CN111235105B

CN111235105B - Method for differentiating human pluripotent stem cells into natural killer cells and application

Info

Publication number: CN111235105B
Application number: CN202010153358.3A
Authority: CN
Inventors: 不公告发明人
Original assignee: Nuwacell Ltd
Current assignee: Nuwacell Ltd
Priority date: 2020-03-06
Filing date: 2020-03-06
Publication date: 2021-08-10
Anticipated expiration: 2040-03-06
Also published as: CN111235105A

Abstract

The invention relates to the field of stem cell biology, in particular to a method and application for differentiating human pluripotent stem cells into natural killer cells. The invention discloses a natural killer cell derived from pluripotent stem cells, which expresses CD56, Nkp30, Nkp44 and Nkp46, and also expresses markers CD16 and CD94 of mature natural killer cells. The invention also discloses a method for preparing natural killer cells, comprising the following steps: S1: forming an embryoid body; S2: differentiating the embryoid body into hematopoietic progenitor cells; S3: differentiating hematopoietic progenitor cells into NK cells; S4: NK cells maturation and expansion. The differentiation method provided by the present invention, based on a well-defined medium and an optimized combination of cytokines, can rapidly, efficiently, simply and inexpensively differentiate induced pluripotent stem cells into natural killer cells.

Description

Method for differentiating human pluripotent stem cells into natural killer cells and application

Technical Field

The invention relates to the field of stem cell biology, in particular to lineage specific differentiation of pluripotent or multipotent stem cells, and specifically relates to a method for differentiating human pluripotent stem cells into natural killer cells and application thereof.

Background

The national latest cancer report in 2018 shows that the national malignancy estimates 380.4 ten thousand new cases in 2014, and more than 1 million people are diagnosed as cancer on average each day. The incidence of the tumor is 278.07/10 ten thousand, the cumulative incidence of the disease is 21.58% between 0 and 74 years old, and the cumulative mortality is 12.00%. In 2013, the journal of science lists tumor immunotherapy as the first ten scientific breakthroughs, and the immunotherapy becomes a new generation of tumor treatment means after surgery, chemotherapy, radiotherapy and tumor targeted therapy, thereby bringing new hopes for treating tumor patients.

Natural killer cells (NK) are the mainstay of the innate immune system, the first line of defense for the body against infection and tumors, and can recognize and kill diseased cells by either the "self-deletion" (missing-self) or "stress-induced" (stress-induced) pattern on the surface of malignant diseased cells. The activation of NK cells is dependent on the result of a balance of activated and inhibitory receptor signals on their cell surface. Activated NK cells can kill tumor cells by a variety of mechanisms: NK cells can release perforin and granzyme, and directly act on target cells to kill cancer cells; cancer cell apoptosis can be promoted by secretion of proinflammatory cytokines; killing can be accomplished by targeting cancer cells that recognize the monoclonal antibody drug marker.

The recognition and killing of NK cells have the characteristics of MHC-I non-restriction, pan-specificity and quick response, have no graft-versus-host reaction (GVHD), can adopt the feedback of allogeneic cells, have short survival period in vivo, have no side effects such as cytokine storm and the like, can be used together with tumor-targeted antibodies to enhance the anti-cancer effect, can be combined with a gene editing technology to develop targeted NK cell products aiming at different tumor targets, and have great application potential in tumor immunotherapy due to the characteristics.

Previous studies have demonstrated that NK cells in tumor patients are often reduced in number and impaired in function. Adoptive therapy of NK cells therefore has the efficacy of restoring immune function to patients against tumors. However, clinical studies after reinfusion of autologous NK cells show that their antitumor effects are very limited. The main reasons may include: (1) the KIR receptor of the autologous NK cells is matched with HLA of the tumor cells, and the activation of the NK cells is inhibited by the self-recognition signal; (2) the function of the patient's own NK cells is damaged, and the ideal killing effect is difficult to generate.

NK cells directly expanded from peripheral blood have certain difficulties, may fail to expand the cells of the patient, and usually the expanded cell mixture contains a certain proportion of T cells, which need to be cleared, otherwise it will cause Graft Versus Host Disease (GVHD). Meanwhile, the NK cytoplasm of different donors has great difference, directly influences the curative effect and is difficult to form a standardized product.

Human pluripotent stem cells (hpscs) including human embryonic stem cells (hescs) and human induced pluripotent stem cells (hipscs) have the ability to proliferate indefinitely and can differentiate into almost all functional cells in vitro, including NK cells. The in vitro large-scale and standardized preparation of the NK cells from the human pluripotent stem cells is an effective solution for the NK cell product patent medicine. However, the differentiation process reported at present has the problems of complex process, long period, feeder layer cell dependence, undefined culture medium components, functional defects of differentiated cells, low purity and activity and the like, so that the differentiation process is not beneficial to large-scale production.

The closest patents to the present invention are: patent CN102388130A discloses differentiation of a pluripotent cell, patent CN107429230A discloses a method and composition for inducing differentiation of hematopoietic cells, and patent CN102822332A discloses a method for generating natural killer cells and dendritic cells from hESC-derived angioblasts.

Patent CN102388130A has at least some of the following problems: only including differentiation of hpscs into hematopoietic precursor cells, but not including the stage of continued differentiation of hematopoietic precursor cells into NK cells. The factor combination used did not bypass the original hematopoietic phase.

Patent CN107429230A has at least some of the following problems: (1) EB 3D was very inefficient. (2) 2D differentiation conditions were used from hPSC to permanent pluripotent blood precursor cells. (3) The whole process uses expensive commercial culture medium (such as Stempro 34) with undefined components, bovine serum and the like, and is not beneficial to the production of clinical grade cell products. (4) The enriched CD34+/CD45+ cells are used for iNK (iPSC-derived NK, NK cells induced and differentiated by iPSC) differentiation, and the process is complicated.

Patent CN102822332A has at least some of the following problems: (1) expensive commercial media with undefined composition were used; (2) the obtained hemangioblast cells mostly belong to primitive hematopoietic cells; (3) the single cell obtained by EB digestion on the differentiation day4 is inoculated into a Methylellulose culture system for further differentiation to obtain vascular cells, and the system is not beneficial to the large-scale production of clinical NK cells; (4) the second and third steps of the method both use a medium containing human serum.

In conclusion, the NK cells have unique advantages and wide application prospects in immunotherapy of cancers, while NK cells of NK cells (PB-NK) from peripheral blood are limited in source and inconsistent in quality and yield, and are often polluted by T cells after being amplified. Therefore, the preparation of NK cells by using hPSC as a source can solve the problems and achieve stable large-scale production. However, the existing differentiation scheme has many problems that serum is an uncontrollable culture factor and further causes unstable factors such as batch difference and the like when a culture medium containing serum is used; the use of feeder layer cells of animal cell origin such as OP9 does not meet clinical production requirements; the commercial serum-free culture medium is expensive and is not suitable for large-scale production; the differentiation process is complex, the dosage and time of the cell factors are not fully optimized and simplified, and the cost is high; the long differentiation period also increases the production cost and improves the uncontrollable process control; the differentiated NK cells have low purity and lack of functions.

Therefore, the invention needs to invent a stable, efficient, low-cost, high-purity and functional NK cell differentiation system, and establishes a foundation for large-scale production and clinical application of NK cells.

Disclosure of Invention

Although the process of in vitro differentiation from pluripotent stem cells into natural killer cells is well-established in principle, and various methods for inducing differentiation are available at present, the existing differentiation methods have the problems of long differentiation period, low differentiation efficiency and purity, unclear differentiation components, feeder layer cell dependence, and complicated operation and high cost, so that it is difficult to expand the production and apply the methods to clinical application. The differentiation program provided by the invention can rapidly, efficiently, simply and conveniently induce the pluripotent stem cells to differentiate into natural killer cells at lower cost based on the culture medium with definite components and the optimized combination of cytokines.

Its advantages are as follows: firstly, the invention realizes the stable and high-efficiency differentiation of natural killer cells, and can obtain NK cells with purity of more than 90% and functionality in about 40 days; secondly, no culture system containing serum or feeder layer cells are used in the whole differentiation process, so that the method is suitable for expanded production and clinical application; third, we try to simplify and optimize the media and cytokines used in the differentiation process, avoiding the use of expensive commercial media such as Stemspan, etc., and thus the cost of the entire differentiation process is greatly reduced.

