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Isolation, culture, and characterization of primordial germ cells in Mongolian sheep [In Vitro Cellular & Developmental Biology](In Vitro Cellular & Developmental Biology Via Acquire Media NewsEdge) Received: 24 February 2013 / Accepted: 13 September 2013 / Published online: 19 October 2013 / Editor: T. Okamoto © The Society for In Vitro Biology 2013 Abstract This study was performed to culture and preliminarily identify the primordial germ cells (PGCs) isolated from the genital ridge of the Mongolian sheep fetus. The growth characteristics of the sheep PGCs were detected in different culture systems such as culture media, resources, and state and passages of feeder cells. The obtained embryonic germ (EG) cells were identified by morphology, enzymology, and immunofluorescence. The results showed that the sheep EG cell colonies were ridgy, typically nest like, and compact, and had regular edges. Alkaline phosphatase staining reaction was weakly positive. EG cells expressed Kit, Rex-1, Nanog, and Oct-4. Immunofluorescence detection was weakly positive for Oct3/4, whereas positive for SSEA-1, SSEA-3, SSEA-4, TRA-1-61, and TRA-1-80. Keywords Sheep . Primordial germ cell . Culture . Characterization Introduction Stem cell lines, though representing an attractive area of research for the scientific community, have many questions that need to be answered before their use as a model for regenerative therapies (Yu and Thomson 2008). Since embryonic stem (ES) cells derived from mice and humans differ considerably, the production of ES cell lines from large mammals may be an advantage as they are more physiologically similar to humans and perhaps more relevant for clinical transplantation studies compared with mice ES cells (Hall 2008). Primordial germ cells (PGCs) from the fetal original gonads are the precursor cells of mature gametes. In the late embryonic developmental stage, the PGCs migrate to the genital ridge through the dorsal mesentery and then proliferate rapidly there. They have developmental totipotency (with the potential for differentiation and development into three germ cell layers). PGCs can form embryonic germ (EG) cells in vitro under certain culture conditions, and ES cells could be obtained from the inner cell mass of the early embryo. Compared to ES cells, EG cells can be obtained more easily, because the number of PGCs is more than ICM; also, isolating the cells is relatively easier. The totipotent cell lines established can be manipulated in vitro, such as culture, cloning, and cryopreservation, and at the same time have the ability of maintaining the undifferentiated state, so they become the important materials in the study of early mammalian cell differentiation. There have been successful reports about PGC culture published, such as mice (De Felici and McLaren 1982;Matsuietal.1992), rats (Jiang 1998), pig (Shim et al. 1997), cattle (Wrobel and Süß 1998), human (Shamblott et al. 1998), goats (Lee et al. 1998; Kühholzer et al. 2000), and rabbits (Lee et al. 1998;Kakegawaetal.2008), etc., but less research on the sheep. In 1996, Ledda et al. (1996)separated the PGCs from 25~35-d-old sheep fetuses with different separation methods and observed many with different vitality in trypsin and collagenase digestion. Dattena et al. (2006)reported that sheep ES cells could be passaged to five generations in vitro. A paper relevant to this research was published by Ledda et al. (2010), who separated PGCs from sheep fetal genital ridge of different ages and obtained ten passages of EG cells. In this study, we isolated PGCs from the sheep genital ridge, cultured EG cells in vitro, and made a preliminary identification. All these will set up the basic method of isolation and culture of PGCs in sheep to investigate the biology of the PGCs, meanwhile forming the basis of establishing EG and ES cell lines in sheep. Moreover, the establishment of large mammal ES cells may provide an alternate source of cells for basic research to understand the mechanism of cell differentiation and repair, for studying diseases and disease mechanisms, and for future cell transplantation, and could overcome the ethical problems related to the use of human embryos to generate ES cell lines. Materials and Methods Reagents. High-quality fetal bovine serum, DMEM/F12, Serum Replacement (SR), and Knockout-DMEM were purchased from Life Technologies (Carlsbad, CA). Mouse ES Cell Marker Sample Kit (SCR002) was from Millipore (Billerica, MA). Mitomycin C, Gelatin, was from Sigma (St. Louis, MO). LIF was from Promega (Fitchburg, WI). Rabbit anti-sheep IgG+ IgM FITC secondary antigen was from KPL (Gaithersburg, MD). PrimeScript® RT Reagent Kit Perfect Real Time (DRR037A) and