CN115216443A

CN115216443A - Method for obtaining NK (natural killer) cells from human pluripotent stem cells through in-vitro rapid and efficient differentiation and application of NK cells

Info

Publication number: CN115216443A
Application number: CN202210865697.3A
Authority: CN
Inventors: 潘光锦; 朱艳玲; 唐俊; 麦玉婵
Original assignee: Guangzhou Institute of Biomedicine and Health of CAS
Current assignee: Guangzhou Institute of Biomedicine and Health of CAS
Priority date: 2022-07-21
Filing date: 2022-07-21
Publication date: 2022-10-21

Abstract

The invention relates to a culture medium for obtaining natural killer cells (NK cells) from human pluripotent stem cells by in vitro rapid and efficient differentiation, application thereof and a method for preparing the NK cells. The culture medium comprises: basal medium, vitamin C, IGF-1, serum, glutamine, diabody, IL2, IL7, IL15, SCF and Flt3-L; the culture medium is used for promoting the differentiation of hematopoietic stem/progenitor cells derived from human pluripotent stem cells into NK cells; the method for producing NK cells includes: culturing hematopoietic stem/progenitor cells derived from human pluripotent stem cells using the medium described above to obtain the NK cells. The culture medium can effectively promote the differentiation of hematopoietic stem/progenitor cells into NK cells, and has the advantages of short culture period, high purity of the obtained NK cells and the like.

Description

Method for obtaining NK (natural killer) cells from human pluripotent stem cells through in-vitro rapid and efficient differentiation and application of NK cells

Technical Field

The invention relates to the field of biology, in particular to a method for obtaining NK cells from human pluripotent stem cells through in-vitro rapid and efficient differentiation and application thereof, and more particularly relates to a culture medium and application thereof as well as a method for preparing the NK cells.

Background

Immune cell therapy is of great significance in the treatment of tumors. Natural Killer (NK) cells have obvious anticancer effect as main cells for clearing canceration cells in a healthy human body, and have important significance for hematological tumors such as acute lymphoblastic leukemia and solid tumors such as liver cancer.

At present, the more mature chimeric antigen receptor T cell (CAR-T) treatment has serious side effects, such as neurotoxicity and 'cytokine storm' (CRS), and the patients have serious illness and poor prognosis. Compared with T cells, the NK cells have higher safety, and at present, no serious side effects such as graft-versus-host disease (GvHD) and CRS generated by the treatment of the adoptive NK cells are reported in clinical tests. Meanwhile, the function of NK cells without MHC restriction determines that the application range of NK cells is wider than that of T cells in the future. At present, the clinical test adopts NK cells separated and amplified from peripheral blood mononuclear cells of healthy people, so that the problems of insufficient donor sources, only one patient can be treated by the NK cells provided by one donor and the like exist, and the problems of inter-individual difference, difficulty in scale and standardization and great difficulty in evaluating the prognosis of the patient exist. The above problems have posed new requirements for providing NK cells with sufficient dose and antitumor activity.

Currently, a large number of CD34+ CD43+ HSPCs are obtained by inducing human induced pluripotent stem cells (hipscs) to differentiate into hematopoietic stem/progenitor cells (HSPCs), which have lymphoid and myeloid differentiation potential; then inducing the HSPCs to differentiate into NK cells to obtain functionally mature NK cells (iPS-NK); and expanding the NK cells to the clinical application scale.

Therefore, there is a high necessity to develop a new method for obtaining NK cells.

Disclosure of Invention

The present invention aims to solve at least to some extent at least one of the technical problems of the prior art. Therefore, the invention provides a culture medium, application thereof and a method for preparing NK cells, the culture medium can lead hematopoietic stem/progenitor cells to differentiate to generate the NK cells, can effectively promote the differentiation of the hematopoietic stem/progenitor cells into the NK cells, and has the advantages of short culture period, high purity of the obtained NK cells and the like.

The present invention has been completed based on the following findings of the inventors:

at present, in a culture method for differentiating hematopoietic stem/progenitor cells to generate NK cells, a culture medium may need to be replaced according to the differentiation state of the hematopoietic stem/progenitor cells, and the operation is complicated; in addition, in the traditional culture method, the time for differentiating the hematopoietic stem/progenitor cells in vitro to form NK cells is generally 14 to 30 days, and the culture period is long.

In order to solve the problems, the inventor adjusts the components and the addition amount of the culture medium through a large number of experiments, and finally discovers that the vitamin C and the IGF-1 are simultaneously added into the culture medium, so that the hematopoietic stem/progenitor cells can be effectively promoted to be differentiated into the NK cells, the culture process only needs 8-10 days, the purity of the obtained NK cells is more than 90%, and the culture medium has the advantages of high purity of the NK cells, short culture period and the like.

In one aspect of the invention, a culture medium is provided. According to an embodiment of the invention, the medium comprises: basal medium, vitamin C, IGF-1, serum, glutamine, diabody, IL2, IL7, IL15, SCF and Flt3-L. The inventor obtains the superior culture medium through a large number of experiments, the culture medium can promote the hematopoietic stem/progenitor cells to be differentiated into the NK cells, and the culture medium has the advantages of high purity of the NK cells, short culture period and the like.

In another aspect of the invention, the invention proposes the use of one of the aforementioned media to promote the differentiation of hematopoietic stem/progenitor cells into NK cells. The inventor finds through experiments that the culture medium can promote the differentiation of the hematopoietic stem/progenitor cells into NK cells, and has the advantages of high purity of the NK cells, short culture period and the like.

