Stem cell-derived myeloid cells, their production and use

Abstract

To provide stem-cell derived hematopoietic cells, in particular, myeloid cells, preferably, macrophages, and their generation and use.SOLUTION: A cell population can be obtained by a method of producing myeloid cells, comprising steps of a) cultivating embryoid bodies in suspension culture in the presence of IL- 3 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 in suspension culture for a sufficient period of time to produce myeloid cells; and c) isolating the myeloid cells. A pharmaceutical composition comprising the cell population for treatment of bacterial infection is also provided.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] The present invention relates to stem cell-derived hematopoietic cells, particularly myeloid cells, preferably macrophages, their generation and use. In particular, the present invention provides a method for producing hematopoietic cells, preferably myeloid cells, which comprises culturing embryoid bodies derivable from pluripotent stem cells, such as induced pluripotent stem cells (iPSCs), under suspension culture to produce a myeloid cell forming complex, and further culturing this under suspension culture to produce myeloid cells, such as macrophages, preferably in a continuous manner. This enables scalable and continuous production, for example, in an industrially compatible stirred tank bioreactor. Macrophages, for example, macrophages having unique characteristics produced using this method, can be used in pharmaceutical compositions for the treatment of patients, for example, for the treatment of infections such as bacterial infections. The present invention further provides an application system suitable for spraying, comprising myeloid cells such as macrophages for use in the treatment of patients for infections, such as bacterial infections, or for wound healing. For example, pharmaceutical compositions are also provided comprising shrunk cells having a reduced size, such as a reduced volume, obtainable by contacting the cells with a hypertonic solution, which are particularly useful for spray application of, for example, macrophages, and the cells having a reduced volume are administered into the lungs.

Background Art

[0002] The worldwide increase in the number of severe infections with multi-drug resistant pathogens emphasizes the need for alternative treatment options. The fact that each pathogen is often refractory not only to standard antibiotic therapies but also to last resort drugs poses a serious clinical problem. Thus, alternative strategies targeting bacterial infections are urgently needed (Freire-Moran et al., 2011, Ventola, 2015, Willyard, 2017).

[0003] One promising alternative to antibiotic therapy could be a cell-based treatment approach that applies stem cell-derived immune cells, particularly phagocytes, as an important component of the innate immune system. However, to date, it has been difficult to generate therapeutically relevant amounts of phagocytes from peripheral blood or other sources. In contrast to somatic cell sources and more specified adult hematopoietic stem cells, human pluripotent stem cells (hPSCs), which have infinite potential for proliferation and differentiation, could in principle enable this therapeutic scenario. Along this line, hematopoietic differentiation of hPSCs has been shown to be achievable (Ackermann et al., 2015) and has been proposed as a promising strategy for future cell-based treatment approaches. However, the clinical application of hPSC-derived hematopoiesis is still hampered by a lack of knowledge regarding in vivo functionality and the absence of therapeutically relevant amounts of effector cells.

[0004] In recent years, the inventors have shown that the potential of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) with respect to unrestricted growth and targeted differentiation is not only a theoretical feature, but can also be applied by developing a scalable and potentially GMP-compliant cell production process in a fully controlled stirred tank bioreactor (Kempf et al., 2015, Kropp et al., 2016a, Zweigerdt et al., 2011). The feasibility of this approach has been demonstrated for hPSC expansion (Kropp et al., 2016b, Olmer et al., 2012) and cardiomyogenic differentiation in stirred suspensions (Kempf et al., 2016, Kempf et al., 2015, Kempf et al., 2014), but the successful use of bioreactor systems for generating hematopoietic cells from pluripotent or multipotent stem cell sources remains difficult to achieve. Similar observations have been made regarding the in vivo applicability of PSC-derived hematopoietic cells. The generation of mature immune cells, including phagocytes, from human PSCs has been demonstrated (Buchrieser et al., 2017, Lachmann et al., 2015, van Wilgenburg et al., 2013, Zhang et al., 2015), but data on their actual in vivo functionality are still lacking to date.

Summary of the Invention

Problems to be Solved by the Invention

[0005] In view of the state of the art, the inventors have addressed the problem of providing a method for producing large amounts of hematopoietic cells, particularly myeloid cells, preferably enabling continuous production. This problem is solved by the present invention, particularly by the subject matter of the claims.

Means for Solving the Problems

[0006] Accordingly, the present invention is a method for producing hematopoietic cells, particularly myeloid cells, comprising a) culturing embryoid bodies (EBs) in suspension culture for a period sufficient to produce a myeloid cell-forming complex, in the presence of IL-3 and optionally at least one additional cytokine; b) culturing the myeloid cell-forming complex in suspension culture for a period sufficient to produce myeloid cells, in the presence of IL-3 and optionally at least one additional cytokine; c) isolating the myeloid cells; and providing a method comprising the same.

[0007] The method allows for continuous production and is preferably used for the continuous production of myeloid cells.

[0008] Embryoid bodies (EBs) are typically round, three-dimensional aggregates derived from pluripotent stem cells (PSCs) that contain cell types from all three germ layers. PSCs are undifferentiated cells that can differentiate into specialized cells, and in particular, can differentiate into cells of all three germ layers under suitable conditions. Examples of pluripotent cell types that can be a source of EBs include embryonic stem cells (ESCs) derived from blastocyst-stage embryos from mouse (mESC), primate, and human (hESC) sources.

[0009] Furthermore, EBs can be formed from PSCs induced by alternative techniques including somatic cell nuclear transfer or reprogramming of somatic cells to yield induced pluripotent stem cells (iPSCs). Methods for generating EBs are well known in the art.

[0010] In the context of the present invention, EBs are preferably not derived from stem cells that require the destruction of a human embryo for their production. Preferably, EBs are derived from iPSCs.

[0011] Similar to stem cells differentiated in monolayer format, the stem cells within EBs undergo differentiation and cell specification along the three germ lines (endoderm, ectoderm, and mesoderm) that include all somatic cell types.

[0012] However, in contrast to monolayer differentiation culture, the structure of spheroids formed when PSCs aggregate enables non-adherent culture of EBs under suspension. This makes EB culture inherently scalable, which is useful for a bioprocess approach where high yields of cells can be produced for potential clinical applications. Furthermore, although EBs predominantly exhibit a heterogeneous pattern of differentiated cell types, they can respond to cues similar to those that direct embryonic development.

[0013] Since stem cells may undergo apoptosis when cultured as single cells, EB formation often requires the use of an inhibitor of the rho-associated kinase (ROCK) pathway, such as Y-27632, HA-100, H-1152, and / or 2,4-disubstituted thiazole (thiazovivin / Tzv), preferably Y-27632, at, for example, about 10 μM. Alternatively, to avoid dissociation into single cells, EBs can be formed from PSCs such as hiPSCs by manual separation of adherent colonies (or regions of colonies), or preferably, by enzymatic treatment with collagenase IV, followed by suspension culture. Formation of EBs under suspension enables the formation of large numbers of EBs, but the size of the resulting aggregates can hardly be controlled, often resulting in EBs of large and irregular shapes. When ESCs are seeded in a bulk suspension, a mixed [or dynamic (moved), e.g., stirred or shaken] culture platform increases the uniformity of EB size.

[0014] Preferably, the embryoid bodies of the present invention are obtained by a method including culturing pluripotent stem cells such as iPSCs under suspension culture for a period sufficient to produce embryoid bodies. For therapeutic use, particularly for use in humans, the use of feeder cells should be avoided. This can be compensated for by adding small molecules including cytokines such as SCF, BMP4, VEGF, or CHIR99021 [(6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile (TOCRIS)] and WNT pathway regulators such as BIO [(2’Z,3’E)-6-bromoindirubin-3’-oxime)(TOCRIS)]. Preferably, the culture conditions include the use of a ROCK inhibitor, but the addition of bFGF is not required.

[0015] Suitable embryoid bodies for use in the method of the present invention can be selected manually, but are preferably selected according to their sedimentation characteristics. For example, EBs that sediment within 10 minutes, preferably within 5 minutes (without centrifugation) can be used (WO2010 / 025776A1).

[0016] The pluripotent stem cells that serve as the source of induction of EBs and to which the resulting myeloid cells belong can be from any species, such as mouse, rat, rabbit, guinea pig, cat, horse, dog, cow, camel, pig, sheep, goat, monkey, or primate animals, such as humans. Human cells are preferred. When the cells are for the purpose of treating humans, human myeloid cells, such as macrophages, are preferably produced. In the method of the present invention, preferably, cytokines derived from the relevant species are used. These can be recombinant cytokines and may contain mutations that do not reduce their functionality in the differentiation processes involved.

[0017] The suspension culture of the present invention prevents adhesion to the surface, particularly to the surface of the container. The suspension culture also does not enable adhesion to the carrier. Preferably, the suspension culture is agitated, for example, shaken, rotated, or stirred, to prevent cell adhesion or sedimentation. The culture can be carried out in a suspension plate, for example, on an orbital shaker or in a roller bottle coated to prevent adhesion.

[0018] In a preferred embodiment, the suspension culture is carried out in a bioreactor that enables suspension culture, such as an Erlenmeyer flask, a spinner flask, a stirred tank bioreactor, a wave bioreactor, and a rotating wall bioreactor, preferably in a stirred tank bioreactor, and most preferably in an instrumented stirred tank bioreactor, that is, a bioreactor equipped with technologies for monitoring and controlling process parameters such as pH, pO2, temperature, and stirring speed. Such a system is scalable. That is, the volume of the culture can be changed without significantly changing the culture conditions. For example, as described below, the DASbox Mini bioreactor system (Eppendorf) can be used.

[0019] Regarding compatibility with industrial processes, relative linear upscaling is probably the most important criterion. Preferred systems may already be used in the industry for other purposes and may meet industry standards. For example, they may have standard ports that allow for the integration of standard probes for process monitoring and control, as well as established sterilization methods and instrument validation procedures. The bioreactor can be GMP compliant.

[0020] The bioreactors used in the present invention, for example, stirred tank bioreactors, may be equipped with ports that enable continuous sampling, monitoring, and harvesting of cells, particularly monocytes and macrophages. They may also be equipped with a suitable cell retention system that enables perfusion-based cell supply and online monitoring of culture medium components such as metabolites, for example, glucose, lactate, and ammonium. The bioreactor can be a single-use (disposable) or reusable bioreactor that utilizes a glass, plastic, or stainless steel container.

[0021] Preferably, the culture in the stirred tank bioreactor is carried out at about 37°C using a headspace gas supply at about 3 L / h with about 21% O2 and about 5% CO2, and stirring at about 50 revolutions per minute using a multi-wing (e.g., 4 - 12 wings) inclined impeller. In the context of the present invention, "about" means + / - 10%, preferably + / - 5% or + / - 2%.

[0022] The culture may also be carried out at 37°C using a headspace gas supply at 3 L / h with 21% O2 and 5% CO2, and stirring at 50 revolutions per minute using an 8-wing inclined 60°C impeller, and real-time monitoring of biomass evaluation based on dissolved oxygen, pH, temperature, and impedance may be performed.

[0023] Preferably, the method of the present invention enables continuous production of myeloid cells, including the repeated batch method. Continuous production in the strict sense (strictu sensu) is also possible and may enable more effective cell production. For example, a cell retention system that enables perfusion can be used in combination with continuous medium exchange to optimize the culture conditions.

[0024] For example, in a stirred tank bioreactor, the volume of suspension culture is scalable, for example, 50 mL to 1000 L, 100 mL to 500 L, 200 mL to 100 L, 500 mL to 50 L, or 1 L to 20 L. Preferably, in particular, with respect to the therapeutic application of the produced cells, the medium used for the culture is a chemically defined medium suitable for application in humans, for example, X-VIVO 15 (Lonza) or APEL (Stem Cell Technologies), preferably X-VIVO 15. Preferably, E8 50 or E6 medium (Stem Cell Technologies) may be used for the suspension culture in step a). Myeloid cell-forming complexes (MCFCs) are known in the art (Lachmann et al., 2015). They contain CD34+ clonogenic progenitor cells. They can continuously produce myeloid cells. In the presence of IL-3, myeloid cells are delivered into the culture medium. Notably, the method of the present invention does not require any purification of MCFCs, only that MCFCs are formed in step a) and that they produce myeloid cells in step b). Steps a) and b) [and further, optionally c)] may be repeated. That is, additional EBs can differentiate into MCFCs and other MCFCs can produce myeloid cells (which may already be isolated).

