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First published online August 4, 2005
Stem Cells Vol. 23 No. 10 November 2005, pp. 1479 -1488
doi:10.1634/stemcells.2004-0369; www.StemCells.com
© 2005 AlphaMed Press

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Generation of Chromosome-Specific Monoclonal Antibodies Using In Vitro–Differentiated Transchromosomic Mouse Embryonic Stem Cells

Ayano Yanagisawaa,b, Chisato Endoa, Katsuya Okawaa, Shingo Shitaraa,b, Hiroyuki Kugohb, Makoto Kakitania, Mitsuo Oshimurab, Kazuma Tomizukaa,b

a Pharmaceutical Research Laboratories, Pharmaceutical Division, Kirin Brewery Co., Ltd., Takasaki, Gunma, Japan;
b Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medicine, Tottori University, Yonago, Tottori, Japan

Key Words. Monoclonal antibody • Embryonic stem cell • Human chromosome • Neural cell • CD133

Correspondence: Kazuma Tomizuka, Ph.D., Pharmaceutical Research Laboratory, Pharmaceutical Division, Kirin Brewery Co., Ltd., 3 Miyahara-cho, Takasaki-shi, Gunma 370-1295, Japan. Telephone: 81-27-346-9934; Fax: 81-27-346-1971; e-mail: ktomizuka{at}kirin.co.jp


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Monoclonal antibodies (MoAbs) recognizing lineage- and stage-specific human cell-surface antigens are valuable reagents for the characterization and isolation of various specialized cell populations derived from human embryonic stem cells (hESCs). In this report, we examined the use of in vitro differentiated transchromosomic mouse embryonic stem cells (TC-ESCs) as immunogens to obtain MoAbs against human cell-surface antigens. Immunization of a neural-cell population derived from differentiating human chromosome 4 and 11 TC-ESCs resulted in two chromosome-specific MoAbs, h4-neural1 and h11-neural1, respectively. The staining profiles of differentiated TC-ESCs and human embryonic carcinoma cells with these MoAbs were similar to the expression profile of nestin, a well-characterized intracellular marker for neural progenitor cells. We also described the successful purification and identification of the gene for h4-neural1 antigen (CD133, 4p15.32) with immunoaffinity chromatography. This procedure may have significant utility in generating MoAbs useful for understanding the mechanism that regulates the in vitro differentiation of hESCs.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The use of monoclonal antibodies (MoAbs) recognizing human cell-surface antigens enables the identification, isolation, and analysis of antigen molecules, thereby revealing their roles in physiological processes. They have also received widespread attention as reagents for the characterization and isolation of specific cell populations expressing lineage- and stage-specific cell-surface markers. However, raising antibodies against membrane proteins is often difficult if conventional protein immunization strategies are used. This is due to the difficulty in the preparation of purified protein antigens with native configuration, and therefore, a substantial fraction of currently available MoAbs against human cell-surface markers has been generated by xenoimmunization with whole human cells. Another practical problem is that the targeted cells in some cases (e.g., cells from early embryos) are scarce, with limited availability for immunization and hybridoma screening. Furthermore, whole cell immunization often results in a biased immune response to a limited number of immunedominant molecules, which prevents the response to a variety of antigens with relatively low immunogenicity.

About 25 years ago, several studies used murine-human somatic cell hybrids with one or a limited number of human chromosomes as immunogens to obtain antisera and MoAbs in mice syngeneic to the parental cells [1–3]. The antibodies generated were specific for a limited number of human antigens coded by a particular human chromosome. Although this "somatic cell hybrid immunization" procedure can facilitate the production of MoAbs against a wide range of human cell-surface antigens, it had not been extensively used because the generation of hybrids retaining a specified human chromosome was laborious and time-consuming.

We previously generated a library comprising approximately 700 human/mouse A9 monochromosomal hybrids, each of which contained single, neor-tagged human chromosome (hChr.) or human chromosome fragment (hCF) derived from normal fibroblasts [4]. The hybrids selected from this library were used as donors for microcell-mediated chromosome transfer (MMCT) to generate a panel of microcell-hybrid mouse embryonic stem cells (mESCs), designated as transchromosomic embryonic stem cells (TC-ESCs), containing an hChr. or hCF derived from hChr.2, 4, 6, 7, 11, 14, 21, or 22 [4–6]. Using these TC-ESCs, we also demonstrated the production of chimeric mice retaining the transferred chromosomes, and the tissue-specific expression of various human genes in adult chimeric tissues [4–6].