The key innovation points of the invention are as follows: 1) the EB 3D differentiation method has low cost, short period and high efficiency, can obtain NK cells which have large quantity, high purity and similar cytotoxicity with NK cells from peripheral blood sources only in about 25 days, and is suitable for large-scale production; 2) the components of the differentiation medium are clear, NK cells can be successfully differentiated under the condition of not using serum and trophoblast cells, and the method is suitable for clinical application; 3) the amplification conditions are optimized, and the NK cells obtained by differentiation can be massively amplified under the condition of no serum and trophoblast cells; 4) the cryopreservation condition is optimized, the NK cells can be stored for a long time, and higher cell activity can be maintained after recovery.

Specifically, the technical scheme of the invention is as follows:

the invention discloses a pluripotent stem cell-derived natural killer cell, which expresses CD56, Nkp30, Nkp44 and Nkp46 and also expresses markers of mature natural killer cells, namely CD16 and CD 94. In the present invention, NKp46, NKp30 and NKp44 all belong to the Natural Cytotoxic Receptor (NCR), and all three are immunoglobulin superfamily (IgSF) members, but have no homology with each other. NCR is expressed only on the surface of NK cells, is a unique marker of NK cells, and usually exerts a killing effect when KIR/KLR loses the ability to recognize "self". NKp30(NCR3) is an important member of the NCR family, expressed on the surface of all NK cells, and plays an important role in the process of NK cell activation and tumor killing. NKp44, CD336, is a member of IgSF, a natural cytotoxic receptor (NCR 2). It is expressed in activated NK cells with the ligand DAP12, involved in mediating NK cell killing activity. The NKp46 extracellular domain contains 2 Ig-like domains, and the NKp30 extracellular domain has only one V-type domain. NKp46 and NKp30 have short cytoplasmic domains, and both transmembrane regions contain positively charged arginine.

In a second aspect of the present invention, there is disclosed a method for preparing natural killer cells, comprising the steps of:

s1: forming an embryoid body;

s2: the embryoid body is differentiated into hematopoietic progenitor cells;

s3: differentiating hematopoietic progenitor cells into NK cells;

s4: maturation and expansion of NK cells.

Preferably, the S1 includes:

s11: placing the cell suspension of the human pluripotent stem cells on a shaking table to shake and culture overnight to form embryoid bodies.

Preferably, the S2 includes:

s21: removing the supernatant of the embryoid body, and adding a fresh first-step differentiation culture medium for cell culture; wherein, the first step differentiation culture medium is obtained by adding a small molecule GSK3 beta inhibitor and at least one of the following components in a basic differentiation culture medium: BMP signaling pathway activators, VEGF (vascular endothelial growth factor), bFGF (basic fibroblast growth factor), SCF (stem cell factor), Flt3L (FMS-like tyrosine kinase 3 ligand), IL3 (interleukin 3), IL6 (interleukin 6), insulin (insulin), IGF-1 (insulin-like growth factor 1), and TPO (human thrombopoietin);

s22: removing the first step differentiation culture medium, and adding the second step differentiation culture medium for cell culture; wherein, the second step differentiation culture medium is prepared by adding VEGF, bFGF and at least one of the following components in a basic differentiation culture medium: BMP signaling pathway activators, Noda inhibitors, SCF, Flt3L, IL15, IL3, IL6, insulin, IGF-1 and TPO.

S23: removing the differentiation culture medium of the second step, and adding the differentiation culture medium of the third step for cell culture to obtain hematopoietic progenitor cells; wherein, the third step differentiation culture medium is to add growth factors and colony stimulating factors in the basic differentiation culture medium.

More preferably, the growth factor is selected from one or more of EGF (epidermal growth factor), VEGF (vascular endothelial growth factor), bFGF, insulin, IGF-1, PGF (platelet growth factor) and PDGF (platelet derived growth factor); the colony stimulating factor is selected from one or more of G-CSF (granulocyte colony stimulating factor), M-CSF (macrophage colony stimulating factor), GM-CSF (recombinant human granulocyte-macrophage colony stimulating factor), multi-CSF (multiple colony stimulating factor, also called IL-3), EPO (erythropoietin), TPO, SCF and Flt-3L.

Preferably, in S3, the third step differentiation medium is removed, cells are seeded in a cell culture vessel coated with matrix protein, and the fourth step differentiation medium is added for cell culture; the matrix protein at least comprises one of Notch pathway activator protein or integrin; wherein the fourth step of differentiation medium is a basal differentiation medium to which colony stimulating factors and interleukins are added.

The cell culture vessel coated with Notch pathway activator protein and integrin is used for culture, so that the growth rate of cells can be increased, and the differentiation efficiency of NK cells can be improved.

In some embodiments of the invention, the cell culture vessel is a cell culture flask.

More preferably, the colony stimulating factor is selected from one or more of G-CSF, M-CSF, GM-CSF, Multi-CSF (IL-3), EPO, TPO, SCF and Flt-3L; the interleukin is selected from one or more of IL-1, IL-2, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21 and IL-27.

More preferably, the Notch pathway activating protein is selected from one or more of DLL1 recombinant protein, DLL4 recombinant protein, Jagged-1 recombinant protein, Jagged-2 recombinant protein and variants thereof, where "they" refers to DLL1 recombinant protein, DLL4 recombinant protein, Jagged-1 recombinant protein or Jagged-2 recombinant protein.

The integrin is selected from one or more of Fibronectin (Fibronectin), Laminin (Laminin), Vitronectin (Vitronectin), MAdCAM-1 (adhesion cell adhesion molecule-1), VCAM-1 (adhesion cell adhesion molecule-1), and ICAM (intercellular adhesion molecule), and variants of these integrins, where "they" refer to Fibronectin (Fibronectin), Laminin (Laminin), Vitronctin (Vitronectin), MAdCAM-1 (adhesion cell adhesion molecule-1), VCAM-1 (adhesion cell adhesion molecule-1), or ICAM (intercellular adhesion molecule).

Preferably, the S4 includes: removing the differentiation culture medium in the fourth step, and adding the differentiation culture medium in the fifth step for cell culture; wherein the fifth step differentiation medium is a basic differentiation medium to which interleukins and a substance promoting the maturation and expansion of NK cells are added.

More preferably, the interleukin is selected from one or more of IL-2, IL-12, IL-18, IL-21, and IL-27 and IL-15; the substance promoting NK cell maturation and expansion is selected from one or more of human AB plasma, human platelet lysate, Vitamin A, nicotinamide, Vitamin E and Heparin.

Preferably, the method further comprises step S5: freezing and storing the NK cells obtained by differentiation by using a freezing and storing solution; the cryopreservation liquid comprises: sodium chloride, sodium gluconate, sodium acetate, potassium chloride, magnesium chloride, human serum albumin and DMSO.

In some embodiments of the invention, the NK cell cryopreservation fluid composition is shown in table 1 below.

TABLE 1

Serial number	Composition (I)	Final concentration
			1	Sodium chloride	1-10mg/ml
2	Sodium gluconate	1-10mg/ml
			3	Sodium acetate	1-10mg/ml
4	Potassium chloride	0-1mg/ml
			5	Magnesium chloride	0-1mg/ml
6	HSA (human serum albumin)	10-30％
			7	DMSO (dimethyl sulfoxide)	5-20％

The steps of the method for preparing the natural killer cells are as follows:

first, Day-1 to Day 0: formation of Embryoid Bodies (EB)

The iPSC which is well undifferentiated and cultured until the polymerization degree is 70-90% is digested into single cell suspension, the single cell suspension is suspended in an iPSC maintenance culture medium according to a certain density, and the iPSC suspension is placed on a shaking table in an incubator at 37 ℃ for shaking culture overnight to form EB with uniform size and shape.

Operation details and optimization of Day-1-Day 0 experiment

1) Culture of human iPSC

The human ipscs used in the experiments were subjected to strict pluripotency validation (expressing various pluripotency markers and forming teratomas comprising three germ layers, inner, middle and outer, in immunodeficient mice). The ipscs are normally cultured in iPSC maintenance medium, and the medium used is E8 or TeSR or other similar medium.

