generation of mouse pluripotent stem cell...

Research Article

Generation of Mouse Pluripotent Stem Cell–DerivedProliferatingMyeloidCellsasanUnlimitedSource of Functional Antigen-Presenting CellsRong Zhang1, Tian-Yi Liu1,2, Satoru Senju3,4, Miwa Haruta3,4, Narumi Hirosawa5,Motoharu Suzuki6, Minako Tatsumi1, Norihiro Ueda1, Hiroyuki Maki1, Ryusuke Nakatsuka7,Yoshikazu Matsuoka7,Yutaka Sasaki7, Shinobu Tsuzuki8, Hayao Nakanishi9, Ryoko Araki10,Masumi Abe10, Yoshiki Akatsuka11, Yasushi Sakamoto5, Yoshiaki Sonoda7,Yasuharu Nishimura3, Kiyotaka Kuzushima1, and Yasushi Uemura1,4

Abstract

The use of dendritic cells (DC) to prime tumor-associatedantigen-specific T-cell responses provides a promising approach tocancer immunotherapy. Embryonic stem cells (ESC) and inducedpluripotent stem cells (iPSC) can differentiate into functional DCs,thusprovidinganunlimited sourceofDCs.However, thepreviouslyestablished methods of generating practical volumes of DCs frompluripotent stem cells (PSC) require a large number of PSCs at thestart of the differentiation culture. In this study,we generatedmouseproliferating myeloid cells (pMC) as a source of antigen-presentingcells (APC) using lentivirus-mediated transduction of the c-Mycgene into mouse PSC-derived myeloid cells. The pMCs couldpropagate almost indefinitely in a cytokine-dependent manner,while retaining their potential to differentiate into functional APCs.After treatment with IL4 plus GM-CSF, the pMCs showed impaired

proliferation and differentiated into immature DC-like cells (pMC-DC) expressing low levels of major histocompatibility complex(MHC)-I, MHC-II, CD40, CD80, and CD86. In addition, exposureto maturation stimuli induced the production of TNFa andIL12p70, and enhanced the expression of MHC-II, CD40, andCD86, which is thus suggestive of typical DC maturation. Similarto bone marrow–derived DCs, they stimulated a primary mixedlymphocyte reaction. Furthermore, the in vivo transfer of pMC-DCspulsed with H-2Kb-restricted OVA257-264 peptide primed OVA-spe-cific cytotoxic T cells and elicited protection in mice against chal-lenge with OVA-expressing melanoma. Overall, myeloid cellsexhibiting cytokine-dependent proliferation and DC-like differ-entiation may be used to address issues associated with the pre-paration of DCs. Cancer Immunol Res; 3(6); 668–77. �2015 AACR.

IntroductionDendritic cells (DC) are commonly used as biologic therapy for

cancer because of their physiologic roles in initiating and mod-ulating the host immune response (1, 2). In many cases, mono-cytes obtained from patients by apheresis have been used as asource of DCs (3, 4). However, the number of monocytesobtained from peripheral blood, their ability to differentiate intoDCs, and the quality of the resulting DCs vary among patients.Therefore, it can be difficult to stably generate a sufficient numberof high-quality DCs for use in cancer therapy. In addition, therequirement to prepare DCs separately for each patient preventsthe broader application of this strategy.

Pluripotent stem cells (PSC), such as embryonic stem cells(ESC) and the recently developed induced PSCs (iPSC), have thepotential to propagate indefinitely and can differentiate intovarious somatic cell types (5–7). Previous studies have establishedmethods of generating DCs from PSCs, and demonstrated theutility of PSC-derived DCs in cancer immunotherapy (8–25).However, generating a large number of DCs from ESCs or iPSCshas required a scaling-up of the initial volume of undifferentiatedPSCs. In addition, these methods are too laborious for practicalapplication in the clinical setting.

We recently developed a simple and efficient method forobtaining a large number of functional antigen-presenting cells(APC) from human iPSCs (26, 27). By introducing the c-Mycgene, alongwith antisenescence factors, such as Bmi-1 or Ezh2, the

1Division of Immunology, Aichi Cancer Center Research Institute,Nagoya, Japan. 2Key Laboratory of Cancer Center, Chinese PLA Gen-eral Hospital, Beijing, China. 3Department of Immunogenetics, Grad-uate School of Medical Sciences, Kumamoto University, Kumamoto,Japan. 4CREST, Japan Science and Technology Agency (JST), Kawa-guchi, Saitama, Japan. 5Department of Biomedical Research Center,Division of Analytical Science, Faculty of Medicine, Saitama MedicalUniversity,Moroyama, Saitama, Japan. 6Department ofObstetrics andGynecology, Faculty of Medicine, Saitama Medical University, Mor-oyama, Saitama, Japan. 7Departmentof StemCell BiologyandRegen-erativeMedicine, Graduate School of Medical Science, Kansai MedicalUniversity, Hirakata, Osaka, Japan. 8Division of Molecular Medicine,Aichi Cancer Center Research Institute, Nagoya, Japan. 9Division ofOncological Pathology, Aichi Cancer Center Research Institute,Nagoya, Japan. 10Transcriptome Research Group, National Instituteof Radiological Sciences, Chiba, Japan. 11Department of Hematologyand Oncology, Fujita Health University, Toyoake, Aichi, Japan.

