Selective induction method for bone marrow-derived neuroprotective microglia-like cells and their application

Abstract

To provide a method for obtaining easily and simply a sufficient amount of microglia in order to achieve a therapy using neuroprotective microglia for neurodegenerative disease and the like, and a pharmaceutical composition containing a microglia-like cell.SOLUTION: Neuroprotective microglia is produced by the following steps 1 and 2. In the step 1, a bone marrow monocytic cell is exposed to GM-CSF. In the step 2, the cell exposed in the step 1 is then exposed to GM-CSF and IL4.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for selectively inducing bone marrow-derived neuroprotective microglia-like cells and use thereof.

Background Art

[0002] Microglia are known as cells involved in immune responses in the nervous system. Microglia include M1 microglia and M2 microglia, where the former is known to induce inflammation and destroy tissues and cells, and the latter is known to suppress inflammation and protect tissues and cells. It is also known that M1 microglia can be converted into M2 microglia by allowing IL-10 or IL-4 to act thereon.

[0003] It is considered effective to treat neurodegenerative diseases and the like using neuroprotective microglia-like cells such as M2 microglia. It is also known that microglia can be obtained from the cerebral cortex.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] In order to realize therapeutic methods that utilize neuroprotective microglia for neurodegenerative diseases, etc., it is necessary to be able to easily and simply prepare a sufficient amount of microglia. However, assuming autologous transplantation, it is difficult to obtain a predetermined amount of microglia from the cerebral cortex as a raw material. [Means for solving the problem]

[0006] As a result of diligent research to solve the above problems, the inventors have discovered that neuroprotective, autotransplantable microglia can be easily induced by applying a predetermined concentration of growth factors to mononuclear cells contained in bone marrow and subjecting them to a specific culture method, thus completing the present invention. That is, the present invention broadly encompasses the inventions shown below.

[0007] Item 1: A method for producing M2 microglia-like cells, comprising the following steps 1 and 2; Step 1: A step in which GM-CSF is applied to bone marrow mononuclear cells. Step 2: A step in which GM-CSF and IL4 are applied to the cells that were treated in Step 1.

[0008] Item 2 The manufacturing method according to Item 1, wherein the duration of action in step 1 is 48 to 120 hours.

[0009] Item 3 The manufacturing method according to Item 1 or 2, wherein the action period in step 2 is 48 to 120 hours.

[0010] The manufacturing method according to any one of items 1 to 3, wherein the concentrations of GM-CSF and IL4 reacted in steps 1 and 2 are greater than 20 ng / ml and 40 ng / ml or less.

[0011] Item 5 M2 microglia-like cells derived from bone marrow mononuclear cells, manufactured by the manufacturing method described in any one of items 1 to 4 above.

[0012] Item 6 A pharmaceutical composition comprising M2 microglia-like cells as described in Item 5 above, for use in the treatment and / or prevention of central nervous system disorders.

[0013] Item 7 The pharmaceutical composition according to Item 6, wherein the central nervous system disorder is Alzheimer's disease, Parkinson's disease, cerebral infarction, hypoxic encephalopathy, spinal cord injury, neuropathic pain, or amyotrophic lateral sclerosis (ALS). [Effects of the Invention]

[0014] According to the manufacturing method of the present invention, a sufficient quantity of M2 microglia-like cells for transplantation can be easily induced. Furthermore, M2 microglia-like cells produced by the manufacturing method of the present invention can be effectively used for the treatment and / or prevention of neurodegenerative diseases such as ALS. [Brief explanation of the drawing]

