Cultivation method for efficiently inducing hiPSC to be differentiated into macrophages based on formation of yolk sac-like organs in vitro

Abstract

The invention discloses a culture method for efficiently inducing hiPSC to be differentiated into macrophages on the basis of formation of yolk sac organs in vitro, and belongs to the technical field of stem cell application. The method comprises the following steps: (1) inducing hiPSC to differentiate to an embryoid body; (2) inducing the embryoid body containing cells determined by the egg sac fate to an egg sac-shaped embryoid body; (3) inducing the formation of a blood island in the yolk sac-shaped embryoid body; (4) inducing the myeloid progenitor cells in the blood island to differentiate into mononuclear cells; and (5) stimulating the mononuclear cells to mature towards macrophages. The method has the beneficial effects that different concentrations and different types of human recombinant cytokines and space-time combinations thereof described in the invention are used in different time periods of differentiating macrophages by hiPSC, and then based on the formation of a yolk sac-shaped embryoid body, the development way of differentiating endogenous macrophages by embryo yolk sacs is simulated; finally, the induced hiPSC has the advantages of high in-vitro yield, high purity and high stability.

Description

Technical Field

[0001] The present invention belongs to the technical field of stem cell application, and specifically relates to a cultivation method for efficiently inducing hiPSC differentiation into macrophages based on the formation of yolk sac organoids in vitro. Background Art

[0002] Cell therapy research has become an important battlefield for the current research of innovative drugs. Among them, macrophages are becoming an ideal target for the release of cell drugs for the treatment of solid tumors and various immune diseases due to their advantages in phagocytosis of abnormal cells in the body, immune regulation, and good infiltration into tissues and solid tumors. However, macrophages obtained from patients themselves are difficult to meet clinical treatment needs and difficult to genetically engineer. Therefore, the development of a program to stably produce safe and reliable macrophages is of extremely high clinical value.

[0003] The differentiation of induced pluripotent stem cells (iPSC) into immune cells is a hot research direction in the field of regenerative medicine. iPSC has the characteristics of stable source, unlimited proliferation, high gene editing efficiency, low cost, etc., and can differentiate into all types of cells in the body. Therefore, it has great potential in the treatment of blood diseases, tumors and autoimmune diseases. At present, the technology of differentiating iPSC into immune cells including T cells, NK cells, macrophages and dendritic cells is maturing. Induced differentiation can be achieved through specific differentiation culture media and conditions.

[0004] Human induced pluripotent stem cells (hiPSC) are a type of pluripotent stem cells that are obtained by re-differentiating highly differentiated human cells through gene editing technology (such as CRISPR-Cas9). At present, the application of macrophages in clinical treatment faces a series of pain points, such as: 1) The number of macrophages derived from the patient's own body is small, and genetic engineering editing is difficult; 2) The existing iPSC differentiation macrophage schemes are not mature and efficient enough, the yield is not high enough, the technology is not stable enough, and the purity is low; 3) Some technical means to obtain macrophages cannot simulate endogenous macrophages well. The methods for inducing the generation of macrophages are mainly divided into two types of schemes: embryoid body (EB) formation and two-dimensional factor-dependent (2D-F) schemes.

[0005] EB solutions (such as publication numbers CN109082411A, CN109266618A, and CN116731967A) simulate the path of hematopoietic stem cell differentiation into macrophages in the body, and the resulting macrophages are closer to endogenous giant cells in expression profile and function, and the process is relatively simple and low-cost. The disadvantage is that the existing technical solutions are not reproducible enough, the cell yield is low, and cannot meet the needs of clinical applications.

[0006] The 2D-F protocol (such as publication number CN115433715A) can produce terminally differentiated cells that meet clinical quantity requirements and achieve better reproducibility. However, the protocol is usually more complicated and expensive, and the terminally differentiated cells produced cannot simulate endogenous macrophages well. Summary of the invention

[0007] The technical problem to be solved by the present invention is how to solve the problems of low efficiency and low purity in the current methods for artificially inducing macrophage generation.