In yet another aspect of the invention, the invention features a method of producing NK cells. According to an embodiment of the invention, the method comprises: culturing the hematopoietic stem/progenitor cells using the aforementioned medium to obtain the NK cells. The method for preparing the NK cells can promote the hematopoietic stem/progenitor cells to be differentiated into the NK cells, and has the advantages of high purity of the NK cells, short culture period and the like.

In yet another aspect of the invention, a culture medium is provided. According to an embodiment of the invention, the medium comprises: HDM medium, BMP signaling pathway activator, activin a, bFGF, and GSK3 inhibitor. The inventors have conducted extensive experiments to obtain the above superior medium which can promote differentiation of pluripotent stem cells into early mesodermal cells.

In yet another aspect of the invention, a culture medium is provided. According to an embodiment of the invention, the medium comprises: HDM medium, BMP signaling pathway activator, TGF- β pathway inhibitor, and WNT pathway inhibitor. The inventors have conducted extensive experiments to obtain the above preferred medium which can promote differentiation of early mesoderm cells into lateral plate mesoderm cells (LM).

In yet another aspect of the invention, a culture medium is provided. According to an embodiment of the invention, the medium comprises: HDM medium, VEGF, bFGF, TGF-beta receptor blockers, thrombopoietin, IL-6, SCF, and IL-3. The inventors have conducted extensive experiments to obtain the above-mentioned superior medium, which can promote the differentiation of hematopoietic endothelial cells into hematopoietic stem/progenitor cells.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 is a schematic diagram of a process from hematopoietic stem cells to NK cells culture according to one embodiment of the present invention;

FIG. 2 is a cell morphology at

days

2, 4, 6, and 8 of the culture process in example 1 according to the present invention;

FIG. 3 shows the results of measurement of differentiation efficiency of early mesoderm cells, lateral mesoderm cells and hematopoietic endothelial cells in example 1 according to the present invention;

FIG. 4 is a result of measurement of generation of NK cells by day 8 of differentiation in example 1 according to the present invention;

FIG. 5 is the production of CD45 and CD56 by NK cells obtained in example 2 according to the present invention and comparison thereof with NK morphology in peripheral blood;

FIG. 6 is the expression of NK cells CD45, CD56, CD16, CD94, NKG2D and NKP46 obtained in example 2 according to the present invention;

FIG. 7 is a graph showing the detection of INF- γ secretion from NK cells after K562 stimulation or PMA stimulation in example 3 of the present invention;

FIG. 8 is a graph showing the detection of NK cells killing tumor cells in example 4 according to the present invention;

FIG. 9 is a graph showing the ability of NK cells to kill different tumor cells at different effective target ratios according to example 4 of the present invention;

FIG. 10 is a result of measurement of NK cell generation on day 10 of differentiation in different groups in example 5 according to the present invention.

Detailed Description

The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention.

It should be noted that the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Further, in the description of the present invention, "a plurality" means two or more unless otherwise specified.

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

In order that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless clearly defined otherwise herein, all other 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.

As used herein, the term "pluripotent stem cell" or "PSC" refers to a cell that is capable of differentiating into a wide variety of specialized cell types under suitable conditions, and is capable of self-renewal and maintenance in a substantially undifferentiated pluripotent state under other suitable conditions. Pluripotent stem cells have the potential to differentiate into various cell tissues, but lose the ability to develop into complete individuals, and the development potential is limited to a certain extent.

As used herein, the term "human induced pluripotent stem cell" or "hiPSC" refers to a stem cell produced from an adult \ neonatal or fetal cell that has been induced or altered to differentiate, i.e., is capable of differentiating into all three germ or dermal layers: cells of mesodermal, endodermal and ectodermal tissues. The hipscs produced do not refer to cells found in nature.

In this context, the term "early mesoderm" refers to one of the three germ layers that occur during early embryogenesis, which give rise to a variety of specific cell types, including blood cells of the circulatory system, muscle, heart, dermis, bone, and other supportive and connective tissues. The term "early mesodermal cells" refers to cells that are capable of producing the early stages of mesoderm.

As used herein, the term "lateral mesoderm" refers to the region of the mesoderm that is located furthest from the spinal cord and that can form the heart, blood vessels, blood cells of the circulatory system, the internal walls of body cavities, other mesoderm components of the limbs other than muscles, and can also help form a series of extraembryonic membranes that allow nutrients to be transferred from the mother to the fetus. The term "flanking mesoderm cells" refers to cells capable of producing flanking mesoderm. It should be noted that the development sequence of the blood lineage is: pluripotent stem cells-early mesoderm cells-lateral mesoderm cells-hematogenic endothelial cells-hematopoietic stem progenitor cells. Reference may be made in particular to the Mapping of the Pairwise choice from Pluripotency to Human Bone, heart, and Other Mesoderm Cell types, cell,2016,166,451-467.

As used herein, the term "hematopoietic endothelial cells" or "HECs" refers to a subpopulation of endothelial cells that have the capacity to produce hematopoietic stem/progenitor cells during the course of an endothelial cell-hematopoietic transition.

As used herein, the term "hematopoietic stem/progenitor cell" or "HPSC" refers to a pluripotent cell with the potential for differentiation of a blood lineage, which can give rise to all blood cell types, including myeloid (e.g., monocytes and macrophages, granulocytes (e.g., neutrophils, basophils, eosinophils, and mast cells), erythrocytes, megakaryocytes/platelets, dendritic cells) and lymphoid lineages (e.g., T cells, B cells, and NK cells) (see, e.g., doultov et al, 2012 notta et al, 2015.

