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BOR - Papers in Press, published online ahead of print August 11, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.032227
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BIOLOGY OF REPRODUCTION 71, 1890–1897 (2004)
DOI: 10.1095/biolreprod.104.032227
© 2004 by the Society for the Study of Reproduction, Inc.

Reproductive Technology

Embryos Derived from Porcine Skin-Derived Stem Cells Exhibit Enhanced Preimplantation Development1

Hai Zhu, Jesse A. Craig, Paul W. Dyce, Nicole Sunnen, and Julang Li2

Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ongoing research to identify the most suitable type of donor cell for nuclear transfer (NT) has suggested that less differentiated stem cells may be better donors than other somatic cell types. Recently, we have reported the isolation and characterization of porcine skin-originated sphere (PSOS) stem cells from fetal skin, making it possible to test this hypothesis in a nonrodent animal model. In the present study, we have investigated and compared the feasibility and preimplantation developmental efficiency of using fetal PSOS cells and fibroblasts as nuclear-transfer donors. The majority of fetal PSOS cells are in the G1/ G0 stage of the cell cycle, which is desirable for NT. During long-term in vitro culture, fetal PSOS cells had greater genome stability, with a lower frequency of abnormal karyotypes than fetal fibroblast cells. Embryos cloned from PSOS cells showed enhanced preimplantation development compared with fibroblast cloned embryos, which is indicated by an increased rate of blastocyst development and a higher total cell number in Day 7 blastocysts. The gene expression profile of genes critical for early development from eight-cell-stage PSOS NT embryos more closely resembled the pattern observed from in vivo-produced embryos compared with that of fibroblast-cloned embryos. Cumulatively, our data suggest that fetal PSOS cells may be better donor cells for NT in the pig.

developmental biology, early development, embryo, gene regulation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Successful nuclear transfer (NT) for production of cloned domestic livestock species such as sheep [1, 2], cattle [3], goats [4], and pigs [5–7] have been reported. However, the efficiency of somatic cell NT remains disappointingly low (reviewed in [8, 9]), with a high proportion of reconstructed embryos lost during the preimplantation and peri-implantation periods. One of the main causes of this failure is suggested to be incomplete or incorrect epigenetic reprogramming of the donor cell nuclei upon transfer to the enucleated oocyte (reviewed in [9, 10]). Little is known about the initial molecular reprogramming events occurring upon transfer of the donor nuclei to the enucleated oocyte, but it is generally believed that this reprogramming must be complete before zygotic transcription commences and that the ease of reprogramming is related to the differentiation status of donor-cell type [10, 11].

Previous studies have suggested that the use of less differentiated donor-cell types may improve the success rate of NT. In the mouse, up to 20% of transferred blastocyst-stage embryos derived from embryonic stem (ES) cells survive to term, compared with less than 3% of cumulus cell-derived blastocysts (reviewed in [10, 11]). A similar result has also been reported comparing the developmental potential of murine ES cell clones with cumulus and tail-tip cell clones [12–14], despite lower blastocyst developmental rates, which is suggested to be due to the fact that a high percentage of ES cells are at S phase of the cell cycle [12, 13].

In livestock species, there has yet to be a comparison of the cloning efficiency using ES and somatic cells as donors, likely from the technical difficulty in obtaining and maintaining an undifferentiated ES cell line [15]. Until recently, there had been no report of a pluripotent ES cell line derived in the pig [15], limiting the investigation of this hypothesis in this species. Cloning efficiency has been compared, however, between various somatic cell types in the pig, with less differentiated fetal fibroblast cells showing enhanced preimplantation development over more highly differentiated cell types, such as cumulus and oviduct cells [16].

Recently, we have reported the isolation and characterization of porcine stem cells from fetal skin. These PSOS (porcine skin-originated sphere) cells are multipotent, and, upon induced differentiation, they are capable of differentiating into cells of the neuronal and mesenchymal lineages [17]. Prior to differentiation, these cells express markers that are related to an undifferentiated state, including STAT3, Oct4, and Nestin, while the expression of these markers is downregulated or absent in differentiated cells [17].

We hypothesize that these stem cells may be better donor cells for NT than somatic cells, as their less-differentiated state may ease epigenetic reprogramming by the oocyte. In this study, the feasibility of using fetal PSOS cells as NT donor cells was investigated. It was found that fetal PSOS cells were primarily in the G0/G1 stage of the cell cycle and maintained their genome stability during long-term in vitro culture. Furthermore, NT embryos reconstructed with fetal PSOS cells showed enhanced preimplantation development compared with fibroblast-derived embryos and exhibited gene expression patterns more characteristic of that of in vivo-produced embryos. These results suggest that fetal PSOS cells provide a viable alternative to fibroblasts as donor cells for NT and may improve the efficiency of NT in the pig.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of Fetal Fibroblasts

All experiments were conducted according to Institutional Animal Care and Use Committee guidelines. A primary cell line of fetal fibroblasts was isolated from Yorkshire pig fetuses as described [18]. Briefly, fetuses were removed from the uterus of 21-day pregnant sows and fetal carcasses were cut into 1–2-mm2 pieces using fine scissors. Tissue pieces were resuspended in Dulbecco minimum Eagle medium (DMEM; Gibco) supplemented with penicillin (100 IU/ml; Gibco), streptomycin (100 µg/ml; Gibco), and 10% fetal bovine serum (FBS; Gibco) and were cultured in 5% CO2 at 38.5°C. Following 5–6 days of culture, unattached clumps of cells and remaining tissue were removed. Attached fibroblast cells were disassociated from the plate with 0.05% trypsin/0.02% EDTA (Gibco) and washed with DMEM and were resuspended in freezing medium (10% dimethyl sulfoxide [DMSO; Sigma], 10% FBS in DMEM), and stored in liquid nitrogen as passage 0 until use.