[0008] The present invention solves the above technical problems through the following technical means:

[0009] The first aspect of the present invention provides a method for efficiently inducing pluripotent stem cells hiPSC to differentiate into macrophages based on the formation of yolk sac organoids, comprising the following steps:

[0010] (1) Inducing hiPSCs to differentiate into embryoid bodies containing mesoderm: After pretreatment, pluripotent stem cells hiPSCs are inoculated into culture medium A to obtain embryoid bodies, which are then replaced with culture medium B to continue culturing the embryoid bodies;

[0011] The components of the culture medium A include: APEL II medium, BMP4, Activin A, TGF-β2 and Y-27632;

[0012] The components of the culture medium B include: APEL II medium, BMP4, Activin A and TGF-β2;

[0013] (2) Inducing embryoid bodies to differentiate into yolk sac organoids: Replace the medium with yolk sac induction medium (YS medium) and continue culturing to obtain yolk sac organoids;

[0014] (3) Inducing yolk sac organoids to generate blood islands: replacing the medium with blood island induction medium (BI medium) and continuing the culture to obtain yolk sac organoids containing blood islands;

[0015] (4) Inducing myeloid progenitor cells in the blood islands to differentiate into monocytes: replacing the medium with monocyte induction medium (MP medium) and continuing the culture to obtain monocytes;

[0016] (5) Stimulating monocytes to mature into macrophages: replacing the medium with macrophage induction medium (MM medium) and continuing the culture to obtain macrophages.

[0017] Preferably, the components of the yolk sac induction medium (YS medium) include APEL II medium, BMP4, VEGF, SCF, IL-3, IL-6, IL-11, Flt-3L and TGF-β2.

[0018] Preferably, the concentration of BMP4 is 30-50 ng / mL, the concentration of VEGF is 50-70 ng / mL, the concentration of SCF is 70-90 ng / mL, the concentration of IL-3 is 10-30 ng / mL, the concentration of IL-6 is 15-35 ng / mL, the concentration of IL-11 is 20-30 ng / mL, the concentration of Flt-3L is 20-30 ng / mL, and the concentration of TGF-β2 is 15-25 ng / mL.

[0019] Preferably, the components of the yolk sac induction medium (YS medium) include: APEL II medium, BMP4 (40 ng / mL), VEGF (60 ng / mL), SCF (80 ng / mL), IL-3 (20 ng / mL), IL-6 (25 ng / mL), IL-11 (25 ng / mL), Flt-3L (25 ng / mL) and TGF-β2 (20 ng / mL).

[0020] Preferably, the components of the blood island induction medium (BI medium) include APEL II medium, bFGF, VEGF, SCF, IGF1, IL-3, TPO, M-CSF, GM-CSF and TGF-β2.

[0021] Preferably, the concentration of bFGF is 15-25 ng / mL, the concentration of VEGF is 55-65 ng / mL, the concentration of SCF is 30-50 ng / mL, the concentration of IGF1 is 5-15 ng / mL, the concentration of IL-3 is 20-40 ng / mL, the concentration of TPO is 25-35 ng / mL, the concentration of M-CSF is 10-30 ng / mL, the concentration of GM-CSF is 15-25 ng / mL, and the concentration of TGF-β2 is 10-30 ng / mL.

[0022] Preferably, the components of the blood island induction medium (BI medium) include: APEL II medium, bFGF (20 ng / mL), VEGF (60 ng / mL), SCF (40 ng / mL), IGF1 (10 ng / mL), IL-3 (30 ng / mL), TPO (30 ng / mL), M-CSF (20 ng / mL), GM-CSF (20 ng / mL) and TGF-β2 (20 ng / mL).

[0023] Preferably, the components of the monocyte induction medium (MP medium) include: XVIVO TM 15medium, bFGF, VEGF, SCF, IGF1, IL-3, M-CSF and GM-CSF.

[0024] Preferably, the concentration of bFGF is 10-30 ng / mL, the concentration of VEGF is 50-70 ng / mL, the concentration of SCF is 35-45 ng / mL, the concentration of IGF1 is 5-15 ng / mL, the concentration of IL-3 is 25-35 ng / mL, the concentration of M-CSF is 35-45 ng / mL, and the concentration of GM-CSF is 30-50 ng / mL.

[0025] Preferably, the components of the monocyte induction medium (MP medium) include: XVIVO TM 15medium, bFGF (20ng / mL), VEGF (60ng / mL), SCF (40ng / mL), IGF1 (10ng / mL), IL-3 (30ng / mL), M-CSF (40ng / mL) and GM-CSF (40ng / mL).

[0026] Preferably, the composition of the macrophage induction medium (MM medium) includes: 1640 (containing 10% FBS) / XVIVO TM 15. IGF1, IL-3, M-CSF and GM-CSF.

