CN117448269A - A method and application for inducing rapid differentiation of human pluripotent stem cells into macrophages - Google Patents

Abstract

The invention relates to a method and application for inducing rapid differentiation of human pluripotent stem cells into macrophages, and relates to the field of biomedical technology. The method includes the following steps: (1) first stage, differentiation to form mesoderm; (2) second stage The first stage differentiates into hematopoietic endothelial cells; (3) the third stage differentiates into immature macrophages; (4) the fourth stage differentiates into mature macrophages. 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 need to go through the differentiation stage of HSPCs and can directly differentiate to obtain functionally mature macrophages. cells; it greatly simplifies the macrophage differentiation system, shortens the differentiation cycle, and significantly reduces production costs; therefore, the macrophage differentiation method provided by the present invention is more safe, reliable and stable, and can produce large quantities of functionally mature cells in a short time of macrophages.

Description

A method and application for inducing rapid differentiation of human pluripotent stem cells into macrophages

Technical field

The invention relates to the field of biomedicine technology, and specifically relates to a method and application for inducing rapid differentiation of human pluripotent stem cells into macrophages.

Background technique

Immune cell therapy is of great significance in tumor treatment, among which T cell therapy has significant anti-cancer effects. With the maturity of this therapy, a variety of T cell therapies have been approved by the US FDA. Although T cell therapy is currently effective against hematological tumors, it is not effective against solid tumors. The main reason is that there are multiple immunosuppressive mechanisms in the tumor microenvironment in solid tumor tissue, and T cells stay in the tumor tissue for a short time and have poor permeability, making it difficult for them to function. Therefore, it is of great significance to explore other potentially feasible immune cell therapies that can effectively combat solid tumors. Macrophages are mononuclear immune cells capable of phagocytosis. They can reside in solid tissues and play an important role in resisting pathogen invasion and maintaining the body's immune homeostasis. The occurrence and development of many diseases, such as tumors, neurodegenerative diseases, autoimmune diseases, etc., are accompanied by abnormal macrophage function. Therefore, macrophages have great potential in the treatment of related diseases. But at present, we first need to develop a differentiation system that is fast, efficient and can produce macrophages on a large scale to meet the demand for macrophage numbers in clinical treatment.

Differentiating macrophages from human pluripotent stem cells (hPSCs) is of special significance because it provides an unlimited source of cells for clinical applications and basic research on disease pathology. Human pluripotent stem cells are a type of cells with the ability to differentiate into various lineages and unlimited proliferation, mainly including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Using human pluripotent stem cells to differentiate to obtain macrophages is expected to solve the problem of limited sources of macrophages. Hematopoietic differentiation of hPSCs is a highly complex and finely regulated process, in which the interaction of different cytokines, stromal factors, signaling pathways, and transcription factors ultimately leads to the specification of the hematopoietic lineage. So far, most hPSCs hematopoietic differentiation protocols in vitro use a variety of cytokines or small molecules to simulate the regulation of signaling pathways at different stages of embryonic development to obtain corresponding blood cells. Domestic and foreign laboratories have now established a variety of differentiation methods from hPSCs to macrophages, which are mainly divided into: (1) embryoid body (EB) differentiation method; (2) trophoblast cell co-culture differentiation method; ( 3) Monolayer differentiation method.

The above three differentiation methods can all obtain macrophages, but the embryoid body (EB) differentiation method has problems such as cumbersome operation and unstable differentiation efficiency. The trophoblast cell co-culture differentiation method relies on heterogeneous materials (stromal cells OP9 (are mouse-derived cells), there are certain safety risks, so there are problems such as limitations that are difficult to overcome; while the monolayer differentiation method is relatively more stable and safe; using the monolayer differentiation method to differentiate to obtain macrophages generally requires first Human pluripotent stem cells are differentiated into hematopoietic stem/progenitor cells (HSPCs), and then the HSPCs undergo myeloid differentiation and finally differentiate into macrophages. This traditional single-layer differentiation method has a long cycle and is too expensive. In view of this, the present invention provides a method and application for inducing rapid differentiation of human pluripotent stem cells into macrophages.

