Method for generating single cell progenitor cells

Abstract

The present application relates to methods for the generation of monocyte progenitor cells and their differentiation into macrophages and microglia, and large-scale cell cultures for producing monocyte progenitor cells.

Description

Technical Field

[0001] Field of the Invention The present application relates to methods for the generation of monocyte progenitor cells and their differentiation into macrophages and microglia, as well as large-scale cell cultures for producing monocyte progenitor cells.

Background Art

[0002] Background Monocytes and macrophages play important roles in the inflammatory process, and their activation and functionality are extremely important in health and disease (Biswas et al. 2012, Mantovani et al. 2013, Sica et al. 2008, Wynn et al. 2013). Diseases in which macrophage involvement has been confirmed include metabolic diseases, allergic disorders, autoimmune diseases, cancer, neurodegenerative diseases, and infections by bacteria, viruses, parasites, and fungi. In addition to mediating acute immune defense in the context of disease, macrophages that are widely distributed throughout tissues are essential for the repair and homeostasis of the surrounding tissues. Thus, impairment of macrophage functionality and subsequent loss of homeostasis are closely related to the pathogenesis of degenerative diseases.

[0003] Important macrophage functions in homeostasis and disease defense include phagocytosis (of pathogens, debris, and dead cells), migration (to sites of injury), and cytokine release to trigger further inflammatory responses or provide trophic support to surrounding tissues (Biswas et al. 2012, Mantovani et al. 2013, Sica et al. 2008, Wynn et al. 2013). For this reason, modulation of monocyte / macrophage function represents a potentially disease-resolving therapeutic strategy. The wide range of disease areas in which macrophages are involved and the functional properties of macrophages result in a very diverse set of potential targets (Tiwari et al. 2008). This creates a high demand for monocytes and macrophages for drug development and screening.

[0004] Previously, macrophage research has been complex and time-consuming due to limitations in the generation of relevant cells. One means of obtaining macrophages that has been predominantly used in the past is to isolate monocytes from PBMCs (peripheral blood mononuclear cells) concentrated from blood donations (Figure 1). However, the use of these primary cells is limited by the limited number of cells per donor, donor-to-donor variability, and the limitations in the possibility of genetic manipulation.

[0005] Recent studies have successfully induced monocyte progenitor cells and macrophages from iPS cells (Ackermann et al. 2018, Hong et al. 2018, Karlsson et al. 2008, Senju et al. 2011, Takamatsu et al. 2014, van Wilgenburg et al. 2013). This approach has several advantages compared to the isolation of primary monocytes (Figure 1). It allows for the use of cells with a disease-related genetic background, enables genetic manipulation (i.e., correction of mutations predisposing to disease in the pluripotent state), and limits donor variability if necessary. The iPS technology provides a virtually unlimited supply of monocytes / macrophages of a defined genotype and function.

[0006] Microglia are a special subtype of tissue-resident macrophages. During embryonic development, two waves of macrophages occur in the blood islands of the yolk sac. These yolk sac-derived macrophages are Myb-independent but PU.1- and IRF8-dependent for proliferation (Haenseler et al. 2016) and give rise to tissue-resident macrophages. In many tissues, this initial macrophage population is partially or completely replaced by bone marrow-derived macrophages, but the brain-resident macrophage population, i.e., microglia, remains of its sole origin.

[0007] Microglia have important homeostatic functions such as clearance of misfolded proteins and dead cells, pruning of synapses, and release of neurotrophic factors. Furthermore, upon inflammatory stimuli, they can be activated to release potentially harmful cytokines and produce reactive oxygen species. High levels of chronic inflammatory activation and expression of some genetic risk factors for neurodegenerative diseases (e.g., LRRK2, TREM2, ASYN, and CD33) have led to high interest in the role of microglia in neurodegenerative diseases and neuroinflammation.

[0008] Previously, due to the low availability of primary human microglia and related human cell models, the study of microglia has been limited to primary rodent cells. Recent protocols for generating monocytes and macrophages from iPS cells (Abud et al. 2017, Ackermann et al. 2018, Brownjohn et al. 2018, Douvaras et al. 2017, Haenseler et al. 2017a, Haenseler et al. 2017b, Hong et al. 2018, Karlsson et al. 2008, Muffat et al. 2016, Senju et al. 2011, Takamatsu et al. 2014, van Wilgenburg et al. 2013) show the correct ontogenetic markers, and the generation of microglia-like cells from their precursors in neuronal cell co-cultures has been recently described (Haenseler et al. 2017a).

[0009] However, the protocols provided by the references have limited cell culture throughput and stability, and thus cannot provide, either qualitatively or quantitatively, the amounts of cells required for high-throughput assays, for example, in drug discovery and development.

[0010] Therefore, there remains a need for an improved protocol for generating large amounts of monocyte progenitor cells from iPS cells in a high-throughput manner. SUMMARY OF THE INVENTION

[0011] A method for producing monocyte progenitor cells, comprising: a) seeding pluripotent stem cells in a pluripotent medium onto a cell culture support coated with laminin; b) harvesting the pluripotent stem cells and contacting the pluripotent stem cells with a mesoderm induction medium in a suspension culture; c) seeding the cells onto a cell culture support suitable for cell attachment; d) harvesting the monocyte progenitor cells from the cell culture supernatant. A method is provided that includes

[0012] In one embodiment, the laminin in step a) includes laminin subunit alpha-5, and specifically, the laminin in step a) includes laminin subunits alpha-5, beta-2, and gamma-1.

[0013] In one embodiment, the cells are contacted in step b) with a defined medium containing BMP4.

[0014] In one embodiment, the cells are contacted in step b) with a defined medium containing VEGF.

[0015] In one embodiment, the cells are contacted in step b) with a defined medium containing SCF.

[0016] In one embodiment, the cells in step b) form embryoid bodies (EBs).

[0017] In one embodiment, the cell culture support in step c) is coated with a basement membrane biomaterial.

[0018] In one embodiment, the cells in step c) are contacted with a myeloid maturation medium.

[0019] In one embodiment, the myeloid maturation medium includes M-CSF.

[0020] In one embodiment, the myeloid maturation medium includes IL-3.

[0021] In one embodiment, the method further includes e) differentiating the collected monocyte progenitor cells into macrophages.

[0022] In one embodiment, the cells in step e) are seeded on an uncoated tissue culture support.

[0023] In one embodiment, the method further comprises e) differentiating the isolated monocyte progenitor cells into microglia.

[0024] An adherent large-scale cell culture for producing monocyte progenitor cells, wherein the adherent cell culture can produce at least about 100,000 monocyte progenitor cells per cm of cell culture area per week, is further provided. 2 The method of the present invention 1011, wherein the cells in step e) are seeded onto an uncoated tissue culture support. [The present invention 1001] A method for producing monocyte progenitor cells, comprising: a) seeding pluripotent stem cells in a pluripotent medium onto a cell culture support coated with laminin; b) harvesting the pluripotent stem cells and contacting the pluripotent stem cells with a mesoderm induction medium in a suspension culture; c) seeding the cells onto a cell culture support suitable for cell attachment; d) harvesting monocyte progenitor cells from the cell culture supernatant and a method comprising the steps of. [The present invention 1002] The method of the present invention 1001, wherein the laminin in step a) comprises laminin subunit alpha-5, and in particular, the laminin in step a) comprises laminin subunits alpha-5, beta-2, and gamma-1. [The present invention 1003] The method of the present invention 1001 or 1002, wherein in step b), the cells are contacted with a defined medium containing BMP4. [The present invention 1004] The method according to any one of the present inventions 1001 to 1003, wherein in step b), the cells are contacted with a defined medium containing VEGF. [The present invention 1005] The method according to any one of the present inventions 1001 to 1004, wherein in step b), the cells are contacted with a defined medium containing SCF. [The present invention 1006] The method according to any one of the present inventions 1001 to 1005, wherein the cells in step b) form embryoid bodies (EBs). [The present invention 1007] The method according to any one of the present inventions 1001 to 1006, wherein the cell culture support in step c) is coated with a basement membrane biomaterial. [The present invention 1008] The method according to any one of the present inventions 1001 to 1007, wherein the cells in step c) are contacted with a myeloid maturation medium. [The present invention 1009] The method according to any one of the present inventions 1001 to 1008, wherein the myeloid maturation medium contains M-CSF. [The present invention 1010] The method according to any one of the present inventions 1001 to 1009, wherein the myeloid maturation medium contains IL-3. [The present invention 1011] e) a step of differentiating the harvested monocyte progenitor cells into macrophages and a method according to any one of the present inventions 1001 to 1010, further comprising the steps of. [The present invention 1012] The method of the present invention 1011, wherein the cells in step e) are seeded onto an uncoated tissue culture support. [The present invention 1013] e) a step of differentiating the harvested monocyte progenitor cells into microglia and a method according to any one of the present inventions 1001 to 1010, further comprising the steps of. [The present invention 1014] An adherent large-scale cell culture for producing monocyte progenitor cells, wherein the adherent cell culture has a cell culture area of 1 cm per week 2 An adherent large-scale cell culture capable of producing at least about 100,000 monocyte progenitor cells per hit. [Invention 1015] The adherent large-scale cell culture of Invention 1014 produced by steps a) to c) of any of the methods of Inventions 1001 to 1013.

