ERas is Expressed in Primate Embryonic Stem Cells but not Related to Tumorigenesis

Cell Culture and Differentiation

A cynomolgus ES cell line (CMK6) (19), its subline (CMK6G) stably expressing enhanced green fluorescent protein (EGFP) (20), and a human ES cell line (SA181, Cellartis AB, Göteborg, Sweden) (10) were maintained on a feeder layer of mitomycin C (Kyowa, Tokyo, Japan)-treated mouse (BALB/c, Clea, Tokyo, Japan) embryonic fibroblasts (MEFs) as previously described (10,19). Confluent ES cells were dissociated from the feeder layer using 0.1% collagenase type IV (Invitrogen, Carlsbad, CA, USA). Cynomolgus stromal cells were obtained from cultured adherent cells of cynomolgus bone marrow.

For neural differentiation from cynomolgus ES cells, astrocyte-conditioned medium (ACM) was prepared by culturing astrocytes obtained from mouse fetal cerebra in Dulbecco's modified Eagle's medium (DMEM)/F12 medium containing an N2 supplement (Invitrogen) (15). Colonies of cynomolgus ES cells (800–1000 μm in diameter) were plucked from the feeder layer using a glass capillary and transferred into nonadhesive bacteriological dishes each containing ACM supplemented with 20 ng/ml of recombinant human fibroblast growth factor-2 (FGF-2) (R&D, Minneapolis, MN, USA). The colonies were cultured for 12 days, giving rise to neural stem cells, which were plated onto poly-L-lysine/laminin (Sigma-Aldrich, St. Louis, MO, USA)-coated dishes and cultivated for 7 days in Neurobasal medium supplemented with 2% B-27 (both from Invitrogen), 20 ng/ml of FGF-2, and 20 ng/ml of recombinant human epidermal growth factor (R&D). After the medium was replaced with ACM and 14 days of culture, the neural stem cells differentiated into neurons (16).

ERas Cloning and Transfection

Based on the human ERas cDNA sequence in Genbank (accession No. NM 181532), the primer set 5′-CAT GGA GCT GCC AAC AAA GCC TG-3′ and 5′-TGT GTC CCT CAA AGC TAG TTG CCT-3′ was designed for the cynomolgus ERas' complete coding sequence. Total RNA was extracted from cynomolgus ES cells using the EZ1 RNA universal tissue kit (Qiagen, Hilden, Germany) with RNase-Free DNase Set (Qiagen), and reverse-transcribed using the RNA LA PCR kit (Takara, Shiga, Japan) with an oligo dT primer. The resulting cDNA was subjected to PCR with this primer set. The PCR product was sequenced with the ABI Prism 310 (Applied Biosystems, Foster, CA, USA). The sequence analysis was performed with Genetyx-Mac software (Genetyx Corporation, Tokyo, Japan). We previously constructed the plasmids pPyCAG-EGFP-gw-IP (expressing EGFP) and pPyCAG-EGFP-gw-IP-mouse ERas (expressing EGFP-mERas) (22). The cDNA encoding the human or cynomolgus ERas gene was inserted into pPyCAG-EGFP-gw-IP to construct pPyCAG-EGFP-gw-IP-human ERas (expressing EGFP-human ERas) or pPyCAG-EGFP-gw-IP-cynomolgus ERas (expressing EGFP-cynomolgus ERas), respectively. The insert was confirmed by DNA sequencing. The plasmids were transfected into cynomolgus stromal cells using Lipofectamin 2000 (Invitrogen). The transfected cells were selected in the presence of puromycin (5 μg/ml).

Transplantation

Cells (1 × 10⁷/site) were transplanted into the thigh muscle of immunodeficient NOD/Shi-scid, IL-2Rγ^null (NOG) mice which were purchased from Central Institute for Experimental Animals (Kanagawa, Japan). Cynomolgus ES cell-derived teratomas were generated after in utero transplantation of cynomolgus ES cells into fetal sheep as described previously (23). Adherent cells from the teratomas were propagated in DMEM (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA). All experiments were performed in accordance with the Jichi Medical University Guide for Laboratory Animals. Experimental procedures were approved by the Animal Care and Use Committee of Jichi Medical University.

Reverse Transcription (RT)-PCR

cDNA was prepared from each sample as mentioned above and subjected to PCR with the following primer sets: for Oct-4, 5′-GGA CAC CTG GCT TCG GAT T-3′ and 5′-TTC GCT TTC TCT TTC GGG C-3′; and for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5′-CCC TGG CCA AGG TCA TCC ATG ACA AC-3′ and 5′-CCA GTG AGC TTC CCG TTC AG-3′. Amplification conditions were 30 cycles of 95°C for 60 s, 58°C for 60 s, and 72°C for 60 s. PCR without the initial RT was also conducted to rule out DNA contamination. For real-time quantitative RT-PCR, a QuantiTect SYBR Green PCR kit (Qiagen) and the ABI Prism 7000 (Applied Biosystems) were used, and amplification conditions were 40 cycles of 95°C for 60 s, 58°C for 60 s, and 72°C for 60 s. The gene expression levels were adjusted based on those of the internal control GAPDH.

