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Keywords:

  • Rabbit embryonic stem cells;
  • Parthenogenetic activation;
  • Stem cell marker self-renewal;
  • Signaling pathway

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

We described the derivation of four stable pluripotent rabbit embryonic stem cell (ESC) lines, one (RF) from blastocysts fertilized in vivo and cultured in vitro and three (RP01, RP02, and RP03) from parthenogenetic blastocysts. These ESC lines have been cultivated for extended periods (RF >1 year, RP01 >8 months, RP02 >8 months, and RP03 >6 months) in vitro while maintaining expression of pluripotent ESC markers and a normal XY or XX karyotype. The ESCs from all lines expressed alkaline phosphatase, transcription factor Oct-4, stage-specific embryonic antigens (SSEA-1, SSEA-3, and SSEA-4), and the tumor-related antigens (TRA-1-60 and TRA-1-81). Similar to human and mouse ESCs, rabbit ESCs expressed pluripotency (Oct-4, Nanog, SOX2, and UTF-1) and signaling pathway genes (fibroblast growth factor, WNT, and transforming growth factor pathway). Morphologically, rabbit ESCs resembled primate ESCs, whereas their proliferation characteristics were more like those seen in mouse ESCs. Rabbit ESCs were induced to differentiate into many cell types in vitro and formed teratomas with derivatives of the three major germ layers in vivo when injected into severe combined immunodeficient mice. Our results showed that pluripotent, stable ESC lines could be derived from fertilized and parthenote-derived rabbit embryos.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Embryonic stem cell (ESC) lines have been successfully established in more than a dozen species, including humans, monkeys, and rats [1, [2], [3]4], since the first successes were reported in the mouse in 1981 [5, 6]. ESCs are important as they provide a model for the study of development biology and a potential source of cells for use in cell- or tissue-based replacement therapy. Human ESCs hold promise in the treatment of degenerative disorders such as Parkinson, Alzheimer, and diabetes. Before clinical applications are begun, however, extensive preclinical studies must be completed in suitable animal models assessing safety, efficacy, and long-term survival of transplanted ESC-derived phenotypes.

The rabbit, as a laboratory animal model, has several advantages in the study of human physiological disorders. Not only can physiological manipulations in the rabbit be more easily carried out than those in mice (because of its larger size), but also it is phylogenetically closer to primates than are rodents. The rabbit has been employed for studying human vascular disease, in invasive hemodynamic measurements, and in investigations of disorders of the central nervous system, abnormalities of lipoprotein metabolism, cardiomyopathy, and pulmonary physiology [7, [8]9]. Unfortunately, to our knowledge, there are currently no rabbit ESC lines available to augment studies in this valuable animal model despite the prior report by the Moreadith group on initial efforts to isolate and maintain rabbit ESCs [10, 11].

Parthenogenesis involves the initiation of embryonic development without a male contribution, and viable pregnancies from parthenotes are precluded based on abnormalities in imprinted gene expression. Nevertheless, parthenote-derived stem cells can contribute to research assessing the importance of imprinting gene expression in early development and to autologous cell-based therapy in the female. That some of the epigenetic abnormalities in parthenotes can be overcome was evidenced by the birth of parthenogenetic mice that were themselves reproductively normal [12, [13], [14]15]. Recent reports in with a nonhuman primate parthenote-derived stem cell line indicated that these cells could differentiated into tyrosine hydroxylase-positive dopaminergic neuronal phenotypes that were capable of long-term survival after engraftment into rat or monkey brains [16, 17]. However, there have been no other reports concerning the derivation of ESC lines from parthenotes. The objectives of this study were to derive ESCs from both fertilized and parthenogenetic rabbit embryos and to study the characteristics of these cells, including marker expression, growth rate, pluripotency, and capacity for self-renewal with preservation of the undifferentiated phenotype over a long culture time.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Embryo Recovery and Culture