Note: Supplementary data for this article are available at Cancer ImmunologyResearch Online (http://cancerimmunolres.aacrjournals.org/).

R. Zhang and T.-Y. Liu contributed equally to this article and share firstauthorship.

Corresponding Authors: Yasushi Uemura, Division of Cancer Immunotherapy,Exploratory Oncology Research & Clinical Trial Center, National Cancer Center,6-5-1 Kashiwanoha, Kashiwa, Chiba 277-8577, Japan. Phone: 81-4-7131-5490;Fax: 81-4-7133-6606; E-mail: [email protected]; and Satoru Senju,Department of Immunogenetics, Graduate School of Medical Sciences, Kuma-moto University, 1-1-1 Honjo, Kumamoto, 860-8556, Japan. Phone: 81-96-373-5313; Fax: 81-96-373-5314; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-14-0117

�2015 American Association for Cancer Research.

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myeloid cells generated from iPSCs could proliferate almostindefinitely while retaining the capacity to differentiate intopotent APCs. In addition, cells derived from transporter asso-ciated with antigen processing 2 (TAP2)–deficient iPSCs down-regulated the expression of endogenous human leukocyteantigen (HLA)-I and avoided recognition by alloreactiveCD8þ T cells, and cells expressing exogenously introducedHLA-I genes stimulated tumor-associated antigen-specificCD8þ T cells in vitro (26). Genetic modification of macrophagesderived from the proliferating myeloid cells (pMC) to expressan anti-HER2 antibody or interferon (IFN) could also exerttherapeutic effects against peritoneally disseminated gastricand pancreatic cancer in xenograft models (27). However, theirin vivo therapeutic potential against cancer and in vivo safetyhave not been examined in syngeneic mouse models. In addi-tion, whether pMCs can be generated from mouse PSCsremains unclear.

In this study, we generated pMCs frommouse ESCs and iPSCs,and showed their potential to differentiate into functionalAPCs. This new method enabled us to obtain a large number ofAPCs from a small number of undifferentiated PSCs. Moreover,once the pMCs were established, it was possible to supply APCsin a few days using the pMCs as a cell source. Using a modelantigen, we demonstrated that vaccination with APCs was safeand feasible for cancer immunotherapy, without any inductionof autoimmunity or leukemia in vivo.

Materials and MethodsMice

C57BL/6 andBALB/cmicewere purchased from Japan SLC, Inc.or CLEA Japan, Inc., and were maintained under specific patho-gen-free conditions. All animal experiments were performed withapproval from the Animal Experiment Committee of the AichiCancer Center.

CellsThe mouse ESC line, B6-ES, derived from C57BL/6 blasto-

cysts (28), and the mouse iPSC lines, 2A-4F-100 and 2A-4F-136(29), derived from C57BL/6 embryonic fibroblasts, were main-tained as described previously (28, 29). The M-CSF–defectivebone marrow–derived stromal cell line, OP9 (30), was main-tained in a-minimum essential medium (a-MEM; Life Tech-nologies) supplemented with 20% fetal bovine serum (FBS),and the cells were seeded onto gelatin-coated dishes beforebeing used as feeder cells. MO4 (31), a C57BL/6-derived B16melanoma cell line expressing OVA, luciferase-transduced MO4(MO4-Luc), and the EL-4 thymoma cell line, were maintainedin RPMI-1640 medium (Sigma-Aldrich) supplemented with10% FBS.

Generation of recombinant lentivirusA lentivirus vector, CSII-EF, and the plasmids used for lentiviral

vector packaging, pCMV-VSV-G-RSV-Rev and pCAG-HIVgp, werekindly provided by Dr. H. Miyoshi (RIKEN BioResource Center;Tsukuba, Japan). CSII-EF containing a human c-Myc cDNA insertwas used as described previously (32). Plasmid constructs wereintroduced into 293T cells using lipofection (Lipofectamine2000; Life Technologies). Three days later, recombinant lenti-viruses were recovered from the culture supernatant using a Lenti-X Concentrator (Clontech).

Generation of pMCs from mouse ESCs or iPSCsESCs or iPSCs were induced to differentiate into myeloid

cells according to an established procedure (Fig. 1; refs. 9, 11–14, 18, 19). Briefly, the ESCs or iPSCs were cultured on feederlayers of OP9 cells for 6 to 7 days in a-MEM supplemented with20% FBS. The mesodermally differentiated cells were thenharvested, reseeded onto fresh OP9 cell layers, and culturedin a-MEM supplemented with 20% FBS, 20 ng/mL GM-CSF,and 50 mmol/L 2-ME. On day 13 to 14, floating cells wererecovered by pipetting. These cells were considered to be ESC-or iPSC-derived myeloid cells (ES-MCs or iPS-MCs, respective-ly). The cells were infected with lentivirus vectors expressing thec-Myc gene in the presence of 8 ng/mL polybrene (Sigma-Aldrich), and were cultured in a-MEM supplemented with20% FBS, 30 ng/mL GM-CSF, and 30 ng/mL M-CSF. After5 to 6 days, proliferating cells appeared and were considered