Immunoblotting

Preparation of cell lysates and Western blot analyses were performed as described previously (22). Briefly, cells (1 × 10⁷) were washed twice with ice-cold phosphate-buffered saline (PBS) and suspended in the buffer containing 10 mM Tris-HCl (pH 7.5), 1 mM MgCl₂, and the 1× Complete Protease Inhibitors (Roche, Basel, Switzerland), followed by incubation on ice for 30 min. These samples were disrupted with Dounce tissue homogenizer (Wheaton, NJ, USA) and added with 1 M NaCl to a final concentration of 150 mM. For lysis, these samples were added with a final concentration of 5% SDS, 1% NP-40, and 1% deoxicorate acids, followed by incubation on ice for 10 min. The samples were then centrifuged at 12,000 × g for 30 min at 4°C, and supernatants were collected. The protein concentrations were measured by the Bio-Rad Bradford assay (BioRad Laboratories, CA, USA). An equal amount of protein (10 μg per lane) was electrophoresed on 12.5% polyacrylamide gel (Atto, Tokyo, Japan) and blotted onto a PVDF membrane (Immobiron; Millipore, MA, USA). The membrane was blocked by TBST containing 5% w/v Amersham ECL-Blocking Agent (GE Healthcare, Buckinghamshire, UK) and then incubated with rabbit antiserum against mouse ERas (22) overnight at 4°C. After washed with TBST, the membrane was incubated with anti-rabbit IgG HRP (Jackson Laboratory, CA, USA) at room temperature for 60 min. Signal was detected using Amersham ECL Plus Western Blotting Detection reagents (GE Healthcare) according to the manufacturer's protocol and visualized on the LAS 3000 mini (Fujifilm, Tokyo, Japan).

Immunohistochemistry

For the immunofluorescent staining of frozen sections, tissues were fixed with 4% paraformaldehyde. The sections were labeled with rabbit anti-serum against mouse ERas (22). The primary antibody (Ab) was detected with a Tyramide Signal Amplification Kit (Invitrogen). After nuclei were stained with DAPI (Donjindo, Kumamoto, Japan), the sections were observed with a confocal laser scanning microscope (Olympus, Tokyo, Japan). For immunohistochemistry, tissues were fixed with 4% paraformaldehyde and embedded in paraffin. To identify GFP-positive cells, the sections were stained with rabbit anti-GFP Ab (Clontech, Palo Alto, CA, USA), reacted with the Dako EnVision+ System HRP (Dako, Copenhagen, Denmark), and visualized with 3,3′-diaminobenzide tetrahydrochloride (Dojindo). Nuclei were counterstained with hematoxylin.

Flow Cytometry

The expression of GFP and ERas was analyzed using a FACS Calibur flow cytometer (BD Pharmingen, San Diego, CA, USA). To detect ERas, cells were fixed using fixation/permeabilization buffer (eBioscience, San Diego, CA, USA) for 2 h at 4°C and then incubated with Alexa Fluor 647 (Invitrogen)-conjugated rabbit antiserum against ERas for 60 min at 4°C. Data acquisition and analysis were performed using CellQuest software (BD Pharmingen). Fluorescence-conjugated, irrelevant Abs served as negative controls.

Cell Proliferation Assay

Total cell numbers and proliferating cell numbers were measured with Cell Counting Kit-8 (Dojindo) and with Cell Proliferation ELISA, BrdU (colorimetric) (Roche), respectively. For Cell Counting Kit-8, cells were seeded in 96-well plates at 5 × 10³ per well and measured after 6, 24, 36, 48, and 80 h of incubation according to the manufacturer's instructions. For Cell Proliferation ELISA, cells were seeded in 96-well plates at 2 × 10³ per well and measured after 24, 36, 48, 72, and 96 h of incubation. Significant differences were examined using the t-test.

Cynomolgus Cells and Tissues Express ERas

We first cloned and sequenced the cynomolgus orthologue of the ERas gene from cDNA of the undifferentiated cynomolgus ES cells (Fig. 1A). The translated amino acid sequence showed a higher degree of homology to human than mouse ERas (99% vs. 75%) (Fig. 1B). We could not detect the ERas protein in cynomolgus ES cells by flow cytometry, implying its weak expression (data not shown).

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Figure 1.