Fertilized and parthenogenetic blastocysts were obtained from Japanese-white breeds (5 months old) by ovarian stimulation with 50 IU/kg pregnant mare serum gonadotrophin (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) followed by 100 IU of human chorionic gonadotropin (hCG) (Sigma-Aldrich) 72 hours later. One-cell fertilized embryos and cumulus-oocyte complexes were collected surgically 13.5 hours after hCG administration and mating to an intact (for fertilized) or a vasectomized (for parthenogenesis) male by flushing oviducts through the uterotubal junction and collecting the fluid through a cannula clamped in the infundibulum. Embryos and oocytes were incubated in 0.5% hyaluronidase (Sigma-Aldrich) to completely remove cumulus cells by gentle pipetting for 2 minutes and 10 minutes, respectively. Three treatments were tested for oocyte parthenogenetic activation. (a) Electrical: oocytes were washed and equilibrated in electrofusion medium (0.3 M mannitol, 0.1 mM MgSO4, and 0.05 mM CaCl2) for 2 minutes, placed in an activation chamber, overlaid with electrofusion medium, and given two direct-current pulses of 2.0 kV/cm for 40 microseconds with BTX ECM2001 electrofusion apparatus (BTX, Harvard Apparatus, San Diego, http://www.btxonline.com). (b) Chemical: oocytes were incubated in 5 μM ionomycin (Sigma-Aldrich) in tyrode lactate-HEPES plus 5 mg/ml bovine serum albumin (Sigma-Aldrich) for 2 minutes followed by exposure to M199 (Sigma-Aldrich) containing 2 mM 6-dimethylaminopurine (Sigma-Aldrich) and 5 μg/ml cytochalasin B (Sigma-Aldrich) for 2 hours. (c) Electrical/chemical: oocytes were sequentially treated by the electrical (first) and chemical (second) protocols as described above. One-cell fertilized embryos and parthenotes were rinsed in TL-HEPES and cultured in M199 microdrops (50 μl, five embryos in a drop) containing 15% newborn calf serum (NCS) (Invitrogen Corporation, Carlsbad, CA, http://www.invitrogen.com), covered with mineral oil (Sigma-Aldrich), in an atmosphere of 5% CO2, 95% humidified air at 38°C for 4 days and developed to the blastocyst stage.

ESC Derivation and Propagation

After zona pellucidae were removed with 0.5% protease (Sigma-Aldrich), zona-free blastocysts were treated with 0.05% trypsin (Sigma-Aldrich)-0.008% EDTA (Sigma-Aldrich) for 3–5 minutes before the inner cell mass (ICM) was mechanically separated from the trophectoderm and seeded on mouse embryonic fibroblast (MEF) feeder layers which were from 13.5-day-old mouse fetuses (129 strain) in gelatin (Sigma-Aldrich)-coated four-well plates (NUNC A/S, Roskilde, Denmark, http://www.nuncbrand.com). MEFs from 13.5-day-old fetuses (129 strain) were inactivated by irradiation (35 Gy, γ-irradiation). The culture medium consisted of Dulbecco's modified Eagle's medium (high glucose, without sodium pyruvate; Invitrogen Corporation) supplemented with 2 mM glutamine (Sigma-Aldrich), 0.1 mM mercaptoethanol (Sigma-Aldrich), 1× nonessential amino acids (Invitrogen Corporation), 1× penicillin-streptomycin (Sigma-Aldrich), and 15% defined fetal bovine serum (HyClone, Logan, UT, http://www.hyclone.com). Approximately 3–5 days after seeding, ICM-derived outgrowths that were treated with 5 mg/ml dispase (Invitrogen Corporation) were mechanically removed from feeder cells after the perimeter of the outgrowths folded back, then treated in a drop exposure to 0.05% trypsin-0.008% EDTA solution for 3–5 minutes at room temperature, and mechanically disaggregated into small clumps. The small ICM cell clumps were reseeded on fresh MEFs, and the ESC-like cell colonies would be seen several days later. The ESC-like cell colonies were mechanically divided into small clumps by micropipette followed by treatment of 5 mg/ml dispase for several minutes and were further propagated in clumps of 5 to approximately 50 cells on fresh MEFs. The stable rabbit ESC lines were obtained after being passaged 5–10 times exactly as in the above methods and were then passaged with 5–10 mg/ml dispase only every 3–5 days. The cells were frozen in liquid nitrogen using freezing solution consisting of 10% dimethyl sulfoxide (Sigma-Aldrich) and 90% NCS according to the freezing protocol as previously described [18].

