Isolation and Characterization of Parthenogenetic Embryonic Stem ( pES ) Cells Containing Genetic Background of the Kunming Mouse Strain *

Parthenogenetic embryonic stem (pES) cells could provide a valuable model for research into genomic imprinting and X-linked diseases. In this study, pES cell lines were established from oocytes of hybrid offspring of Kunming and 129/Sv mice, and pluripotency of pES cells was evaluated. The pES cells maintained in the undifferentiated state for more than 50 passages had normal karyotypes with XX sex chromosomes and exhibited high activities of alkaline phosphatase (AKP) and telomerase. Meanwhile, these cells expressed ES cell molecular markers SSEA-1, Oct-4, Nanog, and GDF3 but not SSEA-3 detected by immunohistochemistry and RT-PCR. The pES cells could be differentiated into various types of cells from three germ layers in vitro by analysis of embryoid bodies (EBs) with immunohistochemistry and RT-PCR, and in vivo by observation of pES cell-derived teratoma sections. Therefore, the established pES cell lines contained all features of mouse ES cells. This work provides a new strategy for isolating pES cells from Kunming mice, and the pES cell lines could be applied as the cell model in research into genomic imprinting and epigenetic regulation of Kunming mice. (


INTRODUCTION
Embryonic stem (ES) cells, which are pluripotent stem cells derived from inner cell mass (ICM) of preimplantation embryos, are capable of proliferating permanently in the undifferentiated state and differentiating into various types of cells from three germ layers in vitro or in vivo (Evans and Kaufman, 1981;Thomson et al., 1998).ES cells could not only provide the unique cell model for many issues of developmental biology and human diseases, but also provide promising sources for cell transplantation and gene therapy (Drukker, 2008).Immune rejection, resulting from expression of the major histocompatibility complex (MHC) genes in ES cell-derived cells, has become one of the main obstacles to application of ES cells in clinical therapy.Parthenogenetically activated oocytes could easily develop to blastocysts which only contain a duplication of maternal genome.ES cells derived from parthenogenetic embryos (pES cells) are either uniformly homozygous or include minimal crossover-associated heterozygosity.So there exists a greater likelihood of obtaining a match between the pES cell derivative and the recipient than that from fertilized embryos.Kim et al. (2007) demonstrated that selected pES cells could serve as a source of histocompatible tissues.Therefore, it would be a feasible alternative to establish pES cell lines for autologous transplantation in females, instead of the ES cell lines from embryos created by somatic cell nuclear transfer with limitations of low success and ethical controversies.Moreover, pES cells could provide a cell model for gene imprinting and X-linked diseases (Allen et al., 1994;Cibelli et al., 2002;Jiang et al., 2007).In recent decades, many pES cell lines had been continuously established from oocytes of monkeys (Cibelli et al., 2002), rabbits (Fang et al., 2006), rats (Sritanaudomchai et al., 2007) and human (Lin et al., 2007;Mai et al., 2007;Revazova et al., 2007).Kaufman et al. (1983) first established pES cell lines from oocytes of 129/SvE female mice and hybrid female mice (C57BL×CBA), and evaluated pluripotency of the pES cells.Other researchers demonstrated that mouse pES cells could undergo extensive differentiation in vitro (Lin et al., 2003), and contribute to germline chimeras (Jiang et al., 2007).However, there were some discrepancies regarding capacities of proliferation and differentiation of mouse pES cells (Jagerbauer et al., 1992;Newman-Smith and Werb, 1995;Park et al., 1998).Up to now, most mouse pES cell lines were derived from oocytes of the 129 mice or other hybrid mice.The Kunming mouse strain (Mus musculus Km) is one of the house-breeding albino mouse strains widely used as the laboratory animal in China.This mouse strain exhibits significant advantages over such inbred strains as 129, C57BL/J, BALB/c, DBA and CBA in respect of environmental resistance, reproduction and breeding, etc. Attempts had been made to isolate ES cells from inbred Kunming mice but few successes were achieved, and especially there are no reports regarding establishment of pES cell lines from them.Kunming mice were generally believed to be unsuitable for isolation of ES cells.129 mice are acknowledged to be the most conducive for isolation of ES cells among many mouse strains, and ES cells could be easily isolated from hybrid embryos containing genetic background of 129 mice (Kress et al., 1998;Brook et al., 2003).Therefore, we hypothesized that oocytes of hybrid offspring of Kunming and 129 mice would acquire higher developmental competence after parthenogenetic activation, increasing the chances of isolating the pES cells.Here, we acquired pES cell lines derived from hybrid offspring of Kunming and 129/Sv mice, and these cells maintained the undifferentiated state in vitro for a long term and could differentiate into various types of cells from three germ layers in vitro or in vivo.

