Generation of dendritic cells and macrophages from human ... - Nature

Gene Therapy (2011) 18, 874–883 & 2011 Macmillan Publishers Limited All rights reserved 0969-7128/11 www.nature.com/gt

ORIGINAL ARTICLE

Generation of dendritic cells and macrophages from human induced pluripotent stem cells aiming at cell therapy S Senju1,2, M Haruta1,2, K Matsumura1,2, Y Matsunaga1,2, S Fukushima1,2, T Ikeda1,2, K Takamatsu1,2, A Irie1,2 and Y Nishimura1 This report describes generation of dendritic cells (DCs) and macrophages from human induced pluripotent stem (iPS) cells. iPS cell-derived DC (iPS-DC) exhibited the morphology of typical DC and function of T-cell stimulation and antigen presentation. iPS-DC loaded with cytomegalovirus (CMV) peptide induced vigorous expansion of CMV-specific autologous CD8+ T cells. Macrophages (iPS-MP) with activity of zymosan phagocytosis and C5a-induced chemotaxis were also generated from iPS cells. Genetically modified iPS-MPs were generated by the introduction of expression vectors into undifferentiated iPS cells, isolation of transfectant iPS cell clone and subsequent differentiation. By this procedure, we generated iPS-MP expressing a membranebound form of single chain antibody (scFv) specific to amyloid b (Ab), the causal protein of Alzheimer’s disease. The scFvtransfectant iPS-MP exhibited efficient Ab-specific phagocytosis activity. iPS-MP expressing CD20-specific scFv engulfed and killed BALL-1 B-cell leukemia cells. Anti-BALL-1 effect of iPS-MP in vivo was demonstrated in a xeno-transplantation model using severe combined immunodeficient mice. In addition, we established a xeno-free culture protocol to generate iPS-DC and iPS-MP. Collectively, we demonstrated the possibility of application of iPS-DC and macrophages to cell therapy. Gene Therapy (2011) 18, 874–883; doi:10.1038/gt.2011.22; published online 24 March 2011 Keywords: dendritic cells; macrophages; induced pluripotent stem cells; HLA; cancer; Alzheimer’s disease

INTRODUCTION Dendritic cells (DCs) are specialized to control T-cell response. They capture and process antigenic proteins, and present antigenic peptides derived from the proteins in the context of major histocompatibility complex molecules to stimulate antigen-specific T cells. Medical application of DC to manipulate immune response has been attracting much attention. In particular, DC-based cellular vaccination is regarded as a powerful means for active immune therapy for cancer. Clinical trials of anticancer therapy with DC loaded with various cancer antigens have been conducted. In most cases, monocytes obtained from the patients by apheresis are used as cell source of DC. The number of monocytes obtained from peripheral blood is limited even with apheresis, and the potential of monocytes to differentiate into DC varies among the blood donors. The limitation of the cell source thus remains one of the major obstacles for broader application of DC therapy. Macrophages have essential roles in the maintenance of homeostasis in many tissues. Macrophages are engaged in the elimination of invading pathogens and clearance of dying cells. The therapeutic application of macrophages, for example, use of macrophages activated with interferon (IFN)-g in the treatment of cancer patients, has been clinically tested.1 As is the case of DC therapy, the issue of a limited supply of the cells is one of the reasons that hamper wide application of macrophage therapy. Embryonic stem (ES) cells possess pluripotency and unlimited capacity to propagate. Several groups including us previously established methods to generate DC or macrophages from mouse or

human ES cells.2–11 Using mouse models, we have demonstrated the usefulness of genetically modified ES cell-derived DC in the induction of anticancer immunity12–17 and also in the negative regulation of immune response to control of autoimmune disease.18,19 ES cell-like pluripotent stem cells can be generated by the simultaneous introduction of several factors, into somatic cells, yielding induced pluripotent stem (iPS) cells.20–24 This technology allows us to generate human pluripotent stem cells without human embryo, and we can overcome the issues of histoincompatibility and ethical problems associated with the use human ES cells. Previously, we have reported generation of DC and macrophages from mouse iPS cells.25 Generation of myeloid-lineage cells including DC and macrophages from human iPS cells has also been reported recently.26 In this study, we generated DC and macrophages from human iPS cells and assessed the possibility of their clinical application. RESULTS Generation of human iPS cells In this study, we established human iPS cell clones by lentivirusmediated transfer of Oct3/4, Sox2, c-Myc and Klf4 transgenes into dermal fibroblasts obtained from a healthy donor. For some iPS cell clones, we removed transgenes for c-Myc and Sox2 by Cre recombinase-mediated DNA deletion, resulting in generation of iPS cell clones with no transgenes for c-Myc and Sox2. The procedure to generate iPS cells and characterization of them are shown in Supplementary Figures S1 and S2. We used both types of iPS cells (c-Myc and Sox2 transgene-deleted or not) in this study.

1Department of Immunogenetics, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan and 2CREST, Japan Science and Technology Agency, Kawaguchi, Japan Correspondence: Dr S Senju, Department of Immunogenetics, Graduate School of Medical Sciences, Kumamoto University, Honjo 1-1-1, Kumamoto 860-8556, Japan. E-mail: [email protected] Received 16 August 2010; revised 16 November 2010; accepted 16 November 2010; published online 24 March 2011

Human iPS cell-derived dendritic cells and macrophages S Senju et al 875

Figure 1 Morphological change of cells during differentiation from human iPS cells to DCs. Phase-contrast images of first step day 1 (a), first step day 6 (b), first step day 15 (c, d), second step day 7 (e), immature iPS-DC (f) and mature iPS-DC (g) are shown. (h) May–Grunwald–Giemsa staining of mature iPS-DC on a glass slide is shown.