In this context, the term "differentiation" refers to the process by which non-specific ("free") or less specific cells acquire the characteristics of a particular cell (e.g., blood cells or muscle cells).

The present invention provides a culture medium, use thereof, and a method of preparing NK cells, which will be described in detail, respectively, below.

Culture medium

In a first aspect of the invention, a culture medium is provided. According to an embodiment of the invention, the medium comprises: basal medium, vitamin C, IGF-1, serum, glutamine, diabody, IL2, IL7, IL15, SCF and Flt3-L.

The inventor adjusts the components and the addition amount of the culture medium through a large number of experiments, and finally discovers that vitamin C and IGF-1 are simultaneously added into the culture medium, the vitamin C and the IGF-1 have a synergistic effect, the hematopoietic stem/progenitor cells can be effectively promoted to be differentiated into NK cells, and the purity of the obtained NK cells is more than 90%; the culture process only needs 8-10 days, and the culture period is short. Furthermore, the inventors have experimentally found that the culture period is increased and the purity of NK cells is decreased when vitamin C and/or IGF-1 is not added to the medium.

In some preferred embodiments, the basal medium is selected from the group consisting of alpha-MEM medium.

In some preferred embodiments, the diabesins are penicillin and streptomycin.

In some preferred embodiments, the final concentration of the vitamin C in the medium is 0.1-100mg/ml, preferably 40-60mg/ml. The inventors have conducted extensive experiments to obtain the above preferred amounts, at which the medium can further improve the efficiency of differentiation of hematopoietic stem/progenitor cells into NK cells.

In some preferred embodiments, the final concentration of said IL2 in said medium is between 0.1 and 100ng/ml, preferably between 40 and 60ng/ml. The inventors have conducted extensive experiments to obtain the above preferred amounts, at which the medium can further improve the efficiency of differentiation of hematopoietic stem/progenitor cells into NK cells.

In some preferred embodiments, the final concentration of IL-7 in the medium is between 0.1 and 100ng/ml, preferably between 40 and 60ng/ml. The inventors have conducted extensive experiments to obtain the above preferred amount at which the medium can further improve the efficiency of differentiation of hematopoietic stem/progenitor cells into NK cells.

In some preferred embodiments, the final concentration of IL-15 in the medium is between 0.1 and 100ng/ml, preferably between 40 and 60ng/ml. The inventors have conducted extensive experiments to obtain the above preferred amount at which the medium can further improve the efficiency of differentiation of hematopoietic stem/progenitor cells into NK cells.

In some preferred embodiments, the final concentration of the SCF in the culture medium is 0.1-100ng/ml, preferably 40-60ng/ml. The inventors have conducted extensive experiments to obtain the above preferred amounts, at which the medium can further improve the efficiency of differentiation of hematopoietic stem/progenitor cells into NK cells.

In some preferred embodiments, the final concentration of Flt3-L in the medium is 0.1-100ng/ml, preferably 40-60ng/ml. The inventors have conducted extensive experiments to obtain the above preferred amount at which the medium can further improve the efficiency of differentiation of hematopoietic stem/progenitor cells into NK cells.

In some preferred embodiments, the final concentration of the serum in the culture medium is 5-25%, preferably 15-25%. The inventors have conducted extensive experiments to obtain the above preferred amounts, at which the medium can further improve the efficiency of differentiation of hematopoietic stem/progenitor cells into NK cells.

In some preferred embodiments, the glutamine is at a final concentration of 0.5-1.5% in the medium. The inventors have conducted extensive experiments to obtain the above preferred amounts, at which the medium can further improve the efficiency of differentiation of hematopoietic stem/progenitor cells into NK cells.

In some preferred embodiments, the diabody is provided in the form of a solution. Illustratively, the double antibody is selected from a double antibody solution manufactured by Hyclone under the trade name SV 30010.

In some preferred embodiments, the double antibody is present at a final concentration of 0.5-1.5% in the culture medium. The inventors have conducted extensive experiments to obtain the above preferred amounts, at which the medium can further improve the efficiency of differentiation of hematopoietic stem/progenitor cells into NK cells.

In a second aspect of the invention, a culture medium is provided. According to an embodiment of the invention, the medium comprises: HDM medium, BMP signaling pathway activator, activin a, bFGF, and GSK3 inhibitor. The inventors have conducted extensive experiments to obtain the above preferred medium, which can promote differentiation of pluripotent stem cells into early mesoderm cells, and the purity of the finally obtained early mesoderm cells can be as high as 100%. More importantly, the differentiation of the pluripotent stem cells into early mesodermal cells can be further promoted by adding the bFGF and the GSK3 inhibitor.

In some preferred embodiments, the BMP signaling pathway activator is present in the first differentiation medium at a final concentration of 0.1-100ng/ml, preferably 40-60ng/ml. The inventors have conducted extensive experiments to obtain the above preferred amounts at which the culture medium can further improve the efficiency of differentiation of pluripotent stem cells into early mesodermal cells.

In some preferred embodiments, said Activin A is present in said first differentiation medium at a final concentration of 0.1-100ng/ml, preferably 40-60ng/ml. The inventors have conducted extensive experiments to obtain the above preferred amount at which the culture medium can further improve the efficiency of differentiation of pluripotent stem cells into early mesodermal cells.

In some preferred embodiments, the final concentration of bFGF in the first differentiation medium is 0.1-100ng/ml, preferably 40-60ng/ml. The inventors have conducted extensive experiments to obtain the above preferred amounts at which the culture medium can further improve the efficiency of differentiation of pluripotent stem cells into early mesodermal cells.

