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

Abstract

The present invention relates to a method for inducing the rapid differentiation of human pluripotent stem cells into macrophages and its application, belonging to the field of biomedical technology. The method comprises the following steps: (1) In the first stage, mesoderm is differentiated; (2) In the second stage, hematopoietic endothelial cells are differentiated; (3) In the third stage, immature macrophages are differentiated; (4) In the fourth stage, mature macrophages are differentiated. 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; it greatly simplifies the macrophage differentiation system, shortens the differentiation cycle, and can significantly reduce the production cost; therefore, the macrophage differentiation method provided by the present invention is safer, more reliable and stable, and can produce functionally mature macrophages in large quantities in a short time.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly relates to a method for inducing rapid differentiation of human pluripotent stem cells into macrophages and applications thereof. Background Art

[0002] Immune cell therapy is of great significance in tumor treatment. Among them, 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 current T cell therapy has good effects on hematological tumors, its efficacy for solid tumors is poor. The main reason is that there are various immunosuppressive mechanisms in the tumor microenvironment of solid tumor tissues, and T cells stay in the tumor tissue for a short time and have poor permeability, making it difficult to play a role. 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 with phagocytic functions, can reside in solid tissues, and play an important role in resisting pathogen invasion and maintaining the immune homeostasis balance of the body. The occurrence and development of many diseases such as tumors, neurodegenerative diseases, and autoimmune diseases are accompanied by abnormal macrophage functions. Therefore, macrophages have great potential in the treatment of related diseases. However, at present, we first need to develop a rapid, efficient and large-scale macrophage differentiation system to meet the demand for the number of macrophages in clinical treatment.

[0003] Differentiating macrophages from human pluripotent stem cells (hPSCs) has special significance because it provides an unlimited cell source 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 ability, mainly including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Differentiating macrophages from human pluripotent stem cells is expected to solve the problem of limited macrophage sources. The 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 specialization of hematopoietic lineages. So far, most in vitro hematopoietic differentiation protocols for hPSCs have used a variety of cytokines or small molecules to mimic the regulation of signaling pathways at different stages of embryonic development to obtain corresponding blood cells. A variety of differentiation methods from hPSCs to macrophages have been established in domestic and foreign laboratories, mainly including: (1) embryoid body (EB) differentiation method; (2) trophoblast cell co-culture differentiation method; (3) monolayer differentiation method.

[0004] The above three differentiation methods can all obtain macrophages. However, the embryoid body (EB) differentiation method has problems such as cumbersome operation and unstable differentiation efficiency. The co-culture differentiation method of trophoblast cells relies on the use of xenogeneic materials (the stromal cell OP9 is a murine cell), which has certain safety risks and thus has problems such as insurmountable limitations. The monolayer differentiation method is relatively more stable and safe. To differentiate macrophages using the monolayer differentiation method, human pluripotent stem cells generally need to be first differentiated into hematopoietic stem / progenitor cells (HSPCs), and then the HSPCs are differentiated myeloidly to finally obtain macrophages. This traditional monolayer differentiation method has a long cycle and high cost. In view of this, the present invention provides a method and application for inducing the rapid differentiation of human pluripotent stem cells into macrophages. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method and application for inducing the rapid differentiation of human pluripotent stem cells into macrophages. The aim is to provide a set of economical, stable, efficient, and rapid monolayer differentiation methods for primitive macrophages. The macrophages produced by this method can normally express the surface markers of classical macrophages, have normal phagocytic function and polarization response ability, and present a typical macrophage morphology.

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] First aspect, a method for inducing the rapid differentiation of human pluripotent stem cells into macrophages is provided, including the following steps:

[0008] (1) In the first stage, differentiating to form mesoderm: Take human pluripotent stem cells and use the first-stage induction medium (HDM1) to induce differentiation to obtain mesoderm; the first-stage induction medium includes HDM, BMP4, Activin A, bFGF, CHIR99021, A8-301, and IWR-1-endo;

[0009] (2) In the second stage, differentiating to form hemogenic endothelium: Replace it with the second-stage induction medium (HDM2), culture the mesoderm, and induce to obtain hemogenic endothelium; the second-stage induction medium includes HDM, VEGF, and bFGF;

[0010] (3) In the third stage, differentiating to form immature macrophages: Replace it with the third-stage induction medium (HDM3), culture the hemogenic endothelium, and induce to obtain suspended immature macrophages; the third-stage induction medium includes StemPro-34 SFM, Flt-3, IL3, and GM-CSF; Starting from the 12th day, immature macrophages can be continuously produced until about the 30th day, and subsequently, the suspended immature macrophages can be collected every 3 - 4 days;

[0011] (4) Fourth stage, differentiating into mature macrophages: Culturing the naive macrophages with the fourth-stage culture medium (HDM4) to obtain mature macrophages (Macrophage); the fourth-stage induction culture medium includes RPMI 1640 and GM-CSF.