ESC Characterization

Cultured rabbit ESC colonies, attached embryoid bodies (EBs), or differentiated cells were fixed in situ with 4% paraformaldehyde in phosphate-buffered saline for 20 minutes at room temperature. For alkaline phosphatase activity analysis, cocultures were stained according to the manufacturer's protocol (Sino-American Biotechnology Company, Luoyang, China, http://www.sabc.com.cn/en). For immunofluorescence, fixed cells were stained with one of the following primary antibodies: stage-specific embryonic antigen (SSEA)-1, SSEA-3, SSEA-4, tumor-related antigen (TRA)-1-60, TRA-1-81, and octamer-binding transcription factor 4 (Oct-4) (all from Chemicon International, Temecula, CA, http://www.chemicon.com) after blocking with 10% goat serum and were then incubated for 30 minutes with fluorescein isothiocyanate- or phycoerythrin-conjugated second antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, http://www.scbt.com) for antibody localization. Cell nuclei were stained with Hoechst 33342 (Sigma-Aldrich). Cells were examined using a confocal laser scanning system (LSM 510 META; Carl Zeiss, Jena, Germany, http://www.zeiss.com). Karyotyping was based on the description by Hayes et al. [19].

ESC Differentiation In Vitro and In Vivo

In vitro differentiation involved adherent differentiation (monolayer cultivation), prolonged cultivation, or EB formation [1, [2]3]. For adherent differentiation, ESCs were dispersed with 10 mg/ml dispase exposure, plated in gelatin-coated six-well dishes (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com), and cultured for 10–30 days. The prolonged cultivation approach involved maintenance of ESCs at high densities for 2–4 weeks without feeder layer replacement. For EB formation, ESCs were digested with 10 mg/ml dispase, resuspended in ESC culture medium, and cultured in hanging drops (30 μl/drop, 40 cells per microliter). Two days later, aggregated simple EBs were transferred to bacteria culture dishes (Becton, Dickinson and Company) coated with agar (Sigma-Aldrich) to maintain continuous suspension cultures. After another 2 days, the resultant cystic EBs were plated into gelatin-coated six-well plates and continuously cultured in ESC culture medium. Cell markers were assessed by immunocytochemistry or reverse transcription-polymerase chain reaction (RT-PCR). The primary antibodies CK7 (DakoCytomation, Glostrup, Denmark, http://www.dakocytomation.com), vimentin (DakoCytomation), α-fetoprotein (AFP) (Sigma-Aldrich), albumin (Sigma-Aldrich), smooth muscle marker actin (DakoCytomation), nestin (Chemicon International), and glial fibrillary acidic protein (GFAP) (Chemicon International) were used for immunocytochemistry as described above. For teratoma formation, 6–8-week-old severe combined immunodeficient (SCID)-beige mice (Charles River Laboratories, Inc., Wilmington, MA, http://www.criver.com) were injected intramuscularly with 2–4 × 106 rabbit ESCs. Tumors were removed (8–14 weeks after injection) and fixed in 4% paraformaldehyde. Paraffin sections were stained with hematoxylin/eosin and processed for histological examination.

RNA Preparation and Gene Expression Analyses

Total RNA was extracted using a TRIzol RNA isolation kit (Invitrogen Corporation). Cytoplasmic RNA from human (BG02, from National Institutes of Health, BresaGen Limited, Thebarton, SA, Autstralia, http://www.bresagen.com.au) [20], mouse (R1, gift from Dr. A. Nagy's laboratory, Mount Sinai Hospital, Ontario, Canada), and rabbit ESCs (RF, RP01, and RP02), and their derived EBs were reverse-transcribed to single-stranded cDNA. Aliquots of cDNA were used as a template for PCR amplification with specific primer sets derived from the conserved sequences of the human, mouse, and rat genes because mRNA sequences of most rabbit genes were unavailable [21]. The sense and antisense primer sequences, corresponding PCR condition, and product sizes are shown in Table 1. Five microliters of PCR products were separated on a 1.5% agarose gel and visualized by ethidium bromide staining.