Pluripotent ESCs possess an unlimited proliferative capacity, and recent advancements in the studies of in vitro differentiation of mESCs have allowed us to enrich various cell populations of embryos and adults, including hematopoietic cells, hepatocytes, germ cells, and melanocytes [7–9]. In this context, we have shown the utilization of an in vitro neuronal or cardio differentiation system of hChr.21 TC-ESCs [10, 11] as a model for the early developmental process of Down’s syndrome. Thus, TC-ESCs can differentiate to various types of cell populations expressing transferred human genes in vivo and in vitro, which makes differentiated TC-ESCs (dTC-ESCs) attractive candidates for use in immunization to obtain specific antibodies against developmentally regulated, stage- and lineage-specific human antigens. Because information about the chromosomal loci and nucleotide sequences of almost all human genes was provided by the Human Genome Project [12, 13], the chromosome-specific MoAbs yielded by immunization of dTC-ESCs may be useful for the identification of antigen genes in a limited number of candidates residing on a specified human chromosome (Fig. 1Go).



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  		Figure 1. The strategy for transchromosomic embryonic stem cell (TC-ESC) immunization. Expansion and differentiation step, immunization step, and screening step are included to obtain human chromosome-specific monoclonal antibodies. TC-ESC library refers to mouse embryonic stem cell lines containing a human chromosome or its fragment [6].

 
Here, we have re-evaluated the utility of "somatic cell hybrid immunization procedure" [1–3] by using in vitro dTC-ESCs as immunogens to generate MoAbs against human cell-surface antigens. In test cases, we attempted to produce MoAbs against human neural progenitor cell (NPC) antigen(s) by using a chemically defined medium (CDM) culture for hChr.4 and hChr.11 dTC-ESCs. Two resulting MoAbs, h4-neural1 and h11-neural1, were shown to specifically recognize the surface of hChr.4 and hChr.11 dTC-ESCs cultured in CDM for 5–6 days, respectively. Particularly, the staining profiles of dTC-ESCs and human embryonal carcinoma (EC) cells with h4-neural1 were similar to the expression profile of nestin, a well-characterized intracellular marker for NPCs [14, 15]. We also described the successful purification and identification of the gene for h4-neural1 antigen with immunoaffinity chromatography.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Culture of Cell Lines
mESCs were maintained following standard techniques by routine culture on mitotically arrested primary embryonic fibroblasts. The ESC culture medium used was Dulbecco’s modified Eagle’s medium (DMEM) (Nissui Pharmaceutical Co. Ltd., Tokyo, http://www.nissui-pharm.co.jp) supplemented with 10% fetal bovine serum (FBS) (Invitrogen Corporation, Carlsbad, CA, http://www.invitrogen.com), ß-2-mercaptoethanol (Sigma, St. Louis, http://www.sigmaaldrich.com), nonessential amino acids (Invitrogen Corporation), and 10 ng/ml leukemia inhibitory factor (Chemicon International, Temecula, CA, http://www.chemicon.com).

The introduction of hChr.4 and hChr.11 into mESCs with MMCT was performed as described previously [4]. Briefly, recipient mESCs (E14) were fused to microcell mixtures prepared from donor A9 cells containing an hChr.4 or hChr.11 tagged with drug-resistant gene (G418r for hChr.4, Hygr for hChr.11). The fused cells were cultured under the nonselective condition for 24 hours and then subjected to the drug selection for 8–9 days. We isolated G418 (hChr.4)– or hygromycin (hChr.11)–resistant colonies and screened them by genomic polymerase chain reaction (PCR) to verify the retention of a transferred chromosome. Retention of each human chromosome in E14/hChr.4 and E14/ hChr.11 clone was confirmed by genomic PCR analyses using DNA markers for hChr.4 (D4S412, HD, D4S418, KIT, D4S395, IL-2, D4S422, FABP2, D4S413, F11, D4S426) and hChr.11 (H19, KVLQT1, P57KIP2, SMS4, TSSC6, H-RAS, NAP2, CARS, D11S133), respectively.

Human cell lines SK-N-MC, Hs 683, SW-1088, MRC-5, NT2/D1, and Tera-2 were obtained from American Type Culture Collection (Manassas, VA, http://www.atcc.org), and HMV-2, NEC-8, and NEC-14 were obtained from the RIKEN BioResource Center (Ibaraki, Japan, http://www.brc.riken.jp/inf/en). SK-N-MC, Hs 683, SW-1088, MRC-5, NT2/D1, and Tera-2 were cultured in DMEM, and MRC-5, U-937, K-562, Ramos, NEC-8, and NEC-14 were cultured in RPMI medium, both supplemented with 10% FBS. HFL-1 was cultured in F-12 supplemented with 15% FBS.

