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Generation and Characterization of Pluripotent Stem Cells from Cloned Bovine Embryos¹

Institute of Zoology, Chinese Academy of Sciences,³ Beijing 100080, People's Republic of China Center for Regenerative Biology/Department of Animal Science,⁴ University of Connecticut, Storrs, Connecticut 06269

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Bovine embryonic stem (ES) cell lines reported to date vary in morphology and marker expression (e.g., alkaline phosphatase [ALPL], stage-specific embryonic antigen 4 [SSEA4], and OCT4) that normally are associated with the undifferentiated, pluripotent state. These observations suggest that the proper experimental conditions for consistently producing bovine ES cells have not been identified. Here, we report three bovine ES cell lines, one from in vitro-fertilized and two from nuclear transfer embryos. These bovine ES cells grew in large, multicellular colonies resembling the mouse ES and embryonic germ (EG) cells and human EG cells. Throughout the culture period, most of the cells within the colonies stained positive for ALPL and the cell surface markers SSEA4 and OCT4. The staining patterns of nuclear transfer ES cells were identical to those of the blastocysts generated in vitro yet different from most previously reported bovine ES cell lines, which were either negative or not detected. After undifferentiated culture for more than 1 yr, these cells maintained the ability to differentiate into embryoid bodies and derivatives of all three EG layers, thus demonstrating their pluripotency. However, unlike the mouse and human ES cells, following treatment with trypsin, type IV collagenase, or protease E, our bovine ES cells failed to self-renew and became spontaneously differentiated. Presumably, this resulted from an interruption of the self-renewal pathway. In summary, we generated pluripotent bovine ES cells with morphology similar to those of established ES cells in humans and mice as well as marker-staining patterns identical to those of the bovine blastocysts.

embryo, in vitro fertilization

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Embryonic stem (ES) cells are derived from the inner cell masses (ICMs) of preimplantation embryos. They can self-renew and are pluripotent; that is, they are capable of proliferating indefinitely and of differentiating into a wide variety of cell types both in vitro and in vivo [1, 2]. Stable ES cell lines have been established in many species, such as chicken [3], mink [4], hamster [5], pig [6], rhesus monkey [7], common marmoset [8], and human [9]. Recently, ES cells also have been derived from nuclear transfer (NT) and parthenogenetic embryos in a few species [10–13].

Establishment of ES cells from bovine embryos has been problematic. Several ES-like cell lines have been reported to exhibit pluripotency both in vitro and in vivo. However, few of them resemble morphologically, or express markers that are normally associated with, ES cell lines generated from other species [14–18]. For example, OCT4, also known as POU-domain transcription factor POU5F1, was found to be associated with the pluripotency of ES cells in many species [19–21]. However, few of the previously reported bovine ES cell lines were OCT4 positive [14–18].

Commonly, ES cells are propagated by enzymatically dissociating colonies and plating individual cells for new colony formation [7, 9, 22, 23]. However, bovine ES-like cells do not form colonies after enzymatic disassociation and replating [14, 16]. Trypsin is the only reported enzyme that dissociates bovine ES cells, but it also causes a failure of bovine ES cells to self-renew and to induce spontaneous differentiation. The effect of other enzymes or buffer-based reagents on the ability of bovine ES cells to self-renew is unclear.

The purpose of the present study was to establish an effective approach to generate bovine ES cells. Specific aims of the present study were as follows: 1) to generate ES cells from in vitro fertilization (IVF), NT, and parthenogenetic embryos; 2) to compare the efficiencies of ES cell derivation from different types of embryos; 3) to characterize ES cell lines developed from these embryos; 4) to test the pluripotency of these cells in vitro through differentiation; and 5) to test the effects of different enzymes and buffer-based reagents on passaging and subsequent propagation of ES cells.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In Vitro Fertilization

The present study used commercially available frozen semen (0.25 ml/ straw) collected from a single ejaculate of a Holstein bull and purchased from Cooperative Resources International (catalog no. 1HO6149; Shawano, WI). After a 10-sec gentle shaking in air (room temperature), the semen straw was thawed for 10 sec in a 37°C water bath. Spermatozoa were washed twice by centrifugation (1000 rpm) for 8 min in 10 ml of Brackett and Oliphant medium (BO medium) [24] containing 3 mg/ml of BSA supplemented with 10 mM caffeine. The washed spermatozoa pellet was resuspended in BO sperm-wash solution at a concentration of 1.0 x 10⁶ sperm/ml for subsequent fertilization. After maturation in vitro, bovine cumulus-oocyte complexes obtained from slaughterhouse ovaries were washed twice and transferred into a 50-µl drop of BO medium (n = 20– 25 oocytes/drop) containing 6 mg/ml of BSA and 10 µg/ml of heparin pre-equilibrated for 2 h at 39°C in 5% CO₂ in humidified air, and 50 µl of sperm suspension were added to each drop. Oocytes were incubated with sperm for 6 h at 39°C in 5% CO₂ in humidified air.