Contents of the invention

The technical problem to be solved by the present invention is to provide a method and application for inducing rapid differentiation of human pluripotent stem cells into macrophages. The purpose is to provide an economical, stable, efficient and rapid monolayer differentiation method of primitive macrophages. The macrophages generated by this method can normally express the surface markers of classic macrophages and have normal phagocytic function and polarization. responsiveness and exhibit typical macrophage morphology.

The technical solutions of the present invention to solve the above technical problems are as follows:

The first aspect provides a method for inducing rapid differentiation of human pluripotent stem cells into macrophages, including the following steps:

(1) The first stage, differentiation to form mesoderm: take human pluripotent stem cells, use the first stage induction medium (HDM1), induce differentiation to obtain mesoderm; the first stage induction medium includes HDM, BMP4, Activin A , bFGF, CHIR99021, A8-301, and IWR-1-endo;

(2) The second stage, differentiation to form hematopoietic endothelial cells: replace the second stage induction medium (HDM2), culture the mesoderm, and induce hematopoietic endothelial cells; the second stage induction medium includes HDM, VEGF and bFGF ;

(3) The third stage, differentiation to form immature macrophages: replace the third stage induction medium (HDM3), culture the hematopoietic endothelial cells, and induce suspended immature macrophages; the third stage induction medium includes StemPro-34 SFM, Flt-3, IL3 and GM-CSF; Naïve macrophages can be continuously produced from day 12 until around day 30, and suspended naïve macrophages can be collected every 3-4 days;

(4) The fourth stage, differentiation to form mature macrophages: the immature macrophages are cultured in the fourth stage medium (HDM4) to obtain mature macrophages (Macrophage); the fourth stage induction medium includes RPMI 1640 and GM-CSF.

The beneficial effects of the present invention are: 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 need to go through the differentiation stage of HSPCs and can directly Functionally mature macrophages are obtained through differentiation; the macrophage differentiation system is greatly simplified, the differentiation cycle is shortened, and the production cost can be significantly reduced; therefore, the macrophage differentiation method provided by the present invention is more safe, reliable and stable, and can be used in a short time. time to produce functionally mature macrophages in large quantities.

On the basis of the above technical solution, the present invention can also make the following improvements.

Further, the conditions for inducing differentiation using the first stage induction medium in step (1) are: the temperature is 37±0.5°C, the volume fraction of _CO2 is 5%, the oxygen content is normoxic, and the time is 1-3 days ;

In step (2), the second stage induction medium is used, and the conditions for culturing the mesoderm to induce hematopoietic endothelial cells are: the temperature is 37±0.5°C, the volume fraction of _CO2 is 5%, and the oxygen content is normoxic. , the time is 3-5 days;

In step (3), the third stage induction medium is used to culture the hematopoietic endothelial cells, and the conditions for inducing immature macrophages are: the temperature is 37±0.5°C, the volume fraction of _CO2 is 5%, and the oxygen content For normoxia, the time is 3-5 days;

The conditions for obtaining mature macrophages using the fourth-stage culture medium in step (4) are: the temperature is 37±0.5°C, the volume fraction of _CO2 is 5%, the oxygen content is normoxic, and the time is 4-6 sky.

Further, the conditions for inducing differentiation using the first-stage induction medium in step (1) are: the temperature is 37±0.5°C, the volume fraction of _CO2 is 5%, the oxygen content is normoxic, and the time is 2 days;

In step (2), the second stage induction medium is used, and the conditions for culturing the mesoderm to induce hematopoietic endothelial cells are: the temperature is 37±0.5°C, the volume fraction of _CO2 is 5%, and the oxygen content is normoxic. , the time is 4 days;

In step (3), the third stage induction medium is used to culture the hematopoietic endothelial cells, and the conditions for inducing immature macrophages are: the temperature is 37±0.5°C, the volume fraction of _CO2 is 5%, and the oxygen content For normoxia, the time is 4 days;

In step (4), the conditions for obtaining mature macrophages using the fourth-stage culture medium are: the temperature is 37±0.5°C, the volume fraction of _CO2 is 5%, the oxygen content is normoxic, and the time is 5 days.

