Expression vector structure and method for producing multilineage hematopoietic stem / progenitor cells

Abstract

The invention relates to the technical field of biology, in particular to an expression vector structure and a method for generating multilineage hematopoietic stem / progenitor cells based on an expression vector. The cDNA sequences of hnCD16 and mbIL-15 are expressed in the same vector in series and integrated into a mammal pluripotent stem cell genome, the pluripotent stem cell capable of stably expressing hnCD16 and mbIL-15 can be obtained, the method is easy and convenient to operate and high in efficiency, hematopoietic stem / progenitor cells capable of expressing mbIL-15-hnCD16 can be obtained through induced differentiation, and the hematopoietic stem / progenitor cells can be used for preparing the hematopoietic stem / progenitor cells. And the mbIL-15-hnCD16-NK cell obtained by further induced differentiation has higher killing activity and longer survival time.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an expression vector structure and a method for producing multi-lineage hematopoietic stem / progenitor cells based on the expression vector. Background Art

[0002] Human natural killer (NK) cells are an important subset of innate lymphocytes and an indispensable part of the innate immune system. NK cells and T cells are derived from the same lymphocyte progenitor cells, so NK cells and T cells are killer immune cells. However, unlike T cells, NK cells are innate immune cells and are the most active type of innate immune cells. In addition, NK cells have a short survival time in the body and cannot form memory cells in adoptive transplantation. NK cells mainly rely on three aspects to exert their functions. First, they release cytokines such as perforin and granzyme to directly kill target cells after direct contact with abnormal cells; second, they eliminate target cells through antibody-dependent cell-mediated killing mechanisms (ADCC); third, they regulate the immune system by synthesizing and releasing cytokines, triggering adaptive immune regulation, and thus systematically enhancing the comprehensive resistance of the body's immune cells. Therefore, how to prolong the survival time of NK cells in the body and improve the function of NK cells is the key to improving NK cells in tumor treatment.

[0003] Currently, there are three sources of NK cells used in clinical treatment: mature NK cells from peripheral blood or umbilical cord blood; NK-92 cell line; and induction of functional NK cells in vitro through regeneration pathways from induced pluripotent stem cells (iPSCs), hematopoietic stem / progenitor cells (HSPCs) from umbilical cord blood or placenta. How to enhance the targeting, effectiveness, and sustainability of NK cells from these sources is currently a key breakthrough in improving clinical efficacy.

[0004] IL-15 is a cytokine similar in structure to IL-2. It plays an extremely important role in the development and survival of lymphocytes, especially the development, maturation and activation of NK cells. Knocking out IL-15 in mice will lead to developmental defects of NK cells. However, due to its small molecular weight and simple structure, IL-15 has a short half-life and limited activity when it exists alone as a soluble factor. Under physiological conditions, IL-15 can fuse with IL-15R on the surface of monocytes to form a membrane fusion protein, which greatly enhances the stimulation of NK progenitor cells, immature and mature NK cells. CD16 is a specific receptor for NK cells to play an ADCC role. It can bind to the Fc end of antibodies, target tumor cells with the help of antibodies, and stimulate NK cells to kill tumor cells. However, after NK cells are activated, the CD16 they express is easily degraded by metalloproteases, and the efficiency of natural CD16 molecules binding antibodies is limited, which greatly limits the ADCC function of NK cells.

[0005] At present, mbIL-15 (Membranebound IL-15, membrane fusion protein of IL-15) is introduced into mature NK cells by lentivirus or retrovirus to prepare NK cells expressing mbIL-15 (mbIL-15-NK). After the expression of mbIL-15, it can significantly enhance the function of NK cells and prolong the survival time in vivo. In addition to the above method to prepare NK cells expressing only hnCD16 (hnCD16-NK), hnCD16 can also be introduced into HSPCs or iPSCs by lentivirus, and then hnCD16-iPSCs are induced to generate hnCD16-NK cells. In addition, mbIL-15 and hnCD16 can be expressed simultaneously in iPSCs by CRISPR technology to prepare mbIL-15-hnCD16-iPSCs, and then mbIL-15-hnCD16-NK cells are induced. However, all three methods have certain drawbacks and cannot be used in clinical treatment efficiently and safely. First, mature NK cells are difficult to be gene-edited or virally infected, and mbIL-15-hnCD16-NK cells cannot be obtained efficiently and stably. Hematopoietic stem / progenitor cells are also difficult to obtain mbIL-15-hnCD16-NK cells by gene editing or viral infection due to their scarcity and strong heterogeneity. The safety of prepared iPSCs is still a controversial issue worldwide, and cell therapy products prepared from iPSCs have exposed a series of problems in clinical trials. Summary of the invention

[0006] The present invention aims to solve one of the technical problems in the related art at least to a certain extent. To this end, one object of the present invention is to provide pluripotent stem cells expressing CD16 mut (hnCD16) and IL-15 / IL-15Rα membrane-bound fusion protein (mbIL-15), and hematopoietic stem / progenitor cells obtained by induction and differentiation thereof. The NK cells derived from the pluripotent stem cells and the hematopoietic stem / progenitor cells induced and differentiated have stronger ADCC ability, stronger cell activity and proliferation ability, more efficient tumor cell killing activity, and longer survival time in vivo.

[0007] To this end, the first aspect of the present invention provides an expression vector. According to some embodiments of the present invention, the expression vector comprises a cDNA sequence encoding a CD16 protein mutant and a mbIL-15 protein, wherein the CD16 protein mutant has p.Phe158Val and p.Ser197Pro mutations relative to the wild-type CD16 protein.