[0027] Preferably, the concentration of IGF1 is 5-15 ng / mL, the concentration of IL-3 is 15-25 ng / mL, the concentration of M-CSF is 55-65 ng / mL, and the concentration of GM-CSF is 50-70 ng / mL.

[0028] Preferably, the composition of the macrophage induction medium (MM medium) includes: 1640 (containing 10% FBS) / XVIVO TM15. IGF1 (10ng / mL), IL-3 (20ng / mL), M-CSF (60ng / mL) and GM-CSF (60ng / mL).

[0029] Preferably, in (1), the pretreatment of the pluripotent stem cells hiPSC comprises: culturing the hiPSC, adding a digestion solution, centrifuging and discarding the supernatant, and collecting the hiPSC after centrifugation.

[0030] The beneficial effects of the present invention are:

[0031] 1. The present invention constructs a method for inducing hiPSCs to differentiate into macrophages in vitro with high yield, high purity and rapidity by using different concentrations and types of human recombinant cytokines and their spatiotemporal combinations as described in the present invention at different time periods of hiPSC differentiation into macrophages, and then simulating the developmental pathway of endogenous macrophage differentiation from blood islands in the embryonic yolk sac based on the formation of yolk sac-like embryoid bodies (Yolksac-EBs).

[0032] 2. The present invention provides a novel monolayer differentiation method to obtain macrophages derived from human pluripotent stem cells. Compared with the existing monolayer differentiation method, this method does not require the differentiation stage of HSPCs, and can directly differentiate to obtain functionally mature macrophages; it greatly simplifies the macrophage differentiation system, shortens the differentiation cycle, and can significantly reduce production costs; therefore, the macrophage differentiation method provided by the present invention is safer, more reliable and more stable, and can produce functionally mature macrophages in large quantities in a short time.

[0033] 3. The cultivation scheme of the present invention simulates the developmental path of macrophages produced by the early yolk sac blood islands of the human body. The macrophages produced have the complete expression spectrum and immunological functions of endogenous giant cells, normally express the classic surface markers of macrophages, and present typical macrophage morphology.

[0034] 4. The present invention provides a novel solution for inducing hiPSC to differentiate efficiently and rapidly into endogenous macrophages with complete immunological functions, which can solve the problems of insufficient quantity, unstable source and unstable technology faced by macrophages in the process of immune cell therapy, and improve the efficiency of iPSC differentiation of macrophages based on yolk sac organoids (yolk sac-like EBs) by more than 10,000 times. At the same time, the macrophages obtained by this method have the characteristics of high editing efficiency.

[0035] 5. The novel hiPSC-derived macrophage differentiation scheme described in the present invention has the advantages of high yield, high purity, and stable technology. It breaks through the bottleneck problem of the existing technology, provides a reliable chassis cell for engineered macrophages used in the treatment of various diseases such as solid tumors, and can achieve large-scale industrial production, with considerable market transformation prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a schematic diagram of the method for inducing hiPSC differentiation into macrophages according to Example 1 of the present invention;

[0037] Figure 2 This is a statistical comparison diagram of the efficiency of hiPSC differentiation into macrophages induced by Example 1 of the present invention and Comparative Example 1;

[0038] Figure 3 This is a comparative diagram of the characteristics of macrophages differentiated from iPSCs and undifferentiated iPSCs in Example 1 of the present invention;

[0039] Figure 4 Graphs for verifying the phagocytic function of macrophages differentiated from iPSCs in Example 1 of the present invention, wherein A is a graph for verifying the phagocytic function of macrophages co-cultured with tumor cells, and B is a statistical comparison graph of the number of tumor cells before and after co-culture;

[0040] Figure 5 : This is a verification diagram of the immune activation characteristics of macrophages differentiated from iPSCs in Example 1 of the present invention, wherein A is a diagram of qPCR test results, and B is a diagram of ELISA test results;

[0041] Figure 6 The figure is a comparison chart of the purity of macrophages obtained in Example 1 of the present invention and Comparative Example 1. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0044] If no specific technology or conditions are specified in the examples, they can be carried out according to the technology or conditions described in the literature in the field or according to the product instructions. Unless otherwise specified, the quantitative tests in the following examples are repeated more than three times, and the results are averaged.

[0045] Cell culture Day 0 refers to the starting day of the cell culture experiment, that is, the first day when cells are inoculated into the culture medium.