Preparation of Fetal PSOS Cells

Cells were isolated as previously described [17]. Briefly, fetuses were removed from the uterus of 35- to 50-day pregnant Yorkshire sows and fetal skin from the back was carefully dissected free from other tissue and cut into 1–2-mm2 pieces. These pieces were washed three times in Hanks balanced salt solution (HBSS; Gibco) and digested with 0.2% trypsin for 35 min at 37°C, followed by 0.1% DNase for 1 min at room temperature. Tissue pieces were washed twice with HBSS, three times with DMEM-F12 (1:1, containing antibiotics; Gibco) and were mechanically dissociated via vortexing and pipetting. The cell suspension was poured through a 40-µm strainer (Falcon), centrifuged, and resuspended in stem-cell medium containing B-27 (Gibco), 20 ng/ml EGF (Sigma), and 40 ng/ml bFGF (Sigma). Cells were cultured in 100-mm tissue-culture dishes (Sarstedt) in a 37°C, 5% CO2 tissue-culture incubator until small floating spheres were formed. Spheres were transferred to a new culture dish containing fresh medium supplemented with growth factors and cultured for 7–10 days. Purified populations of floating spheres were obtained after 3–4 wk. Stem cells were collected by centrifuging culture medium at 500 x g for 6 min to remove spheres from suspension. The resulting cell pellet was resuspended in freezing medium (10% dimethyl sulfoxide [DMSO; Sigma] in DMEM) and stored in liquid nitrogen as passage 0 until use.

Cell Cycle Stage Analysis

Passage 2–3 cells were fixed in 80% methanol (–20°C) overnight. Cells were resuspended in DNA-staining solution containing 1 mg/ml RNase, 20 µg/ml propidium iodide, and 0.1% saponin (Sigma) and incubated at room temperature for 30 min. At least 10 000 cells were analyzed for DNA content per cell type, using a Becton Dickinson FACSCalibur. The percentages of cells at G0/G1, S, and G2/M phases of the cell cycle were calculated using the Modfit cell-cycle analysis program (Verity Software House).

Chromosome Analysis

Chromosomes were analyzed as described [18]. Briefly, 1.2 mg/ml thymidine was added to 60% confluent cell, and the cells were incubated overnight at 5% CO2 at 38.5°C. Cells were then washed with 1x PBS and were cultured in fresh medium for 6 h and trypsinized. The harvested cells were incubated in a hypotonic solution (1:6 dilution of serum in distilled H2O) for 20 min at 38.5°C and were fixed in a 3:1 methanol: acetic acid solution (4°C). Following centrifugation at 500 x g for 7 min, cells were resuspended in the fixative solution and dropped on cold, dry glass slides. Slides were air dried and stained with 1.5% Giemsa (EM Science) for 5 min. Chromosome morphology and number were evaluated using OpenLab image system (Openlab 2.25, Improvision Ltd.). Chromosome number was determined from a minimum of 100 separate spreads from each sample.

In Vitro Oocyte Maturation

Ovaries were collected from gilts at a local abattoir and transported to the laboratory in 1x PBS at 35°C. Cumulus-oocyte complexes (COCs) were isolated from antral follicles between 3–6 mm diameter using a 21-gauge needle. COCs were washed three times with maturation medium (TCM199 [Gibco] supplemented with 5 IU/ml FSH [Sioux Biochemicals], 5 IU/ml LH [Sioux Biochemicals], 0.1 mg/ml cysteine [Sigma], 10 ng/ml EGF [Sigma], and 10% porcine follicular fluid) and incubated in an atmosphere of 5% CO2 in air, 38.5°C for 42–44 h.

Nuclear Transfer

After 42–44 h of maturation, cumulus cells were removed from COCs by gentle vortexing in 0.1% hyaluronidase (Fisher) in TCM199. MII status was confirmed by the presence of the first polar body and was consistently reached by 75–80% of oocytes. MII-stage oocytes were incubated in TCM199 containing 5 µg/ml Hoechst 33342 (VWR) for 10 min and transferred to TCM199 containing 5 µg/ml cytochalasin B (Fisher). Oocytes were enucleated by aspirating the first polar body and metaphase plate with an 18- to 20-µm bevelled glass pipette. Enucleation was confirmed by the absence of the nucleus and first polar body when viewed under ultraviolet light. Following enucleation, a single donor cell (fetal fibroblast or fetal PSOS cell) was placed into the perivitelline space in contact with the oocyte membrane.

Reconstructed oocytes were fused and activated simultaneously by electrical pulse. Oocytes were placed between two electrodes (0.5 mm apart), overlayed with fusion medium (297 mM mannitol [CALB], 0.001 mM CaCl2 [Sigma], 0.05 mM MgCl2 [Sigma], 0.1% BSA [fraction V; Sigma]), and aligned manually. Fusion and activation were induced by two successive direct-current pulses of 2.6 kV/cm for 30 µsec using an Electro-Cell Manipulator 2000 (BTX) as described [19].

Embryo Culture

After fusion and activation, surviving embryos (intact plasma membrane; approximately 76%) were cultured in NCSU23 supplemented with 4 mg/ml BSA and overlayed with light mineral oil (Fisher). Embryos were cultured for 7 days to evaluate preimplantation development of NT embryos. Cleavage and blastocyst rates were calculated as the number of cleaved or blastocyst-stage embryos over the number of surviving embryos. The cell number of blastocysts was counted at Day 7 by staining of the cell nuclei with 1 µg/ml Hoechst 33342 (Sigma) and viewing under ultraviolet light. NT of fetal PSOS and fetal fibroblast cells was performed on seven independent experiments with approximately 300 embryos per cell type.