[0008] According to some embodiments of the present invention, the nucleic acid sequence encoding the CD16 protein mutant is shown as SEQ ID NO:1, and the nucleic acid sequence encoding the mbIL-15 protein is shown as SEQ ID NO:2.

[0009] The sequence of SEQ ID NO:1 is as follows:

[0010] ATGTGGCAGCTGCTCCTCCCAACTGCTCTGCTACTTCTAGTTTCAGCTGGCATGCGGACTGAAGATCTCCCAAAGGCTGTGGTGTTCCTGGAGCCTCAATGGTACAGGGTGCTCGAGAAGGACAGTGTGACTCTGAAGTGCCAGGGAGCCTACTCCCCTGAGGACAATTCCACACAGTGGTTTCACAATGAGAGCCTCATCTCAAGCCAGGCCTCGAGCTACTTCATTGACGCTGCCACAGTCGACGACAGTGGAGAGTACAGGTGCCAGACAAACCTCTCCACCCTCAGTGACCCGGTGCAGCTAGAAGTCCATATCGGCTGGCTGTTGCTCCAGGCCCCTCGGTGGGTGTTCAAGGAGGAAGACCCTATTCACCTGAGGTGTCACAGCTGGAAGAACACTGCTCTGCATAAGGTCACATATTTACAGAATGGCAAAGGCAGGAAGTATTTTCATCATAATTCTGACTTCTACATTCCAAAAGCCACACTCAAAGACAGCGGCTCCTACTTCTGCAGGGGGCTTGTTGGGAGTAAAAATGTGTCTTCAGAGACTGTGAACATCACCATCACTCAAGGTTTGGCAGTGCCAACCATCTCATCATTCTTTCCACCTGGGTACCAAGTCTCTTTCTGCTTGGTGATGGTACTCCTTTTTGCAGTGGACACAGGACTATATTTCTCTGTGAAGACAAACATTCGAAGCTCAACAAGAGACTGGAAGGACCATAAATTTAAATGGAGAAAGGACCCTCAAGACAAA

[0011] The sequence of SEQ ID NO:2 is shown below:

[0012] TCAGGTGGAGGTTCAGGTGGAGGTGGATCAGGTGGAGGTGGATCAGGTGGAGGTGGATCAGGTGGAGGTTCACTGCAAATCACGTGCCCTCCCCCATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCAGGGAGCGGTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACGTC CAGCCTGACGGAGTGCGTGTGAACAAGGCCACGAATGTCGCCCACTGGACAACCCCCAGTCTCAAATGCATTAGAGACCCTGCCCTGGTTCACCAAAGGCCAGCGCCACCCTCCACAGTAACGACGGCAGGGGTGACCCCACAGCCAGAGAGCCTCTCCCCTTCTGGAAAAGAGCCCGCAGCTTCATCTCCCAGCT CAAACAACACAGCGGCCACAACAGCAGCTATTGTCCCGGGCTCCCAGCTGATGCCTTCAAAATCACCTTCCACAGGAACCACAGAGATAAGCAGTCATGAGTCCTCCCACGGCACCCCCTCTCAGACAACAGCCAAGAACTGGGAACTCACAGCATCCGCCTCCCACCAGCCGCCAGGTGTGTATCCACAGGGCCAC AGCGACACCACTGTGGCTATCTCCACGTCCACTGTCCTGCTGTGTGGGCTGAGCGCTGTGTCTCCTGGCATGCTACCTCAAGTCAAGGCAAACTCCCCCGCTGGCCAGCGTTGAAATGGAAGCCATGGAGGCTCTGCCGGTGACTTGGGGGACCAGCAGCAGAGATGAAGACTTGGAAAACTGCTCTCACCACCTA

[0013] The second aspect of the present invention provides a host cell. According to some embodiments of the present invention, the host cell comprises the expression vector described in the first aspect.

[0014] According to some embodiments of the invention, the host cell is a human pluripotent stem cell.

[0015] According to some embodiments of the present invention, the human pluripotent stem cells are embryonic stem cells within 14 days of fertilization that have not undergone in vivo development.

[0016] The safety and omnipotence of iPSCs are not guaranteed. The method of lentiviral editing is time-consuming, labor-intensive, and costly, and the integration is highly random. Gene editing based on CRISPR technology cannot obtain high-copy number expression of exogenous gene fragments, nor can it continuously screen drugs, and the off-target probability is high. The inventors based their work on the following: (1) IL-15 membrane fusion protein (mbIL-15) is expressed on NK cells, which can enhance the cell activity and half-life of NK cells; (2) After the metalloproteinase cleavage site and Fc binding site of CD16 are mutated (F158V, S197P) to form a non-cleavable high-affinity CD16 (High-affinity noncleavable variant of CD16a, hnCD16), the ADCC function of NK cells can be greatly enhanced. By using the Piggybac transposon technology, stably and efficiently expressed mbIL-15 and hnCD16 were knocked into hESCs cells to obtain mbIL-15-hnCD16-hESCs, which in turn induced functional lateral plate mesoderm cells, hemogenic endothelial cells, hematopoietic stem / progenitor cells, NK cell progenitor cells, and myeloid progenitor cells.

[0017] According to some embodiments of the present invention, the host cell is a host cell into which the expression vector described in the first aspect is introduced by Piggybac transposon gene editing.