[0025] The generation of MCFCs from EBs typically takes about 4 to 8 days. The generation of the first myeloid cells from MCFCs starts after the formation of MCFCs, and then continuous generation is observed. Typically, harvesting begins about 3 to 16 days after the generation of MCFCs. The preferred total time taken for the production of MCFCs from EBs and the production of myeloid cells [i.e., the total time of steps a) and b)] is at least about 7 days, for example, 7 to 20 days. The inventors were able to show that at least once a week harvesting of macrophages from a stirred bioreactor system, which shows an increasing yield over time after the 7th to 14th day, is possible. In a 250 mL bioreactor (120 mL culture volume), stable production of about 2 - 3×10 7 macrophages / week is possible as early as the 3rd week, and this can be maintained over time for at least 5 weeks. Naturally, the culture time and conditions may vary depending on the desired cell phenotype. For example, harvesting at continuous or short intervals can result in the production of less mature cells. The harvested cells can then be appropriately further matured as described below.

[0026] Culture in the presence of cytokines such as IL-3 does not require the continuous presence of the cytokine. For example, it is possible to culture cells in the absence of IL-3 for a certain period, but the differentiation, especially the production of myeloid cells by MCFCs, requires the presence of IL-3. A preferred amount of IL-3 is, for example, 10 - 100 ng / mL, preferably 20 - 30 ng / mL (most preferably, about 25 ng / ml) of IL-3.

[0027] For the formation of MCFCs, a differentiation medium I, for example, a basal medium, preferably a chemically defined medium such as X-VIVO 15 (Lonza) or APEL (Stem Cell Technologies) (both are suitable for human cells, for example), may be used. When macrophages are produced, an appropriate amount, for example, 10 - 100 ng / mL, preferably 20 - 30 ng / mL (most preferably about 25 ng / ml) of IL-3 (obtainable from, for example, PeproTech), and optionally at least one other cytokine, for example, 40 - 60 ng / ml (preferably about 50 ng / ml) of M-CSF (obtainable from, for example, PeproTech), are further included. X-VIVO 15 is preferred. The medium may optionally contain an appropriate amount of antibiotics, for example, 1% penicillin-streptomycin.

[0028] If the cells produced are not for use in humans, other basal media may be used, for example, for mouse cells, RPMI1640 supplemented with 10% FCS, 2 mM L-glutamine, optionally 1% penicillin-streptomycin, and at least one cytokine.

[0029] For the production of myeloid cells with a mature phenotype, in at least step b) of the method of the present invention, and optionally also in step a), additional cytokines may be added.

[0030] In a preferred method of the present invention, the additional cytokine is M-CSF (a suitable concentration, for example, 40 - 60 ng / mL, preferably about 50 ng / mL of M-CSF), and the myeloid cells produced are macrophages.

[0031] Alternatively, a further cytokine is G-CSF (a suitable concentration, e.g., 40 - 60 ng / mL, preferably about 50 ng / mL of G-CSF), and the myeloid cells produced are granulocyte precursors, which can be cultured in G-CSF alone (a suitable concentration, e.g., 50 - 200 ng / mL, preferably about 100 ng / mL of G-CSF) to further differentiate into granulocytes.

[0032] Alternatively, a further cytokine is GM-CSF (a suitable concentration, e.g., 40 - 60 ng / mL, preferably about 50 ng / mL of GM-CSF), and the myeloid cells produced are macrophages and granulocyte precursors, which can be cultured in GM-CSF alone (a suitable concentration, e.g., 50 - 200 ng / mL, preferably about 100 ng / mL of GM-CSF) to further differentiate into granulocytes.

[0033] When the further cytokines are SCF and EPO [suitable concentrations, e.g., 80 - 120 ng / mL, preferably about 100 ng / mL of SCF, and 2 - 4 units (U), preferably about 3 U of EPO], the myeloid cells produced include erythroid cells, which can be cultured in SCF and EPO alone [suitable concentrations, e.g., 50 - 200 ng / mL of SCF, preferably about 100 ng / mL of SCF, and 2 - 4 units (U) of EPO, preferably about 3 U of EPO] to further differentiate.

[0034] To further broaden the spectrum of cell types generated to dendritic cells or platelets, other lineage-instructive cytokines may be applied (Ackermann et al., 2015, Choi et al., 2009, Lachmann et al., 2015, Ma et al., 2008).

[0035] Alternatively, additional cytokines are IL-4 and GM-CSF (suitable concentrations, e.g., 40 - 60 ng / mL, preferably about 50 ng / mL of GM-CSF and IL-4), and the myeloid cells produced include dendritic cells, which can be cultured and further differentiated at GM-CSF / IL4 alone (suitable concentration, e.g., 50 - 200 ng / mL, preferably about 100 ng / mL of GM-CSF / IL4).

[0036] Additional cytokines are SCF and TPO (suitable concentrations, e.g., 80 - 120 ng / mL, preferably about 100 ng / mL of SCF, and 2 - 4 U, preferably about 3 U of TPO), and the myeloid cells produced may include megakaryocytes and / or platelets, which can be cultured and further differentiated at SCF / TPO alone [suitable concentration, e.g., 50 - 200 ng / mL of SCF, preferably about 100 ng / mL of SCF, and 2 - 4 units (U) of TPO, preferably about 3 U of TPO]. Since IL-3 may have a negative effect on megakaryocyte maturation, it may be reduced or removed after step a).

[0037] In one embodiment of the method of the present invention, no additional cytokines to IL-3 are added in either step a) or step b), and the myeloid cells produced are immature myeloid cells capable of further differentiation. In such a case, the method is i) culturing the immature cells in the presence of M-CSF (suitable concentration, e.g., 40 - 100 ng / mL, preferably about 50 ng / mL of M-CSF) until macrophages are obtained, or ii) culturing the immature cells in the presence of G-CSF (suitable concentration, e.g., 40 - 120 ng / mL, preferably about 100 ng / mL of G-CSF) until granulocytes are obtained, iii) culturing the immature cells in the presence of GM-CSF (suitable concentration, e.g., 40 - 120 ng / mL, preferably about 100 ng / mL of GM-CSF) until granulocytes and macrophages are obtained, iv) culturing the immature cells in the presence of SCF and EPO [suitable concentrations, e.g., 50 - 200 ng / mL of SCF, preferably about 100 ng / mL of SCF, and 2 - 4 units (U) of EPO, preferably about 3 U of EPO] until erythrocyte cells are obtained; v) culturing the immature cells in the presence of SCF and TPO [suitable concentrations, e.g., 50 - 200 ng / mL of SCF, preferably about 100 ng / mL of SCF, and 2 - 4 units (U) of TPO, preferably about 3 U of TPO] until megakaryocytes and / or platelets are obtained, or vi) GM - CSF and IL - 4 (suitable concentrations, e.g., 40 - 120 ng / mL, preferably about 100 ng / mL of GM - CSF, and 40 - 120 ng / mL of IL - 4, preferably about 100 ng / mL of IL - 4) until dendritic cells are obtained may further be included.

[0038] In a medium compliant with GMP, for the culture of a single iPSC as a monolayer preceding suspension culture, E8 medium (Stem Cell Technologies) supplemented with a ROCK inhibitor and containing 5 - 200 ng / mL, preferably 50 ng / ml of bFGF, for suspension culture, E8 50 or E6 medium (Stem Cell Technologies), and for blood cell differentiation, X - VIVO15 medium supplemented with appropriate cytokines, e.g., 50 ng / ml of hVEGF, 50 ng / ml of hBMP4, and 20 ng / ml of hSCF [subsequently (e.g., on the 4th day of mesoderm priming), the addition of 25 ng / ml of IL - 3 may follow] has shown good results. Subsequent blood cell differentiation of the primed aggregates, for example for macrophage production, can be accomplished by changing the medium to 3 ml of X - VIVO15 supplemented with 25 ng / ml of IL - 3 and 50 ng / ml of M - CSF.

[0039] In the method of the present invention, isolating the produced myeloid cells involves purifying the produced myeloid cells, preferably macrophages, to a purity of at least 50%, or preferably at least 70%, at least 80%, at least 90%, or at least 95%. When the method of the present invention is carried out using, in particular, culturing in the presence of IL-3 and M-CSF to produce macrophages, CD45 + CD11b + CD34 - TRA-1-60 - The purity of the myeloid precursor cells characterized by the expression profile of can preferably be at least 80%, at least 90%, or at least 95%. At least 50% of the cells produced, preferably at least 60%, or 70-90% are CD45 + CD11b + CD34 - TRA-1-60 - CD14 + / CD163 + macrophages. In one embodiment, at least 95% of the cells produced are CD45 + CD11b + CD34 - TRA-1-60 - and 70-90% of the cells produced are CD45 + CD11b + CD34 - TRA-1-60 - CD14 + / CD163 + . Most of the other cells are myeloid precursors of macrophages. If the isolation is continuous or the collection is carried out at shorter intervals, the proportion of precursors may be higher.

[0040] In the method of the present invention, isolating the produced myeloid cells (e.g., macrophages) can be carried out, for example, in a batch continuous method, ci) For example, by sedimentation of the suspension culture for about 5 to 12 minutes, preferably 10 minutes [for example, by temporarily stopping the movement (for example, stirring)], enabling the sedimentation of the majority of MCFCs in the suspension culture, and cii) removing the supernatant, and ciii) filtering the removed supernatant through a filter with a mesh of about 70 to 100 μm, and civ) optionally, returning the residue (mainly containing MCFCs) to the suspension culture and adding fresh culture medium to the sedimented cells of the suspension culture may be included, cv) The myeloid cells produced are contained in the filtrate. The myeloid cells produced may be isolated from the filtrate by sedimentation or centrifugation. They may then be formulated for administration to a patient, for example, incorporated into a pharmaceutically acceptable carrier, such as a buffer like PBS. Alternatively, they may be further cultured, for example, under suspension culture, in the presence of an activator and / or maturating agent selected from the group including cytokines or activators such as IL-3, M-CSF, GM-CSF, TNF-alpha, IFN-gamma, or LPS, and then formulated for later administration.

[0041] Alternatively, for isolation, the medium may be removed from the suspension culture through a filter having a mesh of about 70 to 100 μm, where the macrophages produced can be isolated from the filtrate, for example, by centrifugation, and formulated for administration with or without further maturation. This isolation procedure may be carried out occasionally or continuously to enable batch continuous production of the cells produced.

[0042] Further alternative methods for the isolation of the myeloid cells produced include differential centrifugation or gradient centrifugation using, for example, a sucrose gradient.

[0043] The method of the present invention is after step c, ·Reducing the size of myeloid cells by a method selected from the group consisting of contacting the cells with a hypertonic solution and lyophilization, and / or ·Loading myeloid cells with a therapeutic or diagnostic agent selected from the group consisting of antibiotics, immunomodulators, and dyes may further be included.

[0044] Each or both of these steps are typically carried out after isolation and / or purification of myeloid cells such as macrophages, granulocytes, megakaryocytes, platelets, or dendritic cells. Preferably, the myeloid cells are macrophages.

[0045] In the context of the present invention, reducing the size of a cell, such as a myeloid cell, by a method comprising contacting the cell with a hypertonic solution means contacting a cell typically substantially isolated from a buffer and / or cell culture medium such that the volume of the cell is reduced, with a solution having an osmolarity of more than 300 mosm, such as 310 - 1000 mosm, preferably 350 - 800 mosm, or 400 - 600 mosm, for example a hypertonic sugar solution (preferably one containing sucrose). In the case of myeloid cells, the average size (e.g., average volume) is typically reduced by at least 10%, preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 75%. The size of the cell, e.g., the volume, is preferably determined by FACS based on the forward scatter [mean fluorescence intensity (MFI)] of the cell. Alternatively, the reduction in average diameter may be determined by microscopic observation and computer analysis using, for example, ImageJ. Preferably, the size-reduced macrophages according to the present invention have an average diameter of less than 15 μm, less than 13 μm, less than 12 μm, less than 10 μm, preferably less than 8 μm, less than 6 μm, or less than 4 μm (e.g., in the case of human macrophages). For comparison, human alveolar macrophages not treated by the method of the present invention have been found to have an average size of about 21 μm (Krombach et al., 1997. Environ Health Perspect. 105:1261 - 1263). Preferably, the size-reduced primary mouse macrophages according to the present invention have an average diameter of less than 13 μm, less than 12 μm, less than 10 μm, preferably less than 8 μm, less than 6 μm, or less than 4 μm.

[0046] The contact is carried out for a time sufficient to obtain the desired size reduction, for example, for 30 seconds to 24 hours, 1 minute to 12 hours, preferably 2 minutes to 1 hour, 5 to 30 minutes, 10 to 20 minutes, or about 15 minutes. The time required depends on the cell type and the osmolality of the solution used. The temperature is typically maintained at 4°C to 37°C, for example, room temperature (20 - 25°C), preferably 37°C. The viability of the majority of the cells, or preferably at least 80% of the cells, is maintained. Contact with a hypertonic solution is a preferred method for reducing the cell size in the contact of the present invention. Alternatively, the cell size can also be reduced, for example, by lyophilization or manipulation of the cytoskeleton. For example, methods for reducing the cell size while maintaining the viability of the majority of the cells by lyophilization are known in the art. The inventors have surprisingly found that cells with temporarily reduced size, such as myeloid cells like macrophages, are functional and can be used in pharmaceutical compositions.

