CN117887658A

CN117887658A - Method for differentiating human pluripotent stem cells into natural killer cells and application thereof

Info

Publication number: CN117887658A
Application number: CN202311330015.XA
Authority: CN
Inventors: 范靖; 王安欣; 任芳; 章薇垚
Original assignee: Zhejiang Huode Bioengineering Co ltd
Current assignee: Zhejiang Huode Bioengineering Co ltd
Priority date: 2022-10-14
Filing date: 2023-10-13
Publication date: 2024-04-16

Abstract

The present invention relates to a method for obtaining natural killer cells by differentiation culture using human pluripotent stem cells. The method comprises the step of suspension culture and uses a medium comprising a combination of specific chemical small molecules and cytokines. The invention also provides a cell population comprising a high proportion of natural killer cells obtained by the method, and uses of the method and cell product.

Description

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

Technical Field

The invention relates to the technical fields of stem cell biology and medical bioengineering, in particular to a method for differentiating human pluripotent stem cells into natural killer cells and a preparation method and application thereof.

Background

Pluripotent stem cells (pluripotent stem cells, PSCs) including Embryonic Stem Cells (ESCs) and Induced Pluripotent Stem Cells (iPSCs) are a class of cells with unlimited proliferation potential, differentiated into different cell tissues, and easy to genetically modify, and currently have become a hotspot for scientific research and drug development.

Natural killer cells (natural killer cell, NK cells) are the dominant force of the innate immune system and are widely available, such as peripheral blood, umbilical cord blood, embryonic stem cells, human pluripotent stem cells (hPSCs) and NK cell lines. NK cells from different sources have respective advantages and disadvantages in that primary NK cells can undergo a limited number of cell divisions, are difficult to expand into the large number of cells required for infusion, and different donor NK cells have large differences in quality, rendering it difficult to form standardized products; NK cells obtained by differentiation of hPSCs can provide stable and large-scale cells, and cell products can reach standards in terms of activity, cell phenotype, purity, sterility and the like. Therefore, the large-scale standardized preparation of NK cells from hPSCs in vitro is an effective solution way for NK cell product patent medicine, and has very important application prospect. Currently, NK cells are studied in hematological tumors including acute leukemia, lymphoma, multiple Myeloma (MM), etc., and in solid tumors including prostate cancer, ovarian cancer, pancreatic cancer, non-small cell lung cancer, etc. Results of studies have shown that NK cells are used independently of the occurrence of Cytokine Release Syndrome (CRS), neurotoxicity and phyto-host disease (GVHD), and that the levels of inflammatory cytokines (including IL-3, IL-6) are not increased.

However, the following problems exist in the NK cell system obtained by differentiating human pluripotent stem cells in the prior art: (1) Expensive commercial medium with undefined composition is used; (2) containing animal-derived components; (3) In the second and third steps of the method, matrigel or feeder cells are used, which is not beneficial to the expansion of production; (4) cell killing was weak.

Therefore, there is a need to develop a rapid and efficient NK cell differentiation method which is low in cost and easy for industrial mass production, thus providing a basis for NK cells suitable for clinical treatment.

Disclosure of Invention

The invention develops a method for generating NK cells from human pluripotent stem cells through induced differentiation, and the immune cells generated by the method have great advantages in clinical application and are specifically shown as follows: 1. low immune rejection, no graft versus host disease; 2. can quickly repair the bone marrow suppression phenomenon and restore the blood item to be normal; 3. the anti-radiation reaction capability is strong; 4. improving immunity and hematopoietic function; 5. the implantation rate in the receptor is high, and the focus homing is rapid; 6. intravenous injection is readily accepted by patients. Therefore, the immune cells generated by the invention can enter clinical use as soon as possible through stable mass production.

In a first aspect, the present invention provides a method for the induced differentiation of human pluripotent stem cells into NK cells using a combination of specific small molecule compounds and cytokines that is fast, efficient, low cost and easy for industrial large scale clinical production. The method comprises suspension culture of Embryoid Bodies (EBs) during induced differentiation.

In a second aspect, the present invention provides NK cells or cell cultures comprising NK cells differentiated by the method of the first aspect described above.

In a third aspect, the present invention provides NK cell differentiation medium and NK cell expansion medium for use in the method of the first aspect.

In a fourth aspect, the present invention provides the use of an NK cell or a cell culture comprising an NK cell or a derivative thereof as described in the second aspect above as a medicament.

In a fifth aspect, the present invention provides a genetically modified NK cell obtained from a genetically modified human pluripotent stem cell by the method of the first aspect.

In a sixth aspect, the invention provides a pharmaceutical composition comprising NK cells of the second aspect described above or a cell culture comprising NK cells.

The invention has at least the following advantages:

(1) The method of the present invention uses suspension culture during the induced differentiation period, and can be used for 1×10 in a short period (about 40 days) ⁵ Is greater than 1×10 from iPSCs ⁷ Is suitable for large-scale cell preparation production and clinical application.

(2) The method can be completed rapidly, efficiently and at low cost through the combined use of the specific small molecular compound and the cytokine, is simple and convenient to operate, has stable results, and is beneficial to large-scale NK production.

(3) The method of the present invention uses chemically defined media (chemically defined media), in particular without serum and feeder cells, nor matrigel such as DLL4, and is therefore suitable for expanded production and in particular for the production of NK cells for clinical applications.

(4) NK cells obtained by the method of the invention have high purity and ideal functionality, and CD56 in a final culture system ⁺ The NK cell proportion can reach more than 95%, and only contains a small amount of impurity cells.

(5) The method of the present invention suppresses differentiation of hematopoietic progenitor cells into mast cells and increases the proportion of NK precursor cells obtained by using specific inhibitors in the process of differentiating them into NK cells.

Drawings

FIG. 1 shows a schematic flow chart of the method 1 (example 1) of the present invention for preparing natural killer cells from Induced Pluripotent Stem Cells (iPSCs).

FIG. 2 shows a bright field plot of the process of method 1 of the invention from Induced Pluripotent Stem Cells (iPSCs) to mature NK cells (day 2, day 8, day 17, day 22, day 27 and day 58, respectively).

FIGS. 3A-3C show the differentiation efficiency of hematopoietic progenitor cells of step (3C) of the third stage of method 1 of the invention. Wherein, FIG. 3A shows the results of flow-through detection of Day 8 cells; FIG. 3B shows the open field plots of the cells of stage (3 c) of stage three (day 9, day 12, day 13, respectively); FIG. 3C shows qPCR detection results of Day 14 cells.

FIG. 4 shows the results of a flow assay of 1day 24 cells according to the method of the invention.

FIG. 5 shows the results of a flow assay of 1day 31 cells according to the method of the invention.

FIG. 6 shows the results of a flow assay of 1day 40 cells according to the method of the invention.

FIG. 7 shows the killing efficiency of NK cells prepared by the method 1 of the present invention against Raji cells.

FIGS. 8A-8B show the killing efficiency of NK cells prepared by the method 1 of the present invention against HeLa cells or MKN-7 cells. Wherein, FIG. 8A shows the killing efficiency of NK cells to Hela cells prepared by the method 1 of the present invention; FIG. 8B shows the killing efficiency of NK cells against MKN-7 cells prepared by the method 1 of the present invention.

FIG. 9 shows a bright field plot (day 2, day 8, day 17, day 22, day 27 and day 38, respectively) of the process of method 2 (example 2) of the invention from inducing differentiation of pluripotent stem cells (iPSCs) into mature NK cells.

FIG. 10 shows the results of a flow assay of 2day 13 cells of the method of the invention.

FIG. 11 shows the results of a flow assay of 2day 24 cells according to the method of the invention.

FIG. 12 shows the results of a flow assay of 2day 37 cells of the method of the invention.

FIG. 13 shows a bright field plot of the differentiation process of method 3 (example 3) of the invention starting from Induced Pluripotent Stem Cells (iPSCs) (day 2, day 8, day 14, respectively).

FIG. 14 shows the results of a flow assay of 3day 8 cells according to the method of the invention.

FIG. 15 shows the relative quantitative results of qPCR of cells treated for 12 days with further introduction of 6-thioinosine or AGN193109 based on method 1 in example 8.

Detailed Description

Unless specifically indicated in the present invention, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

"or" is defined herein as "and/or" unless explicitly indicated otherwise.

In the context of the present invention, unless otherwise indicated, "comprising," "including," and "containing" are to be construed as including the listed elements, e.g., a constituent, a feature, a step or a group thereof, but not excluding any other elements, e.g., other components, properties, and steps. As used herein, the term "comprising" or any variation thereof may be substituted with "comprising," "including," or "having" or synonymous variations. In certain embodiments, "comprising" also includes "consisting of … ….

As used herein, the term "about" is used to denote such values: the values include inherent differences in the apparatus, method used to determine the values, or differences that exist between study subjects.

