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STEM-CELL DERIVED MYELOID CELLS, GENERATION AND USE THEREOF

20210155902 · 2021-05-27

    Inventors

    Cpc classification

    International classification

    Abstract

    The present invention relates to stem-cell derived hematopoietic cells, in particular, myeloid cells, preferably, macrophages, their generation and use. In particular, the invention relates to a method of producing hematopoietic, preferably, myeloid cells, comprising cultivating embryoid bodies, which are, e.g., derivable from pluripotent stem cells such as induced pluripotent stem cells (iPSC), in suspension culture, to produce myeloid cell forming complexes, which are further cultivated in suspension culture to produce myeloid cells such as macrophages. This allows for a scalable and continuous production, e.g., in industry-compatible stirred tank bioreactors. Macrophages, e.g., macrophages produced with this method that have unique characteristics, can be used in pharmaceutical compositions for treatment of patients, e.g., for treatment of infection such as bacterial infection. The invention further provides application systems suitable for spraying, comprising myeloid cells such as macrophages, which can have a reduced size, for use in treatment of patients for treatment of infection, e.g., bacterial infection or wound healing.

    Claims

    1. A method of producing myeloid cells, comprising steps of a) cultivating embryoid bodies in suspension culture in the presence of IL-3 and, optionally, at least one additional cytokine, for a sufficient period of time to produce myeloid cell forming complexes; b) cultivating the myeloid cell forming complexes in the presence of IL-3 and, optionally, at least one additional cytokine, in suspension culture for a sufficient period of time to produce myeloid cells; and c) isolating the myeloid cells.

    2. The method of claim 1, wherein the embryoid bodies are derived from pluripotent stem cells, preferably induced pluripotent stem cells.

    3. The method of claim 1, wherein the embryoid bodies are obtained by a method comprising cultivating pluripotent stem cells in suspension culture for a sufficient period of time to produce embryoid bodies.

    4. The method of claim 1, wherein the suspension culture is carried out in a bioreactor allowing suspension culture selected from the group comprising a stirred tank bioreactor, Erlenmeyer flask, spinner flask, wave bioreactor and rotating wall bioreactor, preferably, a stirred tank bioreactor.

    5. The method of claim 1, wherein the cells are human cells.

    6. The method of claim 1, wherein a) the additional cytokine is M-CSF and the produced myeloid cells are macrophages; or b) the additional cytokine is G-CSF and the produced myeloid cells are granulocytes; or c) the additional cytokine is GM-CSF and the produced myeloid cells are macrophages and granulocytes; or d) the additional cytokines are SCF and EPO and the produced myeloid cells are erythroid cells; or e) the additional cytokines are SCF and TPO and the produced myeloid cells are megakaryocytes and/or thrombocytes; or f) the additional cytokine is GM-CSF and IL-4 and the produced myeloid cells are dendritic cells; or g) there is no additional cytokine to IL-3, and the produced myeloid cells are immature cells capable of further differentiation, wherein, optionally, said method further comprises i) cultivating said immature cells in the presence of M-CSF until macrophages are obtained; or ii) cultivating said immature cells in the presence of G-CSF until granulocytes are obtained; iii) cultivating said immature cells in the presence of GM-CSF until granulocytes and macrophages are obtained; iv) cultivating said immature cells in the presence of SCF and EPO until erythroid cells are obtained; v) cultivating said immature cells in the presence of SCF and TPO until megakaryocytes and/or thrombocytes are obtained; wherein, preferably, the additional cytokine is M-CSF and the produced myeloid cells are macrophages.

    7. The method of claim 1, wherein the method allows for a continuous, preferably, a batch-continuous production of said myeloid cells.

    8. The method of claim 1, wherein isolation comprises purifying the produced myeloid cells, preferably, the macrophages, to a purity of at least 50%, wherein the method optionally further comprises, after step c, reducing the size of the myeloid cells by a method selected from the group comprising contacting the cells with a hypertonic solution and lyophilisation and/or loading the myeloid cells with a therapeutic or diagnostic agent selected from the group comprising an antibiotic agent, an immunomodulating agent and a dye.

    9. A suspension culture comprising myeloid cell forming complexes which continuously produce myeloid cells and, optionally, myeloid cells, wherein, optionally, said suspension culture is obtainable from the method of claim 1b.

    10. A cell population obtainable by the method of claim 1, wherein, preferably, the size of the myeloid cells has been reduced by a method selected from the group comprising contacting the cells with a hypertonic solution and lyophilisation and, optionally, the cells are loaded with a therapeutic or diagnostic agent selected from the group comprising an antibiotic agent and an immunomodulating agent.

    11. A cell population comprising CD45+CD11b+/CD14+/CD163+/CD34−TRA1-60− macrophages, wherein, preferably, expression of at least 9 genes selected from a group consisting of DKK1, SEPP1, PITX2, COL3A1, KRT19, A_33_P3221980, CALD1, CYR61, H19, DDIT4L, FRZB, TMEM98, NNMT, NPNT, LUM, DCN, LYVE1, MGP, IGFBP3 and NUAK1 is at least 20 fold upregulated in said macrophages compared to macrophages derived from PBMC; wherein, optionally, said cell population is obtainable by the method of claim 1.

    12. A cell population comprising immature CD45+CD11b+/CD14−/CD163-myeloid cells, wherein, optionally, said cell population is obtainable by the method of claim 1.

    13. A pharmaceutical composition comprising a cell population of claim 11 in a pharmaceutically acceptable carrier.

    14. An application system comprising a) a pharmaceutical composition comprising myeloid cells in a pharmaceutically acceptable carrier, and b) a container suitable for spraying the pharmaceutical composition, wherein, preferably, the myeloid cells are macrophages selected from the group comprising the cell populations of claim 11, and/or the size of the myeloid cells has been reduced by a method selected from the group comprising contacting the cells with an hypertonic solution and lyophilisation, and/or the myeloid cells are loaded with a therapeutic or diagnostic agent selected from the group comprising an antibiotic agent, an immunomodulating agent and a dye.

    15. A pharmaceutical composition comprising a cell population in a pharmaceutically acceptable carrier, wherein the average size of the cells of the population is reduced by at least 10% compared to a population of said cells in a physiologic saline solution, wherein the cell population preferably is a population of myeloid cells selected from the group comprising the population of claim 11.

    16. The pharmaceutical composition of claim 13 for use in treating or preventing an infection in a patient and/or for promoting wound healing, wherein the pharmaceutical composition preferably is for treatment or prevention of a bacterial infection, most preferably, for treatment of a bacterial infection.

    17. Use of the cell population of claim 11, for a) drug screening and/or drug development; b) disease modelling; c) tissue engineering; d) preparation of bioartificial organisms; e) disinfection; f) coating of materials for transplantation; g) development of biomarkers; or quality control of biological products selected from the group comprising antibodies, hormones, cytokines, drugs, culture medium, and serum.

    18. A method of preparing a protein, comprising carrying out a method of claim 1 and isolating a protein produced by the embryoid bodies, the myeloid cell forming complexes and/or the myeloid cells, wherein, preferably, the protein is a protein secreted into the cell culture medium selected from the group comprising a cytokine, chemokine, a growth factor, a S100 protein and a recombinant protein.

    19. A pharmaceutical composition comprising a cell population of claim 10 in a pharmaceutically acceptable carrier.

    20. A pharmaceutical composition comprising a cell population of claim 12 in a pharmaceutically acceptable carrier.

    21. An application system comprising a) a pharmaceutical composition comprising myeloid cells in a pharmaceutically acceptable carrier, and b) a container suitable for spraying the pharmaceutical composition, wherein, preferably, the myeloid cells are macrophages selected from the group comprising the cell populations of claim 10, and/or the size of the myeloid cells has been reduced by a method selected from the group comprising contacting the cells with an hypertonic solution and lyophilisation, and/or the myeloid cells are loaded with a therapeutic or diagnostic agent selected from the group comprising an antibiotic agent, an immunomodulating agent and a dye.

    22. An application system comprising a) a pharmaceutical composition comprising myeloid cells in a pharmaceutically acceptable carrier, and b) a container suitable for spraying the pharmaceutical composition, wherein, preferably, the myeloid cells are macrophages selected from the group comprising the cell populations of claim 12, and/or the size of the myeloid cells has been reduced by a method selected from the group comprising contacting the cells with an hypertonic solution and lyophilisation, and/or the myeloid cells are loaded with a therapeutic or diagnostic agent selected from the group comprising an antibiotic agent, an immunomodulating agent and a dye.