[0015] [Figure 1] Figure 1A shows the culture procedure in Example 1, and transmitted light images (phase contrast) of bone marrow mononuclear cells before treatment (Day 0) and differentiated cells 7 days after treatment (Day 7). Figure 1B shows the relative number (average) of differentiated cells 7 days after treatment to bone marrow mononuclear cells at the start of culture for each Method A, B, and C. ** indicates p<0.01. # indicates p<0.05. Figure 1C shows the expression levels of Arg1 and Nos2 mRNA for GAPDH in differentiated cells 7 days after treatment for each Method A, B, and C. * indicates p<0.05. [Figure 2] Figure 2A shows the flow cytometry results for CD11b in untreated bone marrow mononuclear cells (Day 0) and differentiated cells on day 7 (Day 7) using Method C. Figure 2B shows the flow cytometry results for CD206 in untreated bone marrow mononuclear cells (Day 0) and differentiated cells on day 7 (Day 7) using Method C. Figure 2C shows the flow cytometry results for CD86 in untreated bone marrow mononuclear cells (Day 0) and differentiated cells on day 7 (Day 7) using Method C. [Figure 3]FIG. 3A is a schematic diagram showing the outline of the experiment of Example 2. FIG. 3B shows the results of Example 2. The vertical axis of the graph represents the relative value to the number of viable cells obtained in the case of CTL medium-. * indicates p<0.05. n.s represents that there was no significant difference. [Figure 4] FIG. 4A shows the results of the behavioral evaluation experiment in Example 3. + is the result of a significance test including the temporal changes between the two groups (CTL and BM-iMG), and indicates p<0.05. FIG. 4B shows the relative average value of the mouse body weight in Example 3 with respect to that at the start of transplantation. FIG. 4C shows the survival curve (Kaplan-Meier) in Example 3. + is the result of a significance test between the two groups (CTL and BM-iMG), and indicates p<0.05. [Figure 5] FIG. 5 shows the experimental results confirming the tissue staining experiment performed in Example 4 on the spinal cord of the transplanted mouse produced in Example 3. FIG. 5A is a tissue immunostaining image of β-tubulin. FIG. 5B is a graph obtained by quantifying the results shown in FIG. 5A. FIG. 5C is a tissue immunostaining image of GFAP. FIG. 5D is a graph obtained by quantifying the results shown in FIG. 5C. * indicates p<0.05. # indicates p<0.05. [Figure 6] FIG. 6 shows the experimental results of Example 4. Specifically, it shows the confirmation results of the tissue staining experiment on the spinal cord of the transplanted mouse produced in Example 3. FIG. 6A is a tissue immunostaining image of Iba1. FIG. 6B is a graph obtained by quantifying the results shown in FIG. 6A. * indicates p<0.05. # indicates p<0.05. [Figure 7] FIG. 7 shows the experimental results for confirming the expression level in the spinal cord of the transplanted mouse produced in Example 3, which was tested in Example 5. * indicates p<0.05. [Mode for Carrying Out the Invention]

[0016] Hereinafter, the present invention will be described. In the following, the notation "~" indicating a numerical range means "from or equal to A to or equal to B" unless otherwise specified to mean "less than" or "exceeding". That is, "A to B" means "A or more and B or less".

[0017] In the present specification, the expression "comprising" or "containing" a certain component includes not only the meaning that the component is included and other components may be further contained, but also the concepts of "consisting only of" which means consisting solely of the relevant component, and "consisting essentially of" which means comprising the relevant component as an essential element.

[0018] In the present specification, "neuroprotective microglia-like cells" may be referred to as "M2 microglial cells".

[0019] The method for producing M2 microglia-like cells of the present invention comprises the following steps 1 and 2. Step 1: a step of allowing GM-CSF (granulocyte-macrophage colony-stimulating factor) to act on bone marrow mononuclear cells Step 2: a step of allowing GM-CSF and IL4 to act on the cells after the treatment in Step 1.

[0020] The aforementioned M2 microglia-like cells are not particularly limited, and examples thereof include cells expressing markers such as CD206, Arg1, IRF4, and FIZZ.