Brief Description of the Drawings

[0025] [Figure 1] It is a schematic diagram of a method for inducing monocytes precursors and macrophages from induced pluripotent stem cells (iPSCs). Adult donor cells can be reprogrammed to generate iPSCs. Using the correct combination of differentiation cues (cytokines, morphogens, growth factors, and small molecules), cell lineage development can be induced in vitro and used to generate the desired cell type (i.e., macrophages). This approach provides an unlimited supply of cells from a single donor and enables the use of cells derived from donors with disease-specific genetic backgrounds. Furthermore, iPSCs can be genetically modified and clonally selected in a self-renewing pluripotent state. This technique enables the generation of syngeneic iPSC lines and allows their cell derivatives (e.g., macrophages) to be directly compared to their respective healthy or diseased parental iPSC clones. An alternative means of obtaining monocytes and macrophages is isolation from human blood donations. The cells obtained by this means are limited in number per donor, and due to their post-mitotic state, the generation of genetically modified clonal lines is not feasible. Further variations can occur due to various donor states (physiological states) such as infections prior to blood donation. [Figure 2]Schematic diagram of the sequential differentiation process in the generation process of iPSC-derived macrophages. iPSCs are cultured and maintained in a pluripotent state (step 1). When subculturing the maintenance culture, embryoid body (EB) formation is initiated using 2 to 10 million iPSCs (step 2). After 4 days of EB formation, the pre-differentiated EBs are seeded in a cell culture dish and a hematopoietic factory is formed during the subsequent period (step 3). The hematopoietic factory starts producing and releasing the first monocyte precursors at about 14 days after the start of differentiation. These precursors can be collected from the supernatant twice a week for up to 100 days. Monocyte precursors further differentiate into macrophages in 7 days (step 4), and depending on the requirements of the experiment, these macrophages can be further polarized to generate specific inflammatory or regulatory subtypes by the addition of cytokines (step 5). [Figure 3] Schematic diagram of the time series of differentiation. The cytokines, growth factors, morphogens, media, and coatings used in the five sequential differentiation steps (steps 1 to 5) are as shown. [Figure 4] Comparison between the new culture conditions and the previously published method of Wilgenburg et al. (2013). iPSCs were cultured in either Matrigel with reduced growth factors or laminin-521, and the hematopoietic factory was differentiated as shown in Figures 2 and 3 and compared on day 21 of differentiation. [Figure 4A] Shows that the hematopoietic factory (adherent cells) derived from iPSCs cultured in laminin-521 already produces monocyte precursors at day 21 of differentiation. [Figure 4B] Monocyte precursors (non-adherent cells) in the supernatant of the hematopoietic factory derived from iPSCs cultured in laminin-521 at day 21 of differentiation. [Figure 4C] Shows that the hematopoietic factory (adherent cells) derived from iPSCs cultured in Matrigel does not produce monocytes at day 21 of differentiation. [Figure 4D]It shows that there are few monocyte precursors (non-adherent cells) in the supernatant of the hematopoietic factory derived from iPSCs cultured in Matrigel at the 21st day of differentiation. [Figure 5] It is a comparison between the new culture conditions and the method of Wilgenburg et al. (2013) published so far. iPSCs were cultured in either Matrigel or laminin-521, the hematopoietic factory was differentiated as shown in Figures 2 and 3, and compared on the 21st day of differentiation. Monocyte precursors derived from iPSCs cultured in laminin-521 were analyzed by flow cytometry for the myeloid markers CD14 and CD11b. [Figure 5A] Flow cytometry dot plot analysis of CD11b surface staining of monocyte precursors collected from laminin-521-derived cultures and isotype controls. Multiple peaks indicate a heterogeneous CD11b-positive cell population. [Figure 5B] Flow cytometry dot plot analysis of CD14 surface staining of monocyte precursors collected from laminin-521-derived cultures and isotype controls. Multiple peaks indicate a heterogeneous CD14-positive cell population. [Figure 6] It is a comparison between the new culture conditions and the method of Wilgenburg et al. (2013) published so far. iPSCs were cultured in either Matrigel or laminin-521, the hematopoietic factory was differentiated as shown in Figures 2 and 3, monocyte precursors were collected from the supernatant, and compared on the 34th day of differentiation. Monocyte precursors derived from iPSCs cultured in either laminin-521 or Matrigel were analyzed by flow cytometry for the myeloid markers CD14, CD11b, CD68, and the proliferation marker Ki67. The average yield from B10 culture dishes was 36.5×106 viable cells for laminin-521-derived cultures and 1.2×106 viable cells for Matrigel-derived cultures. [Figure 6A]Flow cytometric dot plot analysis of CD11b surface staining of monocyte precursors collected from laminin-521-derived cultures and isotype controls on day 34. A single peak indicates a homogeneous population of CD11b-positive cells. [Figure 6B] Flow cytometric dot plot analysis of CD14 surface staining of monocyte precursors collected from laminin-521-derived cultures and isotype controls on day 34. A single peak indicates a homogeneous population of CD14-positive cells. [Figure 6C] Flow cytometric dot plot analysis of CD68 staining of monocyte precursors collected from laminin-521-derived cultures and isotype controls on day 34. A single peak indicates a homogeneous population of CD68-positive cells. [Figure 6D] Flow cytometric dot plot analysis of the Ki67 proliferation marker of monocyte precursors collected from laminin-521-derived cultures and isotype controls on day 34. A single peak at the intensity of the isotype control indicates low proliferative activity in the cell population. [Figure 6E] Flow cytometric dot plot analysis of CD11b surface staining of monocyte precursors collected from Matrigel-derived cultures and isotype controls on day 34. A single peak indicates a homogeneous population of CD11b-positive cells. [Figure 6F] Flow cytometric dot plot analysis of CD14 surface staining of monocyte precursors collected from Matrigel-derived cultures and isotype controls on day 34. A single peak indicates a homogeneous population of CD14-positive cells. [Figure 6G] Flow cytometric dot plot analysis of CD68 staining of monocyte precursors collected from Matrigel-derived cultures and isotype controls on day 34. A single peak indicates a homogeneous population of CD68-positive cells. [Figure 6H]Flow cytometry dot plot analysis of the Ki67 proliferation marker of monocytes precursors collected from Matrigel-derived cultures and isotype controls on day 34. A single peak at the intensity of the isotype control indicates low proliferative activity in the cell population. [Figure 7] Comparison of the new culture conditions with the previously published method of Wilgenburg et al. (2013). iPSCs were cultured either in Matrigel or laminin-521, the hematopoietic factories were differentiated as shown in Figures 2 and 3, and monocyte precursors were recovered from the supernatant and compared on day 41 of differentiation. Monocyte precursors derived from iPSCs cultured in either laminin-521 or Matrigel were analyzed by FACS for the myeloid markers CD14, CD11b, CD68, and the proliferation marker Ki67. The average yield from B10 culture dishes was 30×106 viable cells for laminin-521-derived cultures and 8.5×106 viable cells for Matrigel-derived cultures. [Figure 7A] Flow cytometry dot plot analysis of CD11b surface staining of monocyte precursors collected from laminin-521-derived cultures and isotype controls on day 41. A single peak indicates a homogeneous population of CD11b-positive cells. [Figure 7B] Flow cytometry dot plot analysis of CD14 surface staining of monocyte precursors collected from laminin-521-derived cultures and isotype controls on day 41. A single peak indicates a homogeneous population of CD14-positive cells. [Figure 7C] Flow cytometry dot plot analysis of CD68 staining of monocyte precursors collected from laminin-521-derived cultures and isotype controls on day 41. A single peak indicates a homogeneous population of CD68-positive cells. [Figure 7D] Flow cytometry dot plot analysis of the Ki67 proliferation marker of monocyte precursors collected from laminin-521-derived cultures and isotype controls on day 41. A single peak at the intensity of the isotype control indicates low proliferative activity in the cell population. [Figure 7E] Flow cytometric dot plot analysis of CD11b surface staining of monocyte precursors collected from Matrigel-derived cultures and isotype controls on day 41. A single peak indicates a homogeneous population of CD11b-positive cells. [Figure 7F] Flow cytometric dot plot analysis of CD14 surface staining of monocyte precursors collected from Matrigel-derived cultures and isotype controls on day 41. A single peak indicates a homogeneous population of CD14-positive cells. [Figure 7G] Flow cytometric dot plot analysis of CD68 staining of monocyte precursors collected from Matrigel-derived cultures and isotype controls on day 41. A single peak indicates a homogeneous population of CD68-positive cells. [Figure 7H] Flow cytometric dot plot analysis of the Ki67 proliferation marker of monocyte precursors collected from Matrigel-derived cultures and isotype controls on day 41. A single peak at the intensity of the isotype control indicates low proliferative activity in the cell population. [Figure 8] Comparison between the new culture conditions and the previously published method of van Wilgenburg et al. (2013). iPSCs were cultured either in Matrigel (van Wilgenburg et al. 2013) or laminin-521, the hematopoietic factories were differentiated as shown in Figures 2 and 3, and monocyte precursors were recovered from the supernatant and compared at days 21, 35, and 41 of differentiation. Monocyte yields and marker expression at different collection days are summarized. Hematopoietic factories derived from iPSCs grown in laminin-521 matured, produced monocyte precursors, and released them into the supernatant faster and with higher yields. [Figure 9]Comparison with novel culture conditions and the previously published method of Wilgenburg et al. (2013). iPSCs were cultured either in Matrigel (van Wilgenburg et al. 2013) or laminin-521, the hematopoietic factory was differentiated as shown in Figures 2 and 3, monocyte precursors were recovered from the supernatant, and comparisons were made every 7 days starting on day 27 of differentiation and up to day 111 of differentiation at most. Monocyte precursors derived from iPSCs cultured in either laminin-521 (9A) or Matrigel (9B) were analyzed by FACS for the myeloid markers CD14, CD11b, CD68, and the proliferation marker Ki67. [Figure 10] Comparison between monocytes derived from iPSCs and CD14+ monocytes isolated from PBMCs. Monocytes obtained from both sources were analyzed by FACS for the myeloid markers CD14, CD11b, CD68, and the proliferation marker Ki67. Cell types derived from either source expressed CD14, CD11b, CD68, and were negative for Ki67. The intensity of the markers differed between cells obtained from the two sources, showing slight differences in the amounts of CD14, CD11b, and CD68, respectively. [Figure 11] Comparison between macrophages derived from iPSCs and CD14+ monocytes isolated from macrophages derived from PBMCs. Monocytes obtained from both sources were differentiated as described in Materials and Methods and analyzed by FACS for the myeloid markers CD14, CD11b, CD68, and the proliferation marker Ki67 on day 7 of macrophage differentiation. Cell types derived from either source expressed CD14, CD11b, CD68, and were negative for Ki67. The intensity of the markers differed between cells obtained from the two sources, showing slight differences in the amounts of CD14, CD11b, and CD68, respectively. [Figure 12]A comparison between the new culture conditions and the method of van Wilgenburg et al. (2013) published so far. Embryoid bodies generated from three different iPSC lines, SFC840 (Figures 12A and D), Gibco episomal (Figures 12B and E), and SA001 (Figures 12C and F), were seeded either in uncoated cell culture dishes (12A - C) or in culture dishes coated with growth factor-reduced (GFR) Matrigel (12D - F). For the adhesion and cell proliferation of embryoid bodies, for all three cell lines tested, GFR Matrigel was better, ensuring stronger development of the culture. The cell layer prevents monocytes precursors from adhering to the surface of the tissue culture dish and further increases the number of monocytes precursors in the supernatant. [Figure 13] A comparison between the new culture conditions and the method of van Wilgenburg et al. (2013) published so far. Embryoid bodies generated from three different iPSC lines, SFC840 (Figures 13A and D), Gibco episomal (Figures 13B and E), and SA001 (Figures 13C and F), were seeded either in uncoated cell culture dishes (13A - C) or in culture dishes coated with growth factor-reduced (GFR) Matrigel (13D - F). At the 21st day of differentiation, already, more monocyte precursors were released into the supernatant by the hematopoietic factories generated by embryoid bodies grown in GFR Matrigel compared to uncoated dishes. [Figure 14] Monocyte precursors originating from three different cell lines (SFC840 - 03 - 01, SA001, and Gibco episomal) were differentiated into macrophages for 7 days as described in the Materials and Methods. Phagocytosis assays were performed by supplying zymosan particles labeled with Alexa488 to three different iPSC-derived macrophage lines. After 1 hour of phagocytosis, the cells were detached and Alexa488-positive cells were measured by flow cytometry. Macrophages originating from all sources exhibited strong phagocytic ability, ranging from 50% to 70% of positive cells after 1 hour. [Figure 15]Shown is a diagram of a differentiation regimen for obtaining microglia-like cells in a neuron-microglia co-culture. Neurons derived from iPSCs are pre-differentiated for 21 days and can be cryopreserved at this differentiation stage. To initiate the co-culture, the neurons are thawed at least one week before seeding the monocyte precursors. GFP-positive iPS cells were used for the generation of hematopoietic factories and monocyte precursors to better visualize the development, migration, and morphological characteristics of microglia. For microglia-like differentiation, GFP-positive monocyte precursors were seeded on the pre-differentiated neuron cultures and matured for 2 weeks. [Figure 16] The distribution and morphology of microglia in the co-culture were observed by fluorescence microscopy for GFP (Figure 16A) expressed by microglia-like cells and neurons labeled with anti-beta III-tubulin (Tuj) antibody (Figure 16B). After 1 week of differentiation, monocyte microglia-like cells spread uniformly in the co-culture and exhibit a branched morphology. [Figure 17] Shown is the cytokine release of macrophages and microglia upon LPS stimulation. One functional property of macrophages and microglia is the ability to release cytokines in response to inflammatory stimuli, such as LPS. To test the differences between macrophages, microglia, and the baseline, the baseline cytokine levels of the nervous system co-culture, the cytokine levels of IL1b (Figure 17A), IL6 (Figure 17B), MCP1 (Figure 17C), IL 10 (Figure 17D), IL8 (Figure 17E), IL12p40 (Figure 17F), MIP1a (Figure 17G), and TNFa (Figure 17H) of unstimulated cells and cells stimulated with 100 ng / ml of LPS were measured by CBA. Microglia showed more release of IL1b, IL6, Il10, TNFa, IL12p40, and MIP1a, and less release of IL8 compared to macrophages. The nervous system monoculture showed the release of TNFa, MCP1, MIP1a, and IL8, indicating the contribution of astrocytes to the inflammatory response in the co-culture. [Figure 18]Phagocytosis is an important functional property of bone marrow-derived cells. Different substrates labeled with the pH-sensitive dye pHrodo (e.g., zymosan, beet coated with Abeta, or apoptotic cells) can be used to monitor this process under different culture conditions, such as macrophages or microglia. These substrates are recognized by bone marrow cells, engulfed into endosomes, and become fluorescent when the pH decreases during lysosome maturation. Representative images of zymosan labeled with pHrodo taken up by either microglia (Figures 18A and B) or macrophages (Figures 18C and D). [Figure 19] Phagocytosis activity can be used for drug screening and image-based readouts using the pHrodo technology. Interference with the functionality of the cytoskeleton can cause a decrease in phagocytosis activity (Figure 19A), while co-incubation or pretreatment with serum (FCS) can result in a concentration-dependent increase in zymosan uptake activity (Figure 19B). [Figure 20] For medium-term storage and large batch generation of monocyte precursors, monocytes collected from the hematopoietic factory can be cultured in suspension culture ("spinner"). Differentiation of stored monocyte precursors into macrophages can be initiated at any time (Figure 20A). Monocyte precursors cultured in suspension culture ("spinner") remain viable for at least 6 weeks (Figure 20B) and retain their marker profile (Figure 20C). Macrophages differentiated from monocyte precursors maintained in suspension culture ("spinner") have similar marker expression compared to macrophages differentiated directly after collection (Figure 20D). [Figure 21] Macrophages differentiated from monocyte precursors in suspension culture ("spinner") exhibit functional properties indistinguishable from macrophages differentiated directly from cells after collection, and they have similar phagocytosis ability (Figure 21A) and migration ability (Figure 21B).