Figure 1.Schematic illustration showing the generation of pMCs frommouse ESCs andiPSCs. A, the protocol for the generation of pMCs. The culture medium,supplements in the medium, and feeder cells are indicated. B, the fold-increase in the number of differentiated cells from PSCs at each stage. pMC(with c-Myc transduction on days 13–14), MC, myeloid cell (without c-Myctransduction). C, phase contrast images of the cells in the stages of eachdifferentiation. Day 0, PSCs (iPSC 2A-4F-100) on primary embryonicfibroblasts. Day 7, mesodermally differentiated cells on OP9 feeder layers.Day 14, myeloid differentiated cells (myeloid cells) on OP9 feeder layers.Day 24, pMCs generated by transduction of the c-Myc gene. Day 27, pMCstreated with IL4 and GM-CSF for 3 days. Detailed information is providedin the legend for Supplementary Fig. S1.

Mouse ESC and iPSC-Derived Proliferating APCs

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to be ESC- or iPSC-derived pMCs (ES-pMC or iPS-pMC, respec-tively). To induce the differentiation of these cells into DC-likecells (pMC-DC), they were cultured in RPMI-1640 supplemen-ted with 20% FBS in the presence of 20 ng/mL IL4 plus 30ng/mL GM-CSF for 3 days.

Flow cytometry and microscopyCell samples were treated with a Fc-receptor blocking

reagent (Miltenyi Biotec) for 10 minutes, stained with thefluorochrome-conjugated monoclonal antibody (mAb; Sup-plementary Materials and Methods) for 20 minutes, andwashed three times with phosphate-buffered saline/2% FBS.The stained cell samples were analyzed on a FACSCalibur flowcytometer, and the data were analyzed using the BD CellQuestPro software program (BD Biosciences) or FlowJo softwareprogram (TreeStar Inc.). For the morphologic analysis, cyto-spin specimens were stained with May-Gr€unwald/Giemsa(Merck).

Cell proliferationCell proliferation was evaluated by the direct enumeration

of cells, the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-nyltetrazolium bromide (MTT) assay, and the [3H]-thymi-dine incorporation assay (Supplementary Materials andMethods).

Cytokine productionCytokine levels in the culture supernatants were evaluated

using enzyme-linked immunosorbent assays (ELISA; mouseTNFa and IL12p70: Affymetrix eBioscience).

Analysis of the in vivo priming of antigen-specific T cells bypMC-derived DC-like cells

ES-pMC- or iPS-pMC–derived DC-like cells (pMC-DC) wereloaded with vehicle or 10 mmol/L OVA257-264 peptide for 3 hours,and were injected i.p. into C57BL/6 mice (1.0 � 105 cells/mouse)twice at 7-day intervals. In one experiment, the pMC-DCswere heat-killed by incubation at 70�C for 20 minutes or were irradiated (45Gy) before injection. Seven days after the second injection, spleencells were isolated from the mice and pooled for each group of 3mice. After hemolysis, the spleen cells were cultured in 24-wellculture plates (2.5 � 106/well) in RPMI-1640 supplemented with10% horse serum, 2-ME (50 mmol/L), IL2 (10 U/mL), andOVA257–264 peptide (0.1 mmol/L). After 5 days, the cells wererecovered, and the OVA-specific cytotoxic T lymphocyte (CTL)activity was analyzed using the 51Cr-release assay using EL-4thymoma cells or MO4 melanoma cells as targets. The percent-age of specific lysis was calculated as: 100 � (experimentalrelease � spontaneous release)/(maximal release � spontane-ous release). The spontaneous release and maximal release weredetermined in the presence of medium alone and with 1%Triton X-100, respectively.

Tumor challenge experimentsThe OVA257–264 peptide (10 mmol/L)- or vehicle-loaded pMC-

DCswere injected i.p. into C57BL/6mice (1.0� 105 cells/mouse)twice at 7-day intervals. Seven days after the second transfer, 2.0�105 luciferase-transduced MO4 cells were inoculated s.c. into theshaved flanks. The mice were monitored for tumor growth andsurvival; tumor sizewasmeasured at 5-day intervals until themiceeither died or were euthanized because of tumor progression.

Figure 2.Characterization of mouse PSC-derived cells in the stages of myeloidcells and pMCs. Representative flowcytometry profiles of iPSC-derivedmyeloid cells (without c-Myctransduction), iPSC-derived pMCs(with c-Myc transduction), andBM-DCs. CD11b, CD11c, DEC205, andGr-1 expression was analyzed usingflow cytometry. GM-CSF–induced BM-DCs andGM-CSF/IL4–treated BM-DCsserved as references. A, iPSC (2A-4F-100)–derived myeloid cells (days13–14) differentiated on OP9 feedersin the presence of GM-CSF. B, top,iPSC (2A-4F-100)–derived pMCscultured in the presence of GM-CSFand M-CSF (days 23–24). Bottom,iPSC (2A-4F-100)–derived pMCscultured with IL4 and GM-CSF (days26–27). C, top, GM-CSF-BM-DCs.Bottom, GM-CSF/IL4-BM-DCs. Toobtain GM-CSF/IL4-BM-DCs, GM-CSF–induced BM-DCs were culturedfor another 2 days in the additionalpresence of IL4.

