Method for efficiently inducing human induced pluripotent stem cells to differentiate into mononuclear cells

Abstract

The invention relates to the technical field of cell biology, and discloses a method for efficiently inducing hiPSCs to differentiate into mononuclear cells, which comprises the following steps: Step 1: culturing and amplifying human induced pluripotent stem cells; step 2: forming embryoid bodies by human-derived induced pluripotent stem cells: at the first stage, digesting hiPSCs to form cell agglomerates, inoculating the cell agglomerates to a cell culture dish, adding mTeSR Plus containing ROCK inhibitor Y-27632, and culturing for 0.5-1 day; in the second stage, the old culture medium is removed, EBM is added, half of the culture medium is changed every day, and culture is conducted for 4-6 days. Step 3: differentiation of the embryoid bodies into mononuclear cells: at the first stage, inoculating the embryoid bodies into a cell culture plate, adding MDM1, changing half of the liquid every 2-3 days, and culturing for 8-9 days; in the second stage, the old culture medium is removed, MDM2 is added, half of the medium is changed every 2-3 days, and culture is performed for 4-6 days; and a third stage: removing the old culture medium, adding MDM2, completely changing the medium every 2-3 days, and collecting the mononuclear cells. By adjusting EB culture conditions and inoculation density, the differentiation efficiency of the mononuclear cells is remarkably improved, the preparation time is shortened, and the production cost is reduced.

Description

Technical Field

[0001] This invention relates to the field of cell biology, specifically to a method for efficiently inducing human induced pluripotent stem cells to differentiate into monocytes. Background Technology

[0002] Monocytes are key effector cells of the innate immune system, playing a central role in host defense, inflammation regulation, and tissue repair. In biomedical research, in vitro induced monocyte populations have multiple values: firstly, they provide an ideal research platform for constructing precise disease models, elucidating immune regulation mechanisms, and developing targeted drugs; secondly, they drive technological innovation in translational medicine fields such as cell immunotherapy and vaccine development. Therefore, developing efficient in vitro monocyte induction and isolation techniques is of great significance for both basic research and clinical translation.

[0003] Isolating monocytes from peripheral blood mononuclear cells (PBMCs) is a common method. However, monocytes only account for 10%–30% of PBMCs, resulting in a limited number and low purity of isolated monocytes. Furthermore, significant individual differences exist between blood samples from different donors, making it difficult to achieve large-scale and standardized monocyte isolation and induction in vitro. In contrast, induced pluripotent stem cells (iPSCs), with their unlimited proliferative capacity, multipotent differentiation potential, and high homogeneity, have become a powerful tool for monocyte induction in vitro.

[0004] The use of embryoid body (EB) differentiation to convert iPSCs into monocytes is a commonly used technique. For example, patent application CN119662546A discloses a method for differentiating induced pluripotent stem cells into macrophages. This method promotes cell differentiation under conditions closer to nature by simulating the three-dimensional microenvironment of early embryonic development, thereby generating high-purity, high-viability, and functionally complete monocytes. However, because it simulates the natural process of embryonic development, the EB differentiation method has an inherent characteristic: the induction period for monocytes is relatively long, typically requiring several weeks.

[0005] Therefore, in order to meet the needs of large-scale production, it is necessary to improve the differentiation efficiency of monocytes in order to reduce cell preparation time and cost. Summary of the Invention

[0006] To address the low induction efficiency of existing EB differentiation methods, this invention proposes a highly efficient method for inducing human induced pluripotent stem cells (hiPSCs) to differentiate into monocytes. By adjusting EB culture conditions and seeding density, the differentiation efficiency of monocytes is significantly improved, preparation time is shortened, and production costs are reduced.

[0007] This invention provides a method for efficiently inducing hiPSCs to differentiate into monocytes, comprising the following key steps:

[0008] Step 1: Culture and expansion of human induced pluripotent stem cells;

[0009] Step 2: Human induced pluripotent stem cells form embryoid bodies;

[0010] Step 3: The embryoid body differentiates into a mononuclear cell.

[0011] Furthermore, Step 1 includes the following stages:

[0012] Phase 1: hiPSCs were seeded into cell culture plates, mTeSR Plus (containing the ROCK inhibitor Y-27632) was added, and cultured for 0.5-1 days.

[0013] The cell culture plates in this stage were coated with a vitrin (VTN-N) solution at room temperature for 1 hour, wherein the concentration of vitrin was 5–10 μg / mL.