    23. A pharmaceutical composition comprising a cell population in a pharmaceutically acceptable carrier, wherein the average size of the cells of the population is reduced by at least 10% compared to a population of said cells in a physiologic saline solution, wherein the cell population preferably is a population of myeloid cells selected from the group comprising the population of claim 10.

    24. A pharmaceutical composition comprising a cell population in a pharmaceutically acceptable carrier, wherein the average size of the cells of the population is reduced by at least 10% compared to a population of said cells in a physiologic saline solution, wherein the cell population preferably is a population of myeloid cells selected from the group comprising the population of claim 12.

    25. The pharmaceutical composition of 15 for use in treating or preventing an infection in a patient and/or for promoting wound healing, wherein the pharmaceutical composition preferably is for treatment or prevention of a bacterial infection, most preferably, for treatment of a bacterial infection.

    26. The application system of claim 14 for use in treating or preventing an infection in a patient and/or for promoting wound healing, wherein the pharmaceutical composition preferably is for treatment or prevention of a bacterial infection, most preferably, for treatment of a bacterial infection.

    27. Use of the cell population of claim 10, for a) drug screening and/or drug development; b) disease modelling; c) tissue engineering; d) preparation of bioartificial organisms; e) disinfection; f) coating of materials for transplantation; g) development of biomarkers; or h) quality control of biological products selected from the group comprising antibodies, hormones, cytokines, drugs, culture medium, and serum.

    28. Use of the cell population of claim 12, for a) drug screening and/or drug development; b) disease modelling; c) tissue engineering; d) preparation of bioartificial organisms; e) disinfection; f) coating of materials for transplantation; g) development of biomarkers; or h) quality control of biological products selected from the group comprising antibodies, hormones, cytokines, drugs, culture medium, and serum.

    Description

    FIGURE LEGENDS

    [0126] FIG. 1 Hematopoietic differentiation of human iPSC in suspension culture. (A) Representative brightfield image of undifferentiated human iPSC (CD34iPSC16). Scale bar, 500 μm. (B) Representative brightfield image of CD34iPSC16 differentiated towards a myeloid cell forming complex (MCFC) producing iPSC-derived macrophages (iPSC-MACs). Scale bars, 500 μm. (C) Brightfield microscopy (left) and Cytospin staining (right) of iPSC-MACs. Scale bars, 100 μm. (D) IPSC-MACs analyzed by flow cytometry (dashed line: respective isotype control, black line: surface marker). (E) Phagocytosis of pHrodo-labeled E. coli particles by iPSC-MACs at 4° C. (negative control) or at 37° C. (n=2, mean±s.e.m.). See also Figure S1.

    [0127] FIG. 2 Continuous generation of human iPSC-MACs in stirred tank bioreactors. (A) Scheme of hematopoietic differentiation of human iPSC in stirred tank bioreactors (DASbox system). (B) Representative pictures of DASbox bioreactor filled with floating MCFCs. (C) Individual cell counts of viable macrophages produced in bioreactors (n=2 of independent bioreactor runs, mean±s.e.m.). (D) Representative macrophage harvest counts (upper graph) and corresponding data from continuous process monitoring (biomass, temperature, pH and dissolved oxygen (DO) level) for the entire cultivation phase of a 42-day bioreactor run. (E) Analysis of human cytokines in the medium from the bioreactor (technical duplicates). (F) Representative light microscopy of macrophage generation analyzed at harvests 1-5. First row shows brightfield images of non-filtered medium samples from bioreactors. Second row shows brightfield images of freshly harvested macrophages separated from MCFCs by sedimentation. Third and fourth row show cytospin and flow cytometric analysis of iPSC-MACs (dashed line: respective isotype control, blackline: CD45, dotted line CD14). Scale bars, 50, 100 and 500 nm, respectively. See also Figure S2.

    [0128] FIG. 3 Characterization of iPSC-MACs derived from stirred tank bioreactors. (A) Representative flow cytometric analysis of iPSC-MACs derived from bioreactor differentiation (dashed line: respective isotype control, black line: cell surface marker). (B) Representative pictures of either brightfield (left image) or cytospin preparations (right image) of terminally differentiated iPSC-MACs derived from the bioreactor. Scale bars, 200 and 100 nm. (C-E) Transcriptome analysis of iPSC, iPSC-derived macrophages (iPSC-MACs) derived from bioreactors and PBMC-derived macrophages (PBMC-MACs) (n=2, biological replicates). (C) Unbiased hierarchical heatmap clustering. (D) Heatmap of pluripotency-associated genes. (E) Heatmap of differentially regulated genes (p<0.001) associated with activation of innate immune response (GO:0002253). (F) Venn-diagram of upregulated genes in iPSC-MACs and PBMC-MACs (Top 100; compared to undifferentiated iPSC) and cell type assignment based on the upregulated genes for the commonly shared gene set as well as the gene set obtained for iPSC-MACs and PBMBC-Mac, respectively, according to the human gene atlas (EnrichR). (G) Clustergram depicting matched genes from the cell line assignment of the genes upregulated in iPSC-MAC only (EnrichR).

    [0129] FIG. 4 Antimicrobial activity of iPSC-MACs generated in stirred tank bioreactors. (A) Scanning raster electron microscopy depicting phagocytosis of latex beads by iPSC-MACs at different time points (B) Rate of phagocytosis by terminally differentiated iPSC-MACs and PBMC-MACs after 2 h of incubation with GFP-labeled P. aeruginosa (PAO1) at 4 or 37° C. (n=3 of biological replicates, two-way ANOVA with Sidak's multiple comparisons test, ns denotes not significant). (C) Heatmap of differentially regulated genes associated with inflammatory response (GO:0006954, >10-fold upregulated), innate immune response (GO:0045087, >5-fold regulated) and activation of innate immune response (GO:0002253, >2-fold regulated). (D) IPA analysis for disease and function of >5-fold upregulated genes.

    [0130] FIG. 5 Pulmonary infection and simultaneous macrophage transplantation in huPAP mice. (A) Scheme of pulmonary transfer of P. aeruginosa (PAO1) and simultaneous transplantation of iPSC-MACs (PiMT) into huPAP or NSG mice. (B) Time course of rectal temperature and disease score over 24 h after infection of huPAP mice with PAO1 (infected) or infected and transplanted (infected+PiMT) (n=6 animals/group, mean±s.e.m). (C) Change in body weight after 24 h. Values are normalized to the respective weights before infection (n=6 animals/group, mean±s.e.m). (D) Lung function measured by head-out bodyplethysmography (n=6 animals/group, mean±s.e.m). (E) Bacterial colony forming units (CFU) of PAO1 per lung after 24 h (n=6 animals/group, mean±s.e.m). (F) Absorbance of bronchio-alveolar lavage fluid (BALF) samples at 650 nm as a measure for the presence of hemoglobin (n=3 animals/group, mean±s.e.m). (G) Flow cytometric analysis of BALF and lung. Percentage of mouse granulocytes (determined as GR1.sup.+ cells) in BALF and Lung (n=3 animals/group, mean±s.e.m). (H) Right lung histological scoring (n=3 animals/group, mean±s.e.m). (I) Representative diagram of BALF and lung of one animal per group stained with hCD45.

    [0131] FIG. 6 Pulmonary infection and therapeutic transplantation of iPSC-MACs in huPAP mice. (A) Scheme of pulmonary infection with P. aeruginosa (PAO1) and therapeutic transplantation of iPSC-MACs (PiMT) derived from bioreactors into huPAP mice (B) Time course of disease scores of huPAP mice infected with PAO1 (infected), infected and transplanted (infected+PiMT) and control mice receiving PBS twice (control) (n=3 animals/group, mean±s.e.m). (C) Activity 24 h post-infection evaluated by movement of mice analyzed by video documentation and manual tracking (n=1 animal/group). (D) Time course of rectal temperature analyzed over 24 h after infection (n=3 animals/group, mean±s.e.m). (E) Change in body weight after 24 hours. Values are normalized to the respective weights before infection (n=3 animals/group, mean±s.e.m). (F) Bacterial colony forming units (CFU) of PAO1 per lung after 24 hours (n=3 animals/group, mean±s.e.m). (G) Images of BALF samples. (H) Levels of human cytokines in BALF of infected+PiMT mice (n=2 mice). (j) Lung histology images of infected, infected+PiMT and control mice.