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In vivo bioluminescent imagingTumor-bearing mice were injected with 200-mL D-luciferin (15

mg/mL, VivoGlo Luciferin; Promega) under 2% inhaled isoflur-ane anesthesia, and bioluminescence images were obtained usingthe IVIS Lumina II instrument with the Living Image softwareversion 3.2 (Xenogen).

Statistical analysisThe Prism version 6.0e software program (GraphPad Software)

was used for all of the statistical analyses. To compare multipleexperimental groups, a one-way ANOVAwith the Bonferroni posthoc test was used to assess statistical significance. For statisticalanalysis of the Kaplan–Meier survival curves, a log-rank (Mantel–Cox) test was used to calculate P values. P values < 0.05 wereconsidered to denote statistical significance, and are indicated infigures by asterisks (�, P < 0.05; ��, P < 0.01).

ResultsGeneration of pMCs from mouse PSCs

We previously established a method for generating humanmyelomonocytic cells with the capacity for proliferation usingthe simultaneous transduction of c-Myc together with Bmi-1 orEzh2 into human iPSC-derived myeloid cells (26, 27). In thatpreliminary study, mouse ES-MCs were transduced with severalgenes to identify those that induced proliferation. On the basis of

these results, we found that mouse ES-MCs could proliferate afterlentivirus-mediated transduction of c-Myc alone (data notshown). We then attempted to induce the proliferation of mouseES-MCs and iPS-MCs through the transduction of c-Myc alone,and successfully stimulated their differentiation into DC-likeAPCs. The procedure for the generation of proliferating ES- oriPS-MCs (ES- or iPS-pMCs) and the subsequent differentiation ofthese cells intoDC-like cells (pMC-DC) are shown in Fig. 1A. ESCsor iPSCs (day 0) were seeded on the OP9 feeder cell layers. After 6to 7 days, the mesodermally differentiated cells (day 6–7) weretransferred onto newly prepared OP9 cell layers in the presenceof GM-CSF. On days 13 to 14, the differentiated cells (myeloidcells) were transduced with c-Myc and cultured in the presenceof M-CSF and GM-CSF. After about 5 to 6 days, pmCs withprotrusions appeared and propagated continuously (Supple-mentary Fig. S1A). The pMCs were treated with GM-CSF and IL4(days 23–24). After 3 days of culture, the pMCs differentiatedintoDC-like cells (pMC-DC; days 26–27). The fold-increase in thenumber of differentiated cells from PSCs at each stage and thephase contrast micrographs of the cells at each differentiationstage are shown in Fig. 1B and C, respectively. The number ofpMC-DCs generated via the c-Myc transduction of myeloidcells ranged from 8,000 to 40,000 times that obtained fromundifferentiated stem cells. This total was estimated to be atleast 160 times more than that of the MC-DCs (without c-Myctransduction).

Figure 3.GM-CSF–dependent proliferation ofmouse ES- or iPS-pMCs. A, ESC (left)-or iPSC (2A-4F-100; right)–derivedpMCs were cultured in the presence ofM-CSF and GM-CSF on 24-well plates(5 � 104 cells/well), and the cells wereenumerated at the indicated timepoints. B, proliferation of iPS-pMCscultured for 1, 2, or 3 months. The cellswere enumerated as in A. C, ES- (left)or iPS- (right) pMCswere seeded in 96-well culture plates (2 � 103 cells/well)in the presence of the indicatedcytokines, and the proliferation wasdetermined at each time point using astandard MTT assay. D, ES-pMCs werecultured in 96-well culture plates(2.0� 103 cells/well) in the presence ofgraded concentrations of the indicatedcytokines for 4 days, and37 kBq/well [3H]-methylthymidinewas added to the culture for the last16 hours. The incorporatedradioactivity was measured. n.s., notstatistically significant. The datarepresent the mean � SD of triplicatecultures. n.s., not statisticallysignificant.






Figure 4.ES- or iPS-pMCs can differentiate into DC-like cells. A, the surface phenotypes of mouse BM-DCs, ES-pMCs, or iPS-pMCs under different culture conditions;BM-DCs were cultured in the presence of 20 ng/mL GM-CSF alone (top), GM-CSF plus 20 ng/mL IL4 (middle), or GM-CSF, IL4, and 1 mg/mL LPS (bottom) for 3 days.ES- or iPS-pMCs (clone 2A-4F-100- or 2A-4F-136–derived iPSCs) were cultured in the presence of 30 ng/mL GM-CSF plus 30 ng/mL M-CSF (top each),GM-CSF alone (top middle each), GM-CSF plus 20 ng/mLIL4 (bottom middle each), or GM-CSF, IL4, and 1 mg/mL LPS (bottom panels each) for 3 days, andthe expression levels of MHC-I (H-2Kb/H-2Db), MHC-II (I-A/E), CD40, CD80, and CD86 were analyzed using flow cytometry. Staining histograms of theindicated surface molecules (open histograms) and isotype-matched controls (filled histograms) are shown. (Continued on the following page.)