[0046] Embodiment 1:

[0047] The culture medium used in this example is described as follows:

[0048] YI medium containing Y-27632 (referred to as medium A):

[0049] APEL II medium, BMP4 (40ng / mL), Activin A (20ng / mL), TGF-β2 (20ng / mL) and 10μMY-27632.

[0050] YI medium without Y-27632 (referred to as medium B):

[0051] APEL II medium, BMP4 (40ng / mL), Activin A (20ng / mL) and TGF-β2 (20ng / mL).

[0052] Yolk sac induction medium (referred to as YS medium):

[0053] APEL II medium, BMP4 (40ng / mL), VEGF (60ng / mL), SCF (80ng / mL), IL-3 (20ng / mL), IL-6 (25ng / mL), IL-11 (25ng / mL), Flt-3L (25ng / mL) and TGF-β2 (20ng / mL).

[0054] Blood island induction medium (referred to as BI medium):

[0055] APEL II medium, bFGF (20ng / mL), VEGF (60ng / mL), SCF (40ng / mL), IGF1 (10ng / mL), IL-3 (30ng / mL), TPO (30ng / mL), M-CSF (20ng / mL), GM-CSF (20ng / mL) and TGF-β2 (20ng / mL).

[0056] Monocyte induction medium (denoted as MP medium):

[0057] XVIVO TM 15medium, bFGF (20ng / mL), VEGF (60ng / mL), SCF (40ng / mL), IGF1 (10ng / mL), IL-3 (30ng / mL), M-CSF (40ng / mL) and GM-CSF (40ng / mL).

[0058] Macrophage induction medium (referred to as MM medium):

[0059] 1640 (containing 10% FBS) / XVIVO TM 15. IGF1 (10ng / mL), IL-3 (20ng / mL), M-CSF (60ng / mL) and GM-CSF (60ng / mL).

[0060] Macrophage maintenance medium (referred to as MS medium):

[0061] 1640(with 10% FBS) / XVIVO TM 15. M-CSF (20 ng / mL) and GM-CSF (20 ng / mL).

[0062] Medium for maintaining macrophage antigen presentation and M1 polarization properties (referred to as MS-M-CSF medium): 1640 (containing 10% FBS) / XVIVO TM 15 and GM-CSF (20 ng / mL).

[0063] Culture medium for maintaining the M2 polarization characteristics of macrophages (referred to as MS-GM-CSF culture medium): 1640 (containing 10% FBS) / XVIVO TM 15 and M-CSF (20 ng / mL).

[0064] A method for efficiently inducing pluripotent stem cells hiPSC to differentiate into macrophages based on the formation of yolk sac organoids (schematic diagram of the method path as shown in Figure 1 As shown), comprising the following steps:

[0065] (1) The first stage (Day 0-Day 1) is to induce hiPSCs to differentiate into embryoid bodies (EBs) containing mesoderm:

[0066] 1) Pretreatment of hiPSCs:

[0067] hiPSCs were cultured in six-well plates coated with Matrigel (cornning, #354277) using ncTarget serum-free medium. When the cell confluence reached 70%, the medium was discarded, and 1×DPBS was added to each well for washing and the DPBS was discarded. Then, TripLE digestion solution was added to completely cover the cells, and the digestion solution was discarded after digestion at room temperature for 1 minute, and the digestion and culture were continued at 37°C for 4 minutes. An appropriate amount of DMEM / F12 medium was added to terminate the digestion, and the adherent hiPSCs were blown off with a 1ml pipette and transferred to a 15ml centrifuge tube. Centrifuge at room temperature for 3 minutes at 1000rpm. The supernatant liquid after centrifugation was discarded and the hiPSCs after centrifugation were collected.

[0068] 2) hiPSC seeding:

[0069] The hiPSCs collected in the previous step were resuspended in YI medium + Y-27632 (i.e., culture medium A) and counted. According to the amount of 5000 cells per well, 100ul of culture medium containing hiPSCs was added to the round-bottom low-adsorption 96-well plate, and then centrifuged at 500g for 3 minutes at room temperature to allow the hiPSCs to completely aggregate at the bottom of the round-bottom low-adsorption 96-well plate. The round-bottom low-adsorption 96-well plate inoculated with hiPSCs after centrifugation was placed in an incubator at a temperature of 37°C and containing 5% carbon dioxide for further culture of the hiPSCs.