    [0132] FIG. 7 Derivation of myeloid cells from hematopoietic differentiation in suspension. (A) Flow cytometric analysis of suspension-based hematopoietic differentiation cultures of cells generated by IL3 and G-CSF supplementation and finally differentiated in the presence of G-CSF only (dashed line: respective unstained control, black line: cell surface marker). (B) Cytospins of cells generated from IL3/G-CSF cultures, terminally differentiated by G-CSF only. Scale bar: 50 μm and 20 μm. (C) Flow cytometric analysis of suspension-based hematopoietic differentiation cultures of cells generated by IL3 and SCF/EPO supplementation and finally differentiated in the presence of SCF and EPO (dashed line: respective isotype control, black line: cell surface marker). (D) Flow cytometric analysis of cells derived from suspension cultures using IL3 only and differentiated for more than seven days in M-CSF. Data shown as histogram overlays (dashed line: respective unstained control, black line: cell surface marker). (E) Respective cytospin for IL3-derived cultures which have been terminally differentiated by M-CSF. Scale bar: 50 μm and 20 μm. (F) Flow cytometric analysis of cells derived from suspension cultures using IL3 only and differentiated for more than seven days in G-CSF. Data shown as histogram overlays (dashed line: respective unstained control, black line: cell surface marker. (G) Respective cytospin for IL3-derived cultures which have been terminally differentiated by G-CSF. Scale bar: 50 μm and 20 μm.

    [0133] FIG. 8 Lung histology of transplanted mice. Right lung histology showing macrophages in infected+PiMT animals only (representative image, n=3). Scale bar, 50 μm.

    [0134] FIG. 9 Pulmonary infection and simultaneous macrophage transplantation in NSG mouse models. (A) Rectal temperature and (B) disease score of NSG mice infected with PAO1 (infected) or infected and transplanted (infected+PiMT) 6 h and 24 h post-infection. Disease Score and temperatures measured before the experiment served as control values (n=4-5 animals/group, mean±s.e.m). (C) Colony forming units (CFU) of PAO1 per lung after 24 h in infected and infected+PiMT NSG mice (n=4-5 animals/group, mean±s.e.m).

    [0135] FIG. 10 Spraying applications and reduction of cell size. (A) Numbers of viable and dead cells of a human macrophage cell line (U937) after spraying at a concentration of 6×10.sup.6 cells/ml. (B) Number of cells per puff (spraying) is about 2*10.sup.5 cells, using nebulizer (brown-glass, volume 20 ml, sprayvolume approx 50 μl, company Rixius AG, Mannheim Germany)

    [0136] FIG. 11 Reduction of the size of cells (shrinking) in solutions of different osmolarity. Human macrophage cell line U937 were incubated in sucrose solution having 300, 600, 800 or 1000 mosm for 15 min at room temperature. The control is 300 mosm. The respective data was received from flow cytometric analysis. The plot shows an decrease in FSC (Forwards scatter, indicator of volume) upon incubation in solutions of higher osmolarity. The plot shows the reduction in FSC towards approx. 40% with 600 mosm, 32% with 800 mosm and 21% with 1000 osm hypertonic solution, compared to the control having 300 mosm.

    [0137] FIG. 12 Spraying of primary murine macrophages does not significantly reduce numbers of viable cells. Murine primary macrophages were isolated from murine bone marrow samples using established and well known techniques. In brief, femur and tibia were isolated and flushed to receive bone marrow. Total bone marrow was incubated with M-CSF in respective medium for a minimum of 7 days. Adherent macrophages were harvested by trypsin treatment. They were washed and taken up in PBS solution medium (RPMI) for spraying; after spraying they were centrifuged and taken up in PBS for flow cytometric analysis. Right before analysis propidium iodide (PI) was given to the cells on ice to stain dead cells. The plot shows the number of cells without spraying, and the number of cells after spraying, wherein the white box indicates living cells, and the black box indicates dead cells. The percentage of dead cells is about 5-10%.

    [0138] FIG. 13 Reduction of the size of cells (shrinking) in solutions of different osmolarity. Murine primary macrophages prepared as described for FIG. 12 were incubated in sucrose solution having 300, 600, 800 or 1000 mosm for 1 hour at 4° C. The control is 300 mosm. The respective data was received from flow cytomtric analysis. The plot shows an decrease in FSC (Forwards scatter, indicator of volume) upon incubation in solutions of higher osmolarity. The plot shows the reduction in FSC towards approx. 44% with 600 mosm, 36% with 800 mosm and 24% with 1000 osm hypertonic solution, compared to the control having 300 mosm.

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    EXAMPLES

    Example 1: Methods

    Cell Culture and Differentiation

    [0197] iPSC Cultivation

    [0198] Human iPSC (hCD34iPSC16) have been previously generated from mobilized peripheral blood CD34.sup.+cells (Lachmann et al., 2014) and were culture on irradiated murine embryonic fibroblasts (MEF) in iPSC medium (knock-out DMEM, 20% knock out serum replacement, 1 mM L-glutamine, 1% NEAA, 1% penicillin/streptomycin (all Invitrogen, Karlsruhe, Germany), 0.1 mM β-mercaptoethanol (Sigma-Aldrich, St. Louis, Mo., United States), and 10 ng/ml bFGF (PeproTech). Basic-FGF was omitted from the maintenance medium for the last 4-5 days prior to EB formation.

    Hematopoietic Differentiation in Adherent Culture

    [0199] Classical adherence-based hematopoietic differentiation of human iPSC was performed as previously described (Lachmann et al., 2015). In brief, PSC colonies (3 wells of a 6-well plate, approx. 3×10.sup.6 cells) were disrupted to fragments using collagenase-IV, and EB formation was induced by cultivation for 5 days in iPSC medium supplemented with 10 μM Rock inhibitor (Y-27632; Tocris) in six-well suspension plates on an orbital shaker. Subsequently, EBs were manually selected using a binocular and transferred to tissue culture six-well plates and cultivation in differentiation medium I (X-VIVO 15, Lonza) supplemented with 1% penicillin-streptomycin (Life Technologies), 25 ng/ml human IL3 and 50 ng/ml human M-CSF (both PeproTech). From day 10-15 onwards, iPSC-MACs were harvested from the supernatant once a week.

    Hematopoietic Differentiation in Suspension Culture

    [0200] To achieve hematopoietic differentiation in suspension, largest EBs were selected by sedimentation properties (sedimentation <10 min) and transferred to differentiation medium I in 6-well suspension plates on the orbital shaker (Celltron, Infors HT) at 85 revolutions per minute (rpm). From day 10-15 onwards, iPSC-MACs were harvested from the medium once a week. For differentiation into other myeloid cell types, APEL medium was used as described previously (Ng et al., 2008). For differentiation into granulocytic and erythroid cells, 25 ng/ml hIL3 combined with 50 ng/ml hG-CSF or 100 ng/ml hSCF plus 3U hEPO, respectively, were applied.

    Hematopoietic Differentiation in Stirred Tank Bioreactors

    [0201] The bioreactor (DASbox Mini bioreactor system, Eppendorf) was setup and calibrated as previously described (Kempf et al., 2015). In brief, the 250 ml glass vessel was equipped with an 8-blade impeller (60° pitch) and probes for online monitoring of biomass (Aber Instruments), pH, DO as well as control of temperature. Calibration was performed in 120 ml chemically defined X-VIVO 15 (Lonza).

    [0202] For hematopoietic differentiation in the bioreactor, iPSC were expanded to twenty 6-well plates and cultivated for 3 days in the presence of bFGF. EB formation was performed equivalent to adherent cultures. After 5 days, EBs were selected by sedimentation properties (sedimentation <10 min) and transferred to the equilibrated bioreactor. Cells were cultivated in X-VIVO 15 supplemented with IL3 and M-CSF (differentiation medium I) at 37° C. with constant headspace-gassing at 3 L/h (21% O.sub.2; 5% CO.sub.2) and stirring at 50 rpm. To monitor MCFCs integrity and macrophages formation, 1 ml samples were collected 1-2 times per week via the sampling port without interrupting the culture process. Differentiation medium I was manually replaced every 6-7 days with optional fed of 20 ml after 3-4 days. Macrophages were harvested weekly by separation from MCFC via sedimentation (4-5 min) and subsequent filtering of the medium through a 100 nm strainer. Retained MCFCs were returned to the bioreactor. Macrophages were collected from filtered medium via centrifugation at 300×g for 4 min.

    [0203] Data from online monitoring were processed using Microsoft Excel 2016 and GraphPad Prism 6. Supernatant was analysed for concentration of glucose and lactate using YSI 2700 select biochemistry analyser, for osmolarity using Osmomat 300 (Gonotec) and for concentration of lactate dehydrogenase according to manufacturer's instruction (MAK066, Sigma) using a microplate reader (Paradigm, Beckman Coulter).