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Surface phenotype of PSC-derived cells in the stages ofMCs andpMCs

The myeloid cells (days 13–14) were a heterogeneous popu-lation, almost 90% of which was characterized to be CD11bþ

(Fig. 2A). In this stage, CD11bþGr-1high, CD11cþGr-1low, andDEC205lowGr-1þ/� cells were present as minor populations. ThepMCs (days 23–24) obtained by transduction of the c-Myc geneshowed the upregulation of both CD11c and DEC205, anddecreases in the Gr-1high population (Fig. 2B). The pMC-DCs(days 26–27) obtained after 3 days of culture in the presence ofGM-CSF and IL4 showed an enhanced expression of CD11c anda downregulation of Gr-1 (Fig. 2B). In contrast to the BM-DCs,which were composed of a heterogeneous population (Fig. 2C),the pMC-DCs (GM-CSF/IL4-treated pMCs) were largely com-posed of a relatively homogeneous population, thus indicatingthat they were CD11bþ/CD11cþ/DEC205þ/Gr-1low DC-like cells(Supplementary Fig. S1B).

Cytokine-dependent proliferation of mouse ES- andiPS-pMCs

In the maintenance culture medium containing M-CSF andGM-CSF, mouse ES- and iPS-pMCs proliferated for more than 3months. The doubling times of the ES-pMCs and iPS-pMCsafter 15 days of c-Myc transduction were 16.73 and 17.60 hours,respectively (Fig. 3A). The doubling times of the iPS-pMCscultured for 1, 2, and 3 months were 18.74, 17.71, and 20.06hours, respectively (Fig. 3B). In addition, there were no signi-ficant changes in the phenotypes of these cultured pMCs(Supplementary Fig. S1C).

To increase our understanding of the proliferation of thesecells, we examined their proliferation in the presence orabsence of each cytokine or with combinations of cytokines(Fig. 3C). The results showed that proliferation was dependenton GM-CSF, but not M-CSF. The additional presence of M-CSFhad no effect on the proliferation of these cells. The cellsproliferated in a GM-CSF concentration–dependent manner,and the maximal proliferation was induced by 10 ng/mL GM-CSF (Fig. 3D).

Differentiation of mouse ES- and iPS-pMCs into DC-likecells

In the maintenance culture medium containing M-CSF andGM-CSF, mouse ES- and iPS-pMCs proliferated and expressedmajor histocompatibility complex (MHC)-I, MHC-II, CD80, andCD86 (Fig. 4A). However, CD40 was expressed at a low level.Three days after treatmentwith IL4 plusGM-CSF, theproliferationof pMCs was impaired (Supplementary Fig. S2). In the additionalpresence of lipopolysaccharide (LPS), which is known to be aninducer of DC maturation, the pMCs showed enhanced expres-sion of MHC-II, CD40, and CD86, and their proliferation wasfurther impaired (Fig. 4B and Supplementary Fig. S2). The iPSC(2A-4F-100)–derived pMCs showed a tendency to have a sub-stantially lower expression of MHC-II. This tendency was alsoobserved when the cells were stimulated with LPS.

To determine whether the lower expression of MHC-II is aproperty common to all iPSC-derived pMC lines, we examinedthe expression of surface antigens in other pMCs derived from adifferent iPSC clone (2A-4F-136). The levels ofMHC-II expressionof 2A-4F-136–derived iPS-pMCs were generally low, but werehigher than those from 2A-4F-100–derived iPS-pMCs (Fig. 4A).Therefore, low expression ofMHC-II may be a common property,but levels of expression may differ depending on the parentaliPSC clone. LPS orOK432 (penicillin-killed Streptococcus pyogenes)stimulation induced the production of TNFa and IL12p70in the IL4/GM-CSF–treated pMCs, similar to that observed inBM-DCs (Fig. 4C).

To determine the capacity of the cells to activate na€�ve T cells,primary MLR assays were performed. LPS-treated pMC-DCs stim-ulated proliferative responses in allogeneic T cells, but theresponses were lower than those induced by LPS-treated BM-DCs(Fig. 4D). These findings collectively indicate that pMCs treatedwith IL4 plus GM-CSF exhibited some features of DCs.

In vivo priming of OVA-specific CTLs by adoptive transfer ofOVA peptide-loaded pMC-DCs

To determine whether pMC-DCs could prime antigen-specificT-cell responses in vivo, OVA257–264 peptide- or vehicle-loadedpMC-DCs were i.p. transferred into syngeneic C57BL/6 micetwice at 7-day intervals. Spleen cells were isolated 7 days afterthe second transfer and were cultured in vitro in the presence ofthe OVA257–264 peptide. After 5 days, the cells were recovered andassayed for their capacity to kill OVA257–264 peptide-loaded EL-4thymoma cells or OVA-expressing melanoma cells (MO4; Fig. 5).The CTLs primed in vivo by OVA257–264 peptide-loaded pMC-DCsorOVA257–264peptide-loadedBM-DCskilledOVApeptide-loadedEL-4 cells, but not vehicle-loaded EL-4 cells (Fig. 5A–C). In con-trast, the CTLs primed by vehicle-loaded pMC-DCs killed neitherthe OVA peptide-loaded nor the vehicle-loaded EL-4 cells. Nodifferences in the capacity for antigen-specific CTL priming wereobserved among the DCs derived from pMCs cultured for 1, 2, or3 months (Fig. 5D). In addition, the CTLs primed in vivo byOVA257–264 peptide-loaded pMC-DCs killed OVA-expressingmel-anoma, but the CTLs primed by vehicle-loaded pMC-DCs did not(Fig. 5E). Moreover, the CTLs primed in vivo by OVA257–264