Collection, parthenogenetic activation and development in vitro of mouse MII oocytes
Oocytes of hybrid female offspring produced by Kunming female mice (Mus musculus Km) (the Laboratory Animal Centre, the Fourth Military-Medicine University in R. P. China) and 129/Sv male mice (the Laboratory Animal Centre, Peking University) were used to isolate pES cells.Collection, parthenogenetic activation and development in vitro of mouse MII oocytes were performed as described previously (Nagy et al., 2003).Briefly, the cumulus-oocyte complexes (COCs) were collected from oviducts of female mice 15-17 hours after injection of human chorionic gonadotropin (hCG).The cumulus cells surrounding oocytes were removed by hyaluronidase (Sigma) (1 mg/ml).Then, oocytes were activated with Ionomycin Calcium (Sigma) (2.5 μg/ml) followed by 6-DMAP (Sigma) (2.5 mM) and cultured with droplets of KSOM medium (Chemicon) in a CO 2 incubator (Thermo, Forma 311) at 37.5°C for 4-4.5 days.The blastocysts were harvested to isolate the pES cells.

Isolation and maintenance of the pES cells
The harvested blastocysts were cultured on inactivated mouse embryonic fibroblasts (MEF) with mitomycin C (Sigma) (10 μg/ml).ES cell medium was composed of Knockout TM high glucose DMEM (Invitrogen) supplemented with 11.25% KSR (vol/vol) (Invitrogen), 3.75% FBS (vol/vol) (Hyclone), 20 ng/ml leukemia inhibitory factor (LIF) (Chemicon), 1mM nonessential amino acids (NEAA) (Invitrogen) and 0.1 mM β-mercaptoethanol (Sigma).Within 4-6 days, embryos attached to the feeder layers and formed ICM outgrowths with prominent nucleoli and dense morphology.Subsequently, ICM outgrowths were mechanically dissected into small clumps after treatment with trypsin (Invitrogen) (0.5 mg/ml)/EDTA (Sigma) (0.4 mg/ml) solution for 2-3 min, and individually plated on the MEF feeder layers.The fresh ES cell medium was fed every 2-3 days, and the ES cell-like colonies usually appeared within 3-7 days.At the initial stage, the pES cells were subcultured by trypsinization in combination with mechanical dissection.When a large number of ES cell colonies appeared in the plates with 3.5 cm diameter, pES cell lines were affirmed to be established.The putative pES cells were subcultured or frozen every 60-72 h.

Karyotype analysis of of the pES cells
Karyotype analysis of pES cells was performed as described previously (Nagy et al., 2003).Briefly, after culture in feeder-free conditions for 48 h, pES cells were exposed to colchicine (Sigma) (10 μg/ml) for 1-2 h.Subsequently, these cells were trypsinized into a single-cell suspension followed by suspension for 15 min in 0.075 mM KCl solution.Cells were repeatedly fixed in methanol/ acetic acid mixture, then spread over slides and stained with Giemsa (Sigma) solution.The karyotypes were observed and imaged under a Digital Microscopic Imaging System (Leica, DMIRB).