Culture of Bovine Adult Cumulus Cells In Vitro

Cumulus cells, serving as the donor cells for NT, were removed from bovine cumulus-oocyte complexes following ovarian follicle aspiration. They were cultured in Dulbecco modified Eagle medium (DMEM) (Gibco BRL, Grand Island, NY) supplemented with 10% fetal calf serum (Hyclone, Logan, UT), 0.15 g/ml of glutamine (Sigma, St. Louis, MO), and 1% antibiotic-antimycotic solution (Gibco BRL). For each passage (estimated two cell-doublings per passage), cells were cultured until confluent, disaggregated by incubation in a 0.25% trypsin and EDTA solution for 3–5 min at 37°C, and allocated to three new dishes for further passaging.

Somatic Cell NT

The somatic cell NT was conducted according to the method reported by Kubota et al. [25], and the somatic donor cells (cumulus) selected for the present study have been used successfully for production of cloned embryos and calves. Recipient oocyte collection and maturation were conducted as described previously [26]. After 24 h of maturation, oocytes were enucleated by micromanipulation. Successful enucleation was confirmed by Hoechst 33342 staining before transfer of the somatic cell. For donor cells, adult bovine cumulus cells were cultured for six passages and subjected to serum starvation for 5 days after reaching confluency. Immediately before NT, donor cells were trypsinized, washed, and resuspended in PBS supplemented with 0.5% fetal bovine serum. Cells with an approximate diameter of 10–15 mm [27] were transferred to the perivitelline space of the recipient cytoplast using our standard procedure [25]. After transfer, the cell-cytoplast complexes were induced to fuse with two pulses of direct current at 2.5 kV/cm for 10 µsec each by using an Electrocell Manipulator 200 (BTX, San Diego, CA). These electrical pulses also simultaneously induced initial oocyte activation. Fusion was then confirmed by microscopic examination. All fused cell-cytoplast complexes were activated further with 10 µg/ml of cycloheximide (Sigma) in CR1aa medium for 5 h. The cloned embryos were then cultured in potassium simplex-optimized medium (KSOM) plus 0.1% (w/v) BSA for the first 4 days. Then, the medium was switched to KSOM containing 1% BSA for the remaining 3 days required to reach the blastocyst stage. The medium was changed every 2 days during the course of in vitro culture.

Parthenogenetic Activation of Oocytes

The parthenogenetic activation procedure has been described previously [28]. Briefly, matured slaughterhouse bovine oocytes were stripped of cumulus cells, and those with a polar body were selected and subjected to activation. Oocytes were exposed to 5 µM A23187 (Sigma) for 5 min, followed by incubation with 2.5 mM 6-dimethylaminopurine (Sigma) in KSOM containing 0.1% BSA under mineral oil for 3.5 h at 39°C in a 5% CO₂ atmosphere in humidified air. Following the activation treatment, oocytes were washed in KSOM, then cultured in the same manner as the NT embryos described above.

ES Cell Isolation and Maintenance in Culture

A total of 21 NT, 238 IVF, and 101 parthenogenetically activated embryos were used for ES cell isolation as described for humans [9]. The ICMs were isolated by mechanical dissociation and plated on mitomycin C-treated mouse embryonic fibroblasts. Culture medium consisted of 80% knockout DMEM (KO-DMEM) (Gibco BRL) supplemented with 20% fetal bovine serum (Hyclone), 1 mM glutamine, 0.1 mM ß-mercaptoethanol (Sigma), 1% nonessential amino acids (Gibco BRL), 1000 U/ml of leukemia-inhibitory factor (LIF; Sigma), and 4 ng/ml of fibroblast growth factor 2 (Sigma). After 10–14 days, ICM-derived outgrowths were separated into clumps by mechanical dissociation with a micropipette and replated on mitomycin C-treated mouse embryonic fibroblasts in fresh medium. To passage the putative ES cells, individual colonies with a uniform, undifferentiated morphology were selected individually using a micropipette, mechanically dissociated into two to six clumps, and replated. The ES cells were passaged every 14–17 days after replating.