Further, the first stage induction medium includes HDM, 20-60ng/mL BMP4, 15-45ng/mLActivin A, 10-30ng/mL bFGF, 3-9μM CHIR99021, 0.5-1.5μM A8-301 and 0.5-1.5 μM IWR-1-endo;

The second stage induction medium includes HDM, 20-60ng/mL VEGF and 25-75ng/mL bFGF;

The third stage induction medium includes StemPro-34 SFM, 5-15ng/mL Flt-3, 5-15ng/mLIL3 and 2.5-7.5ng/mL GM-CSF;

The fourth stage induction medium includes RPMI 1640 and 5-15ng/mL GM-CSF.

Further, the first stage induction medium includes HDM, 40ng/mL BMP4, 30ng/mL Activin A, 20ng/mL bFGF, 6μM CHIR99021, 1μM A8-301 and 1μM IWR-1-endo;

The second stage induction medium includes HDM, 40ng/mL VEGF and 50ng/mL bFGF;

The third stage induction medium includes StemPro-34 SFM, 10ng/mL Flt-3, 10ng/mL IL3 and 5ng/mL GM-CSF;

The fourth stage induction medium includes RPMI 1640 and 10ng/mL GM-CSF.

Further, the HDM includes DMEM/F12 basic medium, P/S, ITS-G and vitamin C. Specifically, the HDM includes DMEM/F12 basic medium, 1wt.% P/S, 1wt.% ITS-G and 70ug/ml vitamin C.

Further, the seeding density of human pluripotent stem cells described in step (1) is 150,000-175,000/mL. There are no density requirements for other phases.

Further, the human pluripotent stem cells are pre-processed before undergoing the first stage of differentiation to form mesoderm; the pre-processing includes the following specific steps: first digest the human pluripotent stem cells, and then add Y-containing -27632 culture medium for 12-36 hours.

Furthermore, the digestion reagent used in the digestion process is Accutase digestion solution, and the digestion time at room temperature is 3-5 minutes; the culture medium containing Y-27632 includes mTeSR1 and Y-27632. Specifically, the medium containing Y-27632 included 2 mL mTeSR1 and 10 μM Y-27632.

In a second aspect, an application of macrophages is provided, in which the macrophages differentiated by the method are used in medicines for the treatment of diseases with abnormal macrophage function.

Description of the drawings

Figure 1 is a schematic flowchart of the differentiation process from human pluripotent stem cells to macrophages and bright field photos of key nodes according to the present invention. The scale bar is 100 μm;

Figure 2 shows the statistics of macrophage counts for each harvest during the differentiation process of the present invention;

Figure 3 shows the expression of classic surface markers of mature macrophages detected by flow cytometry according to the present invention;

Figure 4 is a morphological display of mature macrophages of the present invention (Giemsa staining), the scale bar is 10 μm;

Figure 5 shows the fluorescent latex microsphere phagocytosis experiment of the present invention, photo display and flow cytometric detection. The latex microsphere fluorescence channel is PE, and the scale bar is 10 μm;

Figure 6 is a bright field photo of mature macrophages before and after polarization of the present invention, the scale bar is 50 μm;

Figure 7 shows the expression of M1 and M2 related surface markers CD80, CD86, CD163 and CD206 detected by flow cytometry of the present invention before and after macrophage polarization;

Figure 8 shows the RNA expression levels of classic genes related to M1 and M2 detected by real-time fluorescence quantitative PCR of the present invention before and after macrophage polarization. The error bars represent the mean ± standard deviation of three repeated experiments. Unpaired two-tailed T test was used for significance analysis, **** means P<0.0001.

Detailed ways

The principles and features of the present invention are described below. The examples cited are only used to explain the present invention and are not intended to limit the scope of the present invention. If specific techniques or conditions are not specified in the examples, the techniques or conditions described in literature in the field shall be followed, or the product instructions shall be followed. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased through regular channels.

