Subculture of Germ Cell-Derived Colonies with GATA4-Positive Feeder Cells from Neonatal Pig Testes

Enrichment of spermatogonial stem cells is important for studying their self-renewal and differentiation. Although germ cell-derived colonies (GDCs) have been successfully cultured from neonatal pig testicular cells under 31°C conditions, the short period of in vitro maintenance (<2 months) limited their application to further investigations. To develop a culture method that allows for in vitro maintenance of GDCs for long periods, we subcultured the GDCs with freshly prepared somatic cells from neonatal pig testes as feeder cells. The subcultured GDCs were maintained up to passage 13 with the fresh feeder cells (FFCs) and then frozen. Eight months later, the frozen GDCs could again form the colonies on FFCs as shown in passages 1 to 13. Immunocytochemistry data revealed that the FFCs expressed GATA-binding protein 4 (GATA4), which is also detected in the cells of neonatal testes and total testicular cells, and that the expression of GATA4 was decreased in used old feeder cells. The subcultured GDCs in each passage had germ and stem cell characteristics, and flow cytometric analyses revealed that ~60% of these cells were GFRα-1 positive. In conclusion, neonatal pig testes-derived GDCs can be maintained for long periods with GATA4-expressing testicular somatic cells.


Introduction
Spermatogonial stem cells (SSCs) are the first precursors to spermatogenic cells (spermatocytes, spermatids, and spermatozoa) and are essential for continued spermatogenesis throughout the life span [1]. Owing to their small population, long-term culture-mediated enrichment of SSCs is important for investigating their self-renewal and differentiation. To date, long-term culture of SSCs has only been reported in rodents [2]. A 5-month culture of mice SSCs has been reported, where mouse embryonic fibroblasts (MEFs) were used as feeder cells [3]. Hamster SSCs cultured with mitomycin-C-treated MEFs for 3 passages have been cultured for 1 year without feeder cells [4]. Additionally, mice SSCs cultured with SIM mouse embryo-derived thioguanine and ouabain-resistant (STO) cell feeders have been shown to proliferate for 7 months [5]. In humans, stage-specific embryonic antigen-4-positive SSCs have been cultured for 4 months without feeder cells [4]. Furthermore, transplantation of mice SSCs results in the production of sperms in recipient mice. In contrast to rodents, long-term culture of SSCs is difficult in domestic animals because of the unavailability of feeder cells (like MEFs or STO cells) for subculture, although shortterm culture (<2 months) has been reported [6]. In our previous study, we reported that male germ cell-derived colonies (GDCs) effectively formed in 10 days at 31 ∘ C and that the transplanted GDCs colonized recipient testes at 8 weeks after transplantation [7]. In this study, we demonstrate a method for the subculture and enrichment of porcine GDCs using feeder cells isolated from neonatal pig testes.

Sample Preparation.
Ten testes from castrated Yorkshireand Landrace-crossed, 5-day-old, hybrid piglets were obtained from the Samwoo Livestock (Yangpyeong, Korea). Tunicae albugineae of the testes were removed for immunocytochemistry and cell culture preparations. The study protocol and standard operating procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Konkuk University (IACUC approval number: KU13035).

