CN117946972A - Method for improving NK cell differentiation efficiency - Google Patents
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Abstract
The invention relates to a method for improving NK cell differentiation efficiency, which relates to the technical field of biological medicines and comprises the following steps: the first stage, differentiation forms mesoderm; a second stage of differentiation to form hematogenic endothelial cells; a third stage of differentiation to form hematopoietic stem/progenitor cells; fourth stage, differentiation into NK cells: changing a fourth-stage induction culture medium, and co-culturing the hematopoietic stem progenitor cells and stromal cells to induce NK cells; the fourth stage induction culture medium comprises DMEM/F12, 500-4000mg/L glucose, FBS, glutaMAXTM-1 and P/S. According to the invention, NK differentiation is performed by co-culturing Hematopoietic Stem Progenitor Cells (HSPCs) derived from human pluripotent stem cells and stromal cells, the NK cell differentiation efficiency is improved by adjusting the glucose concentration in the induction culture medium in the fourth stage, and the optimal glucose concentration suitable for NK differentiation is screened out, so that the NK cell differentiation efficiency is further improved, and the production cost is reduced.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a method for improving NK cell differentiation efficiency.
Background
Immune cell therapy is of great importance in tumor therapy. Natural killer cells (Nature KILLER CELLS, NK) are taken as main cells for clearing cancerated cells in healthy human bodies, have remarkable anticancer effect, and have important significance on hematological tumors such as acute leukemia and solid tumors such as liver cancer. Compared to the currently more mature chimeric antigen receptor T cells (CHIMERIC ANTIGEN receptor T cells, CAR-T), T cell therapy has serious side effects such as neurotoxicity and "cytokine storm" (cytokine release syndrome, CRS), patient's disease is dangerous, and the prognosis is poor [Schubert,M.L.,et al.,Side-effect management of chimeric antigen receptor(CAR)T-cell therapy.Ann Oncol,2021.32(1):p.34-48.]. In contrast, NK cells have higher safety, and clinical trials have not reported that the subsequent NK cell treatment produces serious side effects [Miller,J.S.,et al.,Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patien ts with cancer.Blood,2005.105(8):p.3051-7. And Romee,R.,et al.,Cytokine-induced memory-like natural killer cells exhibit enhanced responses against myeloid leukemia.Sci Transl Med,2016.8(357):p.357ra123.] such as graft-versus-host disease (GvHD) and CRS. Meanwhile, NK cells function without MHC restriction, which determines that the application range of NK cells is wider than that of T cells [VIVIER,E.,et al.,Innate or Adaptive Immunity?The Example of Natural Killer Cells.Science(American Association for the Advancement of Science),2011.331 (6013):p.44-49.] in the future.
At present, clinical tests adopt NK cells separated and amplified from peripheral blood mononuclear cells of healthy people, the donor source is insufficient, the NK cells provided by one donor can only treat one patient and the like, the individual differences exist, the large-scale and standardization are difficult to achieve, and the evaluation of the prognosis of the patient is difficult [Passweg,J.R.,et al.,Purified donor NK-lymphocyte infusion to consolidate engraftment after haploidentical stem cell transplantation.Leukemia,2004.18(11):p.1835-8.]. The above problems place new demands on NK cells that provide adequate doses and have anti-tumor activity. NK cells are obtained by differentiating human induced pluripotent stem cells (hiPSC), and then a large number of NK cells (iPS-NK) with cytotoxicity and secretion activity are obtained by amplification, so that the problems of insufficient source of NK donors and standardized cell functions can be effectively solved, and the method is favorable for making clinical diagnosis and treatment schemes and prognosis evaluation of patients. Therefore, a scheme for obtaining a large number of NK cells by in vitro induction by utilizing the hiPSCs is developed, and the method has important application value and significance.
