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EMBRYONIC STEM CELLS |
aDepartment of Immunogenetics, Graduate School of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan;
bLaboratory of Embryonic Stem Cell Research, Stem Cell Research Center, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan;
cLaboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, University of Tokyo, Tokyo, Japan;
dDepartment of Development and Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
Key Words. Dendritic cells • Embryonic stem cells • Cell differentiation • Cell therapy
Correspondence: Satoru Senju, M.D., Ph.D., Department of Immunogenetics, Graduate School of Medical and Pharmaceutical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556, Japan. Telephone: 81-96-373-5313; Fax: 81-96-373-5314; e-mail: senjusat{at}gpo.kumamoto-u.ac.jp
Received on April 30, 2007;
accepted for publication on July 27, 2007.
First published online in STEM CELLS EXPRESS August 9, 2007.
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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Disclosure of potential conflicts of interest is found at the end of this article.
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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In the present study, looking toward future clinical application of ES-DC technology, a method was developed to generate ES-DC from human ES cells. The morphology and the results of functional and flow cytometric analyses indicate that human ES-DC possess the characteristic features of DC. cDNA microarray analysis revealed that the change of gene expression profile during generation and maturation of human ES-DC partially mimics that of monocyte-derived DC (Mo-DC). The currently established method was also applicable to cynomolgus monkey (Macaca fascicularis) ES cells.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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(TNF-), and soluble CD40-ligand were purchased from Peprotech (London, http://www.peprotech.com). Lipopolysaccharide (LPS) from Escherichia coli and OK-432 were purchased from Sigma-Aldrich (St. Louis, http://www.sigmaaldrich.com) and Chugai Pharmaceutical (Tokyo, http://www.chugai-pharm.co.jp/hc/chugai_top_en.jsp), respectively.
Induction of Differentiation of ES Cells into ES-DC
The procedure for differentiation culture was composed of three steps (Fig. 1 A). Step 1 was as follows: undifferentiated ES cells maintained on PEF were rinsed with phosphate-buffered saline (PBS) and treated with dissociation solution containing 1 mg/ml collagenase, 0.25% trypsin, and 20% knockout serum replacement (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) in PBS [10] and cultured on OP9 feeder cell layers in minimum essential medium- supplemented with 20% fetal calf serum (FCS) and 2-mercaptoethanol (50 µM). Culture of cells was continued for 14–18 days with human ES cells and for 11–13 days with cynomolgus monkey ES cells, and the medium was changed once every 3 days. At the end of this step, the cells were rinsed with PBS, treated with trypsin-EDTA (PBS containing 0.25% trypsin and 1 mM EDTA) for 30–40 minutes, and recovered. After resuspension in culture medium, the cells were plated onto culture dishes and incubated for 2–4 hours. Thereafter, floating or weakly adherent cells were recovered by pipetting, and any firmly adherent cells were discarded. Step 2 was as follows: after being passaged through nylon mesh (Cell Strainer 100 µm; BD Biosciences, Bedford, MA, http://www.bdbiosciences.com), cells recovered from one 90-mm dish were plated in two dishes with freshly prepared OP9 feeder layers. On the following day, the culture medium was exchanged with a medium containing GM-CSF (100 ng/ml) and M-CSF (50 ng/ml). The culture was continued for 7–10 days, depending on the propagation of floating cells on the feeder layers. Step 3 was as follows: ES cell-derived floating cells were recovered by pipetting; resuspended in RPMI 1640 medium containing 10% FCS, GM-CSF (100 ng/ml), and IL-4 (10 ng/ml); and cultured in Petri dishes (3–5 x 105 cells per dish) without a feeder layer (Locus, Tokyo). To induce maturation, IL-4 (10 ng/ml), TNF- (10 ng/ml), LPS (3 µg/ml), and, in some experiments, soluble CD40-ligand (20 ng/ml) or OK-432 (10 µg/ml) were simultaneously added on day 3 or 5 of this step, and the culture was continued for an additional 2–3 days. Differentiating cells were microscopically analyzed on an inverted microscope (IX70; Olympus, Tokyo, http://www.olympus-global.com).