2) Formation of EB

The formation experiment of the embryoid body is carried out when the iPSC is cultured to 70-90% of polymerization degree according to the method. The specific operation is as follows: use ofTrypLE or accutase digests ipscs into a complete single cell suspension, resuspended in iPSC maintenance medium, and Rock inhibitor added to the medium. Placing the cell suspension on a 3D shaking table in an incubator at 37 ℃ for shaking culture, wherein the rotation speed of the shaking table is 10-100 rpm; the shaking culture time in this step is 8-32 hours; at the end of the culture, EBs of more uniform size and morphology were obtained. For example, when T25 flask is used, the Rock inhibitor is Y-27632 at a concentration of 10. mu.M; the cell density was 0.1X 10⁶-5×10⁶Per mL; the rotating speed of the shaking table is 10-20 rpm; the culture time is 8-32 hours.

(II) Day 0 to Day 6-12: differentiation of pluripotent stem cells into hematopoietic progenitor cells

The EBs from D0 were changed to differentiation medium at different time points and EBs containing highly expressing CD34+ cells were harvested at D6-12.

Experimental details and optimization of Day 0-Day 6-12

1) Day 0: replacing with the first step differentiation culture medium

The flask containing the D0 EB was removed from the incubator, the flask was tilted to allow the EB to settle to the bottom, the supernatant was removed, and fresh first step differentiation medium was added. The first step differentiation medium is a basal differentiation medium supplemented with a small molecule GSK3 β inhibitor and one or more cytokines selected from BMP signaling pathway activators, VEGF, bFGF, SCF, Flt3L, IL3, IL6, insulin, IGF-1, and TPO. BMP signal pathway activators are BMP2, BMP4, SB4, ventromorphins (SJ000291942, SJ000063181, SJ000370178), Isoliquiritigenin, diosmetin (diosmin), apigenin (apigenin), biochanin (biochanin) and the like, wherein the isoliquirigenin is a flavonoid compound separated from glycyrrhiza glabra roots and has antitumor activity; the GSK3 beta inhibitor is NP031112, TWS119, SB216763, CHIR-98014, AZD2858, AZD1080, SB415286, LY2090314, CHIR99021, etc. For example, when the culture is carried out using T25 flask, in which the GSK3 beta inhibitor is CHIR99021, at a concentration of 0.5-20. mu.M; the BMP signal pathway activator is BMP4, and the concentration is 0-200 ng/mL; the cytokine comprises insulin, IGF-1, VEGF, bFGF, the concentration is 0-10 mug/mL, 0-100ng/mL, 5-100ng/mL and 0-100ng/mL respectively.

The time for culturing the EBs in the first-step differentiation medium was (2-4) days.

The composition of the basal differentiation medium is shown in Table 2 below.

TABLE 2

2) Day 2-4: replacing with the second step differentiation culture medium

Taking out the culture bottle filled with EB from the culture box, inclining the culture bottle to make EB sink to the bottom, removing the supernatant of the first step differentiation culture medium, and adding fresh second step differentiation culture medium. The second step differentiation medium is a basal differentiation medium supplemented with VEGF, bFGF, and one or more cytokines selected from the group consisting of BMP signaling pathway activators, Noda inhibitors, SCF, Flt3L, IL15, IL3, IL6, insulin, IGF-1, and TPO. BMP signaling pathway activators were selected similarly to D0; the Noda inhibitor is selected from the group consisting of Lefty-A, Lefty-B, Lefty-1, Lefty-2, SB431542, SB202190, SB505124, NPC30345, SD093, SD908, SD208, LY2109761, LY364947, LT580276, A83-01, and derivatives thereof. For example, when the culture is carried out using a T25 flask, wherein the BMP signaling pathway activator is BMP4 at a concentration of 0-200 ng/mL; the Noda inhibitor is SB431542 at a concentration of 0-20 μ M; the cytokine comprises insulin, IGF-1, VEGF, bFGF, IL3 and IL6, and the concentration is 0-10. mu.g/mL, 0-100ng/mL, 5-100ng/mL, 0-20ng/mL and 0-20ng/mL respectively.

The fluid change time point of the differentiation medium of the second step may be between Day2 and Day 4.

3) Day 3-6: changing into a third step differentiation culture medium

Taking out the culture bottle filled with EB from the culture box, inclining the culture bottle to make EB sink to the bottom, removing the supernatant of the differentiation culture medium of the second step, and adding fresh differentiation culture medium of the third step. The third step is to add growth factor and colony stimulating factor into the basic differentiation culture medium. The growth factor is one or more selected from EGF, VEGF, bFGF, insulin, IGF-1, PGF, PDGF and the like. The colony stimulating factor is one or more selected from the group consisting of G-CSF, M-CSF, GM-CSF, Multi-CSF (IL-3), EPO, TPO, SCF, Flt-3L, and the like. For example, when the culture is carried out using T25 flask, wherein the growth factors are insulin, VEGF, IGF-1 and bFGF, at concentrations of 0.5-10. mu.g/mL, 5-100ng/mL, 0-100ng/mL and 0.5-100ng/mL, respectively; colony stimulating factors are TPO, SCF and Flt-3L at concentrations of 0-100ng/mL, 0-100ng/mL and 0-100ng/mL, respectively;

the liquid changing time point of the differentiation medium of the third step may be between Day3 and Day 6.

4) Day 6-12: detecting the obtained hematopoietic progenitor cells

The diameters of the embryoid bodies were measured on

days

0, 5 and 12 of the differentiation culture.

Selecting a proper time point during the period from Day6 to Day12, detecting the cell phenotype of the hematopoietic progenitor cells in the embryoid bodies by using a flow cytometer, and proving that the obtained cells contain the hematopoietic progenitor cells expressing CD34, and the percentage of the hematopoietic progenitor cells which are positive to CD34 in the total cells is between 20% and 80%.

Wherein, the information of the flow-type antibody is as follows:

APC Mouse Anti-Human CD34 Clone 581(RUO)，BD，#555824；

FITC anti-human CD45，Biolegend，#304006。

(III) 1-2 weeks after EB inoculation (Day6-12 to Day 20-26): differentiation of hematopoietic progenitors into NK cells

The above-mentioned EB containing CD34+ hematopoietic progenitor cells of Day6-12 was resuspended in differentiation medium of the fourth step, inoculated in a cell matrix-coated flask and cultured for 2 weeks, and the medium was changed at the end of

week

1 and 2 to maintain the following factors for different periods of action. NK cells containing high CD3-CD56+ were harvested at the end of week 2 (Day 20-26).

Details of experimental procedures at 1-2 weeks after EB inoculation (Day6-12 to Day 20-26):

1) day 6-12: replacing with the fourth step differentiation medium

The flasks were tilted to sink the EBs to the bottom, the third step differentiation medium supernatant was removed, and the EBs were resuspended in fresh fourth step differentiation medium. The fourth step is to add colony stimulating factors and interleukins into the basic differentiation medium. The colony stimulating factor is one or more selected from G-CSF, M-CSF, GM-CSF, multi-CSF (IL-3), EPO, TPO, SCF, Flt-3L, etc. The interleukin is one or more selected from IL-1, IL-2, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, IL-27, etc. For example, when cultured using T25 flasks, the colony stimulating factors are TPO, SCF, Flt-3L and IL-3 at concentrations of 0-100ng/mL, 5-100ng/mL and 5-100ng/mL, respectively, for a period of time of 1 week, 2 weeks or 1-2 weeks after inoculation. The interleukins are IL-2, L-7 and IL-15 at concentrations of 100-1000IU/mL, 5-100ng/mL and 5-100ng/mL, respectively, for periods of time 1 week, 2 weeks or 1-2 weeks after EB inoculation. The liquid changing time point of the differentiation medium of the fourth step may be between Day6 and Day 12.