Superovulation and In Vivo-Produced Embryo Collection

Selected Yorkshire gilts between 70 and 80 kg were superovulated by intramuscular injection of 10 mg PGF2{alpha}, followed 48 h later by 2000 IU of eCG (Ayerst Veterinary Laboratories). One thousand international units of human chorionic gonadotropin (hCG; Ayerst Veterinary Laboratories) was administered 72 h later to induce ovulation. Gilts were artificially inseminated twice on the day following onset of estrus using semen from a Yorkshire boar. Gilts were slaughtered the next morning, reproductive tracts recovered, and one-cell embryos flushed from the oviduct. Approximately 10 embryos were recovered per pig and were washed three times in TCM199 plus 10% FBS and once in NCSU23. The embryos were cultured in NCSU23 supplemented with 4 mg/ml to the eight-cell stage.

Analysis of Pronuclear Formation

To examine nuclear maturation following fusion and activation, reconstructed one-cell embryos were fixed 12 h following activation as described [20], with modification. Briefly, one-cell embryos were fixed in ethanol:acetic acid (3:1) for 12 h and evaluated using a phase-contrast microscope (400x). The experiment was repeated five times, with 138 embryos examined for each cell type.

Reverse Transcription

Individual in vivo-produced embryos, fibroblasts, and PSOS NT embryos at the eight-cell stage were collected in 0.2-ml tubes in 8 µl lysis buffer containing 2 U/µl porcine RNase Inhibitor (Amersham) and 5 mM DTT (Invitrogen) and were stored at –80°C until use as described, with modifications [21]. Embryos were lysed by boiling followed by vortexing for 2 min each. DNase treatment was performed on the entire cell lysates as directed (Invitrogen). Reverse transcription was carried out in a final volume of 25 µl containing 1x First Strand Buffer (Invitrogen), 2.5 µM random hexamer primers (Applied Biosystems), 0.2 mM dNTP (Invitrogen), 200U Moloney murine leukemia virus (MMLV) (Invitrogen), and the entire DNase-treated lysate. The RT reaction was carried out at 25°C for 10 min and 37°C degrees for 50 min, followed by 15 min at 75°C to inactivate MMLV. A total of 33 in vivo-produced embryos, 57 fetal fibroblast-cloned embryos, and 45 fetal PSOS-cloned embryos were investigated individually for gene expression.

Real-Time PCR

Real-time PCR was preformed using SYBR Green PCR Mix (Qiagen), containing MgCl2, dNTP, and HotStar Taq polymerase. DNase I treated-cDNA (2.5 µl) from individual eight-cell embryos was added to a total volume of 25 µl containing 12.5 µl SYBR Green mix and 0.3 µM each of forward and reverse primers, which were designed based on porcine sequences where possible (Table 1). PCR conditions were as follows: initial activation of HotStar Taq at 95°C for 15 min, followed by 60 cycles of 95°C for 15 s, annealing for 30 s (Table 1), and extension at 72°C for 30 s. Histone 2A (H2A) was amplified for each embryo to confirm the presence of RNA and as a housekeeping gene for normalization. Expression of H2A has previously been shown to be consistent during the preimplantation period [22] and was used as the housekeeping gene for normalization during preimplantation development. Amplification and melt-curve analysis was performed using the Smart Cycler (Cepheid). Melt-curve analysis was conducted to confirm the specificity of each product, and products were electrophoresed on agarose gel to confirm product size. The identity of each product was confirmed by DNA sequence analysis. Real-time PCR data were analyzed using the 2–{Delta}{Delta}PCt method [23] using H2A for normalization. The Ct value for each gene was determined at a threshold of 30 fluorescence units. Real-time PCR efficiencies were acquired by amplification of a dilution series of PCR products and were consistent between genes. Negative controls were performed in which water was substituted for cDNA or reverse transcription was not performed before PCR. The experiment was repeated three times, with each replicate consisting of 10–20 individual embryos.


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  		TABLE 1. Primers used for real-time reverse transcription-PCR

Statistics

Cell cycle analysis was performed via t-test, while karyotype and preimplantation development (fusion, cleavage, and blastocyst rates and total cell number) was analyzed by one-way ANOVA. Normalized gene expression 2–{Delta}{Delta}Ct values were compared using a one-way ANOVA. Significant results from ANOVA were further analyzed by Tukey test. Results were considered significant at P < 0.05. Each experiment was repeated at least three times, and data represents the average of all repeats.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Majority of Fetal PSOS and Fetal Fibroblast Cells Are in G0/G1

Fetal PSOS cells were morphologically distinct from fetal fibroblasts; they grew in a defined serum-free medium as floating spheres (Fig. 1). The cell-cycle distribution of fetal PSOS and fibroblast cells was analyzed using flow cytometry. Following methanol fixation, DNA was labeled with propidium iodide and the relative DNA content of each cell was measured and classified as G0/G1, G2/M, or S phase. The distribution of cells at various phases of the cell cycle was similar between fetal PSOS and fibroblast cells. Seventy-six percent and 69% of fetal PSOS and fibroblast cells are in the G0/G1 stage of the cell cycle, respectively, indicating that the majority of cells in both cell types are at the cell cycle stage that is considered desirable for NT, while 14.9% and 10.1% of fetal PSOS and fibroblast cells are in the G2/M stages, respectively.