[0047] Such pharmaceutical compositions may be freshly prepared or may be stored as long as the majority of the cells, preferably at least 80% of the cells, are viable. Storage of cells in contact with a hypertonic solution may be, for example, for up to 48 hours, up to 24 hours, or overnight. Alternatively, the cells may be stored in an isotonic solution at 4°C for up to 12 hours, up to 24 hours, or up to 48 hours and incubated with a hypertonic solution prior to application.

[0048] Also provided herein is a kit for storing cells, such as myeloid cells, particularly macrophages of the present invention, and reducing their size. For example, one chamber contains cells in an isotonic solution and one chamber contains a hypertonic solution. By mixing the two solutions, the cells come into contact with the hypertonic solution as defined herein, resulting in a reduction in cell size. The sterility of the cells in the kit can be easily maintained. Preferably, the kit can be connected to a device suitable for spraying the cells or is part of a device for spraying the cells.

[0049] Cells that have been reduced in size by lyophilization can be stored for a longer period of time at an appropriately low temperature than cells in a hypertonic solution. The lyophilized cells can be administered directly or after incorporating them into a hypertonic solution. For example, macrophages with reduced size have been found to be active in the phagocytosis of particles or bacteria, such as S. aureus. The reduced size results in deeper penetration into the airways and better distribution of the cells in the lower airways. The inventors were able to show in experiments using tissues explanted from rats that the shrunk macrophages of the present invention can be administered into the alveolar cavity by spraying. Thus, cells with reduced size are particularly suitable for, for example, spraying into the airways, or other applications where the reduced size can result in better penetration or distribution, such as in vitro disinfection applications, or coating of devices for transplantation.

[0050] Preferably, simultaneously with contacting with the hypertonic solution, cells, such as macrophages, may be loaded with a therapeutic agent or a diagnostic agent, preferably an agent that can be a therapeutic agent. Of course, a combination of two or more agents may be used. The agent can be an exogenous agent. An exogenous agent is usually not found in significant amounts in the cells used in the present invention, is typically not derived from human cells, and may not be derivable from human cells. Exemplary agents are antibiotics or immunomodulating agents.

[0051] In the context of treating bacterial infections, loading with antibiotics is particularly useful. Examples of antibiotics are macrolide antibiotics such as azithromycin, clarithromycin, erythromycin, beta-lactam antibiotics such as cephalosporins, amoxicillin, methicillin, penicillin, or flucloxacillin, clindamycin, or fluoroquinolones such as levofloxacin, moxifloxacin, doxycycline, or telithromycin, clavulanic acid, rifampicin, or gentamicin, or glycopeptide antibiotics such as vancomycin or teicoplanin, which are preferred lipophilic antibiotics. The selection depends on the indication, in particular on the bacteria that are the cause of the infection to be treated. Immunomodulators, such as agents that can activate or inhibit immune system cells such as cytokines, chemokines, or corticosteroids, may be used, for example, in cases of inflammation. Bronchospasmolytics loaded intracellularly can help to dilate the airways, which can enable deeper penetration of the second dose of cells administered when the first dose becomes effective. Secretolytics may be loaded intracellularly, which can be particularly useful, for example, in the treatment of cystic fibrosis.

[0052] Dyes loaded into cells, particularly cells with a reduced size, can be convenient as tracers or for diagnostic purposes to confirm cell penetration. Exemplary dyes are fluorescent dyes such as Dil.

[0053] Loading into cells may be carried out before or without contacting the cells with a hypertonic solution. For applications of cells where the reduced size does not play a major role, for example, applications mainly to planar areas such as wounds, typically the cell size is not modified.

[0054] Naturally, loading cells with a drug is not required for applications where macrophages themselves can already be effective.

[0055] The present invention provides a suspension culture containing a myeloid cell-forming complex that continuously produces hematopoietic cells and, optionally, myeloid cells. The suspension culture can be obtained, for example, from the method of the present invention described above, where isolation of the produced myeloid cells is possible but not essential. The suspension culture is preferably contained, for example, in a stirred tank bioreactor as described above. In other words, the present invention provides a stirred tank bioreactor containing a suspension culture containing a myeloid cell-forming complex that continuously produces myeloid cells and, optionally, myeloid cells. Preferably, the myeloid cells are macrophages, and the culture medium contains IL-3 and M-CSF.

[0056] The present invention also provides a population of hematopoietic cells, preferably myeloid cells, obtainable by the method of the present invention.

[0057] In an optional embodiment, the size of the myeloid cells, such as macrophages, is reduced by a method selected from the group including contacting the cells with a hypertonic solution and lyophilization. The cells may be further loaded with the above-mentioned therapeutic or diagnostic agents, such as antibiotics, immunomodulators, and dyes. Thus, the present invention provides a population of myeloid cells with a reduced cell size compared to the corresponding cells cultured under physiological conditions (especially cells not contacted with an isotonic solution). In myeloid cells, the average volume is typically reduced by at least 10%, preferably at least 20%, at least 30%, at least 40%, at least 50%, or at least 60%. The cell size or volume is preferably determined by FACS based on the forward scatter of the cells. Preferably, the macrophages with a reduced size according to the present invention have an average diameter of less than 10 μm, preferably less than 8 μm, less than 6 μm, or less than 4 μm. These cells may further contain a therapeutic or diagnostic agent, preferably a therapeutic agent. The cell population is, for example, more than 1×10 6 macrophages, at least 2×10 7 macrophages, at least 5×10 7macrophages, at least 1×10 8 macrophages, at least 1×10 9 macrophages, or at least 1×10 10 macrophages. Cells having a reduced size may have any of the marker profiles described below.

[0058] Cells obtainable by the method of the present invention may have an unmodified size, for example, when not in contact with a hypertonic solution.

[0059] The present invention provides, for example, CD45 + CD11b + CD14 + CD163 + CD34 - TRA1-60 - a cell population comprising macrophages, preferably at least 50%, at least 60%, or at least 70% of said cells being obtainable, for example, by the method of the present invention. Analysis of the expression of genes related to pluripotency and activation of the innate immune response confirmed efficient differentiation of iPSCs into macrophage-like cells. Importantly, genes related to macrophage function, such as toll-like receptors (TLRs) 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-MAC and PBMC-MAC compared to iPSCs. Morphological analysis after analysis of adhesion and function (e.g., phagocytic uptake of latex beads and bacteria) confirmed that the cells produced were macrophages. The method of the present invention provides more than 1×10 6 macrophages, at least 2×10 7 macrophages, at least 5×10 7 macrophages, at least 1×10 8 macrophages, at least 1×10 9 macrophages, or at least 1×10 10For the first time, it enables the production of a cell population containing individual macrophages.

[0060] Macrophages obtainable from the method of the present invention typically have a significantly higher surface expression level of CD14 and C163 (determined by FACS) and a significantly lower surface expression level of HLA than macrophages obtained from PBMC. The macrophages produced in the examples were shown to produce pro-inflammatory cytokines such as IL-6, IL-8, or TNF-alpha. Therefore, they can be considered pro-inflammatory macrophages rather than anti-inflammatory macrophages. By adding suitable cytokines, such as IL-13, IL-10, IL-4, the phenotype can be redirected to anti-inflammatory macrophages. In addition, other substances, such as corticosteroids, may be used to induce an anti-inflammatory phenotype.

[0061] The inventors, for example, CD45 of the present invention obtainable from the method of the present invention + CD11b + CD14 + CD163 + CD34 - TRA1-60 -It has been found that macrophages have a unique expression profile compared to macrophages isolated according to prior art methods. Preferably, in the macrophages of the present invention, the expression of at least 10 genes selected from the 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, preferably at least 50-fold, at least 200-fold, at least 400-fold, or at least 1000-fold upregulated in said macrophages compared to macrophages derived from PBMC. The expression of at least 12 genes, preferably at least 15 genes, or all genes selected from the 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 may be at least 100-fold, preferably at least 200-fold, at least 400-fold, or at least 500-fold upregulated in said macrophages compared to macrophages derived from PBMC. The expression of CYR61, DDIT4L, KRT19, DCN, LUM, COL3A1 may be at least 200-fold, at least 500-fold, or at least 1000-fold upregulated in said macrophages compared to macrophages derived from PBMC.

[0062] Furthermore, in the macrophages of the present invention, the expression of at least 10 genes selected from the group consisting of ANPEP, CDA, CRTAM, ENST00000390237, FBP1, GBP1, GNLY, HLA_DQA1, HLA_DQA2, HLA_DQB1, HLA_DQB2, HLA_DRA, HLA_DRB1, HLA_DRB3, HLA_DRB4, HLA_DRB5, IL15, LY75, S1PR4, and TNFAIP6 may be downregulated by at least 20-fold, preferably at least 200-fold, at least 400-fold, at least 500-fold, or at least 1000-fold in the macrophages as compared to macrophages induced from PBMC. The expression of at least 10, at least 12, or all genes selected from the group may be downregulated by at least 100-fold, preferably at least 200-fold, at least 400-fold, at least 500-fold, or at least 1000-fold in the macrophages as compared to macrophages induced from PBMC.

[0063] Typical expression profiles are disclosed herein, for example, in Table 1. This comparison is found to result in particularly high differences for genes that are virtually not expressed in one of the cell types being compared. In that case, even very small differences in gene expression can result in high differences in the comparative expression rate. Without intending to be bound by theory, it is believed that shear stress in suspension culture results in different expression of genes in the myeloid cells produced.

[0064] The present invention also provides a cell population comprising at least 50%, preferably at least 60%, or at least 70% of immature CD45+CD11b+ / CD14- / CD163- myeloid cells, and optionally, the cell population can be obtained by the method of the present invention. The immature cells are unique due to their CD45+CD11b+CD14- / CD163- / CD66b- / CD34 dim / - expression profile.

[0065] The present invention also provides a cell population comprising at least 50%, preferably at least 60%, or at least 70% granulocytes, and optionally, said cell population can be obtained by the method of the present invention. Said granulocytes are unique because, due to their expression profile, they express significantly less CD66b on their surface than, for example, granulocytes isolated from PBMC. Said granulocyte cells are characterized by their CD45+CD11b+CD66b dim , CD16 dim expression profile.

[0066] The present invention also provides a cell population comprising at least 50%, preferably at least 60%, or at least 70% erythrocyte cells, and said cell population can be obtained by the method of the present invention. Said cells are thought to be erythrocyte precursors.

[0067] The present invention also provides a cell population comprising at least 10%, preferably at least 20%, or at least 30% megakaryocytes, and said cell population can be obtained by the method of the present invention.

[0068] The present invention also provides a cell population comprising at least 10%, preferably at least 15%, or at least 20% dendritic cells, and said cell population can be obtained by the method of the present invention.

[0069] Such cell populations can be frozen, for example, according to methods known in the art. They are typically thawed before use. For example, populations isolated at one or more time points from different batches of one or more cultures can be combined if a higher cell number is required. Of particular importance can be the differentiation using only IL3 to generate a population of myeloid progenitor cells that can be frozen, in combination with subsequent differentiation towards the optimal therapeutic cell type, according to requirements.

[0070] The present invention also provides a pharmaceutical composition comprising, in a pharmaceutically acceptable carrier, a cell population of the present invention, preferably a cell population comprising macrophages or immature myeloid cells of the present invention, and most preferably macrophages characterized herein. The cell population can be obtainable from the methods of the present invention described herein.

[0071] Preferably, the cells are human cells for the treatment of a human patient, most preferably human macrophages or immature myeloid cells of the present invention, and most preferably macrophages characterized herein.

[0072] The pharmaceutically acceptable carrier is typically a buffer, such as PBS (PBS / EDTA supplemented with about 20% human serum albumin), or citrated plasma, or Plasmalyte-A pH 7.4 (Baxter, supplemented with about 2% human albumin). This is compatible with the survival of the cells. It may contain physiological levels of NaCl.

[0073] In a further embodiment, the present invention provides a pharmaceutical composition comprising a cell population in a pharmaceutically acceptable carrier, wherein the average size of the cells in the population is reduced, for example, by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% compared to the population of such cells in an aqueous saline solution. The cell population can be a population of myeloid cells described herein, such as immature myeloid cells of the present invention characterized herein, macrophages, dendritic cells, granulocytes such as neutrophils, platelets, megacaryocytes or erythrocyte cells, preferably a population of macrophages or immature myeloid cells.

[0074] A pharmaceutically acceptable carrier in the context of a pharmaceutical composition comprising a cell population in which the average size of the cells in the population is reduced is, for example, a hypertonic solution containing sugars and / or salts. In one embodiment, the pharmaceutical composition of the invention comprising macrophages having an unmodified size may be contained in a container suitable for spraying the pharmaceutical composition, for example, by means of an atomizer.

[0075] In another embodiment, the pharmaceutical composition of the invention comprises macrophages having a reduced size as described herein and is contained in a container suitable for spraying the pharmaceutical composition, for example, by means of an atomizer.

