Method for differentiating NK cells

Abstract

The invention relates to the technical field of biomedicine, in particular to a differentiation method of NK cells. By optimizing a differentiation strategy, culture medium components and culture parameters, the NK cell differentiation method of the induction system without the feeder layer is obtained; the differentiation method of the NK cells is low in cost and high in differentiation efficiency, and the obtained NK cells have a high cell killing effect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for differentiating NK cells. Background Technology

[0002] Natural killer (NK) cells, as members of the innate immune system, can rapidly and directly kill target cells without prior sensitization. They exhibit non-specific cell-killing characteristics and can also play a role in adaptive immune responses by producing immunomodulatory cytokines, making them highly promising for applications in anti-tumor therapy. NK cells can be extracted from the blood of patients or healthy donors, but this method presents challenges such as significant individual variability, limited expansion, difficulty in controlling cell quality, and the difficulty of genetic modification. Therefore, the availability of NK cells is one of the bottlenecks limiting the development of NK cell immunotherapy.

[0003] Induced pluripotent stem cells (iPSCs) are a novel type of stem cell obtained by Japanese scientist Shinya Yamanaka through reprogramming human skin cells. They possess similar proliferation and differentiation capabilities to embryonic stem cells. Studies have shown that NK cells differentiated from iPSCs exhibit high homogeneity and are easily manipulated through gene editing, making this differentiation pathway an important way to obtain NK cells. The differentiation of iPSCs into NK cells involves two stages: hematopoietic stem cells (HSCs) and NK cells. Initially, in vitro NK cell induction methods mainly include feeder layer co-culture systems and organoid induction systems. In feeder layer co-culture systems, iPSCs rely on mouse bone marrow stromal cells OP9, M210-B4, or S17 to form hematopoietic stem cells, or, under the influence of cytokines (BMP4, SCF, and VEGF), differentiate into embryoid bodies to obtain hematopoietic stem cells. These hematopoietic stem cells are then co-cultured with mouse bone marrow stromal cells MS-5, AFT024, or EL08-1D2, generating NK cells under the influence of cytokines. The NK cell differentiation cycle obtained by this co-culture method with feeder cells is long, the efficiency is unstable, and the purity is not high. In the organoid induction system, iPSCs are induced to the lateral plate mesoderm (LPM) of embryonic development. Organoid aggregates are prepared by mixing LPM cells with OP9 feeder cells that do not express human NOTCH ligands DLL1 or DLL4. The organoids are then seeded in 0.4 μm Millicell transwells to obtain NK cells. Neither of these methods can be scaled up for production, and both rely on xenogeneic cells. The obtained NK cells may carry pathogens, causing immune rejection, and cannot be directly used for clinical anti-tumor therapy.

[0004] Given that existing NK cell culture methods are inefficient, have low purity, and are prone to causing immune rejection, it is particularly necessary to develop new NK cell culture methods. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a method for differentiating NK cells.

[0006] The present invention provides a composition for NK cell differentiation, comprising: a suspension culture medium, induction medium A, induction medium B, induction medium C, induction medium D, and induction medium E;

[0007] The suspension culture medium includes mTeSR1 and Stempro-34, and the volume ratio of mTeSR1 to Stempro-34 is (4~1):(1~2).

[0008] The induction medium A comprises: StemPro-34 and 10 ng / mL to 40 ng / mL BMP4;

[0009] The induction medium B comprises: StemPro-34, 10 ng / mL to 40 ng / mL BMP4 and 1 ng / mL to 10 ng / mL bFGF;

[0010] The induction medium C includes: StemPro-34, 10 ng / mL to 40 ng / mL BMP4, 1 mM to 10 mM CHIR99021, 1 ng / mL to 10 ng / mL bFGF and 1 mM to 6 mM SB431542;

[0011] The induction medium D comprises: StemPro-34, 10 ng / mL to 50 ng / mL VEGF and 1 ng / mL to 10 ng / mL bFGF;

[0012] The induction medium E comprises: Stempro-34, 1 ng / mL to 10 ng / mL bFGF, 10 ng / mL to 100 ng / mL LSCF, 2 U / mL EPO, 50 ng / mL IGF-1, 20 ng / mL IL-6 and 10 ng / mL IL-11.

[0013] Furthermore, the composition of the present invention further includes at least one of the following: lymphocyte progenitor cell differentiation medium, induction medium, NK cell induction medium 1 and / or NK cell induction medium 2;

[0014] The lymphocyte progenitor cell differentiation culture medium includes: Stemspan SFEM II, Stemspan SFEM II, 10 vt% serum substitute, 2 mM L-glutamine, 50 μg / mL L-ascorbic acid-2-phosphate trisodium salt, 1 mM sodium pyruvate, 55 mM β-mercaptoethanol, 1 mM non-essential amino acids, 30 ng / mL SCF, 20 ng / mL FLT3L, 5 ng / mL interleukin-3, 30 ng / mL interleukin-7 and 5 ng / mL thrombopoietin;

[0015] The induction medium consisted of: Stemspan SFEM II, 10 vt% serum substitute, 2 mM L-glutamine, 50 μg / mL L-ascorbic acid-2-phosphate trisodium salt, 1 mM sodium pyruvate, 55 mM β-mercaptoethanol, 1 mM non-essential amino acids, 30 ng / mL SCF, 20 ng / mL FLT3L, 30 ng / mL interleukin-7, and 5 ng / mL thrombopoietin;