Fluorescence In Situ Hybridization Analysis
Preparation of chromosome samples and fluorescence in situ hybridization (FISH) analysis were carried out by standard methods [4]. Images were captured using a fluorescence microscope (Nikon Corporation, Tokyo, http://www.nikon.com) equipped with a photometric CCD camera and processed using the Cytovision Probe System (Applied Imaging Corporation, San Jose, CA, http://www.aicorp.com). The probes were digoxigenin (Boehringer Mannheim, Mannheim, Germany, http://www.boehringer.com)–labeled human COT-1 DNA (Invitrogen Corporation). Digoxigenin-labeled probes were detected with antidigoxigenin-rhodamine (Boehringer). The chromosomes were counter-stained with DAPI (4,6-diamidino-2-phenylindole) (Sigma).

In Vitro Differentiation Culture of ESCs
Neuronal differentiation of mESCs was performed as described by Johansson et al. [16, 17]. Prior to differentiation, ESCs were trypsinized to single cells by gentle digestion with 0.25% trypsin, and feeder cells were removed by passaging the culture onto 0.1% gelatin-coated dishes. ESCs were then washed in CDM (Iscove’s modified Dulbecco’s medium/F12 1:1 [Invitrogen Corporation], 5 mg/ml bovine serum albumin [Sigma], x1 lipid concentrate [x100 mixture of chemically defined lipid concentrate; Invitrogen Corporation], penicillin/streptomycin [Invitrogen Corporation], 150 µg/ml transferrin [Sigma], 7 µg/ml insulin [Sigma], and 450 µM monothioglycerol [Sigma]) and resuspended with 2 x 105 cells per ml in CDM. They were cultured in suspension culture flasks (Sumitomo Bakelite Co. Ltd., Tokyo, http://www.sumibe.co.jp). CDM was prepared within a week before use. The differentiation properties were determined by fluorescence-activated cell sorting (FACS) analysis with anti-SSEA-1, anti-nestin, and anti-ß-tubulin type III MoAbs.

Reverse Transcription–PCR for NCAM1
Total RNA was prepared from wild-type E14 (E14/wt), E14/ hChr.11 on CDM assay at days 0, 5, and 12 using ISOGEN (Nippon Gene, Tokyo, http://www.nippongene.com). RNA was reverse-transcribed using 2.5 µg total RNA in 20 µl reaction with SuperScript RTase (Invitrogen Corporation) and oligo(dT) primer. One microliter of RNase H (Invitrogen Corporation) was added to each reaction tube, and tubes were incubated for 20 minutes at 37°C, then diluted with 30 µl water before reverse transcription–PCR (RT-PCR) analysis. cDNA was amplified by PCR, using the following primer pairs: mNCAM1 forward, 5'-AGTCGCTGGGCGAAGAATCC-3'; mNCAM1 reverse, 5'-TCTTGGACTCAGATGGCTGC-3'; Tm = 57.4°C, 171 bp product and hNCAM1 forward, 5'-GGAGAGCAGTT-GGTGAAGAAGTA-3'; hNCAM1 reverse, 5'-GTGCACT-GGGTTCCCCTTGG-3'; Tm = 57.9°C, 204 bp product. RNA integrity was controlled by cDNA amplification generated by the murine GAPDH gene.

Flow Cytometry
Before immunostaining, cells were dissociated by incubation for 10 minutes at 37°C in Ca/Mg-free phosphate-buffered saline (PBS) with 0.2% EDTA-2Na. For ESC-derived cells, enzyme-free cell dissociation solution (Sigma) was used for dissociation. Cell suspensions were washed in staining medium containing Ca/Mg-free PBS, 5% serum, 0.1% sodium azide, and 1 mM EDTA. The diluted (1:100) SSEA-1 (clone MC-480, mouse IgM; Chemicon International), (1:500) nestin (clone Rat 401, mouse IgG1; BD Biosciences Pharmingen, San Jose, CA, http://www.bdbiosciences.com/pharmingen), and (1:500) ß-tubulin type III (clone TUJ1, mouse IgG2a; CRP Inc., Denver, PA, http://www.crpinc.com) antibodies were used for primary antibodies. Before the immunostaining of intracellular marker nestin and TUJ1, cells were fixed in 4% paraformaldehyde for 10 minutes and permealized in acetone/ethanol (1:1) for 1 minute. The binding of antibodies to cells was traced with (1:50) fluorescein isothiocyanate–conjugated F(ab’)2 rabbit anti-mouse immunoglobulins (DakoCytomation, Carpinteria, CA, http://www.dakocytomation.us). In addition, (1:2) hybridoma supernatant in hybridoma screening and (1:50) phycoerythrin-conjugated AC133/1 (Miltenyi Biotech, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com) were used for primary antibodies, and dead cells in the preparation were excluded from analysis by propidium iodide staining. FACS analysis was performed using a FACS Calibur flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com). Data analysis was performed using Cell Quest software (Becton, Dickinson and Company).