Cell types at different stages

The term "embryonic stem cells" or "ESCs" as used herein refers to embryonic-derived pluripotent stem cells. The term "induced pluripotent stem cells" or "iPSCs" as used herein refers to pluripotent stem cells produced from reprogramming of differentiated somatic cells. In the context of the present invention, embryonic stem cells and induced pluripotent stem cells are collectively referred to as "pluripotent stem cells" or "PSCs". The methods of the invention may use ESCs or iPSCs as starting materials for directed differentiation. In a particular embodiment of the invention, ESCs or iPSCs are of mammalian origin, in particular of human origin. The invention is not limited to the source of stem cells, including ESCs and iPSCs, as long as they meet the requirements of clinical use. The cells may be directly from commercial sources or may be obtained from preparation, for example, by reprogramming somatic cells to iPSCs.

The term "embryoid bodies" or "EBs" as used herein refers to cell aggregates grown from ESCs and iPSCs by three-dimensional culture. EBs can be formed by suspension culture. However, it is difficult to obtain EBs of uniform size and shape in the manner of conventional culture of EBs.

"hematopoietic progenitor cells" or "HPCs" refer to cells that have the potential to differentiate into hematopoietic cells, but are not fully differentiated. Hematopoietic progenitor cells have self-replicating capacity and multipotent differentiation potential, including potential to differentiate into the myeloid and lymphoid lineages.

"NK cells" are "natural killer cells". As a cytotoxic lymphocyte, NK cells are a very important member of the innate immune system. Unlike the dependence of immune cells such as T cells on Major Histocompatibility Complex (MHC), NK cells can recognize stressed cells in the absence of MHC and antibodies, thereby inducing immune responses more rapidly, exhibiting killing effects. Changes in NK cell levels are often used as indicators for assessing and monitoring immune function, therapeutic efficacy, disease progression, particularly malignancy and viral infection.

NK cell surface expression CD56 in human blood can be used as marker of NK cells. Unlike NKT cells (natural killer T cells), human NK cells do not express CD3, so CD3 can be a marker for distinguishing between NKT and NK cells. Thus, the art will pass through CD3 ^- CD56 ⁺ To characterize and identify NK cells in blood.

The determination of whether a cell has differentiated or transformed into a particular type of cell can be made by immunostaining for protein markers that are specifically expressed in the particular cell type. Markers commonly used in neuromics are well known to those skilled in the art.

The term "NKP" or "NKPs" (NK Cell Precursors) refers to NK cell precursor cells. CD122 can be used as a cellular marker for detection of NKPs.

Culture method

The present invention provides a method for obtaining NK cells from human pluripotent stem cells, wherein NK cell differentiation medium and NK cell expansion medium comprising specific small molecule compounds and cytokine combination are used, and comprising a suspension culture step.

The various stages of the cultivation process according to the invention are described with reference to FIG. 1. It should be understood that the flow scheme shown in FIG. 1 is merely one representative example and that the methods of the present invention are not limited to the particular media or to the particular durations shown in FIG. 1.

In one aspect, the methods of the invention are distinguished by cell morphology and can be divided into:

(1) A pluripotent stem cell pre-culture stage;

(2) An Embryoid Body (EB) forming stage;

(3) A stage of differentiation from EB into hematopoietic progenitor cells;

(4) A stage of differentiation from hematopoietic progenitor cells into NK cells; and

(5) NK cell expansion phase.

In each of the above stages, a specific medium and culture conditions are used to complete the present invention.

(1) Pluripotent stem cell preculture stage

As the step (1), the pluripotent stem cell pre-culture stage aims to maintain and expand pluripotent stem cells such as iPSCs and ESCs as starting materials to a certain number. In this step, multipotency of pluripotent stem cells should be maintained without inducing differentiation.

In step (1) a pluripotent stem cell medium (hPSC medium) was used. The pluripotent stem cell medium may be selected and adjusted according to the type of pluripotent stem cells. In one embodiment, the pluripotent stem cells used are induced pluripotent stem cells and the medium used may be CTS-E8, teSR-AOF or other basal medium of similar efficacy.

The culture apparatus used in the culture in the step (1) may be an orifice plate, a petri dish, a culture flask, a cell factory or an automated closed reactor. The well plate may be any well plate such as a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, or a 96-well plate. In a preferred embodiment, the orifice plate is a low or ultra low adsorption orifice plate. For example, an ultra low adsorption 96 well plate or a low adsorption 6 well plate may be used.

The culture in the step (1) may be suspension culture or adherent culture. The suspension culture or the adherent culture may be carried out using a method well known to those skilled in the art.

In one embodiment, the method uses the method disclosed by CN113454230a to prepare iPSCs. In this case or the like, it is preferable to perform preculture for about 6 to 8 days, for example, preculture for about 7 days on the iPSCs. When the preculture is an adherent culture, it is preferable that the confluence of cells in the adherent culture is brought to about 70% to 90% by preculture for the subsequent step.

It should be understood that the preculture of the pluripotent stem cells in step (1) is not a core step of the present invention, and the present invention may use pluripotent stem cells cultured or obtained in any manner to perform the subsequent steps. For example, the pluripotent stem cells may be commercially available or prepared, for example, by reprogramming methods. The pluripotent stem cells may be embryonic stem cells or induced pluripotent stem cells. The embryonic stem cells are stem cells isolated or obtained from human embryos that have never undergone fertilization for less than 14 days of in vivo development.

(2) Embryoid Body (EB) formation stage

As step (2), the EB formation phase aims at forming pluripotent stem cells into embryoid bodies for subsequent differentiation.

In step (2) EB medium is used, which comprises pluripotent stem cell medium as basal medium, and further comprises ROCK inhibitor (ROCK i). As described above with respect to step (1), the pluripotent stem cell medium may be CTS-E8, teSR-AOF or other basal medium of similar efficacy.

The ROCK inhibitors are a class of protein kinase inhibitors that inhibit the activity of rho-associated protein kinases (ROCK) belonging to the serine-threonine protein kinase family and prevent apoptosis of cells after digestion or resuscitation. ROCK is involved in regulating the shape and movement of cells by acting on the cytoskeleton, and also regulates immortalization and differentiation of cells. Exemplary ROCK inhibitors include, but are not limited to, Y-27632, thiazovivin, fasudil, AR122-86, Y27632H-1152, Y-30141, or pyrintegrin. For differentiation to give cells suitable for clinical use, it may be preferable to use clinical grade, preferably GMP grade, cGMP grade or CTS grade ^TM A grade ROCK inhibitor. These ROCK inhibitors are commercially available.

The culture apparatus used in the culture in the step (2) may be an orifice plate, a petri dish, a culture flask, a cell factory or an automated closed reactor. The well plate may be any well plate such as a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, or a 96-well plate. In a preferred embodiment, the orifice plate is a low or ultra low adsorption orifice plate. For example, an ultra low adsorption 96 well plate or a low adsorption 6 well plate may be used.

The culture in the step (2) is suspension culture. The suspension culture may be performed using means well known to those skilled in the art.

The seeding density of the cells at the beginning of step (2) can be adjusted depending on the culture vessel used. For example, it may be 1X 10 ⁵ -1×10 ⁷ Density per mL pluripotent stem cells are inoculated into EB medium, preferably 1×10 ⁵ -1×10 ⁶ Density per mL.

In a preferred embodiment, the pluripotent stem cells provided in step (1), such as induced pluripotent stem cells, are subjected to the culture of step (2) after being dispersed by mechanical or chemical means. The purpose of the enzymatic treatment is to separate or disperse the cells from the culture surface. In a preferred embodiment, such separation or dispersion using digestive enzymes enables the cells that would otherwise have been packed together to be dispersed into more uniform individual discrete cells, as compared to mechanical means. In particular embodiments, the enzyme may digest the cell colony or aggregate into discrete single cells. An exemplary enzyme may be Accutase, dispase, versene (EDTA) or TrypLE. In a preferred embodiment, the enzyme is CTS ^TM TrypLE ^TM Select enzyme. Preferably, the digestive enzyme or enzyme solution is clinical grade, GMP grade, cGMP grade or CTS ^TM The grade, or the digestive enzyme, is suitable for preparing cells for clinical use. The enzyme may be used in the doses recommended by its manufacturer.

In one embodiment of step (2), the following EB culture is performed using 96-well plates. The iPSCs are digested by CTS Tryple, resuspended in a pluripotent stem cell medium (i.e., EB medium) containing a ROCK inhibitor, and the resulting cell suspension is placed in a 37℃incubator for stationary culture to obtain EBs of relatively uniform size and morphology. Preferably, the pluripotent stem cell medium is CTS-E8. Preferably, the 96-well plate isLow adsorption 96 well plates. Preferably, the ROCK inhibitor is Y-27632. Preferably, the ROCK inhibitor such as Y-27632 is used at a concentration of about 10 μm. Preferably, the iPSCs are used in a 1×10 ratio ⁵ -1×10 ⁶ Per mL, more preferably 0.3X10 ⁶ -0.8×10 ⁶ Density inoculation per mL. Preferably, the incubation is for a period of 16-24 hours.