[0016] The NK cell induction medium 1 comprises: Stemspan SFEM II, 10 vt% serum substitute, 2 mM L-glutamine, 50 μg / mL L-ascorbic acid-2-phosphate trisodium salt, 1 mM sodium pyruvate, 55 mM β-mercaptoethanol, 1 mM non-essential amino acids, 20 ng / mL FLT3L and 15 ng / mL interleukin-7;

[0017] The NK cell induction medium 2 comprises: Stemspan SFEM II, 10 vt% serum substitute, 2 mM L-glutamine, 50 μg / mL L-ascorbic acid-2-phosphate trisodium salt, 1 mM sodium pyruvate, 55 mM β-mercaptoethanol, 1 mM non-essential amino acids, 20 ng / mL FLT3L, 15 ng / mL interleukin-7 and 5 ng / mL interleukin-15.

[0018] The present invention provides a method for differentiating NK cells, which involves differentiating NK cells using the composition described in claim 1 or 2.

[0019] Furthermore, the differentiation method of the present invention includes the following steps:

[0020] Step 1: After digestion and resuspension, induced pluripotent stem cells with a confluence rate of 80%~90% are transferred to ultra-low adsorption culture dishes for suspension culture to form embryoid bodies;

[0021] Step 2: The embryoids are cultured sequentially in induction medium A to induction medium E to obtain hematopoietic endothelial cells;

[0022] Step 3: Seed the hematopoietic endothelial cells into culture dishes coated with human recombinant protein DLL4-Fc and non-tissue treated, and culture them sequentially using the lymphocyte progenitor cell differentiation medium and induction medium to obtain lymphocyte progenitor cells;

[0023] Step 4: The lymphocyte progenitor cells are cultured sequentially in NK cell induction medium 1 and NK cell induction medium 2 to obtain NK cells;

[0024] The culture medium used in the suspension culture is a suspension culture medium; the rotation speed of the suspension culture is 50 rpm; the cell concentration during the suspension culture is 4e4 / mL to 4e5 / mL; the CO2 concentration and O2 concentration of the suspension culture are 5%.

[0025] In the differentiation method described in this invention, the induction medium A to induction medium E are cultured for 1 day for each medium.

[0026] The culture time for the lymphocyte progenitor cell differentiation medium is 7 days; the culture time for the induction medium is 7 days.

[0027] The NK cell induction medium 1 is cultured for 7 to 14 days, specifically 14 days.

[0028] In the differentiation method described in this invention, the induction medium A to induction medium E further include a cell digestion step. Digesting the cultured cells to obtain single cells to facilitate the next culture step is a conventional step taken by those skilled in the art, and this invention will not elaborate on it.

[0029] In the differentiation method described in this invention, the induced pluripotent stem cells are mesenchymal-derived induced pluripotent stem cells.

[0030] This invention provides a method for NK cell differentiation using a feeder-free induction system. In a specific embodiment, the differentiation strategy was screened, using hypoxia to rapidly obtain hematopoietic endothelial cells and then NK cells (culture method 1) and normoxic system induction to obtain hematopoietic stem cells and then NK cells (culture method 2). Experimental results showed that the cell differentiation efficiency was higher when using hypoxia to rapidly obtain hematopoietic endothelial cells and then NK cells (culture method 1). Furthermore, the culture medium and culture parameters involved in the process of rapidly obtaining hematopoietic endothelial cells and then NK cells (culture method 1) were optimized to obtain the NK cell differentiation method described in this invention. It is evident that the culture medium components and culture parameters in the differentiation method of this invention interact and influence the differentiation efficiency and NK cell killing efficiency of the NK cells, giving the differentiation method of this invention the advantages of high differentiation efficiency and high NK cell killing efficiency.

[0031] Meanwhile, this invention was compared with existing technologies. Patent CN115896019B involves a technique for inducing pluripotent stem cells to differentiate into NK cells, but this technique still suffers from interference from CD3 cells, resulting in a low purity of approximately 82%. These contaminated cells may have unknown effects on NK cell function, increasing batch-to-batch variability and hindering industrialization. The cells obtained by this invention do not express CD3 at all, achieving a purity of over 90%. Furthermore, compared to CN115896019B, which requires a 20:1 ratio to achieve a 90% kill rate, this invention achieves a 90% kill rate with a ratio of only 10:1, representing a significant increase in kill rate.

[0032] This invention provides a method for differentiating NK cells in an induction system without a feeder layer by optimizing differentiation strategies, culture medium components, and culture parameters. The method for differentiating NK cells is low-cost, highly efficient, and produces NK cells with high cell-killing effects. Attached Figure Description

[0033] Figure 1 The effects of mTeSR1 and Stempro-34 in the culture medium for suspension culture on embryoid viability;

[0034] Figure 2 Morphological diagrams of embryoids formed at different rotation speeds (0, 50, 100 rpm);

[0035] Figure 3 The results show the morphological results of embryoids obtained with different cell amounts using 96-well plates to create embryoids.

[0036] Figure 4 The results show the morphological results of embryoids obtained with different cell amounts using 6-well plates to create embryoids.

[0037] Figure 5 The images show the white light spectrum and flow cytometry results of embryoids after treatment with different concentrations of BMP4; where A is the white light spectrum and B is the flow cytometry result.

