Method for differentiating induced pluripotent stem cells into macrophages

Abstract

The invention discloses a method for differentiating induced pluripotent stem cells into macrophages. The method comprises the following steps: S1, inducing pluripotent stem cells to form embryoid bodies; s2, differentiating the embryoid body into a monocyte with CD14 expression; s3, the mononuclear cells are differentiated into macrophages with CD14 / CD11b expression; and S4, polarizing the macrophages into M1 type macrophages and / or M2 type macrophages. According to the method for differentiating the induced pluripotent stem cells into the macrophages, the cells can be effectively induced to differentiate towards the macrophages, the induced pluripotent stem cells are subjected to directional differentiation culture in a short time to obtain the macrophages, and the generated macrophages express CD14 and CD11b and have remarkable phagocytic activity on yeast glycan. The method is of great significance to large-scale production of macrophages.

Description

Technical Field

[0001] The invention relates to a method for differentiating induced pluripotent stem cells into macrophages, belonging to the technical field of cell differentiation. Background Art

[0002] Macrophages (M) are a type of white blood cells located in peripheral blood and inflammatory tissues. They are mainly found in organs such as lymph nodes, alveolar walls, bone marrow, liver and spleen, and are key components of the innate immune system. Their main function is to phagocytose cell debris and pathogens in the form of fixed cells or free cells, and activate lymphocytes or other immune cells to respond to pathogens. In addition to initiating immune and inflammatory responses to pathogens, macrophages also maintain tissue homeostasis and repair and remodeling of tissues.

[0003] There are two main sources of human macrophages: tumor-derived cell lines such as U937 and THP-1 cells, or primary cells such as peripheral blood mononuclear cells (PBMC) and human-monocyte derived macrophages (HMDM). Although the former has the ability to proliferate indefinitely, it has abnormal karyotype and immature phenotype, and cannot be used to study specific genotypes. The latter is a widely used macrophage function experimental model. Although it is relatively easy to obtain, HMDM is terminally differentiated, cannot self-renew, lacks proliferation ability, and is difficult to edit its genes.

[0004] Induced pluripotent stem cells (iPSCs) have the ability to proliferate indefinitely and can differentiate into almost all functional cells in vitro. Differentiating induced pluripotent stem cells into macrophages is a feasible solution. Summary of the invention

[0005] In view of the above-mentioned prior art, the present invention provides a method for differentiating induced pluripotent stem cells into macrophages. The present invention uses IPSC to induce the generation of macrophages, which can solve the problem of obtaining macrophages, and can use the previously constructed genotype-specific iPSC to derive macrophages with normal karyotype and stable genotype. The method of the present invention provides a good tool for studying the specific functions of macrophages, immune response mechanisms, and the treatment of macrophage-related diseases or tumors.

[0006] The present invention is achieved through the following technical solutions:

[0007] A method for differentiating induced pluripotent stem cells into macrophages, comprising the following steps:

[0008] S1: Induced pluripotent stem cells to form embryoid bodies (EBs);

[0009] S2: embryoid bodies differentiated into monocytes expressing CD14;

[0010] S3: Monocytes differentiate into macrophages with CD14 / CD11b expression;

[0011] S4: Macrophage polarization into M1 macrophages and / or M2 macrophages.

[0012] Furthermore, the step S1 specifically comprises: culturing induced pluripotent stem cells in the presence of ROCK inhibitor Y-27632 to form embryoid bodies.

[0013] Furthermore, the concentration of Y-27632 is 10 μM.