At present, the induction scheme of the hiPSC to NK cell differentiation established in laboratories at home and abroad mainly comprises the following steps: (1) Embryo (EB) differentiation; (2) bone marrow stromal cells co-culture differentiation method. NK cells can be obtained by both of the above differentiation methods. The NK cell differentiation from EB to NK cell better simulates the cell and physicochemical microenvironment required by in vivo NK differentiation, and the NK cell induced by EB in vitro test has good cytotoxicity to various tumor cells. But the EB differentiation process generates cells with complex types, which is unfavorable for the mass acquisition and subsequent clinical application of NK cells. In addition, the differentiation period of EB to NK cells is long, generally, the hiPSCs need to induce EB for 1 week and then differentiate for 4-5 weeks to obtain NK cells, and the technology difficulty is high and the cost is high. Although both of the above methods can finally differentiate to obtain NK cells, the differentiation efficiency is low, resulting in high production costs of NK cells. In view of this, the present invention provides a method for improving NK cell differentiation efficiency.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for improving NK cell differentiation efficiency, which aims to improve NK cell differentiation efficiency and reduce production cost.
The technical scheme for solving the technical problems is as follows:
A method for improving NK cell differentiation efficiency, comprising the steps of:
(1) In the first stage, differentiation forms mesoderm: taking human pluripotent stem cells, and performing induced differentiation by using a first-stage induction culture medium to obtain mesoderm, wherein the first-stage induction culture medium comprises HDM, BMP4, activin A, bFGF, CHIR99021, A8-301 and IWR-1-endo;
(2) In the second stage, differentiation into hematogenic endothelial cells: changing a second-stage induction culture medium, culturing the mesoderm, and inducing to obtain hematogenic endothelial cells; the second stage induction medium comprises HDM, VEGF, bFGF;
(3) In the third stage, hematopoietic stem/progenitor cells are differentiated: changing a third-stage induction culture medium, culturing the hematogenic endothelial cells, and inducing to obtain suspended hematopoietic stem progenitor cells; the third stage induction medium comprises HDM, SCF, TPO, IL and IL6;
(4) Fourth stage, differentiation into NK cells: changing a fourth-stage induction culture medium, and co-culturing the hematopoietic stem progenitor cells and stromal cells to induce NK cells; the fourth stage induction culture medium comprises DMEM/F12, 500-4000mg/L glucose, FBS, glutaMAXTM-1 and P/S.
The beneficial effects of the invention are as follows: according to the method, NK differentiation is carried out by co-culturing Hematopoietic Stem Progenitor Cells (HSPC) derived from human pluripotent stem cells and stromal cells, the NK cell differentiation efficiency is improved by adjusting the glucose concentration in a fourth-stage induction culture medium, namely an NK differentiation culture medium, and the optimal glucose concentration suitable for NK differentiation is screened out, so that the NK cell differentiation efficiency is further improved, and the production cost is reduced.
Further, the first stage induction medium comprises HDM、38-42ng/mL BMP4、28-32ng/mL Activin A、18-22ng/mL bFGF、5-7μM CHIR99021、0.8-1.2μM A8-301、0.8-1.2μM IWR-1-endo;
The second-stage induction culture medium comprises HDM, 8-12ng/mL VEGF, 8-12ng/mL bFGF;
The third stage induction culture medium comprises HDM, 8-12ng/ml SCF, 48-52ng/ml TPO, 8-12ng/ml IL3, 48-52ng/ml IL6;
The fourth stage induction culture medium comprises DMEM/F12, 1000-3000mg/L glucose, 8-12% FBS, 0.8-1.2% GlutaMAXTM-1, 0.8-1.2% P/S, 0-100mg/ml ascorbic acid, 0-100ng/ml IL-7, 0-100ng/ml IL-15, 0-100ng/ml Flt-3L, 0-100ng/ml IGF-1. Wherein% is by volume, for example, 8-12% FBS specifically refers to 8-12% FBS by volume, and the like are not described one by one.