[0026] Cited References TIFF0007701879000001.tif216156TIFF0007701879000002.tif198156

Mode for Carrying Out the Invention

[0027] Detailed Description As used herein, the terms "defined medium" or "chemically defined medium" refer to a cell culture medium in which all individual components and their respective concentrations are known. A defined medium may contain recombinant and chemically defined components.

[0028] As used herein, the terms "differentiate", "differentiation", and "differentiating" refer to one or more processes of converting less differentiated cells into somatic cells, e.g., converting pluripotent stem cells into monocytes or converting monocytes into macrophages. Differentiation is achieved by methods known in the art and described herein.

[0029] As used herein, a "monocyte progenitor cell" is a cell that expresses the specific surface markers CD14 (cluster of differentiation 14, also known as myeloid-specific leucine-rich glycoprotein, official symbol CD14), CD11b (cluster of differentiation 11B, also known as integrin alpha M (ITGAM), macrophage-1 antigen (Mac-1), and complement receptor 3 (CR3 / CR3A), official symbol ITGAM), CD68 (cluster of differentiation 68, also known as GP110, macrosialin, scavenger receptor class D member 1 (SCARD1), and LAMP4, official symbol CD68), is in suspension, and has the ability to give rise to adherent macrophages and microglia.

[0030] As used herein, "macrophage" expresses specific markers CD14 (cluster of differentiation 14, also known as myeloid cell-specific leucine-rich glycoprotein, official symbol CD14), CD11b (cluster of differentiation 11B, also known as integrin alpha M (ITGAM), macrophage-1 antigen (Mac-1), and complement receptor 3 (CR3 / CR3A), official symbol ITGAM), CD68 (cluster of differentiation 68, also known as GP110, macrosialin, scavenger receptor class D member 1 (SCARD1), and LAMP4, official symbol CD68), is adhesive, can phagocytize different substrates, responds to various inflammatory stimuli, and can be polarized by the presence of endogenous cytokines (e.g., IL-4 and INFg).