[0070] 3) Embryoid body (EB) medium change:

[0071] Further induce the formation of embryoid bodies (EBs) with yolk sac fate determination. After the hiPSCs collected in the previous step are cultured in a round-bottom low-adsorption 96-well plate for 24 hours, each well of the round-bottom low-adsorption 96-well plate will have only one spherical EB formed. At this time, discard the supernatant and add fresh YI medium without Y-27632 (i.e., medium B). Continue to culture the EBs for 24 hours.

[0072] (2) The second stage (Day 2-Day 7): Inducing the production of yolk sac organoids:

[0073] After the first stage of EB induction is successful, discard the medium B and replace it with the second stage yolk sac induction medium (YS medium) to induce yolk sac organoids (yolk sac-like EBs). Renew the YS medium once on Day 2, Day 4, and Day 6, with 100ul YS medium in each well. (During this period, vesicular yolk sac organoids will appear. The appearance of vesicular yolk sac organoids indicates that there will be a higher and more reliable monocyte / macrophage yield in the future.)

[0074] (3) The third stage (Day 8-Day 10), inducing the formation of blood islands in yolk sac organoids:

[0075] On Day 8, the second-stage yolk sac induction medium (YS medium) was replaced with blood island induction medium (BI medium), and the culture was continued to obtain yolk sac organoids containing blood islands, wherein the blood islands contained myeloid progenitor cells. 100ul BI medium was added to each well.

[0076] (4) The fourth stage (Day 11 and later) is to induce myeloid progenitor cells in the blood islands to differentiate into monocytes:

[0077] The yolk sac organoids containing myeloid progenitor cells were transferred to a six-well plate coated with Matrigel (cornning, #354277). Twelve yolk sac EBs were placed in each well. Yolk sac EBs were continuously cultured with MP medium, 3 ml of medium was added to each well, and mononuclear cells were collected and the medium was replaced every two days until no mononuclear cells were produced.

[0078] (5) The fifth stage (Day 13 and beyond) stimulates monocytes to mature into macrophages:

[0079] The collected monocytes can be completely induced into macrophages after being cultured for 5 days in 6-well plates treated with TC surface (Tissue Culture Treated) or non-TC surface treated plates using MM medium.

[0080] Maintenance of macrophages:

[0081] Induced mature macrophages can maintain their survival and functional characteristics for a certain period of time in the corresponding culture medium. The culture medium used in this stage is MS culture medium. If antigen presentation and M1 polarization characteristics of macrophages are required, MS-M-CSF culture medium is required for maintenance culture. If the M2 polarization characteristics of macrophages need to be maintained, MS-GM-CSF culture medium is required.

[0082] The characteristics of macrophages differentiated from iPSCs in this example were identified, and undifferentiated iPSCs were used as controls. Figure 3 As shown, flow cytometry analysis showed that CD11B, CD14, CD68, CD86, CD206, CD163 and other innate macrophage marker membrane proteins were significantly highly expressed in macrophages differentiated from iPSCs induced in Example 1. However, undifferentiated iPSCs did not express this series of marker proteins.

[0083] The verification diagram of the phagocytic function of macrophages differentiated from iPSCs in this example is as follows Figure 4 As shown in the figure, confocal scanning detection showed that macrophages differentiated from iPSC (green fluorescent marker) and tumor cells (U87MG cells, red fluorescent marker) showed significant phagocytosis after being co-cultured in vitro at a ratio of 10 / 1 for 48 hours ( Figure 4 A). Flow cytometry analysis showed that co-culture of iPSC-derived macrophages and tumor cells significantly reduced the number of tumor cells ( Figure 4 B) The above results indicate that the macrophages obtained in this example have the function of phagocytizing abnormal cells as endogenous immune cells.

[0084] The verification diagram of the immune activation characteristics of macrophages differentiated from iPSCs in this example is shown in the figure Figure 5 As shown in the figure, the expression and secretion of inflammatory factors by iPSC-derived macrophages under immune stimulation. qPCR detection showed that endogenous macrophages and iPSC-derived macrophages highly expressed inflammatory factors IL1A, IL1B, IL6, IL12, IL23, TNF-α ( Figure 5 A). ELISA assays showed that after stimulation with 100 mg / ml IFN-gamma for 24 hours and 100 mg / ml LPS for 20 minutes, iPSC-derived macrophages secreted high levels of validation-related factors ( Figure 5 B) The above test results indicate that the macrophages obtained in this example have the immune activation potential of endogenous macrophages.