In one embodiment of step (2), the culture of EBs is performed using a 6-well plate as follows. The iPSCs are digested into single cells by Ackutase, resuspended in a pluripotent stem cell culture medium containing a ROCK inhibitor, and the obtained cell suspension is placed on a 3D shaker in a 37 ℃ incubator for shaking culture, so as to obtain EBs with uniform size and morphology. Preferably, the pluripotent stem cell medium is CTS-E8. Preferably, the 6-well plate is a low adsorption 6-well plate. Preferably, the ROCK inhibitor is Y-27632. Preferably, the ROCK inhibitor such as Y-27632 is used at a concentration of about 10 μm. Preferably, the cell density is 0.1X10 ⁶ -0.8×10 ⁶ /mL. Preferably, the rotation speed of the shaking table is 10-100rpm, more preferably 30-70rpm. Preferably, the incubation is for a period of 8-12 hours. After EB formation, the liquid was continuously changed for 4 days, and the EB was slowly increased. The medium used in the liquid exchange was CTS-E8. Preferably, the EB diameter reaches about 130-250mm before proceeding to the next stage of differentiation. For the following description, the shaker was recorded as Day2 after half-shaking for 4 days.

(3) Stage of differentiation from EB into hematopoietic progenitor cells

As step (3), EB induction was aimed at differentiating into hematopoietic progenitor cells. This step includes three subdivision steps ((3 a), (3B), (3C)), and different media (hematopoietic progenitor cell differentiation medium a, hematopoietic progenitor cell differentiation medium B, and hematopoietic progenitor cell differentiation medium C) are used, respectively.

Step (3 a) can be considered as further culturing of EB, thereby differentiating ipscs into mesoderm. Hematopoietic progenitor differentiation medium a is used in step (3 a).

In one embodiment, the hematopoietic progenitor cell differentiation medium a comprises a pluripotent stem cell medium as a basal medium, and further comprises CHIR99021, BMP4, and VEGF. CHIR99021 is a potent inhibitor of glycogen synthase kinase 3 (GSK 3), and functions as a Wnt activator. BMP4 refers to bone morphogenic protein 4, which is a BMP activator. VEGF refers to vascular endothelial growth factor. In one embodiment, the basal medium in hematopoietic progenitor cell differentiation medium A is CTS-E8. In one embodiment, the hematopoietic progenitor differentiation medium a contains about 1 to about 6 μΜ, preferably about 2.5 μΜ CHIR99021; about 5ng/mL to about 200ng/mL, preferably about 25ng/mL to about 100ng/mL, more preferably about 50ng/mL BMP4; and about 5ng/mL to about 300ng/mL, preferably about 25ng/mL to about 100ng/mL, more preferably about 50ng/mL VEGF.

The culture apparatus used in the culture in the step (3 a) may be an orifice plate, a petri dish, a culture flask, a cell factory or an automated closed reactor. The well plate may be any well plate such as a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, or a 96-well plate. In a preferred embodiment, the orifice plate is a low or ultra low adsorption orifice plate. For example, an ultra low adsorption 96 well plate or a low adsorption 6 well plate may be used.

The number of EBs per culture vessel at the beginning of step (3 a) can be adjusted depending on the culture vessel used. For example, where a 6-well plate is used, the inoculation can be performed in an amount of about 12 to 40 EBs per well. In another embodiment, the culture may also be continued directly using the plates of the previous step without transferring the EBs.

Step (3 a) generally lasts about 22 to 48 hours, preferably about 1 day.

The purpose of the step (3 b) is to further differentiate mesodermal cells obtained by the differentiation of the step (3 a) into hematogenic endothelial cells. In step (3B), hematopoietic progenitor differentiation medium B is used, which comprises CTS-E6 or a medium having similar differentiation promoting effect as a basal medium, and further comprises SCF, SB431542 and VEGF. SCF is a stem cell factor. SB431542 is a selective TGF-beta receptor inhibitor. VEGF refers to vascular endothelial growth factor. In one embodiment, the basal medium in hematopoietic progenitor cell differentiation medium B is CTS-E6. In one embodiment, the hematopoietic progenitor differentiation medium B contains about 5 to 200ng/mL, preferably about 25 to 100ng/mL, more preferably about 30ng/mL SCF; SB431542 in the range of about 1 to 10. Mu.M, preferably about 2 to 6. Mu.M, more preferably about 3. Mu.M; and about 5 to 300ng/mL, preferably about 20 to 60ng/mL, more preferably about 40ng/mL VEGF.

The culture apparatus used in the culture in the step (3 b) may be selected from a low-adsorption culture plate, a culture flask or an automated closed reactor. The culture plate may be any multi-well plate, such as a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, or a 96-well plate. For example, an ultra low adsorption 96 well plate or a low adsorption 6 well plate may be used.

The culture in the step (3 b) is suspension culture.

The culture in the step (3 b) may be performed in the culture vessel used in the step (3 a) or may be performed in a new culture vessel.

Step (3 b) generally lasts about 46 to 74 hours, preferably about 3 days.

The purpose of step (3 c) is to promote differentiation of the cells into hematopoietic progenitor cells. In step (3C), hematopoietic progenitor differentiation medium C is used, which comprises Stemline II or a similar medium capable of promoting hematopoietic progenitor expansion as a basal medium, and further comprises SCF, FLT3L and TPO. SCF is a stem cell factor. FLT3L refers to the ligand of FLT3 (Fms-like tyrosine kinase 3), a member of the tyrosine kinase receptor family, which promotes human hematopoietic progenitor cell differentiation in the present invention. TPO refers to thrombopoietin. In one embodiment, the basal medium in hematopoietic progenitor differentiation medium C is Stemline II. In one embodiment, the hematopoietic progenitor differentiation medium C contains about 5 to 200ng/mL, preferably about 20 to 100ng/mL, more preferably about 40ng/mL SCF; about 5 to 200ng/mL, preferably about 20 to 100ng/mL, more preferably about 40ng/mL of FLT3L; and about 5 to 200ng/mL, preferably about 20 to 100ng/mL, more preferably about 50ng/mL of TPO.

The culture apparatus used in the culture in the step (3 c) may be selected from a low-adsorption culture plate, a culture flask or an automated closed reactor. The culture plate may be any multi-well plate, such as a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, or a 96-well plate.

The culture in the step (3 c) is suspension culture.

The culture in the step (3 c) may be performed in the culture vessel used in the step (3 b) or may be performed in a new culture vessel.

Step (3 c) generally lasts about 5 to 12 days, for example about 10 days. Preferably, the half-change is performed every 2 to 3 days.

During step (3), successful differentiation of hematopoietic progenitor cells can be confirmed by detecting hematopoietic progenitor cell specific markers. Representative markers may use, for example, CD34. For example, the proportion of CD34 positive cells in culture is detected by flow cytometry. The detection may be performed after a period of time from the beginning of step (3), in particular after a period of time from the beginning of step (3 c), for example after 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or more from the beginning of step (3 c). For example, the detection is performed when a medium change is performed. In a preferred embodiment, the percentage of CD34 positive hematopoietic progenitor cells determined by the assay is between 40% and 80% of the total cells. Optionally, after confirming the presence of CD34 positive hematopoietic progenitor cells and reaching the desired ratio, the culture may be continued for a period of time, i.e., step (3 c) is continued for a period of time.

(4) Stage of differentiation from hematopoietic progenitor cells into NK

As the step (4), induction differentiation of hematopoietic progenitor cells into NK cells is aimed at.

NK cell differentiation medium containing DMEM or the like as a basal medium and further containing SCF, FLT3L, IL-7, IL-15, immune Cell Serum Replacement was used in the step (4). In one embodiment, the basal medium in the NK cell differentiation medium is DMEM. In one embodiment, the NK cell differentiation medium contains about 5 to 200ng/mL, preferably about 20 to 100ng/mL, more preferably about 40ng/mL SCF; about 5 to 200ng/mL, preferably about 20 to 100ng/mL, more preferably about 40ng/mL of FLT3L; about 5 to 200ng/mL, preferably about 20 to 100ng/mL, more preferably about 40ng/mL IL-7; about 5 to 200ng/mL, preferably about 20 to 100ng/mL, more preferably about 40ng/mL IL-15. In one embodiment, the NK cell differentiation medium contains 10% Immune Cell Serum Replacement.

The culture apparatus used in the culture in the step (4) may be selected from a low-adsorption culture plate, a culture flask or an automated closed reactor. The culture plate may be any multi-well plate, such as a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, or a 96-well plate. For example, an ultra low adsorption 96 well plate or a low adsorption 6 well plate may be used.

The culture in the step (4) is suspension culture.

The culture in the step (4) may be performed in the culture vessel used in the step (4), or may be performed using a new culture vessel.

Step (4) generally lasts about 20 to 30 days, for example about 22 to 28 days, for example about 24 days. Preferably, the half-change is performed every 3 to 5 days.

At the end of step (4), harvesting a fraction containing high CD3 ^- CD56 ⁺ Is a NK cell of (C). NK cells exhibit an elongated morphology.

In particular, conventional NK differentiation methods add DLL4 or to promote differentiation of hematopoietic progenitor cells into NK cells by co-culturing with trophoblast cells. However, the introduction of DLL4 or trophoblast cells increases costs, which is disadvantageous for expanded production.