[0014] Furthermore, the step S2 comprises the following steps:

[0015] S2-1: After embryoid bodies are formed, the supernatant is removed, the first differentiation medium is added, and the cells are induced for 1 to 2 days;

[0016] The first differentiation medium is an APEL medium supplemented with BMP4, VEGF, SCF, FGF2, activin A, and CHIR99021, wherein the concentration of BMP4 is 20 ng / mL, the concentration of VEGF is 40 ng / mL, the concentration of SCF is 40 ng / mL, the concentration of FGF2 is 5 ng / mL, the concentration of activin A is 5 ng / mL, and the concentration of CHIR99021 is 0.2 μM;

[0017] S2-2: remove the first differentiation medium, add the second differentiation medium, and induce culture for 1 to 2 days;

[0018] The second differentiation medium is an APEL medium supplemented with BMP4, VEGF, SCF, FGF2, and CHIR99021, wherein the concentration of BMP4 is 20 ng / mL, the concentration of VEGF is 40 ng / mL, the concentration of SCF is 40 ng / mL, the concentration of FGF2 is 5 ng / mL, and the concentration of CHIR99021 is 0.2 μM;

[0019] S2-3: remove the second differentiation medium, add the third differentiation medium, and induce culture for 4 to 8 days;

[0020] The third differentiation medium is an APEL medium supplemented with BMP4, VEGF, SCF, and FGF2, wherein the concentration of BMP4 is 20 ng / mL, the concentration of VEGF is 40 ng / mL, the concentration of SCF is 40 ng / mL, and the concentration of FGF2 is 5 ng / mL;

[0021] S2-4: remove the third differentiation medium, add the fourth differentiation medium, and induce culture for 7 to 14 days;

[0022] The fourth differentiation medium is a medium of X-VIV0 15 supplemented with VEGF, SCF, FGF2, IL-3, and M-CSF, wherein the concentration of VEGF is 20 ng / mL, the concentration of SCF is 20 ng / mL, the concentration of FGF2 is 10 ng / mL, the concentration of IL-3 is 25 ng / mL, and the concentration of M-CSF is 50 ng / mL.

[0023] Furthermore, the step S3 specifically comprises: collecting suspension cells containing CD14+ monocytes, adding macrophage differentiation medium, and inducing culture for 5 to 10 days;

[0024] The macrophage differentiation medium is X-VIV0 15 medium added with GlutaMax and M-CSF, wherein the concentration of GlutaMax is 2 mM and the concentration of M-CSF is 100 ng / mL.

[0025] Furthermore, the step S4 specifically includes: replacing the macrophage differentiation medium with an M1 macrophage polarization medium and / or an M2 macrophage polarization medium, and culturing for 24 to 48 hours.

[0026] Furthermore, the M1 macrophage polarization medium is an X-VIV0 15 medium supplemented with GlutaMax, LPS and IFN-γ, wherein the concentration of GlutaMax is 2 mM, the concentration of LPS is 100 ng / mL, and the concentration of IFN-γ is 20 ng / mL.

[0027] Furthermore, the M2 macrophage polarization medium is an X-VIV015 medium supplemented with GlutaMax and IL-4, wherein the concentration of GlutaMax is 2 mM and the concentration of IL-4 is 50 ng / mL.

[0028] Furthermore, the culture system used in steps S1 and S2 is an ultra-low adsorption six-well plate; and the culture system used in steps S3 and S4 is a culture bottle treated with tissue culture (TC).

[0029] Furthermore, the culture conditions are: 37°C, 5% CO2.

[0030] The macrophages cultured by the above method expressed CD14 and CD11b and had significant phagocytic activity to zymosan.

[0031] The method for differentiating induced pluripotent stem cells into macrophages of the present invention comprises adding specific key components necessary for maintaining cell growth, such as GlutaMax and cytokines, to the culture medium, and each component acts in a directional manner, and can effectively induce cells to differentiate into macrophages. The induced pluripotent stem cells can be cultured for directional differentiation to obtain macrophages in a relatively short period of time, and the generated macrophages express CD14 and CD11b, can be polarized into M1 macrophages and M2 macrophages, and have significant phagocytic activity to zymosan.

[0032] The method of differentiating induced pluripotent stem cells into macrophages of the present invention has the following advantages:

[0033] 1. The amount of raw materials is not limited, and induced pluripotent stem cells can be expanded indefinitely, which can avoid the ethical controversy over the use of embryonic stem cells.

[0034] 2. High differentiation efficiency. Macrophages can be obtained after inducing differentiation for about 22 days. The purity of macrophages is greater than 99%, and they have significant polarization ability and phagocytic activity.