    Terminal Differentiation

    [0204] For further maturation, cells freshly harvested from MCFCs, were cultured in differentiation medium II (RPMI1640 medium supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 1% penicillin-streptomycin) containing 50 ng/ml hM-CSF for macrophage, 50 ng/ml hG-CSF for granulocyte, and 100 ng/ml SCF and 3U/ml EPO for erythroid differentiation for at least 7 days.

    Isolation of Peripheral Blood Mononuclear Cells (PBMCs) and Differentiation to Macrophages

    [0205] All healthy donors gave written informed consent according to the local ethical committee at Hannover Medical School. Peripheral blood mononuclear cells (PBMC) were isolated from the peripheral blood of healthy volunteers by gradient centrifugation using Biocoll Separating Solution (40 min, 400×g; Biochrome, Billerica, Mass.). Subsequently, cells were cultured in RPMI1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 1% penicillin-streptomycin (all Invitrogen), and hIL3 and hM-CSF (50 ng/ml each, PeproTech) for one week. After this, PBMC-MACs were cultivated for further 3-4 days in differentiation medium containing 50 ng/ml M-CSF only.

    Phenotypic and Functional Characterization

    Flow Cytometry

    [0206] Flow cytometric analysis of myeloid cells was performed as described (Lachmann et al., 2015; Lachmann et al., 2014). For macrophages, PBS supplemented with 10% FCS was used to prevent unspecific binding. Cells were analyzed with a FACScalibur cytometer (Beckton & Dickinson, Heidelberg, Germany) and analyzed with FlowJo software (TreeStar, Ashland, Oreg.). Antibodies were purchased from eBioscience: hTRA-1-60-PE (Cat-No: 12-8863-80), hCD11b-APC (Cat-No: 17-0118-41), hCD14-PE (Cat-No: 12-0149-42), hCD163-APC (Cat-No: 17-1639-41, hCD16-FITC (Cat-No: 11-0168-41), hCD34-FITC (Cat-No: 11-0349-41) and isotype-controls: mouse-IgG1a-PE (Cat-No: 12-4714-41), FITC (Cat-No: 11-4714-41) or APC (Cat-No: 17-4714-41), and rat-IgG2a-PE (Cat-No: 12-4321-81). Antibodies from Biolegend San Diego, Calif., United States: hCD86-APC (Cat-No: 305411), hCD66b-FITC (Cat-No: 305104) or hCD45-PE (Cat-No: 304007).

    [0207] For flow-cytometric analysis of mouse lung and BALF, samples were fixed using 4% PFA. After this, samples were incubated with fc receptor blocking antibodies (CD16/CD32, eBioscience, Cat-No: 14-0161-81) for 20 min to prevent unspecific binding. Used antibodies were purchased from Biolegend San Diego, Calif., United States: hCD45-PeCy7, and eBioscience: mGR1-eFluor450.

    Cytospin Preparation

    [0208] 20,000-50,000 cells were spun on glass slides at 600×g for 7 min and stained for 5 min in 0.25% May-Grunwald and 20 min in 0.4% Giemsa stain modified solution (Sigma).

    Phagocytosis Assays

    [0209] The phagocytic activity of iPSC-MACs derived from suspension or adherent cultures and terminally differentiated on tissue-culture plates was assessed by flow cytometry. Therefore, 1×10.sup.5 cells were incubated with pHrodo™ Red E. coli BioParticles® Conjugate (MolecularProbes/Thermo Fisher Scientific, Schwerte, Germany) or medium for 2 h at 37° or 4° C. as a negative control. After incubation the cells were put on ice for 10 min. Analysis was performed using a Beckman Coulter FC500 flow cytometer.

    [0210] The functional capacity of iPSC-MACs and PBMC-MACs to phagocytose vital P. aeruginosa was assessed using GFP-PAO1 (wild-type P. aeruginosa PAO1 tagged with green fluorescent protein (GFP) by Tn7 transformation (Bjarnsholt et al., 2005), (kindly provided by Thomas Bjarnsholt, University of Copenhagen) and compared to PBMC-derived Macrophages (PBMC-MACs). For the phagocytosis assay, 1×10.sup.5 iPSC-MACs were incubated for 2 h with 6×10.sup.5 CFU GFP-PAO1 at 37° C. or 4° C. as a negative control. Medium controls were treated similar. After incubation, cells were put on ice for 10 min and subsequently fixed with 2% paraformaldehyde (PFA) solution for 30 min. Analysis was performed using a Beckman Coulter FC500 flow cytometer.

    Electron Microscopy

    [0211] Macrophages were grown on 1 cm diameter round coverslips. Latex beads (1 μm diameter) were added to cells at 4° C. and adhesion of latex beads to the surface of the macrophages was allowed for 5 min. Following samples were washed with cold PBS and warmed up with fresh culture medium to 37° C. Phagocytosis was allowed for up to 1 h and samples were fixed at different time points using 1.5% Paraformaldehyde, 1.5% Glutaraldehyde in 150 mM Hepes pH 7.35. Samples were then dehydrated using an increasing methanol series. Critical point drying was performed using a CPD030 critical point dryer (Balzers, Lichtenstein) following manufacturer instructions. Coverslips were then sputtered with gold (Sem Coating System, Polarion) and SEM was carried out using a Philips SEM 505 (Eindhoven, The Netherlands).

    Bacterial Culture

    [0212] For experiments P. aeruginosa laboratory strain PAO1 (Klockgether et al., 2010) was taken from a stock culture kept at −80° C. and grown in Luria Broth (LB) overnight. After washing with sterile PBS the desired infectious dose was extrapolated from a standard growth curve. For the determination of the actual dosage, inoculates were serially plated on LB agar plates via the drop-plate method (Herigstad et al., 2001) and CFU determined after 16-18 h incubation at 37° C.

    Collection of Microarray Samples

    [0213] Terminally differentiated human iPSC-MACs or human PBMC-MACs were seeded on 24-well plates (500.000 cells/well) and cultured overnight. On the next day, cells were washed three times with PBS. Subsequently, P. aeruginosa laboratory strain PAO1 in RPMI medium without antibiotics (MOI10) was centrifugated onto the cells (600×g) and incubated at 37° C. Cells with medium only served as non-infected controls. After 1 h cells were de-attached, washed and resuspended in RNA lysis buffer. RNA isolation was performed with RNAeasy micro Kit (Quiagen) according to manufacturer's instructions. Human iPSC samples were obtained after sorting of iPSC for TRA-1-60.sup.+to separate iPSC from feeder cells.

    Microarray Experiments (Single Colour Mode)

    [0214] The Microarray utilized in this study represents a refined version of the Whole Human Genome Oligo Microarray 4×44K v2 (Design ID 026652, Agilent Technologies), called ‘054261On1M’ (Design ID 066335) developed in the Research Core Unit Transcriptomics (RCUT) of Hannover Medical School. Microarray design was created at Agilent's eArray portal using a 1×1M design format for mRNA expression as template. All non-control probes of design ID 026652 have been printed five times within a region comprising a total of 181560 Features (170 columns×1068 rows). Four of such regions were placed within one 1M region giving rise to four microarray fields per slide to be hybridized individually (Customer Specified Feature Layout). Control probes required for proper Feature Extraction software operation were determined and placed automatically by eArray using recommended default settings.

    [0215] 30 ng of total RNA were used to prepare aminoallyl-UTP-modified (aaUTP) cRNA (Amino Allyl MessageAmp™ II Kit; #AM1753; Life Technologies) as directed by the company (applying one-round of amplification). The labelling of aaUTP-cRNA was performed by use of Alexa Fluor 555 Reactive Dye (#A32756; LifeTechnologies).

    [0216] cRNA fragmentation, hybridization and washing steps were carried-out as recommended in the ‘One-Color Microarray-Based Gene Expression Analysis Protocol V5.7’, except that 500 ng of each fluorescently labelled cRNA population were used for hybridization.

    [0217] Slides were scanned on the Agilent Micro Array Scanner G2565CA (pixel resolution 3 μm, bit depth 20). Data extraction was performed with the ‘Feature Extraction Software V10.7.3.1’ using the extraction protocol file ‘GE1_107_Sep09.xml’, except that ‘Multiplicative detrending’ algorithm was inactivated.

    [0218] Measurements of on-chip replicates (quintuplicates) were averaged using the geometric mean of processed intensity values of the green channel, ‘gProcessedSignal’ (gPS) to retrieve one resulting value per unique non-control probe. Single Features were excluded from averaging, if they i) were manually flagged, ii) were identified as Outliers by the Feature Extraction Software, iii) lie outside the interval of ‘1.42× interquartile range’ regarding the normalized gPS distribution of the respective on-chip replicate population, or iv) showed a coefficient of variation of pixel intensities per Feature that exceeded 0.5.