[0031] As used herein, "microglia" expresses specific markers CD14 (cluster of differentiation 14, also known as myeloid cell-specific leucine-rich glycoprotein, official symbol CD14), CD11b (cluster of differentiation 11B, also known as integrin alpha M (ITGAM), macrophage-1 antigen (Mac-1), and complement receptor 3 (CR3 / CR3A), official symbol ITGAM), CD68 (cluster of differentiation 68, also known as GP110, macrosialin, scavenger receptor class D member 1 (SCARD1), and LAMP4, official symbol CD68), IBA1 (ionized calcium-binding adapter molecule 1, also known as allograft inflammatory factor 1AIF1, official symbol AIF1), has a branched morphology, can phagocytize different substrates, responds to various inflammatory stimuli, and expresses at least one additional marker protein, for example, TMEM119 (transmembrane protein 119, also known as osteoclast-inducing factor (OBIF), official symbol TMEM119), P2RY12 (P2Y purinergic receptor 12, also known as ADP-glucose receptor, official symbol P2RY12), or PROS1 (protein S, also known as PSA, PROS, PS21, PS22, PS23, PS24, PS25, THPH5, THPH6, official symbol PROS1), and / or is in a branched form, and is a cell.

[0032] "Mesoderm induction medium", as used herein, refers to any medium useful for the induction of mesoderm in pluripotent stem cells, preferably a chemically defined medium. One example of such a medium is a defined medium supplemented with human recombinant bone morphogenetic protein-4 (BMP4), human vascular endothelial growth factor (VEGF), and human stem cell factor (SCF), such as MTeSR1 medium. Markers suitable for determining mesoderm induction are MIXL, EOMES, and T-brachyury.

[0033] "Myeloid maturation medium", as used herein, refers to a medium useful for the maturation of cells along the myeloid lineage, preferably a chemically defined medium. One example of such a medium is a defined medium, such as XVIVO15 medium, supplemented with macrophage colony-stimulating factor (M-SCF) and interleukin 3 (IL-3). Suitable markers for determining maturation along the myeloid lineage are CD14, ITGAM, and / or CD68.

[0034] "Macrophage differentiation medium", as used herein, refers to any medium useful for the differentiation of monocyte progenitor cells into macrophages, preferably a chemically defined medium. One example of such a medium is a defined medium, such as XVIVO15 medium, supplemented with macrophage colony-stimulating factor (M-CSF). Suitable macrophage markers for identifying macrophages are CD14, ITGAM, and / or CD68, as well as adhesion to cell culture substrates, phagocytosis, responses to various inflammatory stimuli, and polarization upon treatment with, for example, IL-4 and / or INFγ.

[0035] As used herein, the term "growth factor" means a biologically active polypeptide or small molecule compound that causes cell proliferation, and includes both growth factors and their analogs.

[0036] It should be understood that "high-throughput screening", as used herein, means analyzing and comparing a large number of different disease model conditions and / or chemical compounds in parallel. Typically, such high-throughput screening (assays) are performed in multi-well microtiter plates, such as 96-well plates or 384-well plates, or plates having 1536 or 3456 wells.

[0037] "Large-scale cell culture" as used herein refers to a cell culture (system) in which a large number of cells are confined under conditions (e.g., medium supply, gas exchange, available surface area) to maintain cell viability, and the amount of such cells is suitable for high-throughput screening (assay). In certain embodiments, a large-scale cell culture storage container (e.g., a vessel, container, flask) contains more than 10 6 cells, more than 10 7 cells, more than 10 8 cells, more than 10 9 cells, more than 10 10 cells, more than 10 11 cells, more than 10 12 cells, more than 10 2 cells, more than 500 cm 2 cells, more than 1,000 cm 2 cells, more than 2,000 cm 2 cells, more than 5,000 cm 2 cells, more than 10,000 cm 2 of cell culture area. In one embodiment, a large-scale cell culture (system) is inoculated with at least 1, 2, 3, 4, 5 embryoid bodies per cm 5 corresponding to at least 10 6 cells, 10 7 cells, 10 8 cells, 10 9 cells, 10 2 on day 1. In one embodiment, one embryoid body (corresponding to about 13,000 cells) is seeded per cm 2 of cell culture area.

[0038] As used herein, a "single layer of cells" means that the cells are substantially in a single layer of single cells, as opposed to non-confluent single cells, and also as opposed to (a plurality of) three-dimensional layered or non-layered forms (e.g., embryoid bodies) in which a plurality of cells are either attached or not attached to an adhesive substrate, attached to an adhesive substrate (e.g., a cell culture support).

[0039] As used herein, a "pluripotent medium" refers to any chemically defined medium useful for pluripotent stem cells to adhere as single cells in a single layer while maintaining their pluripotency. Useful pluripotent media are well known in the art and are also described herein. In certain embodiments described herein, the pluripotent medium contains at least one of the following growth factors: basic fibroblast growth factor (bFGF, also referred to as fibroblast growth factor 2, FGF2), and transforming growth factor β (TGFβ).

[0040] As used herein, the term "reprogramming" refers to one or more steps required to convert somatic cells into less differentiated cells, e.g., to convert fibroblasts, adipocytes, keratinocytes, or leukocytes into pluripotent stem cells. "Reprogrammed" cells refer to cells derived by reprogramming somatic cells as described herein.

[0041] The term "small molecule", or "small compound", or "small molecule compound", as used herein, generally refers to an organic or inorganic molecule, either synthetic or found in nature, having a molecular weight of less than 10,000 grams per mole, optionally less than 5,000 grams per mole, and optionally less than 2,000 grams per mole per mole.

[0042] As used herein, the term "somatic cell" refers to any cell that forms the body of an organism and is not a germline cell (e.g., sperm and eggs, which are the cells from which they are made (germ cells)) or an undifferentiated stem cell.

[0043] As used herein, the term "stem cell" refers to a cell having the ability to self-renew. As used herein, an "undifferentiated stem cell" refers to a stem cell having the ability to differentiate into diverse cell types. As used herein, a "pluripotent stem cell" refers to a stem cell that can give rise to cells of multiple cell types. Pluripotent stem cells (PSCs) include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Human induced pluripotent stem cells can be induced from reprogrammed somatic cells, for example, by transduction of four defined factors (Sox2, Oct4, Klf4, c-Myc) by methods known in the art and further described herein. Such human somatic cells can be obtained from healthy individuals or patients. These donor cells can be obtained from any suitable source. Sources that allow isolation of donor cells without using invasive procedures on the human body, for example, cells that can be obtained from human skin cells, blood cells, or urine samples, are preferred herein.

[0044] As used herein, the term "suspension culture" refers to a cell culture system in which cells (single cells or cell aggregates, such as embryoid bodies) are not substantially attached or minimally attached to the surface of the cell culture vessel used to incubate the cells. In a suspension culture, the cells or cell aggregates are floating with minimal or no contact with the surface of the cell culture vessel (e.g., the tissue culture support of a flask). Minimally attached cells or cell aggregates in a suspension culture can be easily detached by using weak or moderate physical forces, for example, by gentle shaking, tapping, or horizontal movement of the cell culture.

[0045] As used herein, the term "adherent cell culture" refers to a cell culture system in which, in contrast to a suspension culture, the cells are attached to the surface of the cell culture storage vessel used to incubate the cells. Minimally adherent cells or cell aggregates of a suspension culture that can be easily detached by the use of weak or moderate physical forces described herein are not considered adherent cell cultures.

[0046] Human cells are preferred, but the methods described herein are also applicable to non-human cells such as primate, rodent (e.g., rat, mouse, rabbit), and canine cells.

[0047] Methods for producing monocyte progenitor cells are provided herein. Prior to the present invention, the use of monocytes and macrophages in drug discovery was limited due to several technical problems. Factors such as cell number, expandability, reproducibility, and phenotypic relevance are essential to ensure that projects meet their deadlines. The inventors were able to modify the published protocol (van Wilgenburg et al. 2013) to increase yield and reproducibility while reducing the differentiation time. In a preferred embodiment, embryoid bodies (EBs) are generated from induced pluripotent stem cells (iPSCs) seeded on a laminin-coated cell culture support. These EBs are similar to early embryogenesis and initiate the formation of the three germ line layers (primitive streak). The EBs are then pre-differentiated by contacting the cells with a defined medium containing BMP4, inducing the cell fate determination towards the mesoderm lineage. After being formed and pre-differentiated, the EBs are seeded and further differentiated along the myeloid lineage to form a hematopoietic factory, which produces monocyte precursors and releases them into the supernatant (Figure 2). The hematopoietic factory can be maintained for over 100 days, and the monocyte precursors can be harvested from the culture supernatant (up to twice a week). After harvesting, these precursors can differentiate into unpolarized macrophages within one week or be further polarized by the addition of specific cytokines that promote either a pro-inflammatory or anti-inflammatory subtype. By increasing the expandability of the hematopoietic factory to a culture area of 10 to 1000 cm 2 the present invention has achieved cell collection and handling times that meet the needs of drug discovery and development projects as well as medium-scale drug screening programs. In another aspect, a novel co-culture setting for the generation of microglia-like cells has been established.

