Improved Establishment of Embryonic Stem (ES) Cell Lines from the Chinese Kunming Mice by Hybridization with 129 Mice

Chinese Kunming mice (Mus musculus Km), widely used as laboratory animals throughout China, remain very refractory for embryonic stem (ES) cell isolation. The present study was aimed to evaluate the effects of hybridization with 129/Sv mice, and culture media containing fetal bovine serum (FBS) or Knockout serum replacement (KSR) on ES cell isolation from Kunming mice. The results demonstrated that ES cells had been effectively isolated from the hybrid embryos of Kunming and 129/Sv mice using all three media containing 15% FBS, 15% KSR and their mixture of 14% KSR and 1% FBS, individually. These isolated ES cells had maintained in vitro undifferentiated for a long time, exhibiting all features specific for mouse ES cells. In addition, the rates of ES cell isolation in the medium containing 14% KSR and 1% FBS, was 46.67% and significantly higher than those in another two media containing only FBS or KSR (p < 0.05). Contrarily, no ES cell line had been established from Kunming mouse inbred embryos using the same protocols. These results suggested that ES cells with long-term self-renewal ability could be efficiently generated from hybrid embryos of Kunming and 129/Sv mice, and a small volume of FBS was necessary to isolate ES cells in the KSR medium when embryos and early ES cells cultured.

Abstract: Chinese Kunming mice (Mus musculus Km), widely used as laboratory animals throughout China, remain very refractory for embryonic stem (ES) cell isolation. The present study was aimed to evaluate the effects of hybridization with 129/Sv mice, and culture media containing fetal bovine serum (FBS) or Knockout serum replacement (KSR) on ES cell isolation from Kunming mice. The results demonstrated that ES cells had been effectively isolated from the hybrid embryos of Kunming and 129/Sv mice using all three media containing 15% FBS, 15% KSR and their mixture of 14% KSR and 1% FBS, individually. These isolated ES cells had maintained in vitro undifferentiated for a long time, exhibiting all features specific for mouse ES cells. In addition, the rates of ES cell isolation in the medium containing 14% KSR and 1% FBS, was 46.67% and significantly higher than those in another two media containing only FBS or KSR (p < 0.05). Contrarily, no ES cell line had been established from Kunming mouse inbred embryos using the same

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individually. As shown in Table 1, it took significantly longer to achieve embryo attachment in the 15% KSR medium, in which the number of attached embryos was significantly lower than that in another two media. All primary ES cells produced from the picked ICM outgrowths, persisted the undifferentiated state and generated the ES cell lines in the two media containing 15% KSR and the mixture of 1% FBS + 14% KSR. By contrast, only a small amount of ICM outgrowths and primary ES cells further formed ES cell clones due to death or differentiation in the medium containing 15% FBS, although embryos attached to the feeder layers as efficiently as that in the medium containing 14% KSR + 1% FBS (Table 2). Finally, ES cell lines had been established in the medium containing 14% KSR and 1% FBS with the higher efficiency of 46.67%, compared with those in another two media ( Table 2).  In addition, when individually plated in the 96-well plates, 14.2% of single ES cells formed cell clones in the 15% KSR medium, which was significantly lower than those in another two media (Table 3). However, ES cell clones in the two media containing KSR ( Figure 1A,B), maintained morphologically undifferentiated for a longer time, and ES cell clones in the 15% FBS medium ( Figure 1C) exhibited morphologically the aging signs with many dark granules. These results suggested that KSR was preferable to FBS for culturing ES cells, and recombined supplement with KSR and a small amount of FBS contributed to improvement of ES cell isolation when embryos and primary ES cells were cultured.

Effect of Genetic Background on ES Cell Derivation
We had efficiently established several ES cell lines from hybrid embryos of Kunming and 129/Sv mice as shown in Table 2. These ES cells derived from hybrid embryos, usually formed a great number of ES cell clones in the plates with 3.5 cm diameter after the second trypsinization, with the long-term self-renewal. We attempted to isolate ES cells from inbred blastocysts of Kunming mice, but the results demonstrated that under the same condition, the vast majority of picked ICM outgrowths and primary ES cells could not further form ES cell clone after dissociation even though embryos could normally attach and form ICM outgrowths as the hybrid blastocysts. Furthermore, we had increased LIF concentration to 100 ng/mL in the media, which had not significantly promoted in vitro propagation of ES cells. As the best results, ES cells derived from only two inbred blastocysts had been maintained in the four-well plates until the seventh passage when ES cell clone disappeared, and ES cell clones gradually decreased due to death or differentiation after being trypsinized. Consequently, no ES cell line had been established from 391 Kunming mouse inbred blastocysts (Table 4).