    [0219] Averaged gPS values were normalized by quantile normalization approach first. Subsequently, values were additionally processed by global linear scaling: All gPS values of one sample were multiplied by an array-specific scaling factor. This factor was calculated by dividing a ‘reference 75th Percentile value’ (set as 1500 for the whole series) by the 75th Percentile value of the particular Microarray to be normalized (Array I′ in the formula shown below). Accordingly, normalized gPS values for all samples (microarray data sets) were calculated by the following formula:


    normalized gPS.sub.Array i=gPS.sub.Array i×(1500/75.sup.th Percentile.sub.Array i)

    [0220] Finally, a lower intensity threshold (surrogate value) was defined based on intensity distribution of negative control features. This value was fixed at 15 normalized gPS units. All of those measurements that fell below this intensity cutoff were substituted by the respective surrogate value of 15.

    [0221] Normalized microarray data of all non-control features were imported into Omics Explorer software v3.2 (Qlucore) under default import settings for Agilent One Color mRNA Microarrays, except that any normalization option was deselected. Accordingly, data processing steps during import were: 1) log base 2 transformation, 2) baseline transformation to the median.

    [0222] Heatmap clustering analysis and generation of GO-based heatmaps were performed in Omics Explorer. Top 100 upregulated genes were calculated using the RCUTAS tool (V1.7; Hannover Medical School) and processed using Venny 2.1 (http://bioinfogp.cnb.csic.es/tools/venny). Gene set enrichment analysis for cell type classification based on the human gene atlas and gene ontology analysis of biological processes and molecular function were conducted using Enrichr (https://amp.pharm.mssm.edu/Enrichr). Gene analysis for disease and function were performed using Ingenuity pathway analysis (Qiagen).

    [0223] Microarray data were deposited under accession number E-MTAB-5436 in the ArrayExpress database (www.ebi.ac.uk/arrayexpress).

    Cytokine Secretion Assays (Luminex)

    [0224] In order to analyze the secretion of human cytokines in bioreactor samples or BALF samples, Luminex® analysis with a Cytokine Human 14-Plex Panel (Millipore, Schwalbach, Germany) was performed as described before (Lachmann et al., 2015). Data were acquired on a Luminex-200 System and analyzed with the Xponent software v.3.0 (Life Technologies).

    In Vivo Experiments

    Animal Maintenance and Infection

    [0225] HuPAP (129S4-Rag2.sup.tm1.1Flv Csf2/Il3.sup.tm1.1(CSF2,IL3)Flv Il2rg.sup.tm1.1Flv/J) mice (Willinger et al., 2011) were obtained from the Jackson Laboratory and housed in the central animal facility of Hannover Medical School. NSG mice (NOD.Cg-Prkdc.sup.scid Il2.sup.tm/Wj1SzJZtm) were obtained from the central animal facility of Hannover Medical School. Both immunodeficient mouse strains were maintained under pathogen free conditions in individually ventilated cages (IVC) with free access to food and water. All animal experiments were approved by the Lower Saxony State animal welfare committee and performed according to their guidelines.

    [0226] For infection with P. aeruginosa or pulmonary iPSC-MAC transplantation, anesthetized (ketamine/midazolam) mice were instilled via the trachea after oral intubation. For the simultaneous infection experiments, iPSC-MAC (4×10.sup.6/animal) and PAO1 (NSG: 5×10.sup.5 and huPAP 0.2×10.sup.5 CFU) were resuspended in PBS and mixed in a total volume of 60 μl. For solely infected mice the same CFU was applied in 60 μl PBS. To avoid phagocytosis before instillation, cell/bacteria mixes were kept on ice all the time. For the therapeutic PiMT experiments, huPAP animals were infected with 0.3×10.sup.5 CFU PAO1 (in a volume of 30 μl PBS) after anesthesia with ketamine/midazolam. Control mice received the same volume PBS. After 4 h, mice were anesthetized by isoflurane inhalation and the second instillation with 50 μl PBS or 4×10.sup.6 iPSC-MACs in PBS was performed. Control mice again received the same volume PBS. To determine the disease score of the animals we used a scoring matrix as described previously (Munder et al., 2005). After 24 h, animals were sacrificed and end-analysis was performed.

    Murine Lung Function

    [0227] Non-invasive head-out spirometry investigating 14 lung function parameters was performed on conscious restrained mice as previously described (Wolbeling et al., 2010). Mice were positioned in glass inserts, their breathing causes air to flow through a pneumotachograph. A pressure transducer creates an electrical signal, which is analysed using NOTOCORD HEM software (Version 4.2.0.241, Notocord Systems SAS, Croissy Sur Seine, France). The parameters of tidal volume (measured in ml), expiratory time, inspiratory time, time of inspiration+expiration, relaxation time and the flow at 50% of the expiratory tidal volume (EF50) were selected to characterize murine lung function during infection.

    Broncho-Aleveolar Lavage (BAL) and Measurement of Hemoglobin Levels

    [0228] Broncho-alveolar lavage was performed by cannulating the murine trachea post-mortem. The right lung was rinsed with 1 ml of PBS for three times. Fresh BALF was used for photometric analysis of haemoglobin. After this, BALF samples were centrifuged and supernatants were stored at −80° C. for Luminex analysis. Pellets were fixed and stained for flow cytometry

    Lung Bacterial Numbers (CFU)

    [0229] The right lungs of the euthanized mice were ligated, resected and homogenized with a tissue homogenizer (Polytron PT 1200, Germany). Total bacterial numbers were assessed from serial dilutions of the homogenates which were cultured on Luria-Bertani plates using the drop plate method (Herigstad et al., 2001).

    Histology

    [0230] At the dedicated time points the animals were sacrificed. Right lungs were filled with OCT buffer and fixed in neutral buffered 4% PFA for 3 days at 4° C. For control animals, left lungs were used. Tissues were trimmed according to the RITA-Guidelines (Ruehl-Fehlert et al., 2003), dehydrated (Shandon Hypercenter, XP) and subsequently embedded in paraffin (TES, Medite). Sections (2-3 μm thick, microtom Reichert-Jung 2030) were deparaffinized in xylene and H&E stained according to standard protocols. Blinded evaluation (Axioskop 40, Zeiss microscope) and histological scoring of the sections was performed as described before (Dutow et al., 2013) by a trained pathologist.

    Statistics

    [0231] GraphPad Prism 6 and 7 was applied to perform unpaired Student's T test or analysis of variance (ANOVA). Unless otherwise stated, mean±s.e.m. is plotted. Asterisks denote: *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

    Results

    [0232] Derivation of Multiple Human iPSC-Derived Myeloid Lineages in Dynamic Suspension Culture

    [0233] Although the generation of different mature hematopoietic cell types from PSC has been proven successful using classical two-dimensional (2D) differentiation cultures (Ackermann et al., 2015; Choi et al., 2009; Dias et al., 2011; Feng et al., 2014; Sturgeon et al., 2014), these systems do not allow for the generation of iPSC-derived cells in clinically relevant quantities. Thus, the inventors developed a suspension-based (3D) hematopoietic differentiation protocol, suitable for process upscaling in industry-compatible stirred tank bioreactors (Kropp et al., 2016b; Zweigerdt, 2009). Using a well-characterized hiPSC line (hCD34iPSC16) (Lachmann et al., 2014) with classical brightfield morphology (FIG. 1A), they first induced the formation of embryoid bodies (EBs) in small-scale suspension culture on an orbital shaker. After 5 days, they transferred the EBs to differentiation medium containing IL3 and M-CSF to induce hematopoietic specification and the formation of myeloid cell forming complexes (MCFCs) (FIG. 1B). After 10-15 days of continued suspension culture, MCFCs continuously produced iPSC-derived macrophages (iPSC-MACs) (4D-differentiation) that could be harvested weekly for up to three months. Generated iPSC-MACs displayed typical macrophage-like morphology, exhibited a highly pure surface marker profile of CD45.sup.+CD11b.sup.+CD14.sup.+CD163.sup.+CD34.sup.−TRA1-60.sup.−, and efficiently phagocytosed fluorescently labeled E. coli particles (FIG. 1C-1E).

    [0234] Of note, different myeloid subsets were generated in following the same suspension-based protocol but simply switching the cytokine composition. Thus, IL3 and G-CSF allowed continuous generation of CD16.sup.+CD66b.sup.+iPSC-granulocytes (FIGS. 7A and 7B), whereas CD34.sup.−CD71.sup.+CD235a.sup.+CD36.sup.+ erythroid cells were derived by the use of IL3/SCF/EPO (FIG. 7C). In contrast to cells generated by IL3 in combination with lineage instructive cytokines, employing IL3 only led to display of a more immature CD45.sup.+/CD11b.sup.+/CD14.sup.−/CD163.sup.− marker profile (data not shown). Of note, further differentiation of these immature cells in the presence of either M-CSF or G-CSF led to the generation of CD45.sup.+CD11b.sup.+CD14.sup.+CD66b.sup.−CD34.sup.− macrophage-like (FIGS. 7D and 7E) or CD16.sup.+CD661).sup.+, granulocyte-like cells (in the following also designated macrophages and granulocytes) (FIGS. 7F and 7G).