[0076] Thus, the present invention also provides a) a pharmaceutical composition comprising myeloid cells such as macrophages in a pharmaceutically acceptable carrier, and b) a container suitable for spraying the pharmaceutical composition, for example, by means of an atomizer in the form of, for example, a kit.

[0077] Preferably, the macrophages are macrophages obtainable by the method of the invention having, for example, the characteristics described herein. However, myeloid cells, particularly macrophages, generated by other methods can also be used in the application system of the invention, for example, for the treatments described herein.

[0078] In one embodiment, the myeloid cells of the application system are myeloid cells, for example, macrophages, which do not have a modified size and can express the markers described herein. They can be obtained by the method of the invention.

[0079] In a second embodiment, the myeloid cells of the application system are myeloid cells having a reduced size, such as macrophages. For example, the size of the myeloid cells may be reduced by a method selected from the group comprising contacting the cells with a hypertonic solution and lyophilization. The cells preferably express the markers described herein. They can be obtained by the methods of the present invention. In a third embodiment, the myeloid cells of the application system are myeloid cells, such as macrophages, comprising a therapeutic or diagnostic agent selected from the group comprising antibiotics, immunomodulators, and dyes, and this agent may be exogenous to the cells. This may be loaded into the cells as described herein. The cells may have a reduced size as described herein, and more preferably, express markers typical of cells obtained by the methods of the present invention.

[0080] Suitable containers are known in the art, for example, from EP2743343A1. For example, the Skin Gun of RenovaCare may be used (RenovaCare), which can spray a liquid suspension of cells, for example, onto a wound. A gentle positive airflow may be used to spray the cell suspension. Such an application system is particularly useful for spraying a cell population of the present invention, including, for example, macrophages, onto a surface, such as a wound, such as a burn and / or an infected wound. They are also useful for treatment in the lungs. They may be used for treatment of a surface in vitro, for example, for disinfection or for reducing the number of pathogens, in particular for reducing the number of bacteria on the surface.

[0081] The cells obtainable from the methods of the present invention may alternatively be administered using a bronchoscope or to an intubated patient.

[0082] The present invention also provides a pharmaceutical composition or application system of the present invention, preferably comprising macrophages such as macrophages obtainable by the methods of the present invention, for use in treating or preventing infection in a patient, such as a human patient.

[0083] The inventors have found that the cells of the present invention can be advantageously used as a cell therapy agent for the treatment of various tissues or organs, such as the brain, lung, skin, intestine, peritoneum, liver, spleen, or pancreas, and particularly for the treatment of macrophage dysfunction in the brain, lung, skin, intestine, peritoneum, or liver. Macrophages genetically engineered to express a protein can also be used for the treatment of other diseases. For example, macrophages expressing insulin can be used for the treatment of diabetes. The treatment of genetic diseases involving protein deficiency or dysfunction can also be treated by administration of macrophages expressing the protein. Due to the relatively low turnover rate and tissue adaptation of macrophages, a single administration can be effective for at least 1, 2, or 3 months, or for several years, optimally for life. If repeated treatment is necessary, the intervals can be longer accordingly, for example, every 1, 2, 3, or 6 months, or every 1, 2, 3, 4, 5, 10, or 20 years.

[0084] The inventors have shown that the pharmaceutical composition can be advantageously used for the treatment or prevention of bacterial infections, most preferably for the treatment of bacterial infections. Bacterial infections can be, for example, infections with P. aeruginosa, S. pneumoniae, S. aureus, and M. tuberculosis. This can be a lung infection. For example, the treatment of these bacteria for lung infection can be performed in the lung, for example, by local cell administration via intubation. Alternatively, an inhalation-based system such as an aerosol-based spray or systemic administration by intravenous (i.v.) injection can be applied. Local administration can also be used. In an inhalation system, when deep penetration into the airways is desired, the use of cells having a reduced size can be advantageous, as described herein.

[0085] Lung infections by bacteria, such as P. aeruginosa, are particularly relevant in patients such as those with cystic fibrosis. Patients who may be particularly relevant for treatment or prevention are immunosuppressed patients, such as transplant patients, patients undergoing intensive care (e.g., ventilator-associated pneumonia patients), neonates, elderly patients, patients with chronic lung diseases such as cystic fibrosis, CGD (or other hereditary diseases causing immunodeficiency), chronic obstructive pulmonary disease, or patients suffering from hPAP, etc., who are immunocompromised patients.

[0086] The treatment of bacterial infections can also be the treatment of sepsis. In this context, macrophages can not only help reduce the bacterial load, but also function as a sink (macrophage sponge) to reduce the load of pro-inflammatory cytokines, cells and bacterial debris, and / or toxins.

[0087] The treatment or prevention of infection may also be the treatment of infected wounds or the prevention of wound infections. In that case, for example, topical administration by spraying the wound with the administration device of the present invention is useful. The wound can be cleaned, for example, by removing coagulated blood to facilitate access before treatment. The treatment or prevention of infection may be, for example, the treatment before or after surgery with the composition of the present invention by spraying the composition of the present invention on the surgical site during surgery or after closing the surgical site.

[0088] The present invention also provides a pharmaceutical composition or application system of the present invention for use in promoting wound healing in a patient, preferably comprising macrophages. The wound can be, for example, a burn, particularly a second-degree or third-degree burn. The promotion of wound healing may or may not be related to infection. Macrophages not only contribute to the elimination of pathogens but also assist in tissue remodeling. These characteristics make the pharmaceutical composition of the present invention useful, for example, for the treatment of ulcers or the removal of necrotic tissue. Further uses can be, for example, in promoting transplant success or in the treatment of periodontitis. The composition may be used to prevent or minimize scar formation.

[0089] In the context of the present invention, treatment means that at least one symptom of the disease is alleviated. Preferably, in the context of a bacterial infection, the bacterial load is also reduced, and most preferably, all bacteria are eliminated.

[0090] Prevention means that the patient to be treated is not yet ill, i.e., does not show symptoms of the disease and may be uninfected. Prevention reduces the likelihood of the occurrence of a disease (especially an infection) and / or reduces the severity of the symptoms of the disease.

[0091] Typically, the present invention is used in the context of treatment, but for example, in the context of an increase in multi-resistant bacteria and / or in patients, such as immunocompromised patients undergoing intensive treatment, who are at high risk of contact with such bacteria, or in the context of solid organ transplantation or blood / bone marrow transfusion, prevention may also be the goal.

[0092] One particular advantage of the present invention is that it can be used to treat or prevent bacterial infections with bacteria that are resistant to at least one antibiotic, or multi-resistant bacteria.

[0093] Alternatively, treatment and / or prevention of viral infections, such as influenza (Cardani et al. 2017), preferably treatment, using a pharmaceutical composition comprising the cell population of the present invention (preferably comprising macrophages) is envisaged. For the treatment of influenza, for example, intrapulmonary application, such as those described herein, or aerosol application can be performed. If the viral pathogen is known, an antibody directed against the surface antigen of that virus (of a suitable species that can be recognized by the macrophages in the composition) may further be included in the pharmaceutical composition. Antibodies directed against the surface antigens of bacteria may be added to the composition intended for the treatment of bacteria.

[0094] Typically, administration of the pharmaceutical composition is carried out at the site of infection or at a potential site of infection. For example, for lung infections, pulmonary (intrapulmonary) administration is optimal. However, other application forms are envisaged when the cells are unlikely to reach the site of infection and / or the intubation is too risky for the patient, i.e., when the lung disease is severe, such as in the case of severe COPD or severe lung infection. Other suitable administration forms include i.v. administration, inhalation-based systems (sprays, e.g., similar to asthma sprays), topical administration, e.g., for wounds such as infected wounds or wounds at risk of infection, ocular administration (e.g., during eye infections), nasal administration, administration to organs by transplantation, or administration near the infected side (e.g., near the infected area associated with a graft).

[0095] Generally, iPSCs derived from a patient (e.g., a human patient) can be used for the generation of myeloid cells and thus the autologous myeloid cells (preferably macrophages) of the present invention. However, this is not essential since the elimination of bacteria by allogeneic macrophages administered to the patient may sometimes be faster than the rejection reaction of the macrophages. Allogeneic macrophages that can be produced as off-the-shelf products in a cost-effective manner by an industrial-scale bioreactor system may be applied when long-term survival of the transplanted macrophages is not required or, even more, is undesirable, e.g., in acute bacterial infections without a genetic disease background. In particular, in the context of pulmonary administration, the post-administration survival and functionality of the administered macrophages have been demonstrated.

[0096] Notably, since allogeneic cells are expected to be immunologically rejected within the first two weeks, the use of the allogeneic myeloid cells of the present invention minimizes the risk of tumor formation, at least in immunocompetent recipients. That being said, repeated application of off-the-shelf preparations is possible when different HLA haplotypes are applied. It is also possible to use myeloid cells (e.g., macrophages) generated from pluripotent stem cells (e.g., iPSCs) that have been selected (or engineered) with respect to either immunocompetence or lack of HLA molecule expression.

[0097] Based on experiments conducted by the inventors, approximately 4×10 6 macrophages (calculated with respect to the total cell number in the cell population of the present invention) were successful in treating lung infections in approximately 25 g mice. Lower cell numbers, e.g., approximately 4×10 5 to approximately 4×10 6 per mouse, or approximately 1×10 6 to approximately 2×10 6 per mouse, are expected to be sufficient for treatment. This corresponds to approximately 1.6×10 7 / kg to approximately 1.6×10 8 / kg, or approximately 5×10 7 / kg to 1×10 8 / kg. As a result, for example, a 60 kg human patient can be treated with approximately 1×10 9 to 1×10 10 macrophages. The number of cells required for treatment success will, of course, depend on several factors that can be evaluated by the clinician, such as the overall health of the patient, particularly the immune status, the severity of the disease, and the bacterial load. For prevention, significantly fewer cells than for treatment are thought to be required. In the case of wound healing or wound infection, a similar evaluation by the clinician can be performed to elucidate the therapeutic cell number. For example, 4×10 5 to approximately 8×10 6 macrophages / cm 2 may be sufficient for treatment.

[0098] In addition to the application of iPSC-derived macrophages, for example, for the treatment of infectious diseases, other iPSC derivatives may alternatively or additionally be applied to target various diseases. Suspension-based differentiation culture also enables the generation of other iPSC-derived myeloid cells, and stirred tank bioreactor technology is also suitable for the generation of, for example, iPSC-derived granulocytes. Such granulocytes can be used, for example, for the improved treatment of bacterial infections, as listed above, or for the treatment of fungal infections, for example, in combination with macrophages and / or alone.

[0099] Platelets obtainable according to the method of the invention can be suitable for use, for example, in the treatment of thrombocytopenia, in particular prophylactically (especially to prevent intracranial hemorrhage) in bleeding episodes, preemptively (for example, when major surgery is expected), or therapeutically, particularly after anticancer chemotherapy and / or hematopoietic stem cell transplantation. Similarly, such platelets can be used in functional platelet deficiencies, such as drug-induced and here particularly aspirin-induced ones, or congenital ones such as Glanzmann thrombasthenia or Bernard-Soulier syndrome.

[0100] Dendritic cells obtainable according to the method of the invention can be suitable for use, for example, in (i) vaccine administration strategies against tumors and other malignant lesions, or (ii) pathogens containing viruses such as EBV or CMV.

[0101] Alternatively, large-scale differentiation using only IL3 can be used to generate a myeloid progenitor cell population suitable for cryopreservation and subsequent differentiation according to requirements towards the optimal therapeutic cell type.

[0102] The cell population of the invention, preferably containing macrophages, can be used, for example, in in vitro a) drug screening and drug development, b) disease modeling, c) tissue engineering (for example, blood production for trauma therapy), d) Preparation of bioartificial organisms, e) Disinfection and / or elimination of pathogens (e.g., for water purification or surface disinfection), f) Materials for transplantation, e.g., coating of stents, g) Development of biomarkers for monitoring the physiological functions and pathophysiology of cells in the hematopoietic differentiation, development, and maturation of, for example, blood cells, preferably macrophages [where, for example, the expression profiles of cells derived from patients and healthy individuals (or standard cells for comparison) can be screened], or h) Quality control of biological products selected from the group consisting of antibodies, hormones, cytokines, drugs, culture media, and sera and are also useful for applications such as the following.

[0103] For some of these in vitro applications, for example, with respect to disinfection or coating of materials for transplantation, spraying of cells may be effective. Thus, the cell population of the present invention may be in the form of an administration system or kit suitable for spraying cells, for example, with cells in a container suitable for spraying that may be connected to an atomizer.