[0021] The bone marrow mononuclear cells in the above Step 1 are not particularly limited as long as they are mononuclear lineage cells derived from hematopoietic stem cells contained in bone marrow. For example, the bone marrow mononuclear cells can be an aggregate of hematopoietic cells comprising an undifferentiated cell population such as lymphocytes, monocyte-lineage leukocytes, their progenitor cells, and mesenchymal stem cells, which is obtained by removing erythrocytes, platelets and granulocyte-lineage cells from bone marrow by a concentration gradient method or the like. More specifically, examples include immunocompetent cells such as T cells / NK cells, B cells, monocytes, dendritic cells and macrophages, their progenitor cells, and mesenchymal stem cells having the ability to differentiate into vascular endothelium, bone, cartilage and the like.

[0022] The "step of allowing to act" in the above Step 1 is not particularly limited as long as the effects of the present invention are exhibited. For example, an embodiment can be mentioned in which the substance to be acted is contained in a culture medium to be used for culturing, and culturing is carried out for a predetermined period.

[0023] The origin of the bone marrow mononuclear cells in step 1 described above is not particularly limited, as long as the effects of the present invention are achieved. Specifically, examples include rodents such as rats and mice, pigs, monkeys, and humans. Among these, it is preferable to use animals that are suitable for transplantation into the target of treatment (e.g., humans).

[0024] The duration of action in step 1 described above is not particularly limited, as long as the effects of the present invention are achieved. For example, it can be 48 to 120 hours. Preferably, it is 60 to 108 hours, and more preferably 60 to 96 hours. By setting the duration of action in step 1 to 48 hours or more, the number of cells necessary for transplantation can be secured. By setting the duration of action in step 1 to 120 hours or less, the proliferation of fractions that do not exhibit neuroprotective effects can be avoided.

[0025] The duration of action in step 2 described above is not particularly limited, as long as the effects of the present invention are achieved. For example, it can be 48 to 120 hours. Preferably, it is 60 to 108 hours, and more preferably 72 to 96 hours. By setting the duration of action in step 2 to 48 hours or more, neuroprotective microglia can be efficiently induced, and by setting the duration of action in step 2 to 120 hours or less, stable neuroprotective microglia suitable for transplantation can be obtained.

[0026] The concentrations of GM-CSF and IL4 applied in steps 1 and 2 above are not particularly limited, as long as the effects of the present invention are achieved. For example, the concentration can be greater than 20 ng / ml and 40 ng / ml or less. Preferably, the concentration can be greater than (or greater than) 30 ng / ml and 40 ng / ml or less. By setting the concentrations of GM-CSF and IL4 to greater than 20 ng / ml, more than half of the cultured cells can be differentiated into neuroprotective microglia, and by setting the concentrations of GM-CSF and IL4 to 40 ng / ml or less, neuroprotective microglia can be obtained even more efficiently. Note that the concentrations of GM-CSF and IL4 may be the same or different, and the concentration of GM-CSF may be the same or different in steps 1 and 2.

[0027] In steps 1 and 2 described above, GM-CSF or IL4 can be applied in the culture medium. The specific culture medium is not particularly limited as long as it exhibits the effects of the present invention. Examples include basal culture media such as DMEM, MEM, RPMI1640, IMDM, and StemSpan®. Among these, StemSpan is preferred in view of efficiently differentiating undifferentiated cells. StemSpan contains insulin, transferrin, 2-mercaptoethanol, etc.

[0028] The above culture medium may contain known serums used for cell culture, such as FBS or FCS, or it may contain antibiotics such as penicillin / streptomycin, kanamycin, hygromycin, gentamicin, or G418.

[0029] Furthermore, the above serum can be inactivated serum, or serum substitutes such as albumin, transferrin, fatty acids, 2-mercaptoethanol, 3'-thiolglycerol, and StemSure® can be used instead of serum.

[0030] Furthermore, the culture medium may also contain known components commonly used in cell culture, to the extent that the effects of the present invention can be achieved. Examples include amino acids such as L-glutamine, vitamins such as ascorbic acid, biotin, glucose, B27 supplements, N2 supplements, growth factors, cytokines, inorganic salts, and other nutritional additives.