    Up-Scaling Continuous Production of hiPSC-MACs is Enabled in Stirred Tank Bioreactors

    [0235] The inventors then translated the suspension-based differentiation into stirred tank bioreactors using an industry-compatible system (DASbox Mini Bioreactor System) (Olmer et al., 2012) previously applied for the efficient cultivation of human iPSC and their differentiation into cardiomyocytes (Kempf et al., 2014) (FIG. 2A, 2B). Bioreactors were equipped with probes for real-time monitoring of dissolved oxygen (DO), pH, temperature and impedance-based biomass assessment. Process parameters were set to 37° C., headspace-gassing at 3 L/h (21% O.sub.2; 5% CO.sub.2) and stirring at 50 revolutions per minute (rpm) using an 8 blade-pitched (60°) impeller (Kempf et al., 2015). With respect to future clinical scale-up, it is noteworthy that a chemically defined culture medium (X-Vivo15), which is also applied in clinical trials, was used. From day 7-10 onwards, weekly harvest of iPSC-MACs from the uninterrupted bioreactor process showed an increase in cell yield over time, reaching stable production of approx. 2-3×10.sup.7 iPSC-MACs/week as early as week three, which was maintained for more than five weeks in two independent process runs (FIG. 2C). The efficient generation of iPSC-MACs in both bioreactor experiments was reflected by the weekly increase in biomass, particularly during the first days after full medium refreshment. DO and pH monitoring revealed expected zigzag-like patterns typical for repeated batch cultures. The pH was ranging from 7.25 (fresh medium) down to 6.5, resulting from typical medium acidification by the cells' metabolic activity, particularly by lactate release (Kropp et al.). Notably, all process parameters showed maintenance of repetitive patterns after reaching the steady state of macrophage production around d15-20, confirming the overall stability of the process (FIG. 2D). This finding is further supported by stable values for glucose, lactate, lactate dehydrogenase, and osmolality, which were determined weekly in parallel to macrophage harvests. Similar, secretion of cytokines/chemokines associated with the activation of macrophages, such as IL2, IL6, IL8, MCPJ, TNFα, and IFNα2, was detected from the first harvest (week 2) onwards (FIG. 2E) and corresponded to the appearance of CD45.sup.+ iPSC-MACs. Notably, MCFCs cultivated in the bioreactor sustained their morphology during the entire process and continuously generated iPSC-MACs of typical morphology and a CD45.sup.+CD14.sup.+ surface marker profile, which increased in purity over time (FIG. 2F). Moreover, the extended cultivation of MCFCs derived from the bioreactor in 6-well suspension plates on an orbital shaker resulted in continued production of iPSC-MACs for additional 8 weeks (data not shown).

    Bioreactor-Derived iPSC-MACs Recapitulate Phenotypic and Transcriptional Characterization of PBMC-MACs

    [0236] Detailed characterization of bioreactor-derived iPSC-MACs revealed a CD45.sup.+CD11b.sup.+CD163.sup.+CD14.sup.+CD34.sup.−TRA1-60.sup.− phenotype, and a typical morphology after adherence to tissue culture plates (FIGS. 3A and 3B). Comparison of iPSC-MACs to non-differentiated hiPSC and Peripheral Blood Mononuclear Cells (PBMC)-derived macrophages (PBMC-MACs) by unsupervised, hierarchical heatmap clustering of whole transcriptomes revealed close proximity of iPSC-MACs and PBMC-MACs when compared to iPSC (FIG. 3C). Analysis of genes associated with pluripotency and activation of innate immune response confirmed efficient differentiation of iPSC into macrophage-like cells (FIGS. 3D and 3E). Importantly, genes associated with macrophage function such as the toll-like receptors (TLR) 1 and 4, CD14, or components of the NF-κB signaling pathway (gene ontology (GO) Activation of innate immunity: 0002218) were significantly upregulated in iPSC-MACs and PBMC-MACs versus iPSCs (FIG. 3E).

    [0237] Comparison of the top 100 upregulated genes in iPSC-MACs versus pluripotent hiPSC and PBMC-MACs versus pluripotent hiPSC revealed a common gene set of 54 upregulated genes. These transcripts including CD14, CD68, CSFR1, CCR1 and CYBB are assigned to CD14.sup.+monocytes, whole blood and CD33.sup.+ myeloid cells (FIG. 3F). Of note, genes of this set exclusively upregulated in iPSC-MACs (46 genes) were also assigned to CD14.sup.+ monocytes and CD33+myeloid cells and included genes such as CD163, the leukocyte immunoglobulin like receptor A6 (LILRA6), stabilin 1 (STAB1) or the formyl peptide receptor 1 (FPR1) (FIG. 3G). In contrast, 46 genes of this set upregulated in PBMC-MACs only revealed the highest score in gene sets associated with CD56.sup.+ natural killer (NK) cells, whole blood and dendritic cells (e.g. human leukocyte antigens (HLA), CCL5/RANTES or granzyme (GZM) A and B), an observation maybe explained by contaminating CD56.sup.+/CD3.sup.−NK-cells (data not shown).

    [0238] Table 1 shows the top 100 genes which are differentially regulated (either upregulated or downregulated) in the macrophages of the invention directly compared to macrophages derived from PBMC.

    In Vitro Antimicrobial Activity of Bioreactor-Derived iPSC-MACs

    [0239] Next, bioreactor-derived iPSC-MACs were evaluated for their in vitro antimicrobial activity. Scanning electron microscopy (SEM) at different time points after incubation of iPSC-MACs with fluorescently labeled latex beads revealed typical changes in the overall cell morphology early after the stimulus and efficient phagocytic uptake of beads over time (FIG. 4A). Even more important, iPSC-MACs also phagocytosed GFP-labeled P. aeruginosa at 37° C. in comparable efficiency to PBMC-MACs, whereas no phagocytosis was observed at 4° C., confirming active phagocytosis (FIG. 4B). To gain insights into the ability of iPSC-MACs to remodel their transcriptome towards the signature of activated macrophages, whole transcriptome analysis of iPSC-MACs was performed before and after contact with P. aeruginosa (PAO1).

    TABLE-US-00001 TABLE 1 Characteristic gene expression pattern of iPS-Mac (compared to PBMC-Mac) upregulated downregulated DKK1 ANPEP SEPP1 CDA PITX2 CRTAM COL3A1 ENST00000390237 KRT19 FBP1 A_33_P3221980 GBP5 CALD1 GNLY CYR61 HLA-DQA1 H19 HLA-DQA2 DDIT4L HLA-DQB1 FRZB HLA-DQB2 TMEM98 HLA-DRA NNMT HLA-DRB1 NPNT HLA-DRB3 LUM HLA-DRB4 DCN HLA-DRB5 LYVE1 IL15 MGP LY75 IGFBP3 S1PR4 NUAK1 TNFAIP6

    [0240] Hierarchical cluster analysis of gene ontologies (GOs) associated with inflammatory response or (activation of) innate immune response showed marked upregulation of cytokines (e.g. IL23A, TNFα, ILIA, IL6, INFG1), chemokines (e.g. CCL5, CCL20, CCL4, CXCL3) and molecules involved in NFκB signaling in response to pathogen contact (FIG. 4C). Similarly, gene ontology enrichment analysis of >5 fold upregulated genes after pathogen contact revealed high scores for GOs associated with biological processes, including inflammatory response, response to lipopolysaccharide (LPS) and molecules of bacterial origin as well as response to wounding. Moreover, GOs associated with molecular function revealed enrichment of GO terms such as cytokine/chemokine activity and receptor binding or G-protein coupled receptor binding. Enrichment analyses of these upregulated genes for disease and function revealed ontologies including inflammatory disease and response, immune cell trafficking, antimicrobial response and free radical scavenging (FIG. 4D).

    iPSC-MACs Prevent Respiratory Infections by Pseudomonas aeruginosa

    [0241] To evaluate the therapeutic efficacy of iPSC-MACs in vivo, a humanized mouse model C; 129S4-Rag2.sup.tm1.1Flv Csf2/Il3.sup.tm1.1(CSF2,IL3)Flv Il2rg.sup.tm1.1Flv/J (huPAP mice), an immunodeficient strain with impaired alveolar macrophage development (Willinger et al., 2011) was utilized. HuPAP mice recapitulate hallmarks of the human disease pulmonary alveolar proteinosis (PAP), including the susceptibility to pulmonary infections. In addition, the iPSC-MACs were utilized in a second immunodeficient mouse model: NOD.Cg-Prkdc.sup.scid Il2rg.sup.tm/Wj1/SzJZtm (NSG), which have impaired lymphopoiesis, but show normal alveolar macrophage development.

