary fibroblast culture, no LIF was included in the culture medium. Still, no differentiation was observed. This leads to the conclusion that goat fibroblasts, when inactivated, could provide a suitable feeder-layer for the culture of PGCs, and that at least for the first day of culture supplementation of the culture medium with LIF does not seem to be necessary. The pluripotency of cultured and frozen-thawed PGCs was demonstrated by their ability to differentiate spontaneously into various cell types (neuronal, epithelial-like polygonal cells), also observed for porcine PGCs by Shim et al. (13). To further characterize PGCs, staining to AP, SSEA-1 and EMAwas performed Alkaline phosphatase, a specific marker for undifferentiated PGCs and ES-cells in mice (7) failed to be specific for goat PGCs due to the inconsistency of staining. No staining of goat PGCs was observed after differentiation. However, AP negative cells, morphologically determined as PGCs, did show positive reaction to SSEA-1 or EMA-1, a phenomenon also observed for porcine PGCs by Tagaki et al. (15). As soon as PGCs started to differentiate they lost their positive reaction to both antibodies, which emphasizes that SSEA-I and EMAcan be used as specific markers for the identification of undifferentiated caprine PGCS. In mice the injection of cultured PGCs succeeded in the production of germ line chimera (7). Recently, the production of porcine chimera after injection of transfected PGCs into blastocysts was reported (9) also the nuclear transfer using PGCs as donor karyoplast in cattle resulted in the birth of live offspring (14, 18). Due to the limited success in the culture of embryonic stem cells in livestock and the very low efficiency of nuclear transfer using differentiated somatic cells, PGCs provide a promising karyoplast source for goat nuclear transfer technologies. In conclusion, goat PGCs from Day 32 fetuses can be successfully isolated from the dorsal mesenterium and the gonads. The culture system used in this study was suitable to keep PGCs in their undifferentiated stage. This suggests that the ST0 feeder could be replaced by inactivated goat fibroblasts with supplementation of LIF and SCF to the culture medium. The ability to keep PGCs in long-term culture (3 mo in this study) and to cryopreserve them provides an alternative source of donor karyoplasts for nuclear transfer procedures. Due to their origin the use of PGCs might improve the efficiency of further efforts in cloning goats.

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