Table Table 1.. Polymerase chain reaction primers and conditions for gene analysis
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Statistical Analysis

The results were presented as means ± SEM. Statistical analysis was performed using the least significant difference test. Statistical significance was defined as p < .05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Generation of Rabbit ESC Lines

One ESC line (RF) was isolated in 10 attempts from blastocysts that were fertilized in vivo and cultured in vitro. Three parthenote ESC lines (RP01, RP02, and RP03) were recovered from 10 parthenogenetic blastocysts. In both instances, the blastocyst morphology was comparable with distinct ICMs (Fig. 1A, 1E, respectively). Parthenote ESC lines were derived only from blastocysts generated following a combined electrical-chemical activation protocol despite our ability to produce blastocysts by electrical or chemical activation alone (Table 2). The ability of isolated ICMs to support outgrowth within 3–5 days of seeding varied with the treatment protocol. The levels were 30% (3/10) for sperm-fertilized blastocysts, 60% (6/10) from parthenote-derived blastocysts following electrical/chemical activation (Fig. 1B, 1F, respectively), 17% (2/12) by electrical activation alone, and 20% (3/15) in the case of chemical activation. The small cells in the ICM outgrowths in the last two cases differentiated or arrested (died) within several days of plating. Furthermore, oocytes with electrical or chemical activation alone displayed lower developmental competence to morula (50.0% and 39.3%, respectively) and blastocysts (35.0% and 28.3%, respectively) compared with oocytes subjected to the combination of electrical/chemical activation (morula: 65.0%, blastocysts: 53.3%; p < .05). The cell numbers of the parthenogenetic blastocysts showed significant differences (p < .01) as a function of the activation methods (electrical/chemical activation: 99 ± 2 cells per average blastocyst; chemical activation: 85 ± 2 cells; electrical activation: 59 ± 1 cells) (Table 2). The blastocysts from combined electrical-chemical activation were as good as the fertilized blastocysts based on the cell numbers of blastocysts (fertilized: 100 ± 2 per average blastocyst), but the embryo development competences of all three parthenogenetic activation methods were lower compared with the fertilized embryos (Table 2). The ESCs from both fertilized embryos and parthenotes were comprised of cells with a high nucleus/cytoplasm ratio, prominent nucleoli, and distinct cell borders, which formed flat cell colonies similar to human ESCs (Fig. 1C, 1D, 1G, 1H) [1, [2]3] but unlike the mouse ESCs that grew into compact, piled-up colonies with indistinct cell borders [5, 6].

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Figure Figure 1.. Morphological characteristics of rabbit RF (A–D) and RP01 (E–H) embryonic stem cells (ESCs). (A, E): Rabbit blastocysts with distinct inner cell masses (ICMs). (B, F): ICM outgrowths after 4 days of culture on mouse embryonic fibroblasts. (C, G): Rabbit ESC colonies after ICM outgrows were passaged. (D, H): Higher magnification of rabbit ESCs after 4 days of culture. Scale bars = 100 μm (B, F), 50 μm (A, C, E, G), 25 μm (D, H).

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Table Table 2.. Effect of activation methods on rabbit oocyte activation and development in vitro
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The rabbit ESCs grown on the feeders without exogenous leukemia inhibitory factor (LIF) remained undifferentiated and continued to proliferate, but spontaneous differentiation and death occurred rapidly when ESCs were cultured without feeder layer, regardless of the presence or absence of exogenous human or mouse LIF. All rabbit ESC lines showed similar growth rates, with generation doubling times of 24 hours. RF has been grown for 120 passages, RP01 for 72 passages, RP02 for 69 passages, and RP03 for 52 passages in vitro, corresponding to a minimum of approximately 420, 252, 242, and 182 population doublings, respectively, based on the average increase in cell numbers determined during routine passaging. All ESC lines that retained a predominate morphology of undifferentiated cells were successfully cryopreserved, thawed, and recovered.

Marker Expression and Karyotype of Rabbit ESCs

Marker analysis was carried out on RF ESC line at passages 10, 49, and 117, on RP01 at passages 25, and 58, on RP02 at passage 27, and on RP03 at passage 30. All lines shared similar marker expression, including alkaline phosphatase activity (Fig. 2A, 2I), OCT-4 (Fig. 2B, 2J), SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 (Fig. 2D–2G). SSEA-1, which is present in mouse but not primate ESCs, was also detected (Fig. 2C) [22, [23]24]. Karyotype analysis was performed on RF at passages 25, 47, and 102, RP01 at passage 46, RP02 at passage 39, and RP03 at passage 35. All expressed normal 44XY or 44XX karyotypes (Fig. 2H, 2P).

