CD30 is a survival factor and a biomarker for transformed human pluripotent stem cells

Daniella Herszfeld^1, 5, Ernst Wolvetang^1, 5, Emma Langton-Bunker¹, Tung-Liang Chung¹, Adam A Filipczyk¹, Souheir Houssami¹, Pegah Jamshidi¹, Karen Koh¹, Andrew L Laslett¹, Anna Michalska¹, Linh Nguyen¹, Benjamin E Reubinoff^1, 4, Irene Tellis¹, Jonathan M Auerbach², Carol J Ording², Leendert H J Looijenga³ & Martin F Pera¹

¹ Monash Institute of Medical Research, Monash University, and the Australian Stem Cell Centre, Bldg. 75 STRIP, Wellington Road, Clayton, Victoria 3800 Australia.

² Stem Cell Center, American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110, USA.

³ Pathology/Lab. Exp. Patho-Oncology, Erasmus MC–University Medical Center Rotterdam, Daniel den Hoed Cancer Center, Josephine Nefkens Institute, Building Be, Room 430b, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands.

⁴ Present address: The Hadassah Human Embryonic Stem Cell Research Center, The Goldyne Savad Institute of Gene Therapy and The Department of Gynecology, Hadassah University Hospital, Ein-Kerem, P.O.B. 12000, Jerusalem 91120, Israel.

⁵ These authors contributed equally to this work.

To understand the role of CD30 in pluripotent stem cell growth and differentiation, we studied its expression in more detail. Northern blot analysis of mRNA from human EC cell lines showed that, in addition to the main band of 3.8 kilobases (kb) seen in the EC cell lines corresponding to the canonical transcript, a fainter band of 2.6 kb was expressed (Fig. 1a). Both the 3.8-kb and the 2.6-kb bands were downregulated during differentiation of EC cells in vitro (Fig. 1b). This 2.6-kB band was similar in size to a variant CD30 transcript described previously in myeloid leukemia cell lines⁹. This transcript, originating from alternate promoter usage, encodes a truncated variant form of CD30 consisting only of its cytoplasmic domain, and this truncated protein constitutively activates signaling pathways in the absence of ligand. Reverse transcriptase–polymerase chain reaction (RT-PCR) using a forward primer specific for the variant transcript confirmed its expression in EC cells (Fig. 1c). Immunoblots of EC cell lysates were probed with antisera raised against a domain in the cytoplasmic portion of the protein (C-terminal epitope). The C-terminal antisera reacted with bands of 120, 55 and 25 kilodaltons (kDa) (Fig. 1d); the size of the 25-kDa band is that expected of the variant form of CD30 protein, and it was not detected in blots probed with antibodies against the N-terminal ectodomain (not shown).

Figure 1. Expression of CD30 transcripts and protein in human EC and ES cells. (a) Northern blot of polyA+ mRNA from EC cell lines GCT 27C-4, GCT 35 and GCT 48, probed with full-length cDNA for CD30 or GAPDH as a loading control. (b) Northern blot of polyA+ mRNA from control GCT 27X-1 EC cells and from cells treated with 50 ng/ml BMP-2 for 24 or 48 h, probed with full-length cDNA for CD30 or GAPDH probes. Scanning densitometry of these Northern blots showed a 3.2-fold reduction in CD30 expression following this treatment. (c) RT-PCR analysis of expression of CD30 canonical and variant transcripts in HES-2 P15, HES-3 P65 and EC cell line GCT 27X-1. Oct-4 products shown as stem cell marker, GAPDH is control for RT-PCR. (d) Immunoblot analysis of GCT 27X-1 cell lysate using antibody to C-terminal region of CD30. Position of molecular weight markers shown to right. The 120-kDa band is the canonical form, the low-molecular-weight band is the variant form and the 57-kDa band in the middle results from known cross-reactivity of the antibody with another cellular protein. Full Figure and legend (19K)

HES cells share many properties with pluripotent EC cells, including morphology, cell-surface antigen and gene expression profile, growth requirements and differentiation capacity. To investigate whether CD30 expression is tumor specific or a general property of human pluripotent cells, we examined expression of CD30 in colonies of hES cells using immunocytochemistry for full-length CD30 protein and RT-PCR for both the canonical and variant cytoplasmic forms of CD30. Antibodies Ber-H2 or M67, both of which react with the ectodomain of CD30, yielded background staining only on colonies of hES cell lines HES-2, HES-3 and HES-4 that were positive for the stem cell antigen GCTM-2 (Ber-H2 staining of HES-4, Supplementary Fig. 1 online) but stained cultured human EC cells in a characteristic pattern decorating the cell border (Ber-H2 staining of GCT 27X-1, Supplementary Fig. 1 online). Whereas RT-PCR products representing both forms of CD30 transcript were obtained from human EC cell line GCT 27X-1, none were obtained from mRNA from the hES cell lines (Fig. 1c and below). The presence of mRNA for the transcription factor Oct-4 and the housekeeping gene -actin confirmed the integrity of EC and hES cell RNA.