Hybridoma Production
Six-week-old female C57BL/6J mice (CLEA Japan Inc., Tokyo, http://www.clea-japan.com) were immunized subcutaneously in a series of five weekly immunizations. Each immunization contained 5 x 106 TC-ESCs cultured in CDM for 5–6 days. A final intravenous injection of 1 x 106 cells was given 3 days before fusion. Immunized animals were sacrificed, and the spleens were removed under sterile conditions. A lymphocyte suspension was prepared and fused to SP2/O-Ag14 mouse myeloma cells. Fused cells were plated on 96-well plates in DMEM supplemented with 10% heat-inactivated FBS (HyClone, Logan, UT, http://www.hyclone.com) and 2 mM L-Glutamine (Invitrogen Corporation) and placed in a 37°C incubator with 6.5% CO2. Hybridoma supernatants were screened on dTC-ESCs using flow cytometric analysis. Selected antibodies used later were derived from cells subcloned by limiting dilution. The isotypes of new antibodies were determined using Isostrip (Roche, Basel, Switzerland, http://www.roche.com). An h4-neural1 antibody column and anti–trinitrophenyl (TNP) column were prepared by the following method: pure h4-neural1 was prepared from a protein G affinity column (Amersham, Piscat-away, NJ, http://www.amersham.com) and linked to an N-hydroxysuccinimide (NHS)–activated column (Amersham).

In Vitro Differentiation of NT2/D1 Cells
Differentiation culture of NT2/D1 cells was performed as previously described by Andrews [18]. NT2/D1 cells were trypsinized to single-cell suspension, and 1 x 106 cells were plated on a 100-mm cell culture–treated plate in DMEM supplemented with 10% FBS and 10 µM all-trans retinoic acid (ATRA) (Sigma).

Purification of h4-neural1 Antigen
h4-neural1 antigen was isolated from NEC-8 cells. The cells were lysed in buffer containing 2% NP40 for 30 minutes at 4°C. Cell nuclei and debris were removed by centrifugation at 10,000g for 10 minutes at 4°C. The lysate was loaded onto a 1-ml anti-TNP column as an h4-neural1 isotype control column before loading onto a 1-ml h4-neural1 affinity column equilibrated in PBS. The column was then washed extensively with PBS, and the antigen was eluted in elution buffer. The pH was immediately adjusted to neutral with glysin-HCl buffer (pH 9.0).

Mass spectrometric identification of proteins was performed as previously described [19]. Briefly, after SDS-PAGE, proteins were visualized by silver staining and excised separately from gels, followed by in-gel digestion with trypsin (Promega Corporation, Madison, WI, http://www.promega.com) in a buffer containing 50 mM ammonium bicarbonate (pH 8.0) and 2% acetonitrile overnight at 37°C. Molecular mass analyses of triptic peptides were performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) using an ultraflex TOF/TOF (Bruker Daltonics, Billerica, MA, http://www.bdal.de). Proteins were identified by comparison of the molecular weights determined by MALDI-TOF/MS and theoretical peptide masses from the proteins registered in NCBInr [19].

Expression of Human AC133-1 and AC133-2 Antigen in Transfected COS-7 Cells
Total RNA was isolated from Tera-2 using ISOGEN (Nippon Gene). RT-PCR was performed as described above using forward primer 5'- TTGGAGGATCTTGCTAGCTATGGCCCTCGT-3', and reverse primer 5'-GCCTTGGTGCCCGCCTGAGTAACTA-3'. PCR was performed using KOD+ Taq polymerase (TOYOBO Co., Ltd., Osaka, Japan, http://www.toyobo.co.jp) as follows: 94°C for 5 minutes followed by 30 cycles of 94°C for 30 seconds and 70°C for 3 minutes. A fragment of AC133-2 [20] was cloned into pGEM-T-Easy vector (Promega Corporation), and a NotI fragment of AC133-2 was introduced into the EcoRV site of the pLP-IRES neo-expression vector (BD Biosciences Clontech, Mountain View, CA, http://www.clontech.com/clontech). For the expression of human AC133-1, an NheI-EcoRV fragment of AC133-1 cloned into the pGEM-T-Easy vector was replaced in the AC133-2 expression vector.

Subconfluent COS-7 cells in a 35-mm cell culture dish were transfected with 4 µg of vector DNA by Lipofection (Lipofectamine 2000; Invitrogen Corporation) and incubated for 36 hours before flow cytometric analysis. Transfected COS-7 cells were stained with 1 µg MoAb per 4 x 106 cells and analyzed with a FACS Calibur flow cytometer.