[0035] 3. The technical route is stable, the differentiation method is simple, and commonly used cytokines are used to induce differentiation into macrophages.

[0036] 4. Safe and reliable, the induced differentiation system contains no serum, no animal cell-derived components, and is independent of feeder cells, which is conducive to clinical transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 : Schematic diagram of the cell states of induced pluripotent stem cells, where A: 4×; B: 10×.

[0038] Figure 2 : Morphological images of monocytes formed on the 16th day of differentiation culture, where A: 10×; B: 20×.

[0039] Figure 3 : Flow cytometry identification results of monocytes, where A: isotype control; B: CD14 antibody staining.

[0040] Figure 4 : Schematic diagram of the state of macrophages formed on the 23rd day of differentiation culture, where A: 10×; B: 20×.

[0041] Figure 5 : CD14 / CD11b detection results of macrophages, where A: CD11b antibody staining; B: CD14 antibody staining.

[0042] Figure 6 : Cell morphology of macrophages after polarization, where A: M0 type (before polarization) cell morphology; B: M1 type cell morphology; C: M2 type cell morphology. All images are 10×.

[0043] Figure 7 : Flow cytometry results after macrophage polarization, including: A: M0 cells stained with CD80 and CD86 antibodies; B: M1 cells stained with CD80 and CD86 antibodies; C: M0 cells stained with CD163 and CD206 antibodies; D: M2 cells stained with CD163 and CD206 antibodies.

[0044] Figure 8 : Flow cytometry results of phagocytosis experiment, where A: iPSC phagocytosis result; A: iPSC-derived macrophage phagocytosis result.

[0045] Fig. 9 : Schematic diagram of cytokine release experimental results. DETAILED DESCRIPTION

[0046] The present invention will be further described below in conjunction with the embodiments. However, the scope of the present invention is not limited to the following embodiments. It will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0047] The instruments, reagents, and materials involved in the following examples, unless otherwise specified, are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through regular commercial channels. The experimental methods and detection methods involved in the following examples, unless otherwise specified, are all conventional experimental methods and detection methods already available in the prior art.

[0048] The ReleSR (ReLeSR human ES / iPS digestion solution) used in the present invention is a commercial product already available in the prior art and can be purchased conventionally.

[0049] The mTeSR-1 complete culture medium used in the present invention is a serum-free complete culture medium, which is a commercial product available in the prior art and can be purchased conventionally.

[0050] The TrypLE (pancreatic enzyme substitute) used in the present invention is a commercial product available in the prior art and can be purchased conventionally.

[0051] The ROCK inhibitor Y-27632, CAS: HY-10583, used in the present invention is a commercial product available in the prior art and can be purchased conventionally.

[0052] The CHIR99021 used in the present invention is a highly specific glycogen synthase kinase-3 (GSK-3) inhibitor, which is a commercial product available in the prior art and can be purchased conventionally.

[0053] The BMP4 used in the present invention is a recombinant bone morphogenetic protein 4, which is a commercial product available in the prior art and can be purchased conventionally.

[0054] The activin A used in the present invention is Activin A, which is a commercial product available in the prior art and can be purchased conventionally.

[0055] The VEGF used in the present invention is vascular endothelial growth factor, which is a commercial product available in the prior art and can be purchased conventionally.

[0056] The FGF2 used in the present invention is fibroblast growth factor 2, which is a commercial product available in the prior art and can be purchased conventionally.

[0057] The APEL culture medium used in the present invention is a culture medium that does not contain serum and animal components. It is a commercial product available in the prior art and can be purchased conventionally.

[0058] The SCF used in the present invention is a stem cell growth factor, which is a commercial product available in the prior art and can be purchased conventionally.

[0059] The IL-3, interleukin 3, used in the present invention is a commercial product available in the prior art and can be purchased conventionally.

[0060] The M-CSF used in the present invention is a macrophage colony stimulating factor, which is a commercial product available in the prior art and can be purchased conventionally.