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  		FIG. 1. Representative image. (A) Fetal fibroblast and (B) fetal PSOS donor cells. Both types of donor cells were isolated from Yorkshire fetuses collected at ~45 days of gestation (x100)

Fetal PSOS Cells Show Greater Genome Stability Than Fetal Fibroblast Cells During Long-Term Culture

Central to the success of NT is the ability of the donor cell to retain a normal karyotype during in vitro culture. To compare the stability of fetal PSOS and fibroblast cell genomes during in vitro culture, karyotype analysis was performed at passages 1, 10, and 15, representing a total of 5 mo (PSOS) and 2 mo (fibroblasts) cultures. Conventional staining of metaphase-arrested chromosomes showed that, although both normal and abnormal karyotypes were apparent in both cell types (Fig. 2, A and B), there were a significantly lower proportion of abnormalities in fetal PSOS cells at each passage investigated (P < 0.01). As shown in Figure 2C, the proportion of fetal PSOS cells showing abnormal karyotypes following 5 mo in vitro culture (passage 15) remained below 3%, while this proportion increased to approximately 5% in fibroblast cells. The majority of abnormal karyotypes observed in fetal PSOS cells were in the form of monosomy or trisomy. This result suggests that fetal PSOS cells have greater genome stability than fetal fibroblast cells, potentially making them better suited to the long-term culture necessary for gene modification before NT.



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  		FIG. 2. Karyotype analysis of fetal PSOS and fetal fibroblast donor cells during in vitro culture. Cells were arrested at metaphase, fixed, and dropped on glass slides to generate smears. Cell-smear morphology was analyzed for ploidy at passage 2, 10, and 15. Representative chromosome spreads from fibroblasts, with (A) normal (n = 38) or (B) polyploidy (triploid; n = 57) karyotypes (x1000). (C) Percentage of cells with abnormal karyotypes at passage 2, 10 and, 15. Chromosome number was determined from a minimum of 100 separate spreads for each sample for each experiment. Data represents the mean ± SEM of three experiments. **, P < 0.01

Comparison of the Developmental Potential of Fetal PSOS and Fetal Fibroblast NT Embryos

To investigate the feasibility of using fetal PSOS cells as donor cells for NT, the in vitro preimplantation development of NT embryos reconstructed with fetal PSOS cells was compared with NT embryos reconstructed with fetal fibroblasts. The formation of pronuclear and polar body structures following activation is dependent on the cell cycle stage of the donor cell [20]. Embryos reconstructed with cells in G0/G1 are characterized by the presence of a single pronuclei (PN) and no polar body (PB), while cells in G2 or M contribute an extra set of chromosomes to the zygote and these embryos commonly extrude a second polar body to retain the correct ploidy, resulting in a conformation of 1 PN and 1 PB [20]. As shown in Table 2, fetal PSOS- and fibroblast-derived NT embryos showed a variety of PN and PB combinations following activation, including the expected combinations of 1 PN + 0 PB (Fig. 3A) and 1 PN + 1 PB (Fig. 3B). Other combinations observed included 2 PN + 0 PB (Fig. 3C), 2 PN + 1 PB (Fig. 3D), 3 PN + 0 PB (Fig. 3E), and 3 PN + 1 PB (Fig. 3F). These conformations are suggested to arise from abnormalities of the spindle-like structure that forms during premature chromatin condensation following fusion [20]. Significantly less abnormal combinations were observed in fetal PSOS embryos compared with fibroblast embryos (Table 2; P < 0.01), which may suggest that fetal PSOS cells are more capable of correct remodeling after fusion and activation.


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  		TABLE 2. Comparison of reconstructed one-cell fetal fibroblast and fetal PSOS cell NT embryos with different numbers of PN at 12 h following activation.a



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  		FIG. 3. Representative one-cell cloned embryos, with (A) 1 PN, (B) 1 PN + 1 PB, (C) 2 PN, (D) 2 PN + 1 PB, (E) 3 PN, (F) 3 PN + 1 PB (x400)

To study the influence of donor cell type on early embryo development, cloned embryos were cultured for 7 days in vitro to evaluate fusion, cleavage, blastocyst development, and total cell number. While there was no difference in either fusion (data not shown) or cleavage rates (Fig. 4A) between the two donor-cell types, embryos reconstructed with fetal PSOS cells showed enhanced preimplantation development. As shown in Figure 4B, a significantly higher percentage of embryos derived from fetal PSOS cells developed to blastocyst compared with fetal fibroblast-derived embryos (P < 0.01). In addition, the total cell number of the fetal PSOS-cloned embryos at the blastocyst stage was significantly higher than those of the fibroblast-cloned embryos (Fig. 4C; P < 0.05), suggesting that fetal PSOS-derived embryos are of higher quality than NT fibroblast embryos.



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  		FIG. 4. Preimplantation development of fetal PSOS and fetal fibroblast NT embryos. Oocytes from large antral follicles were in vitro matured to MII, enucleated, reconstructed with fetal PSOS or fetal fibroblast cells, and cultured in vitro for 7 days. (A) Cleavage rate (Day 2), (B) blastocyst rate (Day 7), and (C) total cell number per blastocyst (Day 7) were evaluated. Data is the mean ± SEM of seven independent experiments, with more than 300 embryos analyzed for each cell type. *, P < 0.05; **, P < 0.01

Gene Expression Pattern of Fetal PSOS-Cloned Embryos More Closely Resembles In Vivo-Produced Embryos