[0018] The third aspect of the present invention provides a lateral plate mesoderm cell. According to some embodiments of the present invention, the lateral plate mesoderm cell is obtained by inducing differentiation of the host cell described in the second aspect, wherein the host cell is a human pluripotent stem cell, and the lateral plate mesoderm cell is capable of expressing the CD16 protein mutant and the mbIL-15 protein.

[0019] The amino acid sequence of the CD16 protein mutant is shown in SEQ ID NO: 3:

[0020] MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVPTISSFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK

[0021] The amino acid sequence of mbIL-15 protein is shown in SEQ ID NO:4:

[0022] *

[0023] The fourth aspect of the present invention provides a method for obtaining lateral plate mesoderm cells. According to some embodiments of the present invention, the expression vector described in the first aspect is introduced into human pluripotent stem cells by piggyback transposon gene editing, and the human pluripotent stem cells are induced to differentiate to obtain the lateral plate mesoderm cells.

[0024] The human embryonic stem cells (ESCs) used in the present invention have a relatively stable genome, and their epigenetic information is not interfered by traces of adult cells. The gene editing technology used in the present invention is the Piggybac transposon technology, which can stably insert the genome, and the number of copies of the exogenous gene expression is large.

[0025] The fifth aspect of the present invention provides a hemogenic endothelial cell. According to some embodiments of the present invention, the hemogenic endothelial cell is obtained by mixing the lateral plate mesoderm cells described in the third aspect with feeder layer cells to differentiate into the hemogenic endothelial cell, wherein the hemogenic endothelial cell is capable of expressing the CD16 protein mutant and the mbIL-15 protein.

[0026] According to some embodiments of the present invention, the feeder layer cells include at least one of monocytes, macrophages, dendritic cells and B cells.

[0027] According to some embodiments of the present invention, the feeder layer cells are macrophages and / or dendritic cells.

[0028] A sixth aspect of the present invention provides a method for obtaining hemogenic endothelial cells. According to some embodiments of the present invention, the method comprises co-culturing the lateral plate mesoderm cells described in the third aspect with feeder layer cells to differentiate and obtain the hemogenic endothelial cells.

[0029] According to some embodiments of the present invention, the feeder layer cells include at least one of monocytes, macrophages, dendritic cells and B cells.

[0030] According to some embodiments of the present invention, the feeder layer cells are macrophages and / or dendritic cells.

[0031] The seventh aspect of the present invention provides a hematopoietic stem / progenitor cell. According to some embodiments of the present invention, the hematopoietic stem / progenitor cell is obtained by inducing differentiation of hemogenic endothelial cells as described in the fifth aspect, wherein the hematopoietic stem / progenitor cell can express the CD16 protein mutant and mbIL-15 protein.

[0032] The eighth aspect of the present invention provides a method for obtaining hematopoietic stem / progenitor cells. According to some embodiments of the present invention, the method comprises obtaining the hematopoietic stem / progenitor cells by inducing differentiation of the hemogenic endothelial cells described in the fifth aspect.

[0033] The ninth aspect of the present invention provides a NK cell progenitor cell or myeloid progenitor cell. According to some embodiments of the present invention, the NK cell progenitor cell or myeloid progenitor cell is obtained by the induced differentiation of the hematopoietic stem / progenitor cells described in the seventh aspect, wherein the NK cell progenitor cell or myeloid progenitor cell can express the CD16 protein mutant and mbIL-15 protein.

[0034] The present invention provides pluripotent stem cells expressing CD16 mut (hnCD16) and IL-15 / IL-15Rα membrane-bound fusion protein (mbIL-15), and hematopoietic stem / progenitor cells obtained by induction and differentiation thereof. The pluripotent stem cells and NK cells derived from the induced differentiation of hematopoietic stem / progenitor cells have stronger ADCC ability, stronger cell activity and proliferation ability, more efficient tumor cell killing activity, and prolonged in vivo existence duration.

[0035] The tenth aspect of the present invention provides a method for obtaining NK cell progenitor cells or myeloid progenitor cells. According to some embodiments of the present invention, the method comprises obtaining the NK cell progenitor cells or myeloid progenitor cells by inducing differentiation of hematopoietic stem / progenitor cells as described in the seventh aspect.

[0036] Embryonic stem cells (ESCs) have not undergone any genetic (engineering) modification, and their genome and epigenetic characteristics are very stable. The present invention uses the piggyback transposon technology to knock in stably and efficiently expressed mbIL-15 and hnCD16 in hESCs cells to obtain mbIL-15-hnCD16-hESCs, and then induce functional mbIL-15-hnCD16-NK.

[0037] The eleventh aspect of the present invention provides the use of the NK cell progenitor cells or myeloid progenitor cells described in the ninth aspect, and the NK cell progenitor cells or myeloid progenitor cells obtained by the method described in the tenth aspect in the preparation of CAR-related cell products.

[0038] According to some embodiments of the present invention, the CAR-related cell products include CAR-iNK cells and CAR-T cells.

[0039] The twelfth aspect of the present invention provides a CAR-iNK cell. According to some embodiments of the present invention, the CAR-iNK cell comprises an iNK cell obtained by differentiation of the NK cell progenitor cell or myeloid progenitor cell described in the ninth aspect and a chimeric antigen receptor.

