Small-molecule combination, kit and method for directed differentiation of lymphohematopoietic precursor cells

Abstract

The present invention relates to the field of molecular biology, and in particular to a small-molecule combination, kit and method for directed differentiation of lymphohematopoietic precursor cells. A culture system comprises a sphere-forming medium, a mesoderm induction medium, a hematopoietic endothelial cell induction medium, a hematopoietic stem / progenitor cell induction medium, and a lymphohematopoietic precursor cell induction medium. In the present invention, by modulating the composition of culture medium and the formulation of growth factors, cells can obtain sufficient nutrients and signals during induction, thereby improving the differentiation efficiency and stability. Using full suspension culture to simulate a three-dimensional in vivo environment avoids the problems of cell morphology change and stress while enhancing the stability and growth rate of cells.

Description

Small molecule combinations, kits and methods for directed differentiation of lymphohematopoietic precursor cells TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology, in particular to small molecule combinations, kits and methods for directed differentiation of lymphohematopoietic precursor cells. BACKGROUND

[0002] Hematopoietic precursor cells have extensive differentiation potential in vivo and can differentiate into various hematopoietic cells such as red blood cells, white blood cells and platelets. They are expected to provide new treatment approaches for various hematopoietic system diseases, including treatment of genetic blood diseases, regenerative anemia, and bone marrow transplantation. In addition, hematopoietic precursor cells not only have important significance for the treatment of hematopoietic system diseases, but also play an important role in the treatment of immune system related diseases. Lymphocytes are a key component of the immune system and can recognize and eliminate pathogens and abnormal cells in the body, while lymphohematopoietic precursor cells can be directed to differentiate into specific types of lymphocytes such as T cells, B cells and natural killer cells, providing new directions for precise treatment of immune system related diseases and application of tissue regeneration medicine.

[0003] Currently, the differentiation of induced pluripotent stem cells into hematopoietic precursor cells usually needs to be completed in multiple stages. The reasons for using a staged strategy are as follows: (1) dividing the differentiation process of induced pluripotent stem cells into mesoderm, hematopoietic and other stages can better simulate the sequence of cell differentiation and signal pathway activity during embryonic development; (2) using different components of the culture medium, adding specific growth factors and chemical small molecules at each stage can better promote the differentiation of cells into target cell types; (3) different stages and different types of cells may have different requirements for culture conditions, including cell density, culture medium composition and cell-cell interactions, such as using three-dimensional culture systems (such as embryonic in vitro culture or scaffold material culture) can better simulate cell interactions and tissue structure during embryonic development, providing a more complex cell signal and growth environment, which helps to increase the efficiency and purity of differentiation. Therefore, this multi-stage, scientifically rigorous induction process provides an important basis and new direction for the treatment of hematopoietic system diseases, precise intervention of immune system diseases and application of tissue regeneration medicine.

[0004] Although, at present, the technology of inducing pluripotent stem cells to differentiate into hematopoietic precursor cells in vitro has made significant progress, and important success has been achieved in the treatment of blood system diseases such as leukemia, regenerative anemia; however, in the process of inducing pluripotent stem cells to differentiate into hematopoietic precursor cells in vitro, the culture method of first suspension and then adhesion or the method of forming a ball by relying on a rotating device is adopted, these methods have the problem of complex operation, at the same time, the method of suspension and adhesion culture may cause cell morphological change and cell damage, and the method of forming a ball by relying on a rotating device may cause cell stress and oxygen and nutrient deficiency, which will affect the differentiation efficiency and stability of hematopoietic precursor cells. In view of this, the present application provides a small molecule combination, kit and method for directional differentiation of lymphoid hematopoietic precursor cells.

[0005] SUMMARY

[0006] The technical problem to be solved by the present application is to provide a small molecule combination, kit and method for directional differentiation of lymphoid hematopoietic precursor cells. The purpose is to improve the differentiation efficiency and stability of pluripotent stem cells in directional differentiation of lymphoid hematopoietic precursor cells.

[0007] The technical solution of the present application to solve the above technical problem is as follows:

[0008] In a first aspect, the small molecule combination for directional differentiation of lymphoid hematopoietic precursor cells comprises a sphere culture medium, a mesoderm induction culture medium, a hematopoietic endothelial cell induction culture medium, a hematopoietic stem / progenitor cell induction culture medium and a lymphoid hematopoietic precursor cell induction culture medium.

[0009] The hematopoietic endothelial cell induction culture medium comprises the following components in the following amounts: 5-100ng / mL VEGF, 1-25ng / mL BMP4, 10-250ng / mL FGF2, 2-50μM SB431542;

[0010] The hematopoietic stem / progenitor cell induction culture medium comprises the following components in the following amounts: 1-25ng / mL BMP4, 2-50ng / mL VEGF, 15-60μM NAC, 1-4μM Minocycline, 4-100ng / mL SCF, 2-50ng / mL FLT3L, 2-50ng / mL IL-15, 2-50ng / mL IL-7, 1-25ng / mL IL3;

[0011] The lymphohematopoietic precursor cell induction medium comprises the following components in the following amounts: 1-25 ng / mL BMP4, 2-50 ng / mL VEGF, 15-60 mM NAC, 1-4 mM Minocycline, 10-100 nM UM171, 5-20 mM SR1, 4-100 ng / mL SCF, 2-50 ng / mL FLT3L, 2-50 ng / mL IL-15, 2-50 ng / mL IL-7.