Abnormal variants of cell lines HES-1, HES-2 and HES-3 showed a faster population doubling time compared with diploid cells (30 h versus 72 h under these growth conditions, data not shown). To assess differentiation and growth requirements in vitro, we compared the effect of withdrawal of FGF-2 on the proportion of stem cell marker–positive cells 15 d after subculture of HES-3 bearing trisomy 12 to the effect of factor withdrawal on diploid HES-3 cells passaged only twice in the serum-free system. Withdrawal of FGF-2 induced differentiation in diploid ES cells under these conditions but had no effect on the abnormal cells in this assay (Supplementary Fig. 3 online). Alterations in differentiation capability of the abnormal cell lines were also apparent in transplantation assays. When injected into immunodeficient mice, the abnormal cell lines did not form malignant ECs, unlike EC cells (Supplementary Fig. 4 online), and still showed differentiation into various types of tissue. However, compared with teratomas formed by diploid ES cells (Supplementary Fig. 4 online), teratomas formed by the abnormal cells (Supplementary Fig. 4 online) contained a much greater proportion of primitive, undifferentiated cells. Seven mice were injected, each at two sites, with cells from two of these karyotypically abnormal lines, and 14 tumors obtained showed similar histology with a preponderance of poorly differentiated cells.

The karyotypically abnormal ES cell sublines expressed CD30 transcripts (Fig. 2a,b). The expression of CD30 was not an inevitable consequence of growth in the absence of serum; diploid cultures of cell line HES-4 grown for as many as 20 passages in serum-free medium were negative for CD30 transcripts or surface staining (Fig. 2a, lane 3, immunostaining not shown). Karyotypically abnormal cells also expressed transcripts encoding the variant form of CD30, similar to EC cells (HES-2, Fig. 2c). Expression of this transcript was downregulated following treatment of abnormal cell line BG01V with 50 ng/ml bone morphogenetic protein 4 (BMP-4) for 4 d (Supplementary Fig. 5 online). All abnormal lines derived from HES-1, HES-2 and HES-3 showed typical surface staining for CD30, as did an independently derived line containing extra copies of 12, 17 and X (ref. 3; Fig. 2d–h). That the abnormal HES-3 subline was mosaic for trisomy 12 when the abnormality was first detected permitted us to evaluate the relationship between karyotype and CD30 expression within a mixed population of cells grown under identical conditions. Combined indirect immunofluorescence analysis for CD30 and fluorescence in situ hybridization with an -centromeric probe for chromosome 12 (Fig. 2i) showed a clear concordance between CD30 expression and the presence of an extra copy of chromosome 12 (quantitative analysis, Fig. 2j).

Figure 2. Expression of CD30 in diploid or karyotypically abnormal hES cell lines. (a) RT-PCR analysis of CD30 expression in HES-2, HES-3 and HES-4. Following reverse transcription, two PCR reactions were carried out for CD30, along with reactions for Oct-4 and -actin. Lanes are as follows: lane 1, HES-2 passage 51+36, bearing partial duplication of the long arm of chromosome 1; lane 2, HES-3 passage 55+18, mosaic for trisomy 12; lane 3, HES-4 passage 41+19, diploid; lane 4, HES-2 passage 92, diploid; lane 5, HES-3 passage 78, diploid; lane 6, HES-4 passage 65, diploid. Molecular weight markers: CD30 840-bp amplicon, 650- and 850-bp markers; CD30 463-bp amplicon, 400-, 500- and 650-bp markers; Oct-4, 500- and 600-bp markers; -actin, 200- and 300-bp markers. (b) RT-PCR for 840-bp amplicon of CD30 on HES-1 cells. Lanes 1–2, two samples of HES-1 P54 from Hadassah University Hospital laboratory bearing rearrangement of 1q; lanes 3–4, diploid HES-1 passages 39 and 40, respectively. (c) RT-PCR for variant form of CD30 in HES-2 passage 28+30 bearing duplication in 1q. Lane 1, 642-bp amplicon; lane 2, no-template control. (d–h) Staining of CD30 in HES-2 passage 51+28 with duplication in the long arm of chromosome 1 (d, scale bar 50 m) or HES-3 passage 55+32 with trisomy 12 (e, scale bar 50 m; preparation counterstained with Hoechst 33258), HES-3 passage 37+33 bearing (1,6) translocation (f, scale bar 200 m), HES-1 passage 55 with rearrangement in 1q (g, same magnification as f) and BG01V with trisomy 12, 17 and X (h, scale bar 200 m). (i) Triple-label staining for CD30 (red), -centromeric probe for chromosome 12 (green) and DNA (blue) in HES-3 passage 55+20 mosaic for trisomy 12 (scale bar 50 m). (j) Quantitative analysis of relationship between chromosome 12 copy number and CD30 staining in HES-3 hES cell cultures mosaic for trisomy 12. Full Figure and legend (96K)