[0031] The actions in steps 1 and 2 described above can be carried out in a manner in which the culture medium is cultured in a predetermined container. Such a container is not particularly limited as long as it can exert the effects of the present invention. Examples include petri dishes, flasks, culture plates, bottles, bags, etc.

[0032] The operating temperature in steps 1 and 2 described above is not particularly limited, as long as the effects of the present invention are achieved. For example, it can be 35 to 39°C, and more preferably 36 to 38°C. The operating environment in steps 1 and 2 described above is also not particularly limited, as long as the effects of the present invention are achieved. For example, it can be in the atmosphere or in a CO2 environment. Preferably, it is in a 5% CO2 environment. The oxygen concentration is not particularly limited, and for example, it can be about 20%.

[0033] The culture medium described above can be replaced with a fresh medium as needed during the culturing process. The interval between such replacements is not particularly limited, as long as the effects of the present invention are achieved. For example, it can be every 1 day, 1.5 days, 2 days, 2.5 days, 3 days, 3.5 days, or 4 days. The culture medium may be replaced entirely or only partially.

[0034] The M2 microglia-like cells obtained by the manufacturing method of the present invention can be used in a pharmaceutical composition for the treatment and / or prevention of central nervous system diseases. That is, the pharmaceutical composition contains the M2 microglia-like cells obtained by the manufacturing method of the present invention. The M2 microglia-like cells contained in the pharmaceutical composition may also be contained together with the culture medium and / or its culture supernatant used in its production. That is, the pharmaceutical composition of the present invention may also contain the culture medium and / or its culture supernatant of the M2 microglia-like cells obtained by the manufacturing method of the present invention as an active ingredient.

[0035] The above-mentioned central nervous system diseases are not particularly limited. Examples include cerebral infarction, hypoxic encephalopathy, spinal cord injury, neuropathic pain, encephalitis, myelitis, and neurodegenerative diseases. The aforementioned neurodegenerative diseases are not particularly limited. Examples include Alzheimer's disease, Parkinson's disease, multiple sclerosis, multiple system atrophy, spinocerebellar degeneration, and amyotrophic sclerosis.

[0036] The amount of M2 microglia-like cells contained in the above-mentioned pharmaceutical composition is not particularly limited. For example, it can be 0.001 to 100 parts by mass per 100 parts by mass of the pharmaceutical composition. In addition to the M2 microglia-like cells, such a pharmaceutical composition may contain buffers, antioxidants, preservatives, surfactants, hydrophilic polymers, chelating agents, etc. Furthermore, it may also contain extracellular matrix or extracellular substrate as a scaffold for the M2 microglia-like cells.

[0037] The administration route of the above-mentioned pharmaceutical composition is not particularly limited, but is limited to the extent that the effects of the present invention are exerted. Examples include direct intratissue administration, intrathecal administration, intravenous administration, intraperitoneal administration, subcutaneous administration, etc.

[0038] The target population for the above-mentioned pharmaceutical composition is not particularly limited. For example, mammals suffering from or potentially suffering from the above-mentioned central nervous system disease can be included. Such mammals are not particularly limited. For example, mice, rats, pigs, cattle, goats, horses, sheep, monkeys, humans, etc. Among these, humans are preferred.

[0039] The dosage of the above-mentioned pharmaceutical composition cannot be determined in general, as it varies depending on the age of the recipient and the desired degree of effect. For example, if the recipient is a human male weighing 60 kg, the dosage per dose would be 10 M2 microglia-like cells contained in the pharmaceutical composition. 4 ~10 10 A pharmaceutical composition can be administered in an amount sufficient to form cells.