[0012] Further, the hemogenic endothelial cell induction medium comprises the following components in the following amounts: 25-100 ng / mL VEGF, 2.5-10 ng / mL BMP4, 25-100 ng / mL FGF2, 5-20 mM SB431542;

[0013] The hemogenic endothelial cell induction medium comprises the following components in the following amounts: 2.5-10 ng / mL BMP4, 5-20 ng / mL VEGF, 15-60 mM NAC, 1-4 mM Minocycline, 10-40 ng / mL SCF, 5-20 ng / mL FLT3L, 5-20 ng / mL IL-15, 5-20 ng / mL IL-7, 2.5-10 ng / mL IL3;

[0014] The lymphohematopoietic precursor cell induction medium comprises the following components in the following amounts: 2.5-10 ng / mL BMP4, 5-20 ng / mL VEGF, 15-60 mM NAC, 1-4 mM Minocycline, 10-100 nM UM171, 5-20 mM SR1, 5-20 ng / mL SCF, 5-20 ng / mL FLT3L, 5-20 ng / mL IL-15, 5-20 ng / mL IL-7.

[0015] Further, the spheroidation medium comprises the following components: Y27632;

[0016] The mesoderm induction medium comprises the following components: BMP4, CHIR99021.

[0017] Further, the spheroidation medium comprises the following components in the following amounts: 1-20 mM Y27632;

[0018] The mesoderm induction medium comprises the following components in the following amounts: 1-100 ng / mL BMP4, 1-12 mM CHIR99021.

[0019] Further, the spheroidation medium comprises the following components in the following amounts: 5-20 mM Y27632;

[0020] The mesoderm induction medium comprises the following components in the following amounts: 10-40 ng / mL BMP4, 1.5-10 μM CHIR99021.

[0021] Further, the sphere formation medium comprises the following components in the following amounts: 10 μM Y27632;

[0022] The mesoderm induction medium comprises the following components in the following amounts: 20 ng / mL BMP4, 3-5 μM CHIR99021;

[0023] The hemogenic endothelial cell induction medium comprises the following components in the following amounts: 50 ng / mL VEGF, 5 ng / mL BMP4, 50 ng / mL FGF2, 10 μM SB431542;

[0024] The hemogenic endothelial cell induction medium comprises the following components in the following amounts: 50 ng / mL VEGF, 5 ng / mL BMP4, 50 ng / mL FGF2, 10 μM SB431542;

[0025] The lymphohematopoietic precursor cell induction medium comprises the following components in the following amounts: 5 ng / mL BMP4, 10 ng / mL VEGF, 30 μM NAC, 2 μM Minocycline, 30 nM UM171, 10 μM SR1, 20 ng / mL SCF, 10 ng / mL FLT3L, 10 ng / mL IL-15, 20 ng / mL IL-7.

[0026] In a second aspect, a kit for iPSC directed differentiation of lymphohematopoietic precursor cells, the kit comprises the sphere formation medium, the mesoderm induction medium, the hemogenic endothelial cell induction medium, the hemogenic endothelial cell induction medium, and the lymphohematopoietic precursor cell induction medium.

[0027] In a third aspect, a method for iPSC directed differentiation of lymphohematopoietic precursor cells, the method comprises the following steps: using the sphere formation medium, the mesoderm induction medium, the hemogenic endothelial cell induction medium, the hemogenic endothelial cell induction medium, and the lymphohematopoietic precursor cell induction medium for directed differentiation culture of pluripotent stem cells, or using the kit for directed differentiation culture, to obtain lymphohematopoietic precursor cells.

[0028] Further, the method comprises the following specific steps:

[0029] (1) the first stage, the spheroid culture: the pluripotent stem cells are suspended and cultured for 1 day by using the spheroid culture medium to form the embryoid bodies;

[0030] (2) the second stage, the mesoderm differentiation: the embryoid bodies are induced and differentiated by using the mesoderm induction medium, and are suspended and cultured for 1-2 days to form the mesoderm cells;

[0031] (3) the third stage, the mesoderm cell differentiation into hematopoietic endothelial cells: the mesoderm cells are induced and differentiated by using the hematopoietic endothelial cell induction medium, and are suspended and cultured for 2-4 days to form the hematopoietic endothelial cells;

[0032] (4) the fourth stage, the hematopoietic endothelial cell differentiation into hematopoietic stem / progenitor cells: the hematopoietic endothelial cells are induced and differentiated by using the hematopoietic stem / progenitor cell induction medium, and are suspended and cultured for 5-7 days to form the hematopoietic stem / progenitor cells;

[0033] (5) the fifth stage, the hematopoietic stem / progenitor cell differentiation into lymphoid hematopoietic precursor cells: the hematopoietic stem / progenitor cells are induced and differentiated by using the lymphoid hematopoietic precursor cell induction medium, and are suspended and cultured for 7-21 days to form the lymphoid hematopoietic precursor cells.