Figure 3. Flow-cytometric analysis of CD30 expression in abnormal and diploid hES cell lines. (a) BG01V; (b) HES-3 passage 37+49 (46XX, t(1;6)(p22;q15)); (c) HES-2 passage 31+11 (46XX). In a and b, top panels show actual profiles for isotype control and CD30, and bottom panels show dot plots for double labeling with GCTM-2. In c, dot plot for double label of Oct-4 and CD30 shown at top, with raw profiles for isotype control and CD30 below. Full Figure and legend (61K)

Figure 4. Combined analysis of CD30 expression and apoptosis in HES-2 serum replacement cultures by flow cytometry and inhibition of apoptosis by expression of CD30 variant. (a,b) Typical flow cytometry profiles for spontaneous apoptosis and H2O2-induced apoptosis, respectively. Gates for isotype control stainings are indicated within the panels. (c) Quantitation of data from three experiments as shown in a and b. Bars show percentage of TUNEL-positive cells for (left to right) control CD30-positive cells, control CD30-negative cells, CD30-positive cells treated with 200 M hydrogen peroxide, CD30-negative cells treated with 200 M hydrogen peroxide. Values are mean s.e.m. of three separate experiments. (d) Inhibition of spontaneous apoptosis in diploid HES-2 cells following transfection of cDNA encoding variant CD30. Level of apoptosis in transfected cells (identified by EGFP expression, light gray bars) is compared to that in nontransfected cells (dark gray bars) in cultures transfected with bicistronic vector containing EGFP only (empty vector) or EGFP and CD30 variant (variant). Values are mean s.e.m. of three separate experiments. (e) Activation of NF-B signaling by CD30 in hES cells. Basal reporter activity was higher in aneuploid HES-3 cells bearing (1,6) translocation, compared with diploid hES cells, and cotransfection with the CD30 variant greatly increased reporter activity. Values are mean s.e.m. of three separate experiments. Full Figure and legend (42K)

We also considered whether CD30 signaling activates the NF-B pathway in hES cells. Abnormal cell line HES-3 with a (1;6) translocation showed a higher basal level of reporter activity when transfected with a construct incorporating NF-B binding sites driving luciferase expression, compared with diploid cells (Fig. 4e). The reporter activity was greatly enhanced when the cells were cotransfected with the CD30 variant construct along with the luciferase plasmid. Incubation with an inhibitor of NF-B transcriptional activity showed 95% inhibition of reporter activation, confirming the specificity of the assay (data not shown).

As we have shown, CD30 expression provides a significant survival advantage for pluripotent stem cells that express it. Evidence that CD30 might function in an autocrine, cell-autonomous fashion to provide a selective advantage for transformed human pluripotent cells was obtained in the present study, which showed that a variant form of CD30 was expressed in EC cells and karyotypically abnormal hES cells. This variant receptor was previously shown to transactivate gene expression through NF-B and to induce differentiation in HL-60 cells in a ligand-independent fashion⁹. Our results confirm activation of the NF-B signaling pathway by the variant form of CD30 in hES cells, and they also show that the basal level of this pathway is higher in aneuploid cells expressing CD30 than in their normal counterparts. The sensitivity to spontaneous or induced apoptosis of hES cells transfected with a bicistronic expression vector containing CD30 variant cDNA and enhanced GFP (EGFP) was reduced, compared with cells expressing EGFP alone, as measured by TUNEL assay. The survival effect was apparent under conditions that drive death or differentiation of human pluripotent stem cells, namely dissociation to single cells and subcultivation in the absence of a feeder cell layer²¹. Such effects of CD30 overexpression are consistent with its postulated action in promoting the survival of other types of transformed cells, for example, lymphomas²².