Differentiation of human iPS cells into DCs We tried to induce differentiation of the established iPS cell clones into DCs (iPS-DC). Schematic depiction of the differentiation culture is in Supplementary Figure S3. To induce hematopoietic differentiation, we cultured iPS cells on the feeder layers of mouse OP9 cells. The shape of the iPS cell colonies on OP9 feeders was dome-like until days 3–4 (Figure 1a). After then, the propagated cells formed large flat cell clusters (Figure 1b). Cystic structures (Figure 1c) and clusters of cobble stone-like cells (Figure 1d) appeared after day 10. At around day 17, cells were recovered and dissociated into single cells. Then, adherent cells were removed and non-adherent cells were cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and M-CSF without OP9 cell layers, to begin the second step culture. Thereafter, small round floating cells and also adherent cells appeared (Figure 1e). Floating cells gradually increased and most of them expressed CD45, CD31 and CD43, and about half of them expressed CD11b at this time point (Figure 2a). For generation of DCs (iPS-DC), the floating or loosely adherent cells were harvested by pipeting, transferred to Petri dishes, and cultured in the presence of GM-CSF and interleukin (IL)-4 (the third step). Cells with protrusions appeared in 3–5 days (Figure 1f). They expressed CD40, CD86 and human leukocyte antigen (HLA) class I, but did not express CD80, CD83 and HLA class II (Figure 2b). After stimulation with tumor necrosis factor (TNF)-a, lipopolysaccharide (LPS) and IL-4, iPS-DC showed morphology of mature DC (Figures 1g and h), and expressed CD80, CD86, CD40, HLA class I and HLA class II (Figure 2b). iPS-DC produced TNF-a and IL-12 upon stimulation with LPS and OK432, respectively (Figures 2c and d). Collectively, we generated iPS-DC with DC-like morphology, surface molecules and cytokine production. Antigen-presentation and T-cell stimulation by human iPS-DC The capacity of the human iPS-DC to stimulate naive T cells was examined based on the stimulation of allogeneic T cells (Figure 3a). In this assay, iPS cell-derived cells recovered from the second step (myeloid-lineage precursor cells) and immature iPS-DC induced low magnitudes of proliferation of the T cells. In contrast, mature iPS-DC induced a robust proliferative response of allogeneic T cells, indicating their potent capacity to stimulate T cells.

Next, HLA class II-restricted antigen presentation by iPS-DC was examined. The iPS cells were positive for the HLA-DRB4*0103 gene encoding b-chain of the HLA-DR53 molecule, and thus DR53 (HLA-DRB4*0103)-restricted antigen presentation by iPS-DC was examined. We used a GAD65-specific DR53-restricted human T-cell clone, SA32.5, as responder T cells. As shown in Figure 3b, iPS-DC pre-loaded with GAD65 peptide specifically induced proliferative response SA32.5T cells. To examine the capacity to process antigenic protein and present resulting peptides, recombinant GAD65 protein was also used as the antigen (Figure 3c). The SA32.5T cell clone co-cultured with the iPS-DC pre-incubated with recombinant GAD65 protein showed dose-dependent response, indicating that iPS-DC were capable of processing antigenic protein. Next, stimulation of antigen-specific CD8+ T cells by iPS-DC was examined. iPS-DC, positive for HLA-A*2402, were loaded with HLAA*2402-restiricted cytomegalovirus (CMV) pp65 peptide and co-cultured with autologous peripheral blood T cells. After 9 days of culture, cells were harvested and stained with tetramer of HLAA*2402-CMV peptide complex. As shown in Figure 3d, 3.8% of the T cells cultured with peptide-loaded iPS-DC were positively stained with both anti-CD8mAb and the HLA-peptide tetramer, while 0.1 % of the T cells cultured with non-peptide-loaded iPS-DC were so. These results indicate that peptide-loaded iPS-DC stimulated antigenspecific T cells in autologous T cells and induced their expansion. Collectively, iPS-DC exhibited not only morphology and surface phenotype of DC but also functional characteristics as DC to stimulate T cells. Characterization of macrophages generated from iPS cells We generated macrophages (iPS-MP) by culturing the myeloid-lineage cells recovered from step 2 in the presence of GM-CSF and M-CSF in Petri dishes. The resultant iPS-MP showed macrophage-like morphology (Figures 4a and b) and expressed CD11b, CD14 and CD68 (Figure 4c), the molecules expressed by physiological macrophages. We added fluorescein isothiocyanate (FITC)-labeled zymosan particles to iPS-MP culture and microscopically observed the phagocytosis (Figure 4d). iPS-MP ingested both immunoglobulin G (IgG)-opsonized and unopsonized zymosan particles (Figure 4e). In addition, iPS-MP showed chemotactic response to C5a, with a peak response at Gene Therapy


Figure 2 Cell surface molecules and cytokine production of differentiated cells. (a) iPS cell-derived floating cells (precursor cells) at day 8 in the second step were analyzed for the expression of CD31, CD34, CD45, CD43 and CD11b. (b) iPS-DC before (immature iPS-DC) or after (mature iPS-DC) exposure to maturation stimuli, TNF-a plus LPS, were analyzed on the expression of CD80, CD86, CD83, CD40, HLA class I and HLA class II. Staining profiles with specific mAb (thick lines) and isotype-matched control mAb (thin lines) are shown. (c, d) iPS-DC (1105 cells per 200 ml) were added with TNF-a (10 ng ml–1), LPS (3 mg ml–1), or OK432 (10 or 20 mg ml–1) as indicated, and concentration of TNF-a (c) and IL-12 p70 (d) in the culture supernatant after 60 h was measured by enzyme-linked immunosorbent assay (ELISA). Data represent the mean value + s.d. of triplicate cultures.