[0012] The beneficial effects of the present invention are as follows: 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; 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 stable, and can produce functionally mature macrophages in large quantities in a short time.

[0013] Based on the above technical solutions, the present invention can also be improved as follows.

[0014] Further, the conditions for inducing differentiation with the first-stage induction culture medium in step (1) are: temperature is 37 ± 0.5 °C, the volume fraction of CO2 is 5%, the oxygen content is normal oxygen, and the time is 1 - 3 days;

[0015] The conditions for culturing the mesoderm with the second-stage induction culture medium to induce the generation of hematopoietic endothelial cells in step (2) are: temperature is 37 ± 0.5 °C, the volume fraction of CO2 is 5%, the oxygen content is normal oxygen, and the time is 3 - 5 days;

[0016] The conditions for culturing the hematopoietic endothelial cells with the third-stage induction culture medium to induce the generation of naive macrophages in step (3) are: temperature is 37 ± 0.5 °C, the volume fraction of CO2 is 5%, the oxygen content is normal oxygen, and the time is 3 - 5 days;

[0017] The conditions for culturing with the fourth-stage culture medium to obtain mature macrophages in step (4) are: temperature is 37 ± 0.5 °C, the volume fraction of CO2 is 5%, the oxygen content is normal oxygen, and the time is 4 - 6 days.

[0018] Further, the conditions for inducing differentiation with the first-stage induction culture medium in step (1) are: temperature is 37 ± 0.5 °C, the volume fraction of CO2 is 5%, the oxygen content is normal oxygen, and the time is 2 days;

[0019] The conditions for culturing the mesoderm with the second-stage induction culture medium to induce the generation of hematopoietic endothelial cells in step (2) are: temperature is 37 ± 0.5 °C, the volume fraction of CO2 is 5%, the oxygen content is normal oxygen, and the time is 4 days;

[0020] In step (3), when culturing the hematopoietic endothelial cells with the third-stage induction medium to induce naive macrophages, the conditions are as follows: the temperature is 37 ± 0.5 °C, the volume fraction of CO2 is 5%, the oxygen content is normal oxygen, and the time is 4 days;

[0021] In step (4), when culturing to obtain mature macrophages with the fourth-stage medium, the conditions are as follows: the temperature is 37 ± 0.5 °C, the volume fraction of CO2 is 5%, the oxygen content is normal oxygen, and the time is 5 days.

[0022] Furthermore, the first-stage induction medium includes HDM, 20 - 60 ng / mL BMP4, 15 - 45 ng / mL Activin A, 10 - 30 ng / mL bFGF, 3 - 9 μM CHIR99021, 0.5 - 1.5 μM A8 - 301, and 0.5 - 1.5 μM IWR-1-endo;

[0023] The second-stage induction medium includes HDM, 20 - 60 ng / mL VEGF, and 25 - 75 ng / mL bFGF;

[0024] The third-stage induction medium includes StemPro-34 SFM, 5 - 15 ng / mL Flt-3, 5 - 15 ng / mL IL3, and 2.5 - 7.5 ng / mL GM-CSF;

[0025] The fourth-stage induction medium includes RPMI 1640 and 5 - 15 ng / mL GM-CSF.

[0026] Furthermore, the first-stage induction medium includes HDM, 40 ng / mL BMP4, 30 ng / mL Activin A, 20 ng / mL bFGF, 6 μM CHIR99021, 1 μM A8 - 301, and 1 μM IWR-1-endo;

[0027] The second-stage induction medium includes HDM, 40 ng / mL VEGF, and 50 ng / mL bFGF;

[0028] The third-stage induction medium includes StemPro-34 SFM, 10 ng / mL Flt-3, 10 ng / mL IL3, and 5 ng / mL GM-CSF;

[0029] The fourth-stage induction medium includes RPMI 1640 and 10 ng / mL GM-CSF.