Gene expression was analyzed in cloned and in vivo-produced embryos at the eight-cell stage, as this is the earliest stage to detect zygotic gene expression in the pig, with the maternal-to-zygotic transition occurring at the four-cell stage. Following real-time reverse transcription-PCR amplification of Oct4, STAT3, FGFR, Rad50, Tsg101, Bub3, and DNMT1 and normalization with H2A, the 2-{Delta}{Delta}Ct method [23] was applied to calculate the relative expression level of each gene in fetal PSOS and fibroblast embryos relative to their in vivo-produced counterparts. Interestingly, while variable expression was observed for each gene investigated, the highest degree of variability was seen in fibroblast NT embryos compared with either fetal PSOS NT embryos or in vivo-produced embryos (Fig. 5). Significantly higher expression of STAT3 (Fig. 5C; 111-fold; P < 0.01), DNMT1 (Fig. 5G; 59.6-fold; P < 0.01), and Tsg101 (Fig. 5H; 66.9-fold; P < 0.01) was observed in fibroblast-cloned embryos compared with in vivo-produced and fetal PSOS-cloned embryos, while there was no significant difference in the expression level between fetal PSOS and in vivo-produced embryos. As shown in Figure 5, no significant differences were detected in Oct4, FGFR, Rad50, and Bub3 expression between in vivo-produced embryos and fetal PSOS- or fibroblast-cloned embryos.



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  		FIG. 5. Early gene expression in fetal PSOS and fetal fibroblast cloned embryos and in vivo-produced eight-cell embryos. (A) Representative agarose gel image of real-time reverse transcription-PCR amplification to confirm the size of H2A (226 bp; lane 1), Oct4 (230 bp; lane 2), STAT3 (129 bp; lane 3), FGFR2 (121 bp; lane 4), Rad50 (122 bp; lane 5), Bub3 (169 bp; lane 6), DNMT1 (213 bp; lane 7), Tsg101 (179 bp; lane 8) from an individual eight-cell PSOS embryo. Relative expression of (B) Oct4, (C) STAT3, (D) FGFR2, (E) Rad50, (F) Bub3, (G) DNMT1, and (H) Tsg101. Data is the mean ± SEM of three experiments with 10–20 embryos per group per experiment. **, P < 0.01. Different letter subscripts indicate statistically significant differences (P < 0.01)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Successful NT requires the recipient oocyte to convert the genome of the differentiated somatic cell back to an undifferentiated state, thus renewing its totipotency in a narrow time window [10]. It has been hypothesized that the genome of undifferentiated cells, such as stem cells, may be more easily reprogrammed by the recipient oocyte (reviewed in [10, 11]). The relationship between donor cell differentiation status and NT success has been demonstrated in mice, with NT embryos derived from ES cells showing significantly enhanced survival to term compared with those derived from somatic cell nuclei (reviewed in [10, 11]). In contrast with the low success rate of NT from somatic cells in mice [14, 24, 25], up to one third of blastocysts cloned using ES cells develop to term (reviewed in [10]). These results support the hypothesis that undifferentiated stem cells may be more easily reprogrammed to an embryonic state. We have previously reported the isolation of stem cells from fetal porcine skin, which proliferate actively in vitro and express markers that are related to an undifferentiated state [17]. Through differentiation studies, these cells have demonstrated differentiation potential for cells from two different lineages [17]. Thus, fetal PSOS cells allow the investigation of using stem cells as NT donors in a nonrodent animal model such as the pig.

Recently, successful preimplantation development of NT embryos reconstructed with adult bovine mesenchymal stem cells has been reported [26]. Although no comparison was made regarding the developmental potential between NT embryos reconstructed with these cells and other somatic cell types, this report suggests that stem cells from adult animals can be reprogrammed and developed to term. In the present study, we report that the cell-cycle distribution of fetal PSOS cells is similar to fetal fibroblast cells and that fetal PSOS cells have greater genome stability during long term in vitro culture. NT embryos reconstructed with fetal PSOS cells show enhanced developmental potential compared with those reconstructed with fetal fibroblast cells, with a significantly higher proportion of embryos reaching the blastocyst stage following 7 days in vitro culture. Moreover, the expression pattern of developmentally important genes more closely resembles that of in vivo-produced embryos, suggesting that the genome of fetal PSOS cells may be easier to reprogram. Cumulatively, this evidence suggests that fetal PSOS cells may be better donor cells for NT in the pig.

Donor cells in the G0/G1 stage are commonly held to be more desirable for NT, resulting in a higher proportion of live births than cells in other stages of the cell cycle (reviewed in [11]). In the present study, we have demonstrated that the cell-cycle distribution of fetal PSOS cells is not significantly different from fetal fibroblast cells, with over 75% of cells in the G0/G1 phase (Table 2). In comparison, approximately 60% of ES cells are in the S phase of the cycle, which is thought to contribute to the high rate of developmental failure of ES NT clones before the blastocyst stage [10]. ES cell clones that reach the blastocyst stage are suggested to be derived from cells in the G1 phase and show enhanced postimplantation development compared with somatic cell clones (reviewed in [10]).

The cell-cycle stage of the donor cell is also thought to impact postfusion remodeling events. Embryos derived from G1/G0 cells typically exhibit 1 PN + 0 PB, while embryos from cells in G2/M show 1 PN + 1 PB [20]. In the present study, significantly more embryos reconstructed with fetal PSOS cells showed the nuclear conformation of 1 PN or 1 PN + 1 PB than those from fetal fibroblast cells (Table 2). Abnormal nuclear conformations of porcine NT embryos (2 PN + 0 PB, 2 PN + 1 PB, 3 PN + 0 PB and 3 PN + 1 PB) have previously been reported and are thought to result form abnormal formation of spindle-like structures following fusion [20]. The lower incidence of nuclear abnormalities in PSOS NT embryos may suggest that fetal PSOS-cloned embryos are more competent to undergo correct remodeling during early embryo development.