[0040] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0042] Figure 1The map of the Piggybac-CD16 mut-mbIL-15 expression vector constructed in Example 1 is shown;

[0043] Figure 2 The base mutation sequencing results of the CD16 gene in Example 1 are shown;

[0044] Figure 3 The flow cytometric diagram of mbIL-15-hnCD16 hESC sorting in Example 2 is shown;

[0045] Figure 4 The flow chart of the secondary purification and sorting of mbIL-15-hnCD16 hESC in Example 2 is shown;

[0046] Figure 5 A schematic diagram of a method for preparing hematopoietic seed cells from stem cells according to an embodiment of the present invention is shown. In which, PS refers to primitive streak; LM refers to lateral plate mesoderm cells; hematopoietic seed cells, i.e., hematopoietic progenitor cells;

[0047] Figure 6 The lateral plate mesoderm cells induced by flow cytometry in Example 3 are shown;

[0048] Figure 7 The flow cytometric analysis of induced hemogenic endothelial cells in Example 4 is shown;

[0049] Figure 8 The flow cytometry analysis of induced hematopoietic seed cells in Example 4 is shown;

[0050] Fig. 9 Figure A shows the NK cells induced by flow cytometry in Example 5; Figure B shows the proliferation results of mbIL-15-hnCD16-NK cells in Example 5 under culture conditions with / without IL-15 factor;

[0051] Fig.10 The results of Example 6 showing the enhanced anti-tumor activity of mbIL-15-hnCD16-NK cells combined with monoclonal antibodies mediated by ADCC;

[0052] Fig.11 The figure shows the survival time of the mbIL-15-hnCD16-NK cells in Example 7 in the animal model. DETAILED DESCRIPTION

[0053] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0054] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0055] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0056] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms and laboratory procedures used herein are terms and routine procedures widely used in the corresponding fields. For example, the standard recombinant DNA and molecular cloning techniques used in the present invention are well known to those skilled in the art and are more fully described in the following documents: Sambrook, J., Fritsch, EF and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter referred to as "Sambrook").

[0057] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0058] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0059] As used herein, the term "and / or" encompasses all combinations of items connected by the term, and each combination should be considered to have been listed separately herein. For example, "A and / or B" encompasses "A," "A and B," and "B." For example, "A, B, and / or C" encompasses "A," "B," "C," "A and B," "A and C," "B and C," and "A and B and C."

[0060] The stem cells described herein include "all-potent stem cells" or "pluripotent stem cells". "All-potent stem cells" or "pluripotent stem cells" are stem cells that can proliferate for a long time in vitro while retaining the potential to differentiate into multiple (pluripotent stem cells) or even all (all-potent stem cells) cell types. In some specific embodiments, the stem cells are human stem cells, i.e., stem cells of human origin.

[0061] The stem cells described herein include, but are not limited to, embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC). Preferably, the stem cells described herein are human pluripotent stem cells, preferably embryonic stem cells within 14 days of fertilization that have not undergone in vivo development.

[0062] Since the lentiviral editing method is time-consuming, labor-intensive, and costly, and the integration is highly random, gene editing that relies on CRISPR technology cannot obtain high-copy number expression of exogenous gene fragments, nor can it continuously screen drugs, and has a high off-target probability. The inventors expressed the cDNA sequences of hnCD16 and mbIL-15 in tandem in the same vector and integrated them into the genome of mammalian pluripotent stem cells, and obtained pluripotent stem cells that stably expressed hnCD16 and mbIL-15. This method is simple to operate and has high efficiency. Hematopoietic stem / progenitor cells are obtained by inducing differentiation, and the NK cells obtained by further inducing differentiation have stronger killing activity and longer in vivo existence time.

[0063] According to a specific embodiment of the present invention, the present invention provides an expression vector, comprising a cDNA sequence encoding a CD16 protein mutant and a mbIL-15 protein, wherein the CD16 protein mutant has p.Phe158Val and p.Ser197Pro mutations relative to the wild-type CD16 protein. The nucleic acid sequence encoding the CD16 protein mutant is shown in SEQ ID NO: 1, and the nucleic acid sequence encoding the mbIL-15 protein is shown in SEQ ID NO: 2.

[0064] The amino acid sequence of the wild-type CD16 protein is shown in SEQ ID NO:5:

[0065] MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLR CHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLFGSKNVSSETVNITITQGLAVSTISSFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK

[0066] According to a specific embodiment of the present invention, the present invention provides a host cell comprising the aforementioned expression vector. The host cell is a human pluripotent stem cell. The human pluripotent stem cell is preferably an embryonic stem cell within 14 days of fertilization that has not undergone in vivo development. The host cell is a host cell into which the aforementioned expression vector is introduced by piggyback transposon gene editing.

[0067] According to a specific embodiment of the present invention, the present invention provides a lateral plate mesoderm cell. The lateral plate mesoderm cell is obtained by inducing differentiation of the host cell described above, wherein the host cell is a human pluripotent stem cell, and the lateral plate mesoderm cell can express the CD16 protein mutant and the mbIL-15 protein.

[0068] There is no particular limitation on the method of obtaining lateral plate mesoderm by inducing differentiation of the host cells. All methods and conditions that are suitable for the growth and differentiation of the host cells and can differentiate into lateral plate mesoderm are within the protection scope of the present invention.

[0069] For example, stem cells can be cultured in a medium I containing a first basal medium and BMP4, ACTIVIN A, bFGF, a Wnt agonist (e.g., CHIR-99021), and a PI3K inhibitor (e.g., PIK-90, LY294002, etc.) for 1-3 days, preferably 24 hours, and then cultured in a second culture stage using medium II for 1-3 days. The culture time can be, for example, 1 day, 1.5 days, 2 days, 2.5 days, or 3 days. Preferably, the stem cells are cultured in medium I for 24 hours, and then cultured in medium II for 24 hours in the second culture stage.