30 ng ml–1 of C5a (Figure 4f). Collectively, iPS-MPs were functional in phagocytosis and chemotaxis. Amyloid b (Ab)-specific phagocytic activity of iPS-MP expressing Ab-specific antibody The accumulation of Ab peptide in the brain is causatively involved in the patho-physiology of Alzheimer’s disease. Macrophages with a capacity to efficiently clear Ab are expected to be useful for the treatment of this disease. We generated iPS-MP expressing membranebound form of single chain antibody (scFv) specific to Ab and tested their Ab-specific phagocytic activity. The scFv was linked to mouse Fcg-receptor I (FcgRI), and c-Myc-tag was added to detect the expression of scFv on the cell surface (Figure 5a). The expression vector for the scFv was introduced into undifferentiated iPS cells by electroporation, and subsequently the transfectant iPS cell clones were induced to differentiate. The expression of the scFv by the cells recovered from the second step of the differentiation culture was analyzed by flow cytometry to detect the c-Myc-tag (Figure 5b). We coated fluorescent micro beads with Ab and subsequently with bovine serum albumin (BSA) (Ab-beads). When the Ab-beads were added to the iPS-MP expressing scFv specific to Ab, many fluorescent microbeads were observed inside of the iPS-MP after overnight culture, indicating efficient phagocytosis (Figures 5c and d). Flow cytometry analysis of iPS-MP recovered from the culture with fluorescent beads indicated that iPS-MP expressing the scFv ingested Gene Therapy

Ab-beads more efficiently than those coated with BSA only (BSAbeads) (Figure 5e). The phagocytosis of Ab-beads and BSA-beads by the transfectant iPS-MP expressing scFv and non-transfectant iPS-MP was comparatively analyzed. In consistent with a previous report showing enhancement of phagocytosis activity by Ab,27 even nontransfectant iPS-MP ingested Ab-beads more efficiently than BSAbeads (Figure 5f). However, the difference between the efficiency of phagocytosis of Ab-beads and that of BSA-beads was more dramatic in case of iPS-MP expressing the scFv than in case of non-transfectant iPS-MP (Figure 5g). These results indicate that the expression of Ab-specific scFv enhanced the Ab-specific phagocytosis activity of iPS-MP. Anticancer activity of iPS-MP expressing cancer antigen-reactive scFv CD20 is expressed on most of human B-cell leukemia and lymphoma cells. We generated iPS-MP expressing scFv specific to human CD20 (Figures 6a and b). iPS-MP expressing scFv were co-cultured with carboxyfluorescein succinimidyl ester-labeled BALL-1, a human B-cell leukemia cell line. Many iPS-MP engulfed BALL-1 cells and most of the engulfed BALL-1 cells were digested in inside of the iPS-MP during overnight co-culture (Figure 6c). To quantitatively evaluate the effect of iPS-MP on BALL-1 cells, BALL-1 cells expressing firefly luciferase (5103 cells per well in 96-well plates) were cultured with iPS-MP (2.5104 cells per well)


Figure 3 T-cell stimulation and antigen presentation by iPS-DC. (a) Indicated numbers of mature iPS-DC (diamonds), immature iPS-DC (triangles), or myeloid-lineage cells isolated from second step culture (circles) were X-ray-irradiated (40 Gy) and co-cultured with allogeneic peripheral blood T cells (4104 cells per well) in a 96-well round-bottomed culture plate for 5 days. The proliferation of T cells was measured based on [3H]-thymidine-uptake in the last 16 h of the culture. The data are indicated as the mean value ±s.d. of duplicate cultures. (b) The indicated numbers of iPS-DC pre-loaded with human GAD65111131 peptide (open circles) or those left un-pulsed (closed circles) were X-ray irradiated and co-cultured with a GAD65-specific HLA-DR53restricted human CD4+ T-cell clone, SA32.5 (3104 cells per well) for 3 days. The proliferation of the T cells in the last 16 h of the culture was measured by [3H]-thymidine uptake. (c) iPS-DC (1104 cells per well) were cultured in the presence of indicated concentrations of glutathione S-transferase (GST)GAD65 recombinant protein (open circles) or GST protein (closed circles). After 16 h, they were X-ray irradiated, added with SA32.5 cells (3104 cells per well), and then were cultured for an additional 3 days. The proliferation of the T cells was measured by [3H]-thymidine uptake in the last 16 h of the culture. (d) iPS-DC loaded with HLA-A*2402-restricted CMV pp65-derived peptide were X-ray irradiated and co-cultured with autologous peripheral blood T cells in the presence of IL-2 and IL-7. The cells were recovered after culture for 9 days, stained with phycoerythrin (PE)-conjugated tetramer of HLA-A*2402/CMVpeptide-complex and FITC-conjugated anti-CD8mAb, and analyzed by flow cytometry. The results of T cells cultured with peptide-loaded or -unloaded iPS-DC are shown. The numbers in the figures indicate the frequency of the T cells positive for both the HLA-peptide tetramer and CD8.