[0038] Figure 6 The white light spectrum and flow cytometry results of embryoids after treatment with different concentrations of bFGF are shown; where A is the white light spectrum and B is the flow cytometry result.

[0039] Figure 7 The images show the white light spectrum and flow cytometry results of embryoids after treatment with different concentrations of CHIR99021; where A is the white light spectrum and B is the flow cytometry result.

[0040] Figure 8 The images show the white light spectrum and flow cytometry results of embryoids after treatment with different concentrations of SB43542; where A is the white light spectrum and B is the flow cytometry result.

[0041] Figure 9 The images show the white light spectrum and flow cytometry results of embryoids after treatment with different concentrations of VEGF; where A is the white light spectrum and B is the flow cytometry result.

[0042] Figure 10 The images show the white light spectrum and flow cytometry results of embryoids after treatment with different concentrations of SCF; where A is the white light spectrum and B is the flow cytometry result.

[0043] Figure 11 The proportion of hematopoietic stem cells obtained under culture protocol 1 is shown.

[0044] Figure 12 The proportion of hematopoietic stem cells obtained under culture protocol 2 is shown.

[0045] Figure 13 The expression results of CD3 were analyzed by flow cytometry using culture method 1.

[0046] Figure 14 The results of NK cell detection obtained by culture method 1 and culture method 2 are shown; where A represents the purity of NK cells obtained by culture method 1 and B represents the purity of NK cells obtained by culture method 2. Detailed Implementation

[0047] This invention provides a method for culturing NK cells. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0048] Terminology Explanation:

[0049] Interferon-gamma (IFN-γ): Interferon-gamma belongs to the type II interferon family and is a glycoprotein. It is also an important cytokine, mainly produced by activated T cells and NK cells. It plays multiple roles in the immune system, particularly in antiviral, antitumor, and immunomodulatory functions.

[0050] Tumor necrosis factor-alpha (TNF-α): Tumor necrosis factor-alpha belongs to the tumor necrosis factor superfamily and plays a key role in immune regulation, inflammatory response and apoptosis.

[0051] Chimeric Antigen Receptor T-cell (CAR-T): CAR-T is a personalized cell immunotherapy that introduces chimeric antigen receptors (CARs) into the patient's own T cells, enabling them to recognize and attack specific tumor cells.

[0052] Major Histocompatibility Complex Class I (MHC-I): Major Histocompatibility Complex Class I molecules are a class of cell surface molecules responsible for presenting intracellular antigens to CD8+ T cells.

[0053] Graft-versus-host disease (GVHD): GVHD occurs when the donor's immune cells recognize the recipient's tissue antigens as "foreign objects" and launch an immune attack, resulting in damage to the recipient's tissues and organs.

[0054] Adoptive NK cell therapy: This involves expanding and activating NK cells in vitro and then reinfusing them into the patient to enhance the immune system's ability to kill tumor or virus-infected cells.

[0055] Chimeric Antigen Receptor Natural Killer Cells (CAR-NK): By introducing chimeric antigen receptors (CARs) into NK cells, they are able to specifically recognize and kill tumor cells.

[0056] Monoclonal antibodies are highly homogeneous antibodies produced by a single B cell clone that target only a specific antigenic epitope.

[0057] NK cell adaptor: It is an artificially designed molecule, usually a bispecific or multispecific antibody, that can bind to both NK cells and tumor cells simultaneously, forming an immune synapse, thereby activating NK cells and inducing them to kill tumor cells.

[0058] EB: Embryoid refers to an embryonic structure similar to a zygote that differentiates from somatic cells or sex cells without fertilization in the in vitro culture of plant cells, tissues or organs.

[0059] Spin EB: Single-cell cluster embryoids are a technique for forming embryoids by centrifuging single cells together.

[0060] Induced pluripotent stem cells (iPSCs): Induced pluripotent stem cells are a type of stem cell that has been converted into pluripotent stem cells through gene reprogramming technology. They have a similar differentiation potential to embryonic stem cells (ESCs).

[0061] Human pluripotent stem cells (hPSCs) are a type of stem cell with self-renewal capacity and the potential to differentiate into multiple cell types. They mainly include human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs).

[0062] Hematopoietic progenitor cells (HPCs) are a type of cell with limited self-renewal capacity and multi-lineage differentiation potential, capable of differentiating into all types of mature blood cells.

[0063] Lateral Plate Mesoderm (LPM): The lateral plate mesoderm is a transient embryonic tissue that forms during the development of vertebrate embryos. It is located on both sides of the embryo and lies between the ectoderm and the endoderm.

[0064] NOTCH ligand: The NOTCH signaling pathway is an evolutionarily conserved cell signaling pathway that regulates cell fate determination and maintains homeostasis in adult tissues during the development of multicellular organisms.

[0065] APEL / BPEL: APEL (albumin-polyvinyl alcohol-essential lipid medium) and BPEL (basal medium-polyvinyl alcohol-essential lipid medium) are two commonly used culture media for stem cell differentiation and culture.

[0066] Bone morphogenetic protein 4 (BMP4): Bone morphogenetic protein 4 is a member of the transforming growth factor-β (TGF-β) superfamily and is an important signaling molecule that plays a key role in embryonic development, cell differentiation and tissue regeneration.

[0067] Stem Cell Factor (SCF): Stem cell factor is an important cytokine that is widely used in research on stem cell differentiation and expansion.