[0104] The present invention also provides a method for preparing a protein, which includes performing the method of the present invention for preparing myeloid cells, such as macrophages, and isolating embryoid bodies, myeloid cell forming complexes, and / or proteins produced by myeloid cells, preferably proteins produced by myeloid cells. The protein can be an intracellular protein or a membrane protein. Preferably, the protein is a protein secreted into the cell culture medium. Advantageously, this can be prepared from the cell culture medium, for example, at the end of the suspension culture of the present invention, for example, when isolating cells from the medium for cell harvesting. The protein can be, for example, a cytokine, chemokine, growth factor, S100 protein, and a recombinant protein. For the expression of the recombinant protein, the myeloid cells are genetically engineered to express the protein, for example, by stable transfection or transient transfection. The inventors have found that, for example, macrophages cultured according to the method of the present invention produce a high amount of S100 protein, also called S100 alarmin, which has medical uses as an immunomodulatory agent or for the treatment of cardiovascular disorders, for example, in the treatment of sepsis in neonates.

Brief Description of the Drawings

[0105]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

[0106] References

Table A

Table B

Table C

Table D

Mode for Carrying Out the Invention

Example

[0107] Example 1 Method Cell Culture and Differentiation Culture of iPSCs Mobilized peripheral blood CD34 + Human iPSCs (hCD34iPSC16) were generated in advance from mobilized peripheral blood CD34 cells (Lachmann et al., 2014) and cultured on irradiated mouse embryonic fibroblasts (MEF) in iPSC medium (knockout DMEM, 20% knockout serum replacement, 1 mM L-glutamine, 1% NEAA, 1% penicillin / streptomycin, all from Invitrogen, Karlsruhe, Germany), 0.1 mM β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, United States), and 10 ng / ml bFGF (PeproTech). Basic FGF was removed from the maintenance medium during the last 4 - 5 days before EB formation.

[0108] Hematopoietic Differentiation in Adherent Culture As previously described (Lachmann et al., 2015), hematopoietic differentiation of human iPSCs based on conventional adhesion was performed. Briefly, collagenase-IV was used to disrupt PSC colonies (3 wells out of 6-well plates, approximately 3×10 6 cells) into fragments, and EB formation was induced by culturing in iPSC medium supplemented with 10 μM Rock inhibitor (Y-27632; Tocris) for 5 days in a 6-well suspension plate on an orbital shaker. Subsequently, EBs were manually selected using a binocular microscope and transferred to a tissue culture 6-well plate and cultured 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 from PeproTech). After day 10 - 15, iPSC-MACs were collected from the supernatant once a week.

[0109] Hematopoietic differentiation in suspension culture To achieve hematopoietic differentiation under suspension, the largest EBs were selected by sedimentation characteristics (sedimentation less than 10 minutes) and transferred to differentiation medium I in a 6-well suspension plate on an orbital shaker (Celltron, Infors HT) at 85 revolutions per minute (rpm). After day 10 - 15, iPSC-MACs were collected 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 granulocytes and erythrocytes, 25 ng / ml of hIL3 was applied in combination with 50 ng / ml of hG-CSF, or 100 ng / ml of hSCF and 3 U of hEPO, respectively.

[0110] Hematopoietic differentiation in a stirred tank bioreactor As described previously (Kempf et al., 2015), the bioreactor (DASbox Mini bioreactor system, Eppendorf) was set up and calibrated. Briefly, a 250 ml glass vessel was equipped with an 8-blade impeller (60° inclined), as well as probes for biomass (Aber Instruments), pH, DO online monitoring, and temperature control. Calibration was performed in 120 ml of chemically defined X-VIVO 15 (Lonza).

[0111] For hematopoietic differentiation in the bioreactor, iPSCs were expanded up to 20 six-well plates and cultured for 3 days in the presence of bFGF. Embryoid body (EB) formation was carried out as in adherent culture. After 5 days, EBs were selected based on sedimentation characteristics (sedimentation less than 10 minutes) and transferred to an equilibrated bioreactor. Cells were cultured at 37 °C using a constant headspace gas supply (21% O2; 5% CO2) at 3 L / h and agitation at 50 rpm in X-VIVO 15 (differentiation medium I) supplemented with IL3 and M-CSF. To monitor the integrity of MCFCs and macrophage formation, 1 ml of samples were collected once or twice a week via a sampling port without interrupting the culture process. Differentiation medium I was manually changed every 6 - 7 days and 20 ml was supplied as appropriate 3 - 4 days later. Macrophages were harvested once a week by sedimentation (4 - 5 minutes) and subsequent separation from MCFCs via filtration of the medium through a 100 μm filter. The retained MCFCs were returned to the bioreactor. Macrophages were collected from the filtered medium by centrifugation at 300×g for 4 minutes.

[0112] Microsoft Excel 2016 and GraphPad Prism 6 were used to process data from online monitoring. For glucose and lactate concentrations, a YSI 2700 select biochemistry analyser was used, for osmolality an Osmomat 300 (Gonotec) was used, and for lactate dehydrogenase concentration, the supernatant was analysed using a microplate reader (Paradigm, Beckman Coulter) according to the manufacturer's instructions (MAK066, Sigma).

[0113] Final differentiation For further maturation, cells freshly harvested from MCFCs were cultured for at least 7 days 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 macrophages, 50 ng / ml hG-CSF for granulocytes, 100 ng / ml SCF and 3 U / ml EPO for erythroid differentiation.

[0114] Isolation of peripheral blood mononuclear cells (PBMCs) and differentiation into macrophages All healthy donors provided written informed consent in accordance with the local ethics committee at Hannover Medical School. Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy volunteers by gradient centrifugation (40 min, 400×g; Biochrome, Billerica, MA) using Biocoll Separating Solution. Cells were then cultured for 1 week in RPMI1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 1% penicillin-streptomycin (all Invitrogen), as well as hIL3 and hM-CSF (50 ng / ml each, PeproTech). Subsequently, PBMC-MACs were cultured for an additional 3 - 4 days in differentiation medium containing only 50 ng / ml M-CSF.

[0115] Phenotypic and functional characterization Flow cytometry As described (Lachmann et al., 2015, Lachmann et al., 2014), flow cytometry analysis of myeloid cells was performed. For macrophages, PBS supplemented with 10% FCS was used to prevent non-specific binding. Cells were analyzed using a FACScalibur cytometer (Beckton & Dickinson, Heidelberg, Germany) and analyzed using FlowJo software (TreeStar, Ashland, OR). The following antibodies: hTRA-1-60-PE (catalog number: 12-8863-80), hCD11b-APC (catalog number: 17-0118-41), hCD14-PE (catalog number: 12-0149-42), hCD163-APC (catalog number: 17-1639-41, hCD16-FITC (catalog number: 11-0168-41), hCD34-FITC (catalog number: 11-0349-41), as well as isotype controls: mouse IgG1a-PE (catalog number: 12-4714-41), FITC (catalog number: 11-4714-41) or APC (catalog number: 17-4714-41), and rat IgG2a-PE (catalog number: 12-4321-81) were purchased from eBioscience. Antibodies obtained from Biolegend San Diego, CA, United States: hCD86-APC (catalog number: 305411), hCD66b-FITC (catalog number: 305104), or hCD45-PE (catalog number: 304007).

[0116] For flow cytometry analysis of mouse lungs and BALF, samples were fixed using 4% PFA. Subsequently, to prevent non-specific binding, samples were incubated with an fc receptor blocking antibody (CD16 / CD32, eBioscience, catalog number: 14-0161-81) for 20 minutes. The antibodies used were purchased from Biolegend San Diego, CA, United States (hCD45-PeCy7) and from eBioscience (mGR1-eFluor450).

[0117] Site spin preparation 20,000 to 50,000 cells were spun on a glass slide at 600×g for 7 minutes and stained in 0.25% May-Gruenwald for 5 minutes and in 0.4% Giemsa staining modified solution (Sigma) for 20 minutes.

[0118] Phagocytosis assay The phagocytic activity of iPSC-MACs derived from suspension culture or adherent culture and terminally differentiated on tissue culture plates was evaluated by flow cytometry. Thus, 1×10 5 cells were incubated with pHrodo™ Red E.coli BioParticles® Conjugate (MolecularProbes / Thermo Fisher Scientific, Schwerte, Germany) or medium for 2 hours at 37°C or 4°C as a negative control. After incubation, the cells were placed on ice for 10 minutes. Analysis was performed using a Beckman Coulter FC500 flow cytometer.

[0119] GFP-PAO1 [wild-type P. aeruginosa PAO1 tagged with green fluorescent protein (GFP) by transformation of Tn7 (kindly provided by Thomas Bjarnsholt, University of Copenhagen)] was used to evaluate the functional ability of iPSC-MACs and PBMC-MACs to phagocytose live P. aeruginosa and compared with PBMC-derived macrophages (PBMC-MACs). In this phagocytosis assay, 1×10 5 iPSC-MACs were incubated with 6×10 5 CFU of GFP-PAO1 for 2 hours at 37°C or 4°C as a negative control. Medium controls were treated similarly. After incubation, the cells were placed on ice for 10 minutes and then fixed with 2% paraformaldehyde (PFA) solution for 30 minutes. Analysis was performed using a Beckman Coulter FC500 flow cytometer.

[0120] Electron microscopy observation Macrophages were grown on a circular coverslip with a diameter of 1 cm. At 4°C, latex beads (diameter 1 μm) were added to the cells, and the latex beads were adhered to the surface of the macrophages over a period of 5 minutes. Subsequently, the samples were washed with cold PBS and warmed to 37°C together with fresh culture medium. Phagocytosis was allowed to proceed for up to 1 hour, and 1.5% paraformaldehyde and 1.5% glutaraldehyde in 150 mM Hepes (pH 7.35) were used to fix the samples at different time points. Then, the samples were dehydrated using a gradually increasing methanol series. Critical point drying was performed using a CPD030 critical point dryer (Balzers, Lichtenstein) according to the manufacturer's instructions. Subsequently, gold was sputtered onto the coverslip (Sem Coating System, Polarion), and SEM was performed using a Philips SEM 505 (Eindhoven, The Netherlands).

[0121] Bacterial culture For the experiment, the P. aeruginosa laboratory strain PAO1 (Klockgether et al., 2010) was taken from a storage culture maintained at -80°C and grown overnight in Luria Broth (LB). After washing with sterile PBS, the desired infectious dose was estimated from a standard growth curve. For the determination of the actual dose administered, inoculates were serially plated on LB agar plates by the drop plate method (Herigstad et al., 2001), and CFUs were determined after incubation at 37°C for 16 - 18 hours.

[0122] Collection of microarray samples Differentiated human iPSC-MAC or human PBMC-MAC were seeded in 24-well plates (500,000 cells / well) and cultured overnight. The next day, the cells were washed three times with PBS. Then, laboratory strain PAO1 of P. aeruginosa (MOI 10) in RPMI medium without antibiotics was centrifuged onto the cells (600×g) and incubated at 37°C. Cells with only medium were used as uninfected controls. After 1 hour, the cells were de-attached, washed, and resuspended in RNA lysis buffer. RNA isolation was performed using the RNAeasy micro Kit (Quiagen) according to the manufacturer's instructions. To separate iPSCs from feeder cells, iPSCs were sorted for TRA-1-60 + After sorting iPSCs for TRA-1-60, human iPSC samples were obtained.

[0123] Microarray experiment (single-color mode) The microarray used in this study corresponds to a refined version of the Whole Human Genome Oligo Microarray 4x44K v2 (Design ID 026652, Agilent Technologies), called "054261On1M" (Design ID 066335), developed at the Research Core Unit Transcriptomics (RCUT) of the Hannover Medical School. The microarray design uses the 1x1M design format as a template for mRNA expression and was created on the Agilent eArray portal. All non-control probes of Design ID 026652 are printed 5 times within a region containing a total of 181,560 features (170 rows × 1068 columns). Four such regions were placed within one 1M region so that four microarray fields per slide were individually hybridized (customer-specified feature layout). The control probes required for appropriate Feature Extraction software operation were determined by eArray using the recommended default settings and automatically placed.

[0124] As directed by the company (applying one round of amplification), 30 ng of total RNA was used to prepare aminoallyl-UTP-modified (aaUTP) cRNA [Amino Allyl MessageAmp (trademark) II Kit, #AM1753, Life Technologies]. Labeling of the aaUTP-cRNA was performed using Alexa Fluor 555 Reactive Dye (#A32756; Life Technologies).

[0125] Fragmentation, hybridization, and washing steps of the cRNA were performed as recommended in the “One-Color Microarray-Based Gene Expression Analysis Protocol V5.7”, except that 500 ng of each fluorescently labeled cRNA population was used for hybridization.

[0126] Slides were scanned on an Agilent Micro Array Scanner G2565CA (pixel resolution 3 μm, bit depth 20). Data extraction was performed using the “Feature Extraction Software V10.7.3.1” with the extraction protocol file “GE1_107_Sep09.xml”, except that the “Multiplicative detrending” algorithm was disabled.