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Enzyme-Linked Immunosorbent Assay to Detect Cytokine Production by ES-DC
Cells were cultured in 96-well flat-bottomed culture plates (1.2 x 105 cells in 150 µl of medium per well) in the presence or absence of soluble CD40-ligand, LPS, or OK432. After 60 hours of culture, supernatant was collected, and the concentration of TNF- and IL-12 p70 was measured by using enzyme-linked immunosorbent assay (ELISA) kits (Pierce, Rockford, IL, http://www.piercenet.com).
Allogeneic T-Cell-Stimulation Assay
Mononuclear cells were isolated from heparinized peripheral blood of a human or a cynomolgus monkey housed in the Chemo-Sero-Therapeutic Research Institute (Kumamoto, Japan), using Ficoll-Paque PLUS (Amersham Biosciences, Uppsala, Sweden, http://www.amersham.com). T cells were purified using the Pan T cell isolation kit for humans or the kit for nonhuman primates (Miltenyi Biotec). The T cells (4 x 104/well) were cocultured with graded numbers of x-ray-irradiated (40 Gy) stimulator cells in RPMI 1640 medium supplemented with 10% human plasma in 96-well round-bottomed culture plates for 5 days. [3H]-Methyl-thymidine (247.9 GBq/mmol) was added to the culture (0.037 MBq/well) for the last 16 hours. At the end this time, the cells were harvested onto glass fiber filters (PerkinElmer Life and Analytical Sciences, Boston, http://www.perkinelmer.com), and the incorporation of [3H]-thymidine was measured by scintillation counting. In the experiment using PD-L1-transfectant ES-DC, anti-PD-L1 blocking Ab (clone MIH1; eBioscience) or control mouse IgG1 Ab (eBioscience) was added to the culture (10 µg/ml).
Recombinant Antigenic Protein
A DNA fragment encoding human glutamic acid decarboxylase (GAD65) p96–174 protein fragment was cloned into the prokaryotic expression vector pGEX-4T-3 (Amersham Biosciences), to generate a vector for glutathione S-transferase-fused GAD65 protein fragment (GST-GAD). The induction of the production of recombinant protein in E. coli (DH5) and the extraction of the recombinant protein from bacterial inclusion bodies was done according to Frangioni and Neel [13]. The purification of the recombinant protein with glutathione-agarose (Sigma-Aldrich) was done as described in our previous report [14, 15]. The purity and integrity of the recombinant protein was confirmed by SDS-polyacrylamide gel electrophoresis. The protein was concentrated and separated from small peptide fragments, if any, with Centricon-10 (Millipore, Bedford, MA, http://www.millipore.com), and the solvent was changed from the elution buffer to the culture medium by dialysis.
Antigen Presentation Assay
A human CD4+ T-cell clone, SA32.5, recognizing GAD65p111–131 in the context of HLA-DR53 molecule (DRA*0101+DRB4*0103) was established and maintained as previously described [16]. In the assay with the synthetic peptide, ES-DC stimulated with TNF- (10 ng/ml) plus LPS (3 µg/ml) were harvested, incubated in the presence of peptide (6 µM) for 3 hours, washed four times with culture medium, and x-ray-irradiated (35 Gy). A T-cell proliferation assay was set up in a 96-well flat-bottomed culture plate with SA32.5 T cells (3 x 104 cells per well) and graded numbers of the peptide-loaded ES-DC in RPMI 1640 medium supplemented with 10% human plasma. In the assay with recombinant protein, the indicated amount of GST or GST-GAD protein was added to the coculture of SA32.5 T cells (3 x 104 cells per well) and irradiated ES-DC (1 x 104 cells per well). After 48 hours of culture, [3H]-thymidine was added, and then after an additional 16 hours of culture, the cells were harvested and the incorporated radioactivity was counted.
Plasmid Construction
cDNA for human PD-L1 was isolated by polymerase chain reaction (PCR) with Pyrobest DNA polymerase (Takara, Osaka, Japan, http://www.takara.co.jp) using cDNA clone CS0DI011, purchased from Invitrogen (Carlsbad, CA, http://www.invitrogen.com), as a template. Double-stranded oligo DNA (5'-atgaacattttacttcagtatgtggtgaaaagtttcgat-3') coding for GAD65p115–127 (the core epitope for SA32.5 T-cell clone) was ligated to human invariant chain (Ii)-based epitope presentation vector pCI [17] to generate GAD65-epitope-fused Ii. A cDNA fragment for HLA-DRB4*0103 was generated by reverse transcriptase (RT)-PCR from RNA isolated from peripheral blood mononuclear cells positive for HLA-DRB4*0103. The coding DNA fragments were cloned into a mammalian expression vector, pCAG-IRES-Neo, which is driven by the CAG promoter and includes an internal ribosomal entry site (IRES)-neomycin-resistance gene cassette [3].