Premix Ex Tag® version 2.0 (loading dye mix, D335A) were from Takala (Tokyo, Japan). TRIzol reagent was purchased from CoWin (Beijing, China). Materials. Mongolian sheep fetuses were collected from a local slaughterhouse (Wuzhumuqin sheep). The conceived 12.5~14.5-d-old Kunming strain mice were bought from Inner Mongolia University animal research center. Preparation of feeder layer cells and cryopreservation. The primary to third passage mouse embryonic fibroblasts (MEF in C57) or Mongolian sheep fetal fibroblasts (SFF), which covered the bottom of the dishes, were incubated with 29.91 mol/L mitomycin for 3 h, on the condition of 37°C, 5% CO2, and 100% humidity. Then the medium containing mitomycin C was discarded completely, and the MEF or the SFF cells were washed seven times with DMEM medium to thoroughly clean the cell surfaces of mitomycin C residues. Then the MEF or the SFF cells inactivated by mitomycin C were digested by 0.25% trypsin-0.04% EDTA solution (TE). The same amounts of the MEF or the SFF culture medium were added to terminate the digestion when the MEF or the SFF cells became retracted as observed in the microscope field. The supernatant was removed after centrifugation of 1, 500 rpm for 5 min; later, a certain concentration of the single cell suspension was made by adding the culture fluid. The freshly collected feeder cells in medium (DMEM/ F12:FBS:DMSO = 7:2:1) were frozen, transferred to vials, placed in a frozen box, and put in a -70°C low-temperature refrigerator and the next day into liquid nitrogen. The feeder cells were thawed at 37~38°C before using, centrifuged to remove DMSO, diluted with culture medium to 1 x 105 cells/ mL, seeded on culture dishes treated by 0.1% gelatin in advance in 100% humidity atmosphere of 5% CO2 at 37°C, and prepared well the day before passage. Isolation and culture of Mongolian sheep. PGCs of 20~40-dold Mongolian sheep embryos, collected from the local slaughterhouse, were brought back to the laboratory, together with the uterus. The uterus was opened, and the gestational sac was taken out carefully. The embryo's age was determined according the length of the embryos. The embryo and amniotic membranes were separated using a sterile scalpel and forceps; the fetus was washed three times with phosphatebuffered saline (PBS) containing antibiotics. The gonadal ridge and its surrounding tissue were separated according the methods of Moreno-Ortiz et al. (2009), and then the genital gonadal ridge was cut into 1-mm3 tissue blocks and transferred into a 7-mL centrifuge tube with 2 mL of prewarmed TE at 37°C. Gently breaking up the pieces by pipetting up and down for less than 10 min, the digestion was terminated by adding an equal volume of serum-containing DMEM medium to the tube. The supernatant was discarded after 2,000 rpm centrifugation for 5 min at room temperature. The pellets were resuspended in EG cell culture medium (see Table 1), then seeded into 30-mm cell culture dishes, and cultured in 5% CO2 and saturated humidity atmosphere at 37°C. After 48 h, the medium was removed from the very top of the culture to avoid disturbing the colony's attachment. The fresh media prewarmed at 37°C were added. This would be done every 2 d for 8-10 d until the colonies formed, but when they could be passaged firstly, they could be cultured for another 20 d. Subculture of PGCs. The primary colonies were dissected mechanically into small colonies, e.g., two, four, six, or eight pieces, according to the size of the dissected colony with a homemade cutting knife, and then the small colonies were transferred to the prepared feeder layer with a fine glass needle. The EG cell culture medium was changed after the pieces adhered to the feeder layer; cell growth was observed and recorded. Generally, colonies could be passaged after 7 d of growth; meanwhile, the culture medium was changed every 24 h. Identification of morphological characteristics of the Mongolian sheep PGCs. Sheep EG cells were identified with reference to the morphological characteristics of other animals. Alkaline phosphatase staining. Briefly, the colonies were fixed in 4% paraformaldehyde fixative solution for 1 h at 37°C, washed three times with PBS, then incubated in the balance of substrate buffer for 20 min, washed three times with PBS again, finally incubated at room temperature for about 45 min of dark staining with the AP staining solution, and rinsed again with PBS. The cells that stained purple, which is an indicator of AP, were considered to be the EG cells. Reverse-transcription polymerase chain reaction. Total RNA was isolated from the seventh passage sheep EG cells using the "TRIzol Reagent" (CoWin) as per manufacturer's instructions. Briefly, the cells were washed