Feeder Cell Preparation.
Feeder cells were prepared according to a previously reported protocol with some modifications [8]. Five volumes (v/w) of enzyme A (0.5 mg/mL collagenase, 0.01 mg/mL DNase I, 0.1 mg/mL soybean trypsin inhibitor, and 0.1 mg/mL hyaluronidase) were added to the decapsulated testes, and the testes were incubated for 10 min at 37 ∘ C. The testes were then washed with phosphate-buffered saline (PBS). Next, 5 volumes (v/w, original testes weights) of enzyme B (5 mg/mL collagenase, 0.01 mg/mL DNase I, and 0.1 mg/mL soybean trypsin inhibitor) were added, and the testes were incubated for a further 10 min at 37 ∘ C. The enzyme-treated testes were then washed with PBS and meshed using a 40 m nylon mesh. Red blood cells were eliminated using a lysis buffer (Sigma-Aldrich, St. Louis, MO, USA). Two Percoll densities, 20% and 40% (Sigma-Aldrich), were used to collect feeder cells. The 40% Percoll suspension was prepared by mixing 40% Percoll solution, 10% PBS (v/w), 1% fetal bovine serum (FBS, v/w), 0.5% antibiotics (v/w, 50 U/mL and 50 g/mL penicillin and streptomycin, resp.), and 48.5% ultrapure water (v/w) in a 15 mL conical tube. The 20% suspension was prepared by diluting the isoosmotic 40% Percoll suspension with PBS supplemented with 1% FBS (v/w) and antibiotics (as above). The cell suspension (2 mL of 5 × 10 6 cells/mL) was loaded on top of the gradient and centrifuged at 600 ×g for 10 min at 4 ∘ C. The cells at top layer (40% Percoll layer) were collected and used as feeder cells. They were analyzed by immunocytochemistry.

Cell Culture for GDCs and Subculture with Fresh Feeder
Cells (FFCs). Total testicular cell (TTC) culture was performed to obtain GDCs. The protocol for single cell preparation was the same as that used for the feeder cells. The isolated cells were seeded onto 0.2% (w/v) gelatin-coated 12well plates and incubated at 31 ∘ C in a 5% CO 2 atmosphere. The StemPro-34 medium (Gibco, Carlsbad, CA, USA) was used for all processes, including derivation and culture of porcine GDCs. The medium was supplemented with the following: insulin-transferrin-selenium (ITS, 25 g/mL, 100 g/mL, or 30 nM), 6 mg/mL glucose, 2 mM L-glutamine, 1% NEAA solution, 1% vitamin solution, 100 units/mL penicillin/streptomycin, 1 mM sodium pyruvate, 0.1 mM vitamin C, 1 g/mL lactic acid, 30 ng/mL estradiol, 60 ng/mL progesterone, 0.2% bovine serum albumin (BSA), 1% knockout serum replacement, 20 ng/mL mEGF, 10 ng/mL bFGF, 10 ng/mL GDNF, and 10 3 U/mL leukemia inhibitory factor. The GDCs were collected on days 10 to 12. They were suspended as single cells using 0.25% trypsin-EDTA and then moved to new gelatin-coated tissue culture plates with feeder cells; in total, 2 × 10 4 GDCs and 1.8 × 10 5 feeder cells were added to each well. To subculture the GDCs, two kinds of feeder cell were used: old feeder cells (OFCs)-those attached to the wells in passages 0 and 1 after colony collection on days 10 to 12-and FFCs-those isolated from the top of the 40% Percoll layer during density separation of the TTCs. The cells from GDCs in passage 13 were frozen with 10% dimethyl sulfoxide (DMSO) and cultured with FFCs 8 months later.

Cell Labeling.
To examine whether GDCs can be maintained in subculture, cells derived from them were labeled with PKH26 red fluorescent dye (Sigma-Aldrich) according to the manufacturer's instructions. The cells were isolated from the colonies in passages 1 and 13 by treatment with 0.25% trypsin for 10 min at 37 ∘ C. The cells from the GDCs at passages 1 and 10 were labeled with PKH26 and then used as germ cells in passages 2 and 11. They were then observed with an excitation filter of 450-560 nm under a fluorescence microscope (Nikon, Tokyo, Japan) at a magnification of 400x.