Transfection of ES Cells
Human ES cells were harvested using CTK solution, dissociated into clusters of 50–100 cells by pipetting, and washed twice with Dulbecco's modified Eagle's medium (DMEM). The cells harvested from two 90-mm culture dishes with subconfluently growing ES cells were suspended in 0.1 ml of DMEM and mixed with 50 µg of linearized plasmid DNA dissolved in 0.1 ml of PBS in a 4-mm-gap cuvette. The electroporation of human ES cells was performed at 150 V and 200 µF on a Gene Pulser (Bio-Rad, Hercules, CA, http://www.bio-rad.com). The transfection of cynomolgus monkey ES cells was done as previously described [18], with some modifications. Cynomolgus monkey ES cells were harvested after treatment with trypsin-EDTA. ES cells (1–1.5 x 107) suspended in 0.7 ml of DMEM were mixed with 50 µg of plasmid DNA in 0.1 ml of PBS in a 4-mm-gap cuvette. Electroporation was done at 250 V and 500 µF. After electroporation, the ES cells were cultured on G418-resistant PEF feeder layers in 90-mm culture dishes or six-well plates. Selection with G418 (150 µg/ml) was done from 2 to 4 days after the transfection, and G418-resistant ES cell colonies were picked up using a micropipette under microscopic observation on days 15–18 for human ES cells and on day 11 for monkey ES cells. The transfectant clones were transferred to 24-well culture plates with PEF and expanded in the presence of G418. ES cell transfectant clones with relatively high levels of expression of the transgene were selected on the basis of the resistance to a high dose (1–3 mg/ml) of G418 and the results of the RT-PCR analysis. Thereafter, the clones were subjected to the differentiation procedures. At the proper stages of differentiation, the cells were screened to select ES cell clones that highly expressed the transgene after differentiation, based on a flow cytometric analysis for PD-L1 and Ii transfectant human ES cells and on the antigen-presenting capacity for HLA-DRB4 transfectant cynomolgus monkey ES cells.
RT-PCR for Detection of the Transgene-Derived Transcripts
cDNA was synthesized from total cellular RNA with random hexamer primers and SuperScript II reverse transcriptase (Invitrogen). The following PCR primer sets were used: 5'-gctggattacatcaaagcactgaa-3' and 5'-caacaaagtctggcttatatccaa-3' for hypoxanthine-guanine phosphoribosyl transferase and 5'-ctgactgaccgcgttactcccaca-3' and 5'-ttggttatagatgtatctgatcaggt-3' for transgene-derived DRB4 transcript.
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RESULTS |
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The protocol for the differentiation culture to generate ES-DC from human ES cells developed in the current study is composed of three steps, as shown in Figure 1A. At the beginning of the differentiation culture, undifferentiated ES cells maintained on mouse PEF feeders (Fig. 1B) were harvested using dissociation solution CTK [9] and plated on OP9 feeder cell layers (step 1). Next, the ES cells grew and formed clusters composed mostly of epithelial cell-like large flat cells (Fig. 1C, 1D). Clusters of round, cobblestone-like cells also appeared at approximately day 8, and those resembled the mesodermally differentiated cell clusters observed in hematopoietic differentiation culture of mouse ES cells [1, 20]. The size and number of round cell clusters gradually increased, and by around day 15, they covered 20%–30% of the surface area (Fig. 1E).