expressing anti-CD20 scFv. After 3 days of co-culture, the number of live BALL-1 cells in each well was determined based on luciferase activity. As shown in Figure 6d, co-culture with iPS-MP expressing anti-CD20 scFv dramatically reduced the number of live BALL-1 cells (Figure 6d). On the other hand, co-culture with non-transfectant iPS-MP without anti-CD20 scFv did not significantly reduced the number of BALL-1 cells. The effect of IFN-g to enhance antitumor activity of macrophages has been reported,28–30 and thus we examined the effect of IFN-g on the anti-BALL-1 activity of iPS-MP in our system. In the presence of IFN-g, even iPS-MP without expression of scFv reduced live BALL-1 cells to less than half (Figure 6d), indicating antitumor effect of iPS-MP was enhanced by IFN-g. Next, the anti-BALL-1 effect of iPS-MP was examined in vivo. BALL-1 cells were injected into the peritoneal cavity of severe combined immunodeficient mice. One group of mice were simultaneously injected with iPS-MP expressing anti-CD20 scFv, and others were injected with non-transfectant iPS-MP. After 3 days, the luciferase activity in lysates of greater omentum of the mice was measured to quantify the number of live BALL-1 cells in the tissue. The results indicate that iPS-MP expressing anti-CD20 scFv but not those without scFv significantly inhibited the growth of BALL-1 cells in vivo (Figure 7a). Antitumor effect of iPS-MP expressing anti-CD20 scFv was demonstrated also by in vivo luminescence imaging analysis detecting live BALL-1 cells in the peritoneal cavity of the mice (Figures 7b and c).

Collectively, these results show antitumor activity of iPS-MP expressing scFv reactive to a tumor cell surface antigen both in vitro and in vivo. The microscopic observation (Figure 6c) suggests a possibility that iPS-MP ingested the live tumor cells and subsequently destroyed them. Generation of iPS-DC and iPS-MP under xeno-free condition For the clinical application, we need to generate iPS-DC and iPS-MP in a xeno-free condition. We tried to establish a differentiation culture procedure without use of feeder cells and serum-containing culture media. We tested various kinds of commercially available serum-free culture media. On the basis of the results of comparative experiments, we established a three-step adherent differentiation culture to generate iPS-DC and iPS-MP from human iPS cells, as follows (see Supplementary Figure S4 for a schematic depiction of this procedure). To initiate differentiation, we started to culture iPS cells in fibronectin-coated culture dishes with the medium used for undifferentiated iPS cells (Supplementary Figure S5A). When the cells grew to sub-confluence, we changed the medium with a 1:1 mixture of AIM-V and Peprogrow III containing BMP-4. In about 10 days, cystic structures and also cobble stone-like round cell clusters appeared, similar to those observed when serum-containing medium was used (Supplementary Figure S5B). Thereafter, the culture medium is replaced with Peprogrow III containing BMP-4 and the cells were cultured for additional 10–14 days. Gene Therapy


Figure 4 Generation of macrophages from iPS cells. (a) A phase-contrast image of iPS-MP is shown. (b) May–Giemsa-stained iPS-MPs on a glass slide are shown. (c) iPS-MPs were analyzed for the expression of CD11b, CD14, and CD68. Staining profiles with specific mAb (thick lines) and isotype-matched control mAb (thin lines) are shown. (d) iPS-MPs were incubated with FITC-labeled zymosan particles and stained with 4,6-diamidino-2-phenylindole (DAPI). A phase-contrast image (upper) and a fluorescence view (lower) are shown. (e) iPS-MP in 48-well culture plates were added with FITC-labeled zymosan particles, opsonized with IgG or non-opsonized. After incubation for the indicated period, cells were harvested by using trypsin/EDTA and then analyzed on a FACScan flow cytometer. Percentages of cells with high fluorescence intensity indicating intracellular zymosans are shown. Data shown are mean±s.d. of duplicate assays. (f) C5a-induced chemotaxis of iPS-MP was analyzed using 24-well culture plates with permeable membrane inserts (pore size¼5 mm).

Figure 5 Enhanced Ab-specific phagocytotic activity of iPS-MP expressing anti-Ab scFv. (a) The structure of FcgRI-fused form of scFv specific to Ab is shown. VH and VL indicate variable regions of heavy chain and light chain, respectively. (b) Cell surface expression of scFv specific to Ab on the genetically modified iPS-MP was detected by staining with anti-cMyc mAb. Histogram profiles of flow cytometric analysis of iPS-MP without (upper) or with (lower) the expression of scFv are shown. The thick lines and the thin lines indicate staining with anti-cMyc mAb and isotype-matched mAb, respectively. (c, d) Fluorescent microbeads coated with Ab were added to iPS-MP expressing scFv specific to Ab. The cells were microscopically observed after incubation for 16 h. Phase-contrast (c) and fluorescence (d) views of the same visual field are shown. (e) iPS-MP expressing Ab-specific scFv incubated with fluorescent microbeads coated with Ab (Ab-beads, thick line) or with BSA only (BSA-beads, thin line) were analyzed by flow cytometry to quantitate the phagocytosis. (f, g) An analysis of phagocytosis of micro beads by iPS-MP expressing Ab-specific scFv (g) or not (f) was done with graded concentrations of the beads. The cell samples were analyzed on a flow cytometer, and geometrical mean fluorescence intensity (Geo MFI) for each sample was calculated. Open and closed circles indicate the results with Ab-beads and BSA-beads, respectively. Mean values of duplicate assays are indicated. Gene Therapy