Subculture of Frozen Colony
Cells. The GDC cells in passage 13 were frozen in 10% DMSO and the StemPro-34 medium for 8 months. These cells were thawed at 37 ∘ C for 1 min, labeled with PKH26 red fluorescent dye (Sigma-Aldrich) (as per manufacturer's instructions), and then cultured with FFCs. They were then observed using an excitation filter of 450-560 nm under a fluorescence microscope (Nikon) at a magnification of 100x.
2.6. Immunocytochemistry. Following Percoll gradient separation, cells from the top layer were fixed with 4% paraformaldehyde (PFA, w/v) in PBS and then washed with PBS. Next, they were attached to amino saline-coated slides (Matsunami, Osaka, Japan) for immunocytochemistry. The fixed cells were incubated with the antibody against protein gene product (PGP 9.5) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), raised in rabbits against human PGP 9.5 (1 : 100; AbD Serotec, Raleigh, NC, USA), at 4 ∘ C overnight. Following washing with PBS, the cells were further incubated with anti-rabbit Alexa 568 (1 : 500; Invitrogen, Carlsbad, CA, USA) against the PGP 9.5 antibody. The cell nuclei were stained with 4 ,6 -diamidino-2-phenylindole (DAPI) staining solution (Vector Laboratories, Burlingame, CA, USA). The same procedure was followed for cells of the colonies in passages 2 and 11, using antibodies against PGP 9.5 and GDNF family receptor alpha-1 (GFR -1) (1 : 50; Santa Cruz Biotechnology). Anti-mouse Alexa 488 (1 : 500; Invitrogen) was used as secondary antibody. Feeder cells attached to the wells at passage 2 were incubated with antibody against GATA-binding protein 4 (GATA4) at 4 ∘ C overnight and then washed with PBS. The cells were then incubated with horseradish peroxidase-(HRP-) conjugated secondary antibody (1 : 500; Santa Cruz Biotechnology) for 1 h at room temperature. This was followed by incubation with 3,3 -diaminobenzidine for staining cell nuclei (Vector Laboratories). TTCs that were collected before cell culture were double-stained with antibodies against PGP 9.5 and GATA4 (1 : 50; Santa Cruz Biotechnology). The procedure Stem Cells International 3 was the same as that for PGP 9.5 and GFR -1 double staining. Finally, the cells were observed using an excitation filter of 450-560 nm under a fluorescence microscope (Nikon).

2.7.
Immunohistochemistry. Briefly, 6 m thick sections of the testis from 5-day-old piglets were deparaffinized with xylene and treated with ethanol. The sections were incubated with a target unmasking fluid (Accurate Chemical & Scientific Corp., Westbury, NY, USA) for 15 min using a microwave oven to retrieve the antigens. The slides were then washed thrice with PBS and blocked with 10% normal goat serum (v/v). For double staining, the slides were incubated with anti-GATA4 (1 : 100; Santa Cruz Biotechnology) and anti-PGP 9.5 antibodies (1 : 100; AbD Serotec, Raleigh, NC, USA) at 4 ∘ C overnight and then washed thrice with PBS. Some of the sections were incubated with 1% BSA as negative controls. Next, the sections were incubated with anti-mouse Alexa 488 and anti-rabbit Alexa 568 (both 1 : 500; Invitrogen) against anti-GFR -1 and anti-PGP 9.5 antibodies, respectively, for 1 h at 25 ∘ C (room temperature). This was followed by incubation with DAPI (Vector Laboratories). The slides were then washed with PBS and observed using an excitation filter of 450-560 nm under a fluorescence microscope (Nikon) at a magnification of 200x.

Alkaline Phosphatase (AP)
Staining. AP staining was performed using a CBA-300 AP staining kit (Cell Biolabs, San Diego, CA, USA) according to the manufacturer's instructions. Briefly, cultured cells including GDCs were fixed in fixation solution, washed thrice with PBS, and incubated with the AP staining solution for 10-20 min. Next, the AP solution was removed and the cells were washed with PBS and observed under a microscope at a magnification of 200x.