Embryonic development requires the correct temporal and spatial expression of many genes, the functions of which are essential for survival. For example, disrupted expression of Oct4, STAT3, FGFR2, Rad50, Bub3, DNMT1, or Tsg101 results in embryonic lethality during early development [27–33]. Abnormal gene expression is frequently observed in NT embryos and is one of the suggested causes of the low success rates of this approach. In the present study, we observed abnormal expression of STAT3, DNMT1, and Tsg101 in fetal fibroblast NT embryos compared with in vivo-produced embryos, while embryos derived from fetal PSOS cells showed expression patterns similar to in vivo-produced embryos.

STAT3 is a cytoplasmic transcription factor important in cytokine signaling, the activation of which leads to activation of transcription of genes with specific STAT3 recognition sites (reviewed in [34]). Unlike other members of the STAT family, ablation of STAT3 in mice leads to embryonic lethality, suggesting STAT3 plays a critical role in embryonic development [28]. In the present study, we observed upregulation of STAT3 mRNA expression in fetal fibroblast NT embryos compared with both fetal PSOS-cloned and in vivo-produced embryos. Due to the pleiotropic effects of STAT3, there are several possible explanations for the upregulation of STAT3 in fetal fibroblast NT embryos. First, upregulation of STAT3 may influence cell proliferation (reviewed in [35]). Interestingly, we did not observe an increase in the number of cells in fibroblast NT blastocysts, which may suggest that other mechanisms are restricting cell proliferation induced by STAT3 upregulation. Alternatively, STAT3 has been described to mediate survival function, due to its induction of the antiapoptotic genes Bcl-2 and Bcl-X (reviewed in [34]). In the embryo, overexpression of STAT3 in fetal fibroblast NT embryos may also play a protective role in the embryo, in that its upregulation may promote survival despite abnormal expression of other genes.

Tumor susceptibility gene 101 (Tsg101) is a putative transcription factor and/or tumor suppressor gene, which may also function outside of the nucleus to influence microtubule dynamics as well as cell growth and proliferation [36, 37]. During mitosis, Tsg101 protein is colocalized with the microtubule-organizing center and mitotic spindle, suggesting it also plays a role in mitotic division [36]. Tsg101 is suggested to play a protective role in the cell, as its deficiency is related to increased abnormalities such as aberrant mitotic spindles, abnormal distribution of metaphase chromatin, and aneuploidy [36]. In the present study, we observed a significant upregulation of Tsg101 in fibroblast NT embryos. It is not known whether its overexpression is a self-protection response or a reprogramming failure of the embryos. Interestingly, overexpression of Tsg101 has also been linked to cell death [36], suggesting a possible detrimental effect of Tsg101 overexpression.

DNA methylation patterns are established and maintained via two DNA methyltransferase (DNMT) enzymes. Hemimethylated DNA is recognized by DNMT1 and converts hemimethylated sites to fully methylated sites. Deletion of DNMT1 leads to genome-wide hypomethylation and results in loss of monoallelic expression of imprinted genes and ultimately embryonic lethality by midgestation [32]. Reduced DNMT1 expression has previously been reported in bovine NT embryos reconstructed from granulosa cells relative to in vitro-produced embryos [38]. Wrenzycki et al. [38] also reported a difference in DNTM1 expression between in vivo- and in vitro-produced embryos, with in vivo-produced embryos expressing significantly less DNMT1, although it was suggested that expression of DNMT1 was more severely affected by in vitro production of embryos via in vitro fertilization. In the present study, we observed a significant increase in DNMT1 mRNA expression in fetal fibroblast NT embryos compared with either in vivo-produced or fetal PSOS NT embryos. Abnormal spatial and temporal expression of DNMT1 protein has also been reported in murine NT embryos derived from cumulus cells, with an increase in DNMT1 protein observed in the eight-cell-stage cloned embryo [39]. Our data are consistent with this observation. Overexpression of DNMTs are suggested to be correlated with methylation errors during both maintenance and de novo methylation, which are transferred to subsequent daughter cells (reviewed in [40]). The possibility that overexpression of DNMT1 alters methylation of the genome and thus expression of other genes in fetal fibroblast-cloned embryos is to be investigated.

In the present study, we did not observe significant differences in expression of Oct4, FGFR2, Rad50, or Bub3. Previously, both normal and abnormal expressions of Oct4 and FGFR2 have been reported. In bovine granulosa cell NT embryos, expression of Oct4 was found to be consistent with in vitro-fertilized embryos, while FGFR2 expression was found to be downregulated [41]. Interestingly, normal FGFR2 expression has also been reported by this group and is suggested to be related to the donor cell line used in the NT experiments [41]. The correct temporal expression of Oct4 has also been reported in murine NT embryos reconstructed with cumulus cell nuclei; however, the spatial expression was found to be abnormal, with expression observed in both inner cell mass and trophectoderm cells [42]. In the present study, we observed similar levels of Oct4, FGFR2, Rad50, and Bub3 expression in NT- and in vivo-produced embryos, but whether the spatial expression of these genes is also consistent bears further investigation.

In conclusion, we have demonstrated that NT embryos reconstructed using fetal PSOS cells as donors show enhanced preimplantation development in vitro, and their gene expression pattern more resembles that of the in vivo-produced embryos compared with fetal fibroblast NT embryos. Our findings suggest that the use of PSOS cells as NT donors may help to increase the efficiency of this technique in the porcine species.