Figure 6 Antitumor activity of iPS-MP expressing anti-CD20 scFv in vitro. (a) The structure of FcgRI-fused form of anti-CD20 scFv is shown. VH and VL indicate variable regions of heavy chain and light chain, respectively. (b) Cell surface expression of scFv on iPS cell-derived myeloid cells was detected by staining with anti-c-Myc-tag mAb. (c) Carboxyfluorescein succinimidyl ester-labeled human B-cell leukemia cells, BALL-1 (green), were added to PKH26labeled iPS-MP expressing CD20-specific scFv (red). The culture was observed after 24 h. Phase-contrast (upper left), fluorescent (upper right: carboxyfluorescein succinimidyl ester, lower left: PKH26), and merged fluorescent (lower right) images are shown. (d) Luciferase gene-introduced BALL-1 cells (5103 cells per well in a 96-well culture plate) were cultured solely or co-cultured with iPS-MP (2.5104 cells per well) expressing anti-CD20 scFv specific to CD20 or not, in the presence (open bars) or absence (closed bars) of IFN-g (500 U ml–1). Number of live BALL-1 cells indicated by luciferase activity was measured after culture for 3 days. The data are indicated as the mean+s.d. of triplicate assays. *Po0.005. The color reproduction of this figure is available on the html full text version of the manuscript.

Figure 7 Antitumor activity of iPS-MP expressing anti-CD20 scFv in vivo. (a) severe combined immunodeficient (SCID) mice were injected with luciferaseexpressing BALL-1 cells (6106 cells per mouse) solely (no iPS-MP, n¼10) or simultaneously with iPS-MP expressing scFv specific to CD20 (iPS-MP-antiCD20, n¼4) or not (iPS-MP, n¼3). IFN-g was injected into all mice simultaneously with the cells. After 3 days, luciferase activity of the tissue lysates of greater omentum was measured. Data are indicated as mean+s.d. for each group. (b, c) SCID mice were injected with luciferase-expressing BALL-1 cells (2107 cells per mouse) solely (no iPS-MP, n¼2) or simultaneously with iPS-MP (6107 cells per mouse) expressing scFv specific to CD20 (iPS-MP-antiCD20, n¼3). IFN-g (1104 U per mouse) and M-CSF (1 mg per mouse) was injected into all mice simultaneously with the cells. After 4 days, the mice were anesthetized, injected with D-luciferin, and analyzed on an in vivo imaging system. Luminescence images (b) and photon-counts per mouse (c) are shown. *Po0.01 and **Po0.05.

After a total of 25–28 days of the first step culture, the differentiating cells were harvested by using trypsin/collagenase/DNase I. Then, after removal of adherent cells, the second step culture was started using a 1:1 mixture of OpTmizer and macrophage serum free medium

(myeloid cell medium) supplemented with GM-CSF and M-CSF. After this passage, we observed many dead cells as well as live cells floating or adherent (Supplementary Figure S5). In the following 7 to 10 days, the floating live cells positive for CD31, CD45, CD43 and Gene Therapy


CD11b (Supplementary Figure S5F) gradually increased. After 10–15 days of the second step culture, we recovered floating cells and proceeded to the third step culture. iPS-DC were generated by culturing the cells recovered from step 2 in myeloid cell medium supplemented with GM-CSF and IL-4. iPSDC exposed to maturation stimuli, TNF-a plus LPS, showed typical morphology of DC with many protrusions (Supplementary Figures S5D and E) and expression of CD80, CD86, CD40, HLA class II (Supplementary Figure S5G). Mature iPS-DC induced robust proliferative response of allogeneic T cells, upon co-culture (Supplementary Figure S6A). They produced TNF-a and IL-12 in response to LPS and OK432, respectively (Supplementary Figures S6B, C). In addition, iPS-MPs were generated by continuing step 2 culture for 415 days (Supplementary Figures S4 and S5H). Although these results show that DCs and macrophages can be generated from human iPS cells in a xeno-free condition, this procedure was not applicable to all of the iPS cell clones that gave rise to iPS-DC and iPS-MP when subjected to the conventional culture procedure. In the xeno-free condition, we could generate iPS-DC and iPS-MP from two out of five iPS cell clones so far tested. On the other hand, other three clones died during the differentiation culture. Thus, further improvement of the method may be necessary. DISCUSSION Alzheimer’s disease is the most common cause of senile dementia. No therapies have been proved to improve long-term prognosis of this disease. Ab a peptide composed of 39–43 amino acids, is the main constituent of amyloid plaques observed in the brain of the patients.31,32 The causative involvement of Ab in the onset of this disease has been established by the results of genetic analysis of hereditary Alzheimer’s disease and by studies using transgenic mouse models.33–37 Soluble oligomers of Ab are reportedly the most toxic to the neurons.38–41 Studies with model mice support the notion that microglias, the phagocytes in the brain, function to protect the neurons through clearance of Ab.42,43 On the other hand, other studies suggest that the stimulated microglias worsened this disease by causing inflammation.44,45 In this study, we demonstrated Ab-specific phagocytic activity of iPS-MP expressing scFv specific to this molecule. Local administration of a sufficient number of macrophages capable of clearing Ab without causing inflammation could provide a promising therapy to treat this disease. Genetically modified iPS cells may well be a useful cell source for such macrophages. A strategy of therapy with macrophage with scFv may also be applied for treatment of other diseases caused by the accumulation of misfolded proteins, such as prion diseases and various types of amyloidosis. Infiltration of macrophages is observed in most cases of cancers and they have either pro- or antitumor effect, depending on various factors such as local cytokine patterns. Anticancer effect of human monocyte-derived macrophages genetically modified to express cancer antigen-specific scFv has been reported by Biglari et al.46 In this study, iPS-MP expressing anti-human CD20 scFv inhibited the growth of human B-cell leukemia cells in vitro and in vivo, suggesting a possibility of anticancer therapy with iPS-MP expressing anticancer antigen scFv. The repetitive administration of a large number of macrophages may be necessary to obtain therapeutic effect in clinical settings. The infinite capacity for proliferation of iPS cells will provide a huge advantage. It is expected that the economic issue associated with the generation of patient-specific iPS cells may be resolved by iPS cell banks covering major HLA-haplotypes in each ethnic group.47 Gene Therapy