[0070] In some specific embodiments, medium II includes a second basal medium as well as BMP4, a TGF-β inhibitor (eg, A-83-01, SB431542, etc.), and a Wnt inhibitor (eg, C59, IWR-1-endo, etc.).

[0071] In some specific embodiments, the first basal medium and the second basal medium are selected from Essential6 TM , DMEM-high glucose, DMEM / F12, α-MEM, F-12, EBM2, MEM, BME, RPMI 1640, and G-MEM.

[0072] In some specific embodiments, in culture medium I, the Wnt agonist is at least one of CHIR-99021; the PI3K inhibitor is at least one of PIK-90 and LY294002. Preferably, the Wnt agonist is CHIR-99021, and the PI3K inhibitor is PIK-90.

[0073] In some specific embodiments, in culture medium I,

[0074] The BMP4 is used at a concentration of 1-100 ng / mL;

[0075] The use concentration of ACTIVIN A is 1-100 ng / mL;

[0076] The bFGF is used at a concentration of 1-100 ng / mL;

[0077] The Wnt agonist is CHIR-99021, and the concentration of CHIR-99021 is 10nM-100μM;

[0078] The PI3K inhibitor is PIK-90, and the concentration of PIK-90 is 10nM-100μM.

[0079] In some specific embodiments, in culture medium I, the concentration of each factor in BMP4, ACTIVIN A, and bFGF is independently 1-100 ng / mL, such as 2-100 ng / mL, 2-50 ng / mL, 5-100 ng / mL, 5-50 ng / mL, or 5-20 ng / mL; for example, 1 ng / mL, 2 ng / mL, 3 ng / mL, 5 ng / mL, 8 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL, 70 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, or 100 ng / mL. Preferably, the concentration of each factor among BMP4, ACTIVIN A and bFGF is independently 20-50 ng / mL.

[0080] In some specific embodiments, in culture medium I, the CHIR-99021 and PIK-90 are used at concentrations of 10nM-100μM, for example, 20nM-90μM, 30nM-80μM, 40nM-70μM, 50nM-60μM, 60nM-50μM, 70nM-40μM, 80nM-30μM, 90nM-20μM or 100nM-20μM; for example, 10-30nM, 10-50nM, 10-80nM, 10-100nM, 100nM-1μM, 1μM-10μM, 10μM-30μM, 10μM-50μM, 10μM-80μM, 30μM-100μM. For example, 20nM, 30nM, 40nM, 50nM, 60nM, 70nM, 80nM, 90nM, 100nM, 200nM, 300nM, 400nM, 500nM, 600nM, 700nM, 800nM, 900nM, 1μM, 2μM, 3μM, 4μM, 5μM, 6μM, 7μM, 8μM, 9μM, 10μM, 20μM, 30μM, 40μM, 50μM, 60μM, 70μM, 80μM, 90μM, 100μM. Preferably, in medium I, the concentrations of CHIR-99021 and PIK-90 are each independently 100nM-20μM.

[0081] In some specific embodiments, in culture medium II, the TGF-β inhibitor is selected from at least one of A-83-01 and SB431542; the Wnt inhibitor is selected from at least one of C59 and IWR-1-endo. Preferably, the TGF-β inhibitor is A-83-01, and the Wnt inhibitor is C59.

[0082] In some specific embodiments, in Medium II,

[0083] The BMP4 is used at a concentration of 1-100 ng / mL;

[0084] The TGF-β inhibitor is A-83-01, and the concentration of A-83-01 is 10nM-100μM;

[0085] The Wnt inhibitor is C59, and the concentration of C59 is 10nM-100μM.

[0086] In some specific embodiments, in medium II, the concentrations of A-83-01 and C59 are independently 10nM-100μM, such as 20nM-90μM, 30nM-80μM, 40nM-70μM, 50nM-60μM, 60nM-50μM, 70nM-40μM, 80nM-30μM, 90nM-20μM or 100nM-20μM. For example, 10-30nM, 10-50nM, 10-80nM, 10-100nM, 100nM-1μM, 1μM-10μM, 10μM-30μM, 10μM-50μM, 10μM-80μM, 30μM-100μM. For example, 20nM, 30nM, 40nM, 50nM, 60nM, 70nM, 80nM, 90nM, 100nM, 200nM, 300nM, 400nM, 500nM, 600nM, 700nM, 800nM, 900nM, 1μM, 2μM, 3μM, 4μM, 5μM, 6μM, 7μM, 8μM, 9μM, 10μM, 20μM, 30μM, 40μM, 50μM, 60μM, 70μM, 80μM, 90μM, 100μM. Preferably, in medium II, the concentrations of A-83-01 and C59 are independently 100nM-20μM. More preferably, 1-10μM.

[0087] In some specific embodiments, in medium II, BMP4 is used at a concentration of 1-100 ng / mL, such as 2-100 ng / mL, 2-50 ng / mL, 5-100 ng / mL, 5-50 ng / mL, or 5-20 ng / mL; for example, 1 ng / mL, 2 ng / mL, 3 ng / mL, 5 ng / mL, 8 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL, 70 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, or 100 ng / mL. Preferably, in medium II, BMP4 is used at a concentration of 5-50 ng / mL.

[0088] In some specific embodiments, Medium I is Essential 6 Medium containing 40 ng / mL BMP4, 30 ng / mL ACTIVIN A, 20 ng / mL bFGF, 6 μM CHIR-99021, and 100 nM PIK-90.