Differentiation experiments of the pES cells
Embryoid bodies (EBs) were prepared as described previously (Nagy et al., 2003).Briefly, ES cells were cultured in ES cell medium without the feeder layers for 24-28 h, then the clumps were mechanically separated and cultured in suspension in LIF-free medium containing 15% FBS for 5-10 days until EBs formed.Subsequently, EBs or their single cells were cultured in a gelatin-pretreated plate with LIF-free medium for 5-7 days until various types of differentiated cells appeared.These differentiated cells were immunohistochemically stained with primary antibodies against the antigens specific for different germ layers (α-fetoprotein (1:100) (AFP) for endoderm cells, α-actin (1:100) for mesoderm cells, Nestin (1:100), β-Tubulin III (1:100) and GFAP (1:100) for ectoderm cells) (all antibodies purchased from Chemicon Co. Ltd).

Statistical analysis
Statistical analyses were performed using SPSS 11.5.0 standard version (SPSS Inc., Illinois, USA).An independent simple T-test was employed to analyze differences of developmental potential to blastocyst (No. of blastocysts/ No. of activated oocytes), attached rate (No. of attached blastocysts/No. of blastocysts cultured), ICM outgrowth rate (No. of ICM outgrowth/No. of blastocysts cultured) and establishing rate (No. of the established pES cell lines/No.of blastocysts cultured) between hybrid and inbred groups.For all tests, statistical significance was taken as p<0.05.

Establishment and characterization of mouse pES cells
The MII oocytes developed in vitro to the blastocysts 4-4.5 days after parthogenetic activation, which showed that developmental competence of oocytes from hybrid mice (129♂×KM♀) was significantly higher than that from inbred Kunming mice (p<0.05)(Figure 1A) (Table 1).Moreover, blastocysts from inbred Kunming mice were morphologically inferior to those from hybrid mice.The blastocysts cultured on the feeder layers successively attached and formed ICM outgrowths with prominent nucleoli (Figure 1B), which showed significantly higher rates of ICM outgrowth formation than those from inbred Kunming mice (p<0.05)(Table 1).The four ICM-derived cell clumps from hybrid mice formed ES cell colonies at the first passage.However, no pES cell clone appeared from inbred Kunming mice.Three putative pES cell lines were established from oocytes of hybrid mice, and one cell line maintained the undifferentiated state for more than 50 passages.
Karyotype analysis of pES cells at the 14 th and 23 rd passage revealed that most cells held 20 pairs of chromosomes with a XX chromosome (83.3% at 14 th passage vs. 70% at 23 rd passage) (Figure 2).During extended culture, pES cells exhibited high activities of AKP, and were immunohistochemically positive for SSEA-1, Nanog, Oct-4 and telomerase but negative for SSEA-3 (Figure 2).They also expressed such genes as Oct-4, nanog and gdf3 by RT-PCR (Figure 3A).Therefore, the pES cells exhibited the same characteristic enzyme activities, antigen profiles and gene expression as mouse ES cells.These Statistically significant differences between the data marked by the different letters.results suggested that the pES cells could be permanently retained in the undifferentiated state.

Pluripotency of mouse pES cells
Some different sizes of EBs continuously appeared 5-10 days after culture in suspension under LIF-and feeder-free conditions (Figure 4).RT-PCR results demonstrated that EBs expressed the specific genes for three germ layers, fgf5 and nf-68 (for ectoderm), BraT (for mesoderm), and Afp (for endoderm) (Figure 3B); meanwhile these EB-derived cells immunohistochemically expressed the specific antigens α-Actin (for mesoderm), AFP (for endoderm), GFAP, β-Tubulin III and Nestin (for neuroectoderm) (Figure 4).Moreover, teratoma sections demonstrated tissue structure resembling connective tissue, blood cells, blood tube, epithelial cells, muscle, lymphatic tissues, skin and endocrine gland (Figure 5).These results suggested that the pES cells could differentiate into various types of cells from three germ layers in vitro or in vivo.