Alkaline Phosphatase and ES Cell-Specific Marker Staining

Alkaline phosphatase (ALPL) was detected histochemically following fixation of cells with 4% paraformaldehyde, and nitro-blue tetrazolium chloride/5-bromo-4-chloro-3-indolylphosphate toluidine (NBT/BCIP) was used as substrate. Thus, ALPL-expressing cells would stain dark blue. Cells also were incubated with monoclonal antibodies against mouse stage-specific embryonic antigen (SSEA) 1 (1:30), SSEA4 (1:30), tumor-rejection antigen gp96 (TRA1; two different antibodies used for TRA1, one recognizing a sialidase-sensitive epitope and one that reacts with an unknown epitope; 1:20), or rabbit OCT4 (1:500). All antibodies were from the ES cells characterization kit purchased from Chemicon International, Inc. (Temecular, CA). The appropriate secondary antibodies, horse anti-mouse immunoglobulin (Ig) G, goat anti-mouse IgM, or sheep anti-rabbit IgG, were used to amplify the signals. Detection of specific binding was performed with an Elite ABC peroxidase staining kit (Vector Laboratories, Inc., Burlingame, CA) and with 3,3'-diaminobenzidine (Vector Laboratories) as substrate. Positive staining was gray-black in color. Staining controls using secondary antibodies alone also were included. The putative ES cells lines were at passages 14–16 at the time that marker expression was analyzed.

The IVF blastocysts also were stained as described above. The primary antibodies for SSEA1, SSEA4, and OCT4 were localized by fluorescein isothiocyanate-conjugated goat anti-mouse IgM, horse anti-mouse IgG, or sheep anti-rabbit IgG (1:200). Finally, the samples were washed, mounted on glass slides, and examined by fluorescence microscopy. Appropriate controls of staining with secondary antibodies alone were included.

In Vitro Differentiation and Tissue-Specific Marker Stains

Mechanically dissected ES cell colonies were plated in a bacteriological dish (Becton Dickinson, Franklin Lakes, NJ) for 5–7 days to induce formation of embryoid bodies or placed onto a tissue culture dish in differentiation medium (KO-DMEM containing 1 mM glutamine, 0.1 mM ß-mercaptoethanol, 1% nonessential amino acids, and 15% fetal bovine serum) for 2–7 wk for random differentiation. Subsequently, indirect immunocytochemistry was performed using markers for all three embryonic layers. These markers were cytokeratin for endoderm [29], actin ₂ smooth muscle aorta (ACTA2) for mesoderm [29], and tubulin ß3 (TUBB3) for ectoderm [21]. For indirect staining, cells were fixed in 4% paraformaldehyde at room temperature for 15 min. Monoclonal antibodies against ACTA2 (1:100; Sigma), TUBB3 (1:200; Sigma), and cytokeratin (1:400; Sigma) were incubated with cells at room temperature for 1 h. Localization of the primary antibodies was determined by a biotinylated secondary antibody and then an avidin-biotinylated horseradish peroxidase complex (Vectastain ABC System; Vector Laboratories) using procedures described by the manufacturer. The putative ES cell lines were at passages 21–23 at the time of induced differentiation.

Enzymatic Dissociation of ES Cell Colonies for Propagation

To dissociate ES cell colonies, Ca²⁺/Mg²⁺-free phosphate saline with 1 mM EDTA, 0.05% trypsin-0.5 mM EDTA, 72-kDa type IV collagenase (1 mg/ml), and protease E (0.3 mg/ml) were tested separately for duration of 5–25 min at 37°C. We found that 1 mM EDTA was insufficient to dissociate colonies. Clumps or individual cells dissociated by trypsin-EDTA, type IV collagenase, or protease E were replated for colony formation.

Karyotyping

The karyotyping procedure has been described previously [14, 29]. Briefly, cells prepared for cytogenetic analysis were incubated in growth media supplemented with 0.08 µg/ml of KaryoMax (Gibco BRL) for 4– 6 h at 37°C. Then, cells were trypsinized and treated with hypotonic KCl (0.57%) for 25 min at 36.5°C and fixed in acetic methanol (1:3, v/v), and drops of cell suspension were spread on clean microscopic slides. The chromosomes were stained with 5% Giemsa for 40 min. The chromosomes were examined at 1000x magnification under oil.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We obtained trophectoderm colonies from IVF, NT, and parthenogenetic embryos generated in vitro. The trophectoderm colonies contained flattened cells in the monolayer as well as multilayer clumps of compact cells that had unclear cell-cell boundaries. The clumps stained positive for ALPL and were presumably undifferentiated. Most of the flattened cells in monolayer stained negative for ALPL and may represent residual trophoblast cells of the embryos or differentiated ES cells (Fig. 1A). Major differences were found in the efficiency of trophectoderm colony formation among the embryo types. The primary trophectoderm colony formation rate was much higher for NT blastocysts (16/ 21, 76%) than for IVF blastocysts (43/238, 18%) or parthenogenetic blastocysts (1/101, 1%; P < 0.01).