[0030] Further, the HDM includes DMEM / F12 basal medium, P / S, ITS-G, and vitamin C. Specifically, the HDM includes DMEM / F12 basal medium, 1 wt.% P / S, 1 wt.% ITS-G, and 70 μg / ml vitamin C.

[0031] Further, in step (1), the seeding density of the human pluripotent stem cells is 150,000 - 175,000 cells / mL. There is no density requirement for other stages.

[0032] Further, the human pluripotent stem cells are pretreated before differentiating into mesoderm in the first stage; the pretreatment includes the following specific steps: first, the human pluripotent stem cells are digested, and then cultured in a medium containing Y-27632 for 12 - 36 hours.

[0033] Further, the digestion reagent used for the digestion treatment is Accutase digestion solution, and the digestion time at room temperature is 3 - 5 minutes; the medium containing Y-27632 includes mTeSR1 and Y-27632. Specifically, the medium containing Y-27632 includes 2 mL mTeSR1 and 10 μM Y-27632.

[0034] In the second aspect, an application of macrophages is provided, and the macrophages differentiated by the method are used in drugs for the treatment of diseases with abnormal macrophage function. Description of the Drawings

[0035] Figure 1 It is a schematic diagram of the process of differentiating human pluripotent stem cells into macrophages and a bright-field photo of key nodes in the present invention, with a scale of 100 μm;

[0036] Figure 2 It is the statistical situation of macrophage counting every time during the differentiation process of the present invention;

[0037] Figure 3 It is the expression situation of classical surface markers of mature macrophages detected by flow cytometry in the present invention;

[0038] Figure 4 It is a morphological display diagram of mature macrophages in the present invention (Giemsa staining), with a scale of 10 μm;

[0039] Figure 5 It is the fluorescence latex microsphere phagocytosis experiment in the present invention, photo display and flow cytometry detection. The fluorescence channel of the latex microsphere is PE, with a scale of 10 μm;

[0040] Figure 6 It is the bright-field photos of mature macrophages before and after polarization in the present invention, with a scale of 50 μm;

[0041] Figure 7 This invention is for detecting the expression of surface markers CD80, CD86 and CD163, CD206 related to M1 and M2 before and after macrophage polarization by flow cytometry;

[0042] Figure 8 This invention is for detecting the expression levels of classical genes related to M1 and M2 before and after macrophage polarization by real-time fluorescence quantitative PCR. The error bars represent the mean ± standard deviation of three repeated experiments. Unpaired two-tailed T-tests were used for significance analysis, and **** indicates P < 0.0001. Detailed implementation manners

[0043] The principles and features of the present invention are described below. The examples given are only used to explain the present invention and are not intended to limit the scope of the present invention. For those not specifying specific techniques or conditions in the examples, the techniques or conditions described in the literature in this field or according to the product specifications are followed. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be purchased through regular channels.

[0044] Source descriptions of the reagents and materials used in the following examples:

[0045] 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).

[0046] Examples

[0047] 1. Inducing rapid differentiation of human pluripotent stem cells into macrophages

[0048] A method for inducing rapid differentiation of human pluripotent stem cells into macrophages, comprising the following steps:

[0049] (1) First stage, differentiation to form mesoderm: Take human pluripotent stem cells and use the first-stage induction medium (HDM1) to induce differentiation to obtain mesoderm; the first-stage induction medium includes HDM, BMP4, Activin A, bFGF, CHIR99021, A8-301, and IWR-1-endo;

[0050] (2) Second stage, differentiation to form hematopoietic endothelial cells: Replace with the second-stage induction medium (HDM2), culture the mesoderm, and induce to obtain hematopoietic endothelial cells; the second-stage induction medium includes HDM, VEGF, and bFGF;

[0051] (3) Third stage, differentiation to form immature macrophages: Replace with the third-stage induction medium (HDM3), culture the hematopoietic endothelial cells, and induce to obtain suspended immature macrophages; the third-stage induction medium includes StemPro-34 SFM, Flt-3, IL3, and GM-CSF; From the 12th day, immature macrophages can be continuously produced until about the 30th day, and then the suspended immature macrophages can be collected every 3-4 days;

[0052] (4) Fourth stage, differentiation to form mature macrophages: Culture the immature macrophages with the fourth-stage medium (HDM4) to obtain mature macrophages (Macrophage); the fourth-stage induction medium includes RPMI 1640 and GM-CSF.