Description of the sources of reagents and materials used in the following examples:

BMP4 (Peprotech); Activin A (Sino Biological); bFGF (Sino Biological); CHIR99021 (selleck); A8-301 (selleck); IWR-1-endo (selleck); VEGF (Sino Biological); StemPro-34 SFM ( gibco); Flt-3 (Peprotech); IL3 (Peprotech); GM-CSF (SinoBiological); RPMI 1640 (gibco); mTeSR1 (gibco); Y-27632 (selleck); DMEM/F12 (gibco); P/S (gibco); ITS-G (gibco); Vitamin C (gibco); LPS-EK (Invivogen); INF-γ (SinoBiological).

Example

1. Inducing human pluripotent stem cells to rapidly differentiate into macrophages

A method for inducing rapid differentiation of human pluripotent stem cells into macrophages, including the following steps:

(1) The first stage, differentiation to form mesoderm: take human pluripotent stem cells, use the first stage induction medium (HDM1), induce differentiation to obtain mesoderm; the first stage induction medium includes HDM, BMP4, Activin A , bFGF, CHIR99021, A8-301, and IWR-1-endo;

(2) The second stage, differentiation to form hematopoietic endothelial cells: replace the second stage induction medium (HDM2), culture the mesoderm, and induce hematopoietic endothelial cells; the second stage induction medium includes HDM, VEGF and bFGF ;

(3) The third stage, differentiation to form immature macrophages: replace the third stage induction medium (HDM3), culture the hematopoietic endothelial cells, and induce suspended immature macrophages; the third stage induction medium includes StemPro-34 SFM, Flt-3, IL3 and GM-CSF; Naïve macrophages can be continuously produced from day 12 until around day 30, and suspended naïve macrophages can be collected every 3-4 days;

(4) The fourth stage, differentiation to form mature macrophages: the immature macrophages are cultured in the fourth stage medium (HDM4) to obtain mature macrophages (Macrophage); the fourth stage induction medium includes RPMI 1640 and GM-CSF.

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 need to go through the differentiation stage of HSPCs and can directly differentiate to obtain functionally mature macrophages. cells; greatly simplifies the macrophage differentiation system, shortens the differentiation cycle, and significantly reduces production costs.

Preferably, the conditions for inducing differentiation using the first stage induction medium in step (1) are: the temperature is 37±0.5°C, the volume fraction of _CO2 is 5%, the oxygen content is normoxic, and the time is 1-3 sky;

In step (2), the second stage induction medium is used, and the conditions for culturing the mesoderm to induce hematopoietic endothelial cells are: the temperature is 37±0.5°C, the volume fraction of _CO2 is 5%, and the oxygen content is normoxic. , the time is 3-5 days;

In step (3), the third stage induction medium is used to culture the hematopoietic endothelial cells, and the conditions for inducing immature macrophages are: the temperature is 37±0.5°C, the volume fraction of _CO2 is 5%, and the oxygen content For normoxia, the time is 3-5 days;

The conditions for obtaining mature macrophages using the fourth-stage culture medium in step (4) are: the temperature is 37±0.5°C, the volume fraction of _CO2 is 5%, the oxygen content is normoxic, and the time is 4-6 sky.

Preferably, the first stage induction medium includes HDM, 20-60ng/mL BMP4, 15-45ng/mL Activin A, 10-30ng/mL bFGF, 3-9μM CHIR99021, 0.5-1.5μM A8-301 and 0.5- 1.5μM IWR-1-endo;

The second stage induction medium includes HDM, 20-60ng/mL VEGF and 25-75ng/mL bFGF;

The third stage induction medium includes StemPro-34 SFM, 5-15ng/mL Flt-3, 5-15ng/mLIL3 and 2.5-7.5ng/mL GM-CSF;

The fourth stage induction medium includes RPMI 1640 and 5-15ng/mL GM-CSF.

Preferably, the HDM includes DMEM/F12 basic medium, P/S, ITS-G and vitamin C.