[0068] Vascular endothelial growth factor (VEGF): VEGF is an important signaling protein that mainly acts on vascular endothelial cells, promoting angiogenesis and increasing vascular permeability.

[0069] Interleukin-7 (IL-7): Interleukin-7 is a pleiotropic cytokine belonging to the IL-2 superfamily. It is mainly produced by bone marrow stromal cells, thymocytes, fetal liver cells, and epithelial cells. It plays a crucial role in the development and maintenance of the immune system.

[0070] Interleukin-15 (IL-15) is an important cytokine primarily secreted by immune cells. It plays a crucial role in regulating the immune system, particularly in the activation and proliferation of T cells and natural killer cells.

[0071] Interleukin-3 (IL-3) is a pleiotropic cytokine mainly produced by activated T cells, playing roles in immune regulation, hematopoietic regulation, and inflammatory responses.

[0072] Interleukin-6 (IL-6) is a multifunctional cytokine belonging to the interleukin family. It plays an important regulatory role in immune regulation, inflammatory responses, cell growth, and differentiation.

[0073] Fms-related Tyrosine Kinase 3 Ligand (Flt3L): Fms-like tyrosine kinase 3 ligand mainly acts on progenitor cells and dendritic cells (DCs) in the hematopoietic system, promoting their proliferation, differentiation and survival.

[0074] Thrombopoietin (TPO): Thrombopoietin mainly acts on megakaryocytes and hematopoietic stem cells to promote platelet production and maintain the homeostasis of the hematopoietic system.

[0075] This invention utilizes a stromal cell-free in vitro induced NK cell method. The differentiation process is safe and controllable, free from mouse-derived contamination, laying the foundation for the clinical translation of stem cells. The process of inducing pluripotent stem cell differentiation to obtain NK cells mainly involves four stages: induced pluripotent stem cell suspension culture to form embryoid bodies; embryoid body suspension culture to generate hematopoietic endothelial cells; hematopoietic endothelial cells further differentiate to generate lymphocyte progenitor cells; and lymphocyte progenitor cells differentiate to obtain NK cells. The differentiation process incorporates a track-type shaker, which greatly increases the differentiation efficiency of hematopoietic endothelial cells and shortens the differentiation time. This invention utilizes multiple methods to induce CD34 cells and compares the efficiency of obtaining NK cells using two different differentiation methods, culture method 1 and culture method 2. The results show that culture method 1 is superior.

[0076] Advantages of this invention compared to existing technologies:

[0077] 1. Improved Differentiation Efficiency: In previous feeder-free cell culture systems, iPSCs required acclimatization from mouse feeder-free cell culture systems, and the differentiation cycle for inducing NK cells took 6-7 weeks. The yield of NK cells was uncertain, generally ranging from 70% to 90%, and a stable high yield could not be maintained. This invention introduces a track-type shaker, shortening the differentiation time to 36 days and obtaining high-purity (>99%) NK cells.

[0078] 2. Previous patents showed that the in vitro induced NK cell killing rate was low. This invention significantly improves the in vitro induced NK cell killing rate to over 90%.

[0079] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:

[0080] Example 1: Culture Protocol 1 - Rapid differentiation of hematopoietic endothelial cells under hypoxia followed by NK cell differentiation.

[0081] I. Suspension Culture of Induced Pluripotent Stem Cells

[0082] Induced pluripotent stem cells (iPSCs) were cultured in 100 mm adherent culture dishes using mTeSR1 medium at 37°C in a 5% CO2, 5% O2 incubator for routine culture, with the medium changed daily. After approximately 4-5 days of culture, iPSCs with a confluence of 80%-90% were selected for digestion (using ReleS R at 37°C for 8-10 minutes), resuspended in mTeSR1, and transferred to ultra-low adsorption culture dishes for suspension culture to form embryoid bodies, which was recorded as day 0.

[0083] The culture medium for suspension culture was mTeSR1 and Stempro-34. The ratio of mTeSR1 to Stempro-34 was 4:1, 3:1, 2:1, 1:1, and 1:2. The results showed that at a ratio of 1:2, the embryoids were observed to have regular shapes, clear cell clusters, and the best embryoid condition. Therefore, a ratio of 1:1 was chosen as the final ratio used. Figure 1 ).

[0084] The suspension culture was performed using a track-type shaker with rotation speeds of 100 rpm, 50 rpm, and 0 rpm. At 50 rpm, the embryoids appeared to have the most regular shape and the cell clusters were most translucent, indicating the best embryoid condition. Therefore, 50 rpm was chosen as the final rotation speed. Figure 2 ).

[0085] The suspension culture involved culturing cells in 96-well or 6-well plates. Cell concentrations in 96-well plates were 1e4 / mL, 4e4 / mL, and 8e4 / mL, while in 6-well plates, concentrations were 1e5 / mL, 2e5 / mL, and 4e5 / mL. The culture environment was 37°C, 5% CO2, and 5% O2. Embryomorphs were created using 96-well plates. The morphology of the embryomorphs obtained with different cell concentrations (1e4 / mL, 4e4 / mL, and 8e4 / mL) was observed. At a concentration of 4e4 / mL, the embryomorphs exhibited the best shape, clear cell clusters, and optimal embryomorph condition. Figure 3 Embryomorphs were prepared using 6-well plates. The morphology of embryomorphs obtained with different cell masses were compared. The cell masses were 1e5 / mL, 2e5 / mL, and 4e5 / mL. Among them, the embryomorphs with 4e5 / mL were observed to have regular shapes and clear cell clusters, and the embryomorphs were in the best condition. Figure 4 ).