In a preferred embodiment, the culturing of step (4) may comprise the use of a C/EBP alpha inhibitor and/or a RAR alpha inhibitor to inhibit differentiation of hematopoietic progenitor cells into a myeloid lineage, e.g., mast cells. For example, the C/ebpα inhibitor and/or rarα inhibitor may be added to the NK differentiation medium used in step (4). Inhibition of myeloid differentiation can be manifested by a decrease in the ratio of myeloid cells, e.g., mast cells, in culture products and/or an increase in the ratio of lymphoid cells, e.g., NK of interest and NK precursor cells (NKPs), in culture products. The cell duty cycle of the different types can be determined by cell markers corresponding to the types of cells. These cell markers are known to those skilled in the art. In a preferred embodiment, by adding a C/ebpα inhibitor and/or a rarα inhibitor in step (4), the expression level of NK precursor cell markers or NK markers can be increased by at least 10-fold, preferably by at least 50-fold, more preferably by at least 100-fold after a period of time after adding the inhibitor and before the end of step (4), compared to the case where either of them is not added; and/or the expression level of myeloid cells, such as mast cell markers, is reduced to less than 1/10, preferably to less than 1/20, more preferably to less than 1/50, even more preferably to less than 1/100.

The C/EBP alpha inhibitor refers to an agent capable of inhibiting the transcriptional activity of C/EBP alpha. Examples of such C/EBP alpha inhibitors include, but are not limited to, fucosterol (Fucosterol), agrimophol B (Agrimol B), and 6-Thioinosine (6-Thioinosine). In a more preferred embodiment, the C/EBP alpha inhibitor is 6-thioinosine. The C/ebpα inhibitor, in particular 6-thioinosine, may be used at a concentration of 10 to 30 μm, preferably about 20 μm.

The rarα inhibitor refers to an agent capable of inhibiting the transcriptional activity of rarα. Examples of such rarα inhibitors include, but are not limited to, AGN192870, AGN193109, and LE135. In a more preferred embodiment, the rarα inhibitor is AGN193109. The rarα inhibitor, particularly AGN193109, may be used at a concentration of 5-20nM, preferably about 10 nM.

In some embodiments, the C/ebpα inhibitor and/or the rarα inhibitor may be used during part of the incubation time of step (4), or may be used throughout the incubation of step (4). For example, the C/ebpα inhibitor and/or rarα inhibitor is added to NK cell differentiation medium during a part of the time in step (4), e.g. from day 5, day 6, day 7, day 8 or later in step (4), preferably from day 8 in step (4). When the two inhibitors are used simultaneously, the C/ebpα inhibitor and/or the rarα inhibitor may be added sequentially or simultaneously. For convenience of operation, simultaneous addition may be preferable. The use of the C/ebpa inhibitor and/or rarα inhibitor may be continued until the end of step (4) or for a certain period of time. In one embodiment, in step (4), the cells are cultured in NK cell differentiation medium containing the C/EBP alpha inhibitor and/or the RAR alpha inhibitor for at least 5 days, e.g. 5-25 days, preferably 10-15 days, e.g. about 12 days.

(5) NK amplification stage

As the step (5), NK cells are further expanded.

In step (5), NK cell expansion medium containing DMEM or the like as a basal medium and further containing various interleukins (IL-2, IL-15 and IL-21) and Immune Cell Serum Replacement is used.

In one embodiment, the basal medium of the NK cell expansion medium is DMEM. In one embodiment, the NK cell expansion medium contains IL-2, IL-15 and IL-21. In one embodiment, the NK cell differentiation medium contains one or more of the following: about 20 to 300ng/mL, preferably about 20 to 50ng/mL, more preferably about 30ng/mL IL-2; about 5 to 200ng/mL, preferably about 20 to 50ng/mL, more preferably about 30ng/mL IL-15; about 5 to 200ng/mL, preferably about 20 to 50ng/mL, more preferably about 30ng/mL IL-21. In one embodiment, the NK cell differentiation medium contains 10% Immune Cell Serum Replacement.

The culture apparatus used in the culture in the step (5) may be selected from a low-adsorption culture plate, a culture flask or an automated closed reactor. The culture plate may be any multi-well plate, such as a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, or a 96-well plate. For example, an ultra low adsorption 96 well plate or a low adsorption 6 well plate may be used.

The culture in the step (5) is suspension culture.

The culture in the step (5) may be performed in the culture vessel used in the step (5), or may be performed using a new culture vessel.

Step (5) generally lasts about 10-25 days, preferably 12-20 days, more preferably about 14 days. Preferably, the half-change is performed every 4 to 6 days.

The application also relates to the following:

1. a method of differentiating human pluripotent stem cells (hPSCs) into hematopoietic progenitor cells, the method comprising the steps of:

(1) Providing a pluripotent stem cell;

(2) Culturing the pluripotent stem cells of step (1) to form Embryoid Bodies (EBs);

(3a) Culturing the EB obtained in step (2) in hematopoietic progenitor cell differentiation medium a, wherein the factor combination of hematopoietic progenitor cell differentiation medium a comprises or consists of CHIR99021, BMP4 and VEGF;

(3b) Culturing the cells cultured in step (3 a) in hematopoietic progenitor cell differentiation medium B, wherein the hematopoietic progenitor cell differentiation medium B comprises or consists of SCF, SB431542, VEGF in combination with a factor; and

(3c) Culturing the cells cultured in step (3 b) in hematopoietic progenitor cell differentiation medium C, wherein the hematopoietic progenitor cell differentiation medium C comprises or consists of TPO, SCF, and FLT3L in combination with a factor.

2. A method of differentiating human pluripotent stem cells (hPSCs) into NK cells, the method comprising the steps of:

(1) Providing a pluripotent stem cell;

(3a) Culturing the EB obtained in step (2) in hematopoietic progenitor cell differentiation medium a, wherein the combination of factors of hematopoietic progenitor cell differentiation medium a comprises CHIR99021, BMP4, and VEGF;

(3b) Culturing the cells cultured in step (3 a) in hematopoietic progenitor cell differentiation medium B, wherein the hematopoietic progenitor cell differentiation medium B comprises a combination of factors comprising SCF, SB431542, VEGF; and

(3c) Culturing the cells cultured in step (3 b) in hematopoietic progenitor cell differentiation medium C, wherein the hematopoietic progenitor cell differentiation medium C comprises a combination of factors comprising TPO, SCF, and FLT3L; and

(4) Culturing the hematopoietic progenitor cells obtained in step (3) in an NK cell differentiation medium to differentiate into NK cells.

3. The method of item 2, further comprising:

(5) Culturing the NK cells obtained in the step (4) in an NK cell expansion medium to expand the NK cells.

4. The method of any one of claims 1-3, wherein the basal medium of hematopoietic progenitor cell differentiation medium a is CTS-E8 medium.

5. The method of any one of claims 1-4, wherein the basal medium of hematopoietic progenitor cell differentiation medium B is CTS-E6 medium.

6. The method of any one of claims 1-5, wherein the basal medium of hematopoietic progenitor cell differentiation medium C is a Stemline II medium.

7. The method of any one of claims 1-6, wherein the hematopoietic progenitor differentiation medium a has a CHIR99021 concentration of 1 to 6 μm, BMP4 concentration of 5ng/mL to about 200ng/mL, and/or VEGF concentration of 5ng/mL to about 300ng/mL.

8. The method of item 7, wherein the hematopoietic progenitor differentiation medium A has a concentration of CHIR99021 of 2.5. Mu.M, a concentration of BMP4 of 50ng/mL, and/or a concentration of VEGF of 50ng/mL.

9. The method of any one of claims 1-8, wherein the hematopoietic progenitor cell differentiation medium B has a SCF concentration of 5 to 200ng/mL, a SB431542 concentration of 1 to 10 μm, and/or a VEGF concentration of 5ng/mL to about 300ng/mL.

10. The method of item 9, wherein the hematopoietic progenitor cell differentiation medium B has a concentration of SCF of 30ng/mL, a concentration of SB431542 of 3 μΜ, and/or a concentration of VEGF of 40ng/mL.

11. The method of any one of claims 1-10, wherein the hematopoietic progenitor differentiation medium C has a SCF concentration of 5 to 200ng/mL, FLT3L concentration of 5 to 200ng/mL, and/or TPO concentration of 5 to about 200ng/mL.

12. The method of item 11, wherein the hematopoietic progenitor differentiation medium C has a SCF concentration of 40ng/mL, a FLT3L concentration of 40ng/mL, and/or a TPO concentration of 50ng/mL.

13. The method of any one of claims 1-12, wherein the hematopoietic progenitor differentiation medium a is comprised of CTS-E8 medium supplemented with CHIR99021 at a concentration of 2.5 μm, BMP4 at a concentration of 50ng/mL, and VEGF at a concentration of 50ng/mL.

14. The method of any one of claims 1-13, wherein the hematopoietic progenitor differentiation medium B is comprised of CTS-E6 medium supplemented with SCF at a concentration of 30ng/mL, SB431542 at a concentration of 3 μm, and VEGF at a concentration of 40 ng/mL.

15. The method of any one of claims 1-14, wherein the hematopoietic progenitor differentiation medium C is composed of Stemline II medium supplemented with SCF at a concentration of 40ng/mL, FLT3L at a concentration of 40ng/mL, and TPO at a concentration of 50ng/mL.