[0061] The LPS used in the present invention is lipopolysaccharide, which is a commercial product available in the prior art and can be purchased conventionally.

[0062] The INF-γ used in the present invention is interferon γ, which is a commercial product available in the prior art and can be purchased conventionally.

[0063] The IL-4, interleukin 4, used in the present invention is a commercial product available in the prior art and can be purchased conventionally.

[0064] The GlutaMAX supplement used in the present invention is a glutamine substitute, which is a commercial product already available in the prior art and can be purchased conventionally.

[0065] The X-VIV0 15 basal culture medium used in the present invention is a commercial product available in the prior art and can be purchased conventionally.

[0066] Example 1 Induced pluripotent stem cells to differentiate into macrophages

[0067] Here are the steps:

[0068] S0: Culture of induced pluripotent stem cells

[0069] The Sendai virus reprogramming kit (Invitrogen TM CytoTune TM -iPS2.0 Sendai virus reprogramming kit, catalog number: A16517), according to the instructions, high-frequency HLA homozygous umbilical cord blood-derived mononuclear cells were obtained by standard reprogramming methods.

[0070] iPSCs were cultured in six-well plates, digested and passaged with ReleSR every 2 to 3 days, and cultured in a 37°C, 5% CO2 incubator using mTeSR-1 complete medium, with the culture medium replaced once a day.

[0071] Schematic diagram of the cell state of induced pluripotent stem cells Figure 1 As shown, it can be seen that the cells grow in aggregates, are round or oval in shape, are tightly packed together and have a high nuclear-to-cytoplasmic ratio.

[0072] S1: Induced pluripotent stem cells to form embryoid bodies (Day -1 to Day 0)

[0073] When the iPSCs with good growth status were cultured to 70% to 80% polymerization degree, TrypLE was used to digest the iPSCs into a single cell suspension and plated into ultra-low adsorption six-well plates at a density of 2×10 6 / well, add culture medium to each well to 2mL, the culture medium is mTeSR-1 medium containing 10μM Y-27632, cross-mix and culture in a 37℃, 5% CO2 incubator for 24h to form embryoid bodies.

[0074] S2: Differentiation of embryoid bodies into monocytes expressing CD14 (differentiation culture day 0 to day 16)

[0075] The specific operations are as follows:

[0076] S2-1: Observe EB formation under a microscope; tilt the cell plate, wait for the EB to sink to the bottom, aspirate the supernatant, add the first differentiation medium, and place the cell plate in a 37°C, 5% CO2 incubator for 1 day (differentiation culture day 0-1).

[0077] The first differentiation medium is an APEL medium supplemented with BMP4, VEGF, SCF, FGF2, Activin A, and CHIR99021, wherein the concentration of BMP4 is 20 ng / mL, the concentration of VEGF is 40 ng / mL, the concentration of SCF is 40 ng / mL, the concentration of FGF2 is 5 ng / mL, the concentration of activin A is 5 ng / mL, and the concentration of CHIR99021 is 0.2 μM.

[0078] S2-2: Take out the culture plate containing EBs from the incubator, tilt the cell plate, wait for the EBs to sink to the bottom, aspirate the first differentiation medium, add freshly prepared second differentiation medium, and place the cell plate in a 37°C, 5% CO2 incubator for 1 day (Day 1 to Day 2 of differentiation culture).

[0079] The second differentiation medium is APEL medium supplemented with BMP4, VEGF, SCF, FGF2, and CHIR99021, wherein the concentration of BMP4 is 20 ng / mL, the concentration of VEGF is 40 ng / mL, the concentration of SCF is 40 ng / mL, the concentration of FGF2 is 5 ng / mL, and the concentration of CHIR99021 is 0.2 μM.

[0080] S2-3: Take out the culture plate containing EBs from the incubator, tilt the cell plate, wait for the EBs to sink to the bottom, aspirate the second differentiation medium, add freshly prepared third differentiation medium, and place the cell plate in a 37°C, 5% CO2 incubator for 6 days (2nd to 8th day of differentiation culture).