DISCUSSION
In this work, we established pES cell lines from parthenogentically activated MII oocytes of hybrid offspring of Kunming and 129/Sv mice, which shared all features of mouse ES cells.
Oct-4 and nanog genes, essential for maintenance of pluripotency in ICM cells and ES cells in vitro (Nichols et al., 1998;Chambers et al., 2003;Mitsui et al., 2003), were generally employed as the molecular markers for characterization of ES cells.Gdf3 is known to be a BMP4 inhibitor and to regulate stem cells to retain the undifferentiated state and differentiate into the full spectrum of cell types, which was also expressed in ES cells in vitro (Levine and Brivanlou, 2005).In this work, the pES cells expressed Oct-4 and nanog in high levels but gdf3 in low levels, which was in accordance with the view that decreased expression of the gdf3 gene permits BMP4 to support maintenance of pluripotency in mouse ES cells  ( Levine and Brivanlou, 2005).These results suggested that the established pES cell lines could maintain the undifferentiated state in vitro for a long time.
Genetic background of mouse was known to influence isolation of ES cells, and there exhibited some differences among different mouse strains in the conditions required for isolation of ES cells (Kawase et al., 1994;Suzuki et al., 1999;Baharvand and Matthaei, 2004).We attempted to isolate pES cells from oocytes of inbred Kunming mice, but the oocytes exhibited significantly lower developmental competence to blastocysts in vitro after parthenogenetic activation.The harvested blastocysts were also morphologically inferior to those from hybrid mice.All these factors constrained isolation of pES cells.Previous reports indicated that rapid loss of Oct-4 gene in ICM cells in vitro was the constraint for isolation of ES cells (Buehr and Smith, 2003;Buehr et al., 2003;Tielens et al., 2006).Similarly, we noticed that ICM outgrowths derived from parthenogenetic embryos of inbred Kunming mice, expressed Oct-4 at even lower level or unexpressed in vitro, as determined by immunohistochemical methods (not presented here).On the contrary, Oct-4 was steadily expressed in ICM outgrowths and pES cells from hybrid mice (not presented).Therefore, we inferred that rapid loss of Oct-4 in ICM outgrowths and pES cells in vitro constrained establishment of the pES cell line from inbred Kunming mice.These results implied that introduction of 129/sv mouse genetic background could significantly improve isolation of pES cells, which was also supported by the findings of Kress et al. (1998) and Brook et al. (2003).Jiang et al. (2007) demonstrated that disruption of genomic methylation and activation of some paternally-  expressed imprinting genes might improve developmental potential of parthenogenetically-activated oocytes and enhance the proliferation and pluripotency of pES cells in vitro.However, further study is necessary to explore the constraints in isolating pES cells from inbred Kunming female mice by investigating genomic methylation and expression patterns of paternal imprinting genes in parthenogenetic embryos and their ICM outgrowths.
Overall, this work may provide new strategy for establishment of pES cell lines from Kunming female mice, and also be a valuable model for investigating differences of epigenetic regulation and gene imprinting between Kunming and other mouse strains.

Figure 3 .
Figure 3. Detection of the ES cell marker genes in pES cells and genes of three germ layers in the pES cell-derived EBs by RT-PCR.A shows that the pES cells at the 45 th passage expressed nanog (Lane 7), Oct-4 (Lane 8), gdf3 (Lane 9), but MEF did not express nanog (Lane 2), Oct-4 (Lane 3), gdf3 (Lane 4).GADPH (Lane 5 and 10) were expressed in MEF and the pES cells as positive control.B shows that the EBs expressed nf68 (Lane 2) and fgf5 (Lane 4) for ectoderm, BraT (Lane 3) for mesoderm, Afp (Lane 5) for endoderm, and GADPH (Lane 7) as positive control, but did not express TTR (Lane 6) for endoderm.

Figure 5 .
Figure 5. Histological sections of teratomas derived from pES cells (H/E staining, 200×).Teratoma sections showed various types of tissue structures resembling connective tissue (the solid line-surrounding region in A), blood cells and blood tube (the dotted linesurrounding region in A), columnar epithelium (the starlike-marked regions in A), muscle (the marked region in B), lymphatic tissues (the marked region in C), skin keratinous layers (the solid line-surrounding region in D) and endocrine gland (the dotted line-surrounding region in D).

Table 1 .
In vitro development of MII mouse oocytes after parthenogenetic activation

Table 2 .
Isolation of the pES cells from parthenogenetic embryos