   RESULTS
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Strategy for the Generation of MoAbs Against Human Antigens by Using In Vitro dTC-ESCs
The outline of our procedure is illustrated in Figure 1Go. To demonstrate the utility of this procedure, we attempted to generate MoAbs against human neural progenitor cell (NPC) antigen(s) by using a CDM culture for the differentiation of TC-ESCs. It has been shown that the suspension culture of mESCs in CDM results in the enhanced survival and proliferation of ESC-derived NPCs, thereby allowing for the enrichment of cell populations exhibiting neuroepithelial morphology and expressing nestin, a well-characterized NPC marker [14, 15]. Two TC-ESC lines, an E14 ESC line retaining a human chromosome 4 (E14/hChr.4) and chromosome 11 (E14/hChr.11), were examined in this study [6]. Chromosome numbers of E14/hChr.4 and E14/hChr.11 revealed that more than 90% of the metaphase spreads contained 41 chromosomes consisting of 40 normal mouse chromosomes and an additional human chromosome. Both hChr.4 and hChr.11 in the spreads are apparently intact, which is consistent with the result that all the tested human DNA markers for each chromosome (11 markers for hChr.4, 9 markers for hChr.11) were detected in each TC-ESC by genomic PCR analysis (data not shown).

Enrichment of Nestin-Positive NPCs in dTC-ESCs
The establishment of a neural-cell lineage has been well characterized at the level of stage-specific marker expression [21]. mESCs and pluripotent cells of the inner cell mass of blastocysts express the embryonic marker, SSEA-1 [22]. The neuroectodermal cells, or NPCs within the neural tube and neurally committed, differentiated ESCs, are characterized by the expression of nestin [14, 15, 23]. The formation of more differentiated neural cells is identified by the expression of TUJ1 (ß-tubulin type III [24]). Figure 2AGo shows the expression profile of these three markers in differentiating wild-type E14 (E14/wt) ESCs by CDM culture. The nestin-positive, NPC population was apparent 3 days after replacing the culture medium with CDM, and showed a maximum expression at day 10 in association with the decrease of the SSEA-1–positive cell fraction. On the other hand, the TUJ1-positive cell population first appeared from day 7 and reached a plateau at day 12. These differentiation properties are consistent with those observed in previous studies using a similar CDM culture condition [25].



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  		Figure 2. Percentage of cells expressing neuronal markers (A–C) and human NCAM1 expression (D) in CDM culture. E14/wt (A), E14/hChr.4 (B), and E14/hChr.11 (C) were cultured in CDM for the indicated times and stained with anti-SSEA-1, nestin, and TUJ1 antibodies. The percentage of positive cells (gate b: determined by the intensity of less than 1% of cells stained by negative control antibodies) is calculated by flow cytometry in gate a, which contains living cells until fixation. (D): Transcripts from E14/hChr.11 were semi-quantified by RT-PCR using primers specific for human and mouse NCAM1. Abbreviations: CDM, chemically defined medium; FITC, fluorescein isothiocyanate.

 
The CDM differentiation of E14/wt, E14/hChr.4, and E14/ hChr.11 cells resulted in the formation of floating, sphere-like colonies at day 3 after medium replacement. The analysis of differentiating E14/hChr.4 cells revealed a marker expression profile (Fig. 2BGo) similar to that of E14/wt cells, suggesting that the introduced hChr.4 did not affect the CDM differentiation of mESCs. On the other hand, although the overall marker expression profile in differentiating E14/hChr.11 cells (Fig. 2CGo) was also similar to that of E14/wt cells, a rapid decrease in the SSEA-1–positive cell fraction and a low percentage of the nestin-positive, NPC fraction were observed only in E14/hChr.11 cells. This suggests that the introduction of hChr.11 has some effect on the CDM differentiation of mESCs. Taken together, the enriched, nestin-positive NPC population can be obtained by the CDM differentiation of TC-ESCs for 5–6 days without the inclusion of the TUJI-positive, differentiated neural-cell population. We therefore used this condition to prepare immunogens for generating MoAbs recognizing TC-ESC-derived NPCs.

To demonstrate the human gene expression in CDM differentiation of TC-ESCs, we performed RT-PCR analysis for a differentiated neural-cell marker, NCAM1 (11q23.1) in E14/hChr.11. As a result, both human and mouse NCAM1 transcripts are increased during this differentiation (Fig. 2DGo). The retention of transferred human chromosomes was also confirmed in differentiated (day 12) E14/hChr.4 (98%) and E14/hChr.11 (99%) cells by FISH analysis using a human-specific COT-1 probe.