On days 15–18 of the first step, cells were recovered from the dishes using trypsin/EDTA and isolated nonadherent cells, and then they were seeded onto freshly prepared OP9 cell layers, to begin the second step. On the next day, the culture medium was exchanged for medium containing GM-CSF and M-CSF. Thereafter, small round cells, floating or loosely adhering to the feeder layer, appeared and gradually increased in number (Fig. 1F). The growth of the round cells depended primarily upon GM-CSF, thus suggesting that they grew in response to that factor. The cells were recovered and analyzed for their expression of hematopoietic cell lineage markers by flow cytometry (Fig. 2A). The cells expressed CD34 and CD45, thus indicating that they followed a hematopoietic cell lineage. They also expressed CD31, CD43, and CD11b, thus collectively indicating a commitment to a myeloid cell lineage. The double peaks seen in the histograms in Figure 2 reflect the heterogeneity of the analyzed cells in size and intensity of autofluorescence.
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Figure 1I and 1J shows the cells after the simultaneous addition of TNF-, LPS, soluble CD40-ligand, and IL-4. Generally, they exhibited longer protrusions than before the stimulation, and some of the protrusions were veil-like. Many of the cells formed aggregates. Flow cytometric analysis showed the increased expression of CD86 and the expression of CD80, CD83, and HLA-DR (Fig. 2B). Collectively, the cells exhibited the characteristics of DC in their morphology and expression of surface molecules, and thus they were designated human ES-DC.
Production of IL-12 and TNF- by ES-DC was measured by ELISA (Fig. 3). Production of TNF- was profoundly induced by either LPS or OK432. OK432, but not LPS, induced the production of IL-12, consistent with the reports that OK432 is an efficient inducer of IL-12 [21, 22]. Addition of CD40-ligand showed little effect on the production of these cytokines by human ES-DC.
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The protocol of differentiation culture described thus far was originally developed using the KhES-1 line of human ES cells. This differentiation procedure was also applied to KhES-3, another human ES cell line. KhES-3 differentiation was similar to KhES-1 except that KhES-3 differentiated slightly more quickly than KhES-1, and a first-step culture of 14–15 days was sufficient for the differentiation of KhES-3.
Function of Human ES-DC
The capacity of the human ES-DC to stimulate T cells was examined based on the proliferative response of allogeneic T cells cocultured with ES-DC (Fig. 4A). ES cell-derived floating cells recovered from the second step (pre-ES-DC) showed little capacity to induce a response of T cells. In contrast, ES-DC following the third step before the addition of maturation stimuli (immature ES-DC) showed a weak but definite stimulation, and following exposure to the maturation stimuli (mature ES-DC) they showed a strong capacity to stimulate allogeneic T cells to proliferate.
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Genetic Modification of Human ES-DC
Previous research established a strategy for the genetic modification of mouse ES-DC [1]. Briefly, the expression vectors were introduced into ES cells by electroporation, and subsequently the transfectant ES cell clones were induced to differentiate to ES-DC. The following experiments were performed to determine whether or not this strategy could be applicable to human ES cells.
PD-L1/B7-H1 is known to downmodulate responses of T cells upon interaction with the ligand, PD-1 on T cells [23]. An expression vector for human PD-L1 was introduced to KhES-1 by electroporation. The expression vector used was pCAG-INeo, driven by the CAG promoter and containing an IRES-neomycin-resistance gene cassette (Fig. 5A). Among the transfectant clones, 23 ES cell clones showing resistance to high does of G418 (2 mg/ml) were selected and subjected to the ES-DC-differentiation culture.
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ES-DC carrying an epitope-presenting vector and expressing recombinant human invariant chain (Ii/CD74), which included GAD65p115–127 in the class II-associated invariant chain peptide region, were also generated (Fig. 5D). It was expected that the epitope could be efficiently targeted to the major histocompatibility complex (MHC) class II pathway [17]. Using a protocol similar to that used for the generation of PD-L1 transfectants, the vector was introduced into KhES-1 ES cells, and a transfectant clone, KhES-1-Ii23, highly expressing transgene-derived recombinant CD74, was selected by a flow cytometric analysis at the pre-ES-DC stage. The expression of CD74 was detected even in the nontransfectant pre-ES-DC, reflecting intrinsic expression of CD74 (Fig. 5E). The transfectant exhibited an increased expression of CD74 in comparison to the nontransfectants, thus indicating additional expression of the molecule derived from the transgene. The ability of the transfectant ES-DC, ES-DC-Ii23, to stimulate the GAD epitope-specific T-cell clone SA32.5 in the absence of antigenic peptide or protein was next examined. As a result, ES-DC-Ii23 stimulated SA32.5 T cells and induced their proliferation, thus demonstrating functional expression of the epitope-presentation vector in the transfectant ES-DC (Fig. 5F). The in vivo transfer of ES-DC transfected with this antigen-presenting vector is therefore expected to be useful for controlling the immune response in an antigen-specific manner [7].