[0081] The third differentiation medium is an APEL medium supplemented with BMP4, VEGF, SCF, and FGF2, wherein the concentration of BMP4 is 20 ng / mL, the concentration of VEGF is 40 ng / mL, the concentration of SCF is 40 ng / mL, and the concentration of FGF2 is 5 ng / mL.

[0082] S2-4: Collect EBs on the 8th day of induction into a 15 mL centrifuge tube, let it stand for 10 min until the EBs sink to the bottom, aspirate the third differentiation medium, add freshly prepared fourth differentiation medium, and place the cell plate in a 37°C, 5% CO2 incubator for 8 days (differentiation culture day 8 to day 16).

[0083] The fourth differentiation medium is a medium of X-VIV0 15 supplemented with VEGF, SCF, FGF2, IL-3, and M-CSF, wherein the concentration of VEGF is 20 ng / mL, the concentration of SCF is 20 ng / mL, the concentration of FGF2 is 10 ng / mL, the concentration of IL-3 is 25 ng / mL, and the concentration of M-CSF is 50 ng / mL.

[0084] The morphology of the monocytes formed on the 16th day of differentiation culture is as follows Figure 2 As shown, many large and plump suspended cells were produced in the cell wells.

[0085] S2-5: Flow cytometry analysis of monocyte marker expression

[0086] On the 16th day of differentiation culture, the suspended cells were collected and centrifuged at 300 g for 5 min, and the supernatant was discarded; the cells were resuspended in DPBS containing 2% BSA, and CD14 antibody was added to perform flow cytometric analysis to detect the expression of CD14.

[0087] The flow cytometry results of monocytes were as follows Figure 3 As shown, the CD14 positivity rate was 70.81%.

[0088] S3: Differentiation of monocytes into macrophages (differentiation culture day 16 to day 23)

[0089] The specific operations are as follows:

[0090] S3-1: Collect the suspended cells expressing CD14, remove the fourth differentiation medium, resuspend the cells in freshly prepared macrophage differentiation medium, and culture the cell plate in a 37°C, 5% CO2 incubator for 7 days (16th to 23rd day of differentiation culture).

[0091] The macrophage differentiation medium is X-VIVO 15 medium added with GlutaMax and M-CSF, wherein the concentration of GlutaMax is 2 mM and the concentration of M-CSF is 100 ng / mL.

[0092] The cell morphology on the 23rd day of differentiation culture was as follows Figure 4 As shown, it can be seen that the cells are round or oval and have short processes.

[0093] S3-2: Flow cytometry detection of macrophage marker expression

[0094] The cells in the culture plate were collected and centrifuged at 300 g for 5 min, and the supernatant was discarded; the cells were resuspended in DPBS containing 2% BSA, and flow cytometry antibodies (CD14, CD11b) were added to detect the expression of CD14 and CD11b by flow cytometry.

[0095] Flow cytometry results of macrophages Figure 5 As shown, it can be seen that the expression rate of CD14 is 99.15%, and the expression rate of CD11b is 98.71%, indicating that macrophages with higher purity are obtained.

[0096] S4: Macrophage polarization experiments

[0097] S4-1: Take the macrophages on the 23rd day of differentiation (obtained in step S3-1), and culture the cells in M1 macrophage polarization medium and M2 macrophage polarization medium respectively for 48 hours.

[0098] The M1 macrophage culture medium is an X-VIV0 15 culture medium supplemented with GlutaMax, LPS and IFN-γ, wherein the concentration of GlutaMax is 2 mM, the concentration of LPS is 100 ng / mL, and the concentration of IFN-γ is 20 ng / mL.

[0099] The M2 macrophage culture medium is an X-VIV0 15 culture medium supplemented with GlutaMax and IL-4, wherein the concentration of GlutaMax is 2 mM and the concentration of IL-4 is 50 ng / mL.

[0100] The morphological changes of macrophages after 48 hours of polarization are as follows: Figure 6 As shown, compared with M0 macrophages (before polarization), M1 macrophages are large and flat, shaped like fried eggs, while M2 macrophages become more slender, which is consistent with the morphological characteristics of M1 and M2 macrophages.