16. The method of any one of claims 1-15, wherein step (1) comprises culturing the pluripotent stem cells in a pluripotent stem cell medium.

17. The method of item 16, wherein the pluripotent stem cell medium is selected from the group consisting of CTS-E8 and TeSR-AOF.

18. The method of any one of claims 1-17, wherein step (2) comprises culturing the pluripotent stem cells in EB medium to form Embryoid Bodies (EBs), the EB medium being a pluripotent stem cell medium comprising a ROCK inhibitor.

19. The method of item 18, wherein the ROCK inhibitor is Y-27632.

20. The method of claim 18 or 19, wherein the ROCK inhibitor is present in the medium at a concentration of 10 μm.

21. The method according to any one of claims 1 to 20, wherein the culture in step (2) is a suspension culture.

22. The method of any one of claims 1-21, wherein step (3 a) is continued for about 22-48 hours.

23. The method according to any one of claims 1 to 22, wherein the culture in step (3 a) is a suspension culture.

24. The method of any one of claims 1-23, wherein step (3 b) is continued for about 46-74 hours.

25. The method of any one of claims 1 to 24, wherein the culturing in step (3 b) is suspension culturing.

26. The method of any one of claims 1-25, wherein step (3 c) is continued for about 5-12 days.

27. The method of any one of claims 1 to 26, wherein the culturing in step (3 c) is suspension culturing.

28. The method of any one of claims 2-27, wherein the combination of factors in NK cell differentiation medium used in step (4) comprises SCF, FLT3L, IL-7, and IL-15.

29. The method of item 28, wherein the SCF concentration is 5 to 200ng/mL, the FLT3L concentration is 5 to 200ng/mL, the IL-7 concentration is 5 to 200ng/mL, and/or the IL-15 concentration is 5 to 200ng/mL.

30. The method of item 29, wherein the SCF concentration is 40ng/mL, the FLT3L concentration is 40ng/mL, the IL-7 concentration is 40ng/mL, and/or the IL-15 concentration is 40ng/mL.

31. The method of any one of claims 2-30, wherein the NK cell differentiation medium used in step (4) comprises Immune Cell Serum Replacement.

32. The method of any one of items 2 to 31, wherein the NK cell differentiation medium used in the step (4) has a composition of DMEM medium supplemented with SCF at a concentration of 40ng/mL, FLT3L at a concentration of 40ng/mL, IL-7 at a concentration of 40ng/mL, IL-15 at a concentration of 40ng/mL, and Immune Cell Serum Replacement at 10%.

33. The method according to any one of items 2 to 32, wherein the step (4) is suspension culture.

34. The method of any one of items 2-33, wherein step (4) is continued for about 20-30 days.

35. The method of any one of claims 2-33, wherein a C/ebpa inhibitor and/or a rara inhibitor is used in step (4), e.g., added to NK cell differentiation medium.

36. The method of item 35, wherein the C/ebpα inhibitor is Fucosterol, agrimol B, or 6-thioinosine.

37. The method of item 35, wherein the rarα inhibitor is AGN192870, AGN193109, or LE135.

38. The method of item 36, wherein the C/ebpα inhibitor is 6-thioinosine.

39. The method of item 37, wherein the rarα inhibitor is AGN193109.

40. The method of claim 38, wherein the concentration of 6-thioinosine in the NK cell differentiation medium is 10 to 30 μΜ, preferably about 20 μΜ.

41. The method of claim 38, wherein the concentration of AGN193109 in the NK cell differentiation medium is 5 to 20nM, preferably about 10nM.

42. The method of any one of claims 35-41, wherein the C/ebpa inhibitor and/or rara inhibitor is used part of the time in step (4).

43. The method of item 42, wherein the portion of time starts on day 5, day 6, day 7, day 8 or 9 days below and/or lasts for 5-25 days, preferably 10-20 days, e.g. 12 days of step (4).

44. The method of any one of claims 3-43, wherein the combination of factors in the NK cell expansion medium used in step (5) comprises IL-2, IL-15 and IL-21.

45. The method of item 44, wherein the IL-2 concentration is 20 to 300ng/mL, the IL-15 concentration is 5 to 200ng/mL, and/or the IL-21 concentration is 5 to 200ng/mL.

46. The method of item 45, wherein the IL-2 concentration is 30ng/mL, the IL-15 concentration is 30ng/mL, and/or the IL-21 concentration is 30ng/mL.

47. The method of any one of claims 2-46, wherein the NK cell expansion medium used in step (5) comprises Immune Cell Serum Replacement.

48. The method of any one of items 2-47, wherein the NK cell expansion medium used in the step (5) has a composition of DMEM supplemented with 30ng/mL of IL-2, 30ng/mL of IL-15, 30ng/mL of IL-21 and 20% of Immune Cell Serum Replacement.

49. The method of any one of claims 2-48, wherein DLL4 and/or feeder cells are not used.

50. The method of any one of claims 1-49, wherein during step (3) the proportion of CD34 positive cells reaches between 40% and 80%.

51. The method of any one of claims 1-50, wherein the human pluripotent stem cells are genetically modified.

52. The method of item 51, wherein the genetic modification causes the human pluripotent stem cell to express one or more elements selected from the group consisting of: chimeric Antigen Receptor (CAR), immunogenicity-related element, or element that enhances cell proliferation and activity (IL 15).

53. A hematopoietic progenitor cell or a population of cells comprising hematopoietic progenitor cells obtained by the method of any one of claims 1-52.

54. The population of cells comprising hematopoietic progenitor cells of item 53, wherein the proportion of CD34 positive cells is between 40% and 80%.

55. An NK cell or a population of cells comprising NK cells obtained by the method of any one of claims 2-52.

56. The NK cell-containing cell population of item 55, having one or more of the following characteristics:

CD45 positive cells reach more than 80%;

CD56 positive cells reached more than 70%; and/or

CD3 positive cells were less than 3%.

57. A cell preparation comprising the hematopoietic progenitor cell or the cell population comprising hematopoietic progenitor cells of item 53 or 54, or the NK cell or the cell population comprising NK cells of item 55 or 56.

58. Use of the hematopoietic progenitor cell or cell population comprising hematopoietic progenitor cells of item 53 or 54 in the manufacture of a medicament for cell therapy.

59. Use of the hematopoietic progenitor cell or cell population comprising hematopoietic progenitor cells of item 53 or 54 for non-therapeutic purposes.

60. The use of claim 59, wherein the hematopoietic progenitor cells or a population of cells comprising hematopoietic progenitor cells are used for cell differentiation.

61. The use of item 60, wherein the hematopoietic progenitor cells or a population of cells comprising hematopoietic progenitor cells are used for T cell, B cell, NKT cell, or NK cell differentiation.

62. Use of the cell or cell population of clause 55 or 56 in the manufacture of a medicament for cell therapy.

63. The use of claim 62, wherein the cell therapy is for the treatment of an autoimmune disease, a tumor, or a viral infection.

64. The use of claim 63, wherein the tumor is a hematological tumor or a solid tumor.

65. The use of claim 63, wherein the viral infection is HIV, RSV, EBV, CMV, adenovirus or BK polyomavirus associated infection.

66. Use of the NK cell or NK cell-containing cell population of clause 55 or 56 for non-therapeutic purposes.

Examples

For a more complete understanding and appreciation of the invention, the invention will be described in detail below with reference to the examples and drawings, which are only intended to illustrate the invention and are not intended to limit the scope of the invention. The scope of the invention is defined in particular by the appended claims.

EXAMPLE 1 differentiation method 1 of pluripotent Stem cells into Natural killer cells

As one embodiment of the present invention, the present example provides a method of differentiating human pluripotent stem cells (hPSCs) into NK cells. In general terms, the differentiation method comprises the following stages or steps:

(1)pluripotent stem cell preculture:providing pluripotent stem cells, for example, culturing pluripotent stem cells in a pluripotent stem cell medium CTS-E8 to a confluency of 70% to 90%;

(2)EB formation:the pluripotent stem cells in step (1) are formed into Embryoid Bodies (EBs) with relatively uniform size and morphology according to any one of the following methods:

the pluripotent stem cells were cultured at 7X 10 ⁵ Inoculating the density of individual cells/mL to a 96-hole cell pore plate with low adsorption, using an EB culture medium and placing the culture medium in a 37 ℃ incubator for standing for 40-48 hours; or (b)

Inoculating the pluripotent stem cells to a 6-hole cell pore plate with low adsorption at a certain density, and shaking and culturing the pluripotent stem cells on a shaking table in a 37 ℃ incubator by using an EB culture medium for 12-24 hours;

(3)differentiation of hematopoietic progenitor cells:culturing the EB obtained in step (2) in one or more hematopoietic progenitor cell differentiation media to committed differentiate into Hematopoietic Progenitor Cells (HPCs);

(4)differentiation of hematopoietic progenitor cells to NK:culturing the hematopoietic progenitor cells obtained in step (3) in NK cell differentiation medium to differentiate into NK cells; and

(5)NK cell expansion:culturing the NK cells obtained in the step (4) in an NK cell expansion medium to further amplify NK cells.

The individual steps of the differentiation method are described in detail below.