In some preferred embodiments, the final concentration of the GSK3 inhibitor in the first differentiation medium is 0.1-100 μ M, preferably 40-60 μ M. The inventors have conducted extensive experiments to obtain the above preferred amounts at which the culture medium can further improve the efficiency of differentiation of pluripotent stem cells into early mesodermal cells.

In some preferred embodiments, the BMP signaling pathway activator is selected from BMP4.

In some preferred embodiments, the GSK3 inhibitor is selected from CHIR99021.

In some preferred embodiments, the HDM medium comprises: DMEM/F12, ITS and vitamin C.

In some preferred embodiments, the ITS is used in an amount of 1mL and the vitamin C is used in an amount of not less than 1mg, preferably 7mg, based on 100mL of the DMEM/F12.

In a third aspect of the invention, a culture medium is provided. According to an embodiment of the invention, the medium comprises: HDM medium, BMP signaling pathway activator, TGF- β pathway inhibitor, and WNT pathway inhibitor. The inventors have conducted extensive experiments to obtain the above-mentioned superior medium, which can promote the differentiation of early mesoderm cells into lateral mesoderm cells, and the purity of the finally obtained lateral mesoderm cells can be as high as 100%.

In some preferred embodiments, the final concentration of the BMP signalling pathway activator in the second differentiation medium is from 0.1 to 100ng/ml, preferably from 40 to 60ng/ml. The inventors have conducted extensive experiments to obtain the above preferred amount at which the medium can further improve the efficiency of differentiation of early stage mesodermal cells into lateral plate mesodermal cells.

In some preferred embodiments, the TGF- β pathway inhibitor is present in the second differentiation medium at a final concentration of 0.1-10 μ M, preferably 4-6 μ M. The inventors have conducted extensive experiments to obtain the above preferred amounts at which the culture medium can further improve the efficiency of differentiation of early stage mesodermal cells into lateral plate mesodermal cells.

In some preferred embodiments, the final concentration of the WNT pathway inhibitor in the second differentiation medium is 0.1-10 μ Μ, preferably 4-6 μ Μ. The inventors have conducted extensive experiments to obtain the above preferred amount at which the medium can further improve the efficiency of differentiation of early stage mesodermal cells into lateral plate mesodermal cells.

In some preferred embodiments, the TGF- β pathway inhibitor is selected from A-83-01.

In some preferred embodiments, the WNT pathway inhibitor is selected from the group consisting of IWR-1-endo.

In a fourth aspect of the invention, a culture medium is provided. According to an embodiment of the invention, the medium comprises: HDM medium, VEGF, bFGF, TGF-beta receptor blocker, thrombopoietin (TPO), IL-6, SCF and IL-3. The inventors have made extensive experiments to obtain the above-mentioned preferable medium which promotes the differentiation of hematopoietic endothelial cells into hematopoietic stem/progenitor cells, shortens the time for obtaining hematopoietic stem/progenitor cells (48 hours), and improves the efficiency of obtaining hematopoietic stem/progenitor cells (90% or more).

In some preferred embodiments, the final concentration of VEGF in the fourth differentiation medium is 0.1-100ng/ml, preferably 40-60ng/ml. The inventors have conducted extensive experiments to obtain the above preferred amounts, at which the culture medium can further improve the efficiency of differentiation of hematopoietic endothelial cells into hematopoietic stem/progenitor cells.

In some preferred embodiments, the final concentration of bFGF in the fourth differentiation medium is 0.1-100ng/ml, preferably 40-60ng/ml. The inventors have conducted extensive experiments to obtain the above preferred amounts, at which the culture medium can further improve the efficiency of differentiation of hematopoietic endothelial cells into hematopoietic stem/progenitor cells.

In some preferred embodiments, the final concentration of the TGF- β receptor blocker in the fourth differentiation medium is 0.1-100 μ M, preferably 4-6 μ M. The inventors have conducted extensive experiments to obtain the above preferred amounts, at which the culture medium can further improve the efficiency of differentiation of hematopoietic endothelial cells into hematopoietic stem/progenitor cells.

In some preferred embodiments, the final concentration of thrombopoietin in the fourth differentiation medium is 0.1-100ng/ml, preferably 40-60ng/ml. The inventors have conducted extensive experiments to obtain the above preferred amounts, at which the culture medium can further improve the efficiency of differentiation of hematopoietic endothelial cells into hematopoietic stem/progenitor cells.

In some preferred embodiments, said IL-6 is present in said fourth differentiation medium at a final concentration of 0.1-100ng/ml, preferably 40-60ng/ml. The inventors have conducted extensive experiments to obtain the above preferred amounts, at which the culture medium can further improve the efficiency of differentiation of hematopoietic endothelial cells into hematopoietic stem/progenitor cells.

In some preferred embodiments, the final concentration of IL-3 in the fourth differentiation medium is between 0.1 and 100ng/ml, preferably between 40 and 60ng/ml. The inventors have conducted extensive experiments to obtain the above preferred amounts, at which the culture medium can further improve the efficiency of differentiation of hematopoietic endothelial cells into hematopoietic stem/progenitor cells.

In some preferred embodiments, the final concentration of the SCF in the fourth differentiation medium is 0.1-100ng/ml, preferably 40-60ng/ml. The inventors have conducted extensive experiments to obtain the above preferred amount, at which the culture medium can further improve the efficiency of differentiating the hematopoietic endothelial cells into hematopoietic stem/progenitor cells.