[0089] Medium II was Essential 6 medium containing 30 ng / mL BMP4, 1 μM A-83-01, and 1 μM C59.

[0090] It should be noted that the medium suitable for the growth of the stem cells is not limited to the composition of medium I and medium II, and can also be any culture condition or medium suitable for the growth of stem cells known in the art. However, preferably, the stem cells are cultured in medium I for 1-3 days, and then cultured in medium II for 1-3 days in the second culture stage. This is further conducive to the differentiation of stem cells to obtain lateral plate mesoderm cells.

[0091] The present invention also provides a hemogenic endothelial cell, which is obtained by mixing the lateral plate mesoderm cells and feeder layer cells mentioned above to differentiate into the hemogenic endothelial cell, wherein the hemogenic endothelial cell can express the CD16 protein mutant and mbIL-15 protein.

[0092] In some specific embodiments, the feeder cells include, but are not limited to, one or more of the following: monocytes, macrophages, dendritic cells, and B cells. In some specific embodiments, the feeder cells are macrophages and / or dendritic cells.

[0093] In some specific embodiments, the human-derived feeder layer cells are differentiated from human stem cells, which are induced pluripotent stem cells or embryonic stem cells within 14 days of fertilization that have not undergone in vivo development.

[0094] In some specific embodiments, the feeder cells of human origin are isolated from human tissues or organs. For example, the feeder cells of human origin are isolated from human blood tissues, such as neonatal umbilical cord blood or peripheral blood.

[0095] It should be noted that there is no particular limitation on the source of human-derived feeder cells. For example, they may be feeder cells directly isolated from human blood tissue as mentioned above, or feeder cells obtained by differentiation of human stem cells.

[0096] In some specific embodiments, the lateral plate mesoderm cells are mixed with human-derived feeder cells at a ratio of 1:5-1:200, such as 1:15, 1:10, 1:20, 1:30, 1:40, 1:60, 1:90, 1:100, 1:110, 1:120, 1:130, 1:140, 1:150, 1:160, 1:170, 1:180, 1:190 or 1:200. Preferably, the lateral plate mesoderm cells are mixed with human-derived feeder cells at a ratio of 1:10-1:50.

[0097] In some specific embodiments, when the lateral plate mesoderm cells are mixed with the human-derived feeder cells, the lateral plate mesoderm cells are a single cell suspension.

[0098] The present invention also provides a hematopoietic stem / progenitor cell, which is obtained by inducing differentiation of the hemogenic endothelial cells described above, wherein the hematopoietic stem / progenitor cell can express the CD16 protein mutant and mbIL-15 protein.

[0099] In some specific embodiments, organoids obtained by mixing lateral plate mesoderm cells with human-derived feeder cells are cultured in medium III for 10-18 days to obtain hemogenic endothelial cells (8-10 days) and hematopoietic stem / progenitor cells (15-18 days).

[0100] Culture medium III includes the third basal medium and TGF-β inhibitor, hydrocortisone, Flt3L, TPO, SCF, EGF, VEGF, bFGF, IGF- 1. In some embodiments, fresh culture medium III is replaced every 1-3 days.

[0101] In some specific embodiments, Medium III further comprises ascorbic acid and serum replacement.

[0102] In some specific embodiments, the third basal medium is selected from Essential 6 TM , DMEM-highglucose, DMEM / F12, α-MEM, F-12, EBM2, MEM, BME, RPMI 1640, and G-MEM.

[0103] In some specific embodiments, in culture medium III, the TGF-β inhibitor is selected from at least one of A-83-01 and SB431542; preferably, the TGF-β inhibitor is SB431542.

[0104] In some specific embodiments, in medium III, the serum replacement is a serum-free additive selected from KOSR, B27 (Gibco 17504-044), Ultroser TM G. (Dakoway, 6122011) at least one of the cell culture supplements.

[0105] In some specific embodiments, in medium III,

[0106] The TGF-β inhibitor is SB431542, and the concentration of SB431542 is 10nM-100μM;

[0107] The hydrocortisone is used at a concentration of 1-100 μM;

[0108] The Flt3L is used at a concentration of 1-100 ng / mL;

[0109] The TPO is used at a concentration of 1-100 ng / mL;

[0110] The SCF is used at a concentration of 1-100 ng / mL;

[0111] The EGF is used at a concentration of 1-100 ng / mL;

[0112] The VEGF is used at a concentration of 1-100 ng / mL;

[0113] The bFGF is used at a concentration of 1-100 ng / mL;

[0114] The IGF-1 is used at a concentration of 1-100 ng / mL;

[0115] The ascorbic acid is used at a concentration of 10-100 μg / mL;

[0116] The serum-free additive is Cell culture supplement, The concentration of cell culture supplements is 1-30% V / V.

[0117] In some specific embodiments, in Medium III, Flt3L, TPO, SCF, EGF, VEGF, bFGF and IGF-1 are each independently used at a concentration of 1-100 ng / mL, such as 2-100 ng / mL, 2-50 ng / mL, 5-100 ng / mL, 5-50 ng / mL, or 5-20 ng / mL; for example, 1 ng / mL, 2 ng / mL, 3 ng / mL, 5 ng / mL, 8 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL, 70 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, or 100 ng / mL. Preferably, in medium III, Flt3L, TPO, SCF, EGF, VEGF, bFGF and IGF-1 are used at a concentration of 5-50 ng / mL each independently.