A Human Antigen-Specific MoAb (h4-neural1) Obtained from E14/hChr.4 Immunization
The differentiating E14/hChr.4 cells cultured in CDM for 5–6 days (CDM 5–6 cells) were injected into C57BL/6J mice subcutaneously at a weekly interval (5 x 106 cells/injection). The inclusion of more than 50% of nestin-positive cells in CDM 5–6 cells was confirmed at each immunization point. After the fourth immunization, specific reactivity to CDM 5–6 cells was detected in 2 of 5 immunized mice with flow cytometry. These two mice were treated with a final intravenous injection and used for hybridoma production. Hybridomas secreting antibodies that bound to surface antigens of E14/hChr.4-derived CDM 5–6 cells were selected by FACS analysis. This primary screening of 461 hybridoma supernatants revealed that 13 recognized cell-surface antigens of E14/hChr.4 -derived CDM5–6 cells. Secondary FACS screening was carried out using E14/wt-derived CDM 5–6 cells, and we identified one hybridoma clone secreting an MoAb (IgG1/{kappa}, designated as h4-neural1) that binds to E14/hChr.4-derived CDM 5–6 cells but not to E14/wt (Fig. 3AGo).



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  		Figure 3. FACS analyses of h4-neural1 antigen expression in E14/ wt and E14/hChr.4 in CDM culture. (A): E14/wt and E14/hChr.4 were cultured in CDM and stained with h4-neural1 (bold gray line) and control mIgG1 (filled in black). (B): E14/hChr.4 were fixed and stained with anti-nestin antibodies (bold gray line) and control mIgG1 (filled in black). Abbreviations: CDM, chemically defined medium; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate.

 
Further analysis demonstrated that the staining profile of differentiating E14/hChr.4 cells with h4-neural1 resembles that with nestin (Figs. 3A, 3BGo). Undifferentiated E14/hChr.4 cells were found to be negative for h4-neural1. The cell population positive for h4-neural1 was apparent 3 days after replacing the culture medium with CDM, and peaked at day 10. The differentiating E14/wt cells were negative throughout the course of CDM differentiation.

Binding Specificity of h4-neural1 to Various Human Cells
FACS analyses using three types of lineage-restricted neural-cell lines, SK-N-MC (neuroblastoma), SW-1088 (astrocytoma), and Hs 683 (glioma), showed that they had no reactivity to h4-neural1 (Table 1Go). On the other hand, EC cell lines NT-2/D1, Tera-2, NEC-8, and NEC-14 were all positive for h4-neural1 (Table 1Go). It should be noted that these four human EC cell lines were also positive for nestin (data not shown). Three cell lines for hematopoietic cell lineage, Ramos (Burkitt lymphoma), K-562 (erythroleukemia), and U937 (histiocytic lymphoma), and two primary fibroblasts, HFL-1 and MRC-5, were negative for h4-neural1 (Table 1Go).


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  		Table 1. h4-neural1 and h11-neural1 staining profile
 
It has been shown that retinoic acid induces the neural differentiation of human EC cell line NT2/D1 [18]. We then examined the h4-neural1 reactivity in differentiating NT2/D1 cells after treatment with ATRA. In the time course of in vitro neural differentiation of NT2/D1 cells, we observed a gradual decrease in the percentage of the nestin-positive cell fraction after day 3 (Fig. 4AGo). As described above, undifferentiated NT2/D1 cells had reactivity to h4-neural1, whereas neuronal cells derived from the NT2/D1 cells were found to have downregulated h4-neural1 antigen expression (Fig. 4BGo). Thus, the staining profile of undifferentiated and differentiated NT2/D1 cells with h4-neural1 was similar to an NPC marker, nestin.



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  		Figure 4. FACS analyses of h4-neural1 antigen expressions in NT2/ D1 ATRA culture. NT2/D1 was cultured in medium containing ATRA and stained with anti-nestin antibodies (A) and h4-neural1 (B). Abbreviations: ATRA, all-trans retinoic acid; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; PE, phycoerythrin.