[0048] Generation of Monocyte Progenitor Cells Pluripotent stem cells have the characteristic of self-renewal and can differentiate into all major cell types of the adult mammalian body. Pluripotent stem cells can be produced in large quantities under standardized cell culture conditions. Thus, in a preferred embodiment, monocyte progenitor cells are generated, i.e., differentiated, from pluripotent stem cells. In one embodiment, monocyte progenitor cells are generated, i.e., differentiated, from embryonic stem cells. In a preferred embodiment, monocyte progenitor cells are generated, i.e., differentiated, from induced pluripotent stem cells (iPSCs). In one embodiment, iPSCs are generated from reprogrammed somatic cells. Reprogramming of somatic cells into iPSCs can be achieved by introducing specific genes involved in maintaining iPSC characteristics. Genes suitable for reprogramming somatic cells into iPSCs include, but are not limited to, Oct4, Sox2, Klf4, and C-Myc, as well as combinations thereof. In one embodiment, the genes for reprogramming are Oct4, Sox2, Klf4, and C-Myc.

[0049] The internal organs, skin, bones, blood, and connective tissues are all made from somatic cells. Somatic cells used to generate iPSCs include, but are not limited to, fibroblasts, adipocytes, and keratinocytes, and can be obtained from a skin biopsy. Other suitable somatic cells are white blood cells, erythroblasts obtained from a blood sample or epithelial cells, or other cells obtained from a blood or urine sample, and are reprogrammed into iPSCs by methods known in the art and described herein. Somatic cells can be obtained from a healthy individual or from an affected individual. In one embodiment, the somatic cells are derived from a subject (e.g., a human subject) suffering from a disease. In one embodiment, the disease is associated with any of chronic inflammation (e.g., inflammatory bowel disease), primary or acquired immunodeficiency (e.g., bare lymphocyte syndrome), or neurodegenerative disease (e.g., multiple sclerosis, Alzheimer's disease, or Parkinson's disease). The genes for reprogramming described herein are introduced into somatic cells by either methods known in the art, delivery to the cells via a reprogramming vector, or activation of the genes via small molecules. Methods for reprogramming include, among others, retroviruses, lentiviruses, adenoviruses, plasmids and transposons, microRNAs, small molecules, modified RNAs, messenger RNAs, and recombinant proteins. In one embodiment, a lentivirus is used for delivery of the genes described herein. In another embodiment, Oct4, Sox2, Klf4, and C-Myc are delivered to somatic cells using Sendai virus particles. Additionally, somatic cells may be cultured in the presence of at least one small molecule. In one embodiment, this small molecule includes an inhibitor of the Rho-associated coiled-coil forming protein serine / threonine kinase (ROCK) family of protein kinases.Non-limiting examples of ROCK inhibitors include fasudil (1-(5-isoquinolinesulfonyl)homopiperazine), thiazovivin (N-benzyl-2-(pyrimidin-4-ylamino)thiazole-4-carboxamide), and Y-27632 ((+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride).

[0050] Providing a defined monolayer of pluripotent stem cells is preferred for the reproducibility and efficiency of the resulting cultures. The inventors have surprisingly found that replacement by using a substrate coated with laminin in stem cell maintenance culture reduces the differentiation time of the hematopoietic factory and increases the throughput of cell cultures. In one embodiment, a monolayer of pluripotent stem cells can be produced by enzymatically dissociating the cells into single cells and seeding them into a cell culture storage container (e.g., a flask) coated with an adhesive substrate, such as a laminin substrate. In a preferred embodiment, the adhesive substrate (coating) is laminin. In one embodiment, the laminin comprises laminin subunit alpha-4. In one embodiment, the laminin comprises laminin subunit alpha-5. In one embodiment, the laminin comprises laminin subunit beta-1. In one embodiment, the laminin comprises laminin subunit beta-2. In one embodiment, the laminin comprises laminin subunit gamma-1. In one embodiment, the laminin comprises laminin subunits alpha-4, beta-1, and gamma-1 (laminin-411). In one embodiment, the laminin comprises laminin subunits alpha-5, beta-1, and gamma-1 (laminin-511). In a preferred embodiment, the laminin comprises laminin subunits alpha-5, beta-2, and gamma-1 (laminin-521, e.g., BioLamina rhLaminin-521).

[0051] Examples of enzymes suitable for dissociation into single cells include Accutase (Invitrogen), trypsin (Invitrogen), and TrypLe Express (Invitrogen). In one embodiment, 20,000 to 60,000 cells per 1 cm 2 are seeded onto the adhesive substrate. The medium used herein is a pluripotent medium that promotes the attachment and growth of pluripotent stem cells as single cells in a monolayer. In one embodiment, the pluripotent medium is a serum-free medium supplemented with a small molecule inhibitor of the Rho-associated coiled-coil forming protein serine / threonine kinase (ROCK) family of protein kinases (referred to herein as a ROCK kinase inhibitor).

[0052] Thus, in one embodiment, the method described herein includes providing a monolayer of pluripotent stem cells in a pluripotent medium on a laminin substrate, where the pluripotent medium is a serum-free medium supplemented with a ROCK kinase inhibitor.

[0053] Examples of serum-free media suitable for the attachment of pluripotent stem cells to a substrate include mTeSR1 or TeSR2 from Stem Cell Technologies, Primate ES / iPS cell medium from ReproCELL, PluriSTEM from Milipore, StemMACS iPS-Brew from Milenyi Biotec, and StemPro hESC SFM from Invitrogen, and X-VIVO from Lonza. Examples of ROCK kinase inhibitors useful herein are fasudil (1-(5-isoquinolinesulfonyl)homopiperazine), thiazovivin (N-benzyl-2-(pyridin-4-ylamino)thiazole-4-carboxamide), and Y27632 ((+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride, for example, catalog number: 1254 from Tocris bioscience). In one embodiment, the pluripotent medium is a serum-free medium supplemented with about 2 - 20 μM of Y27632, preferably about 5 - 10 μM of Y27632. In another embodiment, the pluripotent medium is a serum-free medium supplemented with about 2 - 20 μM of fasudil. In another embodiment, the pluripotent medium is a serum-free medium supplemented with about 0.2 - 10 μM of thiazovivin.

[0054] In one embodiment, the method described herein includes providing a monolayer of pluripotent stem cells in a pluripotent medium on a laminin substrate and growing this monolayer in the pluripotent medium for at least 1 day (24 hours). In another embodiment, the method described herein includes providing a monolayer of pluripotent stem cells in a pluripotent medium and growing this monolayer in the pluripotent medium for 18 hours to 30 hours, preferably 23 - 25 hours. In a further embodiment, the method described herein includes providing a monolayer of pluripotent stem cells in a pluripotent medium on a laminin substrate and growing this monolayer in the pluripotent medium for at least 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or more than 10 days.

[0055] In another embodiment, the method described herein comprises providing a single layer of pluripotent stem cells in a pluripotent medium, which is mTesR1 medium, on a laminin substrate, and growing the single layer in the pluripotent medium for at least 1 day (24 hours). In another embodiment, the method described herein comprises providing a single layer of pluripotent stem cells in a pluripotent medium, which is mTesR1, on a laminin substrate, and growing the single layer in the pluripotent medium for 18 to 30 hours, preferably 23 to 25 hours.

[0056] In the next step b), the pluripotent stem cells are harvested and transferred into a suspension culture. In one embodiment, the pluripotent stem cells are contacted with a mesoderm induction medium. In one embodiment, the mesoderm induction medium contains recombinant bone morphogenetic protein-4 (BMP4). In one embodiment, the mesoderm induction medium is a serum-free medium supplemented with about 10 - 100 ng / ml of BMP4 (e.g., hBMP4), preferably about 50 ng / ml of BMP4.

[0057] In a further embodiment, the mesoderm induction medium further contains vascular endothelial growth factor (VEGF). In one embodiment, the mesoderm induction medium is a serum-free medium supplemented with about 10 - 100 ng / ml of VEGF (e.g., hVEGF), preferably about 50 ng / ml of VEGF.

[0058] In a further embodiment, the mesoderm induction medium further contains stem cell factor (SCF). In one embodiment, the mesoderm induction medium is a serum-free medium supplemented with about 5 - 50 ng / ml of SCF (e.g., hSCF), preferably about 20 ng / ml of SCF.

[0059] In a preferred embodiment, the mesoderm induction medium contains BMP4, VEGF, and SCF, specifically, about 10 - 100 ng / ml of BMP4, about 10 - 100 ng / ml of VEGF, and about 5 - 50 ng / ml of SCF. In a preferred embodiment, the mesoderm induction medium contains about 50 ng / ml of BMP4, about 50 ng / ml of VEGF, and about 20 ng / ml of SCF.

[0060] In one embodiment, the pluripotent stem cells are contacted with a mesoderm induction medium for at least about 1 day (24 hours). In further embodiments, the pluripotent stem cells are contacted with the mesoderm induction medium for about 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or greater than about 10 days. In one embodiment, the pluripotent stem cells are contacted with the mesoderm induction medium for about 24 hours to about 72 hours, preferably about 36 to about 60 hours.