In summary, functional DCs and macrophages were generated from human iPS cells. We demonstrated potential of therapeutic application of genetically modified iPS-MP to Alzheimer’s disease and cancer. In addition, to realize clinical application, we developed a method of xeno-free differentiation culture to generate iPS-DC and iPS-MP. MATERIALS AND METHODS Cells and reagents A human B-cell leukemia cell line, BALL-1, and a mouse bone marrow stromal cell line, OP9, were provided by the RIKEN BioResource Center (Tsukuba, Japan). LPS from Escherichia coli and OK-432 were purchased from Sigma Chemicals (St Louis, MO, USA) and Chugai Pharmaceutical (Tokyo, Japan), respectively. Generation and purification of glutathione S-transferase-fused human glutamic acid decarboxylase 65 protein fragment (GAD65p96–174) was done as described previously.11

Generation of human iPS cells This study was approved by the institutional review board of Kumamoto University. Complementary DNAs coding for human Oct3/4, Sox2, Klf4 and c-Myc were obtained by PCR and cloned into pENTR-TOPO vector (Invitrogen, Carlsbad, CA, USA) and subsequently transferred to a self-inactivating lentiviral vector construct, pCSII-EF.48 The plasmids for lentiviral vector packaging were kindly provided by Dr H Miyoshi (RIKEN BioResource Center). Dermal fibroblasts obtained from a healthy donor positive for HLA-A*2402 and DRB4*0102 were used for generation of iPS cells. The fibroblasts were infected with the recombinant lentivirus to introduce the four reprogramming factor genes. From 6 days after the infection, the cells were cultured on the feeder layers of primary mouse embryonic fibroblasts in the culture medium for human ES cells.21,49 At 25–30 days after the infection, colonies showing human ES cell-like morphology were picked up, expanded, and characterized. For some of the iPS cell clones, transgenes for Sox2 and c-Myc were deleted by CreLox recombinase-mediated DNA excision (Supplementary Figures S1 and S2).

Differentiation culture with OP9 feeder and fetal calf serum The three steps of the culture procedure with the use of OP9 feeder layers and fetal calf serum (FCS) are as follows (schematic depiction shown in Supplementary Figure S3). Step 1: undifferentiated iPS cells maintained on primary mouse embryonic fibroblast were harvested by using dissociation solution, collagenase-trypsin-knockout serum replacement,49 and cultured on OP9 feeder cell layers in a-minimal essential medium supplemented with 20% FCS for 14–18 days. Then, the cells were treated with trypsin/collagenase/ DNase I solution (phosphate-buffered saline containing 0.25% trypsin, 0.1% collagenase type IV, 0.1% DNase I) and recovered. The cells were plated onto culture dishes and incubated for overnight. Thereafter, floating or weakly adherent cells were recovered by pipeting, and firmly adherent cells were discarded. Step 2: the cells were passed through nylon meshes (Cell Strainer 100 mm, BD Falcon, Bedford, MA, USA), suspended in the medium containing GM-CSF (100 ng ml–1) and M-CSF (50 ng ml–1), and cultured in tissue culture dishes for 10–14 days. Step 3: to generate DCs (iPS-DC), the cells recovered from step 2 were cultured in RPMI-1640 medium containing 10% FCS, GM-CSF (100 ng ml–1) and IL-4 (10 ng ml–1) in bacteriological Petri dishes (Locus, Tokyo, Japan) for 3–7 days. IL-4 (10 ng ml–1), TNF-a (10 ng ml–1), LPS (3 mg ml–1) or OK-432 (10 mg ml–1) were added to induce maturation of iPS-DC. For generation of macrophages (iPS-MP), cells recovered from step 2 were cultured in RPMI-1640 medium containing 10% FCS, GM-CSF (100 ng ml–1) and M-CSF (50 ng ml–1).

Xeno-free differentiation culture The xeno-free differentiation culture also involved three steps. Step 1: iPS cells were harvested by collagenase-trypsin-knockout serum replacement, and cultured in human ES cell medium in tissue culture dishes pre-coated with fibronectin (10 mg ml–1) for 60 min. When the cells grew to sub-confluence, culture medium was changed to a 1:1 mixture of Peprogrow III (PeproTech, Rocky Hill, NJ, USA) and AIM-V (Life Technologies, Carlsbad, CA, USA) containing BMP-4 (5 ng ml–1). The medium was changed to Peprogrow III containing BMP-4 after 7–10 days. At days 20–28, the cells were recovered by

Human iPS cell-derived dendritic cells and macrophages S Senju et al 881 treatment with trypsin/collagenase/DNase I. Subsequently, firmly adherent cells were removed by incubation in tissue culture dishes coated with fibronectin. Step 2: after passage through nylon meshes, cells were cultured in myeloid cell medium, 1:1 mixture of macrophage serum free medium (Life Technologies) and OpTmizer T-cell expansion serum free medium, containing GM-CSF (100 ng ml–1) and M-CSF (50 ng ml–1) for 7 to 18 days. Step 3: the cells were recovered from step 2 culture and cultured in myeloid cell medium containing GM-CSF (100 ng ml–1) and IL-4 (10 ng ml–1) for 3–7 days to generate DCs. Macrophages were generated by extension of the step 2 culture for 418 days, or by transferring the cells to Petri dishes and culturing them in the same medium.