In some preferred embodiments, the TGF- β receptor blocker is selected from SB43154.

Use of

In a fifth aspect, the present invention provides a use of the medium of the first aspect for promoting differentiation of hematopoietic stem/progenitor cells into NK cells. The inventors found through experiments that the culture medium of the first aspect can promote the differentiation of hematopoietic stem/progenitor cells into NK cells, and the obtained NK cells have high purity and short differentiation cycle.

It will be appreciated by those skilled in the art that the features and advantages described for the medium of the first aspect apply equally to this use and will not be described in further detail herein.

In a sixth aspect, the present invention provides the use of a medium according to the second aspect to promote differentiation of pluripotent stem cells into early mesodermal cells. The inventors have found through experiments that the use of the medium according to the second aspect can promote differentiation of pluripotent stem cells into early mesoderm cells, and that the obtained early mesoderm cells have high purity.

It will be appreciated by those skilled in the art that the features and advantages described for the medium according to the second aspect apply equally to this use and will not be described in further detail herein.

In a seventh aspect of the invention, the invention provides use of a medium according to the third aspect to promote differentiation of early stage mesoderm cells into lateral plate mesoderm cells. The inventors have found through experiments that the medium according to the second aspect promotes differentiation of early mesoderm cells into flanking mesoderm cells, and that the obtained flanking mesoderm cells have high purity.

It will be appreciated by those skilled in the art that the features and advantages described for the medium of the third aspect are equally applicable to this use and will not be described in further detail herein.

In an eighth aspect, the present invention provides a use of the medium of the fourth aspect for promoting differentiation of hematopoietic endothelial cells into hematopoietic stem/progenitor cells. The inventors have found through experiments that the culture medium according to the second aspect can promote the differentiation of hematopoietic endothelial cells into hematopoietic stem/progenitor cells, and the obtained hematopoietic stem/progenitor cells have high purity.

It will be appreciated by those skilled in the art that the features and advantages described for the medium according to the fourth aspect apply equally to this use and will not be described in further detail here.

Method

At present, the induction scheme of differentiation of hipscs into NK cells established in laboratories at home and abroad is an Embryo Body (EB) differentiation method. Wherein, EB differentiates to NK cells and better simulates cells and physicochemical microenvironment required by NK differentiation in vivo, and the NK cells induced by EB have good cytotoxicity to various tumor cells in vitro tests; however, cells with complex types are generated in the EB differentiation process, so that the mass acquisition and subsequent clinical application of NK cells are not facilitated. In addition, the period of differentiation from EB to NK cells is long, generally, 1 week is needed for inducing EB from hipSCs, and then 4-5 weeks are needed for differentiation to obtain NK cells, and the technical difficulty is high and the cost is high.

In order to solve the problems, the inventor firstly induces hiPSCs to differentiate into CD34+ CD43+ HSPCs, and then co-cultures the suspended HSPCs and mouse bone marrow stromal cells to obtain functional NK cells.

In accordance with this, in a ninth aspect of the invention, a method of producing hematopoietic stem/progenitor cells is provided. According to an embodiment of the invention, the method comprises: subjecting pluripotent stem cells to a differentiation culture treatment in a pluripotent stem cell differentiation medium to obtain said hematopoietic stem/progenitor cells; the differentiation culture medium of the pluripotent stem cells comprises a first differentiation culture medium, a second differentiation culture medium, a third differentiation culture medium and a fourth differentiation culture medium. The inventor obtains the four culture mediums through a large number of experiments, and when the pluripotent stem cells are cultured in a differentiation mode, different culture mediums can be selected according to the differentiation condition of the pluripotent stem cells, so that the development environment of the pluripotent stem cells in vivo can be simulated better. Therefore, the method can promote the differentiation of pluripotent stem cells (especially human pluripotent stem cells) into hematopoietic stem/progenitor cells, and has the advantages of stability, high efficiency, short cycle, high purity of the obtained hematopoietic stem/progenitor cells, and the like.

In some preferred embodiments, the differentiation culture treatment comprises: subjecting said pluripotent stem cells to a first culturing treatment in said first differentiation medium; subjecting the product of the first culture treatment to a second culture treatment in the second differentiation medium; subjecting the product of the second culture treatment to a third culture treatment in the third differentiation medium; subjecting the product of said fourth culture treatment to a fourth culture treatment in said fourth differentiation medium in order to obtain said hematopoietic stem/progenitor cells. The inventor finds through experiments that the first differentiation medium can promote the differentiation of pluripotent stem cells into early mesoderm cells, the second differentiation medium can promote the differentiation of early mesoderm cells into lateral mesoderm cells, the third differentiation medium can promote the differentiation of lateral mesoderm cells into hematogenic endothelial cells, and the fourth differentiation medium can promote the differentiation of hematogenic endothelial cells into hematopoietic stem/progenitor cells.

Specifically, the first differentiation medium is the medium described in the second aspect, the second differentiation medium is the medium described in the third aspect, and the fourth differentiation medium is the medium described in the fourth aspect.

In some preferred embodiments, the third differentiation medium comprises: HDM medium, VEGF and bFGF.

In some preferred embodiments, the final concentration of VEGF in the third differentiation medium is 0.1-100ng/ml, preferably 40-60ng/ml. The inventors have conducted extensive experiments to obtain the above preferred amount, at which the culture medium can further improve the efficiency of differentiation of the collateral mesodermal cells into hematogenic endothelial cells.