peptide-loaded, irradiated pMC-DCs killed OVA-expressing mel-anoma. In contrast, OVA257–264 peptide-loaded, heat-killed pMC-DCs did not result in the priming ofOVA-specific CTLs (Fig. 5E). Itis conceivable that heat-killed pMC-DCsmay fail to induce immu-nity due to their loss of an adjuvant effect,whereas irradiated pMC-DCs survived for a certain period in vivo and were able to primeantigen-specific CTLs. These results suggest that peptide antigen-loaded pMC-DCs could prime the antigen-specific CTL responsein vivo, and their function was maintained for at least 3 months.

Induction of protective immunity against OVA-expressingmelanoma by OVA peptide-loaded pMC-DCs

To determine whether CTLs primed by pMC-DCs adoptivelytransferred into syngeneic C57BL/6 mice could protect against

(Continued.) B, BM-DCs, ES-pMCs, and iPS (2A-4F-100)-pMCs were treated as described in A, and the expression levels of MHC-II, CD40, and CD86 were analyzed.The bar graph indicates the mean fluorescence intensity (MFI) of triplicate cultures. � , P < 0.05; �� , P < 0.01. C, ES- or iPS (2A-4F-100)-pMC-DCs werestimulated with 1 mg/mLLPS or 10 mg/mLOK432 for 24 hours. The TNFa and IL12p70 levels in the culture supernatants were evaluated by ELISA. The mediumcontrol served as the reference for the other culture conditions. D, T cells (1.5 � 105 cells/well) from unprimed female BALB/c mice were cultured with gradednumbers of stimulator cells (BM-DCs, ES-pMC-DCs) and cultured for 6 days. A concentration of 37 kBq/well [3H]-methylthymidine was added to the culturefor the last 16 hours. The incorporated radioactivity was measured. The data represent the mean � SD of triplicate cultures.






a subsequent challenge with OVA-expressing tumor cells,OVA257–264 peptide- or vehicle-loaded pMC-DCs were i.p.transferred into mice twice at 7-day intervals. Seven days afterthe second transfer, the mice were inoculated s.c. with MO4(Fig. 6 and Supplementary Fig. S3). Treatment withOVA257–264 peptide-loaded ES- or iPS-pMC-DCs inhibited thegrowth of the inoculated MO4 for 1 month (Fig. 6A–C andSupplementary Fig. S3A–S3C) and significantly prolonged thesurvival of the mice compared with the vehicle-loaded pMC-DC treatment (Fig. 6D and Supplementary Fig. S3D). Theseobservations indicate that peptide antigen–loaded pMC-DCsinduced antigen-specific protective immunity against melano-ma in vivo.

In vivo safety of pMC-DC treatmentWe next explored whether the mice were adversely affected by

pMC-DC i.p. treatment. Twogroups ofmice treatedwith2.0�105

or 1.0� 107 iPS-pMC-DCs weremonitored. No tumor formationor immune-related toxicities were observed for 3 months, even ata dose that was 100-fold higher than the doses used in theimmunization protocols (Supplementary Fig. S4). Collectively,these observations indicate that peptide antigen-loaded pMC-DCs exhibited antigen-specific antitumor reactivity and were safein vivo.

DiscussionMouse and human ESCs or iPSCs are capable of differentiating

into functional DCs, thus providing an unlimited novel source of

DCs for cancer immunotherapy (9, 17, 19). However, the processof inducing differentiation was relatively complicated, and gen-erating practical volumes of DCs from ESCs or iPSCs has requireda large number of PSCs at the start of the differentiation culture.Therefore, this process has not been suitable for the rapid massproduction of cell preparations. To address this issue, we recentlyestablished amethod for generating human pMCs (the precursorsof DC-like cells) by transduction of c-Myc, together with anti-senescence factors, such as Bmi-1 and Ezh2, into human iPSC-derivedmyeloid cells (26). To evaluate the possible application ofthese cells and their safety, we required an in vivo model notrequiring xenotransplantation, which we developed for thisstudy. Using this model, we demonstrated that pMCs could begenerated from mouse ESCs or iPSCs, and that the cells coulddifferentiate into functional APCs to induce antitumor immuneresponses in vivo without inducing autoimmunity or leukemia.

Mouse ESC-derived myeloid cells could acquire the capacity toproliferate in a cytokine-dependent manner after lentivirus-medi-ated transduction of c-Myc alone. Cell proliferation could also beinduced in mouse iPSC-derived myeloid cells using the sameprocedure (Figs. 1 and 3). Because both the ESC- and iPSC-derivedpMCswere of C57BL/6 origin, it is possible that these observationswere unique to this strain. However, wemade similar observationsfor the 129/Sv strain (data not shown). On the basis of theseresults, it appears that mouse PSC-derived myeloid cells acquirethe capacity to proliferate continuously through the increasedexpression of c-Myc alone, irrespective of the mouse strain.