Cell lines HES-2, HES-3 and HES-4 were all transferred at several passage levels to the culture system described by Amit et al.¹¹ using either collagenase or cell dissociation buffer to disaggregate colonies of ES cells for subculture and then maintained to different passage levels in this system. The culture medium was supplemented with 20% KnockOut Serum Replacement (Invitrogen) and 4 ng/ml FGF-2, and feeder cells were used at a density of 2 10⁴/cm². Cell line HES-2 was also grown in the culture system of Xu et al.¹² on Matrigel (BD-Biosciences)-treated surface in medium supplemented with KnockOut Serum Replacement, 4 ng/ml FGF-2 and 50% mouse embryo fibroblast conditioned medium. Cells were harvested using cell dissociation buffer to disaggregate the colonies for subculture. Cell line BG01V was maintained in the system described by Amit et al.¹¹

PolyA⁺ RNA was isolated from EC cell lines using detergent lysis followed by oligodT Sepharose chromatography; methods for RNA isolation and Northern blot analysis are described in detail elsewhere²⁹. The probe for CD30 was a full-length human cDNA probe (Immunex Corporation). RT-PCR was carried out on polyA⁺ mRNA isolated from hES cells on oligodT magnetic beads as described elsewhere¹⁸ or on total RNA extracted into Trizol and precipitated in isopropanol. PolyA⁺ mRNA from ES cell lines or GCT 27X-1 or total RNA was subjected to RT-PCR to detect full-length and variant CD30 transcripts. The canonical form of CD30 mRNA was amplified using the forward primer 5'-CTGTGTCCCCTACCCAATCT-3' (start, nucleotide position 1119, GenBank accession number M83554) and the reverse primer 5'-CACTGAGAGCATGACATCGC-3' (start, nucleotide position 1959, GenBank accession number M83554) to yield an 840–base pair (bp) amplicon, or the forward primer 5'-AGCTAGAGCTTGTGGATTCCAG-3' (start, nucleotide position 1517, GenBank accession number M83554) and the reverse primer 5'-GTCTTCTTTCCCTTCCTCTTCC-3' (start, nucleotide position 1980, GenBank accession number M83554) to yield a 463-bp amplicon. The variant form was amplified using the forward primer 5'-CAGCAAGGCAAAGAGTGTGG-3' (start, nucleotide position 15, GenBank accession number D86042) and the reverse primer 5'-AGCCAAGCTTTCACTTTCCAGAGGCAGCTGT-3' (start, nucleotide position 687, GenBank accession number D86042) to yield a 672-bp amplicon. Amplifications were carried out over 45 cycles with an annealing temperature of 65 °C. Amplification of Oct-4 and -actin transcripts was carried out as described¹⁸. The identities of all PCR products were verified by automated DNA sequence analysis.

HES-3 cells (passage 55+20) mosaic for trisomy 12 were seeded and cultured for 1 d in medium supplemented with knockout serum replacer and FGF-2. Cells were treated with 75 mM CaCl₂ for 5 min at 37 °C and fixed in methanol–acetic acid (3:1) for 5 min at 4 °C. Slides were next air dried and stored at -20 °C. Slides were incubated in denaturation solution (70% formamide, 2 SSC, pH 7.4) for 5 min at 73 °C and dehydrated by subsequent 1-min incubations in 70%, 85% and 100% ethanol. The green fluorescent chromosome 12 probe was prepared according to the manufacturer's instructions (Vysis), denatured at 73 °C for 5 min and kept at 50 °C. Slides were warmed on a 50 °C heated stage and 10 l of prepared probe applied for 4 h at 42 °C. Next, slides were washed in 0.4 SSC–0.3% NP-40 (pH 7.0) for 2 min at 73 °C and then in 2 SCC–0.1% NP-40 (pH 7.2) for 1 min. Slides were next washed once in PBS and incubated for 1 h at 37 °C in a Ber-H2 antibody to CD30 (DAKO) in PBS, washed three times in PBS, and incubated in a 1:50 dilution of sheep anti-mouse Alexa Fluor 546 (Molecular Probes) in PBS for 1 h at 21 °C. Slides were next washed three times in PBS and counterstained with 4'-6-diamidino-2-phenylindole (DAPI; 0.1 g/ml) for 10 min at 21 °C, washed once in PBS and mounted with VECTASHIELD (Vector Laboratories). The specimens were examined with a DMR Leica fluorescence microscope fitted with a mercury lamp using the ultraviolet, fluorescein and Texas Red channels: images were captured with a Leica DC200 camera using Leica DC200 software. The captured images were superimposed and the number of green spots per blue nucleus of cells that were CD30⁺ and CD^- were scored. A total of 600 cells from randomly selected fields were counted.