[0053] 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; it greatly simplifies the macrophage differentiation system, shortens the differentiation cycle, and can significantly reduce production costs.

[0054] Preferably, the conditions for inducing differentiation with the first-stage induction medium in step (1) are: temperature is 37 ± 0.5 °C, the volume fraction of CO2 is 5%, the oxygen content is normal oxygen, and the time is 1-3 days;

[0055] The conditions for culturing the mesoderm with the second-stage induction medium to induce hematopoietic endothelial cells in step (2) are: temperature is 37 ± 0.5 °C, the volume fraction of CO2 is 5%, the oxygen content is normal oxygen, and the time is 3-5 days;

[0056] The conditions for culturing the hematopoietic endothelial cells with the third-stage induction medium to induce immature macrophages in step (3) are: temperature is 37 ± 0.5 °C, the volume fraction of CO2 is 5%, the oxygen content is normal oxygen, and the time is 3-5 days;

[0057] The conditions for culturing mature macrophages using the fourth-stage medium in step (4) are as follows: the temperature is 37 ± 0.5 °C, the volume fraction of CO2 is 5%, the oxygen content is normoxia, and the time is 4 - 6 days.

[0058] Preferably, the first-stage induction medium includes HDM, 20 - 60 ng / mL BMP4, 15 - 45 ng / mL Activin A, 10 - 30 ng / mL bFGF, 3 - 9 μM CHIR99021, 0.5 - 1.5 μM A8 - 301, and 0.5 - 1.5 μM IWR-1-endo;

[0059] The second-stage induction medium includes HDM, 20 - 60 ng / mL VEGF, and 25 - 75 ng / mL bFGF;

[0060] The third-stage induction medium includes StemPro-34 SFM, 5 - 15 ng / mL Flt-3, 5 - 15 ng / mL IL3, and 2.5 - 7.5 ng / mL GM-CSF;

[0061] The fourth-stage induction medium includes RPMI 1640 and 5 - 15 ng / mL GM-CSF.

[0062] Preferably, the HDM includes DMEM / F12 basal medium, P / S, ITS-G, and vitamin C.

[0063] Taking a 6-well plate (2 mL / well) as an example, the differentiation method of the present invention is specifically described as follows:

[0064] (1) Digest hESCs / iPSCs in good condition with a density of 80 - 90% (the hESCs used are the H1 cell line purchased from weiCell) with Accutase (cell digestive solution, Yeasen Biotech) for 5 minutes, terminate digestion with F12 medium (Procell), collect the cell suspension, remove the supernatant after centrifuging at 500 g for 3 minutes, resuspend with mTeSR1 medium and count, inoculate into a six-well plate at a density of 300,000 - 350,000 cells per well, and culture with 2 mL of mTeSR1 + 10 μM Y-27632 for 24 hours. Y-27632 is a ROCK selective inhibitor, which can effectively reduce cell death and improve the survival rate of stem cells;

[0065] (2) First stage, differentiation into mesoderm: On day 0 (D0), after sucking off the culture medium, wash once with 1 mL of DMEM / F12, replace with human pluripotent stem cells, and use 2 mL of the first-stage induction medium (HDM1) to induce differentiation for 48 hours (2 days) to obtain mesoderm; the first-stage induction medium (HDM1) includes HDM, 40 ng / mL BMP4, 30 ng / mL Activin A, 20 ng / mL bFGF, 6 μM CHIR99021, 1 μM A8-301, and 1 μM IWR-1-endo;

[0066] (3) Second stage, differentiation into hematopoietic endothelial cells: On day 2 (D2), after sucking off the culture medium, wash once with 1 mL of DMEM / F12, replace with 2 mL of the second-stage induction medium (HDM2), and culture the mesoderm for 4 days to induce hematopoietic endothelial cells; the second-stage induction medium (HDM2) includes HDM, 40 ng / mL VEGF, and 50 ng / mL bFGF;