[0014] The initial cell seeding density for this stage is 1–1.5 × 10⁻⁶. 5 cells / mL, preferably 1.25 × 10⁻⁶. 5 cells / mL;

[0015] The concentration of the ROCK inhibitor Y-27632 in this stage is 5–10 μM.

[0016] Second stage: Remove the old culture medium, add mTeSR Plus, change the medium completely every 2-3 days, and culture for 4-6 days.

[0017] Furthermore, Step 2 includes the following stages:

[0018] Phase 1: hiPSCs are digested to form cell clumps, seeded into cell culture dishes, and mTeSR Plus (containing ROCK inhibitor) is added. The cells are then cultured for 0.5-1 days.

[0019] The cell digestion fluid used in this stage is RelesR;

[0020] The cell culture dish used in this stage is an ultra-low adsorption cell culture dish;

[0021] The concentration of the ROCK inhibitor Y-27632 in this stage is 5–10 μM.

[0022] Second stage: Remove the old culture medium, add EBM, change the medium halfway every day, and culture for 4-6 days.

[0023] The EBM used in this stage includes DMEM / F-12, 10% FBS, 20% KOSR, and 1%

[0024] MEM / NEAA, 1% L-Glutamine, 2-Mercaptoethanol (0.1mM).

[0025] Furthermore, Step 3 includes the following steps:

[0026] Phase 1: Seed embryoids into cell culture plates, add MDM1, change the medium every 2-3 days, and culture for 8-9 days.

[0027] The cell culture plates in this stage were coated with Gelatin solution at 37°C for 12-18 hours, wherein the mass concentration of Gelatin was 0.1 wt%.

[0028] The embryoids at this stage were seeded into cell culture plates of the same size with the same number of wells as those for seeding induced pluripotent stem cells.

[0029] The first stage of MDM1 contains DMEM, 10% FBS, 1% MEM / NEAA, 1% L-Glutamine, 2-Mercaptoethanol (0.1mM), M-CSF (100ng / mL), and IL-3 (25ng / mL).

[0030] Second stage: Remove the old culture medium, add MDM2, change the medium every 2-3 days, and culture for 4-6 days.

[0031] The MDM2 in this stage contains DMEM, 10% FBS, 1% MEM / NEAA, 1% L-Glutamine, 2-Mercaptoethanol (0.1mM), M-CSF (25ng / mL), and IL-3 (12.5ng / mL).

[0032] Phase 3: Remove the old culture medium, add MDM2, completely change the medium every 2-3 days, and collect monocytes.

[0033] The number of mononuclear cells collected in this stage was detected using a counter.

[0034] The expression of CD14 in the mononuclear cells collected during this stage was detected by flow cytometry.

[0035] Furthermore, the culture temperature for Step 1, Step 2, and Step 3 is 37°C, and the CO2 concentration is 5%.

[0036] The beneficial effects of this invention are:

[0037] 1. High differentiation rate: Based on the advantages of the EB differentiation method, the differentiation efficiency of monocytes was significantly improved by simply adjusting the embryoid inoculation density, culture medium composition and medium change frequency.

[0038] 2. Stability and reproducibility: The optimized culture conditions and operating procedures have high stability and reproducibility, making them suitable for large-scale production and scientific research.

[0039] 3. Simple and easy to implement: The method provided by this invention is simple to operate, easy to implement, and suitable for laboratory and industrial production environments. Attached Figure Description

[0040] Figure 1 Schematic diagram of iPSC growth at different time points;

[0041] Figure 2 Morphological diagrams of EBs at different time points after their formation;

[0042] Figure 3 Morphological images of EBs at different time points after inoculation;

[0043] Figure 4 Flow cytometry plot of CD14 expression in monocytes collected on day 23 post-EB vaccination;

[0044] Figure 5 A statistical chart of the number of monocytes collected at different time points. Detailed Implementation

[0045] The following detailed embodiments illustrate the implementation of the present invention. It should be noted that the provided embodiments are merely for aiding understanding of the technical solutions of the present invention and do not constitute any limitation on the scope of protection of the present invention. Unless otherwise specified, all reagents used in this experiment are commercially available conventional reagents; unless otherwise specified, all experimental methods involved are performed in accordance with conventional technical specifications in the field or relevant product instructions.