The first stage: pluripotent stem cell preculture

The first stage or step (1) is the stage shown in Day-7 to Day0 in fig. 1. Human iPSCs were prepared by the method disclosed in patent application CN113454230a and subjected to strict pluripotency verification (confirming that the desired pluripotency marker is expressed and that teratomas comprising inner, middle and outer three germ layers can be formed in immunodeficient mice).

The iPSCs are cultured in CTS-E8 which is used as a pluripotent stem cell culture medium, and the confluence reaches 70% -90% under the condition of adherence.

And a second stage: EB formation

The second stage or step (2) is Day0 to Day2 in fig. 1 (for the sake of unification of the following descriptionSecond stage/step (2)The time after the end is noted as stage shown in Day 2). In this stage, well-undifferentiated ipscs were digested and cultured using a multipotent stem cell medium containing ropki to form uniform EBs.

As described above, the present stage of cultivation was performed in accordance with the following procedure (1) or (2) using iPSC cultivated to a confluence of 70 to 90%.

(1) Specific operation of 96 well plate: ipscs were digested with CTS Tryple, resuspended in iPSC maintenance medium, and 10 μm of the Rocki inhibitor Y-27632 was added to medium CTS-E8. The cell density was set to 0.3X10 ⁶ -0.8×10 ⁶ Placing the/mL cell suspension in a 37 ℃ incubator for static culture for 16-24 hours; at the end of the culture, EBs with relatively uniform sizes and morphologies were obtained.

(2) Specific operation of 6 well plate: iPSC single cells were digested with Accutase, resuspended in iPSC maintenance medium, and 10 μm of the Rocki inhibitor Y-27632 was added to the medium. The cell density was set to 0.1X10 ⁶ -0.8×10 ⁶ placing/mL cell suspension on a 3D shaking table in a 37 ℃ incubator for shaking culture, wherein the rotation speed of the shaking table is 30-70rpm; the shaking culture time is 8-12h; at the end of the culture, EBs with relatively uniform sizes and morphologies were obtained. After EB formation, the medium used was CTS-E8 after 4 days of continuous half-change until EB slowly increased.

And a third stage: differentiation of hematopoietic progenitor cells (Day 2-Day 16)

At this stage, EB is cultured as hematopoietic progenitor cells. This stage is divided into three steps, three different media being used respectively.

Step (3 a)

Step (3 a) is the stage shown in Day2 to Day3 in fig. 1.

In step (3 a), the hematopoietic progenitor differentiation medium A uses CTS-E8 as a basal medium, and CHIR99021, BMP4, VEGF are added, wherein the concentration of CHIR99021 is 2.5 mu M, the concentration of BMP4 is 50ng/mL, and the concentration of VEGF is 50ng/mL.

As in the previous stage, the cultivation in this stage can be performed by two operations, the numbers of which correspond to the previous stage.

(1) EBs formed in the previous stage are transferred from an ultra-low adsorption 96-well plate to a low adsorption or non-low adsorption 6-well plate, and the quantity of the EBs in each well is 12-40. The plates were tilted to position the EBs at the bottom and after the supernatant was aspirated, hematopoietic progenitor differentiation medium A was added.

(2) The low-attachment 6-well plate was removed from the shaker in the incubator, the plate was tilted to tilt the EB to the bottom, and after the supernatant was aspirated, hematopoietic progenitor differentiation medium a was added.

In both protocols, cells were incubated at 37℃with 5% CO ₂ Culturing is carried out under conditions for about 22-26 hours, i.e., from Day2 to Day3.

Step (3 b)

Step (3 b) is the stage shown in Day3 to Day6 in fig. 1.

At the beginning of this step, the culture apparatus that can be used in the subsequent differentiation production step is selected from the group consisting of a low-adsorption culture plate, a culture flask, and an automated closed reactor.

The culture supernatant from the previous step was removed and 2mL of fresh hematopoietic progenitor differentiation medium B was added to each well of the 6-well plate. The hematopoietic progenitor cell differentiation medium B is obtained by adding growth factors and colony stimulating factors into a basal differentiation medium with the same efficacy as a basal differentiation medium CTS-E6 and the like. Specifically, the additive was SCF (Stem Cell Factor), SB431542, VEGF. SCF concentration was 30ng/mL, SB431542 concentration was 3. Mu.M, and VEGF concentration was 40ng/mL.

Said step (3 b) lasts about 70-74 hours, i.e. from Day3 to Day6.

Step (3 c)

Step (3 c) is the stage shown in Day6 to Day16 in fig. 1.

The plates containing EB were removed from the incubator, half of the supernatant was removed, and 3mL of freshly prepared hematopoietic progenitor differentiation medium C was added to each well of the 6-well plate. Wherein, the basic culture medium of the hematopoietic progenitor cell differentiation culture medium C is Stemline II, stemspan AOF or DMEM+HAMS-F12, and the additives are SCF, FLT3L and TPO. SCF concentration was 40ng/mL, FLT3L (Fms-related tyrosine kinase 3 ligand) concentration was 40ng/mL, TPO (Thrombopoietin) concentration was 50ng/mL. And then half liquid exchange is carried out every 2-3 days.

Fourth stage: differentiation of hematopoietic progenitor cells into NK

The fourth stage or step (4) is the stage shown in Day16 through Day40 of fig. 1.

In this stage, the medium was replaced with NK cell differentiation medium. The NK cell differentiation medium contained DMEM as a basal medium and contained 40ng/mL SCF, 40ng/mL FLT3L, 40ng/mL IL-7, 40ng/mL IL-15 and Immune Cell Serum Replacement at 10%. The specific operation is as follows.

Half of the old medium was removed and fresh NK cell differentiation medium was added. And then half liquid exchange is carried out every 3-5 days.

Fifth stage: NK cell expansion

The fifth stage or step (5) is the stage following Day40 in fig. 1.

In this stage, the medium was replaced with NK cell expansion medium. The NK cell expansion medium contained DMEM as a basal medium and contained 30ng/mL IL-2, 30ng/mL IL-15 and 30ng/mL IL-21 interleukins and 10% Immune Cell Serum Replacement. The specific operation is as follows.

Half of the old medium was removed and fresh NK cell expansion medium was added. And then, carrying out half liquid exchange every 4-6 days, and carrying out passage according to the cell number.

EXAMPLE 2 differentiation method 2 of pluripotent Stem cells into Natural killer cells

As one embodiment of the present invention, the present example provides a method for differentiating human pluripotent stem cells (hPSCs) into NK cells.

In the method of this embodiment, the first stage and the second stage are the same as those in embodiment 1.

And a third stage: differentiation of pluripotent stem cells into hematopoietic progenitor cells

At this stage, EB is cultured as hematopoietic progenitor cells. Unlike example 1, this stage was divided into two steps, two different media were used separately.

Step (3 a)

Unlike example 1, step (3 a) of this example has a duration of Day 2 to Day 5, i.e., the cells are exposed to 5% CO at 37℃ ₂ The culture was carried out for about 70-74 hours under the conditions, and different hematopoietic progenitor differentiation media A were used. The hematopoietic progenitor cell differentiation medium A was based on a combination of IMDM/HAMS-F12 and further contained 50ng/mL bFGF, 40ng/mL BMP4, 50ng/mL VEGF, 50 μg/mL LAA (L-Ascorbic Acid Free Acid Gamma-Irradiat/VC) and 5mg/mL HSA. The other conditions are the same as in step (3 a) of example 1, and can be performed in two ways in example 1.

Step (3 b)

Unlike example 1, step (3 b) of this example has a duration of Day 5 to Day 15.

The culture supernatant from the previous step was removed and 2mL of fresh hematopoietic progenitor differentiation medium B was added to the 6-well plate. The hematopoietic progenitor cell differentiation medium B is obtained by adding growth factors and colony stimulating factors into a basic differentiation medium Stemspan-AOF. Specifically, the additives are SCF, FLT3L, IL-3 and TPO. SCF concentration was 50ng/mL, FLT3L concentration was 20ng/mL, IL-3 concentration was 20ng/mL, TPO concentration was 30ng/mL. After which half-changing is performed every 2 days.

Said step (3 b) lasts about 238-242 hours, i.e. from Day5 to Day15.

Fourth stage: differentiation of hematopoietic progenitor cells into NK

The fourth stage or step (4) continues from Day15 to Day36. Unlike example 1, this stage was divided into two steps, two different media were used separately.

Step (4 a)

In this step, the medium was replaced with NK differentiation medium A. The basic culture medium of the NK differentiation culture medium A is CellGenix GMP SCGM, and the additives are IL-6, TPO, SCF, FLT L and Immune Cell Serum Replacement. IL-6 concentration was 25ng/mL, TPO concentration was 25ng/mL, SCF concentration was 30ng/mL, and FLT3L concentration was 50ng/mL. Immune Cell Serum Replacement concentration was 5%. After which half-changing is performed every 2 days.

The step (4 a) lasts for about 118-122 hours altogether, i.e. from Day15 to Day20.