Flow cytometric analysis The following mAb conjugated with FITC or PE were purchased from Pharmingen (San Diego, CA, USA) or eBioscience (San Diego, CA, USA): anti-CD31 (clone WM59, mouse IgG1), anti-CD34 (clone 581, mouse IgG1) anti-CD45 (clone HI30, mouse IgG1), anti-CD43 (clone 1G10, mouse IgG1), anti-CD11b (clone ICRF44, mouse IgG1), anti-HLA-DR, DQ, DP (clone TU39, mouse IgG2a), anti-HLA-A, B, C (clone G46–2.6, mouse IgG1), anti-CD80 (clone L307.4, mouse IgG1), anti-CD83 (clone HB15e, mouse IgG1), anti-CD86 (clone FUN-1, mouse IgG1), anti-CD40 (clone 5C3, mouse IgG1), anti-CD14 (clone 61D3, mouse IgG1) and anti-CD68 (clone Y1/82A, mouse IgG2b). As isotype-matched controls, mouse IgG2a (clone G155–178) and mouse IgG1 (clone MOPC-21) were used. The cell samples were treated with FcR-blocking reagent (Miltenyi Biotec, Bergish-Gladbach, Germany) for 10 min, stained with the fluorochrome-conjugated mAb for 30 min, and washed three times with phosphate-buffered saline/2% FCS. The stained cell samples were analyzed on a FACScan (Becton Dickinson, Franklin Lakes, NJ, USA) flow cytometer.

Quantitation of cytokine production The cells were cultured in 96-well flat-bottomed culture plates (1105 cells/200 ml medium per well) in the presence of TNF-a (10 ng ml–1), LPS (3 mg ml–1) or OK432 (10–20 mg ml–1), as indicated. The supernatant was collected after 60 h of culture, and the concentration of TNF-a and IL-12p70 was measured using enzyme-linked immunosorbent assay kits (Pierce, Rockford, IL, USA).

Allogeneic T-cell stimulation assay T cells (4104 per well) were purified from peripheral blood mononuclear cells obtained from an allogeneic donor by using T-cell isolation kit (Miltenyi Biotec) and co-cultured with graded numbers of X-ray-irradiated (45 Gy) stimulator cells in RPMI-1640 medium supplemented with 10% human plasma in 96-well round-bottomed culture plates for 5 days. [3H]-methylthymidine (247.9 Gbq mmol–1) was added to the culture (0.037 Mbq per well) in the last 16 h. The cells were harvested onto glass fiber filters (Wallac, Turku, Finland) and the incorporation of [3H]-thymidine was measured by scintillation counting.

Antigen presentation assay CD4+

A human T-cell clone, SA32.5, recognizing GAD65p111131 (LQDVMNI LLQYVVKSFDRSTK) in the context of HLA-DR53 (DRA*0101+DRB4*0103) was established and maintained as previously described.50,51 In the assay with the synthetic peptide, iPS-DC stimulated with TNF-a plus LPS were incubated with the peptide (20 mM) for 3 h, washed, and X-ray-irradiated (45 Gy). SA32.5T cells were cultured in a 96-well flat-bottomed culture plate (3104 cells per well) with graded numbers of the peptide-loaded iPS-DC in AIM-V medium (Life Technologies) supplemented with 10% human plasma. In the assay with recombinant proteins, glutathione S-transferase or glutathione S-transferase–GAD fusion protein was added to iPS-DC (1104 cells per well) and cultured overnight in a 96-well plate. Thereafter, the culture plate was irradiated and SA32.5T cells (3104 cells per well) were added. Co-culture of T cells and DC was continued for 3 days, and incorporation of [3H]-thymidine during the last 16 h of culture was measured.

Stimulation of CMV-specific human CD8+ T cells iPS-DC stimulated with TNF-a were irradiated, incubated with CMV pp65 peptide (QYDPVAALF, 10 mM) for 3 h, and plated into 48-well culture plates (4104 cells per well). T cells were purified from autologous peripheral blood

mononuclear cells and added to the wells (4105 cells per well). The cells were cultured in AIM-V containing 10% autologous plasma and recombinant human IL-7 (5 ng ml–1), and human IL-2 (20 U ml–1) was added at day 2. At day 9, cells were harvested, stained with phycoerythrin-labeled tetramer of HLA-A*2402/CMV peptide complex in combination with FITC-labeled anti-human CD8 mAb (clone T8), and analyzed by flow cytometry.

Chemotaxis assay A multi-well chemotaxis assay was done by using 24-well Transwell permeable support plates (pore size 5 mm, Corning, NY, USA). Cells were suspended in AIM-V medium and added to the upper compartments (1105 cells /0.1 ml per well). Indicated concentrations of recombinant human C5a (R&D Systems, Minneapolis, MN, USA) in AIM-V medium was added to the lower compartments (0.6 ml per well). Assay plates were incubated at 37 1C for 90 min and then the cells on the upper surface of the microporous membranes were removed by using swabs. Subsequently, the membranes were fixed with methanol for 5 min and stained with Giemsa solution for 15 min. The stained cells on the lower surface of the membranes were counted and the data are indicated as the number of cells per 1 mm2.

Zymosan phagocytosis assay The cell suspensions in Dulbecco’s modified Eagle’s medium/10% FCS were added to 48-well culture plates (2105 cells/200 ml per well) and incubated at 37 1C for 2 h to allow the cells to adhere to the plates. FITC-labeled Zymosan A particles (Molecular Probes, Carlsbad, CA, USA), opsonized or unopsonized, were added to the wells (2106 particles/200 ml per well). After incubation for the indicated period, cells were harvested by using trypsin/EDTA and then analyzed on a FACScan (Becton Dickinson) flow cytometer. In some experiments, cells incubated with FITC-zymosan were stained with 4,6-diamidino-2phenylindole and microscopically analyzed (IX70, Olympus, Tokyo, Japan).