Cloning and sequencing of cynomolgus ERas. (A) The expression of the cynomolgus (cyno) ERas gene is detectable in cynomolgus ES cells (CMK6), but not in human ES cells (SA181), by reverse transcription (RT)-PCR. To exclude the possibility of genomic DNA contamination, PCR without the RT procedure (designated RT -) was also conducted. DW (distilled water) indicates no template in the reaction. RT-PCR of the GAPDH sequence is also shown as an internal control. (B) Amino acid sequences of cynomolgus, human (accession No. NM 181532), and mouse (accession No. NM 181548) ERas are shown. Amino acids identical to cynomolgus ERas are shown as dots and conserved amino acids are encircled with a solid line.

Next, we examined the expression of the ERas gene in adult cynomolgus tissues by RT-PCR. Using a primer set to cover the entire coding region, the amplicons from all nine somatic tissues were the same size as that from cynomolgus ES cells (Fig. 2A). The PCR product was sequenced to confirm that it was the ERas gene. This clearly shows that a full-length version of the ERas gene is transcribed in cynomolgus tissues, despite that ERas is not expressed in mouse tissues (22).

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Figure 2.

Cynomolgus tissues express ERas. (A) The expression of the cynomolgus ERas gene is detectable in various cynomolgus tissues by RT-PCR. To exclude the possibility of genomic DNA contamination, PCR without the RT procedure (designated RT -) was also conducted. DW indicates no template in the reaction. RT-PCR of the GAPDH sequence is also shown as an internal control. (B) Cynomolgus bone marrow stromal cells were transfected with the mouse ERas (mERas), human ERas (hERas), or cynomogus ERas (cyERas) gene fused with the EGFP gene, and immnunoblotted with the polyconal anti-ERas antibody. Although the antibody was generated against recombinant mERas (22), it reacts to hERas and cyERas as well as mERas. In addition, it does not react to cynomolgus other Ras family proteins (N-, H-, or K-Ras; 21 kDa). ERas (25 kDa) fused with EGFP (26 kDa) is detectable at 51 kDa. In mERas-transfected cells, ERas alone released from the fusion protein was also detected. (C) Using the anti-ERas antibody, ERas-positive cells (red) were detected in cynomolgus brain, thymus, intestine, and ovary. Control indicates the staining of cynomolgus brain without the primary ERas antibody. Although the primary antibody to ERas was originally developed for mouse ERas and should react more strongly to mouse than cynomolgus ERas, no positive signals were detected in mouse brain. (D) The ERas fluorescence with or without DAPI is shown at a higher magnification of both cynomolgus brain and thymus. ERas was detected on the cytoplasmic membrane.

We then examined the protein expression in adult cynomolgus tissues by immunohistochemistry. Immunoblotting revealed that the antibody reacts to human and cynomolgus ERas as well as mouse ERas, although the antibody was generated against recombinant mouse ERas (22) (Fig. 2B). In addition, it specifically reacts to ERas (25 kDa) and does not react to other Ras family proteins (N-, H-, or K-Ras; 21 kDa) in cynomolgus cells expressing these Ras genes (Fig. 2B). Using this antibody, we detected ERas-positive cells in all tissues tested (brain, thymus, intestine, and ovary) (Fig. 2C). At a higher magnification, it turned out that ERas is localized on the cytoplasmic membrane as expected (Fig. 2D). We also tested mouse tissues, but ERas-positive cells were not detectable (Fig. 2C). Taken together, the ERas protein is indeed expressed in cynomolgus tissues, unlike in murine tissues.

The expression of the ERas gene becomes undetectable after the differentiation of mouse ES cells (22). We examined the expression of the ERas gene after the differentiation of cynomolgus ES cells. Cynomolgus ES cell-derived neurons and teratoma cells were examined for the expression of ERas as well as Oct-4, a pluripotent marker of ES cells. They were positive for ERas but negative for Oct-4 (Fig. 3A). Although cynomolgus ES cell-derived neurons were fragile and scarcely survived after dissociation from the culture dish, adherent teratoma cells could be cultured for more than six passages at a dilution of 1:4 to 1:8. Quantitative RT-PCR showed that the ERas gene expression levels were even higher in the cultured teratoma cells than in the undifferentiated cynomolgus ES cells (Fig. 3B). Then, we transplanted 1 × 10⁷ cultured teratoma cells expressing the ERas gene (GFP-positive, passage 3) into the thigh muscle of NOG mice (n = 3), and examined the tumorigenicity of the cells. NOG mice were used as recipients, because they are more immunodeficient than other immunodeficient mice and transplanting to NOG mice is the most sensitive assay to detect tumorigenesis (7,14). However, no tumor developed after 2.5 months, although the transplanted cell progeny (GFP positive) were detected in every specimen (Fig. 3C). On the other hand, undifferentiated cynomolgus ES cells formed teratomas in all NOG mice. Taken together, cynomolgus ES cell-derived differentiated progeny and teratoma cells also express the ERas gene, but do not produce tumors in vivo.