The EBs resuspended in the fourth step differentiation medium were inoculated at the appropriate density into medium-protein-coated culture flasks. The matrix protein is at least one of the following components: notch pathway activator proteins and integrins. The Notch pathway activating protein is DLL1 recombinant protein, DLL4 recombinant protein, Jagged-1 recombinant protein, Jagged-2 recombinant protein, and variants of the Notch pathway activating proteins. The integrin is Fibronectin (Fibronectin), Laminin (Laminin), Vitronectin (Vitronectin), MAdCAM-1 (adhesion molecule for adhesion cell-1), VCAM-1 (adhesion molecule for vascular cell-1), ICAM (intercellular adhesion molecule), and variants thereof. For example, when cultured using T25 flasks, the Notch pathway activator protein is a DLL4-Fc recombinant protein. The integrin is VCAM-1.

Half of the fresh fourth step medium was replaced every 3-7 days depending on the cell density.

2) The NK cells obtained were examined 2 weeks after EB inoculation (Day20-26)

At 2 weeks after EB inoculation, the suspension cells in the wells were collected. Detecting the expression of the cell surface related index protein by flow cytometry. The detection indexes comprise: CD56, NKp30, NKp44, NKp 46.

Wherein, the information of the flow-type antibody is as follows:

PE Mouse Anti-Human CD56 Clone B159(RUO)，BD，#555516；

Alexa

647 Mouse anti-Human CD337(NKp30)，BD，#558408；

Alexa

647 Mouse Anti-Human NKp44(CD336)，BD，#558564；

APC Mouse Anti-Human CD335(NKp46)，BD，#558051。

3) 3-4 weeks after EB inoculation (Day 27-Day 40): details of experimental procedures 3-4 weeks after NK cell maturation and expansion EB inoculation (Day 27-Day 40):

after two weeks of EB differentiation, the cell density reached (1-2). times.10⁶Per mL, collecting cells, centrifuging to remove the fourth step differentiation medium at (0.5-1). times.10⁶Cell density of one/mL was resuspended in fresh differentiation medium of the fifth step. The fifth step is to add interleukin and other substances for promoting NK cell maturation and expansion into the basic differentiation medium. The interleukin is one or more selected from IL-1, IL-2, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, IL-27, etc. Other substances promoting NK cell maturation and expansion are selected from human AB plasma, human platelet lysate, Vitamin A, Nicotinamide (NAM, Vitamin B3), Vitamin E and Heparin, etc. For example, when the culture is carried out using a T25 flask, the interleukins are IL-2, IL-12, IL-18, IL-21 and IL-27 and IL-15 at concentrations of 100-1000IU/mL, 0-100ng/mL and 5-100ng/mL, respectively; the additive substances for promoting NK cell maturation and amplification are human platelet lysate, Nicotinamide (NAM), Vitamin E and Heparin, and the concentrations are 1-10%, 1-10mmol/L, 0-10mg/mL and 0-100 μ g/mL respectively.

The NK cells obtained at 3-4 weeks (Day27-40) after EB inoculation were examined

Suspension cells in wells were collected at 3-4 weeks of EB differentiation (Day 27-40). Detecting the expression of the cell surface related index protein by flow cytometry. The detection indexes comprise: CD56, CD94, and CD 16.

Wherein, the information of the flow-type antibody is as follows:

PE Mouse Anti-Human CD56 Clone B159(RUO)，BD，#555516；

APC Mouse Anti-Human CD94，BD，#559876；

APC Mouse Anti-Human CD16 Clone B73.1(RUO)，BD，#561304。

in one embodiment of the present invention, the human pluripotent stem cells are prepared by the method disclosed in patent CN 108085299 a.

It is understood that one skilled in the art can select any commercial cell line or cell strain of human pluripotent stem cells as required to complete the present invention and all fall within the scope of the present invention.

In the invention:

1. the basic differentiation medium comprises the following components: IMDM, F-12, rHSA (recombinant Human serum albumin), MTG-Monothioglycerol (thioglycerol), Ascorbic Acid (Ascorbic Acid), Human Transferrin (Human Transferrin), Na Selenite (sodium Selenite), and Ethanolamine (Ethanolamine). Wherein, IMDM and F12 can be used as the basis for developing serum-free formula, and the culture medium is suitable for mammalian cell culture under the condition of low serum content; in the formula of the culture medium, IMDM and F-12 are mixed in a ratio of 1: 1. The combination and concentration of the other components is optimized, the supplement has the function of replacing serum, and the supplement is an additive for maintaining differentiation to blood progenitor cells and maintaining proliferation of differentiated cells.

GSK-3 inhibitors: substances which block the kinase activity of the GSK3 β protein, such as the GSK3 β inhibitor IX (6-bromoindirubin 3' -oxime), SB216763, the GSK3 β inhibitor VII (4-dibromoacetophenone), L803-mts, and CHIR99021 with high selectivity. CHIR99021 is preferred in the present invention.

The concentration of CHIR99021 in the medium is not particularly limited as long as it inhibits the kinase activity of GSK3 beta protein, but is not limited to, for example, 0.1. mu.M, 0.2. mu.M, 0.3. mu.M, 0.4. mu.M, 0.5. mu.M, 0.6. mu.M, 0.7. mu.M, 0.8. mu.M, 0.9. mu.M, 1. mu.M, 1.1. mu.M, 1.2. mu.M, 1.3. mu.M, 1.4. mu.M, 1.5. mu.M, 2. mu.M, 2.5. mu.M, 3. mu.M, 4. mu.M, 5. mu.M, 6. mu.M, 8. mu.M, or 10. mu.M. Preferably 1 to 8 μ M.

BMP signaling pathway activator: examples of the substance which activates the BMP signaling pathway include BMP2, BMP4, SB4, ventromorphins (SJ000291942, SJ000063181, SJ000370178), isooliquitinin, diosmetin, apigenin, biochaninA, etc. The BMP activator used in the present invention is BMP 4. The concentration of BMP4 in the medium is not particularly limited as long as it activates a BMP signaling pathway, and examples thereof include, but are not limited to, 5ng/ml, 10ng/ml, 20ng/ml, 30ng/ml, 40ng/ml, 50ng/ml, 60ng/ml, 70ng/ml, 80ng/ml, 90ng/ml, 100ng/ml, 110ng/ml, 120dng/ml, 130ng/ml, 140ng/ml, 150ng/ml, 160ng/ml, 180ng/ml and 200 ng/ml. Preferably 5 to 50 ng/ml.

Nodal inhibitors: substances that inhibit Nodal signaling pathways. Lefty-A, Lefty-B, Lefty-1, Lefty-2, SB431542, SB202190, SB505124, NPC30345, SD093, SD908, SD208, LY2109761, LY364947, LT580276, A83-01, and derivatives thereof are selected. The Nodal inhibitor used in the present invention is SB 431542. The concentration of SB431542 in the medium is not particularly limited as long as it blocks the Nodal signaling pathway, and examples thereof include, but are not limited to, 0. mu.M, 1. mu.M, 2. mu.M, 3. mu.M, 4. mu.M, 5. mu.M, 6. mu.M, 7. mu.M, 8. mu.M, 9. mu.M, 10. mu.M, 11. mu.M, 12. mu.M, 13. mu.M, 14. mu.M, 15. mu.M, 20. mu.M, 25. mu.M, and 30. mu.M. Preferably 1-20. mu.M.

5. Colony stimulating factor: cytokines capable of stimulating hematopoietic stem cell proliferation and differentiation. G-CSF, M-CSF, GM-CSF, multi-CSF (IL-3), EPO, TPO, SCF, Flt-3L, and the like may be selected. Colony stimulating factors used in the present invention are G-CSF, GM-CSF, TPO, SCF and Flt-3L and multi-CSF. The concentrations of TPO, SCF and Flt-3L in the medium are not particularly limited as long as they stimulate the proliferation and differentiation of hematopoietic stem cells, and examples thereof include, but are not limited to, TPO at a concentration of 0ng/ml, 5ng/ml, 10ng/ml, 15ng/ml, 20ng/ml, 30ng/ml, 40ng/ml, 50ng/ml, 60ng/ml, 70ng/ml, 80ng/ml, 90ng/ml and 100 ng/ml. Preferably 0 to 100 ng/ml. SCF concentrations of 0ng/ml, 10ng/ml, 20ng/ml, 30ng/ml, 40ng/ml, 50ng/ml, 60ng/ml, 80ng/ml, 100ng/ml, 150ng/ml, 200ng/ml, but are not limited thereto. Preferably 0 to 100 ng/ml. The concentration of Flt-3L is 0ng/ml, 1ng/ml, 5ng/ml, 10ng/ml, 20ng/ml, 30ng/ml, 40ng/ml, 50ng/ml, 80ng/ml, 100ng/ml, 150ng/ml, 200ng/ml, but is not limited thereto. Preferably 0 to 100 ng/ml.