[0118] In some specific embodiments, the SB43154 is used at a concentration of 10 nM-100 μM, such as 20 nM-90 μM, 30 nM-80 μM, 40 nM-70 μM, 50 nM-60 μM, 60 nM-50 μM, 70 nM-40 μM, 80 nM-30 μM, 90 nM-20 μM or 100 nM-20 μM. For example, 10-30 nM, 10-50 nM, 10-80 nM, 10-100 nM, 100 nM-1 μM, 1 μM-10 μM, 10 μM-30 μM, 10 μM-50 μM, 10 μM-80 μM, 30 μM-100 μM. For example, 20nM, 30nM, 40nM, 50nM, 60nM, 70nM, 80nM, 90nM, 100nM, 200nM, 300nM, 400nM, 500nM, 600nM, 700nM, 800nM, 900nM, 1μM, 2μM, 3μM, 4μM, 5μM, 6μM, 7μM, 8μM, 9μM, 10μM, 20μM, 30μM, 40μM, 50μM, 60μM, 70μM, 80μM, 90μM, 100μM. Preferably, in medium III, the concentration of SB43154 used is 100nM-20μM. More preferably, it is 1-10μM.

[0119] In some embodiments, the Medium III is Essential 6 Medium containing 10 μM SB431542, 10 μM hydrocortisone, 5 ng / mL Flt3L, 5 ng / mL TPO, 50 ng / mL SCF, 50 ng / mL EGF, 50 ng / mL VEGF, 50 ng / mL bFGF, 50 ng / mL IGF-1, 50 μg / mL ascorbic acid and 2% SUPERGROW Cell Culture Supplement.

[0120] The present invention also provides a method for obtaining NK cell progenitor cells or myeloid progenitor cells, comprising obtaining the NK cell progenitor cells or myeloid progenitor cells by inducing differentiation of the hematopoietic stem / progenitor cells described above.

[0121] It should be noted that the composition of culture medium I, II, and III, including but not limited to the aforementioned basic culture medium and additive components, may also include the addition of some small molecules or the removal of some small molecules or the addition of non-essential amino acids, as long as the culture medium composition can differentiate stem cells into lateral plate mesoderm cells, it is included in the protection scope of the present invention. Among them, the non-essential amino acids are selected from glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine and any combination thereof.

[0122] In some specific embodiments, all of the above culturing is performed at about 37 degrees Celsius.

[0123] The scheme of the present disclosure will be explained below in conjunction with the examples. Those skilled in the art will appreciate that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. Where specific techniques or conditions are not indicated in the examples, the techniques or conditions described in the literature in this area or the product instructions are used. Where the manufacturers of reagents or instruments are not indicated, they are all conventional products that can be obtained commercially.

[0124] Example 1 Construction of Piggybac-CD16 mut-mbIL-15 expression vector

[0125] Insert the nucleotide sequences of CD16 mut (F158V, S197P, hnCD16) and mbIL-15 cDNA into the piggybac transponson expression vector. The 526th base of CD16 cDNA is mutated from T to G, and the 589th base is mutated from T to C. The mbIL-15 is the cDNA nucleotide sequence of the IL-15 / IL-15Rα membrane-bound interleukin 15 protein. The plasmid map of the successful construction is as follows: Figure 1The nucleic acid sequence encoding hnCD16 is shown in SEQ ID NO: 1, and the nucleic acid sequence encoding mbIL-15 is shown in SEQ ID NO: 2. hnCD16-P2A-mbIL-15 was constructed into the piggybac transponson vector, and the base mutation of the CD16 gene was confirmed by sequencing. Figure 2 As shown. The cDNA sequences of hnCD16 and mbIL-15 are expressed in tandem in the same vector and integrated into the genome of mammalian pluripotent stem cells, thereby obtaining pluripotent stem cells that stably express hnCD16 and mbIL-15.

[0126] The sequence of SEQ ID NO: 1 is as follows:

[0127] ATGTGGCAGCTGCTCCTCCCAACTGCTCTGCTACTTCTAGTTTCAGCTGGCATGCGGACTGAAGATCTCCCAAAGGCTGTGGTGTTCCTGGAGCCTCAATGGTACAGGGTGCTCGAGAAGGACAGTGTGACTCTGAAGTGCCAGGGAGCCTACTCCCCTGAGGACAATTCCACACAGTGGTTTCACAATGAGAGCCTCATCTCAAGCCAGGCCTCGAGCTACTTCATTGACGCTGCCACAGTCGACGACAGTGGAGAGTACAGGTGCCAGACAAACCTCTCCACCCTCAGTGACCCGGTGCAGCTAGAAGTCCATATCGGCTGGCTGTTGCTCCAGGCCCCTCGGTGGGTGTTCAAGGAGGAAGACCCTATTCACCTGAGGTGTCACAGCTGGAAGAACACTGCTCTGCATAAGGTCACATATTTACAGAATGGCAAAGGCAGGAAGTATTTTCATCATAATTCTGACTTCTACATTCCAAAAGCCACACTCAAAGACAGCGGCTCCTACTTCTGCAGGGGGCTTGTTGGGAGTAAAAATGTGTCTTCAGAGACTGTGAACATCACCATCACTCAAGGTTTGGCAGTGCCAACCATCTCATCATTCTTTCCACCTGGGTACCAAGTCTCTTTCTGCTTGGTGATGGTACTCCTTTTTGCAGTGGACACAGGACTATATTTCTCTGTGAAGACAAACATTCGAAGCTCAACAAGAGACTGGAAGGACCATAAATTTAAATGGAGAAAGGACCCTCAAGACAAA

