Generation and Characterization of Rabbit Embryonic Stem Cells - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:58 |只看该作者 |倒序浏览 |打印

作者：Shufen Wanga,b,c, Xianghui Tanga,c, Yuyu Niua,b,c, Hongwei Chena,b,c, Bin Lia,b,c, Tianqing Lia,c, Xiuzhen Zhanga,b,c, Zhixin Hua,b,c, Qi Zhoud, Weizhi Jia,c作者单位：aKunming Primate Research Center, and Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming Yunnan, China;bGraduate School, Chinese Academy of Sciences, Beijing, China;cYunnan Key Laboratory of Animal Reproductive Biology, Kunming Yunnan, China;dInstitute of Zoology, Chinese Academy of
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      【摘要】
   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.
      【关键词】 Rabbit embryonic stem cells Parthenogenetic activation Stem cell marker self-renewal Signaling pathway" g( A0 Q; U% j' c7 B9 O% E
   INTRODUCTION5 z+ ]9 o" {8 D8 @* m
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Embryonic stem cell (ESC) lines have been successfully established in more than a dozen species, including humans, monkeys, and rats . 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.1 }, y9 I( f3 p/ d- p. w

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 .
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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 . 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.% ~# W& ?% _6 a
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MATERIALS AND METHODS

Embryo Recovery and Culture$ t4 f2 |0 m& k
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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¡ãC for 4 days and developed to the blastocyst stage.% \6 m' x2 x2 i+ o# j5 {" }  z
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ESC Derivation and Propagation& g' m4 X+ m+ p9 _
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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¨C5 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, -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), 1x nonessential amino acids (Invitrogen Corporation), 1x penicillin-streptomycin (Sigma-Aldrich), and 15% defined fetal bovine serum (HyClone, Logan, UT, http://www.hyclone.com). Approximately 3¨C5 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¨C5 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¨C10 times exactly as in the above methods and were then passaged with 5¨C10 mg/ml dispase only every 3¨C5 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 .

ESC Characterization
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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. ." f( T; y; s! [  r* @6 ?

ESC Differentiation In Vitro and In Vivo! J5 \$ I; {3 n) h; L+ H  n

In vitro differentiation involved adherent differentiation (monolayer cultivation), prolonged cultivation, or EB formation . 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¨C30 days. The prolonged cultivation approach involved maintenance of ESCs at high densities for 2¨C4 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), -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¨C8-week-old severe combined immunodeficient (SCID)-beige mice (Charles River Laboratories, Inc., Wilmington, MA, http://www.criver.com) were injected intramuscularly with 2¨C4 x 106 rabbit ESCs. Tumors were removed (8¨C14 weeks after injection) and fixed in 4% paraformaldehyde. Paraffin sections were stained with hematoxylin/eosin and processed for histological examination.( j, p5 l4 Y  v: C, D$ X. y' {
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RNA Preparation and Gene Expression Analyses/ y5 O7 I- e& f) Z
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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) . 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.1 {8 E4 z6 r& P/ a2 W  t* W- {

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

Statistical Analysis# o- [4 t5 M: y3 K0 X4 n+ \
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The results were presented as means ¡À SEM. Statistical analysis was performed using the least significant difference test. Statistical significance was defined as p
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RESULTS6 h8 s8 @4 u6 }$ l# [

Generation of Rabbit ESC Lines
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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¨C5 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

Figure 1. Morphological characteristics of rabbit RF (A¨CD) and RP01 (E¨CH) 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).: T* [' H' G! p7 m1 a$ \& X# E

Table 2. Effect of activation methods on rabbit oocyte activation and development in vitro! v, m( _; w8 h6 v4 B; w' o1 w
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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
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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¨C2G). SSEA-1, which is present in mouse but not primate ESCs, was also detected (Fig. 2C) . 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 2. The characteristics of undifferentiated rabbit RF (A¨CH) and RP01 (I¨CP) 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¨CG, I, K¨CO).
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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) . 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).( u% b1 `  P9 k9 i* c/ x" I