[0034] Further, the parameters of the step (1) suspension and culture are as follows: the oxygen volume is 21%, the carbon dioxide volume is 5%, and the temperature is 37 DEG C; the parameters of the step (2) suspension and culture are as follows: the oxygen volume is 21%, the carbon dioxide volume is 5%, and the temperature is 37 DEG C; the parameters of the step (3) suspension and culture are as follows: the oxygen volume is 21%, the carbon dioxide volume is 5%, and the temperature is 37 DEG C; the parameters of the step (4) suspension and culture are as follows: the oxygen volume is 21%, the carbon dioxide volume is 5%, and the temperature is 37 DEG C; the parameters of the step (5) suspension and culture are as follows: the oxygen volume is 21%, the carbon dioxide volume is 5%, and the temperature is 37 DEG C.

[0035] Further, the pluripotent stem cells include chemical-induced pluripotent stem cells (CiPSCs).

[0036] The present application has the following beneficial effects:

[0037] (1) Higher cell differentiation efficiency: By reasonable technical design, a multi-stage differentiation process is set up, which subdivides the hematopoietic stage into two stages of hematopoietic stem / progenitor cells and lymphoid hematopoietic precursor cells. This stage-by-stage regulation method enables cells to activate different signaling pathways in sequence, thereby achieving more precise differentiation and directional guidance. At the same time, it can ensure the introduction of appropriate growth factors and chemical small molecule combinations in each stage. On the one hand, small molecules NAC and Minocycline play an important auxiliary role in the differentiation process of hematopoietic endothelial cells, hematopoietic stem / progenitor cells, and lymphoid hematopoietic precursor cells. Among them, NAC, as an antioxidant, can reduce the level of reactive oxygen species (ROS) generated during differentiation, reducing the impact of oxidative stress on cells. Minocycline has anti-inflammatory and neuroprotective properties, can inhibit the production of inflammatory factors, and can also inhibit the activity of enzymes related to apoptosis, preventing cells from entering the apoptosis pathway. The combination of these two small molecules helps to maintain the health of cells, improve cell survival rate, and ensure that more cells can successfully differentiate into lymphoid hematopoietic precursor cells. In addition, small molecule BMP4 can promote the generation and differentiation of hematopoietic stem cells by activating Smad signaling pathway and other related pathways, while VEGF can promote the expansion of hematopoietic stem / progenitor cells. The combination of these two chemical small molecules plays an important role in the formation of hematopoietic stem / progenitor cells, enabling cells to have higher potential and efficiency when differentiating into lymphoid hematopoietic precursor cells. On the other hand, in the hematopoietic stem / progenitor cell and lymphoid hematopoietic precursor cell induction stage, two new small molecules SR1 and UM171 are added, which participate in the determination of hematopoietic fate and can effectively promote the transformation of hematopoietic stem / progenitor cells into lymphoid hematopoietic precursor cells, significantly increasing the proliferation and differentiation efficiency of hematopoietic stem / progenitor cells, thereby obtaining a larger number of target cell types. Therefore, by subdividing the hematopoietic stage, cells can be more effectively guided to differentiate into specific lymphoid hematopoietic precursor cells.

[0038] (2) More comprehensive immune cell differentiation: Compared with the prior art, the present application can more effectively guide the differentiation of cells to specific lymphohematopoietic precursor cells. In the process of inducing pluripotent stem cells to differentiate into various immune cells in vitro, cytokines such as SCF, IL-3, IL-7, IL-15, and FLT3L are usually used. The functions of these cytokines include maintaining the self-renewal of hematopoietic stem / progenitor cells, promoting the differentiation and maturation of hematopoietic stem / progenitor cells and hematopoietic precursors, and directing the differentiation of cells to various cell lines. Among them, IL-7 is an indispensable cytokine for regulating the development of T cells and B cells, and plays a key role in supporting the survival and development of early T cells; IL-15 is involved in maintaining the long-term survival of memory T cells; BMP4 regulates gene expression related to T cell differentiation by binding to its receptor and activating the SMAD protein signaling pathway, and affects the function of thymic epithelial cells, which is crucial for T cell maturation. In addition, BMP4 is also involved in regulating the balance of helper T cells and regulatory T cells, showing its important potential in immune regulation. Moreover, the present application combines cytokines IL-7, IL-15, and chemical small molecules BMP4, etc., and finally obtains lymphohematopoietic precursor cells stably expressing CD45, CD3, and CD4.

[0039] (3) High cost-effectiveness: In the whole process of induction and differentiation, the present application uses chemical small molecules instead of expensive cytokines for cell culture and manipulation, providing an economical and effective method to enhance the efficiency and quality of cell differentiation; since chemical small molecules usually have the characteristics of low cost, simple preparation, and easy standardization, this greatly reduces the cost of the whole cell production process, and also reduces the bio-safety risk, because the use of chemical small molecules reduces the dependence on animal-derived products.