[0101] S4-2: Flow cytometry detection of the expression of M1 and M2 macrophage markers

[0102] Polarized macrophages (obtained in step S4-1) were collected, centrifuged at 300 g for 5 min, and the supernatant was discarded; the cells were resuspended in DPBS containing 2% BSA, and flow cytometry antibodies (CD80, CD86, CD163, CD206) were added for flow cytometric analysis.

[0103] The flow cytometry results of M1 and M2 macrophages are shown in Figure 7 As shown in the figure, CD80 and CD86 of M0 macrophages are expressed at low levels. After polarization to M1 macrophages, the expression of CD80 and CD86 is significantly upregulated (CD80 expression>90%, CD86 expression>99%); CD163 and CD206 of M0 macrophages are expressed at high levels. According to the literature, when macrophages are in the basal state (M0), they tend to be M2 macrophage phenotypes. After polarization to M2 macrophages, the expression of CD163 and CD206 is upregulated. This shows that macrophages generated by iPSCs have good polarization ability.

[0104] S5: Macrophage phagocytosis assay

[0105] iPSC-derived macrophages were collected and the cells were counted and the number of cells was calculated as 1×10 5The cells were plated into 12-well plates; Alexa488-labeled yeast polysan particles were added to the macrophages at a final concentration of 0.25 mg / mL; the cell culture plate was placed in a 37°C, 5% CO2 incubator for 1 hour; after 1 hour, the cells were collected into a 1.5 mL centrifuge tube, centrifuged at 300 g for 5 min, and the supernatant was discarded; 1 mL of DPBS was added to wash the cells once, and 200 μg / mL of trypan blue was added to wash the cells once. After washing the cells 4 times with DPBS, the expression rate of Alexa488-positive cells was detected by flow cytometry.

[0106] The results of the phagocytosis experiment were as follows Figure 8 As shown in the figure, with iPSC as the negative control, when zymosan A was co-incubated with iPSC, the Alexa488 positive rate was only 7.66%, indicating that iPSC itself had no phagocytic function. After zymosan A was co-incubated with iPSC-derived macrophages, the Alexa488 positive rate was greater than 99%, indicating that the differentiated macrophages had normal phagocytic function.

[0107] S6: Cytokine release assay

[0108] The cell supernatant after polarization of macrophages (obtained in step S4-1) was taken and centrifuged at 2000 rpm for 5 min. The supernatant was transferred to a 1.5 mL centrifuge tube for later use. The IL-6 and TNF-α detection experiments were performed according to the steps described in the ELISA kit instructions.

[0109] The results of cytokine release experiments were as follows Fig. 9 As shown, it can be seen that compared with M0 and M2 macrophages, M1 macrophages secrete higher levels of TNF-α and IL-6, indicating that iPSC-derived macrophages have the ability to release cytokines.

[0110] In summary, the method of differentiating induced pluripotent stem cells into macrophages of the present invention is based on the embryoid body differentiation system and adds a culture medium with clear components and a cytokine combination, so that iPSCs can be differentiated into macrophages quickly and efficiently. The obtained cells not only contain macrophage characteristic proteins such as CD11b, but also have good polarization ability and significant phagocytic activity.

[0111] The above examples are provided to those skilled in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art will fall within the scope of the appended claims.

Claims

1. A method for differentiating induced pluripotent stem cells into macrophages, characterized in that: The following steps are involved: S1: Induced pluripotent stem cells to form embryoid bodies; S2: embryoid bodies differentiated into monocytes expressing CD14; S3: Monocytes differentiate into macrophages with CD14 / CD11b expression; S4: Macrophage polarization into M1 macrophages and / or M2 macrophages.

2. The method for differentiating induced pluripotent stem cells into macrophages according to claim 1, characterized in that: The step S1 specifically comprises: culturing induced pluripotent stem cells in the presence of ROCK inhibitor Y-27632 to form embryoid bodies.