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Figure Figure 2.. The characteristics of undifferentiated rabbit RF (A–H) and RP01 (I–P) embryonic stem cells (ESCs). (A, I): Alkaline phosphatase activity. Shown is immunostaining for (B, J) Oct-4, (C, K) stage-specific embryonic antigen (SSEA)-1, (D, L) SSEA-3, (E, M) SSEA-4, (F, N) tumor-related antigen (TRA)-1-60, and (G, O) TRA-1-81. Normal 44XY and 44XX, G-banded karyotypes for rabbit RF (H) and RP (P) ESCs, respectively. Cell nuclei were counterstained with Hoechst 33342 (blue). Similar results were obtained for RP02 and RP03 ESCs. Scale bars = 100 μm (B, J), 50 (A, C–G, I, K–O).

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All rabbit ESC lines expressed the pluripotency genes OCT-4, NANOG, SOX-2, and UTF-1 (Table 3; supplemental online Table S1A) [22, 25, [26], [27]28]. OCT-4 protein expression was also confirmed in these cell lines with immunostaining (Fig. 2B, 2J). Signal transduction involving the fibroblast growth factor (FGF), transforming growth factor-β (TGF-β)/bone morphogenetic protein (BMP), and WNT pathways has been implicated in the maintenance of ESC pluripotency [29, [30], [31], [32], [33], [34], [35]36]. Therefore, we compared the transcriptional status of these major signaling pathways in rabbit, human, and mouse ESCs. The ESCs from all three species expressed FGF signaling pathway genes, including FGF1, FGF2, four FGF receptors (FGFR1, FGFR2, FGFR3, and FGFR4), and the components of downstream activation cascades (SOS1 and PTPN11) (Table 3; supplemental online Table S1B). WNT signaling pathway genes, the coreceptor LRP6, the key signal transducer β-catenin, and inhibitors Dkk1 and the transmembrane protein Kremen1 were expressed in all three species, whereas WNT3A was not detected in any of these species [37, 38]. However, inhibitors Dkk2 and Gsk3-β were not expressed in the rabbit ESCs but were expressed in human and mouse ESCs, and ligands WNT1, WNT2, WNT4, and WNT5A were expressed in rabbit ESCs, whereas human and mouse ESCs expressed only WNT4 (Table 3; supplemental online Table S1C). In TGF signaling, ligand genes (TGF-β1 and BMP4), LeftyA, as an inhibitor of Nodal, and the important regulator factors SMAD 1, 2, and 4 were detected in all three species, but ligand gene Nodal was not detected in rabbit ESCs but did express in human and mouse ESCs (Table 3; supplemental online Table S1D) [39]. On the other hand, expression levels of FGF1, SOS1, and SMAD 1, 2, and 4, and LeftyA were lower in the rabbit ESCs than in human and mouse ESCs (supplemental online data).

Table Table 3.. Transcriptional characteristics of genes related to pluripotency in rabbit, human, and mouse ESC lines
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Differentiation Capability of Rabbit ESCs In Vitro and In Vivo

All of our rabbit ESC lines differentiated into derivatives of all three embryonic germ layers in vitro and in vivo. The early differentiated markers Pax6 (ectoderm), AFP (endoderm), and BMP4 (mesoderm) were detected by RT-PCR in 6-day EBs (Fig. 3). Specialized cell types were detected by immunofluorescence staining with antibodies when 4-day EBs were cultured for another 10–20 days or longer (Fig. 3). CK7-immunopositive (red for immunopositive), Vimentin-negative (green for immunopositive) cells (CK7+/Vimentin, markers for trophoblast cells, indicating trophoblast differentiation) were detected on day 10, and the percentage of this cell type in all the differentiated cells was approximately 10%–30% (102/1,043, immunopositive cell number/nucleus number; 187/1,421; 342/1,116) (Fig. 3D). AFP-immunopositive cells were detected on day 10, and the percentage of these immunopositive cells was 1%–6% (8/967; 12/801; 36/1,087; 44/987; 98/1,634) (Fig. 3E). Only a small cell fraction (0.3%–4%: 3/967; 6/1,007; 7/763; 15/1,027; 40/1,798) were albumin (hepatocyte marker)-immunopositive on day 15 (Fig. 3F). Actin (smooth muscle marker)-immunopositive cells were only a small cell fraction (0.2%–3.5%: 2/842; 2/536; 12/876; 19/624; 26/749) on day 20 (Fig. 3G). Small elongated cells (neural progenitor-like cells) were selected to culture. At the beginning, 96% of these cells were Nestin-immunopositive when examined in four replicates (1,178/1,240; 1,121/1,162; 1,256/1,328; 1,074/1,097) (Fig. 3H). After continued culture for four passages in ESC medium, GFAP, as an astrocyte marker, was detected as being immunopositive in 40%–55% (132/327; 201/434; 224/407) (Fig. 3I). After ESCs had differentiated for 3 weeks, the presence of progesterone, estrogen, and chorionic gonadotropin in harvested conditioned culture medium also indicated trophoblast differentiation (data not shown). These specialized cell types representing all three germ cell layers were also detected in the adherent differentiation and prolonged cultivation approach, and there was no difference in the three approaches (data not shown). After ESCs were injected intramuscularly into the rear leg of SCID-beige mice for 8–14 weeks, teratoma recovery and analysis were performed including representatives of all three germ layers: bone and muscle (mesoderm); neural rosettes and squamous epithelium (ectoderm); and intestinal, renal epithelia, and gland (endoderm) (Fig. 4A–4H).

