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Generation of Chromosome-Specific Monoclonal Antibodies Using In Vitro–Differentiated Transchromosomic Mouse Embryonic Stem Cells
http://www.100md.com 《干细胞学杂志》
     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@kirin.co.jp

    ABSTRACT

    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 . 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 . 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 . 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 .

    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 . In this context, we have shown the utilization of an in vitro neuronal or cardio differentiation system of hChr.21 TC-ESCs 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 , 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. 1).

    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 .

    Here, we have re-evaluated the utility of "somatic cell hybrid immunization procedure" 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 . We also described the successful purification and identification of the gene for h4-neural1 antigen with immunoaffinity chromatography.

    MATERIALS AND METHODS

    Strategy for the Generation of MoAbs Against Human Antigens by Using In Vitro dTC-ESCs

    The outline of our procedure is illustrated in Figure 1. 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 . 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 . 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 . mESCs and pluripotent cells of the inner cell mass of blastocysts express the embryonic marker, SSEA-1 . The neuroectodermal cells, or NPCs within the neural tube and neurally committed, differentiated ESCs, are characterized by the expression of nestin . The formation of more differentiated neural cells is identified by the expression of TUJ1 (?-tubulin type III ). Figure 2A 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 .

    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. 2B) 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. 2C) 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. 2D). 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/, designated as h4-neural1) that binds to E14/hChr.4-derived CDM 5–6 cells but not to E14/wt (Fig. 3A).

    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, 3B). 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 1). On the other hand, EC cell lines NT-2/D1, Tera-2, NEC-8, and NEC-14 were all positive for h4-neural1 (Table 1). 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 1).

    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 . 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. 4A). 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. 4B). Thus, the staining profile of undifferentiated and differentiated NT2/D1 cells with h4-neural1 was similar to an NPC marker, nestin.

    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 1) 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. 5A). These two products were confirmed to be cell-surface molecules by streptavidin staining of proteins purified from biotinylated NEC8 cells (Fig. 5B). 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 . 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 , whereas COS-7/mock vector transfectants and parental COS-7 cells were negative (Fig. 5C). From these results, we concluded that the gene encoding the h4-neural1 antigen is CD133 (prominin 1).

    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. 2) 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/, designated as h11-neural1) that had the staining profile similar to that with an anti-nestin MoAb up to day 10 (Fig. 6). 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. 6). 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 1).

    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

    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.

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