[0067] (4) Third stage, differentiation into immature macrophages: On day 8 (D8), after sucking off the culture medium, wash once with 1 mL of DMEM / F12, replace with 2 mL of the third-stage induction medium (HDM3), and culture for 4 days (until day 12) to induce the hematopoietic endothelial cells to obtain suspended immature macrophages; the third-stage induction medium (HDM3) includes StemPro-34 SFM, 10 ng / mL Flt-3, 10 ng / mL IL3, and 5 ng / mL GM-CSF; From day 12, immature macrophages can be continuously produced until about day 30, and subsequently, the suspended immature macrophages can be collected every 3 - 4 days;

[0068] (5) Fourth stage, differentiation into mature macrophages: Collect the suspended immature macrophages, centrifuge at 500 g for 3 minutes, remove the supernatant, transfer to a new 6-well plate, and use 2 mL of the fourth-stage medium (HDM4) to culture the immature macrophages. After 5 days, mature macrophages (Macrophage) are obtained; the fourth-stage induction medium (HDM4) includes RPMI 1640 and 10 ng / mL GM-CSF.

[0069] Among them, the HDM includes DMEM / F12 basal medium, 1 wt.% P / S, 1 wt.% ITS-G, and 70 μg / ml vitamin C.

[0070] 2. Experimental detection

[0071] 2.1 Cell morphological observation

[0072] The morphological observation of macrophages was performed by Giemsa staining. First, 500,000 - 800,000 cells were collected for cytocentrifugation, washed three times with FACS, centrifuged at 500 g for 3 minutes each time. After centrifugation, the supernatant was removed, and the cell pellet was resuspended with 200 μL of FACS. A clean blank glass slide and absorbent paper were placed in the slide trough together. The cell suspension was added to the sample chamber and centrifuged at 300 g for 5 minutes.

[0073] The cytocentrifuged slide was taken out, air-dried naturally, and the cell attachment area was observed under light. The area with cell attachment was circled with a hydrophobic pen to prepare for Giemsa staining. Solution A was added dropwise to the pre-drawn circle and reacted for two minutes. Then, an equal volume of Solution B was added, gently shaken evenly, and left standing for 10 minutes. The slide was slowly rinsed with running water from top to bottom to remove the excess stain. After air-drying, it could be observed and photographed. (Wright-Giemsa staining solution 3 * 250 mL Baso / Besol, BA-4034)

[0074] 2.2 Flow cytometry

[0075] The sample was digested according to the cell type to ensure a single-cell suspension was obtained before finally staining with antibodies. Suspended cells could be directly sampled, and adherent cells were digested with Accutase for 5 minutes. The cells to be detected were collected in a 1.5 mL EP tube, resuspended and washed with FACS (DPBS + 2% FBS), and centrifuged at 500 g for 3 minutes. The supernatant was removed, and the cell pellet was resuspended with 100 μL of FACS (control the cell density not exceeding 10 per 100 μL of FACS) 6Add the flow cytometry antibodies to be detected to the cells in a ratio of 1:100 in sequence. When multiple samples need to be detected simultaneously, a mix can be prepared in advance. Incubate at 4°C in the dark for 20 - 30 minutes or at room temperature for 15 minutes. After incubation, add 1 mL of FACS to the EP tube to terminate the staining and wash away non-specific binding. Centrifuge at 500 g for 3 minutes, then discard the supernatant. Resuspend the cell pellet with 100 - 200 μL of FACS and perform detection on the 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-human CD80 (Biolegend, 305219); APC anti-human CD86 (BD, 555660). Latex beads (sigma, L3030-1ML).

[0076] 2.3 Fluorescent latex microsphere phagocytosis experiment

[0077] Add the red fluorescent latex microspheres (Beads) to the culture dish containing macrophages in a ratio of 1:100, mix well, and incubate Beads and macrophages together for 2 hours. Then collect the cells for flow cytometry detection and wash three times with FACS before loading onto the machine. Beads emit red light and are detected using the PE channel.

[0078] (Taking the 24-well plate as an example, the number of macrophages can be 200,000 - 500,000 per well)

[0079] 2.4 Polarization of macrophages

[0080] Seed mature macrophages in a 24-well plate one day in advance (500,000 per well). Add 1 μg / mL of LPS-EK and 40 ng / mL of INF-γ to the culture medium to polarize macrophages towards M1; polarize macrophages towards M2 by adding 20 ng / mL of IL4 to the culture medium. After 24 hours of polarization, collect the cells. Perform flow cytometry detection of M1 / 2-related surface markers on a part of the cells, and extract RNA from the other part. After reverse transcription, detect the expression levels of M1 / 2-related genes by Q-PCR.