Plasmid construction The nucleotide sequences of the complementary DNAs for heavy and light chain (appeared in United States Patent Application 20070238154) of a hybridoma clone 3D6 were used for the design of single chain variable region fragment (scFv) specific to Ab. The double-stranded DNA of the designed sequence was generated by gene synthesis (Genscript, Piscataway, NJ, USA). Complementary DNA fragments for light and heavy chain of hybridoma clone 1F5 producing mAb specific to human CD20 was isolated by reverse transcriptase-PCR and fused by PCR. scFv sequences were linked to nucleotide sequences for a c-Myc tag (EQKLISEEDL) and a C-terminal fragment of mouse Fcg receptor I (FcgRI) by PCR. The protein-coding DNA fragments were first cloned into pENTR-D-Topo (Invitrogen) and subsequently transferred to a mammalian expression vector, pCAG-internal ribosomal entry site-Puro,52 which is driven by the CAG promoter and includes an internal ribosomal entry site-puromycin-resistance gene cassette.

Transfection of iPS cells Single cell suspension of iPS cells (1.5107 cells/0.4 ml culture medium) were mixed with plasmid DNA (35 mg per 100 ml phosphate-buffered saline) in a 4 mm-gap cuvette. Electroporation of iPS cells was performed at 200 V and 800 mF using an electroporation system, Gene Pulser (Bio-Rad, Hercules, CA, USA). After electroporation, the cells were plated on primary mouse embryonic fibroblast feeder layers in 90-mm culture dishes in the presence of Rhoassociated kinase (Rock)-inhibitor (Y27632, Wako, Osaka, Japan), to inhibit death of dissociated cells.53 Selection with puromycin (2–5 mg ml–1) was done from 2 to 4 days after the transfection and puromycin-resistant iPS cell colonies were picked up under microscopic observation at 15–18 days after transfection. Transfectant clones with relatively high levels of expression of the transgene were selected based on their resistance to high dose (5–20 mg ml–1) of puromycin. Thereafter, the clones were subjected to the differentiation procedures. At the second step of differentiation culture, the cells were stained with anti-c-Myc mAb and analyzed by flow cytometry to selected high expresser clones.

Phagocytosis assay with Ab-coated microbeads Fluorescent micro beads (sulfate microspheres, yellow–green, 0.2 mm, Molecular Probes #F8828) were incubated with 30 mg ml of Ab1–42 peptide (Wako Gene Therapy

Human iPS cell-derived dendritic cells and macrophages S Senju et al 882 Chemicals, Osaka, Japan) for 24 h, and subsequently with 2% bovine serum albumin for 24 h. The beads were added to cultures of iPS-MP. After incubation for 16 h, cells were observed on a microscope (Axio Observer Z1, Carl Zeiss, Oberkochen, Germany). For quantitative measurement of phagocytosis, the cells were recovered from the culture plates by using trypsin/EDTA and subjected to flow cytometry analysis.

Fluorescence microscopy analysis of phagocytosis of BALL-1 cells by iPS-MP BALL-1 cells labeled with carboxyfluorescein succinimidyl ester (Dojindo, Kumamoto, Japan) and iPS-MP labeled with PKH-26 (Sigma, St Louis, MO, USA) were co-cultured in 12-well culture plates. After culture for 24 h, the wells were gently rinsed and microscopically analyzed.

Analysis of antitumor activity of iPS-MP For in vitro analysis, BALL-1 cells (5103 cells per well) introduced with firefly luciferase gene were added to iPS-MP (2.5104 cells per well) cultured in 96well flat-bottomed culture plates in the presence or absence of IFN-g. After culture for 3 days, the cell suspensions were recovered by pipeting and transferred into assay plates (B&W Isoplate, Wallac, Waltham, MA, USA) and mixed with substrate solution (SteadyLite Plus, Perkin-Elmer, Waltham, MA, USA). Luminescence activity was measured on a microplate reader (TriStar, Berthold Technologies, Bad Wildbad, Germany). For in vivo assay, BALL-1 cells (6106 cells per mouse) and IFN-g (1104 U per mouse) were injected into the peritoneal cavity of severe combined immunodeficient mice with or without iPS-MP (2.4107 cells per mouse). The greater omentum was isolated from each mouse after 3 days and tissue lysate was prepared. The luciferase activity of the 1/60 (25 ml out of 1.5 ml) of the tissue lysate for each sample was measured. For in vivo imaging analysis, mice were injected i.p. with BALL-1 cells (2107 cells per mouse) and iPS-MP (6107 cells/mouse). After 4 days, D-luciferin (4 mg per mouse) was injected i.p. into the anesthetized mice and luminescence images were obtained by using an in vivo imaging system (NightOWL II, Berthold Technologies).

CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENTS We thank Dr H Miyoshi for a lentivirus vector system and Dr H Niwa for a mammalian expression vector pCAGGS-IRES-PuroR. This work was supported in part by Grants-in-Aid 18014023, 19591172 and 19059012 from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan, the Program of Founding Research Centers for Emerging Infectious Diseases launched as a project commissioned by MEXT, Research Grant for Intractable Diseases from Ministry of Health and Welfare, Japan, grants from Japan Science and Technology Agency (JST), the Uehara Memorial Foundation, and the Takeda Science Foundation.

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Supplementary Information accompanies the paper on Gene Therapy website (http://www.nature.com/gt)

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