[0040] The following are examples to further illustrate the present invention. However, it goes without saying that the present invention is not limited to the scope shown in the examples below. [Examples]

[0041] Statistical analyses in the following examples are expressed as mean ± SD. One-way ANOVA and Scheffe tests were used for comparisons between multiple data sets. Two-way ANOVA was used for behavioral analysis. Log-rank tests were used for the statistical analysis of Kaplan-Meier curves. P-values ​​less than 0.05 are considered statistically significant. These statistical analyses were performed using IBM SPSS Statistics version 25 (International Business Machines Corporation). [Examples]

[0042] Whole bone marrow cells were collected from C57BL / 6 mice (Jackson Laboratories). Subsequently, mononuclear cells were isolated from the whole bone marrow cells using Ficoll-Paque Plusgradient separation (GE Healthcare Biosciences). These isolated mononuclear cells were then cultured for 7 days with GM-CSF or IL-4 in StemSpan® (Stem Cell Technologies), a serum-free growth medium, using the three culture methods described below.

[0043] Method A: Culturing for 7 days in a medium containing GM-CSF at a final concentration of 40 ng / ml. Method B: Culturing for 7 days in a medium containing GM-CSF at a final concentration of 40 ng / ml and IL-4 at a final concentration of 40 ng / ml. Method C: Culture for 3 days in a medium containing GM-CSF at a final concentration of 40 ng / ml, followed by 4 days of culture in a medium containing GM-CSF at a final concentration of 40 ng / ml and IL-4 at a final concentration of 40 ng / ml.

[0044] In all of these methods, the culture medium was replaced with fresh medium on the third day after the start of culture. Then, the cultured cells were observed under a light microscope on the seventh day after the start of culture. These cells were then treated with trypsin-EDTA (Life Technology Inc.) for 10 minutes and the cell count was measured. The results are shown in Figures 1A and 1B.

[0045] As shown in Figure 1B, methods A and C showed a significant increase in cell count after 7 days, whereas the increase in cell count after 7 days in method B was less than that of methods A and C. From these results, it can be inferred that stimulating with IL-4 from the beginning may not yield a sufficient number of cells.

[0046] Furthermore, the expression levels of Arg1 and Nos2 mRNA expressed in cells 7 days after the start of culture were confirmed by quantitative RT-PCR. Specifically, total RNA was extracted from these cells using the RNeasy kit (QIAGEN). Reverse transcription was performed on this total RNA using the TAKARA PrimeScript RT reagent kit (Takara Bio), followed by quantitative RT-PCR using LightCycler 480 SYBR Green I Master (Roche Diagnostics) and Light-Cycler 480 (Roche Diagnostics). The base sequences of the primers used in quantitative RT-PCR are shown in Table 1 below. GAPDH was used as an internal standard. The results are shown in Figure 1C.

[0047] [Table 1]

[0048] As shown in Figure 1C, in Method A, neither Arg1, an M2 microglia marker, nor Nos2, an M1 microglia marker, was expressed. On the other hand, in Methods B and C, both Arg1 and Nos were expressed. In particular, Arg1 expression was higher in Method C than in Method B, and Nos2 expression was higher in Method B than in Method C. From these results, it can be inferred that M2 microglia were most effectively induced in Method C, and M1 microglia were most effectively induced in Method B.

[0049] Furthermore, the expression levels of CD11b, CD206, and CD86 in cells 7 days after the start of culture were confirmed by flow cytometry. Specifically, measurements were performed using FACS Calibur and FACS Aria flow cytometers (BD Biosciences), and FITC-labeled anti-CD11b antibody (BD Biosciences), PE-labeled anti-CD86 antibody (BD Biosciences), and Alexaflora 647-labeled anti-CD206 antibody (Biolegend). The results are shown in Figure 2.