hES cell line HES-2 was grown in serum replacement medium containing FGF-2 for eight passages on a mouse feeder cell layer and was either not treated or incubated with 200 M H₂O₂ for 24 h. The culture medium was collected and centrifuged at 3,220g for 4 min to collect the apoptotic bodies. The cells were then harvested with Cell Dissociation Solution (Sigma) and made into a single-cell suspension by careful trituration before combining the cells with the resuspended apoptotic bodies. The cell suspension was subsequently fixed in 2% paraformaldehyde for 1 h at 21 °C and TUNEL labeled according to the manufacturer's protocol (Roche Diagnostics GmbH). The TUNEL-labeled cell suspension was then incubated with a 1:30 dilution of CD30 antibody (DAKO) for 1 h at 37 °C, washed with PBS and incubated with 1:500 dilution of anti-mouse Alexa Fluor 647 (Molecular Probes) for 1 h at 37 °C. Finally the cell suspension was washed in PBS and analyzed by flow cytometry (TMC MoFlo Cytomation Co.).

Diploid cultures of HES-2 were expanded under serum-free conditions for five passages in the presence of FGF-2 on mouse embryonic fibroblast feeder layers and determined to be CD30^-. Cells were harvested with Cell Dissociation Solution and seeded at 50% confluence in eight-well chamber slides (Nalgene-Nunc International) coated with Matrigel (BD-Biosciences) in the presence of mouse embryonic fibroblast–conditioned medium, as described by Xu et al.¹². The next day the cells were transiently transfected with 2 g of DNA per well of pBOS-EGFP (empty vector control), pBOS-CD30-EGFP or pBOS-CD30 variant-EGFP using Fugene (Roche) at a Fugene-to-DNA ratio of 3:2 according to the manufacturer's protocol. On the second day the transfection efficiency was judged by the number of cells showing GFP fluorescence (15–20% of all cells). The cell cultures were next fixed with 1% paraformaldehyde, and apoptosis was quantified using a commercially available TUNEL assay that labels the DNA of apoptotic cells with fluorescein isothiocyanate (Chemicon). Subsequently the fixed cell preparations were incubated with a 1:30 dilution of CD30 antibody (DAKO) overnight at 37 °C, washed three times for 15 min each with PBS and incubated with a 1:200 dilution of anti-mouse Alexa Fluor 546 (H+L) (Molecular Probes) for 1 h at 37 °C. After another three washes in PBS, nuclei were stained with DAPI (1 g/ml) for 10 min, washed in PBS and mounted with VECTASHIELD. The slides were subsequently observed with a BX-51 Olympus fluorescent microscope and images captured with an Olympus DP-70 digital camera. Transfected cells were identified as cells that showed GFP fluorescence (and red fluorescent CD30 staining in the case of full-length CD30), and the proportion of TUNEL-positive (apoptotic) cells within this population was quantified by scoring a minimum of 500 transfected cells in random fields.

Activation of NF-B signaling in hES cells by CD30 variant expression.

HES-3 cells propagated on serum-free medium supplemented with FGF-2 in the presence of a feeder cell layer were harvested with Cell Dissociation Buffer and seeded at a density of 60% confluence onto 96-well plates coated with Matrigel. The next day the cells were overlaid with 100 l mouse embryo fibroblast conditioned medium. The cells were subsequently transfected with 100 ng of plasmid DNA consisting of 33 ng PCS-2 lacZ with the cytomegalovirus 1E94 promoter driving -galactosidase, 33 ng NF-B-luciferase reporter (5 B-luciferase construct from Stratagene) and 33 ng of either BOS-empty vector or 33 ng of BOS-CD30 variant, using 15 l Fugene-6 per well. The next day the medium was changed again and cells grown for an additional 2 d. Then cells were lysed using 100 l of passive lysis buffer (Promega) according to the manufacturer's instructions. To assess -galactosidase activity (for normalization of well-to-well differences in transfection efficiency) 40 l of lysate was used; another 40 l was used for measurement of luciferase activity using the Promega luciferase assay system. Each condition was performed in quintuplicate on each plate, and three separate experiments were performed. The NF-B-luciferase reporter activities were divided by the -galactosidase activities and normalized relative to the empty vector control.

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