    [0242] First, huPAP mice were infected with the P. aeruginosa laboratory strain PAO1 and co-administered with 4×10.sup.6 iPSC-MACs in the same instillation process (solely PAO1 infected mice served as controls). The infection course was closely monitored and mice were sacrificed 24 h post-infection for end analysis (FIG. 5A). Infected huPAP mice displayed clinical symptoms already 4 h post-infection as indicated by an elevated disease score of 3.5±0.9, which gradually increased over time up to 6.1±0.4 after 24 h. In addition, a decrease in rectal temperature to 35.0±0.3° C. and body weight loss of 2.6±0.5 g was observed in infected animals 24 h post-infection (all mean±s.e.m., n=6) (FIGS. 5B and 5C). In contrast, animals receiving a simultaneous pulmonary iPSC-MACs transplantation (PiMT) showed only mild symptoms of infection (FIG. 5B). This was in line with normal rectal temperature of 37.1±0.2° C., a very low disease score of 0.6±0.2 and only little loss of body weight of −0.89±0.2 g 24 h post-infection in PiMT treated animals (all mean±s.e.m., n=6) (FIGS. 5B and 5C). Similar beneficial effects of PiMT were demonstrated by head-out body-plethysmography to measure lung function (Wolbeling et al., 2010). While infected animals showed a decrease in tidal volume as well as expiratory and inspiratory time and an increase in breathing rate, animals from the infected+PiMT group showed normal values for all parameters analyzed (FIG. 5D). As a consequence of PiMT, transplanted mice showed significantly reduced bacterial numbers in the lung 24 h post-infection compared to their non-transplanted controls (FIG. 5E). Moreover, erythrocytes were present only in broncho-alveolar lavage fluid (BALF) from infected mice not receiving PiMT (FIG. 5F). Lung inflammation was furthermore indicated by an increase in murine GR1.sup.+ granulocytes in the BALF and lungs of infected animals, but not infected+PiMT or control mice (FIG. 5G). In addition, histological evaluation revealed massive granulocyte infiltration, severe hemorrhage and alveolar edema in infected mice (score: 13.7±0.3), whereas lungs from infected+PiMT animals showed merely slight changes (score: 2.0±1.2, both mean±s.e.m., n=3) (FIG. 5H). Reduced inflammation in infected+PiMT animals was associated with the detection of hCD45.sup.+ cells in lung and BALF as well as presence of macrophages in histological sections of the right lung (FIGS. 5I and 8).

    [0243] Similar results were obtained in NSG mice, a second immunodeficient mouse strain with normal alveolar macrophage development. Infected NSG animals developed profound symptoms of infection, such as a high disease score and a profound drop in rectal temperature 6 h and 24 h post-infection. In clear contrast, animals simultaneously receiving iPSC-MACs displayed normal body temperature and only minimally increased disease scores 6 h and 24 h post-infection, respectively (FIGS. 9A and 9B). Moreover, analysis of bacterial numbers 24 h post-infection revealed a significantly reduced bacterial load in the lungs of infected+PiMT mice (FIG. 9C).

    Therapeutic PiMT Rescues Mice from Severe Respiratory Infections

    [0244] After demonstrating the efficacy of iPSC-MACs in simultaneous infection experiments, the inventors evaluated a clinically more relevant therapeutic PiMT treatment approach. In these experiments, huPAP mice were infected intra-pulmonary with P. aeruginosa and closely monitored for 3-4 h until first disease symptoms developed (determined by a disease score >5). Subsequently, in this model, mice received 4×10.sup.6 iPSC-MACs (infected+PiMT) only after the infection-related symptoms were readily manifested. Of note, infected control mice received PBS only instead of PiMT (infected) (FIG. 6A).

    [0245] In this infection and treatment schedule, therapeutic PiMT treated mice showed a decrease in the disease score as well as a normalization of rectal temperature and body weight to a degree comparable to non-infected animals already within 4-8 hours post treatment. In contrast, infected mice receiving PBS showed pronounced disease progression over time (FIGS. 6B, 6D and 6E). Of note, 24 h post-infection, infected mice showed marked disease symptoms, whereas animals that received a therapeutic PiMT showed a significantly reduced disease score (1.8±0.2 infected+PiMT vs 8.1±0.2 for infected animals, mean±s.e.m., n=3). In addition, the elevated disease score in infected mice was associated with clearly restricted activity when compared to control animals and mice receiving the therapeutic PiMT (FIG. 6C). Efficiency of therapeutic PiMT was further documented by normalized rectal temperature and body weight values 24 h post-infection, and a profound reduction in lung bacterial numbers in infected+PiMT mice (FIG. 6B, 6D, 6F). Recapitulating our findings in the simultaneous transplant model, BALF of animals from the infected+PiMT group showed reduced erythrocyte levels compared to infected, non-transplanted controls. This observation was accompanied by the detection of important pro-inflammatory human cytokines/chemokines such as hIL6, hIL8, hINFa2, hMCP-1 and TNFα (FIGS. 6G and 6H). Inflammation was furthermore assessed by lung histology sections. Here, infected mice showed several areas of massive granulocytic infiltration, severe hemorrhage and alveolar edema, which were barely detectable in mice treated with iPSC-MACs from the bioreactor (FIG. 6I).

    DISCUSSION

    [0246] In the present study, the inventors have evaluated a treatment concept that utilizes macrophages as a cellular therapeutic for bacterial infections. Since clinically-relevant numbers of autologous or donor derived macrophages can hardly be produced from somatic sources, the inventors investigated the possibility to utilize hPSCs for generating substantial quantities of functional macrophages.

    [0247] The use of iPSC derivatives as a cellular approach to target infections has not been seriously considered so far. This might be explained by the fact that the supposed potential of hPSC for the production of specific progenies in therapeutically relevant numbers has not yet been fully translated into practice. Taking advantage of recent advances in culture medium formulations, the inventors tried to culture and differentiate human PSCs as floating aggregates, and adapted stirred tank bioreactor technology to stem cell requirements, allowing for clinical scale-up of iPSC and their derivatives. In order to develop the application of iPSC-macrophages as a cellular therapeutic against bacterial infections, and more specifically to clear pulmonary infections triggered by P. aeruginosa, they first established a scalable and continuous hematopoietic differentiation process for human iPSC in fully equipped bioreactors.

    [0248] For in vivo studies, huPAP mice were employed, an immunodeficient strain which lacks alveolar macrophages. This is of clinical relevance, as this mouse model recapitulates important hallmarks of pulmonary alveolar proteinosis (PAP) (Willinger et al., 2011), including the susceptibility to pulmonary infections typically observed in PAP patients (Trapnell et al., 2003). In this mouse model, prevention of acute P. aeruginosa infection by simultaneous PiMT, or, even more important, therapeutic PiMT was highly effective within a short time frame. These proof-of-concept experiments were performed with a high cell dose. Similar therapeutic effects may be achievable with lower cell numbers. However, even with this maximum cell dose, no apparent adverse events were observed in either treatment scenario, which concurs with observations from pulmonary macrophage transplantation therapy studies employing bone marrow-derived macrophages (BMDM) (Happle et al., 2014; Suzuki et al., 2014).

    [0249] Considering a body weight of 25 g per mouse, clinical translation of iPSC-MACs for the treatment of respiratory infections would require approximately 1×10.sup.10 iPSC-MACs for a 60 kg patient. Even without further process optimization, this would translate to a 40-60 L production scale, which is in principle feasible with current bioreactor technologies (Kropp et al., 2016b). Of note, continuous process monitoring of the bioreactor-based differentiation of the invention revealed a substantial increase in biomass especially in the first 2-3 days of the repeated batch culture. This was followed by partial recovery of dissolved oxygen levels and a profound drop in pH in the last days before medium refreshment. These observations suggest the presence of process-limiting factors and highlight the potential for further process optimization. This can be achieved by cultivation at higher cell densities combined with the application of perfusion systems and feedback control of oxygen, pH and other process parameters, which can multiple the increases in cell yields of hPSCs and their progenies (Kropp et al., 2016a).