Still further, the first stage induction medium comprises HDM, 40ng/mL BMP4, 30ng/ML ACTIVIN A, 20ng/mL bFGF, 6. Mu.M CHIR99021, 1. Mu. M A8-301, 1. Mu.M IWR-1-endo;
the second-stage induction culture medium comprises HDM, 10ng/mL VEGF, 10ng/mL bFGF;
The third stage induction medium comprises HDM, 10ng/ml SCF, 50ng/ml TPO, 10ng/ml IL3, 50ng/ml IL6;
The fourth-stage induction culture medium comprises DMEM/F12 and 2000mg/L glucose 、10％ FBS、1％GlutaMAXTM-1、1％P/S、0-100mg/ml ascorbic acid、0-100ng/ml IL-7、0-100ng/ml IL-15、0-100ng/ml Flt-3L、0-100ng/ml IGF-1., and the DMEM/F12 in the fourth-stage induction culture medium is sugar-free, so that the problem that the sugar content of the culture medium cannot be accurately regulated by adding glucose due to the occurrence of a small amount of sugar is avoided, and the differentiation efficiency of NK cells cannot be accurately regulated. Where% is by volume, for example, 10% FBS specifically refers to 10% by volume FBS, and the like are not described again. Further, the HDM includes DMEM/F12, 0.9-1.1% P/S, 0.9-1.1% ITS-G, 68-72 μg/ml vitamin C. Wherein% is by volume, for example, 0.9-1.1% P/S specifically refers to 0.9-1.1% P/S by volume, and the like are not described in any more detail.
Further, the conditions for inducing differentiation using the first-stage induction medium in step (1) are: the temperature is 37+/-0.5 ℃, the volume fraction of CO 2 is 5%, the oxygen content is normal oxygen, and the time is 1-3 days;
In the step (2), the conditions for culturing the mesoderm to induce the hematogenic endothelial cells are as follows: the temperature is 37+/-0.5 ℃, the volume fraction of CO 2 is 5%, the oxygen content is normal oxygen, and the time is 1-3 days;
In the step (3), the third stage induction culture medium is adopted to culture the hematogenic endothelial cells, and the conditions for inducing to obtain the suspended hematopoietic stem progenitor cells are as follows: the temperature is 37+/-0.5 ℃, the volume fraction of CO 2 is 5%, the oxygen content is normal oxygen, and the time is 4-8 days;
The conditions for obtaining NK cells by culturing in the fourth-stage culture medium in the step (4) are as follows: the temperature is 37+/-0.5 ℃, the volume fraction of CO 2 is 5%, the oxygen content is normal oxygen, and the time is 9-12 days.
Further, the conditions for inducing differentiation using the first-stage induction medium in step (1) are as follows: the temperature is 37+/-0.5 ℃, the volume fraction of CO 2 is 5%, the oxygen content is normal oxygen, and the time is 2 days;
In the step (2), the conditions for culturing the mesoderm to induce the hematogenic endothelial cells are as follows: the temperature is 37+/-0.5 ℃, the volume fraction of CO 2 is 5%, the oxygen content is normal oxygen, and the time is 2 days;
In the step (3), the third stage induction culture medium is adopted to culture the hematogenic endothelial cells, and the conditions for inducing to obtain the suspended hematopoietic stem progenitor cells are as follows: the temperature is 37+/-0.5 ℃, the volume fraction of CO 2 is 5%, the oxygen content is normal oxygen, and the time is 8 days;
the conditions for obtaining NK cells by culturing the NK cells in the fourth-stage induction medium in the step (4) are as follows: the temperature was 37.+ -. 0.5 ℃, the volume fraction of CO 2 was 5%, the oxygen content was normoxic, and the time was 10 days.