Characterization of mES Cells
Microsatellite marker-primed PCR results summarized in Table 5, demonstrated that at all examined microsatellite loci except D14Mit113, the tested mES cells exhibited the identical PCR products to the F 1 hybrid embryos from the Kunming females and 129/Sv males. Moreover, the mES cells and the F 1 embryos contained the identical PCR products to the Kunming females at the microsatellite loci D1Mit15, D2Mit30, D11Mit4, D11Mit167, D12Mit56 and D14Mit129, and to the 129/Sv males at the microsatellite loci D2Mit30, D11Mit4, D11Mit167, D14Mit129, and D17Mit36, respectively. The results could verify that the tested mES cells contain genetic background of the Kunming strain and 129/Sv strain. Table 5. Genotype analysis of the established mES cells by microsatellite marker-based PCR.

Microsate-llite markers KM females 129/Sv males F 1 embryos (KM × 129) The tested mES cells
D1Mit15 Note: "a", "b" and "c" refered to the bands of PCR products, and "-"represented no PCR products on the agarose gel. During the extended culture, the novel ES cells produced from hybrid embryos, always formed morphologically compacted cell clones (Figure 2A) in the presence of MEF feeder layer and LIF, and most of the checked cells (89.5% at the 22th passage vs. 75.0% at the 33th passage) maintained diploid karyotypes with 19 pairs of autosomes and XY chromosomes ( Figure 2I). These cells were positively stained for AKP ( Figure 2B), and by immunohistochemical staining for telomerase ( Figure 2C), Oct4 ( Figure 2D), Nanog ( Figure 2E) and SSEA-1 ( Figure 2F), but negatively by immunohistochemical staining for SSEA-3 ( Figure 2G) and SSEA-4 ( Figure 2H). In addition, RT-PCR analysis showed that the tested ES cells positively expressed Oct4, Nanog and Gdf-3 ( Figure 3A), while Gdf-3 expression level was relatively lower compared with those of Oct4 and Nanog ( Figure 3A).  After removing feeder layer and LIF, ES cell clumps in suspension formed embryoid bodies (EBs) ( Figure 4A) in the 15% FBS medium, which differentiated morphologically into various types of cells. These EB-derived cells immunohistochemically presented the specific antigen phenotypes of differentiated cells derived from mesoderm ( Figure 4B,C), endoderm ( Figure 4D) and neuroectoderm ( Figure 4E-G), while EBs expressed the specific marker genes Fgf-5 and Nf-68 for ectoderm, BraT for mesoderm and Afp for endoderm ( Figure 3B). In addition, the ES cell suspension was subcutaneously injected into nude mice and formed teratomas, the sections of which demonstrated such resembling tissue structure as lymphatic tissues ( Figure 5A,C), blood tube ( Figure 5A), epithelium ( Figure 5B), fat tissue ( Figure 5B), gland tissue ( Figure 5B), muscle ( Figure 5B,C) and connective tissue ( Figure 5D).
Together, these results suggested that the isolated ES cells could permanently retain undifferentiated and differentiate into various types of cells from three germ layers in vitro or in vivo.