[0081] The Q-PCR primers are shown in Table 1:

[0082] Table 1 Q-PCR primers

[0083]

[0084]

[0085] Note: -q- represents the q-PCR primer

[0086] 2.5 RNA extraction and reverse transcription

[0087] Collect the cells to be processed, and extract RNA according to the RaPure Total RNA Micro Kit (R4012-03) for microextraction of tissue cell RNA. Reverse transcribe 2 μg of RNA according to the TOYOBO ReverTra Ace kit.

[0088] 2.6 Detection of the RNA expression level of classical genes of M1 / 2 macrophages by Q-PCR

[0089] Dilute the cDNA obtained by reverse transcription 50-fold with ddH2O, 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. Specifically, refer to the chamQ SYBR qPCR master mix (Novizan, Q311-03) kit.

[0090] Table 2 Q-PCR reaction system

[0091] Component Volume 10 μL 2×SYBR Green Mix 5 μL Target gene-Q-F / R MIX 0.8 μL cDNA 4 μL <![CDATA[ddH2O]]> 0.2 μL

[0092] 3. Experimental results

[0093] Among them, Figure 1 is the flow chart of the differentiation of human pluripotent stem cells into macrophages and the bright-field photo of the key nodes in the present invention, with a scale of 100 μm; Figure 1 It shows that human pluripotent stem cells can be successfully induced to differentiate into macrophages quickly by the method of the present invention.

[0094] Figure 2 is the statistical situation of the macrophage count at each harvest during the differentiation process of the present invention; Figure 2 It shows that a large number of macrophages can be harvested in multiple batches finally by the method of the present invention.

[0095] Figure 3 is the expression situation of the classical surface markers of mature macrophages detected by flow cytometry in the present invention; Figure 3It is shown that the macrophages derived from human pluripotent stem cells differentiated by the method of the present invention highly express the classical surface markers of macrophages, demonstrating that they are mature macrophages.

[0096] Figure 4 This is the morphological display diagram (Giemsa staining) of the mature macrophages of the present invention, scale bar: 10 μm; by Figure 4 It is shown that the macrophages derived from human pluripotent stem cells differentiated by the method of the present invention exhibit the morphology of normal macrophages.

[0097] Figure 5 This is the phagocytosis experiment of fluorescent latex microspheres of the present invention, photo display and flow cytometry detection. The fluorescence channel of the latex microspheres is PE, scale bar: 10 μm; by Figure 5 It is shown that the macrophages derived from human pluripotent stem cells differentiated by the method of the present invention have phagocytic ability.

[0098] Figure 6 These are the bright-field photos of the mature macrophages of the present invention before and after polarization, scale bar: 50 μm; by Figure 6 It is shown that the macrophages derived from human pluripotent stem cells differentiated by the method of the present invention have the ability to polarize.

[0099] Figure 7 This is the flow cytometry detection of the expression of surface markers CD80, CD86 and CD163, CD206 related to M1 and M2 before and after macrophage polarization of the present invention; by Figure 7 It is shown that the macrophages derived from human pluripotent stem cells differentiated by the method of the present invention can respond to polarization signals and have the ability to polarize into M1 and M2 macrophages.

[0100] Figure 8 This is the real-time fluorescence quantitative PCR detection of the expression levels of classical genes related to M1 and M2 before and after macrophage polarization. The error bars represent the mean ± standard deviation of three repeated experiments. Unpaired two-tailed T-tests were used for significance analysis, **** indicates P < 0.0001; by Figure 8 It is shown that the macrophages derived from human pluripotent stem cells differentiated by the method of the present invention can polarize into M1 and M2 macrophages and express related genes.

[0101] In summary, the present invention provides a novel monolayer differentiation method to obtain macrophages derived from human pluripotent stem cells. This method does not require the differentiation stage of HSPCs and can directly differentiate into functionally mature macrophages; it greatly simplifies the macrophage differentiation system, shortens the differentiation cycle, and can significantly reduce the production cost.