Further, the human pluripotent stem cells are pre-treated prior to undergoing the first stage of differentiation to form mesoderm; the pretreatment comprises the following specific steps: the human pluripotent stem cells are subjected to digestion treatment and then cultured in a medium containing a Y-27632 inhibitor for 12-36 hours. Wherein the culture medium containing the Y-27632 inhibitor comprises mTESR1 and 8-12 mu M Y-27632.
Further, the human pluripotent stem cells in step (1) are seeded at a density of 30 to 35 ten thousand per well.
Further, the stromal cells in step (4) are mouse bone marrow stromal cells OP9-DLL1; the ratio of the number of hematopoietic stem progenitor cells to the number of stromal cells in co-culture is 1:1.
The invention has the further beneficial effects that: the use of mouse bone marrow stromal cells OP9-DLL1 can accelerate the rate of obtaining NK cells by induced differentiation, thereby improving the directional induced differentiation efficiency of NK cells.
Drawings
FIG. 1 is a flow chart of NK cell differentiation in accordance with the present invention;
FIG. 2 shows NK cell differentiation efficiency under different glucose concentration conditions detected by cell flow technique in experimental example of the present invention; wherein CD45 is used as a blood cell surface marker, CD56 is used as an NK cell surface marker, and the cell population of cd45+cd56+ represents NK cells obtained by differentiation;
FIG. 3 is statistical data of NK cell differentiation efficiency under different glucose concentrations in the experimental example of the present invention showing that 0.001 < P < 0.01 and showing that P < 0.001;
FIG. 4 shows statistics of NK cell number obtained by differentiation under different glucose concentrations in experimental examples of the present invention, wherein 0.001 < P < 0.01, and P < 0.001.
Detailed Description
The principles and features of the present invention are described below with examples given for the purpose of illustration only and are not intended to limit the scope of the invention. The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.
In embodiments of the invention, the reagents used are of the origin
BMP4(Peprotech);Activin A(Sino Biological);bFGF(Sino Biological);CHIR99021(selleck);A8-301(selleck);IWR-1-endo(selleck);VEGF(Sino Biological);StemPro-34 SFM(gibco);Flt-3(Peprotech);SCF(Peprotech);TPO(Sino Biological);IL3(Sino Biological);IL6(Sino Biological);mTeSR1(gibco);Y-27632(selleck);DMEM/F12(gibco);P/S(gibco);ITS-G(gibco); Vitamin C (gibco).
Examples
1. Method for improving NK cell differentiation efficiency
A method for improving NK cell differentiation efficiency comprises the following steps, as shown in FIG. 1
(1) In the first stage, differentiation forms mesoderm: taking human pluripotent stem cells, and performing induced differentiation by using a first-stage induction culture medium to obtain mesoderm, wherein the first-stage induction culture medium comprises HDM, BMP4, activin A, bFGF, CHIR99021, A8-301 and IWR-1-endo;
(2) In the second stage, differentiation into hematogenic endothelial cells: changing a second-stage induction culture medium, culturing the mesoderm, and inducing to obtain hematogenic endothelial cells; the second stage induction medium comprises HDM, VEGF, bFGF;
(3) In the third stage, hematopoietic stem/progenitor cells are differentiated: changing a third-stage induction culture medium, culturing the hematogenic endothelial cells, and inducing to obtain suspended hematopoietic stem progenitor cells; the third stage induction medium comprises HDM, SCF, TPO, IL and IL6;
(4) Fourth stage, differentiation into NK cells: changing a fourth-stage induction culture medium, and co-culturing the hematopoietic stem progenitor cells and stromal cells to induce NK cells; the fourth stage induction culture medium comprises DMEM/F12, 500-4000mg/L glucose, FBS, glutaMAXTM-1 and P/S.