[0040] (4) Simplified operation, improved production efficiency, and easy expansion: The whole process of induction and differentiation adopts the method of suspension culture, especially the method of spontaneous ball formation, which brings many significant advantages in cell culture; first, it can effectively avoid the cell morphological changes and damage commonly seen in the process of suspension and adhesion conversion, thereby protecting the natural state of the cells and improving their growth and differentiation efficiency; second, suspension culture simplifies the equipment requirements, only requiring a conventional bioreactor, which greatly reduces the cost and operational complexity; third, the spontaneous ball formation method allows continuous cultivation in the same reactor without the need for frequent environmental changes, significantly improving production efficiency and batch consistency; finally, this culture mode is easy to expand and automate, and is very suitable for large-scale production, providing an effective solution for the production of commercial cell products.

[0041] (5) High safety: Using chemical small molecules to induce pluripotent stem cells as the source of initial cells reduces the risk of genetic instability and improves cell safety. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 is a phenotype chart of each stage of Example 1;

[0043] Figure 2 is a flow cytometry result chart of Example 1;

[0044] Figure 3 is a phenotype chart of each stage of Example 2;

[0045] Figure 4 is a flow cytometry chart of Example 2;

[0046] Figure 5 is a phenotype chart of each stage of Example 3;

[0047] Figure 6 is a flow cytometry chart of Example 3;

[0048] Figure 7 is a phenotype chart of each stage of Comparative Example 1;

[0049] Figure 8 is a flow cytometry result chart of Comparative Example 1;

[0050] Figure 9 is a phenotype chart of each stage of Comparative Example 2;

[0051] Figure 10 is a flow cytometry result chart of Comparative Example 2. DETAILED DESCRIPTION

[0052] The principles and features of the present application are described below, and the examples are used only to explain the present application and are not intended to limit the scope of the present application. If a specific technique or condition is not specified in the examples, it is performed according to the technique or condition described in the literature in the art or according to the product manual. If the manufacturer of the reagent or instrument is not specified, it is a conventional product that can be commercially available through a regular channel.

[0053] The CiPSC used in the following examples was prepared by referring to the research results published by Jingyang Guan, Guan Wang, Jinlin Wang, et al. in Nature (Nature, 2022). The specific preparation process is achieved by chemical reprogramming of human somatic cells. In this method, suitable human somatic cells are first selected as starting materials, which are processed through the following stages:

[0054] (1) Cell dedifferentiation stage: small molecule compounds CHIR99021, 616452 and TTNPB are used in combination to convert human fibroblasts into epithelial-like cells; then, Y27632, ABT869 and SAG in the chemical library are screened to further promote the generation of such epithelial-like cells;

[0055] (2) Epigenetic regulation stage: Epigenetic regulators 5-azacytidine and tranylcypromine are added to regulate the global DNA methylation state, thereby inducing a hypomethylation state, which is crucial for losing somatic cell identity;

[0056] (3) Enhance cell plasticity stage: Further add JNK pathway inhibitors (such as JNKIN8) in combination with 5-azacytidine and tranylcypromine to enhance cell plasticity and thus initiate regenerative cell programs.

[0057] (4) Induced pluripotency stage: A series of small molecules, including epigenetic regulators and cell signaling inhibitors (e.g., valproic acid, DZNep, EPZ004777, and UNC0379), are used to activate the pluripotency gene OCT4. This induced pluripotency state is then maintained and promoted by compounds such as CHIR99021 and PD0325901 to obtain CiPSCs. These CiPSCs exhibit key characteristics similar to human embryonic stem cells and are able to expand long-term, showing a stable diploid karyotype.

[0058] Example 1

[0059] This embodiment relates to a method for the directed differentiation of iPSCs into lymphohematopoietic progenitor cells, including the following specific steps:

[0060] (1) First stage, spheroidization culture: pluripotent stem cells are suspended and statically cultured for 1 day using the spheroidization culture medium to form embryoid bodies;

[0061] The specific steps for CiPSC spheroidization are as follows: Take a CiPSC cultured in a 6-well plate with 80-90% cell confluence and place it in a biosafety cabinet. Discard the culture medium and add 1 mL of DPBS along the wall of the plate to wash the cells, repeating twice. Add 200 μL of ReleSR cell digestion solution, shake the plate to wet the entire cell surface with the digestion solution, and then incubate the cells in the biosafety cabinet for 1 min. Discard 180 μL of the digestion solution and place the plate in a 37°C incubator for 3-5 min. Remove the cells from the incubator and observe under a microscope that most cells have become round and shiny spherical. Gently tap the edge of the plate to detach most of the cells. Add 2 mL of mTeSR. TM Digestion was terminated in Plus medium. Cells were pipetted into a single-cell suspension and transferred to 15 mL centrifuge tubes. The cells were centrifuged at 300 g for 5 min. The cell pellet was resuspended in spheroidizing medium and counted. Cells were seeded into 6-well plates at a density of 5 × 10⁶ cells / well. 4Cells were resuspended in 2 mL of the sphere formation medium per well, and the target number of cells were inoculated in a 6-well plate and placed in an incubator for static culture for 1 day.