[0050] As shown in Figure 2, increased expression of CD11b, CD206, and CD86 was observed in cells from Method C 7 days after the start of culture. The CD11b positivity indicated that over 80% of the cells were induced as microglia, with more than half of these being positive for CD206, an M2 microglia marker. On the other hand, less than 20% of the microglia were positive for CD86. [Examples]

[0051] An experiment was conducted to confirm the degree of recovery from nerve cell damage. First, bone marrow-derived mononuclear cells were cultured using method C described above, and the supernatant was collected as the conditioned medium. In addition, Stemspan medium (without cells) containing 40 ng / ml GM-CSF and 40 ng / ml IL-4 was incubated at 37°C in a CO2 environment for 4 days, and this was used as the CTL medium.

[0052] NSC-34 cells (CELLutions BIOSYSTEMS) were placed in DMEM high-glucose medium containing 10% FBS (Fujifilm Wako Pure Chemical Industries) in 3 × 10⁶ cells. 4 Cells were seeded in a 96-well plate at a concentration of cells / well and cultured for 3 hours. The supernatant was then replaced with DMEM high-glucose medium mixed to contain 40% Conditioned Medium or CTL Medium. Subsequently, 400 μM NOC-18 (1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene) was added to induce neuronal cell death, and the cells were incubated at 37°C for 48 hours under a CO2 environment. Cell counts were then measured using Cell Counting Kit-8 (Dojin Chemical Laboratories). The results are shown in Figure 3B.

[0053] As shown in Figure 3B, the results demonstrate that the culture medium for M2 microglia-like cells, using Method C, exhibits a reversal effect against the induction of neuronal cell death. This suggests that components secreted into the culture medium by the induced M2 microglia-like cells have a neuroprotective effect. [Examples]

[0054] We conducted an experiment in which M2 microglia-like cells induced from bone marrow mononuclear cells using method C described above were transplanted into ALS model mice.

[0055] Specifically, as bone marrow mononuclear cells, GFP-tg mouse (C57BL / 6-Tg(UBC-GFP) 30Scha / J From Jackson Laboratories, bone marrow mononuclear cells were collected in the same manner as shown in Example 1, and then the mononuclear cells were isolated. These were then used to induce M2 microglia-like cells using the method described in C above, and these 1.0 × 10 5 A PBS suspension of cells / μl was prepared for transplantation.

[0056] Next, 10-week-old male ALS model mice (hSOD1(G93A)1Gur / J: Jackson Laboratories) were anesthetized by intraperitoneal injection of 0.3 mg / kg medetomidine, 4 mg / kg midazolam, and 5 mg / kg butorphanol. Subsequently, 1 μl of the M2 microglia-like cell suspension prepared above was slowly injected into one side of the spinal cord between the 12th and 13th thoracic vertebrae of the anesthetized mice, and then the same amount of suspension was similarly injected into the other side of the spinal cord. As a control, ALS model mice were prepared in which PBS was injected instead of M2 microglia-like cells. These mice were given free access to water and specialized feed and were housed in a 12-hour light-dark cycle.

[0057] The motor skills of transplanted ALS model mice were analyzed weekly using the Rota-Rod test (Ugo Basile, Comerio-Varese), and body weight was measured at the same frequency. The Rota-Rod test described above was performed at 5-50 rpm (with an acceleration of 9 rpm). 2The experiment was conducted for 5 minutes within the specified range, and the mean of the three medians of the five trials for each mouse was calculated.

[0058] Furthermore, physiological death was defined as a Rota-Rod test result of "0 seconds" or a weight loss of more than 15% from the maximum weight in each mouse, and this was analyzed using the Kaplan-Meier method. These results are shown in Figure 4. The mouse experiment protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Shiga University of Medical Science, and was conducted in accordance with its guidelines.