View larger version (157K): [in this window] [in a new window]   FIG. 1. Morphology of colonies derived from NT and PA embryos. A) A representative primary trophectoderm colony. After ALPL staining, part of the colony was stained positive (dark), whereas the rest was not. B) Colonies selected for replating from passage-1 ICM cultures. The colonies were surrounded by cells growing in monolayer, which may be residual trophoblast cells. C) Multiple cell colonies derived from bovine cloned embryos growing on a feeder layer of mitotically inactivated mouse fetal fibroblasts. Note that the colonies are large and have clear boundaries with the feeder layer. Cells in the colonies are compact, with indistinguishable cell-cell boundaries. The morphology of these colonies is very similar to that of established ES/EG cell lines of the mouse/human. D) ICM culture from parthenotes. Signs of differentiation are visible. The colony boundary becomes unclear. The surface of the colony is not smooth, and cell-cell boundaries within the colony become obvious. Only few cells are ALPL positive (arrows). Bar = 1 mm (A) and 100 µm (B–D)

By passaging the multilayered cell clumps in trophectoderm colonies, we established three putative ES lines, one from IVF and two from NT embryos (NTL-1 and NTL-2). All three ES cell lines showed typical ES cell morphology similar to those of the mouse ES cells and EG cells and the human EG cells. The bovine putative ES cells were large and tightly packed, and they had indistinguishable cell-cell boundaries. The cells were in multicellular colonies with a smooth surface and a distinct colony boundary from the surrounding feeder layers (Fig. 1C).

Our bovine putative ES cells were similar to the established mouse ES cells not only in morphology but also in the expression of specific cell markers for totipotent cells. High levels of ALPL activity were detected in most of the cells within colonies of all three cell lines throughout the culture period (Fig. 2A). We further characterized NTL-1 and NTL-2 with a bank of five monoclonal antibodies: SSEA1, SSEA4, TRA1 (two antibodies), and OCT4. Immunostaining showed that colonies express SSEA4 and OCT4 but not SSEA1 (Fig. 2, B–D). Feeder cells gave no signal (data not shown). The TRA1 was not expressed in these colonies (Fig. 2, E and F). Because these marker-staining patterns were different from most of the marker-staining results of previously reported bovine ES-like cells, we used the same set of markers to stain bovine IVF blastocysts, which surely contain pluripotent stem cells and previously showed comparable expression of stem cell markers to ES cells in mice and humans [30–34]. The IVF blastocysts displayed an identical staining pattern to that our putative ES cell colonies (i.e., SSEA1 negative, SSEA4 positive, and OCT4 positive) (Fig. 2, G–I).

View larger version (139K): [in this window] [in a new window]   FIG. 2. Characterization of putative ntES (NTL-1 and NTL-2) cell lines derived from cloned embryos. A) The colony stained positive for ALPL. B) The ntES cells stained negative for SSEA1. C) The ntES cells stained positive (brown) for SSEA4. D) The ntES cells also stained strongly positive for OCT4. E and F) The ntES cells stained negative for TRA1 using two different monoclonal antibodies. G–I) Bovine in vitro-produced blastocysts were used as positive controls for staining pattern and were stained for SSEA1 (negative; G), SSEA4 (positive, H), and OCT4 (positive; I). We used different secondary antibodies for the blastocysts stain. These antibodies were conjugated with fluorescein isothiocyanate, because the staining kits used for ES colonies show dark stain, which is difficult to visualize in small embryos. Bar = 100 µm (A–F), x200 (G–I)

We then determined the differentiation potential of these putative NT ES (ntES) cells by embryoid body (EB) formation and random differentiation using NTL-1. Without feeder cells and LIF, NTL-1 cells spontaneously generated embryoid bodies (Fig. 3A). For random differentiation, we replated these cells on a tissue culture dish by mechanical dissection. The cells gave rise to a wide variety of differentiated cell types, including derivatives of the three embryonic germ (EG) layers. We observed ectodermal derivatives, such as neurofilament TUBB3-reactive cells (Fig. 3, C and D), and mesodermal derivatives, such as smooth muscle specific ACTA2-reactive myocytes (Fig. 3E). Endodermal derivatives were observed as well, such as several types of cytokeratin-reactive epithelia, including simple cuboidal epithelial cells (Fig. 3F) and net-like epithelial cell structures (Fig. 3G). The putative ntES cells gave rise to cells of different lineages of tissue differentiation. Therefore, we surmise that they are pluripotent ES cells, and we refer them as such in the rest of the text.