Table 3. Transcriptional characteristics of genes related to pluripotency in rabbit, human, and mouse ESC lines/ I& \: {( n; a4 G
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Differentiation Capability of Rabbit ESCs In Vitro and In Vivo
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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¨C20 days or longer (Fig. 3). CK7-immunopositive (red for immunopositive), Vimentin-negative (green for immunopositive) cells (CK7 /Vimentin¨C, 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%¨C30% (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%¨C6% (8/967; 12/801; 36/1,087; 44/987; 98/1,634) (Fig. 3E). Only a small cell fraction (0.3%¨C4%: 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%¨C3.5%: 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%¨C55% (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¨C14 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¨C4H).7 X& |; i* m2 e) C' U5 C, m0 Z

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¨CI): Specialized cell types were detected by immunofluorescence staining with antibodies when 4-day EBs were cultured for another 10¨C20 days or longer. (D): CK7-immunopositive (red for immunopositive), Vimentin-negative (green for immunopositive) cells (CK7 /Vimentin¨C, 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%¨C55%) 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¨CI). Abbreviations: AFP, -fetoprotein; BMP4, bone morphogenetic protein 4; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.9 B1 R+ u8 N* V/ W! _5 m+ W+ e
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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).8 A! J* S8 k  U8 I# c
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DISCUSSION5 A! z5 e1 d( f& E$ v
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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 . 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 . 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-ß 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-ß 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./ E; `8 y# e1 @6 T( F, f+ ~
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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.

DISCLOSURES

W.J. owns stock in Kumming Institute of Zoology.% w' W% s% z/ M0 @- I: ~- w

ACKNOWLEDGMENTS
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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.
      【参考文献】
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Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts. Science 1998;282:1145¨C1147.
* _( q: S! {0 l- ?
Thomson JA, Kalishman J, Golos TG et al. Isolation of a primate embryonic stem cell line. Proc Natl Acad Sci U S A 1995;92:7844¨C7848.+ l9 y/ X; p. A5 e; b0 Y8 L
% W" J- O0 i) L& Z" D3 ?& ]% d
Thomson JA, Kalishman J, Golos TG et al. Pluripotent cell lines derived from common marmoset (Callithrix jacchus) blastocysts. Biol Reprod 1996;55:254¨C259.

Iannaccone PM, Taborn GU, Garton RL et al. Pluripotent embryonic stem cells from the rat are capable of producing chimeras. Dev Biol 1994;163:288¨C292.

Evans MJ, Kaufman M. Establishment in culture of pluripotential stem cells from mouse embryos. Nature 1981;292:151¨C156.( ^! i! Q  `3 }7 W
( K  Q; r6 H, o3 ^, x/ n  |. S1 s
Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci U S A 1981;78:7634¨C7638.

Chowdhury JR, Grossman M, Gupta S et al. Long-term improvement of hypercholesterolemia after ex-vivo gene therapy in LDLR-deficient rabbits. Science 1991;254:1802¨C1805.& f* f7 `& F- Q* H  }6 c& O7 y
3 s  U% [- |% W% ^
Hoeg JM, Santamarina-Fojo S, Berard AM et al. Overexpression of lecithin:cholesterol acyltransferase in transgenic rabbits prevents diet-induced atherosclerosis. Proc Natl Acad Sci U S A 1996;93:11448¨C11453.
( F" Y$ h6 [6 J  f; }
Chen JM, Cutler C, Jacques C et al. A combined analysis of the cystic fibrosis transmembrane conductance regulator: Implications for structure and disease models. Mol Biol Evol 2001;18:1771¨C1788.
" M" v: L" `5 I- L- t# q2 I
Graves KH, Moreadith RW. Derivation and characterization of putative pluripotential embryonic stem cells from preimplantation rabbit embryos. Mol Reprod Dev 1993;36:424¨C433.
. Y2 F9 W* ~. r, A4 \
Schoonjans L, Albright GM, Li JL et al. Pluripotential rabbit embryonic stem (ES) cells are capable of forming overt coat color chimeras following injection into blastocysts. Mol Reprod Dev 1996;45:439¨C443.1 v# I" x4 h/ L. j( ^
* N3 O% d, b8 r( Z# s' N
Kaufman MH, Barton SC, Surani MA. Normal postimplantation development of mouse parthenogenetic embryos to the forelimb bud stage. Nature 1977;265:53¨C55., p  ~# s+ B* \, e' _