[0061] In one embodiment, the cells are seeded in step c) onto a cell culture support suitable for attachment of the cells after mesoderm induction. In a preferred embodiment, the cells are seeded onto a cell culture support coated with a basement membrane biomaterial such as Matrigel, Cultrex BME, Geltrex Matrix, etc. In one embodiment, the basement membrane biomaterial comprises laminin, type IV collagen, heparan sulfate proteoglycan, and entactin / nidogen-1,2. In a preferred embodiment, the cells are seeded onto a cell culture support coated with Matrigel.

[0062] In one embodiment, the cells are seeded in step c) into a large-scale cell culture vessel. In certain embodiments, greater than 10 6 greater than 10 7 greater than 10 8 greater than 10 9 greater than 10 10 greater than 10 11 greater than 10 12 greater than 10 cells are seeded into each large-scale cell culture storage vessel. In one embodiment, the large-scale cell culture comprises one single cell culture storage vessel. In another embodiment, the large-scale cell culture comprises an assembly of a plurality of cell culture storage vessels. In a further embodiment, the large-scale cell culture (storage vessel) is at least 100 cm 2 500 cm 2 1,000 cm 2 2,000 cm 2 5,000 cm 2, a cell culture area of 10,000 cm 2 is included. In one embodiment, a large-scale cell culture (system) has at least 10 6 cells, 10 7 cells, 10 8 cells, 10 9 cells, 10 10 cells, 10 11 cells, 10 12 cells seeded.

[0063] In the next step, the cells in the large-scale cell culture are further differentiated along the myeloid lineage. In one embodiment, the seeded cells are contacted with a myeloid maturation medium in step c). Suitable myeloid maturation media are known in the art and are also described herein. In one embodiment, the myeloid maturation medium contains interleukin 3 (IL-3). In one embodiment, the myeloid maturation medium is a serum-free medium supplemented with about 1-50 ng / ml of IL-3 (e.g., hIL-3), preferably about 25 ng / ml of IL-3. In one embodiment, the cells are contacted with the myeloid maturation medium for about 4 days (about 96 hours). In a further embodiment, the cells are contacted with the myeloid maturation medium for about 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or more than about 10 days. In one embodiment, the cells are contacted with the myeloid maturation medium for about 72 hours to about 120 hours, preferably about 84 to about 108 hours. During the myeloid maturation process, the large-scale cell culture begins to produce monocyte progenitor cells. Monocyte progenitor cells can be harvested from the adherent cell culture by collecting the supernatant of the cell culture after myeloid maturation. In one embodiment, the large-scale cell culture of step c) according to the present invention can produce monocyte progenitor cells for more than about 10 days, more than 15 days, more than 20 days, more than 25 days, more than 30 days, more than 40 days, more than 50 days, more than 60 days, more than 70 days, more than 80 days, more than 90 days, or more than 100 days. In one embodiment, the large-scale culture of step c) produces, per week, a cell culture area of 1 cm 2can produce at least about 100,000 monocyte progenitor cells.

[0064] Differentiation of monocyte progenitor cells into macrophages Monocyte progenitor cells can be differentiated into macrophages by methods known in the art and described herein. In one embodiment, the monocyte progenitor cells are contacted with macrophage differentiation medium. In one embodiment, the cells are contacted with macrophage differentiation medium for about 1 to 10 days, 4 to 8 days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or more than about 10 days. In one embodiment, the macrophage differentiation medium contains macrophage colony-stimulating factor (M-CSF). In one embodiment, the macrophage differentiation medium is a serum-free medium supplemented with 10 to 200 ng / ml of M-CSF (e.g., hM-CSF), preferably 100 ng / ml of M-CSF. In a preferred embodiment, the cells are contacted with macrophage differentiation medium for about 6 days. In one embodiment, the cells are seeded onto an uncoated tissue culture support before or simultaneously with contacting the cells with macrophage differentiation medium. In one embodiment, the macrophages are reseeded onto an uncoated tissue culture support. In one embodiment, the macrophages are reseeded in a high-throughput plate format. In one embodiment, the macrophages are reseeded in a 24-well plate format, 96-well plate format, or 384-well plate format.

[0065] Differentiation of monocyte progenitor cells into microglia Monocyte progenitor cells can be differentiated into microglia by methods known in the art and described herein. In one embodiment, the monocyte progenitor cells are contacted with neurons. In one embodiment, the neurons are generated using the method described in International Publication No. 2017081250. In some embodiments, the neurons are differentiated for (at least) about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks. In a preferred embodiment, the neurons are differentiated for about 2 - 5 weeks. In some embodiments, the cells are contacted with the neurons for about 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or greater than about 10 days. In some embodiments, the cells are contacted with the neurons for about 5 - 20 days or about 10 - 18 days. In one embodiment, the cells are co-cultured with the neurons in a co-culture differentiation medium. In one embodiment, the co-culture differentiation medium contains granulocyte macrophage colony-stimulating factor (GM-CSF) and / or interleukin 34 (IL-34). In one embodiment, the co-culture differentiation medium is a serum-free medium supplemented with 10 - 200 ng / ml of GM-CSF (e.g., hGM-CSF), preferably 100 ng / ml of GM-CSF. In one embodiment, the co-culture differentiation medium is a serum-free medium supplemented with 1 - 500 ng / ml of IL-34 (e.g., hIL-34), preferably 100 ng / ml of IL-34. In a preferred embodiment, the cells are contacted with the neurons and the co-culture differentiation medium for about 14 days in a serum-free medium supplemented with 10 - 200 ng / ml of GM-CSF (e.g., hGM-CSF) and 1 - 500 ng of IL-34, preferably 100 ng / ml of GM-CSF and 100 ng / ml of IL-34.