[0062] (2) The second stage is mesoderm differentiation: the mesoderm induction medium is used to induce differentiation of the embryoid bodies, and the mesoderm cells are formed after 1-2 days of suspension and static culture.

[0063] The mesoderm induction stage is specific: after 1 day, the embryoid bodies are spontaneously formed from CiPSCs, and 2 mL of mesoderm induction medium is slowly added to each well of the 6-well plate, which is placed in an incubator for static culture for 2 days.

[0064] (3) The third stage is mesoderm cell differentiation into hematopoietic endothelial cells: the hematopoietic endothelial cell induction medium is used to induce differentiation of the mesoderm cells, and the hematopoietic endothelial cells are formed after 2-4 days of suspension and static culture.

[0065] The hematopoietic endothelial induction stage is specific: after 2 days of induction, 2 mL of hematopoietic endothelial induction medium is slowly added to each well of the 6-well plate, which is placed in an incubator for static culture for 4 days.

[0066] (4) The fourth stage is hematopoietic endothelial cell differentiation into hematopoietic stem / progenitor cells: the hematopoietic stem / progenitor cell induction medium is used to induce differentiation of the hematopoietic endothelial cells, and the hematopoietic stem / progenitor cells are formed after 5-7 days of suspension and static culture.

[0067] The hematopoietic stem / progenitor cell induction stage is specific: after 4 days of continuous induction, 2 mL of hematopoietic stem / progenitor cell induction medium is slowly added to each well of the 6-well plate, which is placed in an incubator for static culture for 7 days.

[0068] (5) The fifth stage is hematopoietic stem / progenitor cell differentiation into lymphoid hematopoietic precursor cells: the lymphoid hematopoietic precursor cell induction medium is used to induce differentiation of the hematopoietic stem / progenitor cells, and the lymphoid hematopoietic precursor cells are formed after 7-21 days of suspension and static culture.

[0069] The lymphoid hematopoietic precursor cell induction stage is specific: after 7 days, 2 mL of lymphoid hematopoietic precursor cell induction medium is slowly added to each well of the 6-well plate, which is placed in an incubator for static culture for 7-21 days.

[0070] The technical solutions of Example 1 are repeated three times, and the results of each time are detected.

[0071] The sphere formation medium, mesoderm induction medium, hematopoietic endothelial cell induction medium, hematopoietic stem / progenitor cell induction medium, and lymphoid hematopoietic precursor cell induction medium used above are as follows:

[0072] ①Spheroid formation medium is mTeSRTM Plus (Stemcell, 100-0276) medium added with 10 μM Y27632 (Selleck, S1049);

[0073] ②Mesoderm induction medium is 1640 (Gibco, 22400089) complete medium added with 20 ng / mL BMP4 (Stemimmune LLC, HST-B4-0100), 3 μM CHIR99021 (Selleck, S1263);

[0074] ③Hematovascular endothelial cell induction medium is 1640 complete medium added with 50 ng / mL VEGF (Stemimmune LLC, HVG-VF5-1000), 50 ng / mL FGF2 (MCE, HY-P7331), 10 μM SB431542 (Selleck, S1067), 5 ng / mL BMP4;

[0075] ④Hematopoietic stem / progenitor cell induction medium is IMDM (Gibco, 12440053) complete medium added with 20 ng / mL SCF (PeproTech, 300-07), 10 ng / mL FLT3L (PeproTech, 300-19), 10 ng / mL IL-15 (PeproTech, 200-15), 20 ng / mL IL-7 (PeproTech, 200-07), 5 ng / mL IL3 (PeproTech, 200-03), 5 ng / mL BMP4, 10 ng / mL VEGF, 30 μM NAC (Sigma, A7250), 2 μM Minocycline (Selleck, S4226);

[0076] ⑤Lymphohematopoietic precursor cell induction medium is IMDM complete medium added with 20 ng / mL SCF, 10 ng / mL FLT3L, 10 ng / mL IL-15, 20 ng / mL IL-7, 5 ng / mL BMP4, 10 ng / mL VEGF, 30 μM NAC, 2 μM Minocycline, 30 nM UM171 (Aladdin, U421660), 10 μM SR1 (Selleck, S2858);

[0077] wherein, the 1640 complete medium comprises 2% of B-27 without vitamin A in the medium by volume percentage TMAdditives (Gibco, A3353501), MEM Non-Essential Amino Acids Solution (Gibco, 11140050) at 1% of the volume of the medium, GlutaMAX at 1% of the volume of the medium TM Additives (Gibco, 35050061) and 20 ng / mL Ascorbic acid (Sigma, PHR1008).

[0078] IMDM complete medium, including B-27 without vitamin A at 2% of the volume of the medium TM Additives (Gibco, A3353501), MEM Non-Essential Amino Acids Solution (Gibco, 11140050) at 1% of the volume of the medium, GlutaMAX at 1% of the volume of the medium TM Additives (Gibco, 35050061) and 20 ng / mL Ascorbic acid (Sigma, PHR1008).