[0086] II. In vitro induction of hematopoietic endothelial cells

[0087] 1. On day one, embryoids were transferred to induction medium A. Induction medium A consisted of StemPro-34 and BMP4, with final BMP4 concentrations of 10 ng / mL, 20 ng / mL, and 40 ng / mL. Based on the flow cytometry results, the yield of CD34 was highest at 40 ng / mL; therefore, this concentration was selected as the optimal concentration. Figure 5 ).

[0088] 2. On the second day, the embryoids were transferred to induction medium B, which consisted of StemPro-34, BMP4, and bFGF. The final concentration of BMP4 was 40 ng / mL. The final concentrations of bFGF were 1 ng / mL, 5 ng / mL, and 10 ng / mL. Based on the differentiation effect, the yield of CD34 was highest at 10 ng / mL, therefore this concentration was selected as the optimal concentration. Figure 6 ).

[0089] 3. On the third day, the embryoids were transferred to induction medium C for further differentiation induction. Induction medium C consisted of StemPro-34 supplemented with BMP4, CHIR99021, bFGF, and SB431542. The final concentration of BMP4 was 40 ng / mL, and the treatment times were 72 h and 96 h, respectively; 96 h was selected based on experimental results. The final concentration of bFGF was 10 ng / mL; the final concentrations of CHIR99021 were 1 mM, 5 mM, and 10 mM. Based on experimental results, the highest yield of CD34 was observed at 10 mM, therefore this concentration was selected as the optimal concentration. Figure 7The final concentrations of SB431542 were 1 mM, 3 mM, and 6 mM. According to the experimental results, the yield of CD34 was highest at 6 mM, therefore this concentration was selected as the optimal concentration. Figure 8 ).

[0090] 4. On the fourth day, the embryoids were transferred to induction medium D for further induction differentiation. Induction medium D consisted of StemPro-34, VEGF, and bFGF. The bFGF concentration was 10 ng / mL, and the VEGF concentrations were 10 ng / mL, 20 ng / mL, and 50 ng / mL. Based on the experimental results, the yield of CD34 was highest at 50 ng / mL; therefore, this concentration was selected as the optimal concentration. Figure 9 ).

[0091] 5. On the sixth day, the embryoids were transferred to induction medium E for further differentiation induction. Induction medium E consisted of Stempro-34, bFGF, SCF, EPO, IGF-1, IL-6, and IL-11. The concentrations were as follows: bFGF 10 ng / mL; SCF 50 ng / mL; EPO 2 U / mL; IGF-1 50 ng / mL; IL-6 20 ng / mL; IL-11 10 ng / mL; and SCF concentrations of 10 ng / mL, 50 ng / mL, and 100 ng / mL. Based on experimental results, the highest yield of CD34 was observed at 50 ng / mL, therefore this concentration was selected as the optimal concentration. Figure 10 ).

[0092] 6. On day 8, resuspend the cells with Accutase, gently pipette to mix, and then incubate in a 37°C water bath. Every 10 minutes, gently pipette 1 mL for 30 seconds to promote the digestion and dissociation of cell clumps. After digestion for 20-60 minutes, centrifuge at 400g for 10 minutes to collect the cells. Then resuspend the cells with DPBS, filter through a 70-micron filter to remove clumps, and obtain a single-cell suspension of hematopoietic endothelial cells for subsequent flow cytometry analysis and purification.

[0093] III. Induction of Lymphocyte Progenitor Cells

[0094] 1. Coat non-tissue-treated culture dishes with human recombinant protein DLL4-Fc and incubate at 4°C for 2 hours. Wash once with DPBS before use.

[0095] 2. The purified hematopoietic endothelial cells were transferred to culture dishes coated with human recombinant protein DLL4-Fc and cultured in lymphocyte progenitor cell differentiation medium for 7 days, with the medium changed every 3 days. The lymphocyte progenitor cell differentiation medium consisted of Stemspan SFEM II, 10 vt% serum substitute (StemCell Tech, 09500), 2 mM glutamine (Gibco, 35050061), 50 μg / mL L-ascorbic acid-2-phosphate trisodium salt, 1 mM sodium pyruvate (Gibco, 11360070), 55 mM β-mercaptoethanol (Gibco, 21985023), 1 mM non-essential amino acids (Gibco, 11140050), SCF, FLT3L, interleukin-3, interleukin-7, and thrombopoietin. The concentrations of SCF, FLT3L, interleukin-3, interleukin-7, and thrombopoietin were 30 ng / mL, 20 ng / mL, 5 ng / mL, 30 ng / mL, and 5 ng / mL, respectively.

[0096] 3. Centrifuge to collect the cell pellet, replace with fresh induction medium, and culture for 7 days, changing the medium every 3 days to obtain lymphocyte progenitor cells. The induction medium consisted of Stemspan SFEM II, 10 vt% serum substitute (StemCell Tech, 09500), 2 mM glutamine (Gibco, 35050061), 50 μg / mL L-ascorbic acid-2-phosphate trisodium salt, 1 mM sodium pyruvate (Gibco, 11360070), 55 mM β-mercaptoethanol (Gibco, 21985023), 1 mM non-essential amino acids (Gibco, 11140050), SCF, FLT3L, interleukin-7, and thrombopoietin. The concentrations of SCF, FLT3L, interleukin-7, and thrombopoietin were 30 ng / mL, 20 ng / mL, 30 ng / mL, and 5 ng / mL, respectively.