 
Identification of a Gene Encoding h4-neural1 Antigen
Because FACS analyses showed better reactivity of h4-neural1 to NEC8 (Table 1Go) cells than to E14/hChr.4-derived CDM 5–6 cells, we used NEC8 cells as a source for the purification of h4-neural1 antigen. An affinity column of h4-neural1 was used to purify its antigen from a solubilized membrane fraction of NEC8 cells. Examination of the purification product by SDS-PAGE electrophoresis showed 200-kDa and 120-kDa components specific for h4-neural1 (Fig. 5AGo). These two products were confirmed to be cell-surface molecules by streptavidin staining of proteins purified from biotinylated NEC8 cells (Fig. 5BGo). Mass spectrometric analyses were performed as described in Materials and Methods, and the results indicated that the 120-kDa product is CD133 (prominin 1, ~120-kDa, 4p15.32) and the 200-kDa product is DOCK7 (dedicator of cytokinesis 7, ~180-kDa, 1p31.3). As h4-neural1 specifically recognizes the surface of differentiated E14/ hChr.4 cells, it was supposed that the antigen molecule is encoded by the gene residing on hChr.4. We therefore selected the CD133 gene for further studies. Human CD133 has two isoforms (AC133-1, AC133-2) and their differential tissue distribution was reported previously [20]. FACS analyses of COS-7 cells transiently transfected with the cDNA of these two CD133 isoforms showed that both transfectants were positive for h4-neural1 and previously reported anti-CD133 MoAb, AC133/1 [26], whereas COS-7/mock vector transfectants and parental COS-7 cells were negative (Fig. 5CGo). From these results, we concluded that the gene encoding the h4-neural1 antigen is CD133 (prominin 1).



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  		Figure 5. Isolation and determination of h4-neural1 antigen from the human embryonal carcinoma cell line. (A): Silver-stained gel of purified h4-neural1 antigen using an IgG1 control column and h4-neural1 column for protein mass analysis (B). Western blotting of purified antigen from a control IgG1 affinity column and h4-neural1 column using AP-conjugated streptavidin. Cell-surface proteins were biotinylated and detected by this procedure. (C): FACS analysis for the binding activity of h4-neural1 to two AC133 isoforms, AC133-1 and AC133-2. Cos-7 cells transfected with intact human AC133-1 and AC133-2 cDNA were stained with h4-neural1 and AC133/1 (bold gray line) or control antibody (filled in gray). Abbreviations: AP, alkaline phosphatase; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; PE, phycoerythrin.

 
A MoAb (h11-neural1) Obtained from the Immunization of E14/hChr.11
The immunization of E14/hChr.11 cells cultured in CDM for 5–6 days (Fig. 2Go) was also carried out as for the experiment with E14/ hChr.4. Two of six immunized mice were sacrificed for hybridoma production, and the primary screening of 935 hybridoma supernatants revealed that five recognized cell-surface antigens of E14/hChr.11-derived CDM 5–6 cells. Secondary FACS screening using E14/wt-derived CDM 5–6 cells identified three hybridoma supernatants that bind to E14/hChr.11-derived CDM 5–6 cells but not to E14/wt. Time-course analyses in CDM differentiation of E14/hChr.11 revealed one MoAb (IgM/{kappa}, designated as h11-neural1) that had the staining profile similar to that with an anti-nestin MoAb up to day 10 (Fig. 6Go). On the other hand, at days 12–14, differentiated E14/hChr.11 remained reactive to h11-neural1, whereas the nestin-positive fraction already disappeared. Another interesting feature in the staining profile with h11-neural1 was the cross-reactivity to the murine antigen expressed in differentiated E14/wt at days 12–14 (Fig. 6Go). The reactivity against human cell-surface antigen(s) was also evidenced by the result that NT2/D1 and G361 were positive for h11-neural1 (Table 1Go).



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  		Figure 6. FACS analysis of h11-neural1 antigen expression in E14/wt and E14/hChr.11 in CDM culture. FACS analysis was performed on E14/wt, E14/hChr.11 using primary antibody h11-neural1 (bold gray line) and control IgM (filled in black) after differentiation. Abbreviations: CDM, chemically defined medium; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Here, we demonstrated the utility of dTC-ESC immunization to obtain MoAbs against human cell-surface antigen(s) by showing the generation of two MoAbs, h4-neural1 and h11-neural1, each of which recognizes antigen(s) expressed on the surface of the NPC population derived from hChr.4 and hChr.11 TC-ESCs, respectively. These MoAbs were able to bind to nestin-positive NPCs derived from TC-ESCs in CDM differentiation, but had no reactivity to wild-type ESC-derived NPCs, indicating that they recognize antigens the expression of which relies on the presence of transferred human chromosomes. The reactivity of these MoAbs to human antigens was evidenced as they also bound to the cell surface of human EC cells.