[0079] 2. Detection method

[0080] Flow cytometry detection of cell surface markers: aspirate the suspended cells in the 6-well plate into a 15 mL centrifuge tube, centrifuge at 300g for 3 min, then discard the supernatant, add 1-2 mL DPBS (Gibco, 14190144) to resuspend the cell pellet, and transfer the cell suspension to a 5 mL flow cytometry sample tube, centrifuge at 300g for 3 min; discard the supernatant, add DPBS (containing 0.5% BSA, Sigma, A1933) to resuspend the cells as needed, 100 μL / tube; set up the blank control group, test group, isotype control group, single dye compensation group, etc. in advance, add antibodies, vortex to mix for 3-5 s, and incubate at 4°C in the dark for 15 min; after staining, wash with 1 mL DPBS, centrifuge at 300g for 3 min, repeat twice, and finally resuspend the cells in 200-300 μL DPBS per tube to start flow cytometry detection.

[0081] Flow cytometry detection antibodies include PE / Cyanine7 anti-human CD4 Antibody (Biolegend, 300511), PE anti-human CD45 (Biolegend, 304007), FITC anti-human CD3 (Biolegend, 300306), APC anti-human CD8 Antibody (Biolegend, 344721).

[0082] As can be seen from FIGS. 1 and 2, under the suspension culture condition, the CiPSCs can spontaneously form embryoid-like bodies and migrate out hematopoietic cells, which further proliferate and differentiate to produce lymphoid hematopoietic precursor cells expressing CD45, CD3 and CD4.

[0083] Example 2

[0084] Compared with Example 1, the rest are the same as Example 1 except that the adopted spheroid formation medium, mesoderm induction medium, hemogenic endothelial cell induction medium, hematopoietic stem / progenitor cell induction medium and lymphoid hematopoietic precursor cell induction medium are different. The above-mentioned adopted spheroid formation medium, mesoderm induction medium, hemogenic endothelial cell induction medium, hematopoietic stem / progenitor cell induction medium and lymphoid hematopoietic precursor cell induction medium are as follows:

[0085] ①The spheroid formation medium is mTeSR Plus (Stemcell, 100-0276) medium added with 1 μM Y27632 (Selleck, S1049); TM

[0086] ②The mesoderm induction medium is 1640 (Gibco, 22400089) complete medium added with 1 ng / mL BMP4 (Stemimmune LLC, HST-B4-0100), 1 μM CHIR99021 (Selleck, S1263);

[0087] ③The hemogenic endothelial cell induction medium is 1640 complete medium added with 5 ng / mL VEGF (Stemimmune LLC, HVG-VF5-1000), 50 ng / mL FGF2 (MCE, HY-P7331), 10 μM SB431542 (Selleck, S1067), 5 ng / mL BMP4;

[0088] ④The hematopoietic stem / progenitor cell induction medium is IMDM (Gibco, 12440053) complete medium added with 20 ng / mL SCF (PeproTech, 300-07), 10 ng / mL FLT3L (PeproTech, 300-19), 10 ng / mL IL-15 (PeproTech, 200-15), 20 ng / mL IL-7 (PeproTech, 200-07), 5 ng / mL IL3 (PeproTech, 200-03), 5 ng / mL BMP4, 10 ng / mL VEGF, 15 μM NAC (Sigma, A7250), 1 μM Minocycline (Selleck, S4226);

[0089] ​The lymphoid hematopoietic precursor cell induction medium is IMDM complete medium added with 20 ng / mL SCF, 10 ng / mL FLT3L, 10 ng / mL IL-15, 20 ng / mL IL-7, 5 ng / mL BMP4, 10 ng / mL VEGF, 15 μM NAC, 1 μM Minocycline, 10 nM UM171 (Aladdin, U421660), 5 μM SR1 (Selleck, S2858);

[0090] The 1640 complete medium includes 2% B-27 without vitamin A in the medium TM additives (Gibco, A3353501), 1% MEM non-essential amino acid solution (Gibco, 11140050) in the medium, and 1% GlutaMAX in the medium TM additives (Gibco, 35050061) and 20 ng / mL Ascorbic acid (Sigma, PHR1008);

[0091] The IMDM complete medium includes 2% B-27 without vitamin A in the medium TM additives (Gibco, A3353501), 1% MEM non-essential amino acid solution (Gibco, 11140050) in the medium, and 1% GlutaMAX in the medium TM additives (Gibco, 35050061) and 20 ng / mL Ascorbic acid (Sigma, PHR1008).

[0092] As can be seen from FIGS. 3 and 4, under the suspension culture condition, the CiPSCs can spontaneously form morphologically uniform embryoid-like bodies, and the embryoid-like bodies increase in diameter, and cells can be observed to migrate out of the embryoid-like bodies into the hematopoietic stage, the cells proliferate at a relatively high speed, and the hematopoietic precursor cells induced by differentiation have a large number of cells and can form cell clusters, and the lymphoid hematopoietic precursor cells expressing CD45, CD3 and CD4 are generated.