Discussion
Here, we have efficiently isolated ES cells from hybrid embryos of Kunming and 129/Sv mice. The established ES cell lines contained the genetic background of Kunming and 129/Sv mice by microsatellite marker-primed PCR analysis, and shared all features of mES cells including normal karyotypes, high activity of AKP and telomerase, SSEAs antigen profile, differentiated potential into various types of cells from three germ layers in vitro or in vivo, and expression of two key transcription factors of pluripotency, namely Oct4 and Nanog [25,26]. Moreover, RT-PCR results demonstrated in the isolated ES cells that the gene Gdf-3 had been transcribed in a relatively lower level compared with mRNA levels of the genes Oct4 and Nanog, which accorded with the finding that low expression of the BMP4 inhibitor GDF3 facilitated BMP4 to maintain pluripotency of mES cells [27]. This suggested that the isolated ES cells could maintain the long-term self-renewal ability in vitro.
As the vital component of ES cell medium, FBS is not an optimal supplement to maintain pluripotency of ES cells since it harbors some undefined factors which induce differentiation or apoptosis [5,[7][8][9]12]. KSR, an artificial substitute for serum, had been proven preferable to FBS in maintaining pluripotency of mES cells in these mouse strains C57BL/6, BALB/cAJ, CBA/Ca and NOD [5][6][7][8][9]12]. However, some reports pointed out that partial replacement of KSR with FBS contributed better to increasing the efficiencies of ES cell isolation than complete replacement, when culturing embryos and initial ES cells [8,10,12]. Similarly, the results from our work indicated that in comparison with the sole use of FBS or KSR, recombined use of KSR and a small volume of FBS significantly contributed to increasing the efficiencies of ES cell isolation from hybrid embryos, and to formation of ES cell clones when being plated by the single ES cells. Also, we recommended that KSR and FBS would be supplemented together in the medium to efficiently establish ES cells when embryos and initial ES cells were cultured.
It has been proved that, in the presence of FBS or KSR, mES cells could be successfully isolated from the recalcitrant strains such as BALB/c, BALB/k, CBA/Ca and NOD by increasing LIF concentration [4,7,9,11,17]. The results from Peng et al.'s work on Kunming inbred mice [18] indicated that ES cells maintained undifferentiated in vitro in the feeder cell-and LIF-free condition by supplement with KSR, PD0325901 and CHIR99021, and contrarily lost the self-renewal capacity in the medium consisting of LIF and serum. Likewise, our present results indicated that ES cells from Kunming inbred mice could not propagate extendedly in the culture system composed of feeder cells, FBS, KSR and high concentration of LIF (100 ng/mL) since the vast majority of ICM outgrowths and subsequent ES cells quickly disappeared when being plated after the second digestion. This is in contrast with the fact that ES cells from hybrid embryos of Kunming and 129/Sv mice exhibited the powerful self-renewal capacity. Therefore, we presumed that establishment of ES cell lines from hybrid embryo of Kunming and 129/Sv mice, should be attributed to heterosis or 129/Sv genetic background, as the previous reports suggested [19,20], and LIF was insufficient to maintain the self-renewal ability of ES cells from Kunming inbred mice in the presence of serum or KSR, and feeder cells. Thus, it remains necessary to explore the genetic/epigenetic mechanism of hampering pluripotency of Kunming mouse-derive ES cells.

Animals
Kunming mice (Mus musculus Km) and 129/Sv mice (purchased from the Laboratory Animal Centre in West-China Center of Medical Sciences, Sichuan University (Chengdu, China)) used in this study with ethical approval from the Animal Research Ethical Committee, were kept in temperatureand light-controlled rooms. Adult Kunming females injected by equine chorionic gonadotrophin (eCG) and human chorionic gonadotrophin (hCG) (purchased from Ningbo Second Harmone Factory, Ningbo, China), were mated with 129/Sv males or Kunming males overnight.

Isolation and Culture of ES Cells
Blastocysts for ES cell isolation, were flushed from uteri of the pregnant Kunming females, and cultured on the inactivated mouse embryonic fibroblasts (MEF) with 5 µg/mL mitomycin C (Sigma, Kanagawa, Japan). After embryos attached and formed ICM outgrowths with prominent nucleoli and dense morphology, ICM outgrowths were mechanically dissected into small cell clumps after treatment with trypsin (Invitrogen, Carlsbad, CA, USA) (0.5 mg/mL)/EDTA (Sigma, Kanagawa, Japan) (0.2 mg/mL), and individually plated on MEF feeder layers. The initial ES 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, ES cell lines were ascertained.

Karyotype Analysis of ES Cells
Karyotype analysis of ES cells was performed as described previously [28]. Briefly, after cultured in feeder-free conditions for 24-36 h, ES cells were exposed to 10 µg/mL colchicine (Sigma, Kanagawa, Japan) for 1-2 h, and 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, Kanagawa, Japan) solution. Karyotypes were observed and imaged under a Digital Microscopic Imaging System (Leica, DMIRB, Wetzlar, Gemany).