with ice-cold PBS, 20- 50 µL of cold cell lysis buffer was added, and the reaction mixture was incubated in a programmable temperature controller (PolyScience, Warrington, PA) at 75°C for 10 min. The cell lysate was treated with DNase-I and RNase inhibitor at 37°C for 20 min to degrade genomic DNA and then heated at 75°C for 5 min to inactivate DNase-I. The total RNA/cell lysate (6.5 µL) + 0.5 µL of 1x Oligo dTPrimer (50 µM) + 0.5 µL of 1x Random 6 mers (100 µM) + 2 µLof5x primescriptTM buffer + 0.5 µLofPrimescriptTMRT Enzyme mix was added, and the mixture was incubated at 37°C for 15 min and then at 85°C for 5 s. The PCR cycle included heating to 94°C for 5 min, followed by repeated cycles of 94°C for 30 s, annealing temperature 56-59°C for 30 s, and extension at 72°C for 40 s; this cycle was repeated 35 times with a final extension at 72°C for 7 min. The PCR primers were listed in Table 2. GAPDH was amplified as a housekeeping marker gene. Feeder layer cells were used as negative control for EG-like cells. The PCR products were examined by 1.5% agarose gel electrophoresis, and the fragment size was determined based on the MW Marke. Immunofluorescence identification. For immunocytochemistry studies, the 8th, 10th, and 13th passages of EG cells were grown on a four-well culture plate coated with 0.1% gelatin, rinsed for 5 min in PBS, and then were fixed by 20-min incubation in PBS containing 4% paraformaldehyde, rinsed in PBS, and stored at 4°C in this buffer until use. All subsequent steps of permeabilization, washing, and incubation with antibodies were performed at room temperature. Fixed cells were permeabilized for 10 min in PBS containing 0.1% Triton X, blocked for 30 min in PBS goat serum (PBS containing 1% goat serum), and incubated for 60 min with the primary antibody diluted 1:10-1:100 (Oct-4, SSEA-1, SSEA-3, SSEA-4, TRA-1-61, and TRA-1-80, Millipore) and then for 60 min with a rabbit anti-sheep IgG + IgM FITC-labeled secondary antigen (KPL) diluted 1:10-1:100. PBS was used for washing between incubations, rinsed twice, each time for 10 min. The control group did not include primary antibody, and the other manipulation was similar to the experimental group. The cells were examined under a fluorescence microscope immediately. Results Growth behavior of PGC clones and AP staining results. Sheep PGCs exhibited morphological features similar to those of mouse ES cells and appeared round or oval, larger than the surrounding cells. Typically nest-like EG cell clones appeared when cultured for 7 to 10 d. The clones were ridgy with regular edges, and the cells were compactly linked. The primary colonies generally looked dark brown (maybe due to more nonhomogeneous cells) and showed hill-like, bread-like morphology (Fig. 1A). At the early stage of passages, cells at the top were black (Fig. 1B). With the increase of culture time, gradually, the new cells grew up from the black area. Typically, nest-like EG cell colonies relatively increased and showed different sizes companied with the increasing passages (Fig. 1C). After the AP staining, the fourth passage colonies were weakly positive (Fig. 1D). Growth behavior of PGCs in different culture systems. The growth state of sheep EG cells in different culture systems is showninFig.1C, E, F. The sheep PGCs grew equally well in medium A and B(see Table 1). When the first-to-third passage feeder layers were fresh or frozen, the MEF or the SFF could better support the growth of the sheep EG cells, and there were no significant differences, as shown in Fig. 1G, H.Withthe increasing passages, the EG cells proliferated slowly; meanwhile, the phenomenon of differentiation appeared gradually. The 20th-passage EG cell colony is presented in Fig. 1I. RT-PCR detection of EG cell-specific genes. Sheep EG cellspecific genes were detected by reverse-transcription polymerase chain reaction (RT-PCR). The electrophoresis results are shown in Fig. 2. The obtained fragments were approximately 258 bp (Kit), 297 bp (Rex-1), 449 bp (Nanog), and 571 bp (Oct-4) and were consistent with the expected sizes, so sheep EG cells expressed the pluripotent genes of Kit, Rex-1, Nanog, and Oct-4. Immunofluorescence identification. The 8th, 10th, and 13th Mongolian sheep PGCs were identified by cell-specific markers; the immunofluorescence results are shown in Fig. 3. It showed that sheep PGC cell-specific markers Oct3/ Oct4 were weakly positive, whereas SSEA-1, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 were strongly positive. Discussion Sheep PGC growth behaviors were compared in two kinds of medium. A medium was equivalent to B medium when knockout serum in B medium was replaced by the highquality fetal bovine serum. It was found that both media supported the growth