In some preferred embodiments, the final concentration of bFGF in the third differentiation medium is 0.1-100ng/ml, preferably 40-60ng/ml. The inventors have conducted extensive experiments to obtain the above preferred amount, at which the culture medium can further improve the efficiency of differentiation of the collateral mesodermal cells into hematogenic endothelial cells.

In some preferred embodiments, the first culturing treatment is carried out for a period of 22 to 24 hours. The inventors have found through experiments that the purity of early mesodermal cells differentiated by the above culture time can reach as high as 100%.

In some preferred embodiments, the second culturing treatment is carried out for a period of 22 to 24 hours. The inventors found through experiments that the purity of the lateral plate mesoderm cells differentiated in the above culture time can reach as high as 100%.

In some preferred embodiments, the time of the third culturing treatment is 45-48h. The inventor finds out through experiments that the purity of the hematopoietic endothelial cells obtained by differentiation in the culture time can reach more than 70%.

In some preferred embodiments, the fourth culturing treatment is for a period of 4 to 5 days. The inventor finds out through experiments that the purity of the hematopoietic stem/progenitor cells obtained by differentiation in the culture time can reach more than 90%.

It will be appreciated by those skilled in the art that the features and advantages described for the medium according to the second, third and fourth aspects are equally applicable to the method for producing hematopoietic stem/progenitor cells and will not be described in further detail herein.

In a tenth aspect of the present invention, the present invention provides a method for preparing NK cells. According to an embodiment of the invention, the method comprises: culturing hematopoietic stem/progenitor cells using the medium of the first aspect, so as to obtain the NK cells. The method for preparing the NK cells can promote the differentiation of the hematopoietic stem/progenitor cells into the NK cells, the purity of the obtained NK cells is more than 90%, the culture process only needs 8-10 days, and the culture period is short. The method has the advantages of stability, high efficiency, short period, complete function of the obtained NK cells, high purity and the like.

In some preferred embodiments, as shown in fig. 1, the method further comprises: the mouse bone marrow stromal cells are added to the culture medium in advance before the hematopoietic stem/progenitor cells are cultured. The inventor finds that the mouse bone marrow stromal cells can be co-cultured with the hematopoietic stem/progenitor cells, and the mouse bone marrow stromal cells can be used as the nutritive cells of the hematopoietic stem/progenitor cells, can rapidly induce the hematopoietic stem/progenitor cells to be differentiated into NK, and can improve the differentiation efficiency of the hematopoietic stem/progenitor cells.

In some preferred embodiments, the culturing is for a period of 8 to 10 days. Thus, the method has the advantage of short culture period.

In some preferred embodiments, the hematopoietic stem/progenitor cells are obtained by subjecting pluripotent stem cells to differentiation culture.

In some preferred embodiments, the hematopoietic stem/progenitor cells are obtained by differentiating pluripotent stem cells in a pluripotent stem cell differentiation medium.

The pluripotent stem cell differentiation medium and the method for differentiating pluripotent stem cells in the pluripotent stem cell differentiation medium are specifically described in the ninth aspect of the present invention.

It will be appreciated by those skilled in the art that the features and advantages described in the medium of the first aspect and the method for producing hematopoietic stem/progenitor cells of the ninth aspect are equally applicable to the method for producing NK cells, and will not be described herein again.

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

In the embodiment of the invention, the double antibody is selected from double antibody solution with the manufacturer of Hyclone and the product number of SV 30010.

Example 1: monolayer hematopoietic differentiation

First, hiPSCs were differentiated into HSPCs using the constructed monolayer hematopoietic differentiation system. The method specifically comprises the following steps: on day 0, hiPSCs were cultured in the first differentiation medium (HDM medium +50ng/ml BMP4+50ng/ml Activin a +50ng/ml bFGF +50 μ M CHIR 99021) and induced to differentiate into early mesodermal cells. On day 1, the early mesoderm cells were induced into Lateboard Mesoderm (LM) cells by replacing the first differentiation medium with the second differentiation medium (HDM medium +50ng/ml BMP4+ 5. Mu.M A-83-01+ 5. Mu.M IWR-1-endo). On day 2, the second differentiation medium was changed to a third differentiation medium (HDM medium +50ng/ml VEGF +50ng/ml bFGF), and the collateral mesodermal cells were induced to differentiate into hematogenic endothelial cells. After day 4, the third differentiation medium was changed to a fourth differentiation medium (HDM medium +50ng/ml VEGF +50ng/ml bFGF + 50. Mu.M SB43154+50ng/ml thrombopoietin (TPO for short) +50ng/ml IL6+50ng/ml SCF +50ng/ml IL 3), inducing the conversion of the hematopoietic endothelial cells to HSPC. Taking a picture by using a microscope and observing cell morphology diagrams of

days

2, 4, 6 and 8 in the process of differentiating the hiPSCs into the HSPCs, and particularly referring to FIG. 2; detecting differentiation efficiency of early mesoderm cells, lateral plate mesoderm cells and hematogenic endothelial cells by flow cytometry, specifically referring to fig. 3; suspension cells were detected on day 8 using flow cytometry, see in particular figure 4. As shown in FIG. 2, production of HSPCs began on day 6 and a large amount of suspended HSPCs could be collected on day 8. As shown in FIG. 3, the differentiation efficiency of T + early mesoderm cells was close to 100%, the differentiation efficiency of HAND1+ flanking mesoderm cells was also close to 100%, and the differentiation efficiency of CD34+ hematopoietic endothelial cells was 80% or more. As shown in fig. 4, the day 8 cells were mostly hematopoietic stem/progenitor cells of CD34+ CD43+ CD44 +.