[0046] The reagents used in the embodiments and comparative examples of this invention were sourced from the following sources: mTeSR Plus (Stemcell); Y-27632 (Tocris); VTN-N (Gibco); TrypLE (Gibco); DMEM / F12 (Gibco); FBS (Hyclone), KOSR (Gibco); MEM / NEAA (Gibco); L-Glutamine (Gibco); 2-Mercaptoethanol (Sigma-Aldrich); RelesR (Stemcell); DMEM (Gibco); RPMI 1640 (Gibco); rhM-CSF (Servicebio); rhIL-3 (R&D Systems); rhIL-4 (R&D Systems); rhIL-13 (R&D Systems); rhIL-6 (R&D Systems); P / S (Gibco); 7-AAD (BioLegend); Trustain FcX (BioLegend); anti-human CD14 (BioLegend) examples

[0047] A method for inducing hiPSCs to differentiate into monocytes includes the following steps:

[0048] (1) Cultivation and amplification of hiPSCs

[0049] Phase 1: Using mTeSR Plus medium containing the ROCK inhibitor Y-27632 (5 μM), iPSCs were seeded at a density of 1.25 × 10^5 cells / mL in 6-well plates coated with 5 μg / mL fibronectin, with 2 mL of cell suspension in each well, and cultured for 1 day.

[0050] Phase 2: Remove the old culture medium and continue using mTeSR Plus medium, changing the medium completely every 2 days, and culture for 4-6 days until the cell density reaches more than 80%.

[0051] Schematic diagram of hiPSC growth at different time points as follows Figure 1 As shown, cells grow, proliferate, and form cell clumps, which gradually increase in size over time.

[0052] (2) hiPSCs form embryoid bodies

[0053] Phase 1: hiPSCs were digested with RelesR to form cell clumps. The cell clumps were collected with a 5ml pipette and seeded into a 10cm ultra-low adsorption cell culture dish. mTeSR Plus medium containing the ROCK inhibitor Y-27632 (5μM) was added and cultured for 1 day.

[0054] Phase 2: Remove the old culture medium, add EBM, and change the EBM medium halfway daily for 5 days. The composition of EBM medium includes DMEM / F-12, 20% KOSR, 1% MEM / NEAA, 1% L-Glutamine and 2-Mercaptoethanol (0.1mM).

[0055] Schematic diagram of EB growth at different time points as follows Figure 2 As shown, in the early stages of cultivation, EBs appear as regular, uniform spheres, with a predominantly gray-black color. As the cultivation time increases, the edges of some EBs begin to become irregular, and the color gradually darkens.

[0056] (3) The embryoid body differentiates into mononuclear cells.

[0057] Phase 1: Embryomorphs were seeded into 6-well plates coated with 0.1% Gelatin, and MDM1 was added. The MDM1 medium was changed halfway every 3 days, and the plates were cultured for 9 days. The number of wells for embryomorphs was the same as the number of wells for iPSCs, meaning the number of embryomorphs generated from iPSCs in one 6-well plate was evenly distributed among the wells. The MDM1 medium consisted of DMEM, 10% FBS, 1% MEM / NEAA, 1% L-Glutamine, 2-Mercaptoethanol (0.1 mM), M-CSF (100 ng / mL), and IL-3 (25 ng / mL).

[0058] Phase 2: Remove the old culture medium and replace it with MDM2. Change the medium halfway every 3 days and culture for 6 days. The composition of MDM2 medium includes DMEM, 10% FBS, 1% MEM / NEAA, 1% L-Glutamine, 2-Mercaptoethanol (0.1mM), M-CSF (25ng / mL) and IL-3 (12.5ng / mL).

[0059] Morphological images of EBs at different time points after 6-well plate inoculation are shown below. Figure 3 As shown, EB adheres to the bottom of the cell culture plate and gradually grows and spreads spontaneously to the surrounding area over time, accompanied by the proliferation and expansion of mesodermal cells (pale yellow).

[0060] Phase 3: Remove the old culture medium, continue using MDM2, and completely change the medium every 3 days. Collected mononuclear cells are analyzed for cell count and CD14 expression using a cell counter and flow cytometry, respectively.

[0061] Flow cytometry analysis results of mononuclear cells collected on day 23 post-EB vaccination are as follows: Figure 4As shown, the CD14 positivity rate was 81.2%.

[0062] Compare with Example 1

[0063] Unlike the examples, in steps (1) and (2), the EB medium used in Control Example 1 [STAR Protoc.2024Mar15;5(1):102827.doi:10.1016 / j.xpro.2023.102827] was entirely EBM and did not contain mTeSRPlus, and the EB inoculation density was 8-10 EB / well (6-well plate).