[0127] The geometric mean of the processed intensity values of the green channel, “gProcessedSignal” (gPS), was used, and the measurements of the on-chip replicates (5 replicates) were averaged to derive one value per unique non-control probe as a result. A single feature was excluded from the averaging if i) it was manually flagged, ii) it was identified as an outlier by the Feature Extraction Software, iii) it was outside the interval of “1.42 × interquartile range” with respect to the normalized gPS distribution of each on-chip replicate population, or iv) it showed a coefficient of variation of pixel intensity per feature exceeding 0.5.

[0128] The averaged gPS values were first normalized by a quantile normalization approach. Subsequently, the values were further processed by a global linear scaling. All the gPS values of one sample were multiplied by an array-specific scaling factor. This factor was calculated by dividing the “reference 75th Percentile value” (set to 1500 for the whole series) by the 75th percentile value of the specific microarray to be normalized (Array I in the formula shown below). Thus, the normalized gPS values for all samples (microarray dataset) were calculated by the following formula: Normalized gPS アレイi = gPS アレイi × (1500 / 75th percentile アレイi )

[0129] Finally, a lower intensity threshold (proxy value) was defined based on the intensity distribution of the negative control features. This value was fixed at 15 normalized gPS units. All measurements below this intensity cut-off were replaced by each proxy value of 15.

[0130] For Agilent One Color mRNA Microarrays, normalized microarray data for all non-control features were imported into Omics Explorer software v3.2 (Qlucore) using the default import settings, except that all normalization options were left unselected. Thus, the data processing steps during import were 1) log base 2 transformation and 2) baseline transformation to the median.

[0131] Heatmap clustering analysis and generation of heatmaps based on GO were performed in Omics Explorer. The 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). Enrichr (https: / / amp.pharm.mssm.edu / Enrichr) was used to perform gene set enrichment analysis for cell type classification based on the Human Gene Atlas, as well as gene ontology analysis of biological processes and molecular functions. Ingenuity Pathway Analysis (Qiagen) was used to perform gene analysis related to diseases and functions.

[0132] The microarray data were deposited in the ArrayExpress database (www.ebi.ac.uk / arrayexpress) under accession number E-MTAB-5436.

[0133] Cytokine secretion assay (Luminex) To analyze the secretion of human cytokines in bioreactor samples or BALF samples, as previously described (Lachmann et al., 2015), Luminex® analysis using the Cytokine Human 14-Plex Panel (Millipore, Schwalbach, Germany) was performed. Data were acquired on a Luminex-200 System and analyzed using Xponent software v.3.0 (Life Technologies).

[0134] In vivo experiments Maintenance and infection of animals HuPAP[129S4-Rag2 tm1.1Flv Csf2 / Il3 tm1.1(CSF2,IL3)Flv Il2rg tm1.1Flv / J] mice (Willinger et al., 2011) were obtained from the Jackson Laboratory and housed in the central animal facility of the Hannover Medical School. NSG mice (NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJZtm) were obtained from the central animal facility of the Hannover Medical School. Both immunodeficient mouse strains were maintained under pathogen-free conditions in individually ventilated cages (IVCs) and allowed free access to food and water. All animal experiments were approved by the Animal Welfare Committee of the Lower Saxony State and performed in accordance with its guidelines.

[0135] For infection with P. aeruginosa or lung transplantation of iPSC-MACs, anesthetized (ketamine / midazolam) mice were intubated orally and then instilled via the trachea. In coinfection experiments, iPSC-MACs (4×10 6 / animal) and PAO1 (NSG: 5×10 5 and huPAP 0.2×10 5(CFU) were resuspended in PBS and mixed to a total volume of 60 μl. For mice with infection alone, the same CFU were applied in 60 μl of PBS. Prior to instillation, to avoid ingestion, the cell / bacteria mix was kept on ice at all times. In the therapeutic PiMT experiment, after anesthesia with ketamine / midazolam, huPAP animals were infected with 0.3 × 10 5 CFU of PAO1 (in 30 μl volume of PBS). Control mice received the same volume of PBS. Four hours later, the mice were anesthetized by isoflurane inhalation and a second instillation was performed using 50 μl of PBS or PBS containing 4 × 10 6 individual iPSC-MAC. Control mice again received the same volume of PBS. To determine the disease score of the animals, we used the scoring matrix described previously (Munder et al., 2005). Twenty-four hours later, the animals were sacrificed and a final analysis was performed.

[0136] Mouse lung function As described previously (Wolbeling et al., 2010), non-invasive head-out plethysmography to investigate 14 lung function parameters was performed on conscious restrained mice. The mice were positioned inside a glass insert. With the mouse breathing, air flowed through a pneumotachograph. A pressure transducer provided an electrical signal which was analyzed using NOTOCORD HEM software (version 4.2.0.241, Notocord Systems SAS, Croissy Sur Seine, France). To characterize the lung function of the mice during infection, parameters of tidal volume (measured in ml), expiratory time, inspiratory time, inspiratory + expiratory time, relaxation time, and flow at 50% of the expiratory tidal volume (EF50) were selected.

[0137] Bronchoalveolar lavage (BAL) and measurement of hemoglobin levels Bronchoalveolar lavage was performed by cannulating the trachea of mice after death. The right lung was rinsed three times with 1 ml of PBS. Fresh BALF was used for photometric analysis of hemoglobin. Thereafter, the BALF samples were centrifuged and the supernatant was stored at -80 °C for Luminex analysis. The pellet was fixed and stained for flow cytometry.

[0138] Lung bacterial count (CFU) The right lung of euthanized mice was ligated, excised, and homogenized using a tissue homogenizer (Polytron PT 1200, Germany). The total bacterial count was evaluated from serial dilutions of the homogenate cultured on Luria-Bertani plates using the drop plate method (Herigstad et al., 2001).

[0139] Tissue diagnosis Animals were sacrificed at a dedicated time point. The right lung was filled with OCT buffer and fixed in 4% neutral buffered PFA for 3 days at 4 °C. For control animals, the left lung was used. Tissues were trimmed, dehydrated (Shandon Hypercenter, XP), and then embedded in paraffin (TES, Medite) according to the RITA-Guidelines (Ruehl-Fehlert et al., 2003). Sections (2 - 3 μm thick, microtome Reichert-Jung 2030) were deparaffinized in xylene and stained with H&E according to standard protocols. Blind evaluation of the sections (Axioskop 40, Zeiss microscope) and histological scoring were performed by a trained pathologist as previously described (Dutow et al., 2013).

[0140] Statistics GraphPad Prism 6 and 7 were applied to perform independent Student's T-tests or analysis of variance (ANOVA). Unless otherwise specified, mean ± s.e.m. is plotted. Asterisks indicate * P < 0.05; ** P < 0.01; *** P < 0.001;**** P < 0.0001 means.

[0141] Result Induction of multiple human iPSC-derived myeloid lineages in dynamic suspension culture The generation of different mature hematopoietic cell types from PSCs has been demonstrated to succeed using conventional 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), but these systems do not enable the generation of iPSC-derived cells in clinically relevant amounts. Thus, the inventors developed a suspension-based (3D) hematopoiesis differentiation protocol suitable for process upscaling in an industrially compatible stirred tank bioreactor (Kropp et al., 2016b, Zweigerdt, 2009). The inventors first induced the formation of embryoid bodies (EBs) in small-scale suspension culture on an orbital shaker using a well-characterized hiPSC line (hCD34iPSC16) with a conventional brightfield morphology (Figure 1A) (Lachmann et al., 2014). After 5 days, the inventors transferred the EBs to a differentiation medium containing IL3 and M-CSF to induce hematopoiesis specification and the formation of myeloid cell-forming complexes (MCFCs) (Figure 1B). After 10 - 15 days of continuous suspension culture, the MCFCs continuously produced iPSC-derived macrophages (iPSC-MACs) that could be harvested once a week for up to 3 months (4D differentiation). The generated iPSC-MACs showed a typical macrophage-like morphology and presented a highly pure surface marker profile of + CD11b + CD14 + CD163 + CD34 - TRA1-60 - and efficiently phagocytosed fluorescently labeled E. coli particles (Figures 1C - 1E).

[0142] Notably, following protocols based on the same suspension, different subsets of myeloid were generated simply by changing the cytokine composition. Thus, IL3 and G-CSF enabled the continuous generation of CD16 + CD66b + iPSC-granulocytes (Figures 7A and 7B), while CD34 - CD71 + CD235a + CD36 + erythroid cells were induced by the use of IL3 / SCF / EPO (Figure 7C). In contrast to the cells generated by IL3 in combination with lineage-indicative cytokines, the use of IL3 alone resulted in a more immature CD45 + / CD11b + / CD14 - / CD163 - marker profile presentation (data not shown). Notably, further differentiation of these immature cells in the presence of either M-CSF or G-CSF resulted in the generation of CD45 + CD11b + CD14 + CD66b - CD34 - macrophage-like cells (Figures 7D and 7E) or CD16 + CD66b + granulocyte-like cells (hereinafter also referred to as macrophages and granulocytes) (Figures 7F and 7G).

[0143] Enables the upscaling of continuous production of hiPSC-MAC in a stirred tank bioreactor The inventors then applied suspension-based differentiation to a stirred-tank bioreactor using an industrially compatible system (DASbox Mini bioreactor system) (Olmer et al., 2012) that had been previously applied for efficient culture of human iPSCs and their differentiation into cardiomyocytes (Kempf et al., 2014) (Figure 2A, 2B). The bioreactor was equipped with probes for real-time monitoring of dissolved oxygen (DO), pH, temperature, and biomass assessment based on impedance. The process parameters were set at 37 °C, headspace gas supply at 3 L / h (21% O2; 5% CO2), and agitation at 50 revolutions per minute (rpm) using an 8-blade inclined (60°) impeller (Kempf et al., 2015). It is noteworthy that a chemically defined culture medium (X-Vivo15), which is also applicable in clinical trials, was used for future clinical scale-up. Weekly harvesting of iPSC-MAC from the continuous bioreactor process after day 7–10 showed an increase in cell yield over time, reaching approximately 2–3×10 as early as week 3. 7Reached a stable production level of individual iPSC-MACs per week, which was maintained for more than 5 weeks in two independent process runs (Figure 2C). The efficient generation of iPSC-MACs in both bioreactor experiments was reflected in the weekly biomass increase, especially during the first few days after a full medium exchange. Monitoring of DO and pH revealed the expected zigzag-like pattern typical of repeated batch cultures. The pH ranged from 7.25 (fresh medium) to 6.5 due to acidification of the medium typical of cell metabolic activity, especially due to the release of lactate (Kropp et al.). Notably, all process parameters showed a repetitive pattern after reaching a steady state of macrophage production around days 15 - 20, confirming the overall stability of the process (Figure 2D). This finding was further supported by the stable values of glucose, lactate, lactate dehydrogenase, and osmotic pressure, which were determined weekly in parallel with macrophage harvesting. Similarly, the secretion of cytokines / chemokines such as IL2, IL6, IL8, MCP1, TNFα, and IFNα2, which are related to macrophage activation, was detected from the first harvest (week 2) onwards (Figure 2E), corresponding to the emergence of CD45 + iPSC-MAC. Notably, MCFCs cultured in the bioreactor maintained their morphology throughout the process, generating iPSC-MACs with a typical morphology and CD45 + CD14 + surface marker profile continuously, and their purity increased over time (Figure 2F). Furthermore, long-term culture of MCFCs derived from the bioreactor in a 6-well suspension plate on an orbital shaker resulted in continuous production of iPSC-MACs for an additional 8 weeks (data not shown).

[0144] Bioreactor-derived iPSC-MACs reproduce the phenotypic and transcriptional characterization of PBMC-MACs Detailed characterization of bioreactor-derived iPSC-MACs was performed for CD45 + CD11b + CD163 + CD14 +CD34 - TRA1-60 - The phenotype and typical morphology after adhesion to tissue culture plates were revealed (Figs. 3A and 3B). Comparison of iPSC-MAC with undifferentiated hiPSC and macrophages derived from peripheral blood mononuclear cells (PBMC-MAC) by unsupervised hierarchical heatmap clustering of the whole transcriptome revealed the proximity of iPSC-MAC and PBMC-MAC when compared with iPSC (Fig. 3C). Analysis of genes related to pluripotency and activation of the innate immune response confirmed efficient differentiation of iPSC into macrophage-like cells (Figs. 3D and 3E). Importantly, genes related to macrophage function, such as 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-MAC and PBMC-MAC compared to iPSC (Fig. 3E).

