Pluripotency of ES cells derived from tetraploid embryo complemented male mice

Summary

Abstract:
Pluripotent mouse embryonic stem (ES) cells have the ability to generate completely ES cell-derived mice by tetraploid (TE-4N) complementation although; derivation of ES cells from TE-4N animals has not been well defined. In this study, we generated TE-4N mice from high passage number ES cells and, in turn, determined the possibility of deriving ES cells from the TE-4N mice. The results showed that adult fertile TE-4N mice could be generated by aggregation of tetraploid embryos using ES cells passaged 23 times. Furthermore, ES cells could be derived from the TE-4N male mice and as judged by molecular characterisation and a study of chimeras, these ES cells were pluripotent. The findings demonstrate that tetraploid complementation is an efficient way to produce fertile mice, which can then give rise to pluripotent ES cells.

Keywords:
embryonic stem cells; pluripotency; tetraploid embryos

Introduction

Mouse embryonic stem (ES) cells have been derived from cultured mouse blastocysts (Evans and Kaufman, 1981; Bryja et al., 2006; Ying et al., 2008). Although the efficiency of such ES cell derivation differs between mouse strains, authentic ES cells are defined by three cardinal properties: (i) unlimited symmetrical self-renewal in vitro; (ii) comprehensive contribution to primary chimeras and (iii) generation of functional gametes for genome transmission (Buehr et al., 2008). Such pluripotent ES cells are used widely both in mouse genetics and developmental biology, and they also have therapeutic potential in regenerative medicine.

In addition to germline contribution in chimeras, pluripotent ES cells could support full-term development of tetraploid (TE) embryos (Nagy et al., 1990) and produce completely ES cell-derived (TE-4N) mice. Conventionally, ES cells were either injected into, or aggregated with, the tetraploid embryos produced by electrofusion at the two-cell stage (Eggan et al., 2001), and then transferred to the uterus of a pseudopregnant recipient mouse. These TE-4N mice frequently survive to birth but the efficiency of their production is markedly influenced by such factors as the cell line (Nagy et al., 1993), their genotype (Eggan et al., 2001; Li et al., 2005) and the passage number (Li et al., 2007) of the donor ES cells. Furthermore, the survival rate of TE-4N pups to adulthood is low (Wang et al., 1997; Li et al., 2005; Li et al., 2007). While there are numerous studies which have elucidated the isolation of ES cells from different wild type mouse strains (Kawase et al., 1994; McWhir et al., 1996; Brook and Gardner, 1997; Ying et al., 2008) and species (Buehr et al., 2008) the possibility of deriving ES cells from tetraploid complemented mice still remains unclear.

In this study, we aimed to generate TE-4N mice from high passage number ES cells, and then derive ES cells from the TE-4N mice created. We anticipated that the TE-4N mice would be fertile and that ES cells derived from them would be pluripotent.

Materials and methods

Mice and ES cell line

All the recipient (CD-1) and donor (C57BL/6) female mice were purchased from Beijing Vitalriver Laboratory Animal Technology (Beijing, China). They were housed in a 12 h light–12 h dark photoperiod regimen and fed with pelleted food under approval of the Laboratory Animal Administration Committee of Inner Mongolia. The original ES cell line, which contained a mixed genetic background of MF1, C57BL/6 and 129/sv, was cultured under standard conditions as described previously (Nagy et al., 2003).

Generation of TE-4N mice

CD-1 females were mated with CD-1 males and the resulting 2-cell embryos were flushed from their oviducts next day using KSOM-HEPES medium (Nagy et al., 2003). After several washes in drops of KSOM the embryos were transferred in KSOM to a 4-well plate (Nunc, USA). Next the embryos were placed in a 592-µm gap electrode chamber filled with electrofusing solution containing 0.3 M mannitol, 0.1 mM calcium chloride, 0.1 mM magnesium sulphate and 0.3% BSA (all from Sigma, USA). The blastomeres were then fused by two short electric pulses (60 V for 60 µs) applied by a cell fusion machine (Cellfusion, CF-150/B, BLS, Hungary). After electrofusion, the embryos were rinsed through drops of KSOM-HEPES, transferred into KSOM medium under mineral oil (Sigma) and cultured to 4-cell stage in an incubator. The zonae pellucida of the resulting tetraploid embryos were then removed by treatment with acidic Tyrode's medium. ES cells were trypsinised briefly before being assembled carefully with two tetraploid embryos in a small depression created artificially in the surface of a 4-well plate. They were incubated with KSOM overnight and after 24 h culture most of the aggregates had formed a single embryo, which had progressed to the blastocyst stage. These were transferred into the uterine lumen of 2.5-day pseudopregnant Kunming female mice to be carried to term.