Taking a 6-well plate (2mL/well) as an example to specifically illustrate the differentiation method of the present invention, it includes the following steps:

(1) Digest hESCs/iPSCs with a density of 80-90% in good condition (the hESCs used are H1 cell lines purchased from weiCell) with Accutase (cell digestion solution, Yisheng Biotechnology) for 5 minutes, and use F12 medium (Punuo) race) to stop digestion, collect the cell suspension, centrifuge at 500g for 3 minutes, remove the supernatant, resuspend in mTeSR1 culture medium and count, inoculate into a six-well plate at a density of 300,000-350,000 cells per well, use 2mL mTeSR1+ Culture for 24 hours with 10 μM Y-27632. Y-27632 is a selective ROCK inhibitor that can effectively reduce cell death and improve stem cell survival rate;

(2) The first stage, differentiation to form mesoderm: On day 0 (D0), aspirate the medium and wash it with 1mL DMEM/F12, replace it with human pluripotent stem cells, and use the first stage induction medium (HDM1) 2mL, induce differentiation for 48 hours (2 days) to obtain mesoderm; the first stage induction medium (HDM1) includes HDM, 40ng/mL BMP4, 30ng/mL Activin A, 20ng/mL bFGF, 6μM CHIR99021, 1μM A8- 301 and 1 μM IWR-1-endo;

(3) The second stage, differentiation to form hematopoietic endothelial cells: On day 2 (D2), absorb the medium and wash it with 1mL DMEM/F12, replace the second stage induction medium (HDM2) with 2mL, and culture as described Hemogenic endothelial cells were induced 4 days after the germ layer; the second stage induction medium (HDM2) included HDM, 40ng/mL VEGF and 50ng/mL bFGF;

(4) The third stage, differentiation to form immature macrophages: on day 8 (D8), remove the medium and wash it with 1mL DMEM/F12, replace with 2mL of the third stage induction medium (HDM3), and culture for 4 days (To day 12) The hematopoietic endothelial cells were induced to obtain suspended naive macrophages; the third stage induction medium (HDM3) included StemPro-34 SFM, 10ng/mLFlt-3, 10ng/mL IL3 and 5ng/mL GM. -CSF; Naïve macrophages can be continuously produced from day 12 until about day 30, and suspended naïve macrophages can be collected every 3-4 days;

(5) The fourth stage, differentiation to form mature macrophages: collect the suspended immature macrophages, centrifuge at 500g for 3 minutes, remove the supernatant, transfer to a new 6-well plate, and use the immature macrophages in the fourth stage. Culture medium (HDM4) 2mL, culture, and mature macrophages (Macrophage) are obtained after 5 days; the fourth stage induction medium (HDM4) includes RPMI 1640 and 10ng/mL GM-CSF.

Wherein, the HDM includes DMEM/F12 basic medium, 1wt.% P/S, 1wt.% ITS-G and 70ug/ml vitamin C.

2. Experimental testing

2.1 Cell morphology observation

Macrophage morphology was observed using Giemsa stained slides. First, collect 500,000-800,000 cells, spin them out, wash them three times with FACS, and centrifuge them at 500g for 3 minutes each time. After centrifugation, remove the supernatant and resuspend the cell pellet in 200 μL FACS. Put the clean blank slide and absorbent paper into the slide slot, add the cell suspension into the sample chamber, and centrifuge at 300g for 5 minutes.

Take out the shaken slide and let it dry naturally. Observe the cell attachment area under the light. Use a hydrophobic pen to circle the cell attachment area to prepare for Giemsa staining. Add liquid A dropwise to the pre-drawn circle and let it react for two minutes. Then add an equal volume of liquid B dropwise. Shake gently and let it sit for 10 minutes. Rinse the slide slowly with running water from top to bottom to take away the excess dye. . After drying, you can observe and take pictures. (Wright-Giemsa staining solution 3*250mL Baso/Basso, BA-4034)