[0145] Comparison of the top 100 upregulated genes in iPSC-MAC compared to pluripotent hiPSC, and in PBMC-MAC compared to pluripotent hiPSC, revealed a common gene set of 54 upregulated genes. These transcripts, including CD14, CD68, CSFR1, CCR1, and CYBB, were assigned to CD14 + monocytes, whole blood, and CD33 + and myeloid cells (Fig. 3F). Notably, this set of genes (46 genes) that were upregulated exclusively in iPSC-MAC were also assigned to CD14 + monocytes and CD33+ myeloid cells and included genes such as CD163, leukocyte immunoglobulin-like receptor A6 (LILRA6), stabilin 1 (STAB1), or formyl peptide receptor 1 (FPR1) (Fig. 3G). In contrast, this set of 46 genes that were upregulated only in PBMC-MAC were CD56 +Identified the highest scores in gene sets related to natural killer (NK) cells, whole blood, and dendritic cells [e.g., human leukocyte antigen (HLA), CCL5 / RANTES, or granzyme (GZM) A and B]. This observation is CD56 + / CD3 - which may be explained by the contamination of CD56

[0146] In vitro antibacterial activity of bioreactor-derived iPSC-MAC Next, bioreactor-derived iPSC-MAC were evaluated for their in vitro antibacterial activity. Scanning electron microscopy (SEM) at different time points after incubating iPSC-MAC with fluorescently labeled latex beads revealed typical changes in the overall cell morphology early after stimulation and efficient phagocytic uptake of the beads over time (Figure 4A). Even more importantly, iPSC-MAC also showed no phagocytosis at 4°C, but at 37°C, they phagocytosed GFP-labeled P. aeruginosa with an efficiency comparable to PBMC-MAC, and active phagocytosis was confirmed (Figure 4B). To gain insights into the ability of iPSC-MAC to remodel their transcriptome towards the characteristics of activated macrophages, whole transcriptome analysis of iPSC-MAC was performed before and after contact with P. aeruginosa (PAO1).

[0147]

Table 1

[0148] Hierarchical cluster analysis of gene ontology (GO) related to the activation of inflammatory or innate immune responses showed significant upregulation of cytokines (e.g., IL23A, TNFα, IL1A, IL6, INFG1), chemokines (e.g., CCL5, CCL20, CCL4, CXCL3), and molecules involved in NFκB signaling in response to pathogen contact (Figure 4C). Similarly, gene ontology enrichment analysis of genes upregulated more than 5-fold after pathogen contact revealed high scores for GO related to biological processes, including inflammatory response, response to lipopolysaccharide (LPS), and molecules of bacterial origin, as well as response to injury. Furthermore, GO related to molecular function revealed enrichment of GO terms such as cytokine / chemokine activity and receptor binding or G-protein-coupled receptor binding. Enrichment analysis of diseases and functions of these upregulated genes revealed ontologies including inflammatory diseases and responses, immune cell trafficking, antibacterial response, and free radical scavenging (Figure 4D).

[0149] iPSC-MAC prevents respiratory infection by Pseudomonas aeruginosa To evaluate the in vivo therapeutic efficacy of iPSC-MAC, the humanized mouse model C;129S4-Rag2 (Willinger et al., 2011), an immunodeficient strain with impaired alveolar macrophage development, was used. HuPAP mice recapitulate the characteristics of the human disease pulmonary alveolar proteinosis (PAP), including susceptibility to lung infection. In addition, a second immunodeficient mouse model, NOD.Cg-Prkdc tm1.1Flv Csf2 / Il3 tm1.1(CSF2,IL3)Flv Il2rg tm1.1Flv / J (huPAP mice), was used. HuPAP mice recapitulate the characteristics of the human disease pulmonary alveolar proteinosis (PAP), including susceptibility to lung infection. In addition, a second immunodeficient mouse model, NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJZtm (NSG), was used to utilize iPSC-MAC.

[0150] First, huPAP mice were infected with P. aeruginosa laboratory strain PAO1, and 4×10 6 individual iPSC-MACs were co-administered in the same intravenous injection process (mice infected with PAO1 alone served as controls). The course of infection was carefully monitored, and the mice were sacrificed for final analysis 24 hours after infection (Figure 5A). Infected huPAP mice already showed clinical symptoms 4 hours after infection, as indicated by an elevated disease score of 3.5±0.9, and the disease score gradually increased over time to a maximum of 6.1±0.4 at 24 hours after infection. In addition, a decrease in rectal temperature to 35.0±0.3°C and a weight loss of 2.6±0.5 g were observed in infected animals 24 hours after infection (all mean±s.e.m., n = 6) (Figure 5B and 5C). In contrast, animals that received simultaneous lung transplantation of iPSC-MACs (PiMT) showed only mild infection symptoms (Figure 5B). This was consistent with a normal rectal temperature of 37.1±0.2°C, a very low disease score of 0.6±0.2, and only a slight weight loss of -0.89±0.2 g in PiMT-treated animals 24 hours after infection (all mean±s.e.m., n = 6) (Figure 5B and 5C). A similar beneficial effect of PiMT was demonstrated by head-out body plethysmography for measuring lung function (Wolbeling et al., 2010). Infected animals showed a decrease in tidal volume as well as expiratory and inspiratory times, and an increase in respiratory rate, while animals in the infection+PiMT group showed normal values for all parameters analyzed (Figure 5D). As a result of PiMT, transplanted mice showed significantly reduced bacterial counts in the lungs 24 hours after infection compared to their non-transplanted controls (Figure 5E). Furthermore, red blood cells were present only in the bronchoalveolar lavage fluid (BALF) of infected mice that did not receive PiMT (Figure 5F). Furthermore, mouse GR1 was seen in the BALF and lungs of infected animals but not in infected+PiMT mice or control mice +An increase in granulocytes indicated pulmonary inflammation (Figure 5G). In addition, histological evaluation revealed extensive granulocyte infiltration, severe hemorrhage, and alveolar edema in infected mice (score: 13.7 ± 0.3), while the lungs of infected + PiMT animals showed only minor changes (score: 2.0 ± 1.2, both mean ± s.e.m., n = 3) (Figure 5H). The reduced inflammation in infected + PiMT animals was associated with the detection of hCD45 + cells in the lungs and BALF, and the presence of macrophages in histological sections of the right lung (Figures 5I and 8).

[0151] Similar results were obtained in NSG mice, a second immunodeficient mouse strain with normal alveolar macrophage development. Infected NSG animals exhibited marked symptoms of infection, such as high disease scores and a significant decrease in rectal temperature, 6 and 24 hours after infection. In contrast, animals that received iPSC-MAC simultaneously showed normal body temperature and only slightly increased disease scores 6 and 24 hours after infection, respectively (Figures 9A and 9B). Furthermore, analysis of bacterial counts 24 hours after infection revealed a significantly reduced bacterial burden in the lungs of infected + PiMT mice (Figure 9C).

[0152] Therapeutic PiMT rescues mice from severe respiratory infection After demonstrating the efficacy of iPSC-MAC in co-infection experiments, the inventors evaluated a more clinically relevant therapeutic PiMT treatment approach. In these experiments, huPAP mouse lungs were infected with P. aeruginosa and carefully monitored over 3 - 4 hours until the first disease symptoms appeared (determined by a disease score ≥ 5). Subsequently, mice in this model received 4 × 10 6 individual iPSC-MAC only after the infection-related symptoms became apparent (infected + PiMT). Notably, infected control mice received only PBS instead of PiMT (infected) (Figure 6A).

[0153] In this infection and treatment schedule, the therapeutic PiMT-treated mice already showed a decrease in disease score, as well as normalization of rectal temperature and body weight to a comparable extent as non-infected animals, within 4 - 8 hours after treatment. In contrast, infected mice receiving PBS showed obvious disease progression over time (Figures 6B, 6D, and 6E). Notably, 24 hours after infection, infected mice showed significant disease symptoms, while animals receiving therapeutic PiMT showed significantly reduced disease scores (8.1 ± 0.2 in infected animals vs. 1.8 ± 0.2 in infected + PiMT, mean ± s.e.m., n = 3). Additionally, the elevated disease scores in infected mice were clearly associated with restricted activity compared to control animals and mice receiving therapeutic PiMT (Figure 6C). The efficiency of therapeutic PiMT was further evidenced by the normalized rectal temperature and body weight values 24 hours after infection in infected + PiMT mice, as well as a marked reduction in the number of lung bacteria (Figures 6B, 6D, 6F). Reproducing our findings in the co-transplantation model, the BALF of animals in the infected + PiMT group showed reduced red blood cell 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 (Figures 6G and 6H). Lung tissue sections were used to further evaluate the inflammation. Here, infected mice showed extensive granulocyte infiltration, severe bleeding, and alveolar edema in several areas, which were hardly detectable in mice treated with iPSC-MAC obtained from the bioreactor (Figure 6I).

[0154] Discussion In this study, the inventors have evaluated the concept of treatment using macrophages as a cell therapy for bacterial infection. Since a clinically significant number of autologous or donor-derived macrophages can hardly be produced from somatic cell sources, the inventors investigated the possibility of using hPSCs to generate a substantial amount of functional macrophages.

[0155] The use of iPSC derivatives as a cellular approach to target infection has not been seriously considered heretofore. This can be explained by the fact that the putative potential of hPSCs for the generation of specific progeny in therapeutically relevant numbers has not yet been applied in practice. Leveraging recent advances in culture medium formulation, the inventors attempted to culture and differentiate human PSCs as floating aggregates and adapted stirred tank bioreactor technology to the requirements of stem cells to enable the clinical scale-up of iPSCs and their derivatives. To develop the application of iPSC-macrophages as a cell therapy for bacterial infection and, more specifically, to eliminate lung infections induced by P. aeruginosa, the inventors first established a scalable and continuous hematopoiesis differentiation process of human iPSCs in a well-equipped bioreactor.

[0156] In in vivo studies, huPAP mice, an immunodeficient strain lacking alveolar macrophages, were used. This mouse model is clinically relevant as it recapitulates key features of pulmonary alveolar proteinosis (PAP), including susceptibility to lung infections typically observed in PAP patients (Trapnell et al., 2003; Willinger et al., 2011). In this mouse model, prevention of acute P. aeruginosa infection by simultaneous PiMT, or more importantly therapeutic PiMT, was highly effective within a short time frame. These proof-of-concept experiments were conducted using high cell doses. Similar therapeutic effects may be achievable using lower cell numbers. However, no obvious adverse events were observed in any treatment scenario, even using this maximum cell dose, which is consistent with observations in macrophage lung transplantation therapy studies using bone marrow-derived macrophages (BMDM) (Happle et al., 2014; Suzuki et al., 2014).

[0157] Considering a body weight of 25 g per mouse, the clinical application of iPSC-MAC for the treatment of respiratory infections would be approximately 1×10 10This would require individual iPSC-MACs. Even without further process optimization, this corresponds to a production scale of 40 - 60 L and is in principle achievable using current bioreactor technology (Kropp et al., 2016b). Notably, continuous process monitoring of differentiation based on the bioreactor of the present invention revealed a substantial increase in biomass, especially during the first 2 - 3 days of repeated batch culture. This was followed by a partial recovery of dissolved oxygen levels and a significant decrease in pH during the last few days before medium exchange. These observations suggest the presence of process-limiting factors and emphasize the potential for further process optimization. This can be achieved by culturing at higher cell densities, in combination with the application of perfusion systems and feedback control of oxygen, pH, and other process parameters, which can double the increase in cell yield of hPSCs and their progeny (Kropp et al., 2016a).

[0158] Given the excellent in vivo functionality of iPSC-MACs in the acute infection model, the therapeutic efficacy of PiMT in chronic infections with P. aeruginosa, which are frequently observed in patients with chronic obstructive pulmonary disease (COPD) (Rakhimova et al., 2009) or cystic fibrosis (CF) (Oliver et al., 2000), is expected. In particular, CF represents an interesting disease scenario regarding the application of PiMT, as pathogenic mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene also interfere with the functionality of professional phagocytic cells and thus the host defense against infection (Bonfield et al., 2012, Bruscia et al., 2009). Although a single PiMT showed therapeutic efficacy against acute infections, repeated therapeutic interventions or the application of genetically enhanced cells (Pasula et al., 2016) may be required to eliminate established infections in chronic diseases.

[0159] Long-term engraftment and sustained functionality of transplanted iPSC-MACS are expected. In the transplantation scenarios tested, iPSC-MACs were still detected at the 24-hour time point, but later time points were not investigated in this study. Previous studies have demonstrated engraftment of BMDMs for more than one year (Suzuki et al., 2014). Furthermore, lung transplantation of mouse macrophages derived from different pluripotent progenitor cells has shown chromatin remodeling, adaptation to the local tissue environment, and the ability for long-term integration (Happle et al., 2014, Lavin et al., 2014, Suzuki et al., 2014, van de Laar et al., 2016). Accordingly, the iPSC-MACs of the present invention demonstrated rapid upregulation of pro-inflammatory gene expression after pathogen contact in vitro and efficient antibacterial activity in vivo, suggesting their ability to respond to the inflammatory environment in the lung after transplantation.