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Figure 3.

Cynomolgus ES cell-derived cells express ERas but do not form tumors in vivo. (A) The ERas gene was expressed in cynomolgus ES cell-derived neurons and teratoma cells as assessed by RT-PCR, whereas the Oct-4 gene was expressed in neither. DW indicates no template in the reaction. RT-PCR of the GAPDH sequence is also shown as an internal control. (B) The ERas gene expression level was nearly five times higher in the cultured teratoma cells than in the undifferentiated cynomolgus ES cells as assessed by quantitative RT-PCR. The gene expression level was adjusted using the internal control GAPDH. (C) The cultured teratoma cells were transplanted into the thigh muscles of NOG mice and examined for tumorigenicity in vivo after 2.5 months. Staining of the specimen with anti-GFP is shown. Although the transplanted cell progeny (GFP positive, brown) were detected, no tumor was observed.

ERas Overexpression in Cynomolgus Cells

To examine whether the cynomolgus ERas contributes to cell proliferation, we transfected cynomolgus stromal cells with a plamid expressing the cynomolgus or mouse ERas, EGFP, and puromycin resistance genes. Transfectants were obtained by treatment with puromycin and more than 90% of the cells expressed EGFP (Fig. 4A). These transfectants did not show significant morphological changes. Quantitative RT-PCR showed that the transfected cells expressed approximately 1000 times more ERas than undifferentiated cynomolgus ES cells (Fig. 4B). These cells expressing either cynomolgus or mouse ERas showed larger total cell numbers or proliferating cell numbers after plating than the mock-transfected cells, but did not show any more rapid proliferation thereafter (Fig. 4C). Thus, cynomolgus ERas improves plating efficiency but does not promote cell proliferation, even when it is expressed at high levels.

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Figure 4.

Overexpression of ERas does not promote cell proliferation but improves plating efficiency. (A) The plasmids expressing the cynomolgus or mouse ERas, EGFP, and puromycin resistance genes were transfected into cynomolgus stromal cells. After puromycin selection, more than 90% of cells expressed EGFP (upper). The ERas expression was also detected by flow cytometry (lower). (B) The level of cynomolgus ERas gene expression was 1000 times higher in the cynomolgus ERas-transfected cells (cyERas) than in naive cynomolgus ES cells (CMK6) or mock-transfected cells by quantitative RT-PCR. The gene expression levels were adjusted using the internal control GAPDH. (C) The mock-, cynomolgus ERas (cyERas)-, and mouse ERas (mERas)-transfected cells were plated at 5 × 10³ per well and total cell numbers were measured after 6, 24, 36, 48, and 80 h of incubation (upper). The mock-, cyERas-, and mERas-transfected cells were plated at 2 × 10³ per well and proliferating cell numbers were measured after 24, 36, 48, 72, and 96 h of incubation (lower). The cyERas- and mERas-transfected cells showed larger total cell or proliferating cell numbers after plating than the mock-transfected cells, but did not show any more rapid proliferation thereafter. Statistical differences with the t-test are indicated: ^∗p < 0.01 for the cyERas- versus mock-transfected cells, ^∗∗p < 0.01 for the mERas- versus mock-transfected cells.

In this report, we showed that the cynomolgus ERas gene is expressed in cynomolgus ES cells and tissues. Its expression pattern is quite different from that of mouse ERas, which is not expressed in mouse ES cell-derived differentiated progeny or mouse tissues. Although cynomolgus ERas improved the plating efficiency when overexpressed, its expression did not promote cell proliferation or induce tumor formation in vivo (Figs. 3C, 4C). Thus, cynomolgus ERas might only suppress the apoptosis of cynomolgus cells (5). Because the formation of teratomas is one of the greatest obstacles to the clinical application of human ES cells (3,8,18), it is important to elucidate whether the ERas gene expressed in cynomolgus ES cells is related to teratoma development in vivo in order to tell whether nonhuman primate models are really suitable for preclinical research. From our study, it is at least suggested that cynomolgus ES cells are more similar to human than mouse ES cells in that ERas does not contribute to the formation of teratomas in vivo.

To date, the pluripotent marker Oct-4 has been used to predict the formation of teratomas (4) and the removal of Oct-4-positive cells from ES cell-derived progenitor preparations is reported to prevent teratomas from developing posttransplant (2). However, Oct-4-negative immature cells are also reported to contribute to the formation of teratomas (6). Therefore, although Oct-4 could be used to predict whether teratomas develop to some extent, it does not regulate the developmental process. For future clinical applications, the mechanism by which primate ES cells form teratomas should be studied in more detail.