Notch signaling pathway activators: recombinant proteins that activate the Notch signaling pathway. The recombinant DLL1 protein, the recombinant DLL4 protein, the recombinant Jagged-1 protein, the recombinant Jagged-2 protein, and variants of these Notch pathway activating proteins, etc. can be selected. The Notch activator used in the present invention is DLL4-Fc recombinant protein. The density of the coating of the coated DLL4-Fc recombinant protein is not particularly limited as long as it can activate the Notch signaling pathway, and for example, the density of the coating of the DLL4-Fc recombinant protein is 0. mu.g/cm²、0.1μg/cm²、0.2μg/cm²、0.3μg/cm²、0.4μg/cm²、0.5μg/cm²、0.6μg/cm²、0.7μg/cm²、0.8μg/cm²、0.9μg/cm²、1μg/cm²、2μg/cm²、3μg/cm²、4μg/cm²、5μg/cm²But is not limited thereto. Preferably 0.5 to 2 mu g/cm²。

7. Growth factor: a native protein that stimulates cell proliferation and cell differentiation. EGF, VEGF, bFGF, insulin, IGF-1, PGF, PDGF, and the like may be selected. The growth factors used in the present invention are insulin, VEGF, IGF-1 and bFGF. The concentration of the insulin, VEGF, and bFGF in the medium is not particularly limited as long as it can stimulate cell proliferation and cell differentiation, and examples thereof include concentrations of insulin of 0. mu.g/ml, 0.5. mu.g/ml, 1. mu.g/ml, 2. mu.g/ml, 3. mu.g/ml, 4. mu.g/ml, 5. mu.g/ml, 6. mu.g/ml, 7. mu.g/ml, 8. mu.g/ml, 9. mu.g/ml, 10. mu.g/ml, 20. mu.g/ml, and 30. mu.g/ml. Preferably 0 to 10. mu.g/ml. VEGF concentrations are 5ng/ml, 10ng/ml, 15ng/ml, 20ng/ml, 30ng/ml, 40ng/ml, 50ng/ml, 60ng/ml, 70ng/ml, 80ng/ml, 90ng/ml, 100ng/ml, 150ng/ml, 200ng/ml, but are not so limited. Preferably 5 to 100 ng/ml. The concentration of IGF-1 is 5ng/ml, 10ng/ml, 15ng/ml, 20ng/ml, 30ng/ml, 40ng/ml, 50ng/ml, 60ng/ml, 70ng/ml, 80ng/ml, 90ng/ml, 100ng/ml, 150ng/ml, 200 ng/ml. Preferably 0 to 100. mu.g/ml. The concentration of bEGF is 5ng/ml, 10ng/ml, 15ng/ml, 20ng/ml, 30ng/ml, 40ng/ml, 50ng/ml, 60ng/ml, 70ng/ml, 80ng/ml, 90ng/ml, 100ng/ml, 150ng/ml, 200ng/ml, but is not limited thereto. Preferably 0 to 100 ng/ml.

8. Interleukin (IL): cytokines that mediate immune cell activation, proliferation and differentiation. One or more of IL-1, IL-2, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, IL-27, etc. may be selected at different differentiation stages.

The interleukins used in the present invention D6-12 to D20-26 (i.e., within two weeks after EB inoculation) were IL-2, IL-7 and IL-15. The concentration of IL-2, IL-7 and IL-15 in the medium is not particularly limited as long as it can mediate immune cell activation, proliferation and differentiation, and examples thereof include, but are not limited to, IL-2 concentrations of 0IU/ml, 50IU/ml, 100IU/ml, 200IU/ml, 300IU/ml, 400IU/ml, 500IU/ml, 700IU/ml, 800IU/ml, 1000IU/ml and 2000 IU/ml. Preferably 0 to 1000 IU/ml. IL-7 concentrations are, but not limited to, 10ng/ml, 20ng/ml, 30ng/ml, 40ng/ml, 50ng/ml, 60ng/ml, 70ng/ml, 80ng/ml, 90ng/ml, 100ng/ml, 150ng/ml, 200 ng/ml. Preferably 5 to 100 ng/ml. IL-15 concentrations are, but not limited to, 1ng/ml, 5ng/ml, 10ng/ml, 15ng/ml, 20ng/ml, 25ng/ml, 30ng/ml, 40ng/ml, 50ng/ml, 60ng/ml, 80ng/ml, 100ng/ml, 150ng/ml, 200 ng/ml. Preferably 5 to 100 ng/ml.

ROCK inhibitor: a substance which inhibits Rho kinase (ROCK) function. For example: y-27632, HA100, HA1152, and Blebbistatin. Y-27632 is preferred in the present invention.

The concentration of Y-27632 in the medium is not particularly limited as long as it is a concentration that inhibits Rho kinase, and examples thereof include, but are not limited to, 1. mu.M, 1.5. mu.M, 2. mu.M, 2.5. mu.M, 3. mu.M, 3.5. mu.M, 4. mu.M, 4.5. mu.M, 5. mu.M, 5.5. mu.M, 6. mu.M, 6.5. mu.M, 7. mu.M, 7.5. mu.M, 8. mu.M, 9. mu.M, 10. mu.M, 15. mu.M, 20. mu.M, 30. mu.M, and 50. Preferably 10. mu.M.

In a third aspect, the invention discloses a natural killer cell prepared by the above method.

In a fourth aspect, the invention discloses a cell population enriched for the above natural killer cells.

In the fifth aspect of the invention, the invention discloses a medicament for preventing and/or treating tumors, which comprises the natural killer cells.

The key points and points to be protected of the invention are as follows:

1) based on the rapid, efficient and low-cost EB 3D differentiation method (including the types, combination modes, adding time, concentration and the like of the small molecular compounds and the cytokines) combined by the small molecular compounds and the cytokines, the method has the advantages of simpler operation, more stable result and lower cost, and is beneficial to large-scale iNK production.

2) NK cell differentiation and expansion media components. The key innovation points are as follows: the components of the differentiation and amplification culture medium are clear, NK cells can be successfully differentiated and amplified under the condition of not using serum and trophoblast cells, and the method is suitable for production and clinical application of large-scale cell preparations.

3) The cryopreservation condition is optimized, the NK cells can be stored for a long time, and higher cell activity can be maintained after recovery.

On the basis of the common general knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily without departing from the concept and the protection scope of the invention.

Compared with the prior art, the invention has the following remarkable advantages and effects:

first, the method uses 3D suspension culture during induced differentiation, which can be performed in a short period (about 40 days) of 1X 10⁵The hiPSC gain of is greater than 1 x 10⁷The NK cells of (1), are suitable for large-scale cell preparation production;

secondly, the differentiation process is finely regulated and controlled by the combined use of small molecular compounds and cytokines, so that stable and efficient differentiation is realized, and the proportion of CD56+ NK cells in a final culture system can reach more than 90%;

thirdly, the NK cell prepared by the method has similar cell phenotype and functionality with PB-NK cell, and the preparation cost is low, so that the NK cell has great clinical and scientific research application potential;

fourth, the method uses serum-free media and no feeder cells during both induction and expansion, and is suitable for the production and use of subsequent clinical-grade cell preparations.

Drawings

FIG. 1 shows a flow chart for preparing natural killer cells disclosed in the present invention.

FIG. 2 shows the process of induced differentiation from pluripotent stem cells into mature NK cells in the method disclosed in the present invention (day-1, day 0, day 5, day12, after 2 weeks of EB inoculation and after 4 weeks of EB inoculation, respectively).

FIG. 3 shows that the specificity of D8 EB was measured by flow cytometry, which indicates that hematopoietic stem cells (CD34+) reached a high level.