The presence of the intermediate filament protein, nestin, has been the predominant marker used to describe self-renewing, multipotent NPCs that give rise to neurons, astrocytes, and oligodendrocytes in the central nervous system [14, 15]. However, antibodies against this intracellular protein cannot be used to isolate living NPCs. The staining profile with h4-neural1 in dTC-ESCs resembled that with an anti-nestin antibody throughout the course of CDM differentiation. In the neural differentiation of human EC cells with retinoic acids, the staining profile of h4-neural1 was also consistent with that of an anti-nestin. It should be noted that undifferentiated human EC cells are positive for both h4-neural1 and anti-nestin in contrast to the result in undifferentiated TC-ESCs. Taken together, these results suggest that h4-neural1 could be useful to isolate nestin-positive, living NPCs.

Antibodies are valuable tools that allow us to isolate and purify antigen proteins. In addition, recent advances in mass spectrometry combined with progress in the Human Genome Project [12, 13] have facilitated the rapid identification of these proteins by peptide mass fingerprinting. In this study, purification with an h4-neural1 (IgG1) immunoaffinity column resulted in 120-kDa and 200-kDa products in the solubilized membrane fraction of h4-neural1–positive NEC8 cells, and mass spectrometric analysis showed that each product is CD133 and DOCK7, respectively. It was anticipated that immunization of hChr.4 dTC-ESCs could yield MoAbs specific for the antigens coded by hChr.4. As expected, CD133 was shown to be a gene encoding the h4-neural1 antigen with the result that transfection of the human CD133 expression vector conferred reactivity to h4-neural1 on h4-neural1–negative COS-7 cells. Although the fact that human DOCK7 protein was copurified with CD133 (Fig. 5Go) presumably reflects the interaction of DOCK7 with h4-neural1 or CD133, we have not carried out further examinations on these possibilities in the present study. The use of chromosome-specific MoAbs obtained by TC-ESC immunization has an advantage over the use of MoAbs by whole-cell immunization in identifying antigen molecules, because we can focus on the search for only a limited number of candidate gene sequences residing on the predetermined human chromosome. This may help to identify a true gene for antigens among nonspecific signals. An attempt to identify antigen(s) for h11-neural1 (IgM) is now under way, but it might be more difficult than the case using IgG MoAbs because the IgM MoAb has relatively low affinity and specificity to antigen(s) when compared with IgG.

Human CD133 antigen, also known as AC133, was a recently identified cell-surface marker for hematopoietic stem cells and neural stem/progenitor cells with the ability of neurosphere formation, self-renewal, and multilineage differentiation at the single-cell level [26, 27]. Examination of the binding specificity of h4-neural1 to various cell types, including COS-7/CD133 transfectants deglycosilated with tunicamycin and human cord blood cells (data not shown), revealed that h4-neural1 has similar characteristics to previously described anti-human CD133 MoAb (AC133/1) [26]. Although the expression of the CD133 marker on undifferentiated hESCs has been reported by several groups [28–30], its expression in differentiated hESCs has not been described to date. The above report [30] also described that CD133 is expressed only by 50%–60% of the hESCs in culture, whereas other embryonic markers such as SSEA-4, TRA-1-60, and TRA-1-81 are expressed by 70%–100% of the population. Therefore, h4-neural1-negative, undifferentiated TC-ESCs (Figs. 2BGo, 3AGo) presumably represent the CD133-negative fraction [30] of hESCs. As hChr.4 TC-ESCs contain an apparently intact hChr.4 and entire CD133 locus, it can be expected that the expression profile of human CD133 in hChr.4 dTC-ESCs may reflect that in differentiating hESCs. Thus, our present data showing the behavior of human CD133 on hChr.4 dTC-ESCs in CDM culture may provide insights regarding the mechanism that regulates the in vitro differentiation of hESCs.

The differentiation capacity of mESCs in vitro has been studied extensively over the past 20 years, and many technologies pioneered in the mouse are being assessed for their efficacy with human and primate ESCs. In this context, we believe that this procedure using dTC-ESCs as immunogens is an important addition to the currently available repertoire of techniques for generating MoAbs against human cell-surface antigens. Although optimizing the protocol for dTC-ESC immunization (e.g., injection route, adjuvant, mouse strain) is clearly the next step for more efficient hybridoma production, systematic screening of MoAbs against TC-ESCs differentiated in various conditions should provide a panel of MoAbs, which may be useful to generate a greater and more in depth understanding of human and mouse ESC biology.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We thank Wakako Yamada for the anti-mCD133 antibody, Naoko Tago for technical assistance, and Hitoshi Yoshida for the transfer of hybridoma procedures. We are grateful to Dr. Motonobu Kato, Dr. Yasuaki Shirayoshi, Dr. Mitsuo Nishikawa, and Dr. Shin-Ichi Hayashi for technical advice and valuable comments. These studies were supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

DISCLOSURES
The authors indicate no potential conflicts of interest.


   REFERENCES
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received December 24, 2004; accepted for publication May 23, 2005.




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