[0097] IV. Acquisition of NK Cells

[0098] 1. Coat non-tissue-treated culture dishes with human recombinant protein DLL4-Fc and incubate at 4°C for 2 hours. Wash once with DPBS before use.

[0099] 2. Transfer lymphocyte progenitor cells to newly coated culture dishes and induce them using NK cell induction medium 1. Culture for 14 days, changing the medium every 5 days. After 14 days, replace the medium with NK cell induction medium 2. NK cell induction medium 2 consists of Stemspan SFEM II, 10 vt% serum substitute (StemCell Tech, 09500), 2 mM glutamine (Gibco, 35050061), 50 μg / mL L-ascorbic acid-2-phosphate trisodium salt, 1 mM sodium pyruvate (Gibco, 11360070), 55 mM β-mercaptoethanol (Gibco, 21985023), 1 mM non-essential amino acids (Gibco, 11140050), FLT3L, interleukin-7, and interleukin-15. The concentration of FLT3L was 20 ng / mL, the concentration of interleukin-7 was 15 ng / mL, and the concentration of interleukin-15 was 5 ng / mL.

[0100] Example 2: Culture Method 2 - Obtaining NK cells after inducing hematopoietic stem cells using a normoxic system

[0101] I. Suspension Culture of Induced Pluripotent Stem Cells

[0102] Induced pluripotent stem cells (iPSCs) with a confluence of 80%–90% were selected for single-cell digestion, resuspended in Stempro-34 containing BMP4, and transferred to ultra-low adsorption culture dishes for suspension culture to form embryoid bodies, which was recorded as day 0. The concentration of BMP4 was 40 ng / mL. The culture environment was 37°C, 5% CO2, and 20% O2. The concentrations of the same substances used in Method 2 and Method 1 were taken as the optimal values ​​for Method 1.

[0103] II. In vitro induction of hematopoietic stem cells

[0104] 1. On day one, the embryoids were transferred to induction medium A for resuspension. Induction medium A consisted of StemPro-34, BMP4, and bFGF, with a final concentration of 40 ng / mL for BMP4 and 10 ng / mL for bFGF.

[0105] 2. On the third day, the embryoids were transferred to induction medium B for resuspension. Induction medium B consisted of stempro-34, bFGF, VEGF, SCF, FLT3, and IL-3. The final concentrations of bFGF, VEGF, SCF, FLT3, and IL-3 were 10 ng / mL, 50 ng / mL, 50 ng / mL, and 1-10 ng / mL, respectively.

[0106] 3. On day 7, the embryoids were transferred to induction medium C for resuspension. Induction medium C consisted of stempro-34, VEGF, SCF, FLT3, and IL-3, with a final concentration of VEGF of 50 ng / mL, SCF of 50 ng / mL, FLT3 of 50 ng / mL, and IL-3 of 10 ng / mL.

[0107] 4. On day 12, cells were resuspended in Accutase, gently pipetted to mix, and then incubated in a 37°C water bath. Every 10 minutes, cells were gently pipetted for 30 seconds using a 1mL pipette to promote cell dissociation. After digestion for 20-60 minutes, cells were collected by centrifugation at 400g for 10 minutes. Cells were then resuspended in DPBS and filtered through a 70-micron filter to remove clumps, obtaining a single-cell suspension of hematopoietic stem cells for subsequent flow cytometry analysis of hematopoietic stem cell surface marker proteins and further purification.

[0108] III. Induction of Lymphocyte Progenitor Cells

[0109] 1. Coat non-tissue-treated culture dishes with human recombinant protein DLL4-Fc and incubate at 4°C for 2 hours. Wash once with DPBS before use.

[0110] 2. The purified hematopoietic stem cells were transferred to lymphocyte progenitor cell differentiation medium and cultured for 7 days, with the medium changed every 3 days. The lymphocyte progenitor cell differentiation medium consisted of Stemspan SFEM II, 10 vt% serum substitute (StemCell Tech, 09500), 2 mM glutamine (Gibco, 35050061), 50 μg / mL L-ascorbic acid-2-phosphate trisodium salt, 1 mM sodium pyruvate (Gibco, 11360070), 55 mM β-mercaptoethanol (Gibco, 21985023), 1 mM non-essential amino acids (Gibco, 11140050), SCF, FLT3L, interleukin-3, interleukin-7, and thrombopoietin. The concentrations of SCF, FLT3L, interleukin-3, interleukin-7, and thrombopoietin were 30 ng / mL, 20 ng / mL, 5 ng / mL, 30 ng / mL, and 5 ng / mL, respectively.

[0111] 3. Centrifuge to collect the cell pellet, replace with fresh induction medium, and culture for 7 days, changing the medium every 3 days to obtain lymphocyte progenitor cells. The induction medium consisted of Stemspan SFEM II, 10 vt% serum substitute (StemCell Tech, 09500), 2 mM glutamine (Gibco, 35050061), 50 μg / mL L-ascorbic acid-2-phosphate trisodium salt, 1 mM sodium pyruvate (Gibco, 11360070), 55 mM β-mercaptoethanol (Gibco, 21985023), 1 mM non-essential amino acids (Gibco, 11140050), SCF, FLT3L, interleukin-7, and thrombopoietin. The concentrations of SCF, FLT3L, interleukin-7, and thrombopoietin were 30 ng / mL, 20 ng / mL, 30 ng / mL, and 5 ng / mL, respectively.