Genotype Analysis of mES Cells
To determine genetic source of the established mES cells, the microsatellite markers listed in Table 6, were examined by PCR for these samples including mES cells, tail tissues from Kunming female and 129/Sv male, and the F 1 hybrid (KM × 129/Sv) embryos as controls. The microsatellite markers and their corresponding primer sequences were selected and obtained from Mouse Genome Database (http://www.informatics.jax.org) and the documents [29]. Genomic DNA of all samples were prepared according to instructions of the QIAamp DNA Mini Kit (Qiagen Co. Ltd., Shanghai, China). PCR reaction were carried out as follows: initial denaturation at 94 C for 2 min, followed by 35 cycles of 15 s at 94 C, 25 s at 60 C, 35 s at 72 C, and finally 10 min extension at 72 C. PCR products were separated by electrophoresis on 4% agarose gel and imaged under the Gel Image System (Syngene Co. Ltd., Karnataka, UK). The genotypes of the tested samples were scored according to the traveling distance of PCR products of every microsatellite loci primers on the 4% agarose gel, with the shortest being given the genotype "a", followed by "b", "c", and so on. Table 6. The primer sequences of microsatellite markers used in the present experiment.

Microsatellite markers
Forward primer Reverse primer

Genes
Forward primer Reverse primer Product Size (bp) Note: bp = base pairs.
RT-PCR was performed to examine expression of the genes Oct4, Nanog, Gdf-3 (a direct BMP4 inhibitor) and GAPDH as the inner control in ES cells. Total RNA was extracted according to instructions of the RNeasy Mini kit (Qiagen Co. Ltd., Shanghai, China) and reversely transcribed into cDNA using RevertAidTM First Strand Kits (Fermentas Co. Ltd., Waltham, MA, USA). PCR reaction were carried out as follows: 2 min denaturation at 94 C, followed by 35 cycles of 30 s at 94 C, 30 s at 55 C, 60 s at 72 C, and finally 10 min extension at 72 C. PCR products were separated by electrophoresis and imaged under the Gel Image System (Syngene Co. Ltd., Shanghai, China). PCR primers for amplifying the genes Oct4, Nanog, Gdf-3 and GAPDH (as the inner control) listed in Table 7, were designed according to the corresponding gene sequences published in GenBank.

Pluripotency of mES Cells
Embryoid bodies (EBs) were prepared as described previously [27]. Briefly, ES cells were cultured in ES cell medium without the feeder layers for 24-28 h, then the cell clumps were mechanically separated and cultured in suspension in LIF-free medium containing 15% FBS until EBs formed. Subsequently, EBs were cultured in a gelatin-coated plate with LIF-free medium until various types of differentiated cells appeared. These cells were immunohistochemically stained with primary antibodies against the marker antigens specific for different germ layers, namely, α-fetoprotein (AFP) (1:50) for endoderm cells, α-Actin (1:100) for mesoderm cells, Nestin (1:100), β-Tubulin III (1:50) and GFAP (1:100) for ectoderm cells (all purchased from Chemicon Co. Ltd., Billerica, MA, USA), and would be dyed as red-brown or yellow-brown if positive. Meanwhile, osteoblasts from the mesoderm layer were stained as yellow-brown by Alizarin Red.
Moreover, EBs were harvested to examine expression of Fgf-5 and Nf-68 specific for ectoderm, BraT specific for mesoderm, Afp specific for endoderm, and GAPDH as the inner control by RT-PCR. PCR primers for amplifying the genes Fgf-5, Nf-68, BraT and Afp and GAPDH listed in Table 5, were designed according to the corresponding gene sequences published in GenBank.
ES cells (about 5 × 10 6 cells/mouse) were subcutaneously injected into nude mice. After 4-6 weeks, the harvested teratomas were fixed in 4% formaldehyde to prepare paraffin sections. Sections were stained by hematoxylin/eosin (HE) and observed under a Digital Microscopic Imaging System (Olympus Co. Ltd., Tokyo, Japan).

Statistical Analysis
Statistical analyses were performed using SPSS11.5.0 Version (SPSS Inc., Chicago, IL, USA). An independent simple t-test was employed to analyze differences of 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 ES cell lines/No. of blastocysts cultured) between different groups. Statistical significance was taken as p < 0.05.

Conclusions
In summary, ES cell lines had been efficiently established from Kunming female mice by hybridization with 129/Sv male mice. Meanwhile KSR in conjunction with a small amount of FBS (1%) facilitated mES cell isolation, when blastocysts and primary ES cells were cultured. The results provided a new approach to ES cell isolation from Kunming mice and facilitated the exploration of the genetic/epigenetic mechanism related to Kunming mouse development.