Generation and Genetic Modification of Cynomolgus Monkey ES-DC
The differentiation protocol established using human ES cells was then applied to nonhuman primate ES cells. An ES cell line derived from cynomolgus monkey, CMK6 [11], was subjected to the ES-DC differentiation culture. Following the transfer to OP9 feeder layers, CMK6 cells grew and differentiated more rapidly than did human ES cells KhES-1 and KhES-3. The optimal duration of the first step of the differentiation culture for CMK6 was 11–13 days, whereas the duration ranged from 14 to 18 days for human ES cells. Figure 6A–6C illustrates the morphological changes of CMK6-derived cells following the second step of the differentiation culture. The surface phenotypes of the CMK6-derived pre-ES-DC, immature ES-DC, and mature ES-DC were then analyzed by flow cytometry (Fig. 6D). The double peaks seen in the histograms in Figure 6D reflect the heterogeneity of the analyzed cells in size and intensity of autofluorescence. Cynomolgus monkey ES-DC had the capacity to stimulate allogeneic cynomolgus monkey T cells (Fig. 6E), as human ES-DC did.
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chain is very similar to that of HLA-DR, with only one amino acid difference in 1 domain (GenBank accession no. AY591919). This suggested that the transgene-derived HLA-DRβ chain could associate with the intrinsic CyLA-DR chain expressed in cynomolgus ES-DC and present an antigen to human T cells. The expression of the transgene before and after the ES-DC differentiation was confirmed by an RT-PCR analysis (Fig. 7B). ES-DC derived from a transfectant ES cell clone, cES-53-23, were prepulsed with synthetic GAD65 peptide and cocultured with the HLA-DR53-restricted, GAD65-specific T-cell clone SA32.5. Figure 7C shows that the GAD65 peptide-pulsed transfectant ES-DC stimulated the T cells to proliferate. In contrast, ES-DC originating from parental ES cells prepulsed with the peptide could not stimulate the T-cell clone. In addition, DR53-transfectant cynomolgus ES-DC had the capacity to process and present a protein antigen to the T cells (Fig. 7D). These results demonstrate the antigen-processing and presenting capacity of cynomolgus ES-DC and also the functional expression of the transgene that had been introduced into the ES cells before the differentiation. Thus, the effect and safety of the immune therapy by the in vivo transfer of ES-DC can be examined by preclinical studies using cynomolgus monkeys.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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Previously, two other groups reported the generation of functional antigen-presenting cells or DC from human ES cells. Zhan et al. adapted embryoid body-based induction of hematopoietic differentiation [24]. Slukvin et al. recently reported a method using OP9 [25]. Although there are some similarities between the method of Slukvin et al. [25] and the one reported here, the two methods differ in the following points.
In both methods, human ES cells were cocultured with OP9 feeder cells at the initial differentiation step (the first step). However, the duration of this culture step in our method (14–18 days) is significantly longer than the 10 days in the method of Slukvin et al. [25]. In our system, cells with morphology indicating mesodermal differentiation first appeared on day 8 or 9, and the extension of the first step of culture to days 14–18 significantly improved the yield of hematopoietically differentiated cells (Fig. 1D, 1E). In addition, we pretreated OP9 cells with mitomycin C before use as feeder cells, and this was essential for efficient generation of hematopoietic cells. Treatment with mitomycin C may not only inactivate the mitosis of OP9 but also enhance the capacity of OP9 to support hematopoietic differentiation [26].
In the method of Slukvin et al., cells harvested from the first step of culture were directly transferred to 2-hydroxyethyl methacrylate-coated culture containers for the second step of culture [25]. In our method, cells harvested from the first step of culture were incubated in tissue culture-coated dishes for 2–5 hours to remove adherent cells. Removal of cells committed to nonmesodermal lineages by this procedure is essential. In addition, the second step of culture was also done with OP9 feeder in our method.