In a preferred embodiment, the first stage induction medium comprises HDM、38-42ng/mL BMP4、28-32ng/mL Activin A、18-22ng/mL bFGF、5-7μM CHIR99021、0.8-1.2μM A8-301、0.8-1.2μM IWR-1-endo;
The second-stage induction culture medium comprises HDM, 8-12ng/mL VEGF, 8-12ng/mL bFGF;
The third stage induction culture medium comprises HDM, 8-12ng/ml SCF, 48-52ng/ml TPO, 8-12ng/ml IL3, 48-52ng/ml IL6;
The fourth stage induction culture medium comprises DMEM/F12, 1000-3000mg/L glucose, 8-12% FBS, 0.8-1.2% GlutaMAXTM-1, 0.8-1.2% P/S, 0-100mg/ml ascorbic acid, 0-100ng/ml IL-7, 0-100ng/ml IL-15, 0-100ng/ml Flt-3L, 0-100ng/ml IGF-1.
Preferably, the HDM comprises DMEM/F12, 0.9-1.1% P/S, 0.9-1.1% ITS-G, 68-72 μg/ml vitamin C.
In this embodiment, preferably, the conditions for inducing differentiation in the step (1) using the first-stage induction medium are as follows: the temperature is 37+/-0.5 ℃, the volume fraction of CO 2 is 5%, the oxygen content is normal oxygen, and the time is 1-3 days;
In the step (2), the conditions for culturing the mesoderm to induce the hematogenic endothelial cells are as follows: the temperature is 37+/-0.5 ℃, the volume fraction of CO 2 is 5%, the oxygen content is normal oxygen, and the time is 1-3 days;
In the step (3), the third stage induction culture medium is adopted to culture the hematogenic endothelial cells, and the conditions for inducing to obtain the suspended hematopoietic stem progenitor cells are as follows: the temperature is 37+/-0.5 ℃, the volume fraction of CO 2 is 5%, the oxygen content is normal oxygen, and the time is 4-8 days;
The conditions for obtaining NK cells by culturing in the fourth-stage culture medium in the step (4) are as follows: the temperature is 37+/-0.5 ℃, the volume fraction of CO 2 is 5%, the oxygen content is normal oxygen, and the time is 9-12 days.
Preferably, in this embodiment, the human pluripotent stem cells are pre-treated before undergoing the first stage of differentiation to form mesoderm; the pretreatment comprises the following specific steps: the human pluripotent stem cells are subjected to digestion treatment and then cultured in a medium containing a Y-27632 inhibitor for 12-36 hours. Wherein the culture medium containing the Y-27632 inhibitor comprises mTESR1 and 8-12 mu M Y-27632.
Preferably, the human pluripotent stem cells in step (1) are seeded at a density of 30-35 ten thousand per well.
Preferably, in the present embodiment, the stromal cells in step (4) are mouse bone marrow stromal cells OP9-DLL1; the ratio of the number of hematopoietic stem progenitor cells to the number of stromal cells in co-culture is 1:1.
Specifically, the differentiation of the 6-well plate is described in detail:
(a) hESCs/iPSCs with good density of 80-90% (the hESCs used are the H1 cell line purchased from weiCell) are digested for 5 minutes by Ackutase (cell digest, the other holocellum), the digestion is stopped by F12 (pramoxine), cell suspension is collected, 500g is centrifuged for 3 minutes, supernatant is removed, mTESR1 culture medium is used for resuspension and counting, 30-35 ten thousand cells per hole are inoculated into six-hole plates, 2mL mTESR1+10 mu M Y-27632 is used for culturing for 24 hours, and Y-27632 is a ROCK selective inhibitor, so that cell death can be effectively reduced, and the survival rate of stem cells can be improved;
(b) Day 0 (D0), after the medium was aspirated, it was washed with 1mL DMEM/F12, 2mL of the first stage induction medium (HDM 1) was replaced, and differentiation into mesoderm was induced for 48 hours (2 days), the first stage induction medium (HDM 1): HDM+40ng/mL BMP4+30ng/ML ACTIVIN A +20ng/mL bFGF+6. Mu.M CHIR 99021+1. Mu. M A8-301+1. Mu.M IWR-1-endo;
(c) On day 2 (D2), the medium was aspirated and washed once with 1mL of DMEM/F12, 2mL of second-stage induction medium (HDM 2) was replaced, and the culture was performed for 48 hours (2 days) to induce endothelial cells of birth, and the second-stage induction medium (HDM 2): HDM+10ng/mL VEGF+10ng/mL bFGF;
(d) On day 4 (D4), the medium was aspirated and washed once with 1mL of DMEM/F12, 2mL of third stage induction medium (HDM 3) was replaced, and after 4 days of culture (to day 8) suspension of Hematopoietic Stem Progenitor Cells (HSPCs) was induced, third stage induction medium (HDM 3): HDM+10ng/ml SCF+50ng/ml TPO+10ng/ml IL3+50ng/ml IL6; HSPCs can be produced continuously from day 8 up to around day 30, and suspended HSPCs can be collected every two days later for NK cell differentiation.