[0064] Compare with Example 2

[0065] Unlike the examples, the EB seeding density used in Comparative Example 2 [Front Immunol.2020Jun 4:11:1016.doi:10.3389 / fimmu.2020.01016] was 20-30 EBs / well (6-well plate).

[0066] Compare with Example 3

[0067] Unlike the examples, Comparative Example 3 [PLoS One.2020Dec 17;15(12):e0243807.doi:10.1371 / journal.pone.0243807] used mTeSR Plus and StemPro-34 EB culture medium, which did not contain EBM, and the EB inoculation density was 5 EB / well (6-well plate).

[0068] The number of mononuclear cells collected at different time points in the examples and control examples are as follows: Figure 5 As shown, the number of mononuclear cells collected in the examples was generally significantly higher than that in the control examples.

[0069] The culture conditions for the above cells or embryoids were: temperature 37℃ and CO2 concentration 5%.

[0070] It should be noted that the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention. Those skilled in the art should understand that technical solutions obtained through equivalent substitution, parameter adjustment, or process optimization without departing from the core concept and basic principles of the present invention should all be covered within the protection scope of the present invention.

Claims

1. A method for efficiently inducing hiPSCs to differentiate into monocytes, comprising the following steps: Step 1: Culture and expansion of human induced pluripotent stem cells; Step 2: Human induced pluripotent stem cells form embryoid bodies; Step 3: The embryoid body differentiates into a mononuclear cell; characterized by, Step 2 includes the following stages: Phase 1: hiPSCs are digested to form cell clumps, seeded into cell culture dishes, and mTeSR Plus containing the ROCK inhibitor Y-27632 is added. The cells are then cultured for 0.5-1 days. Second stage: Remove the old culture medium, add EBM, change the medium halfway every day, and incubate for 4-6 days; The EBM in this stage contains DMEM / F-12, 20% KOSR, 1% MEM / NEAA, 1% L-Glutamine, and 0.1 mM 2-Mercaptoethanol; Step 3 includes the following steps: Phase 1: Seed the embryonic bodies into cell culture plates, add MDM1, change the medium every 2-3 days, and culture for 8-9 days; The MDM1 contains DMEM, 10% FBS, 1% MEM / NEAA, 1% L-Glutamine, 0.1 mM 2-Mercaptoethanol, 100 ng / mL M-CSF, and 25 ng / mL IL-3; Second stage: Remove the old culture medium, add MDM2, change the medium every 2-3 days, and incubate for 4-6 days; The MDM2 contains DMEM, 10% FBS, 1% MEM / NEAA, 1% L-Glutamine, 0.1 mM 2-Mercaptoethanol, 25 ng / mL M-CSF, and 12.5 ng / mL IL-3; Phase 3: Remove the old culture medium, add MDM2, completely change the medium every 2-3 days, and collect monocytes.

2. The method according to claim 1, characterized in that, The steps include: Step 1 includes the following stages: Phase 1: hiPSCs were seeded into cell culture plates, mTeSR Plus was added, and cultured for 0.5-1 days; Second stage: Remove the old culture medium, add mTeSR Plus, change the medium completely every 2-3 days, and culture for 4-6 days.

3. The method according to claim 2, characterized in that, In the first stage of Step 1, the initial cell seeding density is 1–1.5 × 10⁻⁶. 5 cells / mL.

4. The method according to claim 3, characterized in that, In the first stage of Step 1, the initial cell seeding density is 1.25 × 10⁻⁶. 5 cells / mL.

5. The method according to claim 1, characterized in that, The concentration of the ROCK inhibitor Y-27632 is 5–10 μM.

6. The method according to claim 2, characterized in that, In the first stage of Step 1, the cell culture plate is coated with a hydrin solution at room temperature, wherein the concentration of hydrin is 5-10 ug / mL.

7. The method according to claim 1, characterized in that, In Step 2, the cell digestion solution used for hiPSCs digestion is RelesR.

8. The method according to claim 1, characterized in that, In the first stage of Step 3, the cell culture plate is coated with Gelatin solution at 37°C for 12-18 hours.

9. The method according to claim 8, characterized in that, The mass concentration of gelatin is 0.1 wt%.

10. The method according to claim 1, characterized in that, The incubation temperature for Step 1, Step 2, and Step 3 is 37°C, and the CO2 concentration is 5%.

Images