[0102] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for inducing the rapid differentiation of human pluripotent stem cells into macrophages, characterized in that, It includes the following steps: (1) In the first stage, mesoderm is differentiated: Take human pluripotent stem cells and use the first-stage induction medium for 1 - 3 days to induce and differentiate into mesoderm; (2) In the second stage, hematopoietic endothelial cells are differentiated: Replace the second-stage induction medium and culture the mesoderm for 3 - 5 days to induce hematopoietic endothelial cells; (3) In the third stage, immature macrophages are differentiated: Replace the third-stage induction medium and culture the hematopoietic endothelial cells for 3 - 5 days to induce suspended immature macrophages; (4) In the fourth stage, mature macrophages are differentiated: Culture the immature macrophages with the fourth-stage medium for 4 - 6 days to obtain mature macrophages; This method for inducing the rapid differentiation of human pluripotent stem cells into macrophages does not go through the differentiation of hematopoietic stem / progenitor cells; The first-stage induction medium includes HDM, 20 - 60 ng / mL BMP4, 15 - 45 ng / mL Activin A, 10 - 30 ng / mL bFGF, 3 - 9 μM CHIR99021, 0.5 - 1.5 μM A8 - 301, and 0.5 - 1.5 μM IWR - 1 - endo; The second-stage induction medium includes HDM, 20 - 60 ng / mL VEGF, and 25 - 75 ng / mL bFGF; The third-stage induction medium includes StemPro - 34 SFM, 5 - 15 ng / mL Flt - 3, 5 - 15 ng / mL IL3, and 2.5 - 7.5 ng / mL GM - CSF; The fourth-stage induction medium includes RPMI 1640 and 5 - 15 ng / mL GM - CSF; HDM includes DMEM / F12 basal medium, P / S, ITS - G, and vitamin C.

2. The method for inducing the rapid differentiation of human pluripotent stem cells into macrophages according to claim 1, characterized in that, The conditions for inducing differentiation with the first-stage induction medium in step (1) are: temperature is 37 ± 0.5 °C, the volume fraction of CO2 is 5%, the oxygen content is normoxia, and the time is 2 days; The conditions for culturing the mesoderm with the second-stage induction medium to induce hematopoietic endothelial cells in step (2) are: temperature is 37 ± 0.5 °C, the volume fraction of CO2 is 5%, the oxygen content is normoxia, and the time is 4 days; The conditions for culturing the hematopoietic endothelial cells with the third-stage induction medium to induce immature macrophages in step (3) are: temperature is 37 ± 0.5 °C, the volume fraction of CO2 is 5%, the oxygen content is normoxia, and the time is 4 days; The conditions for culturing with the fourth-stage medium to obtain mature macrophages in step (4) are: temperature is 37 ± 0.5 °C, the volume fraction of CO2 is 5%, the oxygen content is normoxia, and the time is 5 days.

3. The method for inducing the rapid differentiation of human pluripotent stem cells into macrophages according to claim 1, characterized in that, The first-stage induction medium includes HDM, 40 ng / mL BMP4, 30 ng / mL Activin A, 20 ng / mL bFGF, 6 μM CHIR99021, 1 μM A8 - 301, and 1 μM IWR - 1 - endo; The second-stage induction medium comprises HDM, 40 ng / mL VEGF, and 50 ng / mL bFGF; The third-stage induction medium comprises StemPro-34 SFM, 10 ng / mL Flt-3, 10 ng / mL IL3, and 5 ng / mL GM-CSF; The fourth-stage induction medium comprises RPMI 1640 and 10 ng / mL GM-CSF.

4. The method for inducing the rapid differentiation of human pluripotent stem cells into macrophages according to claim 1, characterized in that, In step (1), the seeding density of the human pluripotent stem cells is 150,000-175,000 cells / mL.

5. The method for inducing the rapid differentiation of human pluripotent stem cells into macrophages according to claim 1, characterized in that, The human pluripotent stem cells are pretreated before differentiating into mesoderm in the first stage; the pretreatment comprises the following specific steps: first, the human pluripotent stem cells are digested, and then cultured in a medium containing Y-27632 for 12-36 hours.

6. The method for inducing the rapid differentiation of human pluripotent stem cells into macrophages according to claim 5, characterized in that, The digestion reagent used for the digestion treatment is Accutase digestion solution, and the digestion time at room temperature is 3-5 minutes; the medium containing Y-27632 comprises mTeSR1 and Y-27632.

Images