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Figure Figure 3.. Rabbit embryonic stem cells (ESCs) differentiated into many cell types representing all three germ cell layers in vitro. RF ESCs were used in these representative micrographs. Similar results were obtained for the other three RP ESC lines. (A): Simple representative embryoid bodies (EBs) in hanging-drop culture for 2 days and suspension culture for another 2 days. (B): A typical cystic EB cultured for 7 days. (C): Markers representing the three germ layers (AFP, Pax6, and BMP4) were expressed in rabbit RF, RP01, and RP02 EBs by reverse transcription-polymerase chain reaction. (D–I): Specialized cell types were detected by immunofluorescence staining with antibodies when 4-day EBs were cultured for another 10–20 days or longer. (D): CK7-immunopositive (red for immunopositive), Vimentin-negative (green for immunopositive) cells (CK7+/Vimentin, markers for trophoblast cells) on day 10. (E): AFP-immunopositive cells on day 10. (F): Albumin (hepatocyte marker)-immunopositive cells on day 15. (G): Actin (smooth muscle marker)-immunopositive cells on day 20. (H): Nestin-immunopositive cells; most (96%) selected small elongated cells are Nestin-positive. (I): Glial fibrillary acidic protein (as an astrocyte marker)-immunopositive cells; many cells (40%–55%) were positive when the selected small elongated cells were cultured continuously for four passages. Cell nuclei were counterstained with Hoechst 33342 (blue). Scale bars = 100 μm (A), 50 μm (B, D–I). Abbreviations: AFP, α-fetoprotein; BMP4, bone morphogenetic protein 4; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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Figure Figure 4.. Rabbit embryonic stem cells (ESCs) differentiated into tissues representative of all three germ cell layers in vivo in severe combined immunodeficient-beige mice. Simple teratomas were examined histologically. RF ESCs were used in these representative micrographs. Similar results were obtained for RP ESC lines. (A): Neural rosettes. (B): Squamous epithelium. (C): Ostiod island showing bony differentiation. (D): Bone. (E): Striated muscles. (F): Gastrointestinal epithelium. (G): Renal tissues. (H): Gland. Scale bars = 50 μm (A, C, D, F), 100 μm (B, E, G, H).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

To our knowledge, this was the first time that stable rabbit pluripotent ESC lines have been successfully derived from parthenotes and that the overall characteristics of rabbit ESCs from both fertilized and parthenogenetic embryos have been described. In previous studies, the isolation of putative rabbit ESCs was reported by the Moreadith group [10, 11], but there were no results about the ability of forming different tissues representing three primitive germ layers from different tissues in vivo and no marker expression characteristics. Afterward, there was no other report about these putative ESCs. The characteristics of the rabbit ESC lines derived from fertilized and parthenogenetic blastocysts had no differences among them. Unfortunately, we could not obtain an ESC line from the parthenotes with electrical or chemical activation alone but we could get through the combined electrical-chemical parthenogenetic activation method. This might be related to the developmental competence of the parthenogenetic activation embryos and the quality of the parthenogenetic blastocysts (Table 2); however, we do not know the reason. Previous reports indicated that artificial activation of oocytes not only might perturb the ratio of ICM to trophectoderm cells number at the blastocyst stage [40] but also might result in anomalies that were not apparent until later, when postimplantation development was well underway [41]. Therefore, parthenotes that derived from different activation treatments may have undergone different development in vitro. The results of our study showed that the electrical/chemical parthenogenetic activation treatment was an efficient way to get excellent blastocysts that were fit to establish the ESC lines.