The HDM medium of the present application includes DMEM/F12 (Hyclone), ITS (insulin transferase-selenium, GIBCO) and vitamin C (Vc, GIBCO), wherein the amount of ITS is 1mL and the amount of vitamin C is 7mg, based on 100mL of the DMEM/F12.

Example 2: preparation of NK cells

The HSPCs obtained in example 1 were subjected to NK cell differentiation. The method comprises the following specific steps: laying mouse bone marrow stromal cells (OP 9-DLL 1) on a medium (α -MEM medium +20% FBS +1% GlutaMAXTM-1+1% P/S +50mg/ml ascobic acid +50ng/ml IL-2+50ng/ml IL-7+50ng/ml IL-15+50ng/ml SCF +50ng/ml Flt-3L +50ng/ml IGF-1) one day ahead of time, collecting HSPCs obtained in example 1 next day, co-culturing the HSPCs with the medium on which the mouse bone marrow stromal cells are laid, collecting suspended cells for 10 days, and then performing cell morphology observation, flow cytometry detection, immunofluorescence technical detection, specifically see FIGS. 5-6.

As shown in FIG. 5, iPS-NK cells cultured at day 10 were functionally matured (i.e., terminated in differentiation), iPS-NK cells differentiated from human pluripotent stem cells were similar in morphology to NK cells in peripheral blood (PB-NK cells), and NK marker genes of CD45 and CD56 were highly expressed, and the purity of the obtained iPS-NK cells was 90% or more.

In FIG. 6, the expression of CD45 and CD56 in the differentiated iPS-NK cells was detected by immunofluorescence technique (left side of FIG. 6), and the expression of NK maturation-related genes such as CD16/CD94/NKG2D/NKP46 in the differentiated iPS-NK cells was detected by flow cytometry (right side of FIG. 6), respectively.

Example 3: detection of secretion function of NK cell

The secretory function of the iPS-NK cell obtained in example 2 was detected by a cell flow meter. The condition that iPS-NK cells secrete INF-gamma after being stimulated by K562 or PMA for 4 hours is detected by a flow cytometer, and the result particularly shows that the iPS-NK cells obtained through differentiation have a perfect interferon secretion function as shown in figure 7.

Example 4: detection of tumor killing function of NK cells

The iPS-NK cells obtained in example 2 were mixed with leukemia cells (K562) according to 1:1 for 24 hours, and then adopting a microscope to perform cell morphology observation and a flow cytometer to detect the capability of killing tumor cells, and particularly referring to fig. 8, the result shows that iPS-NK cells can obviously kill K562 tumor cells (annexinV is apoptotic cells).

The iPS-NK cell obtained in example 2 and different tumor cells expressing luciferase genes are co-cultured according to different effective target ratios, a fluorogenic substrate is added in 24 hours, the fluorescence intensity is detected by an enzyme-labeling instrument, and the killing activity is calculated, and the result shows that the iPS-NK cell has strong killing effect on different tumor cells, particularly as shown in FIG. 9.

Example 5: preparation of NK cells in different media

In the embodiment, the experimental group, the control group, the + Vc group and the + IGF-1 group are compared, and the addition of vitamin C and IGF-1 can obviously improve the NK differentiation efficiency. Specifically, the medium of the experimental group (i.e., "+ Vc + IGF-1") is α -MEM medium +20% FBS +1% GlutaMAXTM-1+1% P/S +50mg/ml ascorbic acid +50ng/ml IL-2+50ng/ml IL-7+50ng/ml IL-15+50ng/ml SCF +50ng/ml Flt-3L +50ng/ml IGF-1, to which vitamin C and IGF-1 are added simultaneously; the control group differed from the experimental group only in that vitamin C and IGF-1 were not added to the medium; the + Vc group differed from the experimental group only in that IGF-1 was not added to the medium; the + IGF-1 group differed from the experimental group only in that no vitamin C was added to the medium. Then, the HSPCs in the experimental group, the control group, the + Vc group and the + IGF-1 group are subjected to NK cell differentiation under the specific differentiation culture conditions shown in example 2 until the cells are collected at day 10, and the NK cells of CD45+ CD56+ are detected by a flow cytometer, which is shown in FIG. 10. The result shows that the addition of vitamin C and IGF-1 can obviously improve the NK differentiation efficiency.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Moreover, various embodiments or examples and features of various embodiments or examples described in this specification can be combined and combined by one skilled in the art without being mutually inconsistent.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims

1. A culture medium, comprising:

basal medium, vitamin C, IGF-1, serum, glutamine, diabody, IL2, IL7, IL15, SCF and Flt3-L.

2. The culture medium of claim 1, wherein the basal medium is selected from the group consisting of alpha-MEM medium;

optionally, the diabesin is penicillin and streptomycin;

optionally, the final concentration of said vitamin C in said medium is 0.1-100mg/ml;

optionally, the final concentration of said IL2 in said medium is 0.1-100ng/ml;

optionally, the final concentration of said IL7 in said medium is 0.1-100ng/ml;

optionally, the final concentration of said IL15 in said medium is 0.1-100ng/ml;

optionally, the final concentration of the SCF in the culture medium is 0.1-100ng/ml;

optionally, said Flt3-L is present in said culture medium at a final concentration of 0.1 to 100ng/ml;

optionally, the final concentration of the serum in the culture medium is 5-25%;

optionally, the glutamine has a final concentration in the medium of 0.5-1.5%;

optionally, the diabody is provided in the form of a solution;

optionally, the double antibody is at a final concentration of 0.5-1.5% in the culture medium.