(E) Hematopoietic differentiation HSPC cells can be harvested for NK differentiation to day 12: one day in advance, preparing six-well plates to pave and differentiate required mouse bone marrow stromal cells OP9-DLL1 (ATCC cell resource center), the number of cells per well is twenty-ten thousand, and collecting suspended HSPCs generated by hematopoietic differentiation and prepared stromal cells according to the cell number of 1:1 co-cultivation was performed with 2mL of fourth stage medium (HDM 4) of different glucose concentration, NK cells were induced after 10 days of cultivation, fourth stage induction medium (HDM 4): DMEM/F12 (sugar-free) +glucose (500/1000/2000/3000/4000mg/L)+10％FBS+1％GlutaMAXTM-1+1％ P/S+0-100mg/ml ascorbic acid+0-100ng/ml IL-7+0-100ng/ml IL-15+0-100ng/ml+Flt-3L+0-100ng/ml IGF-1.
After 10 days of NK differentiation, the stromal cells were completely killed and lysed, and NK cell differentiation was completed. The cell suspension was collected, centrifuged at 500g for 3 minutes, and the supernatant was removed to obtain cells, namely NK cells differentiated from human pluripotent stem cells. NK differentiation efficiency was detected by cell flow technique and CD45, CD56 antibodies, and NK cell differentiation efficiency and NK cell number were counted.
Wherein, the culture conditions of all culture stages in the specific example are 37 ℃,5% CO 2 and normoxic; the culture medium dosage is 2 mL/hole by taking six pore plates as columns; HDM is self-prepared culture medium in laboratory, the preparation method uses DMEM/F12 as basic culture medium, and 1% P/S, 1% ITS-G, and 70ug/ml vitamin C are added; wherein% by volume of each medium is, for example, 1% P/S, specifically 1% P/S by volume, and the like are not described one by one.
2. Differentiation efficiency assay
NK differentiation efficiency was detected by cell flow technique and CD45, CD56 antibodies, and NK cell differentiation efficiency and NK cell number were counted. The results are shown in FIGS. 2-4.
The sample is subjected to digestion treatment according to the cell type selection, so that single cell suspension is obtained before final antibody staining, suspended cells can be directly sampled, and adherent cells are digested with Ackutase for 5 minutes. Cells to be detected were collected with a 1.5mL EP tube and washed by FACS (DPBS+2% FBS) resuspension, and centrifuged at 500g for 3 min. The supernatant was removed and the cell pellet was resuspended with 100. Mu.L FACS (10 6 cells per 100. Mu.L FACS) and the flow antibody to be detected was added sequentially at a ratio of 1:100, and mix was pre-formulated when multiple samples were to be detected simultaneously. Incubation at 4℃for 20-30 min or 15 min at ambient temperature was performed in the absence of light, after incubation was completed 1mL of FACS was added to the EP tube to stop staining and wash away non-specific binding, after centrifugation at 500g for 3 min the supernatant was removed and 100-200. Mu.L of FACS was used to resuspend cell pellet for on-line detection using flow cytometer CytoFLEX. The flow-through antibodies used were as follows: PC7-CD45, BV421-CD56.