McGrath J, Solter D. Completion of mouse embryogenesis requires both the maternal and paternal genomes. Cell 1984;37:179¨C183.

Boediono A, Suzuki T, Li LY et al. Offspring born from chimeras reconstructed from parthenogenetic and in vitro fertilized bovine embryos. Mol Reprod Dev 1999;53:159¨C170.

Kono T, Obata Y, Wu Q et al. Birth of parthenogenetic mice that can develop to adulthood. Nature 2004;428:860¨C864.
' ?: a) K. j7 G! D5 `  H1 g5 x( G
Vrana KE, Hipp JD, Goss AM et al. Nonhuman primate parthenogenetic stem cells. Proc Natl Acad Sci U S A 2003;100 (suppl 1):11911¨C11916.

Sanchez-Pernaute R, Studer L, Ferrari D et al. Long-term survival of dopamine neurons derived from parthenogenetic primate embryonic stem cells (cyno-1) after transplantation. STEM CELLS 2005;23:914¨C922.
; K. h2 n% W( f( D+ {
Turksen K. Embryonic Stem Cells Methods and Protocols. Totowa, NJ: Humana Press,2002;1¨C16.  E$ f, D3 f/ a* z' `2 r1 y5 H6 y- c4 k

Hayes H, Rogel-Gaillard C, Zijlstra C et al. Establishment of an R-banded rabbit karyotype nomenclature by FISH localization of 23 chromosome-specific genes on both G- and R-banded chromosomes. Cytogenet Genome Res 2002;98:199¨C205., _+ i4 U5 D5 E
  D" ^1 j0 P8 b+ J9 q' j# t
Mitalipova M, Calhoun J, Shin S et al. Human embryonic stem cell lines derived from discarded embryos. STEM CELLS 2003;21:521¨C526.
% v4 J  v1 ]! n+ p  @
Kayo T, Aillison DB, Weindruch R et al. Influences of aging and caloric restriction on the transcriptional profile of skeletal muscle from rhesus monkey. Proc Natl Acad Sci U S A 2001;98:5093¨C5098.

Nichols J, Zevnik B, Anastassiadis K et al. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 1998;95:379¨C391.
1 J2 b7 x  W# l% H. |9 V
Kannagi R, Levery SB, Ishigami F et al. New globoseries glycosphingolipids in human teratocarcinoma reactive with the monoclonal antibody directed to a developmentally regulated antigen, stage-specific embryonic antigen 3. J Biol Chem 1983;258:8934¨C8942./ e' x+ g9 `' V

Solter D, Knowles BB. Monoclonal antibody defining a stage-specific mouse embryonic antigen (SSEA-1). Proc Natl Acad Sci U S A 1978;75:5565¨C5569.$ C) l1 E1 c4 X3 N0 L

Mitsui K, Tokuzawa Y, Itoh H et al. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ESCs. Cell 2003;113:631¨C642.8 n* T, u9 h5 V8 H
# w  V1 r1 R- C- f1 t/ N# N
Chambers I, Colby D, Robertson M et al. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 2003;113:643¨C655.