Subculture of GDCs Using FFCs.
In a previous report, we showed that the germ cell-rich layer can be collected by density separation using 20% and 40% Percoll [8]. In this study, we observed only few PGP 9.5-positive cells among those isolated from the top of the 40% Percoll layer upon density separation of the TTCs (Figure 1(a)). A previous study reported the formation of porcine germ cell colonies in StemPro-34 medium at 31 ∘ C within days 10 and 12 [7]. In the present study, cells isolated from the top of the 40% Percoll layer were cultured in StemPro-34 medium at 31 ∘ C and did not form colonies until day 12, and only the cells attached to the wells showed proliferation (Figure 1(b)). These results indicate that these isolated cells are mostly PGP 9.5 negative. GDCs were observed in passage 0 on day 10 ( Figure 2(A)). When cells prepared from GDCs in passage 0 were cultured with OFCs, some colonies formed in passage 1 were smaller than those in passage 0 ( Figure 2(B)). Colonies similar to the initial colony in Figure 1 (Table 1).

Maintenance and Characterization of GDCs in Subculture.
Red-labeled GDC cells, from passages 1 and 10, were cultured ). In neonatal testis tissues, GATA4, which is involved in mammalian testis development, was detected in the Sertoli and interstitial cells, but not in testicular germ cells that were stained with PGP 9.5 (Figures 3(E) and 3(F)). Further, immunocytochemistry of TTCs revealed that testicular germ cells were positive for PGP 9.5 antibody and GATA4 (Figure 3(G)). PGP 9.5-and GATA4negative cells were also detected in the TTCs (Figure 3(G)). Nuclei of the cells in Figure 3(G) were stained with DAPI ( Figure 3(H)). Bright-field microscopic examination revealed the germ cells to be rounded, measuring around 10 m in diameter, while the testicular somatic cells appeared to have irregular forms (Figure 3(I)). In addition, immunocytochemistry was performed for feeder cells that were attached to the well on day 10 at passage 2. No protein was detected in the negative controls (Figure 3(J)). Among OFCs, both GATA4-positive and GATA4-negative nuclei were detected (Figure 3(K)). In contrast, all attached FFCs were GATA4 positive (Figure 3(L)). GATA4 transcript levels were also higher in the attached cells in FFCs in passage 2 than in passage 2 OFCs (Figure 3(M)). To investigate the characteristics of the colonies in each passage, immunocytochemistry, RT-PCR, and flow cytometry were performed. PGP 9.5 and GFR -1 were detected in GDC cells collected in passages 2 and 11. Both the nuclei and morphology of these cells were normal (Figures 4(a) and 4(b)). Further, our RT-PCR results showed that the germ cell markers PGP 9.5, GFR -1, and PLZF were expressed in the GDCs in passages 0 through 14; GDCs cultured with FFCs from frozen GDC cells at passage 13 had the same characteristics as those cultured from GDC cells at passages 0 to 10. Furthermore, GDCs at all the passages were found to express the stem cell markers Oct4 and Nanog (Figure 4(c)). Our indirect flow cytometry analyses revealed 63.2 ± 4.51% and 67.31 ± 6.73% GFR -1-positive GDC cells at passages 0 and 8, respectively (Figure 4(d)); GFR -1-negative cells were also observed, but these were considered somatic cell contamination that occurred during GDC collection. To confirm long-term maintenance, cells labeled red from the frozen GDCs at passage 13 were subcultured with FFCs. On