2.2 Flow cytometry

Digest the sample according to the cell type selection to ensure that a single cell suspension is obtained before final staining with antibodies. Suspended cells can be sampled directly, and adherent cells are digested with Accutase for 5 minutes. Collect the cells to be detected in a 1.5 mL EP tube, resuspend and wash in FACS (DPBS + 2% FBS), and centrifuge at 500g for 3 minutes. Remove the supernatant and resuspend the cell pellet in 100 μL FACS (control the cell density to not exceed 10 ⁶ cells per 100 μL FACS). Add the flow cytometry antibodies to be detected in a ratio of 1:100. If multiple samples need to be detected at the same time, they can be prepared in advance. mix. Incubate in the dark at 4°C for 20-30 minutes or at room temperature for 15 minutes. After the incubation, add 1mL of FACS to the EP tube to terminate the staining and wash away non-specific binding. Centrifuge at 500g for 3 minutes, remove the supernatant, and resuspend the cell pellet with 100-200μL FACS. On-machine detection was performed using flow cytometer CytoFLEX. The flow cytometry antibodies used are as follows: PE-Cy7 anti-human CD45 (Biolegend, 304016); APC anti-human CD11b (Biolegend, 301310); FITC anti-human CD14 (BD, 555397); PE anti-human CD163 (BD, 560933 ); APC/Cyanine7 anti-human CD11b (Biolegend, 301342); BV421 anti-human CD206 (BD, 566281); PE-Cyanine7 Anti-Human CD14 (eBioscience, 25-0149-42); APC anti-humanCD80 (Biolegend, 305219); APC anti-human CD86 (BD, 555660). Latex beads (sigma, L3030-1ML).

2.3 Fluorescent latex microsphere phagocytosis experiment

Add red fluorescent latex microspheres (Beads) to the culture dish cultured with macrophages at a ratio of 1:100, mix well, and incubate the Beads and macrophages for 2 hours. Then collect the cells for flow cytometry and put them on the machine. Washed three times with FACS before use. Beads have red light and are detected using the PE channel.

(The number of macrophages in a 24-well plate can range from 200,000 to 500,000/well)

2.4 Polarization of macrophages

Mature macrophages were seeded in a 24-well plate one day in advance (500,000/well), and 1 μg/mL LPS-EK and 40 ng/mL INF-γ were added to the culture medium to polarize the macrophages toward M1; Macrophages were polarized toward M2 by adding 20 ng/mL of IL4 to the culture medium. Cells were collected after 24 hours of polarization. One part was used for flow cytometric detection of M1/2-related surface markers, and the other part was RNA extracted. After reverse transcription, the expression levels of M1/2-related genes were detected by Q-PCR.

The Q-PCR primers are shown in Table 1:

Table 1Q-PCR primers

Note: -q- indicates q-PCR primers

2.5RNA extraction and reverse transcription

Collect the cells to be processed and extract RNA according to the RaPure Total RNA Micro Kit (R4012-03) tissue cell RNA microextraction kit. Reverse 2ug RNA according to TOYOBO ReverTra Ace kit.

2.6 Q-PCR detection of RNA expression levels of classic genes in M1/2 macrophages

Dilute the cDNA obtained by reverse transcription 50 times with ddH ₂ O, configure the Q-PCR reaction system according to Table 2, and use the program set in the BIO-RAD instrument to perform real-time fluorescence quantitative PCR to detect the mRNA expression level of the specified gene. For details, refer to chamQSYBR qPCR master mix (Norvizan, Q311-03) kit.

Table 2 Q-PCR reaction system

Components Volume 10μL 2×SYBR Green Mix 5μL Target gene-Q-F/R MIX 0.8μL cDNA 4μL ddH ₂ O 0.2μL

3. Experimental results

Among them, Figure 1 is a schematic flow chart of the differentiation of human pluripotent stem cells into macrophages and bright field photos of key nodes according to the present invention. The scale bar is 100 μm. Figure 1 shows that human pluripotent stem cells can be quickly induced to successfully differentiate through the method of the present invention. for macrophages.

Figure 2 shows the statistics of macrophage counts for each harvest during the differentiation process of the present invention; Figure 2 shows that a large number of macrophages can ultimately be harvested in multiple batches through the method of the present invention.

Figure 3 shows the expression of classic surface markers of mature macrophages detected by flow cytometry according to the present invention; Figure 3 shows that macrophages derived from human pluripotent stem cells differentiated by the method of the present invention highly express classic surface markers of macrophages. The cells were found to be mature macrophages.