    [0250] Given the impressive in vivo functionality of iPSC-MACs in the acute infection model, a therapeutic efficacy of PiMT in chronic infections with P. aeruginosa, as frequently observed in patients suffering from chronic obstructive pulmonary disease (COPD) (Rakhimova et al., 2009) or cystic fibrosis (CF) (Oliver et al., 2000) is expected. Especially CF represents an interesting disease scenario for the application of PiMT, as the disease-causing mutations in the cystic fibrosis conductance regulator (CFTR) gene also hamper functionality of professional phagocytes and thus the host's defense against infections (Bonfield et al., 2012; Bruscia et al., 2009). While single PiMT showed therapeutic efficacy for acute infections, repetitive therapeutic intervention or the application of genetically enhanced cells (Pasula et al., 2016) might be required to clear the established infection in chronic diseases.

    [0251] Long term engraftment and sustained functionality of the transplanted iPSC-MACS are expected. In the tested transplantation scenarios, iPSC-MACs were still detected at 24 h, however later time point were not investigated in this study. Earlier studies demonstrated engraftment of BMDM for more than one year (Suzuki et al., 2014). Moreover, pulmonary transplantation of murine macrophages derived from different multipotent progenitor cells showed the capacity for chromatin remodeling, adaption to the local tissue environment and long-term integration (Happle et al., 2014; Lavin et al., 2014; Suzuki et al., 2014; van de Laar et al., 2016). Along this line, the iPSC-MACs of the invention demonstrate rapid upregulation of pro-inflammatory gene expression after pathogen contact in vitro as well as efficient anti-microbial activity in vivo, suggesting their ability to response to the inflammatory environment in the lung after transplantation.

    [0252] The strong and rapid anti-microbial and therapeutic effect observed by the inventors is of particular importance for a broad applicability of iPSC-MACs to target bacterial infections and a quick clinical implementation. The anti-microbial activity of iPSC-MAC against P. aeruginosa was evaluated, however, a phagocyte-based cell therapy might allow for a broad spectrum of applications in a number of different infection scenarios caused by other gram negative or positive bacteria such as Streptococcus pneumoniae or Staphylococcus aureus, or pathogens associated with implant infections that meanwhile pose an immense health and economical problem. This being said, new form of treatment are of specific clinical relevance considering the increasing numbers of pathogens resistant to standard or reserve antibiotic therapy (Aloush et al., 2006; Falgenhauer et al., 2016; Gould, 2013; Hirsch and Tam, 2010; Liu et al., 2016; Schroeder and Stephens, 2016).

    [0253] In summary, the inventors provide the therapeutic application of PSC-derived phagocytes for the treatment of bacterial infections. They demonstrate the feasibility of phagocyte production under defined conditions that allow for clinical and industrial scale up and provide evidence for the efficacy and safety of iPSC-MAC transplantation as a new treatment approach targeting, e.g., severe respiratory infections. This technology, including process upscaling and the generation of additional hematopoietic cell types as well as assessment in other preclinical models allow for innovative cell-based treatment strategies for a wide variety of diseases.

    Example 2: GMP Compliant Suspension iPSC Cultivation and Hematopoietic Differentiation

    [0254] Single iPSC Generation:

    [0255] Single iPSCs were derived directly from iPSCs cultured on murine feeder cells. iPSCs cultured on murine feeder cells were incubated with Accutase (Cell Dissociation Reagent, StemPro™ Thermo Scientific) for maximum 5 min in the cell culture incubator at 37° C. Accutase reaction was stopped by diluting it with either PBS or DMEM/F12 media (Thermofisher Scientific). Pipetting up and down of resuspended cells helped to have dissociation of clumps and getting single iPSCs (not more than 3 times and very slowly). Cells were counted and subjected to monolayer cultivation on defined matrices (see next step). Alternatively, single iPSC can be derived from iPSC cultured on matrix coated dishes (e.g. GelTrex (Thermofisher Scientific), Matrigel (Corning Fisher Scientific), or Laminin (e.g. CellAdhere Laminin-521; Stem Cell Technologies) treating the cells as described before.

    Splitting Cells as Monolayer:

    [0256] 6-well tissue culture plates (e.g. NUNC plates (Thermofisher Scientific)) were coated with a matrix (e.g., GelTrex (Thermofisher Scientific), Matrigel (Corning Fisher Scientific), or Laminin (e.g. CellAdhere Laminin-521; Stem Cell Technologies) for at least one hour. Single iPSCs were seeded in the pre-coated plates in ROCK-Inhibitor (10 μM) supplemented E8.sup.100 or E8.sup.50 media (Stem Cell Technologies) for further expansion. Maximum 2×10.sup.5 generated single iPSCs were cultured as monolayer in each well of 6-well plate. The media was changed on day 2 of and passaged at day 3 or day 4. The media was not changed the day after the seeding. These cultures were maintained for at least 10 passages.

    Aggregate Formation:

    [0257] 5×10.sup.5 single iPSCs cultivated as single cells on matrix for more than two passages were inoculated in 3 ml ROCK-Inhibitor (10 μM) supplemented E8.sup.50 or E6 media (Stem Cell Technologies) in Greiner CELLSTAR multiwell culture plates (Sigma Aldrich) on an orbital shaker (70 rpm) in a suspension culture for aggregate formation. Aggregate formation started within 24 hours. The media was changed at day 2, changing 2 to 2.5 ml of media. Aggregates on day 3 were transferred either for differentiation or passaged as single cell in suspension.

    Hematopoietic Differentiation:

    [0258] Induction of hematopoietic differentiation was started by mesoderm priming.

    [0259] Mesoderm priming was started by transferring about 100 aggregates on 3 days into 3 ml X-VIVO15 supplemented with 50 ng/ml hVEGF, 50 ng/ml hBMP4 and 20 ng/ml hSCF which was followed by later (at day 4 of mesoderm priming) addition of 25 ng/ml of IL3. Subsequent hematopoietic differentiation of primed aggregates was pursued by changing the media to 3 ml of 25 ng/ml IL-3 and 50 ng/ml M-CSF supplemented X-VIVO15. Mesoderm priming and, following that, hematopoetic differentiation were done on orbital shaker of 85 rpm.

    [0260] The resulting cells were suitable for clinical application in humans.

    Example 3: Preparation of Cells Having a Reduced Size

    [0261] Macrophages were produced or isolated, e.g., from mice, according to methods known in the art. Alternatively, the macrophage cell line U937 was used.

    [0262] The cells were washed and incubated with a hypertonic solution (e.g., a sugar solution such as a sucrose solution) for 15 min to 1 h, typically, for about 15 min at 4° C.-37° C., preferably at 37° C. Hypertonic solutions had an osmolarity of more than 300 mosm, preferably, more than 350 mosm. Reduced size, as measured by forward scatter in flow cytometry for incubations with 300 mosm (control), 600 mosm, 800 mosm, and 1000 mosm is shown in FIGS. 11 and 13.

    [0263] For murine primary macrophages, the average diameter was determined by microscopy and computational analysis e.g. with ImageJ after incubation at the defined omolarities:

    300 mosm.fwdarw.mean area 155.7 μm.sup.2, corresponding to an average diameter of 14.08 μm
    600 mosm.fwdarw.mean area 131.6 μm.sup.2, corresponding to an average diameter of 12.94 μm
    800 mosm.fwdarw.mean area 113.9 μm.sup.2, corresponding to an average diameter of 12.04 μm
    1000 mosm.fwdarw.mean area 115.2 μm.sup.2, corresponding to an average diameter of 12.11 μm.

    [0264] For U937 cells, the average diameter was determined by microscopy and computational analysis e.g. with ImageJ after incubation at the defined omolarities:

    300 mosm.fwdarw.mean area 113.8 μm.sup.2, corresponding to an average diameter of 12.04 μm
    600 mosm 94.34 μm.sup.2, corresponding to an average diameter of 10.96 μm
    800 mosm 82.63 μm.sup.2, corresponding to an average diameter of 10.26 μm
    1000 mosm 74.26 μm.sup.2, corresponding to an average diameter of 9.72 μm.

    Example 4: Preparation of Proteins Form the Cell Culture Supernatant

    [0265] Macrophages were produced according the method of the invention as described in Example 1. The inventors found by mass spectrometry and Western blotting that the cells release high levels of S100 protein into the culture supernatant of the suspension culture, including but not limited to S100A7, S100A8 and S100S9 variant. Gene expression of S100A8 and S100A9 showed >500-fold increase compared to hPSCs and comparable expression to PBMC-derived macrophages. The S100 protein is isolated from the cell culture supernatant, e.g., by affinity chromatography, or by other chromatographic methods as known in the art, or a combination thereof.