Discussion
Several groups have reported the culture of SSCs of domestic animals. Aponte et al. reported that the StemPro-34 medium with 4 growth factors was optimal for short-term culture (15 days) of bovine SSCs. They further expanded the culture period up to 33 days, and when the cells cultured for 30 days were transplanted into the seminiferous tubules of nude mice, proliferation of type A spermatogonia was observed [9]. In pigs, short-term culture of gonocytes has been reported. Goel et al. cultured purified gonocytes in DMEM/F12 supplemented with 10% FBS in the absence of specific growth factors for 7 days. They observed cell proliferation and subsequent formation of colonies [10]. Further, Kuijk et al. reported that cell lines obtained from neonatal pig testes and cultured in the StemPro-34 medium could be maintained for up to 9 passages after cessation of proliferation and that growth factors affect the culturing of porcine spermatogonia [11]. However, the lack of established feeder cells limits the longterm culture of SSCs from domestic animals as used in mice Stem Cells International even though growth factors have an important role in shortterm SSC culture. Previously, we found that a temperature of 31 ∘ C, which positively correlates with cell growth, is optimal for GDC formation. In addition, GDCs were not detected in the presence of mitomycin-C-treated feeder cells (like MEFs and STO cells) [7]. In this study, we purified somatic cells from TTCs using density gradient separation and showed that the purified FFCs can be used for subculture of porcine GDCs. The GDCs were maintained in each passage as cell lines despite a few germ cells being present in the isolated somatic cells. PGP 9.5 is known to be specifically expressed in porcine spermatogonia in vivo [12] and is often used as a marker for the same [7,10]. GFR -1, which combines with GDNF, is a cell surface receptor of undifferentiated spermatogonia in rodents, and it is often used to sort undifferentiated spermatogonia [13][14][15]. In previous studies, we found that PGP 9.5 and GFR -1 transcripts are highly expressed in porcine GDCs and spermatogonia, respectively [7,8]. Recent studies have shown that these markers are constitutively expressed in colonies of all passages. It has been reported that Nanog, Oct4, and Sox2, often used as stem cell markers, function cooperatively in the regulatory network of self-renewal and pluripotency [16]. Expression of POU5F1 and NANOG transcripts has also been reported in domestic animals. For instance, higher mRNA expression of both POU5F1 and NANOG was detected in porcine colonies [7]. Further, POU5F1, but not NANOG, was expressed in mouse germinal stem cells cultured under serum-and feeder-free conditions [17] and in porcine germ cell colonies [11]. POU5F1 transcripts were detected in 20-week-old testes but not in neonatal ones (2-7 days) [18]. In our previous results, POU5F1 and NANOG mRNAs were highly expressed in GDCs cultured in the StemPro-34 medium at 31 ∘ C, and GFR -1-positive cells expressed Oct4 and Nanog mRNA [7,8]. Based on these reports, it seems that germ and stem cell markers are expressed in cultured germ cells of domestic animals. In this study, cells of the colonies at each passage were found to be positive for GFR -1 and PGP 9.5, as well as for stem cell markers, in RT-PCR, immunocytochemistry, and flow cytometry analyses. These results suggest that the GDCs stably maintain germ and stem cell characteristics during subculture. GATA4 is known to be involved in the development and function of the mammalian testis [19]. In humans, the p.Gly221Arg mutation in GATA4 leads to anomalous testicular development [20]. GATA4 is also a key regulator of Sertoli cell function in adult mice [21]. It plays an integral role in the development of testicular steroidogenic cells [22]. Furthermore, GATA4 was also detected in the nuclei of mouse and human granulosa and thecal cells [23,24]. In addition, the nuclei of 90-95% granulosa cells of porcine primordial, unilaminar, multilaminar, and antral follicles of different sizes stain positive for GATA4 [25]. McCoard et al. reported that GATA4 localizes to the coelomic epithelium of gonads and to the Sertoli and follicle cells before and after sex differentiation, respectively [26]. It is clear from these reports that GATA4 plays an important role in gonadal somatic cells that are involved in spermatogenesis and oogenesis. In this study, we found GATA4-positive cells in TTCs and testis tissues. FFCs cultured with the colonies, which were attached to the culture dish, were positive for GATA4. Moreover, GATA4 expression was stronger in FFCs than in OFCs, and it was detected in Sertoli cells of neonatal pig testes. These results suggest that GATA4-positive cells support GDC formation in two-dimensional culture and that, during culture with GDCs, the GATA4-expressing FFCs probably play a role similar to that of somatic cells in the testessupporting germ cell growth and proliferation.

Conclusions
In conclusion, isolated GATA4-positive somatic cells can be used as feeder cells for long-term culture of porcine GDCs (>4 months) and the GDCs thus cultured maintain germ and stem cell characteristics during subculture.