FIG. 4 shows that the specificity indexes of iNK cells at 2 weeks and 4 weeks after EB inoculation are detected by a flow cytometer, and that iNK cells which are CD56 positive at 2 weeks after EB inoculation reach more than 80% and simultaneously highly express Nkp30, Nkp44 and Nkp 46. iNK cells simultaneously expressed specific indexes of mature NK cells, CD94 and CD16, at 4 weeks after EB inoculation, indicating that NK cells have matured.

FIG. 5 shows that iNK cells harvested at week 4 after EB inoculation produced a significant cytotoxic effect on target cell K562, with a cell killing capacity similar to that of PB-NK.

FIG. 6 shows that the concentration of CHIR99021 in the first step differentiation medium affects the differentiation efficiency of CD34+ hematologic progenitors.

FIG. 7 shows that the time for the second differentiation medium exchange affects the differentiation efficiency of CD34+ blood progenitor cells.

FIG. 8 shows that the time for the third differentiation medium exchange affects the differentiation efficiency of CD34+ blood progenitor cells.

FIG. 9 shows that EB seeding time points affect the differentiation efficiency of NK cells (CD3-CD56 +).

FIG. 10 shows that the effect concentration of BMP4 affects the differentiation efficiency of CD34+ blood progenitor cells.

FIG. 11 shows that the concentration of SB affects the differentiation efficiency of CD34+ blood progenitor cells.

FIG. 12 shows that the concentration of bFGF acting affects the differentiation efficiency of CD34+ blood progenitor cells.

Detailed Description

The technical solution of the present invention is described in detail below with reference to the drawings and the embodiments, but the present invention is not limited to the scope of the embodiments.

The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions. The reagents and starting materials used in the present invention are commercially available.

Example 1

This example discloses a method for preparing natural killer cells, comprising the steps of:

s1: forming an embryoid body;

s2: the embryoid body is differentiated into hematopoietic progenitor cells;

s3: differentiating hematopoietic progenitor cells into NK cells;

s4: maturation and expansion of NK cells.

Specifically, the flow chart is shown in fig. 1, and the steps are as follows:

first, Day-1 to Day 0: formation of Embryoid Bodies (EB)

1) Culture of human pluripotent stem cells (iPSCs)

The human iPSC was prepared by the method disclosed in patent CN 108085299 a.

2) Formation of EB

The formation experiment of the embryoid body is carried out when the iPSC is cultured to 70-90% of polymerization degree according to the method. The specific operation is as follows: ipscs were digested to a complete single cell suspension using TrypLE or accutase, resuspended in iPSC maintenance medium, and Rock inhibitors were added to the medium. Placing the cell suspension on a 3D shaking table in an incubator at 37 ℃ for shaking culture, wherein the rotation speed of the shaking table is 10-100 rpm; the shaking culture time in this step is 8-32 hours; at the end of the culture, EBs of more uniform size and morphology were obtained. When T25 flask is used, the Rock inhibitor is Y-27632 at a concentration of 10. mu.M; the cell density was 0.1X 10⁶-5×10⁶Per mL; the rotating speed of the shaking table is 10-20 rpm; the culture time is 8-32 hours.

1) Day 0: replacing with the first step differentiation culture medium

The flask containing the D0 EB was removed from the incubator, the flask was tilted to allow the EB to settle to the bottom, the supernatant was removed, and fresh first step differentiation medium was added. The first step differentiation culture medium is to add a small molecule GSK3 beta inhibitor and one or more components selected from the following components in a basic differentiation culture medium: BMP signaling pathway activator, VEGF, bFGF, SCF, Flt3L, IL3, IL6, insulin, IGF-1 and TPO. BMP signal pathway activators are BMP2, BMP4, SB4, ventromorphins (SJ000291942, SJ000063181, SJ000370178), isoliquiritigenin, diosmetin (diosmin), apigenin (apigenin), biochanin (biochanin), and the like; the GSK3 beta inhibitor is NP031112, TWS119, SB216763, CHIR-98014, AZD2858, AZD1080, SB415286, LY2090314, CHIR99021, etc. For example, when the culture is carried out using T25 flask, in which the GSK3 beta inhibitor is CHIR99021, at a concentration of 0.5-20. mu.M; the BMP signal pathway activator is BMP4, and the concentration is 0-200 ng/mL; the cytokine comprises insulin, IGF-1, VEGF, bFGF, the concentration is 0-10 mug/mL, 0-100ng/mL, 5-100ng/mL and 0-100ng/mL respectively.

Wherein Isooliquitinigenin is a flavonoid compound separated from Glycyrrhiza glabra root, and has antitumor activity.

The composition of the basal differentiation medium is shown in Table 2 below.

TABLE 2

2) Day 2-4: replacing with the second step differentiation culture medium

Taking out the culture bottle filled with EB from the culture box, inclining the culture bottle to make EB sink to the bottom, removing the supernatant of the first step differentiation culture medium, and adding fresh second step differentiation culture medium. The second step differentiation medium is a basal differentiation medium supplemented with VEGF, bFGF, and one or more cytokines selected from the group consisting of BMP signaling pathway activators, Noda inhibitors, SCF, Flt3L, IL15, IL3, IL6, insulin, IGF-1, and TPO. BMP signaling pathway activators were selected similarly to D0; the Noda inhibitor is selected from the group consisting of Lefty-A, Lefty-B, Lefty-1, Lefty-2, SB431542, SB202190, SB505124, NPC30345, SD093, SD908, SD208, LY2109761, LY364947, LT580276, A83-01, and derivatives thereof. When the culture is carried out by using a T25 culture flask, wherein the BMP signal pathway activator is BMP4, and the concentration is 0-200 ng/mL; the Noda inhibitor is SB431542 at a concentration of 0-20 μ M; the cytokine comprises insulin, IGF-1, VEGF, bFGF, IL3 and IL6, and the concentration is 0-10. mu.g/mL, 0-100ng/mL, 5-100ng/mL, 0-20ng/mL and 0-20ng/mL respectively.

3) Day 3-6: changing into a third step differentiation culture medium

4) Day 6-12: detecting the obtained hematopoietic progenitor cells

The diameters of the embryoid bodies at day 0, day 5 and day12 of the differentiation culture were 50-120. mu.M, 300-400. mu.M and 400-650. mu.M, respectively. The specific cell morphology is shown in FIG. 3.

Selecting a proper time point during the period from Day6 to Day12, detecting the cell phenotype of the hematopoietic progenitor cells in the embryoid bodies by using a flow cytometer, and proving that the obtained cells contain the hematopoietic progenitor cells expressing CD34, and the percentage of the hematopoietic progenitor cells which are positive to CD34 in the total cells is between 20% and 80%. See figure 5 for specific data.

Wherein, the information of the flow-type antibody is as follows:

APC Mouse Anti-Human CD34 Clone 581(RUO)，BD，#555824；

FITC anti-human CD45，Biolegend，#304006。

week

1) day 6-12: replacing with the fourth step differentiation medium

The flasks were tilted to sink the EBs to the bottom, the third step differentiation medium supernatant was removed, and the EBs were resuspended in fresh fourth step differentiation medium. The fourth step is to add colony stimulating factors and interleukins into the basic differentiation medium. The colony stimulating factor is one or more selected from G-CSF, M-CSF, GM-CSF, multi-CSF (IL-3), EPO, TPO, SCF, Flt-3L, etc. The interleukin is one or more selected from IL-1, IL-2, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, IL-27, etc. When cultured using T25 flasks, the colony stimulating factors were TPO, SCF, Flt-3L and IL-3 at concentrations of 0-100ng/mL, 5-100ng/mL and 5-100ng/mL, respectively, for a period of time of 1 week, 2 weeks or 1-2 weeks after inoculation. The interleukins are IL-2, L-7 and IL-15 at concentrations of 100-1000IU/mL, 5-100ng/mL and 5-100ng/mL, respectively, for periods of time 1 week, 2 weeks or 1-2 weeks after EB inoculation. The liquid changing time point of the differentiation medium of the fourth step may be between Day6 and Day 12.