[0085] Comparative Example 1:

[0086] Macrophages were differentiated using the method described in the literature [Zhang L, Tian L, Dai X, Yu H, Wang J, Lei A, Zhu M, Xu J, Zhao W, Zhu Y, Sun Z, Zhang H, Hu Y, Wang Y, Xu Y, Church GM, Huang H, Weng Q, Zhang J. Pluripotent stem cell-derived CAR-macrophage cells with antigen-dependent anti-cancer cell functions. J Hematol Oncol. 2020 Nov 11; 13(1): 153. doi: 10.1186 / s13045-020-00983-2.].

[0087] The number of macrophages differentiated from Example 1 and Comparative Example 1 was counted, and the results were as follows: Figure 2 shown.

[0088] The number of macrophages produced by each EB formed by inoculating 3000 iPSCs was counted. During the EB induction process, 3000 iPSCs were inoculated in each well of a round-bottom low-adsorption 96-well plate, and then induced into one EB. The statistical data showed that using the traditional differentiation method in Comparative Example 1, each EB could eventually differentiate into approximately 9×10 6 The differentiation efficiency is about 3000 times. However, using the differentiation scheme of Example 1 of the present invention, more than 3×10 7The differentiation efficiency can reach more than 10,000 times.

[0089] Therefore, the differentiation efficiency of Example 1 of the present invention is more than 3 times that of the traditional differentiation method of Example 1, which has a significant efficiency advantage.

[0090] The purity of the macrophages obtained in Example 1 of the present invention is higher than that in Comparative Example 1:

[0091] The ratio of the macrophage marker membrane protein CD11B obtained in Example 1 of the present invention and Comparative Example 1 was detected by flow cytometry (eg Figure 6 The test statistical results show that the proportion of CD11B positive cells produced by the conventional method of comparative example 1 is greater than 83% of all terminally differentiated cells, while the CD11B positive cells obtained by Example 1 of the present invention account for more than 95% of all terminally differentiated cells. This result shows that the macrophages obtained by the novel differentiation scheme of the present invention have a higher purity than the macrophages obtained by the existing differentiation method.

[0092] Embodiment 2:

[0093] The difference between this embodiment and embodiment 1 is that the concentration of the culture medium used is slightly different, and the rest is the same as embodiment 1, as shown below:

[0094] Yolk sac induction medium (referred to as YS medium):

[0095] APEL II medium, BMP4 (30ng / mL), VEGF (70ng / mL), SCF (70ng / mL), IL-3 (30ng / mL), IL-6 (15ng / mL), IL-11 (30ng / mL), Flt-3L (30ng / mL) and TGF-β2 (25ng / mL).

[0096] Blood island induction medium (referred to as BI medium):

[0097] APEL II medium, bFGF (25ng / mL), VEGF (65ng / mL), SCF (50ng / mL), IGF1 (5ng / mL), IL-3 (20ng / mL), TPO (25ng / mL), M-CSF (10ng / mL), GM-CSF (25ng / mL) and TGF-β2 (30ng / mL).

[0098] Monocyte induction medium (denoted as MP medium):

[0099] XVIVO TM15medium, bFGF (10ng / mL), VEGF (70ng / mL), SCF (45ng / mL), IGF1 (5ng / mL), IL-3 (35ng / mL), M-CSF (45ng / mL) and GM-CSF (50ng / mL).

[0100] Macrophage induction medium (referred to as MM medium):

[0101] 1640 (containing 10% FBS) / XVIVO TM 15. IGF1 (5ng / mL), IL-3 (25ng / mL), M-CSF (55ng / mL) and GM-CSF (70ng / mL).

[0102] Embodiment 3:

[0103] Yolk sac induction medium (referred to as YS medium):

[0104] APEL II medium, BMP4 (50ng / mL), VEGF (50ng / mL), SCF (90ng / mL), IL-3 (10ng / mL), IL-6 (35ng / mL), IL-11 (20ng / mL), Flt-3L (20ng / mL) and TGF-β2 (15ng / mL).

[0105] Blood island induction medium (referred to as BI medium):

[0106] APEL II medium, bFGF (15ng / mL), VEGF (55ng / mL), SCF (30ng / mL), IGF1 (15ng / mL), IL-3 (40ng / mL), TPO (35ng / mL), M-CSF (30ng / mL), GM-CSF (15ng / mL) and TGF-β2 (10ng / mL).