3. Results and analysis
The NK cell differentiation efficiency results under different glucose concentrations are shown in FIGS. 2-4, and according to the results shown in FIGS. 2-4, NK cell differentiation efficiency is higher at a glucose concentration of 1000-3000mg/L and highest at a glucose concentration of 2000 mg/L.
In a word, the method utilizes the co-culture of Hematopoietic Stem Progenitor Cells (HSPC) derived from human pluripotent stem cells and stromal cells to conduct NK differentiation, improves NK cell differentiation efficiency by adjusting the glucose concentration in a fourth-stage induction medium, namely an NK differentiation medium, screens out the optimal glucose concentration suitable for NK differentiation, further improves NK cell differentiation efficiency, obviously shortens the stromal cell co-culture differentiation period and reduces production cost compared with an Embryoid Body (EB) differentiation method.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (10)
1. A method for increasing the efficiency of NK cell differentiation comprising the steps of:
(1) In the first stage, differentiation forms mesoderm: taking human pluripotent stem cells, and performing induced differentiation by using a first-stage induction culture medium to obtain mesoderm, wherein the first-stage induction culture medium comprises HDM, BMP4, activin A, bFGF, CHIR99021, A8-301 and IWR-1-endo;
(2) In the second stage, differentiation into hematogenic endothelial cells: changing a second-stage induction culture medium, culturing the mesoderm, and inducing to obtain hematogenic endothelial cells; the second stage induction medium comprises HDM, VEGF, bFGF;
(3) In the third stage, hematopoietic stem/progenitor cells are differentiated: changing a third-stage induction culture medium, culturing the hematogenic endothelial cells, and inducing to obtain suspended hematopoietic stem progenitor cells; the third stage induction medium comprises HDM, SCF, TPO, IL and IL6;
(4) Fourth stage, differentiation into NK cells: changing a fourth-stage induction culture medium, and co-culturing the hematopoietic stem progenitor cells and stromal cells to induce NK cells; the fourth stage induction culture medium comprises DMEM/F12, 500-4000mg/L glucose, FBS, glutaMAXTM-1 and P/S.
2. The method for improving NK cell differentiation efficiency according to claim 1, wherein,
The first stage induction medium comprises HDM、38-42ng/mL BMP4、28-32ng/mL Activin A、18-22ng/mL bFGF、5-7μM CHIR99021、0.8-1.2μM A8-301、0.8-1.2μM IWR-1-endo;
The second-stage induction culture medium comprises HDM, 8-12ng/mL VEGF, 8-12ng/mL bFGF;
The third stage induction culture medium comprises HDM, 8-12ng/ml SCF, 48-52ng/ml TPO, 8-12ng/ml IL3, 48-52ng/ml IL6;
The fourth stage induction culture medium comprises DMEM/F12, 1000-3000mg/L glucose, 8-12% FBS, 0.8-1.2% GlutaMAXTM-1, 0.8-1.2% P/S, 0-100mg/ml ascorbic acid, 0-100ng/ml IL-7, 0-100ng/ml IL-15, 0-100ng/ml Flt-3L, 0-100ng/ml IGF-1.
3. The method for improving NK cell differentiation efficiency according to claim 1, wherein,
The first stage induction culture medium comprises HDM, 40ng/mL BMP4, 30ng/ML ACTIVIN A, 20ng/mL bFGF, 6 μM CHIR99021, 1 μ M A8-301, 1 μM IWR-1-endo;
the second-stage induction culture medium comprises HDM, 10ng/mL VEGF, 10ng/mL bFGF;
The third stage induction medium comprises HDM, 10ng/ml SCF, 50ng/ml TPO, 10ng/ml IL3, 50ng/ml IL6;
The fourth stage induction culture medium comprises DMEM/F12 and 2000mg/L glucose 、10％FBS、1％GlutaMAXTM-1、1％P/S、0-100mg/ml ascorbic acid、0-100ng/ml IL-7、0-100ng/ml IL-15、0-100ng/ml Flt-3L、0-100ng/ml IGF-1.