2) The NK cells obtained were examined 2 weeks after EB inoculation (Day20-26)

Wherein, the information of the flow-type antibody is as follows:

PE Mouse Anti-Human CD56 Clone B159(RUO)，BD，#555516；

Alexa

647 Mouse anti-Human CD337(NKp30)，BD，#558408；

Alexa

647 Mouse Anti-Human NKp44(CD336)，BD，#558564；

APC Mouse Anti-Human CD335(NKp46)，BD，#558051。

3) 3-4 weeks after EB inoculation (Day 27-Day 40): maturation and expansion of NK cells

Details of experimental procedures 3-4 weeks after EB inoculation (Day 27-Day 40):

The NK cells obtained were examined at 4 weeks (Day27-40) after EB inoculation

At the 4 th week of EB differentiation (Day27-40), the suspension cells in the wells were collected. Detecting the expression of the cell surface related index protein by flow cytometry. The detection indexes comprise: CD56, CD94, and CD 16.

Wherein, the information of the flow-type antibody is as follows:

PE Mouse Anti-Human CD56 Clone B159(RUO)，BD，#555516；

APC Mouse Anti-Human CD94，BD，#559876；

APC Mouse Anti-Human CD16 Clone B73.1(RUO)，BD，#561304。

specific cell morphology patterns at day-1, day 0, day 5, day12, week 2 after EB inoculation, and week 4 after EB inoculation in differentiation culture are shown in fig. 2.

Example 2

This example investigates the effect of different EB culture methods on EB differentiation rates. In the experimental group, a culture method of forming an embryoid body through 3D suspension culture is adopted in the whole induced differentiation process, namely, iPSC is directly formed into EB with a smaller volume on a 3D shaking table, and then the EB is changed into different special differentiation culture media at different differentiation stages (the specific steps are shown in example 1). During the culture process, the volume of EB increases gradually and the cells proliferate continuously. Compared with a 2D differentiation scheme (see patent CN107429230A), the 3D differentiation condition greatly saves culture space and culture volume, and the number of cells obtained in the same culture system is remarkably increased, thereby being beneficial to the large-scale production of the hPSC to the multipotential blood precursor cells. Compared with the differentiation scheme of Spin EB (US20180072992A1) and a method for further differentiation after sorting and purifying hematopoietic progenitor cells in EB cells in the literature, the 3D differentiation scheme of the invention is simpler and easier, has higher efficiency, and is more suitable for clinical-level large-scale production. Meanwhile, the present protocol directly uses EBs from day6 to day12 for differentiation, and obtains higher differentiation efficiency (the proportion of NK cells positive to CD56 reaches more than 90% at the fourth cycle of EB differentiation), and the results are shown in FIG. 4.

Example 3

The invention discovers that in the 3D induction culture, the small molecular reagent, the cell factor and the cell matrix can replace serum and trophoblast cells, accelerate the differentiation process, improve the differentiation efficiency and be beneficial to the production of the subsequent clinical cell preparation.

In our differentiation method, in addition to the use of 3D to increase differentiation efficiency, the combined use of small molecule agents, cytokines and cell matrices not only avoids the use of serum and trophoblast cells, but also further increases the proportion of blood precursor cells. The GSK3 β inhibitor selected CHIR99021, BMP signaling pathway activator used BMP4, Nodal inhibitor used SB431542, and Notch signaling pathway activator in the cell matrix used DLL4-Fc recombinant protein. On days 6-12, the proportion of blood progenitor cells (CD34+) obtained can be as high as 20-80%, which is higher than the proportion of positive cells obtained by differentiation method using serum-containing medium and trophoblast cells reported in the literature (for example, in patent CN102822332A, the proportion of CD56+ cells only reaches 20-30%).

Example 4

The invention finds that the cell density and the culture time of the initial human iPSC can influence the size of EB formation, thereby influencing the efficiency of 3D differentiation. On day 0 of differentiation, we digested human ipscs into single cells using accutase (cell digest), initial cell density was controlled at 0.1-5 million cells/T25, hpscs were maintained in medium for 8-32 hours.

Example 5

The invention discovers that the efficiency of differentiating pluripotent stem cells into blood progenitor cells can be obviously improved by optimizing the concentration of CHIR99021 in the differentiation medium of the first step.

When EB differentiation was performed using T25 flask, other conditions were as described in example 1, and concentration of CHIR99021 in the first differentiation step was gradient-optimized by treating cells with 0. mu.M, 1. mu.M, 2. mu.M, 4. mu.M, 6. mu.M, 8. mu.M or 10. mu.M of CHIR99021, respectively, and measuring CD34, which is an index of blood progenitor cells, using a flow cytometer at D12, and the results are shown in FIG. 6.

It can be seen that the addition of optimized CHIR99021 increases the efficiency of differentiation of blood progenitor cells; at lower concentrations, the differentiation rate of blood progenitor cells is low, and at higher concentrations, the differentiation of blood progenitor cells is inhibited; optimizing the concentration of CHIR99021 can improve the purity of the blood progenitor cells at D6-12, namely, the proportion of CD34+ cells is improved; the concentration of CHIR99021 is preferably 1-8 μ M.

Example 6

The invention discovers that the efficiency of differentiating pluripotent stem cells into blood progenitor cells can be obviously improved by optimizing the liquid changing time point of the differentiation culture medium of the second step.

When EB differentiation was performed using T25 flask, other condition methods were as described in example 1, the point of time for changing to the second-step differentiation medium was optimized, i.e., differentiation was continued by changing to the second-step differentiation medium at D1, D2, D2.5, D3, D3.5, D4 or D5, and the index of blood progenitor cells, CD34, was measured using flow cytometry at D12, and the results are shown in FIG. 7. Therefore, the proportion of the obtained blood progenitor cells can be obviously improved by optimizing the liquid changing time point of the differentiation culture medium of the second step; the proportion of blood progenitor cells that differentiate is low when fluid is changed at an earlier or later time point; the liquid change time point of the differentiation medium in the second step is preferably D2 to D4.

Example 7

The invention discovers that the efficiency of differentiating pluripotent stem cells into blood progenitor cells can be obviously improved by optimizing the liquid changing time point of the differentiation culture medium in the third step.

When EB differentiation was performed using T25 flask, other condition method was as described in example 1, the time point for changing to the differentiation medium of the third step was optimized, i.e., differentiation was continued by changing D3, D4, D5, D6, D7 or D8 to the differentiation medium of the fourth step, and the index CD34 of blood progenitor cells was measured using flow cytometry at D12, and the result was shown in FIG. 8. It can be seen that the proportion of blood progenitor cells obtained can be significantly increased by optimizing the time point for changing to the differentiation medium of the third step; at later time points, the proportion of blood progenitor cells differentiated is low; the time point for changing the differentiation medium in the third step is preferably D3-D6.

Example 8

The invention discovers that the optimized EB inoculation time point can obviously improve the differentiation efficiency of NK cells.

In the case of EB differentiation using T25 flasks, other conditions were optimized as described in example 1, i.e., EBs were formed according to the above differentiation method, and EBs were removed on

days

4, 6, 8, 10, 12 and 14 of differentiation, inoculated into matrix protein-coated plates, and subsequently differentiated. The proportion of differentiated NK cells (CD56+) was measured two weeks after inoculation and the results are shown in FIG. 9.

Therefore, optimizing the EB inoculation time point can improve the differentiation efficiency of NK cells; NK cell differentiation efficiency was low for EBs at early stage (day 4) and EBs at late stage (day 14) at time point; the preferred EB inoculation time points should be D6-D12.

Example 9

The invention finds that optimizing the action concentration of BMP4 can obviously improve the proportion of blood progenitor cells.

In EB differentiation using T25 flasks, other conditions were performed as described in example 1, and the concentration of BMP4 in the first and second differentiation media was gradient optimized by treating the cells with BMP4 at 0ng/ml, 10ng/ml, 20ng/ml, 30ng/ml, 40ng/ml, 50ng/ml, 60ng/ml, 70ng/ml, 80ng/ml, 90ng/ml, or 100ng/ml, respectively, and measuring the CD34, which is an index of blood progenitor cells, using a flow cytometer at D12, as shown in FIG. 10. It can be seen that the addition of optimized BMP4 increases the efficiency of differentiation of blood progenitor cells; at lower concentrations, the differentiation rate of blood progenitor cells is low, and at higher concentrations, the differentiation of blood progenitor cells is inhibited; optimizing the concentration of BMP4 can improve the purity of blood progenitor cells at D12, namely, the proportion of CD34+ cells is improved; the concentration of BMP4 is preferably 10-50 ng/ml.