Derivation of putative ES cell lines (hereafter termed teES)

Blastocysts were isolated from C57BL/6 females mated to TE-4N males. Those expressing GFP were selected and placed into 12-well plates containing a layer of mitomycin-C-treated STO cells. The 2i-LIF medium consisted of a 1:1 mixture of DMEM/F12 and Neurolbasal (Gibco, USA) supplemented with N₂ Supplement (Gibco), B27 Supplement (Gibco), 2 mM Gluta-Max (Gibco), 100 µM β-mercaptoethanol (Gibco), penicillin (100 U/mL)/streptomycin (100 µg/mL; Gibco), 1 µM PD0325901 (Selleck, USA), 3 µM CHIR99021 (Selleck) and 1000 U/mL recombinant mouse LIF (Prospec, USA). The blastocysts were allowed to attach to the feeder layer without any further experimental interference for 5 days until they ‘hatched’ and expanded. All the cell clumps, which originated from the blastocysts were then dissociated in drops of 0.25% trypsin-EDTA solution (Gibco) and the resulting cell suspension was replated onto a fresh feeder layer (Li et al., 2007). Until passage 4, the teES cells were maintained on the feeder layer of cells. Then they were either frozen, or they were cultured feeder-free on plates coated with 0.1% gelatin (Sigma).

Alkaline phosphatase staining and RT-PCR analysis

For alkaline phosphatase (AP) staining, the teES cells were washed once in cold PBS and fixed in 4% paraformaldehyde for 10 min at room temperature. A Leukocyte Alkaline Phosphatase Kit (Sigma) was then used to measure the alkaline phosphatase activity. For RT-PCR analysis, total RNA was extracted using an RNeasy Kit (QIAGEN, Germany) and cDNA was synthesized using an oligo(dT) primer and a Reverse Kit (Promega, USA). RT-PCR was performed using the PCR Master Mix (QIAGEN) on PCR instrument (Applied Biosystems, USA). Primer sequences are listed in Table 1.

Immunoflorescent staining and EB formation

Cells were fixed in 4% paraformaldehyde in PBS for 15 min at room temperature, permeabilised for 10 min in PBS containing 0.25% Triton X-100 and blocked with 1% BSA (Sigma) in PBST for 30 min again at room temperature. Primary antibodies used included rabbit anti-Nanog (Abcam), rabbit anti-Oct4 (Abcam), and rabbit anti-Sox2 (Abcam) while the secondary antibody employed was goat anti-rabbit IgG-FITC (Abcam). The samples were counterstained with 1 µ/mL DAPI (Invitrogen). For EB formation, cell suspensions were prepared by trypsinisation and approximately 1,000 cells were aggregated in hanging drops. After 3 days EBs were seeded onto cover slips and cultured in 6-well plates for 1 week. The germ-layer differentiation of the EBs was tested using markers for endoderm (anti-AFP, R&D), mesoderm (anti-Brachyury, Abcam), and ectoderm (anti-NCAM1, Ptlab).

Generation of chimeras from teES cells

Blastocysts were collected from of CD-1 mice and around 15 teES cells were injected into the blastocoelic cavity of each embryo using a piezo-assisted micromanipulator (Prime Tech, Japan) attached to an inverted microscope (Olympus). The injected blastocysts were then held in KSOM medium until transferred, 8–10 per uterine horn, into pseudo-pregnant recipient females at 2.5 days post coitum.

Results

Generation of TE-4N mice from high passage number ES cells

After electrofusion, 2-cell embryos developed into fused embryos. They were cultured further to the 4-cell stage and aggregated with ES cells, which exposed an Oct4-ΔPE-GFP transgene (Bao et al., 2009) being expressed in both the ICM of the blastocysts and in ES cells (Figure 1a). Ten to fifteen ES cells at passage 23 were aggregated with the tetraploid embryos (Figure 1b), which were then transferred into recipient mice. From the 201 aggregates transferred in this manner 16 pups were born. However, only five of these pups developed to adulthood (Figure 1c). Nevertheless all five adult TE-4N mice appeared normal and they showed normal fertility. They all produced normal offspring containing the transmitted ES cell genotype.