View larger version (90K): [in this window] [in a new window]   FIG. 3. Morphology and immunohistological analysis of in vitro differentiated putative bovine ntES (NTL-1) cells. A) A representative embryoid body formed after removal of LIF and feeder cells for 7–10 days. B) Approximately 7 wk after removal of LIF and feeder cells, a variety of cells with different morphologies were observed. C and D) A cell clump differentiated from embryoid bodies (C) and a neuron-like cell (D) stained positive for TUBB3. E) Differentiated cells stained positive for smooth muscle specific actin, ACTA2. F and G) Cuboidal (F) and net-like epithelial cell structures (G) stained positive for cytokeratin. Bar = 100 µm (A– D and F) and 50 µm (E)

We next attempted to propagate these pluripotent ntES cells by enzymatic dissociation of colonies and replating the dissociated cells for the formation of new colonies. After treating the ntES colonies with EDTA, trypsin-EDTA, type IV collagenase, and protease E for a range of durations, we observed that EDTA treatment alone was insufficient to dissociate colonies. Although typsin-EDTA, type IV collagenase, or protease E could disrupt the colonies into clumps or individual cells irrespective of treatment times, the newly plated cells/clumps failed to self-renew, and differentiation was induced in all cases. Therefore, all passaging of these pluripotent ntES cells was conducted by mechanical dissociation.

We also examined the karyotype of the pluripotent ES colonies. The karyotyping results revealed normal female bovine chromosomes in accordance with their donor cell origin (Fig. 4). The pluripotent ES colonies have been maintained in culture without differentiation for more than a year.

View larger version (46K): [in this window] [in a new window]   FIG. 4. A representative metaphase spread of bovine pluripotent ntES cells. Arrows indicate the X chromosomes. Original magnification x1000

For the ICMs isolated from parthenogenetic embryos, developmental defects were observed after 7–10 days in culture. They did not proliferate to maintain an ICM population and differentiated. Additionally, the ICM cultures could not be identified by molecular criteria. Only a few cells within the ICM cultures of the parthenotes were stained positive for ALPL (Fig. 1D), suggesting that only a few cells of the culture maintained an undifferentiated status. The only parthenote-derived colony was lost at passage 1 with signs of differentiation (Fig. 1D).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In the present study, we generated one ES cell line from a bovine IVF embryo and two ES cell lines from cloned embryos. These cells have typical ES cell morphology and high levels of ALPL activity. Expression profiles of stem cell-specific markers in the ntES cell lines (NTL-1 and NTL-2) are similar to those of established mouse and human ES cells. To date, five studies have reported the generation of bovine pluripotent ES-like cells from IVF or NT embryos [14–18]. The bovine ES cells that we generated were different from all previously reported lines either in morphology [14–16] or in marker expression patterns in cell lines for which staining was conducted [14–18]. Our ES cells formed large, round colonies with distinct boundaries, whereas those reported in previous studies formed a monolayer sheet [14–16]. Most cells in our colonies displayed positive ALPL staining throughout the culture period. This has been shown in almost all other mammalian pluripotent ES cells, including those generated from the mouse, monkey, human, and pig [7, 9, 29, 35, 36]. However, three of five previously generated bovine ES-like cell lines were either ALPL negative or not stained for ALPL [14–16]. In addition to ALPL stains, our present work also illustrates, to our knowledge for the first time, the expression of specific molecular markers for totipotent cells in both of our ntES cell lines. These cells are characterized by positive staining for high levels of OCT4 as well as SSEA4 expression and by negative staining for SSEA1. This is similar to the reported characterization of undifferentiated primate ES and parthenogenetic stem cells [7, 13], human ES cells, and ntES cells [9, 11, 20] but is different from the previously established pluripotent stem cell lines produced from bovine IVF embryos, which were SSEA1 positive [14, 17]. Our results showed that the bovine ntES cells expressed an identical marker to that of the ICM cells of the bovine blastocysts. This also is consistent with findings observed in the mouse and human [30–34]. Moreover, like mouse ES and EG cells, our ntES cells proved to be TRA1 negative.