The characteristics of rabbit ESCs, such as morphologies, LIF independence, and trophoblast cell differentiation capability, were similar to those of the primate ESCs but unlike those of the mouse ESCs [1, [2]3, 5, 6, 42]. On the other hand, the rabbit ESC lines proliferated at a high speed of proliferation within 24 hours of the generation doubling time, which was similar to mouse ESCs but not to human ESCs [1, 5, 6]. Although cell surface markers were thought to indicate the ability of ESCs to maintain undifferentiation and pluripotency [43], expression of cell surface markers in ESCs showed species-related differences. In this study, rabbit ESCs expressed SSEA-1, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81, which had been found in both primates and mouse ESCs. But it was not clearly understood why the expression patterns of these antigens were different in the ESCs of different species, because the function of these antigens was unknown. As in human and mouse ESCs, expression of genes such as OCT-4, NANOG, SOX-2, and UTF-1, which are characteristic for ESCs, was detected in rabbit ESCs (Table 3; supplemental online Table S1) [22, 25, [26], [27]28]. It implied that the rabbit ESCs might employ self-renewal and pluripotency pathways similar to those of human and mouse ESCs. Previous studies demonstrated that several signaling pathways, such as FGF, WNT, and TGF-β signaling pathways, played key roles in maintaining growth and self-renewal of human and mouse ESCs [29, [30], [31], [32], [33], [34]35]. Our data showed that many genes of FGF signaling were detected in the rabbit, human, and mouse ESCs, which suggested that rabbit ESCs might require the FGF signaling pathway to maintain the undifferentiated state. A similar situation was found in WNT and TGF-β signaling pathways (Table 3; supplemental online Table S1); however, some difference was found in the expression of some inhibitor and ligand genes. These results suggested that different inhibitors and ligands of WNT and TGF-β signaling performed different roles in the maintenance and differentiation of ESCs, and the expression of these pathway components in conjunction with different regulators suggested that ESCs were competent to respond to different ligands and then balance the self-renewal and differentiation of ESCs. These findings implied that, in rabbit ESCs, the main signaling pathways for self-renewal were similar to those of human and mouse ESCs; however, the differences between species might somehow operate differently during embryo and ESC development.

From our perspective, it looked like it would be difficult to establish rabbit ESC lines because there was no report on the existence of long-term surviving of rabbit ESCs in vitro. In our laboratory, we tried to isolate the rabbit ICMs of blastocysts from the trophectoderm layer by immunosurgery with anti-rabbit serum produced in goat (Sigma-Aldrich), but it did not work well. In the present study, we found that removing ICMs cleanly from the trophectoderm layer with enzymatic and mechanical methods was a key step in deriving the ESC lines. Otherwise, rabbit ICMs were prone to differentiate into trophoblast cells, which suggested that the trophectoderm might strongly induce ICM cells to differentiate into the trophoblast cells. We also found the ICMs and ESC-like cells were very sensitive to enzyme digestion and mechanical damages, because they would rapidly differentiate or die if exposed to higher concentrations of enzyme, longer time, or mechanical damage, especially during the first five passages.

We have established four stable rabbit ESC lines, one derived from blastocysts fertilized in vivo and cultured in vitro and the other three from parthenogenetic blastocysts with electrical/chemical activation, sharing the characteristics of ESCs without any differences among them. Rabbit ESCs shared similar morphologic characteristics with primate ESCs and expressed cell surface markers and the genes of pathways related to the self-renewal of ESCs. As large animals with many characteristics similar to humans, rabbits have advantages over mice in size and physiology, which suggests that rabbit ESCs may be a good model for some specific human diseases in preclinical trials.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

This work was supported by research grants from Major State Research Development Program 2004CCA01300 and 2006CB701500, The Chinese Academy of Sciences KSCX1-05, KSCX1-YW-R-47, Chinese National Science Foundation 30370166 and 30570906. We thank Dr. Wenhui Nie and Jinhuan Wang for analysis of ESC karyotype and Li Jian for assistance in confocal microscopic analysis. We also thank Dr. Don Wolf for deliberated revision of the manuscript. S.W. and X.T. contributed equally to the study.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information
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