Step (4 b)

In this step, the medium was replaced with NK differentiation medium B. The NK cell differentiation medium B comprises CellGenix GMP SCGM as a basal medium, and additives are SCF, FLT3L, IL-15, IL-7 and Immune Cell Serum Replacement. Wherein the concentration of SCF is 30ng/mL, the concentration of FLT3L is 50ng/mL, the concentration of IL-15 is 25ng/mL, the concentration of IL-7 is 50ng/mL, and the concentration of Immune Cell Serum Replacement is 5%. After which half-changing is performed every 2 days.

The step (4 b) lasts for about 382-386 hours, i.e. from Day20 to Day36.

Fifth stage: NK cell expansion

The fifth stage or step (5) is a stage after Day36.

In this stage, the medium was changed to NK cell expansion medium and the cell density was adjusted to 1X 10 ⁵ cells/mL. The NK cell expansion medium was based on SCGM and contained 80ng/mL IL-2, 80ng/mL IL-15 and 80ng/mL IL-21 interleukins and 20% Immune Cell Serum Replacement.

After that, the liquid is half changed every 4 to 6 days. Passaging was performed according to the number of cells.

EXAMPLE 3 differentiation method 3 of pluripotent Stem cells into Natural killer cells

Step (3 a)

Unlike example 1, step (3 a) of this example has a duration of Day2 to Day8, i.e., the cells are cultured for about 144 hours, and a different hematopoietic progenitor differentiation medium a is used. The hematopoietic progenitor cell differentiation medium A was based on CTS-E6 and further contained 50ng/mL SCF, 40ng/mL BMP4, 50ng/mL VEGF. The other conditions are the same as in step (3 a) of example 1, and can be performed in two ways in example 1.

Step (3 b)

Unlike example 1, step (3 b) of this example has a duration of Day8 to Day 15.

The culture supernatant from the previous step was removed and 3mL of fresh hematopoietic progenitor differentiation medium B was added to the 6-well plate. The hematopoietic progenitor cell differentiation medium B is obtained by adding growth factors and colony stimulating factors into a combined differentiation medium DMDM/HAMS-F12. Specifically, the additives are SCF, FLT3L, IL-3, IL-7, L15, LAA and Immune Cell Serum Replacement. SCF concentration was 50ng/mL, FLT3L concentration was 20ng/mL, IL-3 concentration was 20ng/mL, IL-15 concentration was 30ng/mL, LAA concentration was 20. Mu.g/mL, and Immune Cell Serum Replacement concentration was 15%. After which half-changing is performed every 2 days.

The step (3 b) lasts about 166-170 hours, i.e. from Day8 to Day15.

Fourth stage: differentiation of hematopoietic progenitor cells into NK

The fourth stage or step (4) continues from Day15 to Day45.

In this stage, the medium was replaced with NK cell differentiation medium. The NK cell differentiation medium comprises a combination of DMDM/HAMS-F12 as a basal medium, and additives are SCF, FLT3L, IL-15, IL-7 and Immune Cell Serum Replacement. Wherein the concentration of SCF is 20ng/mL, the concentration of FLT3L is 50ng/mL, the concentration of IL-7 is 25ng/mL, the concentration of IL-15 is 10ng/mL, the concentration of LAA is 20 mug/mL, and the concentration of Immune Cell Serum Replacement is 5%. After which half-changing is performed every 3 days.

Fifth stage: NK cell expansion

The fifth stage or step (5) is a stage after Day 45.

In this stage, the medium was changed to NK cell expansion medium and the cell density was adjusted to 1X 10 ⁵ cells/mL. The NK cell expansion medium takes RPMI1640 as a basic differentiation medium and contains 80ng/mL IL-2, 80ng/mL IL-15 and 80ng/mL IL-21 interleukins and 20% Immune Cell Serum Replacement.

EXAMPLE 4 characterization of the differentiation Process and final NK killing Capacity of example 1

Morphology observation and differentiation efficiency measurement were performed on the cells during the culture method of example 1, and morphology, differentiation efficiency and killing efficiency of the finally obtained cells were examined.

Day 2 to Day 58: morphological observation

The cells were examined for morphology during the differentiation culture, and the morphological observations are shown in FIG. 2. As can be seen in fig. 2, the cells gradually grew from round to bar, indicating that the cells differentiated from hematopoietic progenitor cells into NK cells.

Day 8 to Day 16: testing the differentiation efficiency of hematopoietic progenitor cells

The cell phenotype of the bleached single cell suspension differentiated using the Stemline ii medium, which was collected in step (3 c) of example 1, was examined using a flow cytometer to examine the content of hematopoietic progenitor cells expressing CD34 in the resulting cells. Wherein antibody information detected by the flow meter: PE Mouse Anti-Human CD34Clone 563 (RUO) (BD # 550761). The percentage of ideal CD34 positive HPC in total cells should be between 40% and 80%. As can be seen from FIG. 3A, detection of the specific index of Day 8 differentiation intermediate using flow cytometry showed hematopoietic progenitor cells (CD 34 ⁺ ) Higher levels (73.19%) have been reached.

With the same additives as in step (3 c) of example 1, comparative differentiation tests were performed with three basal media, stemline II, stemSpan AOF, DMEM+HAMS-F12, respectively. The plates were photographed under a biological microscope on days 9, 12, and 13 of differentiation, respectively, and as shown in the bright field chart of the differentiation results in fig. 3B, EB expansion was morphologically achieved by using all three basal media, indicating differentiation toward hematopoietic progenitor cells. The single cells that were obtained by the method of collecting the single cells on day 14 of differentiation were subjected to the procedure of total RNA extraction kit (OMEGA) to extract RNA, and then subjected to PrimeScript ^TM RT Master Mix (Takara) procedure reverse transcription to cDNA, finally using TB Green ^TM Premix Ex Taq ^TM II (Takara) qPCR was performed with detection indexes CD34, CD117 and CD122. As a result, as shown in FIG. 3C, the cells differentiated from these three basal media showed significant expression on CD34, a hematopoietic progenitor marker, compared to undifferentiated iPSCs, particularly StemSpan AOF, followed by Stemline II, and most followed by DMEM+HAMS-F12.

Day24 to Day 40: detection of NK differentiation efficiency

The cell phenotype was examined during Day24 to Day40 using a flow cytometer to examine the proportion of NK cells expressing CD45, CD56, CD3 in the resulting cells. The percentage of CD45 positive NK should be between 80% and 100% of total cells and the percentage of CD56 positive NK should be between 70% and 98% of total cells. Flow antibody information: FITC Mouse Anti-Human CD45 (BD # 555482), alexa Fluor 488Mouse Anti-Human CD56 (BD # 561905), PE Mouse Anti-Human CD3Clone UCHT1 (BD # 561808).

As shown in FIG. 4, the specificity index of Day 24 differentiation intermediate was detected by flow cytometry, respectively, showing that CD45 positive NK cells had reached 85% and CD56 positive NK cells had reached 8%.

As shown in FIG. 5, the specificity index of Day 31 differentiation intermediate was detected by flow cytometry, respectively, showing that 90% of CD 45-positive NK cells were reached and 31% of CD 56-positive NK cells were reached.

As shown in FIG. 6, the detection of Day40 specificity index by flow cytometry shows that CD56 positive NK cells have reached 97%, while CD3 positive rate is only 1.6%, which indicates that differentiated cells have almost no T cells and high purity.

⁺ Day40: detection of killing efficiency of NK cells

The killing efficiency of NK against different cells obtained by the method of example 1 was examined.

Killing efficiency against Raji cells

Raji cells (south modular organism #nm-B07-1) were compared to CD56 positive NK cells of example 1 according to 1: 5. 1: 10. 1:15 cells were incubated for 24h and Raji cells were examined for stable transfer of Luciferase fluorescence using One Glo Luciferase Assay System (Promega#E6110).

As shown in fig. 7, the results demonstrate that NK cells differentiated using the method of example 1 have the ability to kill Raji, and the killing efficiency exhibits a concentration dependence.

Killing efficiency against Hela cells

Hela cells (TCHu 187, proc. Natl. Acad. Sci.) were isolated from CD56 in example 1 ⁺ According to 1: 2. 1: 5. 1:10 cells were co-incubated in Axion ZHT real-time label-free analyzer well plates. Hela survival was continuously and dynamically detected using an Axion ZHT real-time label-free analyzer.

As shown in fig. 8A, the results demonstrate that the stronger and concentration-dependent the ability to kill Hela cells with increasing incubation time, the more potent the response in effector cells: target cells (E: T) =10: in the case of 1, the maximum killing rate is up to more than 90 percent.

Killing efficiency against MKN-7 cells

MKN-7 cells (Procell#CL-0574) were compared to CD56 of example 1 ⁺ According to 1: 2. 1: 5. 1:10 cells were incubated separately from the inoculation into Axion ZHT real-time label-free analyzer well plates. The MKN-7 survival rate was continuously and dynamically detected using an Axion ZHT real-time label-free analyzer.

As shown in fig. 8B, the results demonstrate that the stronger and concentration-dependent the ability to kill MKN-7 cells with increasing incubation time, the more potent the response in effector cells: target cells (E: T) =10: 1, the maximum killing rate of NK cells is up to more than 90%, and the maximum killing rate of NK92MI cells is up to more than 70%, which shows that under the effect target ratio, the NK cells have stronger killing ability to MKN-7 cells than the NK92MI cells.