The ES cells are defined functionally by their ability to form representative cell types from three germ layers: the endoderm, mesoderm, and ectoderm, both in vivo and in vitro [9, 22, 23, 29]. The differentiation of NTL-1 cells in vitro into many specific cell types, representative of all three EG cell layers, suggests that these cells are, indeed, pluripotent. Additional studies to demonstrate the ability of these ntES cells to form teratocarcinoma and germ line chimeras in vivo will further support our characterization of pluripotency.

Our studies indicated that unlike the findings in other species, bovine pluripotent ntES cells were sensitive to enzymes, including trypsin, which is used to dissociate mouse ES cells; type IV collagenase, which is used to dissociate human ES cells; and protease E. All these enzymes might interrupt the cells' self-renewal pathways and cause spontaneous differentiation. Previous studies have shown that the self-renewal pathway of ES cells differs between species. More specifically, the LIF/STAT3 pathway is not required to maintain self-renewal of human ES cells, whereas it is critical for that of the mouse ES cells [37–40]. However, self-renewal signal pathways of ES cells might be partially conserved, as was suggested by the fact that transcription factors, such as OCT4, are expressed in human and mouse ES cells [41] as well as in our pluripotent bovine ntES cells. Further studies are needed to understand the self-renewal pathway in bovine pluripotent ES cells to answer the question of why bovine ES-like stem cells are not amendable to enzymes.

In the present study, we found that the rate of primary trophectoderm colony derivation from NT embryos is higher than that from IVF embryos. This may be associated with the observation that IVF can generate polyspermic and mixoploidy embryos. Additionally, oocytes used in IVF were not selected as stringently as in NT, because cumulus cells are required for IVF. However, in NT, we removed the cumulus cells completely before micromanipulation. This distinction in the routine experimental procedure permitted use of the best oocytes in NT, because we can better judge the quality of oocytes after the removal of cumulus cells. All these factors could contribute to the difference in the efficiency of stem cell generation from embryos produced by IVF versus those produced by NT.

We also report the failure to maintain bovine parthenote ICM cultures in vitro. Our results suggest that ICMs of bovine parthenotes had developmental defects in that they spontaneously differentiated in the same culture system that was used in the successful generation of pluripotent IVF and ntES cells. The majority of parthenotes exhibit abnormalities [42, 43], such as high incidence (90%) of polyploid metaphase [44] and abnormal regulation of the IGF1R/ IGF2 mitogenic pathway [45]. Both IGF2 and IGF1R were expressed in normal mouse blastocysts, but neither was detected in parthenotes [42]. However, pluripotent ES cell lines have been derived from mouse and monkey parthenotes. It remains a possibility that the proper sequence of activating stimuli and improved culture conditions are necessary to increase the derivation efficiency of ES cells from bovine parthenotes.

In summary, we generated pluripotent ES cells from bovine IVF and cloned embryos with morphology similar to those of the mouse and human ES cells. The ES marker-staining patterns of the two ntES cell lines are identical to those of the cells derived from the ICMs of bovine IVF blastocysts yet different from all pluripotent bovine ES-like cells reported previously. Moreover, the pluripotency of our NTL-1 cells was proven by the in vitro differentiation study. The fact that the staining pattern of ES cell-specific markers is very similar to that of the human ES cells suggests that bovine ES cells may serve as a better model than mouse ES cells for studies of human ES cell tissue regeneration.

	ACKNOWLEDGMENTS

 
We thank S. Jiang for helping to generate parthenogenetic embryos, Marina Julian and Sadie Smith for critical reading of this manuscript, and Lan Yang for help in the statistical analyses. We also thank Dr. Zhongde Wang for providing helpful scientific suggestions in final manuscript preparation.

	FOOTNOTES

 
¹ Supported in part by the Cooperative State Research, Education, and Extension Service, U.S. Department of Agriculture, under agreement 96-35203-3268, by a grant from Connecticut Innovations, Inc., by the Special Funds for Major State Basic Research Project (2001CB510103), and by the Knowledge Innovation Program of CAS (KSCX2-3-08).

² Correspondence: X. Cindy Tian, Center for Regenerative Biology, University of Connecticut, 1392 Storrs Road, U-4243, Storrs, CT 06269. FAX: 860 486 8809; xiuchun.tian{at}uconn.edu

Received: 16 October 2004.

First decision: 16 November 2004.

Accepted: 16 February 2005.

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