Example 10

The invention discovers that the proportion of the obtained blood progenitor cells can be obviously improved by optimizing the acting concentration of SB 431542.

In EB differentiation using T25 flask, other conditions were as described in example 1, and the concentration of SB431542 in the differentiation medium of the second step was gradient optimized by treating cells with 0. mu.M, 2. mu.M, 4. mu.M, 6. mu.M, 8. mu.M, 10. mu.M, 20. mu.M, 30. mu.M, 40. mu.M or 50. mu.M of SB431542, respectively, and measuring the CD34 index of blood progenitor cells using flow cytometry at D12, and the results are shown in FIG. 11.

It can be seen that the addition of optimized SB431542 increases the efficiency of differentiation of blood progenitor cells, but at higher concentrations, differentiation of blood progenitor cells is inhibited; optimizing SB431542 concentration increases the purity of blood progenitor cells at D6-12, i.e., increases the proportion of CD34+ cells; the preferable concentration of SB431542 is 0-20 μ M.

Example 11

The invention discovers that the proportion of the obtained blood progenitor cells can be obviously improved by optimizing the action concentration of the bFGF.

In EB differentiation using T25 flasks, other conditions were optimized by gradient as described in example 1 for bFGF concentration in the first, second and third differentiation media, i.e., cells were treated with bFGF at 0ng/ml, 0.1ng/ml, 0.5ng/ml, 1ng/ml, 2.5ng/ml, 5ng/ml, 10ng/ml or 20ng/ml, respectively, and the CD34 index of blood progenitor cells was determined by flow cytometry at D6-12, as shown in FIG. 12.

It can be seen that the addition of optimized bFGF increases the efficiency of differentiation of blood progenitor cells, but at higher concentrations, differentiation of blood progenitor cells is inhibited; the optimized bFGF concentration can improve the purity of the blood progenitor cells at D6-12, namely, the proportion of CD34+ cells is improved; the preferable concentration of bFGF is 0.1-5 ng/ml.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. a method for preparing natural killer cells, is characterized in that, comprises the following steps:

S1: form embryoid body;

S2: embryoid body differentiates into hematopoietic progenitor cells;

Wherein, the S2 includes:

S21: remove the embryoid body supernatant, add fresh first-step differentiation medium for cell culture;

Wherein, the first step differentiation medium is to add a small molecule GSK3β inhibitor to the basal differentiation medium, and at least one of the following components: BMP signaling pathway activator, insulin, IGF-1, VEGF, bFGF; GSK3β inhibitor It is CHIR99021 at a concentration of 0.5-20 μM; BMP signaling pathway activator is BMP4 at a concentration of 0-200 ng/mL; insulin, IGF-1, VEGF, bFGF at a concentration of 0-10 μg/mL, 0-100 ng/mL, respectively mL, 5-100 ng/mL and 0-100 ng/mL;

S22: Remove the first-step differentiation medium, and add the second-step differentiation medium for cell culture; wherein, the second-step differentiation medium is the addition of VEGF, bFGF, and at least one of the following components to the basal differentiation medium: BMP signaling pathway activator, Nodal inhibitor, insulin, IGF-1, IL3 and IL6; the concentrations of VEGF and bFGF are 5-100 ng/mL, 0-100 ng/mL and not 0, respectively; BMP signaling pathway is activated The inhibitor is BMP4 at a concentration of 0-200 ng/mL; the Nodal inhibitor is SB431542 at a concentration of 0-20 μM; insulin, IGF-1, IL3 and IL6 at a concentration of 0-10 μg/mL, 0-100 ng/mL, respectively , 0-20 ng/mL and 0-20 ng/mL;

S23: remove the second-step differentiation medium, add the third-step differentiation medium for cell culture, and obtain hematopoietic progenitor cells; wherein, the third-step differentiation medium is the addition of growth factors and colony-stimulating factors to the basal differentiation medium ;

The growth factor is selected from one or more of insulin, VEGF, IGF-1 and bFGF, wherein the concentrations of insulin, VEGF, IGF-1 and bFGF are 0.5-10 μg/mL, 5-100 ng/mL, 0-100ng/mL and 0.5-100 ng/mL; the colony stimulating factor is selected from one or more of TPO, SCF and Flt-3L; wherein TPO, SCF and Flt-3L, the concentrations are respectively 0-100ng /mL, 0-100 ng/mL and 0-100 ng/mL;

S3: hematopoietic progenitor cells differentiate into NK cells;

Wherein, in S3, the third-step differentiation medium is removed, the cells are inoculated into a cell culture container coated with matrix protein, and the fourth-step differentiation medium is added for cell culture;

The matrix protein includes at least one of Notch pathway activating protein or integrin; wherein, the fourth step differentiation medium is to add colony-stimulating factor and interleukin to the basal differentiation medium; the colony-stimulating factor is TPO, SCF, Flt-3L, and IL-3 at concentrations of 0-100 ng/mL, 5-100 ng/mL, 5-100 ng/mL, and 5-100 ng/mL, respectively; interleukins were IL-2, IL-7, and IL-15, at concentrations of 100-1000 IU/mL, 5-100 ng/mL, and 5-100 ng/mL, respectively;

S4: maturation and expansion of NK cells;

Wherein, the S4 includes: removing the fourth step differentiation medium, and adding the fifth step differentiation medium for cell culture; wherein, the fifth step differentiation medium is adding interleukin and promoting NK to the basal differentiation medium Substances for cell maturation and expansion;

The interleukin is selected from one or more of IL-2, IL-12, IL-18, IL-21, IL-27 and IL-15; One or more of human AB plasma, human platelet lysate, Vitamin A, nicotinamide, Vitamin E and Heparin; wherein IL-2, IL-12, IL-18, IL-21, IL-27 and IL-15 at 100-1000 IU/mL, 0-100 ng/mL, 0-100 ng/mL, 0-100 ng/mL, 0-100 ng/mL, and 5-100 ng/mL; human platelet lysate , Nicotinamide (NAM), Vitamin E and Heparin, the concentrations are 1-10%, 1-10mmol/L, 0-10 mg/mL and 0-100 μg/mL, respectively;

The above S2-S4 basal differentiation medium includes IMDM final concentration 50%, F-12 final concentration 50%, 0.2-20mg/ml rHSA, 0-1000 μM thioglycerol, 0-200 μg/ml ascorbic acid, 1-50 μg/ml ml transferrin, 1-50 ng/ml sodium selenite, 5-100 μM ethanolamine.

2. The method according to claim 1, wherein the S1 comprises:

S11: The cell suspension of human pluripotent stem cells is placed on a shaker and shaken overnight to form an embryoid body.

3. The method according to claim 1, wherein, in S3, the Notch pathway activating protein is selected from the group consisting of DLL1 recombinant protein, DLL4 recombinant protein, Jagged-1 recombinant protein, Jagged-2 recombinant protein and their variants. one or more of the body;

The integrin is selected from the group consisting of Fibronectin (fibronectin), Laminin (laminin), Vitronectin (vitronectin), MAdCAM-1 (adhesion cell adhesion molecule-1), VCAM-1 (vascular cell adhesion molecule- 1) and one or more of ICAM (Intercellular Adhesion Molecule) and their variants.

4. The method according to claim 1, further comprising step S5: freezing the differentiated NK cells using a cryopreservation solution; the cryopreservation solution comprises: sodium chloride, sodium dextrose, sodium acetate , potassium chloride, magnesium chloride, human albumin and DMSO.

5. A natural killer cell prepared by the method of any one of claims 1-4.

6 . A cell population characterized in that the natural killer cells of claim 5 are enriched. 7 .

7 . A medicament for preventing and/or treating tumors, characterized in that, the medicament comprises the natural killer cell according to claim 5 .