[0107] Monocyte induction medium (denoted as MP medium):

[0108] XVIVO TM 15medium, bFGF (30ng / mL), VEGF (50ng / mL), SCF (35ng / mL), IGF1 (15ng / mL), IL-3 (25ng / mL), M-CSF (35ng / mL) and GM-CSF (30ng / mL).

[0109] Macrophage induction medium (referred to as MM medium):

[0110] 1640 (containing 10% FBS) / XVIVO TM 15. IGF1 (15ng / mL), IL-3 (15ng / mL), M-CSF (65ng / mL) and GM-CSF (50ng / mL).

[0111] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for cultivating induced pluripotent stem cells hiPSCs to differentiate into macrophages, characterized in that: The following steps are involved: (1) After pretreatment, pluripotent stem cells hiPSCs are inoculated into medium A to obtain embryoid bodies, and then medium B is used to continue culturing the embryoid bodies; The components of the culture medium A include: APEL II medium, BMP4, Activin A, TGF-β2 and Y-27632; The components of the culture medium B include: APEL II medium, BMP4, Activin A and TGF-β2; (2) replacing the medium with yolk sac induction medium and continuing the culture to obtain yolk sac organoids; (3) replacing the medium with blood island induction medium and continuing culturing to obtain yolk sac organoids containing blood islands; (4) replacing the medium with monocyte induction medium and continuing culturing to obtain monocytes; (5) Replace the medium with macrophage induction medium and continue culturing to obtain macrophages.

2. The cultivation method according to claim 1, characterized in that The components of the yolk sac induction medium include APEL II medium, BMP4, VEGF, SCF, IL-3, IL-6, IL-11, Flt-3L and TGF-β2.

3. The cultivation method according to claim 2, characterized in that In the yolk sac induction medium, the concentration of BMP4 is 30-50 ng / mL, the concentration of VEGF is 50-70 ng / mL, the concentration of SCF is 70-90 ng / mL, the concentration of IL-3 is 10-30 ng / mL, the concentration of IL-6 is 15-35 ng / mL, the concentration of IL-11 is 20-30 ng / mL, the concentration of Flt-3L is 20-30 ng / mL, and the concentration of TGF-β2 is 15-25 ng / mL.

4. The cultivation method according to claim 1, characterized in that The components of the blood island induction culture medium include APELII medium, bFGF, VEGF, SCF, IGF1, IL-3, TPO, M-CSF, GM-CSF and TGF-β2.

5. The cultivation method according to claim 4, characterized in that: In the blood island induction culture medium, the concentration of bFGF is 15-25 ng / mL, the concentration of VEGF is 55-65 ng / mL, the concentration of SCF is 30-50 ng / mL, the concentration of IGF1 is 5-15 ng / mL, the concentration of IL-3 is 20-40 ng / mL, the concentration of TPO is 25-35 ng / mL, the concentration of M-CSF is 10-30 ng / mL, the concentration of GM-CSF is 15-25 ng / mL, and the concentration of TGF-β2 is 10-30 ng / mL.

6. The cultivation method according to claim 1, characterized in that: The components of the monocyte induction medium (MP medium) include: XVIVO TM 15medium, bFGF, VEGF, SCF, IGF1, IL-3, M-CSF and GM-CSF.

7. The cultivation method according to claim 6, characterized in that: In the monocyte induction medium, the concentration of bFGF is 10-30 ng / mL, the concentration of VEGF is 50-70 ng / mL, the concentration of SCF is 35-45 ng / mL, the concentration of IGF1 is 5-15 ng / mL, the concentration of IL-3 is 25-35 ng / mL, the concentration of M-CSF is 35-45 ng / mL, and the concentration of GM-CSF is 30-50 ng / mL.

8. The cultivation method according to claim 1, characterized in that: The components of the macrophage induction medium include: 1640 (containing 10% FBS) / XVIVO TM 15. IGF1, IL-3, M-CSF and GM-CSF.

9. The cultivation method according to claim 8, characterized in that: In the macrophage induction medium, the concentration of IGF1 is 5-15 ng / mL, the concentration of IL-3 is 15-25 ng / mL, the concentration of M-CSF is 55-65 ng / mL, and the concentration of GM-CSF is 50-70 ng / mL.

10. The cultivation method according to claim 1, characterized in that: In (1), the pretreatment of the pluripotent stem cells hiPSC includes: culturing the hiPSC, adding a digestion solution, centrifuging and discarding the supernatant, and collecting the hiPSC after centrifugation.

Images