[0093] Example 3

[0094] The rest was the same as Example 1 except for the employed spheroid culture medium, mesoderm induction medium, hemogenic endothelial cell induction medium, hemogenic stem / progenitor cell induction medium, and lymphohematopoietic precursor cell induction medium. The employed spheroid culture medium, mesoderm induction medium, hemogenic endothelial cell induction medium, hemogenic stem / progenitor cell induction medium, and lymphohematopoietic precursor cell induction medium were as follows:

[0095] ① The spheroid culture medium was mTeSR™ Plus (Stemcell, 100-0276) medium added with 20 μM Y27632 (Selleck, S1049);

[0096] ② The mesoderm induction medium was 1640 (Gibco, 22400089) complete medium added with 100 ng / mL BMP4 (Stemimmune LLC, HST-B4-0100), 12 μM CHIR99021 (Selleck, S1263);

[0097] ③ The hemogenic endothelial cell induction medium was 1640 complete medium added with 100 ng / mL VEGF (Stemimmune LLC, HVG-VF5-1000), 50 ng / mL FGF2 (MCE, HY-P7331), 10 μM SB431542 (Selleck, S1067), 5 ng / mL BMP4;

[0098] ④ The hemogenic stem / progenitor cell induction medium was IMDM (Gibco, 12440053) complete medium added with 20 ng / mL SCF (PeproTech, 300-07), 10 ng / mL FLT3L (PeproTech, 300-19), 10 ng / mL IL-15 (PeproTech, 200-15), 20 ng / mL IL-7 (PeproTech, 200-07), 5 ng / mL IL3 (PeproTech, 200-03), 5 ng / mL BMP4, 10 ng / mL VEGF, 60 μM NAC (Sigma, A7250), 4 μM Minocycline (Selleck, S4226);

[0099] The lymphoid hematopoietic precursor cell induction medium is IMDM complete medium added with 20 ng / mL SCF, 10 ng / mL FLT3L, 10 ng / mL IL-15, 20 ng / mL IL-7, 5 ng / mL BMP4, 10 ng / mL VEGF, 60 μM NAC, 4 μM Minocycline, 100 nM UM171 (Aladdin, U421660), 20 μM SR1 (Selleck, S2858);

[0100] The 1640 complete medium comprises 2% B-27 without vitamin A of the medium by volume percentage TM additives (Gibco, A3353501), 1% MEM non-essential amino acid solution (Gibco, 11140050) of the medium by volume percentage, and 1% GlutaMAX of the medium by volume percentage TM additives (Gibco, 35050061) and 20 ng / mL Ascorbic acid (Sigma, PHR1008);

[0101] The IMDM complete medium comprises 2% B-27 without vitamin A of the medium by volume percentage TM additives (Gibco, A3353501), 1% MEM non-essential amino acid solution (Gibco, 11140050) of the medium by volume percentage, and 1% GlutaMAX of the medium by volume percentage TM additives (Gibco, 35050061) and 20 ng / mL Ascorbic acid (Sigma, PHR1008).

[0102] As can be seen from FIGS. 5 and 6, under the suspension culture condition, the CiPSCs can spontaneously form morphologically uniform embryoid bodies, and the embryoid bodies increase in diameter, and cells can be observed to migrate out of the embryoid bodies into the hematopoietic stage, and the cells proliferate at a relatively high speed, and the hematopoietic precursor cells induced by differentiation have a large number of cells and can form cell clusters, and can produce lymphoid hematopoietic precursor cells expressing CD45, CD3 and CD4.

[0103] Comparative Example 1

[0104] Compared with Example 1, the rest is the same as Example 1 except that a different hematopoietic stem / progenitor cell induction medium is used. The hematopoietic stem / progenitor cell induction medium used above is as follows:

[0105] (4) Hematopoietic stem / progenitor cell induction medium is IMDM (Gibco, 12440053) complete medium added with 20 ng / mL SCF (PeproTech, 300-07), 10 ng / mL FLT3L (PeproTech, 300-19), 10 ng / mL IL-15 (PeproTech, 200-15), 20 ng / mL IL-7 (PeproTech, 200-07), 5 ng / mL IL3 (PeproTech, 200-03), 5 ng / mL BMP4, 10 ng / mL VEGF, 30 mM NAC (Sigma, A7250), 2 mM Minocycline (Selleck, S4226), 30 nM UM171, 10 mM SR1.

[0106] As can be seen from FIGS. 7 and 8, during the hematopoietic stem / progenitor cell induction stage, the addition of UM171 and SR1 affects the positive expression of CD3.

[0107] Comparative Example 2

[0108] Compared with Example 1, except that the hematopoietic stem / progenitor cell induction medium and the lymphoid hematopoietic precursor cell induction medium do not contain SCF, the rest is the same as Example 1.

[0109] As can be seen from FIGS. 9 and 10, during the hematopoietic stem / progenitor cell induction stage and the lymphoid hematopoietic precursor cell induction stage, the removal of SCF significantly reduces the proportion of main population cells and the differentiation efficiency of lymphoid hematopoietic precursor cells. The reason is that SCF plays a key role in maintaining the survival of hematopoietic stem / progenitor cells and promoting the directional differentiation of blood cells.