The c-Myc protein is a transcription factor that induces cellproliferation and cell senescence (33). Other than c-Myc,

Figure 5.In vivo priming of antigen-specificCTLs by ES- or iPS-pMC-DCs. Vehicle-or OVA257–264 peptide-loaded DCswere injected i.p. into 3 C57BL/6 micetwice at 7-day intervals. BM-DCs (A),ES-pMC-DCs (B), iPSC (2A-4F-100)-pMC-DCs (1.0� 105 cells/mouse each;C), and iPSC (clone 2A-4F-100)-pMC-DCs (D) cultured for 1, 2, or 3 months.E, irradiated iPS-pMC-DCs or heat-killed iPS-pMC-DCs were used. Sevendays after the second transfer,isolated spleen cells were pooled foreach group and were cultured in thepresence of OVA257–264 peptide. After5 days, the cells were recovered andassayed for their ability to kill vehicle-or OVA257–264 peptide-loadedthymoma cells (EL-4; A–D), or OVAprotein-expressing melanoma cells(MO4; E).

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antisenescence genes, such as Bmi-1, Mdm2, and Ezh2, may beindispensable for the continuous proliferation of human ESC-or iPSC-derived myeloid cells (34–36). However, antisenes-cence factors were not required for acquisition of the capacityfor long-term proliferation in the mouse PSC-derived myeloidcells. In addition, the increased expression of Bmi-1 had noeffect on the cell proliferation, morphology, or surface markersin mouse ES- or iPS-pMCs (data not shown). This may beexplained by differences between species, but the precise mech-anism remains unclear.

When the medium was supplemented with GM-CSF with orwithout M-CSF, the mouse ES- and iPS-pMCs proliferatedindefinitely. In the additional presence of IL4, the proliferationwas impaired, and the cells functioned as APCs. LPS exposureinduced the production of TNFa and IL12p70, and furtherenhanced the expression of antigen-presenting molecules andcostimulatory molecules (Fig. 4). These findings were similar tothe results of DC maturation. However, the T-cell stimulatoryactivity of ES- or iPS-pMC-DCs was slightly inferior to that ofBM-DCs, based on their lower expression of T cell–stimulatingmolecules and the decreased production of cytokines (Fig. 4).

In particular, iPSC-derived pMCs showed a tendency to havesubstantially lower expression of MHC-II than BM-DCs or ESC-derived pMCs (Fig. 4A). At present, the mechanisms by whichiPSC-derived pMCs have downregulated MHC-II expression arenot clear. The expression levels of MHC-II in iPS-pMC-DCsvaried depending on the iPSC lines used as the source, and theepigenetic memory of iPSCs might affect the expression ofMHC-II (37). In any case, despite the relatively lower levelsof MHC-II expression, the iPS-pMC-DCs could prime the anti-gen-specific T cells in vivo (Fig. 5).

We successfully stimulated OVA257–264 peptide-specific CTLsin vivo by administration of OVA257–264 peptide-prepulsed ES- oriPS-pMC-DCs, leading to protection against OVA-expressingmel-anoma (Fig. 6 and Supplementary Fig. S3). DCs transduced withthe entire tumor antigen gene expressed diverse T-cell epitopes viadifferent MHC molecules and efficiently stimulated diverse anti-gen-specific T cells (38). Therefore, vaccination with pMC-DCsexpressing the entire tumor antigen may be more effective forinducing antigen-specific CTLs, which could elicit potent antitu-mor responses. Moreover, pMC-DCs expressing multiple tumorantigensmay exert a higher therapeutic effect (39–41). In addition

Figure 6.Protection against s.c. inoculatedOVA-expressing melanoma by OVA-peptide loaded ES-pMC-DCs. Vehicle-or OVA257–264 peptide-loadedBM-DCs or ES-pMC-DCs (1.0 � 105

cells/mouse each) were injected i.p.into C57BL/6 mice twice at 7-dayintervals. Seven days after the secondtransfer, luciferase-transduced MO4cells (2.0 � 105 cells/mouse) wereinoculated s.c. into the shavedflank. A,tumor growth was monitored at theindicated time points usingbioluminescence imaging (n ¼ 3 pergroup). The bioluminescence imagesof representative mice from theindividual groups are shown. B, totalflux (photon/s) is shown as the mean� SD (n ¼ 3 mice per group). Thecontrol mice were treated withsaline. C, tumor size (mm2:length � width) was measured at5-day intervals (n ¼ 7 per group).D, survival rate (n ¼ 7 per group).�� , P < 0.01.






to manipulation of the tumor antigens, modification of pMC-DCs through the transduction of immunostimulatory molec-ules (such as IL21) may improve the cellular immune responses(25, 42). To this end, genetic modification of pMC-DCs canbe performed by introducing exogenous genes into pMCs vialentivirus transduction, followed by subsequent induction ofpMC-DCs.