of the sheep EG cells. During the sheep PGC culture, the role of the promoting growth factors in serum might be larger than that of the promoting differentiation factors, so we could completely use the conventional serum-containing culture medium instead of serum-free medium. Li et al (2011) reported that compared to knockout serum replacement (KSR) medium, the serum-containing medium better promoted the colony formation of sheep IPS. The appropriate feeder layer was needed in ES cell proliferation culture. MEF feeder layer was used commonly. In the study of ES cells, researchers began to study homologous cells as a feeder layer to avoid the pollution problem of heterogeneous cells as a feeder layer. In this experiment, the feeder layer before the fourth passages of fresh and frozen MEF or SFF could support the growth of the sheep EG cells; at the same time, there were no significant differences in cell morphology and passages. Undoubtedly, the frozen feeder cells were more convenient to use. The method of mechanical dissection was used in this study. Compared to the trypsin digestion method, it was time consuming and laborious, and the different clone sizes were made, but there were smaller damages to the EG cells (De Miguel et al. 2011), so we believed that this was one of the key factors which could make sheep EG cells to be passaged for 20 passages. The results showed that AP staining of sheep PGCs was weakly positive; sheep EG cells expressed Kit, Rex-1, Nanog, and Oct4; immunohistochemistry test indicated Oct3/Oct4 was weakly positive, and SSEA-1, SSEA-3, SSEA-4, TRA-1-60, and TRA1-81 were strongly positive. In particular, Ledda et al. (2010) evidenced that colonies could be negative to AP but could be stained with SSEA-1, while others were negative to SSEA-1 and AP, but expressed the proteins of the pluripotent genes EMA-1 (POU5F1, NANOG,andSOX-2)inthedetectionofsheepEG cells. That was to say the markers tested in the putative EG cells often displayed a pattern not completely coherent with pluripotency. Bao et al (2011) demonstrated that sheep iPS cells expressed ES cell markers, including alkaline phosphatase, Oct4, Nanog, Sox2, Rex1, Ssea-1, TRA-1-60, TRA-1-81, and Ecadherin. While others (Li et al 2011) found they expressed AP, Oct4, Sox2, Nanog, and the cell surface marker SSEA-4. All the inconsistencies may be led by the detection antibodies from different manufacturers. So these results show the most effective method still is to examine the ability to differentiate into athree-germ-layer structureinvitro andinvivoortoparticipate in the formation of chimeras. The sheep EG cells expressed Kit, Rex-1, Nanog, and Oct-4, and the putative gene expressed results were the same as the amniotic epithelial cells from humans and other animals (Miki et al 2005;MikiandStrom2006;Parolini et al 2008;Insaustietal2010; Bao et al 2011). Dattena et al. (2006) isolated ES cells from the inner cell mass from the sheep. The ES cells were positive to AP and could be stained with the ES cell surface antigen of SSEA-1, SSEA-3, and SSEA-4; the test results were similar to ours. The study will continue to optimize the in vitro culture conditions of sheep EG cells; increase the number of test samples; calculate clone formation rate, differentiation rate, and other indexes; expand culture and analyze karyotype; develop induced differentiation experiments in vivo and in vitro; and detect the expressed amount of pluripotent factors in sheep EG cells by real-time RT-PCR and immunofluorescence identification of other antibodies like Nanog. All these will lay a better foundation for the separation, culture, and line establishment and regulation mechanism study of the sheep ES cells. Acknowledgments Funding was supplied by the Chinese National 863 Project (2008AA101005). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. References Bao L.; He L.; Chen J.; Wu Z.; Liao J.; Rao L.; Ren J.; Li H.; Zhu H.; QianL.;GuY.; DaiH.;XuX.;ZhouJ.; WangW.;CuiC.;XiaoL. 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Identification and temporospatial distribution of bovine primordial germ cells prior to gonadal sexual differentiation. Anat Embryol 197: 451-467; 1998. Yu J.; Thomson J. A. Pluripotent stem cell lines. Genes Dev 22: 1987- 1997; 2008. Chun Xia Liu, Wen Long Wang, and Rui Yuan Zhao contributed to the research equally. C.X.Liu * R.Y.Zhao * H.T.Wang * Y.Y.Liu * S.Y.Wang * H. M. Zhou () College of Life Science, Inner Mongolia Agricultural University, 306 Zhao wu da street, Hohhot, Inner Mongolia 010018, China e-mail: [email protected] W. L. Wang College of Veterinary Medicine, Inner Mongolia Agricultural University, 306 Zhao wu da street, Hohhot, Inner Mongolia 010018, China (c) 2014 Society for In Vitro Biology |