4. A method of increasing the efficiency of NK cell differentiation according to claim 2 or 3, wherein said HDM comprises DMEM/F12, 0.9-1.1% p/S, 0.9-1.1% its-G, 68-72 μg/ml vitamin C.
5. The method for improving NK cell differentiation efficiency according to claim 1, wherein,
The conditions for inducing differentiation by using the first-stage induction medium in the step (1) are as follows: the temperature is 37+/-0.5 ℃, the volume fraction of CO 2 is 5%, the oxygen content is normal oxygen, and the time is 1-3 days;
In the step (2), the conditions for culturing the mesoderm to induce the hematogenic endothelial cells are as follows: the temperature is 37+/-0.5 ℃, the volume fraction of CO 2 is 5%, the oxygen content is normal oxygen, and the time is 1-3 days;
In the step (3), the third stage induction culture medium is adopted to culture the hematogenic endothelial cells, and the conditions for inducing to obtain the suspended hematopoietic stem progenitor cells are as follows: the temperature is 37+/-0.5 ℃, the volume fraction of CO 2 is 5%, the oxygen content is normal oxygen, and the time is 4-8 days;
The conditions for obtaining NK cells by culturing in the fourth-stage culture medium in the step (4) are as follows: the temperature is 37+/-0.5 ℃, the volume fraction of CO 2 is 5%, the oxygen content is normal oxygen, and the time is 9-12 days.
6. The method for improving NK cell differentiation efficiency according to claim 1, wherein,
The conditions for inducing differentiation by using the first-stage induction medium in the step (1) are as follows: the temperature is 37+/-0.5 ℃, the volume fraction of CO 2 is 5%, the oxygen content is normal oxygen, and the time is 2 days;
In the step (2), the conditions for culturing the mesoderm to induce the hematogenic endothelial cells are as follows: the temperature is 37+/-0.5 ℃, the volume fraction of CO 2 is 5%, the oxygen content is normal oxygen, and the time is 2 days;
In the step (3), the third stage induction culture medium is adopted to culture the hematogenic endothelial cells, and the conditions for inducing to obtain the suspended hematopoietic stem progenitor cells are as follows: the temperature is 37+/-0.5 ℃, the volume fraction of CO 2 is 5%, the oxygen content is normal oxygen, and the time is 8 days;
the conditions for obtaining NK cells by culturing the NK cells in the fourth-stage induction medium in the step (4) are as follows: the temperature was 37.+ -. 0.5 ℃, the volume fraction of CO 2 was 5%, the oxygen content was normoxic, and the time was 10 days.
7. The method for increasing the differentiation efficiency of NK cells according to claim 1, wherein said human pluripotent stem cells are pre-treated before undergoing said first stage of differentiation to form mesoderm; the pretreatment comprises the following specific steps: the human pluripotent stem cells are subjected to digestion treatment and then cultured in a medium containing a Y-27632 inhibitor for 12-36 hours.
8. The method for improving NK cell differentiation efficiency according to claim 7, wherein the medium containing the Y-27632 inhibitor comprises mTESR1 and 8-12 μ M Y-27632.
9. The method for improving NK cell differentiation efficiency according to claim 1, wherein the seeding density of the human pluripotent stem cells in the step (1) is 30-35 ten thousand/well.
10. The method for improving NK cell differentiation efficiency according to claim 1, wherein the stromal cells in step (4) are mouse bone marrow stromal cells OP9-DLL1; the ratio of the number of hematopoietic stem progenitor cells to the number of stromal cells in co-culture is 1:1.
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