3. Use of the medium of claim 1 or 2 for promoting differentiation of hematopoietic stem/progenitor cells into NK cells.

4. A method of producing NK cells, comprising: culturing hematopoietic stem/progenitor cells using the medium of claim 1 or 2, so as to obtain the NK cells.

5. The method of claim 4, further comprising:

adding mouse bone marrow stromal cells to the culture medium in advance before culturing the hematopoietic stem/progenitor cells;

optionally, the culturing time is 8-10 days;

optionally, the hematopoietic stem/progenitor cells are obtained by subjecting pluripotent stem cells to differentiation culture;

preferably, the hematopoietic stem/progenitor cells are obtained by subjecting pluripotent stem cells to a differentiation culture treatment in a pluripotent stem cell differentiation medium;

optionally, the pluripotent stem cell differentiation medium comprises a first differentiation medium, a second differentiation medium, a third differentiation medium, and a fourth differentiation medium;

optionally, the differentiation culture treatment comprises:

subjecting the pluripotent stem cells to a first culture treatment in the first differentiation medium;

subjecting the product of the first culture treatment to a second culture treatment in the second differentiation medium;

subjecting the product of the second culture treatment to a third culture treatment in the third differentiation medium;

subjecting the product of said third culture treatment to a fourth culture treatment in said fourth differentiation medium in order to obtain said hematopoietic stem/progenitor cells.

6. The method of claim 5, wherein the first differentiation medium comprises:

HDM medium, BMP signaling pathway activator, activin a, bFGF, and GSK3 inhibitor;

optionally, the final concentration of the BMP signaling pathway activator in the first differentiation medium is 0.1-100mg/ml;

optionally, said Activin A is present in said first differentiation medium at a final concentration of 0.1-100ng/ml;

optionally, the final concentration of bFGF in the first differentiation medium is 0.1 to 100ng/ml;

optionally, the final concentration of the GSK3 inhibitor in the first differentiation medium is 0.1-100 μ Μ;

optionally, the BMP signaling pathway activator is selected from BMP4;

optionally, the GSK3 inhibitor is selected from CHIR99021;

optionally, the time of the first culture treatment is 22-24h;

optionally, the second differentiation medium comprises:

HDM medium, BMP signaling pathway activator, TGF- β pathway inhibitor and WNT pathway inhibitor;

optionally, the final concentration of said BMP signaling pathway activator in said second differentiation medium is 0.1-100ng/ml;

optionally, the final concentration of said A8-301 in said second differentiation medium is 0.1-10 μ Μ;

optionally, the final concentration of the WNT pathway inhibitor in the second differentiation medium is 0.1-10 μ Μ;

optionally, the BMP signaling pathway activator is selected from BMP4;

optionally, the TGF- β pathway inhibitor is selected from A-83-01;

optionally, the WNT pathway inhibitor is selected from the group consisting of IWR-1-endo;

optionally, the time of the second culture treatment is 22-24h;

optionally, the third differentiation medium comprises:

HDM medium, VEGF and bFGF;

optionally, the final concentration of the VEGF in the third differentiation medium is 0.1-100ng/ml;

optionally, the final concentration of bFGF in the third differentiation medium is 0.1 to 100ng/ml;

optionally, the time of the third culturing treatment is 45-48h;

optionally, the fourth differentiation medium comprises:

HDM medium, VEGF, bFGF, TGF-beta receptor blockers, thrombopoietin, IL-6, SCF, and IL-3;

optionally, the final concentration of VEGF in the fourth differentiation medium is 0.1-100ng/ml;

optionally, the final concentration of bFGF in the fourth differentiation medium is 0.1 to 100ng/ml;

optionally, the TGF- β receptor blocker is at a final concentration of 0.1-100 μ Μ in the fourth differentiation medium;

optionally, the final concentration of thrombopoietin in the fourth differentiation medium is 0.1-100ng/ml;

optionally, the final concentration of said IL-6 in said fourth differentiation medium is 0.1-100ng/ml;

optionally, the final concentration of said IL-3 in said fourth differentiation medium is 0.1-100ng/ml;

optionally, the final concentration of the SCF in the fourth differentiation medium is 0.1-100ng/ml;

optionally, the TGF- β receptor blocker is selected from SB43154;

optionally, the fourth culturing treatment is for a period of 4-5 days.

7. The method of claim 6, wherein the HDM medium comprises:

DMEM/F12, ITS and vitamin C;

optionally, the ITS is used in an amount of 1mL, and the vitamin C is used in an amount of not less than 1mg, preferably 7mg, based on 100mL of the DMEM/F12.

8. A culture medium, comprising:

optionally, the BMP signaling pathway activator is selected from BMP4;

optionally, the GSK3 inhibitor is selected from CHIR99021;

optionally, the HDM medium comprises:

DMEM/F12, ITS and vitamin C;

9. A culture medium, comprising:

optionally, the TGF- β pathway inhibitor is at a final concentration of 0.1-10 μ Μ in the second differentiation medium;

optionally, the BMP signaling pathway activator is selected from BMP4;

optionally, the TGF- β pathway inhibitor is selected from a-83-01;

optionally, the HDM medium comprises:

DMEM/F12, ITS and vitamin C;

10. A culture medium, comprising:

optionally, the TGF- β receptor blocker is selected from SB43154;

optionally, the HDM medium comprises:

DMEM/F12, ITS and vitamin C;