After the second step of culture, removal of dead cells and aggregated cells may be necessary in the method of Slukvin et al., as described in their report [25]. Indeed, we observed many dead cells, as well as DC-like cells, when we tried that method. In our method, most of recovered cells after the second step were viable, and removal of dead cells was not necessary.
The issues of safety and efficacy are critical for the establishment of ES-DC therapy. It is presumed that preclinical in vivo studies with the nonhuman primates will be required. Therefore, the ability to generate ES-DC from cynomolgus monkey ES cells is also considered to be important. It is probable that ES-DC can be generated from the ES cells of other nonhuman primates used in medical research, such as the rhesus monkey (Macaca mulatta) [27] and the common marmoset (Callithrix jacchus) [28]. For clinical application of the ES-DC technology, development of a feeder-free differentiation method may be required. Embryoid body-mediated differentiation methods may be one way to resolve this issue. In the mouse system, induction of mesodermal differentiation of ES cells using type IV collagen-coated culture plates has been reported [29, 30]. Several molecules have been reported to be involved in support of hematopoietic cell growth or differentiation by stromal cells [31–33]. Information on the molecular basis of the interaction between differentiating ES cells and feeder cells is valuable for the development of a feeder-free differentiation system.
Considering clinical applications, manipulation of function of ES-DC by genetic modification without use of viral vectors, demonstrated in the present study, has a significant advantage. However, random integration of multiple copies of transgenes into various genomic loci of ES cells is accompanied by risks such as activation of cellular oncogenes. Thus, a method to integrate transgenes into intended loci of the genome of human ES cells needs to be established.
Previously, we demonstrated a method for efficient targeted integration of expression vectors into specific genomic sites of mouse ES cells, using exchangeable gene-trap vector with Cre-Lox-mediated recombination system [1]. We are now trying to develop a system for targeted integration of transgenes into human ES cell genome. In this system, at first, gene-targeting vector conveying a drug resistance marker gene flanked by lox sequences is introduced, and then ES cell clones carrying the vector properly integrated by homologous recombination are selected. Subsequently, expression vectors with lox sequences are introduced with the aid of the Cre-Lox recombination system. Integration of a single copy of the transgene into the intended locus can be verified by Southern blot analysis. By this strategy, we can obtain ES cell clones with defined genetic modification, thus avoiding the risks accompanying the random integration of exogenous genes.
Allogenicity caused by differences in the genetic background between human ES cell lines and the recipients is considered to be a critical problem in medical application of ES-DC. We previously reported that mouse ES-DC administered into semiallogeneic recipients, sharing one MHC haplotype with the ES-DC, effectively primed antigen-specific cytotoxic T lymphocytes (CTL), suggesting that ES-DC can survive for a period long enough to stimulate antigen-specific CTL restricted by the shared MHC class I [4]. However, in the same semiallogeneic setting, we also observed five times that injection of no antigen-loaded ES-DC significantly reduced the efficiency of priming of antigen-specific CTL induced by the subsequent injection of antigen-loaded ES-DC (unpublished observations). Thus, repetitive stimulation with ES-DC expressing allogeneic MHC may result in activation and expansion of allogeneic MHC class I-reactive CTL, and in such recipients, subsequently transferred ES-DC may be rapidly eliminated. Repeated immunization may be required in clinical applications, for example, to induce antitumor immunity. Thus, we should resolve the problem of the histoincompatibilty between ES cell lines and recipients.
Methods for targeted gene modification of human ES cells and for targeted chromosome elimination of mouse ES cells have been developed [34–36]. To overcome the problem of histoincompatibility, genetic modification to inhibit expression of endogenous HLA class I in ES-DC may be effective. Deletion of more than 1,000 kilobases of entire HLA class I region of human ES cell genome by gene targeting is infeasible by currently available technology. However, disruption of the genes of molecules necessary for the cell surface expression of HLA class I molecules, such as transporter associated with antigen processing (TAP) or β2-microglobulin (β2M), is presumably feasible. In our plan, we will introduce expression vector encoding the β2M-linked form of recipient-matched HLA class I heavy chain into TAP1- or β2M-deficient human ES cells. We are now testing this strategy by using a mouse system.
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST |
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ACKNOWLEDGMENTS |
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REFERENCES |
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