[0059] As shown in Figure 4A, the ALS model mice transplanted with M2 microglia-like cells induced from bone marrow mononuclear cells using Method C (BM-iMG: black line) showed significantly suppressed decline in motor function compared to the control (CTL: gray line). As shown in Figure 4B, the BM-iMG mice (black line) also experienced a slower weight loss than the control mice (gray line), and as shown in Figure 4C, the survival curve for BM-iMG mice was favorable. [Examples]

[0060] Histological staining experiments of the spinal cord were performed according to standard procedures three weeks after transplantation or in the terminal stage, specifically for β3-tubulin, GFAP, and Iba-1. The antibodies used are listed below. • Rabbit anti-β3 tubulin antibody (Cell Signaling Technologies) • Rabbit anti-GFAP antibody (Cell Signaling Technologies) • Rabbit anti-Iba-1 antibody (Fujifilm Wako Pure Chemical Industries, Ltd.)

[0061] Figure 6A shows the results of staining the spinal cords of ALS model mice (BM-iMG cells) transplanted with M2 microglia-like cells produced by Method C, along with DAPI staining and β3 tubulin, as well as the spinal cords of a control (Buffer CTL). The number of neurons calculated from these results is shown in Figure 5B. Figure 5C shows the results of staining the spinal cords of Buffer CTL and BM-iMG cells with GFAP along with DAPI staining. The GFAP positivity intensity calculated from these results is shown in Figure 5D.

[0062] Figure 6A shows the results of staining the spinal cord of buffer CTLs and BM-iMG cells with Iba-1 along with DAPI staining. Figure 6B shows the number of Iba-1-positive cells calculated from these results.

[0063] These results revealed that BM-iMG cells can suppress the decrease in the number of neurons in the terminal stage of disease. Furthermore, BM-iMG cells showed suppressed GFAP positivity in the terminal stage. Additionally, BM-iMG cells showed a decrease in the number of Iba-1 positive cells in the terminal stage. Based on these results, it is hypothesized that the aforementioned cell transplantation exhibits neuroprotective effects, suppression of astrogliosis, and inhibition of microglia accumulation within the spinal cord. [Examples]

[0064] Next, experiments were conducted to measure the expression levels of Nos2, Arg1, CD86, Mrc1, TNFα, and IL-6 in the spinal cord three weeks or in the terminal stage after transplantation. Specifically, total RNA was collected from the spinal cord three weeks or in the terminal stage after transplantation in the same manner as in Example 2, and the expression levels were confirmed by quantitative RT-PCR. The base sequences of the primers used in quantitative RT-PCR are shown in Table 2 below. The primer sequences for Nos2 and Arg1 are the same as those in Table 1 above. β-actin was used as an internal standard. The results are shown in Figure 7.

[0065] [Table 2]

[0066] As shown in Figure 7, in mice transplanted with M2 microglia-like cells induced from bone marrow mononuclear cells using Method C (BM-iMG), the expression level of Nos2 in the terminal stage was lower than in the control (CTL), and the expression level of Arg1 was higher than in the control. Furthermore, the expression level of Mrc1 was also higher than in the control. In addition, the expression level of IL-6 was lower in BM-iMG compared to the control. The expression levels of CD86 and TNFα remained unchanged. These results suggest that in BM-iMG, the inflammatory markers Nos2 and IL-6 were reduced, while the expression levels of the cytoprotective marker Arg1 and the CD206 gene Mrc1 were increased. Therefore, it is suggested that the transplantation suppressed inflammation in the spinal cord and had a cytoprotective effect.

Claims

1. A method for producing M2 microglia-like cells, comprising the following steps 1 and 2; Step 1: A step in which bone marrow mononuclear cells are treated with GM-CSF in a concentration greater than 20 ng / ml and less than or equal to 40 ng / ml. and Step 2: A step in which the cells treated in Step 1 are treated with GM-CSF in a concentration greater than 20 ng / ml and 40 ng / ml or less, and IL4 in a concentration greater than 20 ng / ml and 40 ng / ml or less.

2. The manufacturing method according to claim 1, wherein the duration of action in step 1 is 48 to 120 hours.

3. The manufacturing method according to claim 1 or 2, wherein the action period in step 2 is 48 to 120 hours.

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