Derivation of ES cells from TE-4N mice

After mating C57BL/6J females mice to the TE-4N males, blastocysts were extracted from their uteri and plated on STO feeders in 2i-LIF medium as described previously (Ying et al., 2008). As the Oct4-ΔPE-GFP transgene is heterogeneous, only green coloured blastocysts indicating their derivation from ES cells were selected (Figure 2A). From 12 such C57BL/6 × TE-4N blastocysts, 9 teES were created representing an efficiency of 75% (Figures 2B–2D). In addition an attempt was made to derive teES cell lines in Knockout Serum Replacement (KSR) medium as described by Bryja et al. (2006). However, in all the attempts, either they could not be passaged further or they lost their ES morphology after freezing and thawing. Furthermore, even using higher LIF concentrations (Nagy et al., 2003) in the KSR medium, could not support the derivation of teES cells.

Molecular characterisation of teES cells

To assess the pluripotency state of the teES cells, AP staining and RT-PCR analyses were carried out using a variety of stem cell markers. All the teES cell colonies were AP positive and they exhibited a dome-like morphology with well-defined edges (Figure 3A). The teES cells expressed five pluripotency markers, including Nanog, Sox2 and Oct4, at high levels (Figure 3B) and immunoflorescent staining showed that all the teES showed this high level of expression of the three markers in all the teES cells (Figure 3C). Differentiation of the embryoid bodies (EB) showed that the teES cells comprised all three germ layers in vitro (Figure 3D).

Germline competency of teES cells

To determine the pluripotency status of the teES cells, male teES cells (Figure 4A) at passage 9 were injected into blastocysts (Figure 4B) and were then transferred into pesudopregnant recipients. Subsequently the gonads of each embryo were recovered surgically on day E12.5 (Figure 4C) and analysed for the presence of teES cells. From 20 transplanted embryos thus examined, eight proved to be germ line transmitted chimeras (Figures 4C and 4D) thereby demonstrating conclusively the incorporation of the teES cells into the germ line during development.

Discussion

Tetraploid complementation is a valuable technology for the generation of mice with targeted mutations and for embryology research in general. However, the efficiency of the tetraploid method is low due to a number of factors and its success rate is highly variable and depends upon the blastocyst ES cell strain, its number of passages and the quality of the in vitro preparation (Kirchain et al., 2008). In the present study 201 aggregates gave birth to only 16 pups with an efficiency that was only a little higher than in previous experiments (Li et al., 2005, 2007); this indicates that hybrid vigour may be detrimental to the survival of TE-4N mice (Eggan et al., 2001; Eggan and Jaenisch, 2003). Furthermore, 9 other pups died for different reasons, including developmental failure and matricide.

The derivation of mouse ES cells from blastocysts is very inefficient and is strongly dependent upon the strain of mouse used (Brook and Gardner, 1997; Bryja et al., 2006). In practice, derivation efficiency is very low in all strains other than strain 129 (McWhir et al., 1996; Bryja et al., 2006). In the present study, 9 teES cell lines were derived from 12 blastocysts to give an efficiency of production that was similar to the norm (Bryja et al., 2006). The result indicates that, like normal and targeted mice, fertile adult TE-4N mice are equally capable of giving rise to ES cells.

We also characterised the teES cells, we generated for ES cell markers by AP staining, RT-PCR, immunocytochemistry and embryoid body formation in vitro. The results all indicated that the teES cells resembled the basic state of naïve mouse ES cells cultured in 2i-LIF medium (Ying et al., 2008). The essential attribute of pluripotent ES cells is their capacity to contribute to all the germinal layers in their progeny, including the germlines of chimeras (Buehr et al., 2008). We injected the teES cells into blastocysts and transferred 20 chimeric embryos to recipient mice. The isolated gonads of the eight chimeric fetuses examined had all been colonised by teES cells, thereby indicating very high-grade germline transmission.

In summary, we generated TE-4N mice by aggregating ES cells with tetraploid embryos and we proved the success of the technique by analysing the derivation of pluripotent teES cells obtained from the TE-4N mice generated. The results indicated that male TE-4N mice have little effect on the derivation of ES cells and it will now be important to determine if teES cells can support the development of tetraploid embryos to full term.

Acknowledgements and funding

This study was supported by the Project of the Establishment of Animal Genetic Resources of Mongolia Plateau (No. 20091801 and No. 20121406), Science and Technology Agency, Inner Mongolia, P.R. China.

Conflict of interest

The authors have no conflicts of interest to declare.

Abbreviations

ES embryonic stem cells

TE-4N tetraploid

EB embryoid bodies

AP alkaline phosphatase

Received 23 June 2014;

Accepted 26 August 2014. 

Final version published online December 2014.

Corresponding author: e-mail: lixh@life.imu.edu.cn