[0066] Exemplary embodiments: 1. A method for producing monocyte progenitor cells, comprising: a) seeding pluripotent stem cells in a pluripotent medium onto a cell culture support coated with laminin; b) collecting pluripotent stem cells and contacting the pluripotent stem cells with a mesoderm induction medium in a suspension culture; c) seeding the cells onto a cell culture support suitable for cell attachment; d) collecting monocyte progenitor cells from the suspension; and A method comprising the steps. 2. The method according to embodiment 1, wherein the cells in step a) are cultured on a cell culture support coated with laminin for at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days, specifically at least about 1 day. 3. The method according to embodiment 1 or 2, wherein the laminin in step a) comprises laminin subunit alpha-5, particularly, the laminin in step a) comprises laminin subunit alpha-5, beta-2, and gamma1. 4. The method according to any one of embodiments 1 to 3, wherein the mesoderm induction medium is a chemically defined medium containing recombinant bone morphogenetic protein-4 (BMP4). 5. The method according to embodiment 4, wherein the medium contains about 10 - 100 ng / ml of BMP4, preferably about 50 ng / ml of BMP4. 6. The method according to embodiment 4 or 5, wherein the mesoderm induction medium further contains vascular endothelial growth factor (VEGF). 7. The method according to embodiment 6, wherein the mesoderm induction medium contains about 10 - 100 ng / ml of VEGF, preferably about 50 ng / ml of VEGF. 8. The method according to any one of embodiments 4 to 7, wherein the mesoderm induction medium further contains stem cell factor (SCF). 9. The method according to embodiment 8, wherein the mesoderm induction medium contains about 5 - 50 ng / ml of SCF, preferably about 20 ng / ml of SCF. 10. The method according to any one of embodiments 1 to 9, wherein the cells are contacted with the mesoderm induction medium for about 1 - 10 days, 2 - 6 days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days. 11. The method according to any one of embodiments 1 to 10, wherein the cells are contacted with the mesoderm induction medium for about 4 days. 12. The method according to any one of Embodiments 1 to 11, wherein the cells in step b) form embryoid bodies (EBs). 13. The method according to any one of Embodiments 1 to 12, wherein the cell culture support in step c) is coated with a basement membrane biomaterial. 14. The method according to Embodiment 13, wherein the basement membrane biomaterial comprises laminin, type IV collagen, heparan sulfate proteoglycan, and entactin / nidogen-1,2. 15. The method according to any one of Embodiments 1 to 14, wherein the cells in step c) are contacted with a myeloid maturation medium. 16. The method according to any one of Embodiments 1 to 15, wherein the myeloid maturation medium comprises macrophage colony-stimulating factor (M-CSF). 17. The method according to Embodiment 16, wherein the myeloid maturation medium comprises about 20 - 200 ng / ml of M-CSF, preferably about 100 ng / ml of M-CSF. 18. The method according to any one of Embodiments 15 to 17, wherein the myeloid maturation medium further comprises IL-3. 19. The method according to Embodiment 18, wherein the medium comprises about 1 - 50 ng / ml of IL-3, preferably about 25 ng / ml of IL-3. 20. The method according to any one of Embodiments 15 to 19, wherein the cells are contacted with the myeloid maturation medium for about 1 - 10 days, 2 - 6 days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days. 21. The method according to any one of Embodiments 15 to 20, wherein the cells are contacted with the myeloid maturation medium for about 4 days. 22. The method according to any one of Embodiments 1 to 21, wherein in step d), monocyte progenitor cells are collected by recovering the supernatant of the cell culture. 23. The method according to any one of Embodiments 1 to 22, wherein in step d), monocyte progenitor cells are batch-collected by recovering the supernatant of the cell culture. 24. The method according to any one of embodiments 1 to 23, wherein the monocyte progenitor cells are collected in batches at regular intervals, specifically, daily, every other day, every three days, every four days, every five days, or every six days. 25. The method according to any one of embodiments 1 to 24, wherein the monocyte progenitor cells are collected continuously. 26. The method according to any one of embodiments 1 to 25, wherein in step d), the monocyte progenitor cells are continuously collected by removing the supernatant from the cell culture and, if necessary, replacing the removed supernatant with fresh medium. 27. The method according to any one of embodiments 1 to 26, further comprising step e) of differentiating the collected monocyte progenitor cells into macrophages. 28. The method according to embodiment 27, wherein the cells in step e) are contacted with a macrophage differentiation medium. 29. The method according to embodiment 27, wherein the macrophage differentiation medium contains macrophage colony-stimulating factor (M-CSF). 30. The method according to embodiment 28 or 29, wherein the macrophage differentiation medium contains about 10 to 200 ng / ml of M-CSF, preferably about 100 ng / ml of M-CSF. 31. The method according to any one of embodiments 28 to 30, wherein the cells are contacted with the macrophage differentiation medium for about 1 to 10 days, 4 to 8 days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days. 32. The method according to any one of embodiments 28 to 31, wherein the cells are contacted with the macrophage differentiation medium for about 6 days. 33. The method according to any one of embodiments 28 to 32, wherein the cells in step e) are seeded on an uncoated tissue culture support. 34. The method according to any one of embodiments 28 to 33, wherein the macrophages are reseeded on an uncoated tissue culture support. 35. The method according to any one of embodiments 28 to 34, wherein the macrophages are reseeded in a 24-well plate format, 96-well plate format, or 384-well plate format. 36. The method according to any one of embodiments 1 to 26, further comprising a step of differentiating monocyte progenitor cells into microglia. 36. The method according to embodiment 35, wherein the monocyte precursor in step e) is co-cultured with nerve cells. 37. The method according to embodiment 35 or 36, wherein the cells in step e) are contacted with a co-culture differentiation medium. 38. The method according to embodiment 37, wherein the co-culture differentiation medium contains granulocyte macrophage colony-stimulating factor (GM-CSF) and / or interleukin 34 (IL-34). 39. The method according to embodiment 38, wherein the co-culture differentiation medium contains about 10 - 200 ng / ml of GM-CSF, preferably about 100 ng / ml of GM-CSF. 40. The method according to embodiment 38 or 39, wherein the co-culture differentiation medium contains about 1 - 500 ng / ml of IL-34 (e.g., hIL-34), preferably about 100 ng / ml of IL-34. 41. The method according to any one of embodiments 38 to 40, wherein the cells are contacted with the co-culture differentiation medium for about 1 - 28 days, 7 - 21 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days. 42. The method according to any one of embodiments 38 to 41, wherein the cells are contacted with the co-culture differentiation medium for about 14 days. 43. The method according to any one of embodiments 36 to 42, wherein the nerve cells are derived from pluripotent stem cells. 44. The method according to any one of embodiments 36 to 43, wherein the nerve cells are produced according to the method for producing a standardized cell culture of uniformly distributed differentiated NCs described in WO 2017 / 081250. 45. The method according to any one of embodiments 1 to 44, wherein the pluripotent stem cells are mammalian cells, particularly human cells. 46. The method according to any one of embodiments 1 to 45, wherein the pluripotent stem cells are embryonic stem cells (ESCs). 47. The method according to any one of embodiments 1 to 45, wherein the pluripotent stem cells are induced pluripotent stem cells (iPSCs). 48. An adherent large-scale cell culture for producing monocyte progenitor cells, which can produce at least about 100,000 monocyte progenitor cells per cm of cell culture area per week. 2 An adherent large-scale cell culture capable of producing at least about 100,000 monocyte progenitor cells per cm of cell culture area per week. 49. The adherent large-scale cell culture according to embodiment 48, produced by steps a) to c) of the method according to any one of claims 1 to 26. 49. The invention described herein.

Examples

[0067] The following are non-limiting examples of the compositions and methods of the present invention. It is understood that various other embodiments can be implemented based on the general description provided above.

[0068] Materials and Methods To generate macrophages from human induced pluripotent stem cells, the inventors adopted a published protocol (van Wilgenburg et al. 2013). This resulted in a multi-step protocol shown in Figure 3. This protocol consists of five steps: iPSC maintenance (step 1), EB formation (step 2), seeding of EBs (step 3), macrophage differentiation (step 4), and macrophage polarization (step 5).

[0069] iPSC Maintenance under Feeder-Free Conditions Culture dishes (Corning) were coated with 12.5 μg / ml of rh laminin-521 (BioLamina) in PBS containing calcium and magnesium for at least 2 hours before use. hiPS cells were seeded in mTesR1 medium (StemCell Technologies) at 37°C in 5% CO 2 and cultured, and the medium was changed daily. The cells were passaged at a 90% confluence density. Then, the medium was removed, the cells were washed once with PBS, and detached with Accutase at 37°C for 2 - 5 minutes. After removing Accutase by centrifugation, the cells were used for either maintenance or the start of differentiation.

[0070] EB Formation and Mesoderm Induction To obtain homogeneous EBs, iPS cells were seeded onto Aggrewell 800 (StemCell Technologies) plates. Then, 10 μM of ROCK inhibitor (Y27632, Callbiochem) was supplemented, and 2 ml of mTesR1 containing 4 × 10 6 individual iPS single cells was added to each Aggrewell and centrifuged at 100 g for 3 minutes to ensure that the iPS cells were evenly and rapidly distributed into the microwells of the Aggrewell. The next day, mesoderm induction was initiated by replacing 75% of the mTeSR1 medium (replacing 1 ml out of 2 ml twice in each well) with fresh mTeSR1 medium supplemented with 50 ng / ml of hBMP4, 50 ng / ml of hVEGF, and 20 ng / ml of hSCF. This was subsequently repeated for 2 more days for further differentiation.

[0071] Seeding of EBs and Continued Maturation along the Myeloid Lineage On day 4 of differentiation, the EBs were harvested by gently removing the EBs by rinsing the Aggrewell with PBS. The EBs were collected through a 40-μm strainer and transferred to a cell culture vessel with a desired surface area (2 - 2000 cm 2 ) pre-coated with Matrigel (354230 Corning) with reduced growth factors diluted in cold DMEM F12 1:1 1× Glutamax (Gibco 31331-028) at room temperature for 1 hour, at a density of 0.8 - 1.5 EBs / cm 2 . To enable EB adhesion, the EBs were evenly distributed by gentle agitation, and the culture vessel was immediately placed at 37°C in 5% CO 2During this period, no further interference was carried out during the first week of differentiation. During the next two weeks of differentiation, fresh factory medium at 50% of the starting volume was added once a week. From the third week of differentiation until the production and release of (CD14+) monocyte precursors in the supernatant became detectable, half of the medium was exchanged. After this point, complete medium exchange with fresh factory medium was carried out twice a week.

[0072] Harvesting of monocytes Monocytes were harvested from the supernatant by centrifugation (4 minutes, 300 g), the cells were resuspended, counted, and quality control of marker expression by flow cytometry (CD68, Ki67, CD11b, and CD14) was performed once a week. Monocyte precursors were transferred to differentiation medium and differentiated into macrophages or differentiated into microglia in co-culture with neurons.

[0073] Differentiation of macrophages According to the application requirements, macrophages were either directly differentiated in the required plate format or pre-differentiated in upcell (trademark) plates for 6 days and then re-seeded into the final plate format one day before the start of the assay according to the manufacturer's protocol. For differentiation, the cells were cultured in either XVIVO 15 (supplemented with 2 mM Glutamax, 1% Penstrep, and 10 - 200 ng / ml M-CSF) or RPMI1640 (supplemented with 1% Penstrep and 10 - 200 ng / ml M-CSF or 1 - 10% fetal bovine serum). The medium was exchanged 3 days after seeding and the cells were differentiated for 7 days.

[0074] Polarization of macrophages For polarization of macrophages into pro-inflammatory (M1) or regulatory (M2) phenotypes, cells were cultured in XIVIVO15 medium supplemented with 2 mM Glutamax, 1% Penstrep, 5 - 100 ng / ml GM-CSF, and 1 - 100 ng / ml INFγ (M1), or 2 mM Glutamax, 1% Penstrep, 5 - 100 ng / ml M-CSF, and 1 - 100 ng / ml IL-4 (M2) for the desired polarization period.

[0075] Generation of microglia-like cells in neuron co-cultures For differentiation of monocytes into microglia-like cells, monocytes were seeded onto pre-differentiated neurons and co-cultured for 2 weeks prior to analysis.

[0076] Neuron generation Neurons were differentiated as described in WO2017081250, and a large stock solution was frozen on day 21. Two weeks prior to the start of co-culture, the neurons were thawed and seeded at a density of 50 - 200,000 cells per cm 2 onto cell culture vessels pre-coated with 5 μg / ml recombinant human laminin-521 (BioLamina). The medium was changed every 3 days (without ROCK inhibitor for further neuron maturation).