[0110] In summary, by precisely regulating the composition of the culture medium and the formula of the growth factors, the present application ensures that the cells obtain sufficient nutrition and signals during the induction process, thereby improving the differentiation efficiency and stability. Compared with the traditional culture method, the present application uses whole-suspension culture, simulates the three-dimensional environment in vivo, avoids the problems of cell morphology change and stress, and at the same time improves the stability and growth rate of the cells. The present application has the characteristics of simple operation and low cost, and is suitable for large-scale production and clinical application. Therefore, the present application provides a more reliable and efficient solution for in vitro induction of pluripotent stem cells into lymphoid hematopoietic precursor cells, and brings new hope for the treatment of blood system diseases and the application of tissue engineering.

[0111] Although the embodiments of the present application have been shown and described above, it should be understood that the above embodiments are exemplary and should not be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above embodiments within the scope of the present application.

Claims

1. A method for the directed differentiation of iPSC into lymphohematopoietic precursor cells, characterized in that, The method comprises the following steps: (1) the first stage, the ball formation culture: the pluripotent stem cells are suspended and cultured for 1 day by using the ball formation culture medium to form the embryoid bodies; (2) the second stage, the mesoderm differentiation: the embryoid bodies are induced and differentiated by using the mesoderm induction culture medium, and are suspended and cultured for 1-2 days to form the mesoderm cells; (3) the third stage, the mesoderm cell differentiation into hematopoietic endothelial cells: the mesoderm cells are induced and differentiated by using the hematopoietic endothelial cell induction culture medium, and are suspended and cultured for 2-4 days to form the hematopoietic endothelial cells; (4) the fourth stage, the hematopoietic endothelial cell differentiation into hematopoietic stem / progenitor cells: the hematopoietic endothelial cells are induced and differentiated by using the hematopoietic stem / progenitor cell induction culture medium, and are suspended and cultured for 5-7 days to form the hematopoietic stem / progenitor cells; (5) the fifth stage, the hematopoietic stem / progenitor cell differentiation into lymphoid hematopoietic precursor cells: the hematopoietic stem / progenitor cells are induced and differentiated by using the lymphoid hematopoietic precursor cell induction culture medium, and are suspended and cultured for 7-21 days to form the lymphoid hematopoietic precursor cells; The lymphoid hematopoietic precursor cells express CD45, CD3 and CD4, and do not express CD8; The ball formation culture medium comprises the following components in the following amounts: mTeSR Plus culture medium and 1-20 μM Y27632; The mesoderm induction culture medium comprises the following components in the following amounts: 1640 complete culture medium, 1-100 ng / mL BMP4 and 1-12 μM CHIR99021; The hematopoietic endothelial cell induction culture medium comprises the following components in the following amounts: 1640 complete culture medium, 5-100 ng / mL VEGF, 1-25 ng / mL BMP4, 10-250 ng / mL FGF2 and 2-50 μM SB431542; The hematopoietic stem / progenitor cell induction culture medium comprises the following components in the following amounts: IMDM complete culture, 1-25 ng / mL BMP4, 2-50 ng / mL VEGF, 15-60 μM NAC, 1-4 μM Minocycline, 4-100 ng / mL SCF, 2-50 ng / mL FLT3L, 2-50 ng / mL IL-15, 2-50 ng / mL IL-7 and 1-25 ng / mL IL3; The lymphoid hematopoietic precursor cell induction culture medium comprises the following components in the following amounts: IMDM complete culture, 1-25 ng / mL BMP4, 2-50 ng / mL VEGF, 15-60 μM NAC, 1-4 μM Minocycline, 10-100 nM UM171, 5-20 μM SR1, 4-100 ng / mL SCF, 2-50 ng / mL FLT3L, 2-50 ng / mL IL-15 and 2-50 ng / mL IL-7.

2. The method of claim 1, wherein the iPSCs are differentiated into lymphohematopoietic precursor cells. The hemogenic endothelial cell induction medium comprises the following components in the following amounts: 25-100 ng / mL VEGF, 2.5-10 ng / mL BMP4, 25-100 ng / mL FGF2, 5-20 µM SB431542; The hemogenic endothelial cell induction medium comprises the following components in the following amounts: 25-100 ng / mL VEGF, 2.5-10 ng / mL BMP4, 25-100 ng / mL FGF2, 5-20 µM SB431542; The hemogenic endothelial cell induction medium comprises the following components in the following amounts: 25-100 ng / mL VEGF, 2.5-10 ng / mL BMP4, 25-100 ng / mL FGF2, 5-20 µM SB431542; 3. The method of claim 1, wherein the iPSCs are differentiated into lymphohematopoietic precursor cells. The hemogenic endothelial cell induction medium comprises the following components in the following amounts: 25-100 ng / mL VEGF, 2.5-10 ng / mL BMP4, 25-100 ng / mL FGF2, 5-20 µM SB431542; The pluripotent stem cells comprise chemically induced pluripotent stem cells.

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