Figure 4 is a diagram showing the morphology of mature macrophages of the present invention (Giemsa staining), the scale bar is 10 μm; Figure 4 shows that the morphology of macrophages derived from human pluripotent stem cells differentiated by the method of the present invention is normal macrophages. form.

Figure 5 shows the fluorescent latex microsphere phagocytosis experiment of the present invention, photo display and flow cytometric detection. The latex microsphere fluorescence channel is PE, and the scale bar is 10 μm. Figure 5 shows that macrophages derived from human pluripotent stem cells differentiated by the method of the present invention are Cells have phagocytic ability.

Figure 6 is a bright field photo of mature macrophages before and after polarization of the present invention, scale bar 50 μm; Figure 6 shows that macrophages derived from human pluripotent stem cells obtained through differentiation by the method of the present invention have polarization ability.

Figure 7 shows the expression of M1 and M2-related surface markers CD80, CD86, CD163, and CD206 detected by flow cytometry before and after macrophage polarization according to the present invention; Figure 7 shows that human pluripotent stem cells derived from differentiation obtained by the method of the present invention Macrophages can respond to polarizing signals and have the ability to polarize into M1 and M2 macrophages.

Figure 8 shows the RNA expression levels of classic genes related to M1 and M2 detected by real-time fluorescence quantitative PCR of the present invention before and after macrophage polarization. The error bars represent the mean ± standard deviation of three repeated experiments. Unpaired two-tailed T test was used for significance analysis, **** means P<0.0001; Figure 8 shows that macrophages derived from human pluripotent stem cells differentiated by the method of the present invention can be polarized into M1 and M2 macrophages and express related genes.

In summary, the present invention provides a novel monolayer differentiation method to obtain macrophages derived from human pluripotent stem cells. This method does not need to go through the differentiation stage of HSPCs and can directly differentiate to obtain functionally mature macrophages; extremely The macrophage differentiation system is simplified, the differentiation cycle is shortened, and the production cost can be significantly reduced.

Although the embodiments of the present invention have been shown and described above, it can be understood that the above-mentioned embodiments are illustrative and should not be construed as limitations of the present invention. Those of ordinary skill in the art can make modifications to the above-mentioned embodiments within the scope of the present invention. The embodiments are subject to changes, modifications, substitutions and variations.

Claims (10)

1. A method for inducing rapid differentiation of human pluripotent stem cells into macrophages, comprising the steps of:

(1) In the first stage, differentiation forms mesoderm: taking human pluripotent stem cells, and adopting a first-stage induction culture medium to induce differentiation to obtain mesoderm; the first stage induction medium comprises HDM, BMP4, activin A, bFGF, CHIR99021, A8-301, and IWR-1-endo;

(2) In the second stage, differentiation into hematogenic endothelial cells: changing a second-stage induction culture medium, culturing the mesoderm, and inducing to obtain hematogenic endothelial cells; the second stage induction medium comprises HDM, VEGF and bFGF;

(3) In the third stage, differentiation into naive macrophages: changing a third-stage induction culture medium, culturing the hematogenic endothelial cells, and inducing to obtain suspended immature macrophages; the third stage induction medium comprises Stempro-34 SFM, flt-3, IL3 and GM-CSF;

(4) Fourth stage, differentiation into mature macrophages: culturing the immature macrophages with a fourth stage medium to obtain mature macrophages; the fourth stage induction medium included RPMI 1640 and GM-CSF.

2. The method of claim 1, wherein the conditions for inducing differentiation of human pluripotent stem cells into macrophages using the first stage induction medium in step (1) are: the temperature is 37+/-0.5 ℃ and CO ₂ The volume fraction of (2) is 5%, the oxygen content is normoxic, and the time is 1-3 days;

in the step (2), the conditions for culturing the mesoderm to induce the hematogenic endothelial cells are as follows: the temperature is 37+/-0.5 ℃ and CO ₂ The volume fraction of (2) is 5%, the oxygen content is normal oxygen, and the time is 3-5 days;

in the step (3), the third stage induction culture medium is adopted to culture the hematogenic endothelial cells, and the condition of inducing to obtain the immature macrophages is as follows: the temperature is 37+/-0.5 ℃ and CO ₂ The volume fraction of (2) is 5%, the oxygen content is normal oxygen, and the time is 3-5 days;

the conditions for obtaining mature macrophages by culturing in the fourth stage medium in step (4) are as follows: the temperature is 37+/-0.5 ℃ and CO ₂ The volume fraction of (2) is 5%, the oxygen content is normoxic, and the time is 4-6 days.