   ACKNOWLEDGMENTS
 
We wish to thank Jennifer Croce for technical assistance in oocyte collection, staff at the Meat Wing at the University of Guelph and at Constoga Farm Fresh Pork for their assistance in collecting porcine ovaries, and the staff at Arkell Research station for their ongoing assistance.


   FOOTNOTES
 
1 Supported by Natural Sciences and Engineering Research Council studentships to J.A.C. and P.W.D., Agriculture and Agri-Food Canada, Ontario Ministry of Agriculture and Food, Ontario Pork, and the Food System Biotechnology Centre at the University of Guelph. H.Z. and J.A.C. contributed equally to this work. Back

2 Correspondence: Julang Li, Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1. FAX: 519 836 9873; jli{at}uoguelph.ca Back

Received: 17 May 2004.

First decision: 11 June 2004.

Accepted: 22 July 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Campbell KH, McWhir J, Ritchie WA, Wilmut I. Sheep cloned by nuclear transfer from a cultured cell line. Nature 1996 380:64-66[CrossRef][Medline]
  2. Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KH. Viable offspring derived from fetal and adult mammalian cells. Nature 1997 385:810-813[CrossRef][Medline]
  3. Cibelli JB, Stice SL, Golueke PJ, Kane JJ, Jerry J, Blackwell C, Ponce de Leon FA, Robl JM. Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science 1998 280:1256-1258[Abstract/Free Full Text]
  4. Baguisi A, Behboodi E, Melican DT, Pollock JS, Destrempes MM, Cammuso C, Williams JL, Nims SD, Porter CA, Midura P, Palacios MJ, Ayres SL, Denniston RS, Hayes ML, Ziomek CA, Meade HM, Godke RA, Gavin WG, Overstrom EW, Echelard Y. Production of goats by somatic cell nuclear transfer. Nat Biotechnol 1999 17:456-461[CrossRef][Medline]
  5. Onishi A, Iwamoto M, Akita T, Mikawa S, Takeda K, Awata T, Hanada H, Perry AC. Pig cloning by microinjection of fetal fibroblast nuclei. Science 2000 289:1188-1190[Abstract/Free Full Text]
  6. Betthauser J, Forsberg E, Augenstein M, Childs L, Eilertsen K, Enos J, Forsythe T, Golueke P, Jurgella G, Koppang R, Lesmeister T, Mallon K, Mell G, Misica P, Pace M, Pfister-Genskow M, Strelchenko N, Voelker G, Watt S, Thompson S, Bishop M. Production of cloned pigs from in vitro systems. Nat Biotechnol 2000 18:1055-1059[CrossRef][Medline]
  7. Polejaeva IA, Chen SH, Vaught TD, Page RL, Mullins J, Ball S, Dai Y, Boone J, Walker S, Ayares DL, Colman A, Campbell KH. Cloned pigs produced by nuclear transfer from adult somatic cells. Nature 2000 407:86-90[CrossRef][Medline]
  8. Niemann H, Wrenzycki C, Lucas-Hahn A, Brambrink T, Kues WA, Carnwath JW. Gene expression patterns in bovine in vitro-produced and nuclear transfer-derived embryos and their implications for early development. Cloning Stem Cells 2002 4:29-38[CrossRef][Medline]
  9. Han YM, Kang YK, Koo DB, Lee KK. Nuclear reprogramming of cloned embryos produced in vitro. Theriogenology 2003 59:33-44[CrossRef][Medline]
  10. Rideout WM 3rd, Eggan K, Jaenisch R. Nuclear cloning and epigenetic reprogramming of the genome. Science 2001 293:1093-1098[Abstract/Free Full Text]
  11. Oback B, Wells D. Donor cells for nuclear cloning: many are called, but few are chosen. Cloning Stem Cells 2002 4:147-168[CrossRef][Medline]
  12. Rideout WM 3rd, Wakayama T, Wutz A, Eggan K, Jackson-Grusby L, Dausman J, Yanagimachi R, Jaenisch R. Generation of mice from wild-type and targeted ES cells by nuclear cloning. Nat Genet 2000 24:109-110[CrossRef][Medline]
  13. Wakayama T, Rodriguez I, Perry AC, Yanagimachi R, Mombaerts P. Mice cloned from embryonic stem cells. Proc Natl Acad Sci U S A 1999 96:14984-14989[Abstract/Free Full Text]
  14. Wakayama T, Yanagimachi R. Cloning of male mice from adult tail-tip cells. Nat Genet 1999 22:127-128[CrossRef][Medline]
  15. Li M, Zhang D, Hou Y, Jiao L, Zheng X, Wang WH. Isolation and culture of embryonic stem cells from porcine blastocysts. Mol Reprod Dev 2003 65:429-434[CrossRef][Medline]
  16. Lee GS, Hyun SH, Kim HS, Kim DY, Lee SH, Lim JM, Lee ES, Kang SK, Lee BC, Hwang WS. Improvement of a porcine somatic cell nuclear transfer technique by optimizing donor cell and recipient oocyte preparations. Theriogenology 2003 59:1949-1957[CrossRef][Medline]
  17. Dyce PW, Zhu H, Craig J, Li J. Stem cells with multilineage potential derived from porcine skin. Biochem Biophys Res Commun 2004 316:651-658[CrossRef][Medline]
  18. Zhu H, Tamot B, Quinton M, Walton J, Hacker RR, Li J. Influence of tissue origins and external microenvironment on porcine fetal fibroblast growth, proliferative life span and genome stability. Cell Prolif 2004 37:255-266[CrossRef][Medline]
  19. Kuhholzer B, Hawley RJ, Lai L, Kolber-Simonds D, Prather RS. Clonal lines of transgenic fibroblast cells derived from the same fetus result in different development when used for nuclear transfer in pigs. Biol Reprod 2001 64:1695-1698[Abstract/Free Full Text]