[0160] The potent and rapid antibacterial and therapeutic effects observed by the inventors are particularly important with regard to the broad applicability and rapid clinical implementation of iPSC-MACs for targeting bacterial infections. Although the antibacterial activity of iPSC-MACs against P. aeruginosa was evaluated, cell-based therapies based on phagocytes could enable broad use in several different infection scenarios caused by other Gram-negative or positive bacteria such as Streptococcus pneumoniae or Staphylococcus aureus, or pathogens associated with graft infections that simultaneously pose significant health and economic problems. However, considering the increasing number of pathogens resistant to standard or reserve antibiotic therapies (Aloush et al., 2006, Falgenhauer et al., 2016, Gould, 2013, Hirsch and Tam, 2010, Liu et al., 2016, Schroeder and Stephens, 2016), new forms of treatment have particular clinical significance.

[0161] In summary, the present inventors provide a therapeutic application of PSC-derived phagocytes for the treatment of bacterial infections. The present inventors demonstrate the feasibility of phagocyte production under defined conditions that enable clinical and industrial scale-up, and provide evidence of the efficacy and safety of iPSC-MAC transplantation as a new treatment approach targeting, for example, severe respiratory infections. This technology enables innovative cell-based treatment strategies for a wide variety of diseases, including process upscaling and the generation of additional hematopoietic cell types, as well as evaluation in other preclinical models.

[0162] Example 2 GMP-compliant suspension iPSC culture and blood cell differentiation Generation of single iPSCs: Single iPSCs were derived directly from iPSCs cultured on mouse feeder cells. The iPSCs cultured on mouse feeder cells were incubated with Accutase [cell dissociation reagent, StemPro™ Thermo Scientific] for up to 5 minutes in a cell culture incubator at 37 °C. The Accutase reaction was stopped by diluting with either PBS or DMEM / F12 medium (Thermofisher Scientific). Dissociation of aggregates was promoted by pipetting the resuspended cells up and down (3 times or less and very slowly) to obtain single iPSCs. The cells were counted and used for monolayer culture on a defined substrate (see next step). Alternatively, single iPSCs may be derived from iPSCs cultured in dishes coated with a substrate [e.g., GelTrex (Thermofisher Scientific), Matrigel (Corning Fisher Scientific), or laminin (e.g., CellAdhere Laminin-521, Stem Cell Technologies)] as previously described.

[0163] Division of monolayer cells: A 6-well tissue culture plate [e.g., NUNC plate (Thermofisher Scientific)] was coated with a substrate [e.g., GelTrex (Thermofisher Scientific), Matrigel (Corning Fisher Scientific), or laminin (e.g., CellAdhere Laminin-521, Stem Cell Technologies)] for at least 1 hour. Single iPSCs were seeded in the pre-coated plate in E8 100 or E8 50 medium (Stem Cell Technologies) supplemented with a ROCK inhibitor (10 μM) for further expansion. The maximum of 2×10 5 single iPSCs generated were cultured as a monolayer in each well of a 6-well plate. The medium was changed on day 2 and passaged on day 3 or 4. The medium was not changed on the day after seeding. These cultures were maintained for at least 10 passages.

[0164] Aggregate formation: 5×10 5 single iPSCs cultured as single cells on the substrate for more than 2 passages were seeded for aggregate formation in suspension culture in a Greiner CELLSTAR multi-well culture plate (Sigma Aldrich) on an orbital shaker (70 rpm) in 3 ml of E8 50 or E6 medium (Stem Cell Technologies) supplemented with a ROCK inhibitor (10 μM). Aggregate formation started within 24 hours. The medium was changed on day 2 to 2 - 2.5 ml. Aggregates on day 3 were transferred for differentiation or passaged as single cells under suspension.

[0165] Hematopoietic differentiation: Induction of hematopoietic differentiation was initiated by mesoderm priming.

[0166] Mesoderm priming was initiated by transferring approximately 100 aggregates into 3 ml of X-VIVO15 supplemented with 50 ng / ml of hVEGF, 50 ng / ml of hBMP4, and 20 ng / ml of hSCF for 3 days, followed by the addition of 25 ng / ml of IL3 (on day 4 of mesoderm priming). Subsequent hematopoietic differentiation of the primed aggregates was performed by changing the medium to 3 ml of X-VIVO15 supplemented with 25 ng / ml of IL-3 and 50 ng / ml of M-CSF. Mesoderm priming and subsequent hematopoietic differentiation were performed on an orbital shaker at 85 rpm.

[0167] The resulting cells were suitable for clinical application in humans.

[0168] Example 3 Preparation of cells with reduced size Macrophages were produced or isolated from, for example, mice, according to methods known in the art. Alternatively, the macrophage cell line U937 was used.

[0169] The cells were washed and incubated with a hypertonic solution (e.g., a sugar solution such as a sucrose solution) for 15 minutes to 1 hour, typically about 15 minutes, at 4°C to 37°C, preferably at 37°C. The hypertonic solution had an osmolarity greater than 300 mosm, preferably greater than 350 mosm. Reduced size, measured by forward scatter in flow cytometry for incubation with 300 mosm (control), 600 mosm, 800 mosm, and 1000 mosm, is shown in Figures 11 and 13.

[0170] For mouse primary macrophages, after incubation at the defined osmolarity, the following mean diameters were determined by microscopic observation and computer analysis using, for example, ImageJ: 300 mosm → mean area 155.7 μm 2 , corresponding to a mean diameter of 14.08 μm, 600 mosm → average area 131.6 μm 2 corresponding to an average diameter of 12.94 μm, 800 mosm → average area 113.9 μm 2 corresponding to an average diameter of 12.04 μm, 1000 mosm → average area 115.2 μm 2 corresponding to an average diameter of 12.11 μm.

[0171] For U937 cells, after incubation at the specified osmolality, the following average diameters were determined by microscopic observation and computer analysis using, for example, ImageJ: 300 mosm → average area 113.8 μm 2 corresponding to an average diameter of 12.04 μm, 600 mosm → 94.34 μm 2 corresponding to an average diameter of 10.96 μm, 800 mosm → 82.63 μm 2 corresponding to an average diameter of 10.26 μm, 1000 mosm → 74.26 μm 2 corresponding to an average diameter of 9.72 μm.

[0172] Example 4 Preparation of proteins from cell culture supernatants As described in Example 1, macrophages were produced according to the method of the present invention. The inventors have found by mass spectrometry and Western blotting that the cells release high levels of S100 proteins, including but not limited to S100A7, S100A8, and S100S9 variants, into the culture supernatant of the suspension culture. Gene expression of S100A8 and S100A9 showed an increase of more than 500-fold compared to hPSCs and expression comparable to PBMC-derived macrophages. S100 proteins are isolated from cell culture supernatants, for example, by affinity chromatography or other chromatography methods known in the art, or combinations thereof.

Claims

1. A cell population obtained by a method for producing myeloid cells, the method comprising: a) culturing embryoid bodies under suspension culture for 4 to 8 days to produce a myeloid cell-forming complex in the presence of 10 to 100 ng / mL of IL-3; b) culturing the myeloid cell-forming complex under suspension culture for a period sufficient to produce myeloid cells in the presence of 10 to 100 ng / mL of IL-3, wherein the production of the myeloid cell-forming complex from the embryoid bodies and the production of myeloid cells together take at least 7 days, and c) isolating the myeloid cells. Including, wherein the suspension culture is agitated to prevent cell adhesion or sedimentation, the cell population comprises CD45+CD11b+ / CD14+ / CD163+ / CD34- / TRA1-60- macrophages, wherein any of said cells are human cells, and wherein the expression of at least 10 genes selected from the group consisting of CRTAM, FBP1, GNLY, HLA_DQA1, HLA_DQB1, HLA_DQB2, HLA_DRA, HLA_DR B1, HLA_DR B3, HLA_DR B4, HLA_DR B5, IL-15, LY75 is at least 20-fold downregulated in said macrophages compared to macrophages derived from PBMCs, wherein said macrophages derived from PBMCs are isolated from the peripheral blood of healthy volunteers by gradient centrifugation using Biocoll Separating Solution at 400×g for 40 minutes, and the cells are cultured in RPMI1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 1% penicillin-streptomycin and hIL3 and hM-CSF (50 ng / mL each) for 1 week, and then cultured in a differentiation medium containing only 50 ng / mL of M-CSF for 3 to 4 days, cell population.

2. The cell population according to claim 1, wherein the culture in step a) is carried out in the presence of at least one additional cytokine and / or the culture in step b) is carried out in the presence of at least one additional cytokine.

3. The cell population according to claim 1 or 2, wherein the embryoid bodies are derived from pluripotent stem cells.

4. The cell population according to claim 3, wherein the pluripotent stem cells are induced pluripotent stem cells.

5. The cell population according to any one of claims 1 to 4, wherein the embryoid body is obtained by a method comprising culturing pluripotent stem cells under suspension culture for a period sufficient to produce the embryoid body.

6. The cell population according to any one of claims 1 to 5, wherein the suspension culture is carried out in a bioreactor enabling suspension culture, selected from the group consisting of a stirred tank bioreactor, an Erlenmeyer flask, a spinner flask, a wave bioreactor, and a rotating wall bioreactor.

7. The cell population according to claim 6, wherein the bioreactor enabling suspension culture is a stirred tank bioreactor.

8. The cell population according to any one of claims 2 to 7, wherein the further cytokine is M-CSF and the myeloid cells produced are macrophages.

9. In step a) or step b), there is no further cytokine for IL-3, and the myeloid cells produced are immature cells capable of further differentiation, wherein the method further comprises culturing the immature cells in the presence of M-CSF until macrophages are obtained. The cell population according to claim 1.

10. The cell population according to any one of claims 1 to 9, wherein the method enables continuous production of the myeloid cells.

11. The cell population according to any one of claims 1 to 10, wherein isolation comprises purifying the myeloid cells produced to a purity of at least 50%.

12. After step c, the method - reducing the size of the cells by a method selected from the group consisting of contacting the myeloid cells with a hypertonic solution and lyophilization, and / or - loading the myeloid cells with a therapeutic or diagnostic agent selected from the group consisting of antibiotics, immunomodulators, and dyes further comprises The cell population according to claim 11.

13. The cell population according to any one of claims 1 to 12, wherein the size of the myeloid cells is reduced by a method selected from the group consisting of contacting the cells with a hypertonic solution and lyophilization.

14. The cell population according to claim 13, wherein the cells are loaded with a therapeutic or diagnostic agent selected from the group consisting of an antibiotic and an immunomodulator.

15. The cell population according to any one of claims 1 to 14, wherein the expression of at least 9 genes selected from the group consisting of DKK1, SEPP1, PITX2, COL3A1, KRT19, A_33_P3221980, CALD1, CYR61, H19, DDIT4L, FRZB, TMEM98, NNMT, NPNNT, LUM, DCN, LYVE1, MGP, IGFBP3, and NUAK1 is upregulated by at least 20-fold in the macrophages as compared to macrophages derived from PBMC.

16. A pharmaceutical composition comprising the cell population according to any one of claims 1 to 15 in a pharmaceutically acceptable carrier.

17. a) A pharmaceutical composition comprising the cell population according to any one of claims 1 to 15 in a pharmaceutically acceptable carrier, b) A container suitable for spraying the pharmaceutical composition An application system comprising the same.

18. - The size of the myeloid cells is reduced by a method selected from the group consisting of contacting the cells with a hypertonic solution and lyophilization, and / or - The myeloid cells are loaded with a therapeutic or diagnostic agent selected from the group consisting of an antibiotic, an immunomodulator, and a dye. The application system according to claim 17.

19. A pharmaceutical composition comprising the cell population according to any one of claims 1 to 13 in a pharmaceutically acceptable carrier, wherein the average size of the cells in the population is reduced by at least 10% as compared to the population of the cells in a physiological saline aqueous solution.

20. Use of the pharmaceutical composition according to claim 16 or 19 or the application system according to claim 17 or 18 for the treatment or prevention of infection in a patient and / or for the promotion of wound healing.

21. The pharmaceutical composition for use according to claim 20, wherein the pharmaceutical composition is for the treatment of a bacterial infection.

22. a) Drug screening and / or drug development, b) Disease modeling, c) Tissue manipulation, e) Disinfection, f) Coating of materials for transplantation, g) Biomarker development, or h) Quality control of biological substances selected from the group consisting of antibodies, hormones, cytokines, drugs, culture media, and sera Use of a cell population according to any one of claims 1 to 12 for [

23. ] A method for producing myeloid cells, comprising: a) culturing embryoid bodies in suspension culture for 4 to 8 days to produce a myeloid cell-forming complex in the presence of 10 to 100 ng / mL of IL-3; b) culturing the myeloid cell-forming complex in suspension culture for a period sufficient to produce myeloid cells in the presence of 10 to 100 ng / mL of IL-3, wherein the production of the myeloid cell-forming complex from the embryoid bodies and the production of myeloid cells together take at least 7 days, and c) isolating the myeloid cells wherein the suspension culture is agitated to prevent cell adhesion or sedimentation. [

24. ] The method according to claim 23, wherein the culture in step a) is carried out in the presence of at least one further cytokine and / or the culture in step b) is carried out in the presence of at least one further cytokine, wherein the suspension culture is agitated to prevent cell adhesion or sedimentation.

Images