3. The method for differentiating induced pluripotent stem cells into macrophages according to claim 2, characterized in that: The concentration of Y-27632 was 10 μM.

4. The method for differentiating induced pluripotent stem cells into macrophages according to claim 1, characterized in that: The step S2 comprises the following steps: S2-1: After embryoid bodies are formed, the supernatant is removed, the first differentiation medium is added, and the cells are induced for 1 to 2 days; The first differentiation medium is an APEL medium supplemented with BMP4, VEGF, SCF, FGF2, activin A, and CHIR99021, wherein the concentration of BMP4 is 20 ng / mL, the concentration of VEGF is 40 ng / mL, the concentration of SCF is 40 ng / mL, the concentration of FGF2 is 5 ng / mL, the concentration of activin A is 5 ng / mL, and the concentration of CHIR99021 is 0.2 μM; S2-2: remove the first differentiation medium, add the second differentiation medium, and induce culture for 1 to 2 days; The second differentiation medium is an APEL medium supplemented with BMP4, VEGF, SCF, FGF2, and CHIR99021, wherein the concentration of BMP4 is 20 ng / mL, the concentration of VEGF is 40 ng / mL, the concentration of SCF is 40 ng / mL, the concentration of FGF2 is 5 ng / mL, and the concentration of CHIR99021 is 0.2 μM; S2-3: remove the second differentiation medium, add the third differentiation medium, and induce culture for 4 to 8 days; The third differentiation medium is an APEL medium supplemented with BMP4, VEGF, SCF, and FGF2, wherein the concentration of BMP4 is 20 ng / mL, the concentration of VEGF is 40 ng / mL, the concentration of SCF is 40 ng / mL, and the concentration of FGF2 is 5 ng / mL; S2-4: remove the third differentiation medium, add the fourth differentiation medium, and induce culture for 7 to 14 days; The fourth differentiation medium is a medium of X-VIV0 15 supplemented with VEGF, SCF, FGF2, IL-3, and M-CSF, wherein the concentration of VEGF is 20 ng / mL, the concentration of SCF is 20 ng / mL, the concentration of FGF2 is 10 ng / mL, the concentration of IL-3 is 25 ng / mL, and the concentration of M-CSF is 50 ng / mL.

5. The method for differentiating induced pluripotent stem cells into macrophages according to claim 1, characterized in that: The step S3 specifically comprises: collecting suspended cells containing CD14+ monocytes, adding macrophage differentiation medium, and inducing culture for 5 to 10 days; The macrophage differentiation medium is X-VIV0 15 medium added with GlutaMax and M-CSF, wherein the concentration of GlutaMax is 2 mM and the concentration of M-CSF is 100 ng / mL.

6. The method for differentiating induced pluripotent stem cells into macrophages according to claim 1, characterized in that: The step S4 specifically includes: replacing the macrophage differentiation medium with an M1 macrophage polarization medium and / or an M2 macrophage polarization medium, and culturing for 24 to 48 hours.

7. The method for differentiating induced pluripotent stem cells into macrophages according to claim 6, characterized in that: The M1 macrophage polarization medium is an X-VIV0 15 medium supplemented with GlutaMax, LPS and IFN-γ, wherein the concentration of GlutaMax is 2 mM, the concentration of LPS is 100 ng / mL, and the concentration of IFN-γ is 20 ng / mL.

8. The method for differentiating induced pluripotent stem cells into macrophages according to claim 6, characterized in that: The M2 macrophage polarization medium is an X-VIV0 15 medium supplemented with GlutaMax and IL-4, wherein the concentration of GlutaMax is 2 mM and the concentration of IL-4 is 50 ng / mL.

9. The method for differentiating induced pluripotent stem cells into macrophages according to claim 1, characterized in that: The culture system used in steps S1 and S2 is an ultra-low adsorption six-well plate; Or: the culture system used in steps S3 and S4 is a culture bottle treated with tissue culture; Alternatively, the culture conditions are: 37° C., 5% CO 2 .

10. Macrophages cultured by the method according to any one of claims 1 to 9.

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