  20. Lai L, Tao T, Machaty Z, Kuhholzer B, Sun QY, Park KW, Day BN, Prather RS. Feasibility of producing porcine nuclear transfer embryos by using G2/M-stage fetal fibroblasts as donors. Biol Reprod 2001 65:1558-1564[Abstract/Free Full Text]
  21. Kumazaki T. Detection of mRNA expression and alternative splicing in a single cell. In: O'Connell J (ed.), RT-PCR Methods, vol. 193. New Jersey: Humana Press Inc.; 2002:59–64
  22. Robert C, McGraw S, Massicotte L, Pravetoni M, Gandolfi F, Sirard M-A. Quantification of housekeeping transcript levels during the development of bovine preimplantation embryos. Biol Reprod 2002 67:1465-1472[Abstract/Free Full Text]
  23. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(–Delta Delta C(T)) Method. Methods 2001 25:402-408[CrossRef][Medline]
  24. Wakayama T, Perry AC, Zuccotti M, Johnson KR, Yanagimachi R. Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 1998 394:369-374[CrossRef][Medline]
  25. Wakayama T, Yanagimachi R. Mouse cloning with nucleus donor cells of different age and type. Mol Reprod Dev 2001 58:376-383[CrossRef][Medline]
  26. Kato Y, Imabayashi H, Mori T, Tani T, Taniguchi M, Higashi M, Matsumoto M, Umezawa A, Tsunoda Y. Nuclear transfer of adult bone marrow mesenchymal stem cells: developmental totipotency of tissue-specific stem cells from an adult mammal. Biol Reprod 2004 70:415-418[Abstract/Free Full Text]
  27. Nichols J, Zevnik B, Anastassiadis K, Niwa H, Klewe-Nebenius D, Chambers I, Scholer H, Smith A. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 1998 95:379-391[CrossRef][Medline]
  28. Takeda K, Noguchi K, Shi W, Tanaka T, Matsumoto M, Yoshida N, Kishimoto T, Akira S. Targeted disruption of the mouse Stat3 gene leads to early embryonic lethality. Proc Natl Acad Sci U S A 1997 94:3801-3804[Abstract/Free Full Text]
  29. Arman E, Haffner-Krausz R, Chen Y, Heath JK, Lonai P. Targeted disruption of fibroblast growth factor (FGF) receptor 2 suggests a role for FGF signaling in pregastrulation mammalian development. Proc Natl Acad Sci U S A 1998 95:5082-5087[Abstract/Free Full Text]
  30. Luo G, Yao MS, Bender CF, Mills M, Bladl AR, Bradley A, Petrini JH. Disruption of mRad50 causes embryonic stem cell lethality, abnormal embryonic development, and sensitivity to ionizing radiation. Proc Natl Acad Sci U S A 1999 96:7376-7381[Abstract/Free Full Text]
  31. Kalitsis P, Earle E, Fowler KJ, Choo KH. Bub3 gene disruption in mice reveals essential mitotic spindle checkpoint function during early embryogenesis. Genes Dev 2000 14:2277-2282[Abstract/Free Full Text]
  32. Li E, Bestor TH, Jaenisch R. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 1992 69:915-926[CrossRef][Medline]
  33. Ruland J, Sirard C, Elia A, MacPherson D, Wakeham A, Li L, de la Pompa JL, Cohen SN, Mak TW. p53 accumulation, defective cell proliferation, and early embryonic lethality in mice lacking tsg101. Proc Natl Acad Sci U S A 2001 98:1859-1864[Abstract/Free Full Text]
  34. Levy DE, Lee CK. What does Stat3 do?. J Clin Invest 2002 109:1143-1148[CrossRef][Medline]
  35. Calo V, Migliavacca M, Bazan V, Macaluso M, Buscemi M, Gebbia N, Russo A. STAT proteins: from normal control of cellular events to tumorigenesis. J Cell Physiol 2003 197:157-168[CrossRef][Medline]
  36. Xie W, Li L, Cohen SN. Cell cycle-dependent subcellular localization of the TSG101 protein and mitotic and nuclear abnormalities associated with TSG101 deficiency. Proc Natl Acad Sci U S A 1998 95:1595-1600[Abstract/Free Full Text]
  37. Li L, Cohen SN. Tsg101: a novel tumor susceptibility gene isolated by controlled homozygous functional knockout of allelic loci in mammalian cells. Cell 1996 85:319-329[CrossRef][Medline]
  38. Wrenzycki C, Wells D, Herrmann D, Miller A, Oliver J, Tervit R, Niemann H. Nuclear transfer protocol affects messenger RNA expression patterns in cloned bovine blastocysts. Biol Reprod 2001 65:309-317[Abstract/Free Full Text]
  39. Chung YG, Ratnam S, Chaillet JR, Latham KE. Abnormal regulation of DNA methyltransferase expression in cloned mouse embryos. Biol Reprod 2003 69:146-153[Abstract/Free Full Text]
  40. Jones PA. DNA methylation errors and cancer. Cancer Res 1996 56:2463-2467[Free Full Text]
  41. Daniels R, Hall V, Trounson AO. Analysis of gene transcription in bovine nuclear transfer embryos reconstructed with granulosa cell nuclei. Biol Reprod 2000 63:1034-1040[Abstract/Free Full Text]
  42. Boiani M, Eckardt S, Scholer HR, McLaughlin KJ. Oct4 distribution and level in mouse clones: consequences for pluripotency. Genes Dev 2002 16:1209-1219[Abstract/Free Full Text]



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