[0112] IV. Acquisition of NK Cells

[0113] 1. Coat non-tissue-treated culture dishes with human recombinant protein DLL4-Fc and incubate at 4°C for 2 hours. Wash once with DPBS before use.

[0114] 2. Transfer lymphocyte progenitor cells to newly coated culture dishes and induce them using NK cell induction medium. Culture for 14 days, changing the medium every 5 days. After 14 days, replace the medium with NK cell induction medium for 7-14 days. The NK cell induction medium consists of Stemspan SFEM II, 10 vt% serum substitute (StemCell Tech, 09500), 2 mM glutamine (Gibco, 35050061), 50 μg / mL L-ascorbic acid-2-phosphate trisodium salt, 1 mM sodium pyruvate (Gibco, 11360070), 55 mM β-mercaptoethanol (Gibco, 21985023), 1 mM non-essential amino acids (Gibco, 11140050), FLT3L, and interleukin-7. The concentration of FLT3L was 20 ng / mL, the concentration of interleukin-7 was 15 ng / mL, and the concentration of interleukin-15 was 5 ng / mL.

[0115] Example 3: Detection of cell performance using culture method 2 and culture method 2

[0116] I. Detection of marker proteins on the surface of hematopoietic endothelial cells or hematopoietic stem cells

[0117] The expression of the surface marker protein CD34 of hematopoietic stem cells (or hematopoietic endothelial cells) was detected by flow cytometry. The analytical steps are as follows:

[0118] 1. To minimize the impact of apoptotic cells on the flow cytometry results, apoptotic cells were first stained with BD Horizon Fixable Viability Stain 510 antibody. BD Horizon Fixable Viability Stain 510 was added to a single-cell suspension of hematopoietic stem cells resuspended in DPBS, mixed, and incubated at room temperature in the dark for 10 minutes. The cells were then centrifuged at 400g for 3 minutes, the supernatant was discarded, and the cell pellet was collected. The cells were then washed once with DBPS and the cell pellet was collected again.

[0119] 2. Resuspend the cells in DBPS, add CD34 antibody and mix well. Incubate on ice for 30 minutes, avoiding light during the process. After incubation, centrifuge at 400g for 3 minutes, discard the supernatant and collect the cell pellet. Then wash twice with DBPS and collect the cell pellet.

[0120] 3. Resuspend cells in DPBS and analyze using a flow cytometer. The analysis results are as follows: Figure 11 and 12 As shown. Figure 11 In the culture protocol 1, the proportion of hematopoietic stem cells obtained and the positive rate of CD34 were both above 60%. Figure 12 In the study, culture protocol 2 showed that the efficiency of obtaining hematopoietic stem cells and the CD34 positivity rate were both below 30%. Based on the experimental results, culture protocol 1 was ultimately selected for differentiation.

[0121] II. Detection of NK cell surface marker proteins

[0122] 1. To reduce the influence of apoptotic cells on the analysis results in flow cytometry, a control sample needs to be set up. The control group is treated as follows: BD Horizon Fixable ViabilityStain 510 is added to the cell suspension resuspended in DPBS, mixed well, and incubated on ice in the dark for 30 minutes. Then, the cells are centrifuged at 400g for 3 minutes, the supernatant is discarded, and the cell pellet is collected. The cells are then washed once with DBPS and the cell pellet is collected.

[0123] 2. Processing methods and procedures: To detect the proportion of viable cells and the expression of the NK cell surface marker protein CD56 in the test samples, NK cells were resuspended in DPBS, and BD Horizon Fixable Viability Stain510 and CD56 were added. The cells were incubated on ice in the dark for 30 minutes, followed by centrifugation at 400g for 3 minutes. The supernatant was discarded, and the cell pellet was collected. The cells were then washed once with DBPS, and the cell pellet was collected again.

[0124] 3. Resuspend the two groups of samples using DPBS and analyze them using a flow cytometer. The analytical results are as follows: Figure 13 and Figure 14 As shown:

[0125] Figure 13 The expression of CD3 was analyzed by flow cytometry using method 1. The results showed that the cells in the differentiation system did not express CD3 at all. Figure 14 The results of NK cell detection obtained by culture method 1 and culture method 2 are shown; where A represents culture method 1, which yields the highest proportion of NK cells, reaching over 99%, and has the best effect. Culture method 2 ( Figure 14 In section B), the purity of the NK cells obtained was low, below 65%.

[0126] III. Validation of NK cell killing function

[0127] 1. Adjust the tumor cell density to 5×10^4 cells / mL one day in advance, seed the cells in 96-well plates, and incubate at 100μL / well (5×10^3 cells / well) in a 37℃ incubator;

[0128] 2. Add NK cells (100 μL / well) according to different effector-to-target ratios and incubate at 37°C.

[0129] 3. After 24 hours of incubation, centrifuge the cell culture plate at 300g for 5 minutes, collect 100μL / well of cell supernatant into another 96-well plate, and freeze at -80℃ for subsequent detection of IL-2 release, etc.