[0077] Co-culture Freshly harvested monocyte precursors were seeded onto mature neurons in N2 medium (consisting of Advanced DMEM F-12, N2 supplement, Glutamax, 50 μM mercaptoethanol, 1% P / S, and 1 - 100 ng / ml GM-CSF, as well as 1 - 500 ng IL-34). Microglial cells were matured in the co-culture with medium changes twice a week for 14 days.

[0078] Recovery of monocytes and storage of intermediates in suspension cultures Freshly isolated monocyte precursors were recovered and cultured in XVIVO15 medium (Lonza) supplemented with 2 mM Glutamax, 1% penicillin / streptomycin, 50 μg / ml mercaptoethanol, M-CSF (20 - 200 ng / ml), and IL3 (1 - 50 ng / ml) in suspension culture, termed a "spinner", for several weeks. The cell number was adjusted to 500,000 - 2,000,000 cells / ml, the medium was changed twice a week, the cells were resuspended, counted, and quality control was performed (CD68, Ki67).

[0079] Example 1 Maintenance of modified stem cells promotes the differentiation of the hematopoietic factory and increases the yield of monocytes Induced pluripotent stem cells (iPSCs) were cultured under feeder-free conditions and differentiated into the hematopoietic factory as described above. Replacement of Matrigel with laminin-521 coating substrate in the stem cell maintenance culture reduced the differentiation time of the hematopoietic factory. Hematopoietic factories derived from iPSCs cultured with laminin-521 initiated the production of monocyte precursors on day 21 of differentiation, while no monocyte precursors were released into the supernatant by hematopoietic factories derived from iPSCs cultured with Matrigel until day 34 of differentiation (Figure 4). Consistent with the early release of macrophages, monocyte marker gene expression also increased early in the differentiation process, and the weekly collection yield was significantly higher in hematopoietic factories derived from iPSCs cultured with laminin-521 (Figures 5 - 9). This observation indicates that iPSC maintenance conditions are highly relevant for an efficient differentiation process.

[0080] Example 2 Monocyte precursors derived from iPSCs differentiate into macrophages with a marker pattern equivalent to that of cultured primary human macrophages. To compare iPSC-derived macrophages with primary macrophages, monocytes precursors derived from iPS cells and CD14-positive blood monocytes obtained from LONZA were differentiated into macrophages as described above. The expression of marker genes in the starting population (monocytes / Figure 10) and macrophages (Figure 11) was evaluated by flow cytometry for CD14, CD11b, CD68, and Ki67. Monocytes derived from iPS cells had higher levels of CD14 and lower CD11b expression but overall had equivalent marker expression patterns (Figure 10). Macrophages differentiated from both sources similarly exhibited similar marker patterns (Figure 11), indicating that iPSC-derived monocyte precursors and macrophages are a valid alternative source for in vitro models of bone marrow biology.

[0081] Example 3 Enhancing the culture conditions of the hematopoietic factories improves the adhesion of EBs and the stability of the hematopoietic factories. EBs derived from three iPSC lines from different donors were generated as described above and seeded into either culture vessels pre-coated with growth factor-reduced Matrigel or untreated culture vessels, and the adhesiveness and culture stability were visually monitored over the differentiation period (Figures 12 and 13). EBs from all donors adhered better to plates coated with growth factor-reduced Matrigel, and cell outgrowths from more EBs were observed on the coated plates. This change in the protocol increased the stability of the cultures, thereby increasing the long-term culture success rate.

[0082] Example 4 The new culture protocol enables the generation of functional macrophages from different iPS cell lines. Monocyte precursors and macrophages were derived from three different iPS cell lines as described above. To evaluate the functionality of the macrophages, the phagocytic ability of these macrophages was tested by incubating them with pHrodo green-labeled zymosan for 120 minutes and subsequently detecting the green cells by flow cytometry analysis (Figure 14). After 120 minutes of incubation, approximately 60% of the cells had taken up zymosan particles as measured by green fluorescence. The differences observed among the three different iPSC donors were minimal (Figure 14), highlighting the robustness of the differentiation protocol.

[0083] Example 5 Differentiation of microglia in co-cultures with neurons As described above, the monocyte precursors derived from the iPS cell line can be co-cultured with neurons derived from human iPSCs to differentiate them into microglia-like cells (Haenseler et al. 2017a) (overview, Figure 15). In this specification, the inventors shortened the published protocol and re-thawed the monocyte precursors at the third week of differentiation when seeding them on neurons (International Publication No. WO2017081250). The use of this frozen neuron stock solution enables high flexibility and throughput in experimental co-culture designs. By using iPS cells with stable GFP expression, GFP-positive monocyte precursors and microglia-like cells can be generated, facilitating live cell imaging and the detection of microglia in co-cultures (Figures 15 and 16). Interestingly, upon LPS stimulation, microglia in co-cultures showed different cytokine release patterns compared to macrophages in monoculture and neuron monocultures (Figure 17), indicating their potential use as a neuroinflammation model. In contrast to the changes in cytokine release, phagocytosis measurements using pHrodo Red zymosan in combination with GFP-positive macrophages and microglia in a high-content imaging setting showed similar uptake of zymosan particles by macrophages and microglia (Figure 18). By utilizing high-content imaging and miniaturizing the assay to 384 wells, this setting can be used to screen for modulators of phagocytosis in macrophages and microglia (Figure 19), as demonstrated herein by the dose-dependent inhibition of phagocytosis by cytochalasin D and the dose-dependent stimulation by serum incubation.

[0084] Example 6 Recovery of Monocyte Precursors in Suspension Cultures for Large-Scale Screening Campaigns Monocyte precursors were harvested from the hematopoietic factory and recovered in suspension culture for several weeks (Figure 20A). The viability of monocyte precursors, as well as marker expression, remained constant in suspension culture for at least 6 weeks (Figures 20B and C). When monocytes were removed from the suspension culture at different time points and differentiated into macrophages, the marker expression of the resulting macrophages showed no difference among cells derived from the suspension culture compared to cells derived directly after harvest (Figure 20D). The ability to generate a large homogeneous population of monocyte precursors is highly suitable for screening applications, and thus, cells stored in such suspension cultures should generate macrophages with functional characteristics equivalent to directly differentiated macrophages. To evaluate the functionality of macrophages, the phagocytic ability of macrophages derived from suspension cultures ("spinner") and macrophages derived from fresh harvests ("harvest") was tested by incubating them with pHrodo Red-labeled zymosan for 120 minutes and subsequently detecting phagocytic cells by high-content-based analysis (Figure 21A). No difference was observed between the two conditions (Figure 21A). A second functional characteristic of macrophages is the ability to migrate towards chemotactic substances, and the inventors evaluated this for two macrophage populations by using the IncuCyte transwell assay (Essen Bioscience) and the chemotactic substance C5a. Also in this setting, cells derived from suspension cultures ("spinner") showed no significant difference in their migratory behavior compared to cells differentiated from freshly harvested monocyte precursors ("harvest") (Figure 21B). Equivalent functional characteristics and marker expression confirmed the phenotype and utility of cells derived from suspension cultures for large-scale functional and phenotypic assays.

[0085] The foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, but the description and examples are not to be construed as limiting the scope of the invention. The disclosures of all patents and scientific literature cited herein are hereby expressly incorporated by reference in their entirety.

Claims

1. A method for producing monocyte progenitor cells, comprising: a) seeding pluripotent stem cells in a pluripotent medium onto a cell culture support coated with laminin; b) harvesting the pluripotent stem cells and contacting the pluripotent stem cells with a mesoderm induction medium in a suspension culture, wherein the cells form embryoid bodies (EBs); c) seeding the EBs onto a cell culture support suitable for EB attachment, which is coated with a basement membrane biomaterial; d) harvesting monocyte progenitor cells from the cell culture supernatant; and

2. The method according to claim 1, wherein the laminin in step a) comprises laminin subunit alpha-5.

3. The method according to claim 1, wherein the laminin in step a) comprises laminin subunits alpha-5, beta-2, and gamma-1.

4. The method according to any one of claims 1 to 3, wherein in step b), the cells are contacted with a defined medium containing BMP4.

5. The method according to any one of claims 1 to 4, wherein in step b), the cells are contacted with a defined medium containing VEGF.

6. The method according to any one of claims 1 to 5, wherein in step b), the cells are contacted with a defined medium containing SCF.

7. The method according to any one of claims 1 to 6, wherein in step c), the EBs are contacted with a myeloid maturation medium.

8. The method according to any one of claims 1 to 7, wherein the myeloid maturation medium contains M-CSF.

9. The method according to any one of claims 1 to 8, wherein the myeloid maturation medium contains IL-3.

10. e) differentiating the harvested monocyte progenitor cells into macrophages; The method according to any one of claims 1 to 9, further comprising.

11. The method according to claim 10, wherein the cells in step e) are seeded onto an uncoated tissue culture support.

12. e) differentiating the harvested monocyte progenitor cells into microglia; The method according to any one of claims 1 to 9, further comprising.

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