3. The method of claim 1, wherein the conditions for inducing differentiation of human pluripotent stem cells into macrophages using the first stage induction medium in step (1) are: the temperature is 37+/-0.5 ℃ and CO ₂ The volume fraction of (2) is 5%, the oxygen content is normoxic, and the time is 2 days;

in the step (2), the conditions for culturing the mesoderm to induce the hematogenic endothelial cells are as follows: the temperature is 37+/-0.5 ℃ and CO ₂ The volume fraction of (2) is 5%, the oxygen content is normoxic, and the time is 4 days;

in the step (3), the third stage induction culture medium is adopted to culture the hematogenic endothelial cells, and the condition of inducing to obtain the immature macrophages is as follows: the temperature is 37+/-0.5 ℃ and CO ₂ The volume fraction of (2) is 5%, the oxygen content is normoxic, and the time is 4 days;

the conditions for obtaining mature macrophages by culturing in the fourth stage medium in step (4) are as follows: the temperature is 37+/-0.5 ℃ and CO ₂ The volume fraction of (2) was 5%, the oxygen content was normoxic, and the time was 5 days.

4. The method of claim 1, wherein the first stage induction medium comprises HDM, 20-60ng/mL BMP4, 15-45ng/mL Activin A, 10-30ng/mL bFGF, 3-9. Mu.M CHIR99021, 0.5-1.5. Mu.M MA8-301, and 0.5-1.5. Mu.M IWR-1-endo;

the second-stage induction culture medium comprises HDM, 20-60ng/mL VEGF and 25-75ng/mL bFGF;

the third stage induction culture medium comprises StemPro-34 SFM, 5-15ng/mL Flt-3, 5-15ng/mL IL3 and 2.5-7.5ng/mL GM-CSF;

the fourth stage induction medium comprises RPMI 1640 and 5-15ng/mL GM-CSF.

5. The method of claim 1, wherein the first stage induction medium comprises HDM, 40ng/mL BMP4, 30ng/mL Activin a, 20ng/mL bFGF, 6 μΜ CHIR99021, 1 μ M A8-301, and 1 μmiwr-1-endo;

the second-stage induction medium comprises HDM, 40ng/mL VEGF and 50ng/mL bFGF;

the third stage induction medium comprises StemPro-34 SFM, 10ng/mL Flt-3, 10ng/mL IL3 and 5ng/mL GM-CSF;

the fourth stage induction medium comprises RPMI 1640 and 10ng/mL GM-CSF.

6. A method of inducing rapid differentiation of human pluripotent stem cells into macrophages according to claim 4 or 5, wherein the HDM comprises DMEM/F12 basal medium, P/S, ITS-G and vitamin C.

7. The method of claim 1, wherein the human pluripotent stem cells in step (1) are seeded at a density of 15-17.5 ten thousand/mL.

8. The method of claim 1, wherein the human pluripotent stem cells are pre-treated prior to undergoing the first stage of differentiation to form mesoderm; the pretreatment comprises the following specific steps: the human pluripotent stem cells are subjected to digestion treatment and then cultured in a medium containing Y-27632 for 12-36 hours.

9. The method for inducing rapid differentiation of human pluripotent stem cells into macrophages according to claim 8, wherein the digestion treatment adopts an Accutase digestive juice, and the digestion time is 3-5 minutes at normal temperature; the medium containing Y-27632 includes mTESR1 and Y-27632.

10. Use of macrophages differentiated according to any one of claims 1 to 9 in a medicament for the treatment of diseases in which macrophages are dysfunctional.