[0130] 4. Detection of Luciferase signal in residual cell solution: Add 50 μL / well of diluted substrate, react at room temperature in the dark for 5 minutes, aspirate 80 μL of liquid into a 96-well opaque white plate, and read the Luciferase signal.

[0131] Table 1

[0132]

[0133] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A composition for NK cell differentiation, characterized in that, include: Suspension culture medium, induction medium A, induction medium B, induction medium C, induction medium D, and induction medium E; The suspension culture medium includes mTeSR1 and Stempro-34, and the volume ratio of mTeSR1 to Stempro-34 is (4~1):(1~2). The induction medium A comprises: StemPro-34 and 10 ng / mL to 40 ng / mL BMP4; The induction medium B comprises: StemPro-34, 10 ng / mL to 40 ng / mL BMP4 and 1 ng / mL to 10 ng / mL bFGF; The induction medium C includes: StemPro-34, 10 ng / mL to 40 ng / mL BMP4, 1 mM to 10 mM CHIR99021, 1 ng / mL to 10 ng / mL bFGF and 1 mM to 6 mM SB431542; The induction medium D comprises: StemPro-34, 10 ng / mL to 50 ng / mL VEGF and 1 ng / mL to 10 ng / mL bFGF; The induction medium E comprises: Stempro-34, 1 ng / mL to 10 ng / mL bFGF, 10 ng / mL to 100 ng / mL SCF, 2 U / mL EPO, 50 ng / mL IGF-1, 20 ng / mL IL-6 and 10 ng / mL IL-11.

2. The composition according to claim 1, characterized in that, The composition further includes at least one of the following: lymphocyte progenitor cell differentiation medium, induction medium, NK cell induction medium 1 and / or NK cell induction medium 2; The lymphocyte progenitor cell differentiation culture medium includes: Stemspan SFEM II, 10 vt% serum substitute, 2 mM L-glutamine, 50 μg / mL L-ascorbic acid-2-phosphate trisodium salt, 1 mM sodium pyruvate, 55 mM β-mercaptoethanol, 1 mM non-essential amino acids, 30 ng / mL SCF, 20 ng / mL FLT3L, 5 ng / mL interleukin-3, 30 ng / mL interleukin-7 and 5 ng / mL thrombopoietin; The induction medium comprises: SFEM II, 10 vt% serum substitute, 2 mM L-glutamine, 50 μg / mL L-ascorbic acid-2-phosphate trisodium salt, 1 mM sodium pyruvate, 55 mM β-mercaptoethanol, 1 mM non-essential amino acids, 30 ng / mL SCF, 20 ng / mL FLT3L, 30 ng / mL interleukin-7 and 5 ng / mL thrombopoietin; The NK cell induction medium 1 comprises: SFEM II, 10 vt% serum substitute, 2 mM L-glutamine, 50 μg / mL L-ascorbic acid-2-phosphate trisodium salt, 1 mM sodium pyruvate, 55 mM β-mercaptoethanol, 1 mM non-essential amino acids, 20 ng / mL LFLT3L and 15 ng / mL interleukin-7; The NK cell induction medium 2 comprises: Stemspan SFEM II, 10 vt% serum substitute, 2 mM L-glutamine, 50 μg / mL L-ascorbic acid-2-phosphate trisodium salt, 1 mM sodium pyruvate, 55 mM β-mercaptoethanol, 1 mM non-essential amino acids, 20 ng / mL FLT3L, 15 ng / mL interleukin-7 and 5 ng / mL interleukin-15.

3. A method for differentiating NK cells, characterized in that, To differentiate NK cells using the composition of claim 1 or 2.

4. The differentiation method according to claim 3, characterized in that, Includes the following steps: Step 1: Induced pluripotent stem cells with a confluence rate of 80%~90% are digested and resuspended, then transferred to ultra-low adsorption culture dishes for suspension culture to form embryoid bodies; Step 2: The embryoids are cultured sequentially in induction medium A to induction medium E to obtain hematopoietic endothelial cells; Step 3: Seed the hematopoietic endothelial cells into culture dishes coated with human recombinant protein DLL4-Fc and non-tissue treated, and culture them sequentially using the lymphocyte progenitor cell differentiation medium and induction medium to obtain lymphocyte progenitor cells; Step 4: The lymphocyte progenitor cells are cultured sequentially in NK cell induction medium 1 and NK cell induction medium 2 to obtain NK cells; The culture medium used in the suspension culture is a suspension culture medium; the rotation speed of the suspension culture is 50 rpm; the cell concentration during the suspension culture is 4e4 / mL to 4e5 / mL; the CO2 concentration and O2 concentration of the suspension culture are 5%.

5. The differentiation method according to claim 4, characterized in that, Induction medium A to induction medium E, the incubation time for each medium is 1 day.

6. The differentiation method according to claim 5, characterized in that, The culture time for the lymphocyte progenitor cell differentiation medium is 7 days.

7. The differentiation method according to claim 6, characterized in that, The induction medium was cultured for 7 days.

8. The differentiation method according to claim 7, characterized in that, The NK cell induction medium 1 was cultured for 14 days.

9. The cultivation method according to claim 8, characterized in that, The NK cell induction medium 1 is cultured for 7-14 days.

10. The differentiation method according to any one of claims 3 to 8, characterized in that, The induced pluripotent stem cells are mesenchymal-derived induced pluripotent stem cells.

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