The human iPS-pMCs proliferated in an M-CSF–dependentmanner (26), whereas the pMCs established frommouse ESCs oriPSCs in this study proliferated in a GM-CSF–dependent manner(Fig. 3C andD). There is a good concordance between the humanand mouse pMCs in terms of their cytokine-dependent prolifer-ation. In either case, the long-term proliferating capacity of pMCsmay lead to thedevelopment of leukemia after administration to apatient. However, proliferation was induced by cytokine concen-trations higher (>10 ng/mL) than the physiologic levels. Theobservation that the administration of a large amount of pMC-DCs (1.0� 107) did not induce the onset of leukemia in this studyis suggestive of their in vivo safety (Supplementary Fig. S4).Although pMCs may not proliferate in the absence of GM-CSF,we can further enhance their safety by irradiating such pMCsbefore they are administered as therapy.

Despite the observed in vivo safety of the treatment, there areseveral challenges to the possible clinical application of this tech-nique in humans. The lentivirus-mediated delivery of the c-Mycgene permanently integrates the transgene into the genome, poten-tially altering the genomic features. In addition, the oncogenicc-Myc gene can lead to the development of certain cancers. In thisregard, a genome-integrating but excisable system (43), and tran-sient expression of c-Myc with Sendai-virus (44), adenoviral vec-tors (45), or nonviral expression [such as transduction of the c-Mycprotein (46) and the use of modified mRNA (47)] may providesafer pMCs in the future. In addition, using other genes that pro-mote the proliferation of pMCs may secure their safety.

Construction of an extensive bank of iPSCs, including themost common HLA haplotypes, is currently in progress. Withthis resource it will be possible to generate pMCs from the cells inthe iPSC bank. When the iPS-pMC bank is established, functionalAPCs can be supplied within a short period, which will permitbroader clinical applications of these cells. Considering the use ofpMC-DCs in an allogeneic or semi-allogeneic setting as a cancervaccine, one potential hurdle is that the alloreactive CTLs mayeliminate pMC-DCs, thereby compromising their efficacy. Toavoid such issues, the genes associated with the cell-surfaceexpression of HLA-I molecules, such as TAP or b2-microglobulin,can be disrupted. Alternatively, recipient-matched HLA-I heavychain or the b2-microglobulin–linked form of the HLA-I heavychain can be transduced into the TAP- or b2-microglobulin gene–deficient iPS-pMCs to induce suitable CTL responses (18).

iPSC-derived DCs may resolve the problems associated withthe number and quality of DCs derived from patients with cancer.

However, considering the cost, labor, and preparation time, iPSC-derived DC vaccine therapy as personalized medicine is not yetsuitable as a widely applicable form of cancer immunotherapy.In contrast, production of iPS-pMCs is easily scalable to rapidlyprovide an unlimited number of functional APCs, which wouldbenefit patients requiring multiple vaccine doses. The iPSC-derived proliferating APCs with the capacity to stimulate tumorantigen–specific T-cell responses and with good in vivo safetyprofiles may provide a novel vaccine strategy for clinical cancertherapy, although further investigation is required to improvetheir effectiveness and to further confirm their safety.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: N. Hirosawa, Y. Sakamoto, Y. UemuraDevelopment of methodology: R. Zhang, T.-Y. Liu, S. Senju, M. Haruta,M. SuzukiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): R. Zhang, T.-Y. Liu, M. Tatsumi, N. Ueda, H. Maki,H. Nakanishi, Y. AkatsukaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): R. Zhang, T.-Y. Liu, N. Hirosawa, Y. Sakamoto,K. Kuzushima, Y. UemuraWriting, review, and/or revision of the manuscript: R. Zhang, T.-Y. Liu,S. Senju, N. Hirosawa, Y. Akatsuka, Y. Sakamoto, Y. UemuraAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): R.Nakatsuka, Y.Matsuoka, Y. Sasaki, S. Tsuzuki,R. Araki, M. Abe, Y. Sonoda, K. KuzushimaStudy supervision: N. Hirosawa, Y. Sakamoto, Y. Nishimura, Y. Uemura

AcknowledgmentspCSII-EF, pCMV-VSV-G-RSV-Rev, and pCAG-HIVgp constructs were kindly

provided by Dr. H. Miyoshi (RIKEN BioResource Center).

Grant SupportThis study was supported by grants from the Japan Science and Technology

Agency (JST), a KansaiMedicalUniversity Internal grant C (9), theOsakaCancerResearch Foundation (9), the Takamatsu Cancer Research Foundation (9), theAichi Cancer Research Foundation (12, 13), the Nagono Medical Foundation(12, 13), the Daiwa Securities Health Foundation (13), the Pancreas ResearchFoundation of Japan (13), and the Foundation for Promotion of CancerResearch in Japan (13). R. Zhang, M. Suzuki, and Y. Uemura were supported,in part, byGrants-in-Aid 25861253, 23791850, or 23592022, respectively, fromthe Ministry of Education, Culture, Sports, Science and Technology (MEXT)of Japan. T.-Y. Liu was supported by the National Natural Science Foundationof China (81101882). K. Kuzushima was supported by the Takeda ScienceFoundation (12–14).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received June 14, 2014; revised January 18, 2015; accepted January 29, 2015;published OnlineFirst February 11, 2015.

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2015;3:668-677. Published OnlineFirst February 11, 2015.Cancer Immunol Res Rong Zhang, Tian-Yi Liu, Satoru Senju, et al. Antigen-Presenting CellsMyeloid Cells as an Unlimited Source of Functional

Derived Proliferating−Generation of Mouse Pluripotent Stem Cell

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