Yuan H, Corbi N, Basilico C et al. Developmental-specific activity of the FGF-4 enhancer requires the synergistic action of Sox2 and Oct-3. Gens Dev 1995;9:2635¨C2645.& L4 r3 z7 E: w: q3 R' ^
; n  C/ o8 ]( S  A1 p
Okuda A, Fukushima A, Nishimoto M et al. UTF1, a novel transcriptional coactivator expressed in pluripotent embryonic stem cells and extra-embryonic cells. EMBO J 1998;17:2019¨C2032.! F" V$ j. E, Z5 S% S8 X" h
: ~5 }" `4 V# ~) f) V. A
Brandenberger R, Wei H, Zhang S et al. Transcriptome characterization elucidates signaling networks that control human ESC growth and differentiation. Nat Biotechnol 2004;22:707¨C716.
5 ~4 l4 L8 d3 ~$ d, d# @/ W
Sato N, Sanjuan IM, Heke M et al. Molecular signature of human embryonic stem cells and its comparison with the mouse. Dev Biol 2003;260:404¨C413.

Xu RH, Peck RM, Li DS et al. Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ESCs. Nat Methods 2005;2:185¨C190.

Sato N, Meijer L, Skaltsounis L et al. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat Med 2004;10:55¨C63.

Willert K, Brown JD, Danenberg E et al. Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 2003;423:448¨C452.! i' r+ N  D, ]- l; e' O
8 I- U, c, c- Z$ t) V) }
Ying QL, Nichols J, Chambers I et al. BMP induction of ID proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 2003;115:281¨C292.0 {+ @4 N' J) d6 i# c. D4 O
, a+ l: n- c, _; I  }3 T# ^& _  U
Qi XX, Li TG, Hao J et al. BMP4 supports self-renewal of embryonic stem cells by inhibiting mitogen-activated protein kinase pathways. Proc Natl Acad Sci U S A 2004;101:6027¨C6032.( ]; H7 ^, a( z& `" C/ G
  v0 \1 c4 j3 U& B. P) h2 P% j- f" H1 v
Li TQ, Wang SF, Xie YH et al. Homologous feeder cells support undifferentiated growth and pluripotency in monkey embryonic stem cells. STEM CELLS 2005;23:1192¨C1199.) C5 ]8 Q( Y9 p
: e! ?$ _* C0 o- d7 k  ?+ X
Mao BY, Wu W, Li Y et al. LDL-receptor-related protein 6 is a receptor for Dickkopf proteins. Nature 2001;411:321¨C325.4 ]0 h9 M) c: v+ q% V

Mao BY, Wu W, Davidson G et al. Kremen proteins are dickkopf receptors that regulate Wnt/&szlig;-catenin signaling. Nature 2002;417:664¨C667.
1 \& g. |! p8 V4 |6 q
Zhao GQ. Consequences of knocking out BMP signaling in the mouse. Genesis 2003;35:43¨C56.

Bos-Mikich A, Whittingham DG, Jones KT. Meiotic and mitotic Ca2  oscillations affect cell composition in resulting blastocysts. Dev Biol 1997;182:172¨C179.8 l6 N6 |3 v% U, ^# z5 W

Ozil JP, Huneau D. Activation of rabbit oocytes: The impact of the Ca2  signal regime on development. Development 2001;128:917¨C929." L6 b5 q* j9 D7 p$ S
' }5 B1 [' c; j3 ?
Beddington RS, Robertson EJ. An assessment of the developmental potential of embryonic stem cells in the midgestation mouse embryo. Development 1989;105:733¨C737.
. }. A/ X, h1 I# M9 n( a) O8 G
Andrews PW, Casper J, Damjanov I et al. Comparative-analysis of cell-surface antigens expressed by cell-lines derived from human germ-cell tumors. Int J Cancer 1996;66:806¨C816.

Lafuste P, Robert B, Mondon F et al. Alphafetoprotein gene expression in early and full-term human trophoblast. Placenta 2002;23:600¨C612.8 J* v9 A% B8 |: E! f
. m8 \, ?3 U+ a- J, n; Q' [' i
Soloviev MM, Ciruela F, Chan WY et al. Mouse brain and muscle tissues constitutively express high levels of homer proteins. Eur J Biochem 2000;267:634¨C639.

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