Example 5 monitoring of the differentiation Process of example 2

Morphological observations and differentiation efficiency measurements were performed on cells during the culture method of example 2.

Day2 to Day 40: morphological observation

The results of morphological examination of cells during differentiation culture are shown in FIG. 9.

Day 8 to Day 14: testing the differentiation efficiency of hematopoietic progenitor cells

The single cell suspension thus rinsed was collected and examined for its cell phenotype using a flow cytometer to examine the content of hematopoietic progenitor cells expressing CD34 in the resulting cells. Wherein antibody information detected by the flow meter: PE Mouse Anti-Human CD34 Clone 563 (RUO) (BD # 550761).

As can be seen from FIG. 10, detection of the specific index of Day 13 differentiation intermediate in example 2 using a flow cytometer showed that hematopoietic progenitor cells (CD 34 ⁺ ) Only 16%, significantly less than example 1.

Day20 to Day 40: detection of NK differentiation efficiency

The morphology and number of single cells that were bleached were observed during Day20 to Day 40, and their cell phenotype was examined using a flow cytometer to examine the proportion of NK cells expressing CD45, CD56 in the resulting cells. Flow antibody information: FITC Mouse Anti-Human CD45 (BD # 555482), alexa Fluor 488Mouse Anti-Human CD56 (BD # 561905).

As shown in FIG. 11, the specificity index of Day 24 differentiation intermediate in example 2 was detected by a flow cytometer, respectively, to show CD45 ⁺ Has reached 82.8%, CD56 ⁺ Has reached 7.35%.

As shown in FIG. 12, the specific index of Day 37 differentiation intermediate in example 2 was detected by flow cytometry, respectively, to show CD45 ⁺ NK cells 82.5%, CD56 ⁺ Only 10.3% of NK cells. In addition, the positive rate of the target cells in the cell population produced by the method of example 2 cannot be increased with further differentiation, and the differentiation effect is significantly inferior to that of example 1, so that the subsequent expansion is stopped, and example 1 is preferred.

Example 6 monitoring of the differentiation Process of example 3

Morphological observations and differentiation efficiency measurements were performed on cells during the culture method of example 3.

Day 2～Day 14: morphological observation

The results of morphological examination of the cells during the differentiation culture are shown in FIG. 13.

The single cell suspension which is collected and bleached out is used for detecting the cell phenotype by a flow cytometry, and the obtained cells are proved to contain hematopoietic progenitor cells expressing CD 34. Wherein antibody information detected by the flow meter: PE Mouse Anti-Human CD34Clone 563 (RUO) (BD # 550761). As can be seen from FIG. 14, detection of the specific index of Day 8 differentiation intermediate in example 3 using a flow cytometer showed that hematopoietic progenitor cells (CD 34 ⁺ ) Only 13%, significantly less than example 1.

The number of subsequent single cells is significantly less than in examples 1 and 2, and hematopoietic progenitor cells are not significantly differentiated, thus terminating subsequent differentiation.

Example 7 alignment of different differentiation methods

The results of the measurements on the cell cultures obtained by the different methods in examples 1 to 3 demonstrate that the final differentiation efficiency is significantly different in the case of using different media, additives, culture conditions. Even in the case where some steps are identical, for example, some NK cell differentiation media are highly similar, it is still difficult to estimate the final culture result in advance.

For ease of comparison, the methods in examples 1 to 3 are summarized in the following table.

TABLE 1 different media used in examples 1-3

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Example 8 inhibition of differentiation into mast cells

Hematopoietic progenitor cells have multipotent differentiation potential, including differentiation in two large directions towards lymphoid precursor cells (T, B, NK lineage) and towards myeloid precursor cells (erythroid, granulocyte-monocyte). This example tests the effect of the addition of a C/ebpα inhibitor and/or a rarα inhibitor on the differentiation of hematopoietic progenitor cells into NK cells, thereby determining that the inhibitor is capable of inhibiting the differentiation of hematopoietic progenitor cells into mast cells (one of the myeloid precursor cells).

In the method of this example, the same procedure as in example 1 was adopted in the first to fifth stages, except that a C/ebpα inhibitor (6-thioinosine group) or rarα inhibitor (AGN 193109 group) was added in the fourth stage.

On day 8 of the fourth phase, 6-thioinosine (6-thioinosine group) or AGN193109 (AGN 193109 group) was added to the differentiation medium of the cells, respectively. The specific method is to culture cells using NK differentiation medium containing 20. Mu.M 6-thioinosine or 10nM AGN193109 for 12 days. A control group was also set up in which the medium used was not supplemented with 6-thioinosine nor AGN193109, and the rest of the procedure was the same as that of the 6-thioinosine group and the AGN193109 group.

On day 20 of the fourth phase, the expression of NKPs marker CD122 and mast cell markers PRG2, TPSB2 in the treated cells was detected by qPCR. Each index was normalized with respect to the control group 1, and the result is shown in fig. 15.

The results show that treatment of cells with 6-thioinosine or AGN193109 increased the expression of the NKPs cell marker CD122 and significantly reduced the expression of the two fecund markers PRG2 and TPSB2, indicating a higher proportion of NKPs cells and a lower proportion of mast cells in the cell population. Experiments have shown that the use of C/ebpα inhibitors or rarα inhibitors increases the differentiation of hematopoietic progenitor cells into lymphoid lineage NKPs and inhibits differentiation into myeloid lineage mast cells during the differentiation stage of hematopoietic progenitor cells into NK cells.

Claims

(1) Providing a pluripotent stem cell;

(3c) Culturing the cells cultured in step (3 b) in hematopoietic progenitor cell differentiation medium C, wherein the hematopoietic progenitor cell differentiation medium C comprises a combination of factors comprising TPO, SCF, and FLT3L.

(1) Providing a pluripotent stem cell;

3. The method of claim 2, further comprising:

7. The method of any one of claims 1-6, wherein the hematopoietic progenitor differentiation medium a has a CHIR99021 concentration of 1 to 6 μm, BMP4 concentration of 5ng/mL to 200ng/mL, and/or VEGF concentration of 5ng/mL to 300ng/mL.

8. The method of any one of claims 1-7, wherein the hematopoietic progenitor cell differentiation medium B has a SCF concentration of 5 to 200ng/mL, a SB431542 concentration of 1 to 10 μΜ, and/or a VEGF concentration of 5 to 300ng/mL.

9. The method of any one of claims 1-8, wherein the hematopoietic progenitor differentiation medium C has a SCF concentration of 5 to 200ng/mL, FLT3L concentration of 5 to 200ng/mL, and/or TPO concentration of 5 to 200ng/mL.

10. The method of any one of claims 1-9, wherein one or more of step (2), step (3 a), step (3 b), step (3 c), step (4) is suspension culture.

11. The method of any one of claims 2-10, wherein the combination of factors in NK cell differentiation medium used in step (4) comprises SCF, FLT3L, IL-7, and IL-15.

12. The method of claim 11, wherein the SCF concentration is 5ng/mL to 200ng/mL, the FLT3L concentration is 5ng/mL to 200ng/mL, the IL-7 concentration is 5ng/mL to 200ng/mL, and/or the IL-15 concentration is 5ng/mL to 200ng/mL.

13. The method of any one of claims 2-12, wherein a C/ebpα inhibitor and/or a rarα inhibitor is used in the culturing of step (4).

14. The method of claim 13, wherein the C/ebpa inhibitor is selected from fucosterol, agrimophol B (Agrimol B) or 6-thioinosine, and/or the rara inhibitor is selected from AGN192870, AGN193109 or LE135.

15. The method of claim 13 or 14, wherein the C/ebpa inhibitor is 6-thioinosine and/or the rara inhibitor is AGN193109.

16. The method of claim 15, wherein the 6-thioinosine is used at a concentration of 10 to 30 μΜ, preferably about 20 μΜ; and/or the AGN193109 is used at a concentration of 5 to 20nM, preferably about 10 nM.

17. The method of any one of claims 3-16, wherein the combination of factors in NK cell expansion medium used in step (5) comprises: IL-2, IL-15 and IL-21.

18. The method of claim 17, wherein the IL-2 concentration is 20 to 300ng/mL, the IL-15 concentration is 5 to 200ng/mL, and/or the IL-21 concentration is 5 to 200ng/mL.

19. The method of any one of claims 1-18, wherein the human pluripotent stem cells are genetically modified.

20. Hematopoietic progenitor cells or cell populations comprising hematopoietic progenitor cells obtained by the method of any one of claims 1-19.

21. NK cells or a population of cells comprising NK cells obtained by the method of any one of claims 2-19.

22. A cell preparation comprising the hematopoietic progenitor cell or population of cells comprising hematopoietic progenitor cells of claim 20, or the NK cell or population of cells comprising NK cells of claim 21.

23. Use of the hematopoietic progenitor cell or cell population comprising hematopoietic progenitor cells of claim 20, or the NK cell or cell population comprising NK cells of claim 21, in the manufacture of a medicament for cell therapy.

24. Use of the hematopoietic progenitor cell or cell population comprising hematopoietic progenitor cells of claim 20, or the NK cell or cell population comprising NK cells of claim 21, for non-therapeutic purposes.