[0128] The sequence of SEQ ID NO:2 is as follows:

[0129] TCAGGTGGAGGTTCAGGTGGAGGTGGATCAGGTGGAGGTGGATCAGGTGGAGGTGGATCAGGTGGAGGTTCACTGCAAATCACGTGCCCTCCCCCATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCAGGGAGCGGTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACGTC CAGCCTGACGGAGTGCGTGTGAACAAGGCCACGAATGTCGCCCACTGGACAACCCCCAGTCTCAAATGCATTAGAGACCCTGCCCTGGTTCACCAAAGGCCAGCGCCACCCTCCACAGTAACGACGGCAGGGGTGACCCCACAGCCAGAGAGCCTCTCCCCTTCTGGAAAAGAGCCCGCAGCTTCATCTCCCAGCT CAAACAACACAGCGGCCACAACAGCAGCTATTGTCCCGGGCTCCCAGCTGATGCCTTCAAAATCACCTTCCACAGGAACCACAGAGATAAGCAGTCATGAGTCCTCCCACGGCACCCCCTCTCAGACAACAGCCAAGAACTGGGAACTCACAGCATCCGCCTCCCACCAGCCGCCAGGTGTGTATCCACAGGGCCAC AGCGACACCACTGTGGCTATCTCCACGTCCACTGTCCTGCTGTGTGGGCTGAGCGCTGTGTCTCCTGGCATGCTACCTCAAGTCAAGGCAAACTCCCCCGCTGGCCAGCGTTGAAATGGAAGCCATGGAGGCTCTGCCGGTGACTTGGGGGACCAGCAGCAGAGATGAAGACTTGGAAAACTGCTCTCACCACCTA

[0130] Example 2 Construction of hnCD16 and mbIL-15 targeted stem cell lines

[0131] The hnCD16-P2A-mbIL-15 expression element was targeted on the human pluripotent stem cell line (hPSC) by piggyback transponson technology. In this example, hES cells were used to construct the targeted stem cell line, and the construction steps were as follows:

[0132] 1) At least 1 hour in advance, apply Vitronectin matrix gel, 1 mL / six wells;

[0133] 2) Discard the supernatant of E8 medium (Gibco, A1517001), wash hES cells with 0.5mL DPBS, discard DPBS, add 0.5mL Tryple and digest in a 37℃ cell culture incubator for 3min. Discard the Tryple digestion solution, add an appropriate amount of E8 medium, use a 1mL pipette to blow the cells into a single cell suspension, then transfer to a 15mL centrifuge tube and count. Take 1 million cells into a 1.5mL EP tube, centrifuge at 300g for 3min, and discard the supernatant;

[0134] 3) Mix electroporation solution A and solution B (Celetrix, Cat. No. 1207) in a 1:1 ratio, then resuspend the cell pellet with 20ul of the mixture, add the required amount (1ug Piggybac-hnCD16-mbIL-15 + 1ug Piggybactransposase) of linearized plasmid, mix well and transfer to the electroporation cup, try not to have bubbles, where the Piggybactransposase vector is purchased from System Biosciences, cat#PB210PA-1. Select the Cell line program, put the electroporation cup into the card slot, select 480V voltage, click OK and run. After hearing the sound, the main interface displays OK, which means that the electroporation is complete and the electroporation cup can be removed. Take out the Vitronectin plate that has been laid in advance, discard the Vitronectin, add an appropriate amount of E8 medium (containing 10uM Y27632), then quickly transfer the liquid from the electroporation cup to the culture dish with a pipette, gently shake to mix, and then place in a 37°C, 5% CO2 cell culture incubator;

[0135] 4) After culturing for 24 hours, change to E8 medium without Y27632, observe the cell growth status every day for 5-7 days, and when the density of clones is close to 80% under the microscope, sorting can be carried out;

[0136] 5) During sorting, discard the supernatant of E8 culture medium, wash hES cells with 0.5mL DPBS, discard DPBS, add 0.5mL Tryple and digest in a 37°C cell culture incubator for 3 minutes. Discard the Tryple digestion solution, add an appropriate amount of E8 culture medium, use a 1mL pipette to blow the cells into a single cell suspension, then transfer to a 5mL flow tube and count. After centrifugation at 300g for 3 minutes, discard the culture medium, add 200ul 2% FBS+DPBS to resuspend the cells, add appropriate amounts of anti-human CD16 antibody and anti-human mbIL-15 antibody, and incubate on ice in the dark for 15-30 minutes. Then add 1mL 2% FBS+DPBS, centrifuge at 300g for 3 minutes, discard the supernatant, resuspend with DAPI solution, and sort using a Sony MA900 sorter;

[0137] 6) Sorting out CD16 + mbIL-15 + The cells were centrifuged at 300 g for 3 min, resuspended in 2 mL of E8 medium at a volume of 50,000 cells / well, and the pre-plated Vitronectin was discarded, and seeded in one well of a six-well plate. Placed in a cell culture incubator at 37°C and 5% CO2 for 5-7 days;

[0138] 7) After 5-7 days of culture, the clone density was close to 80% under microscope observation, and sorting was performed again. + mbIL-15 + The cells were centrifuged at 300 g for 3 min, resuspended in 2 mL E8 medium at 300,000 to 100,000 cells / well, and the pre-plated Vitronectin was discarded. The cells were seeded in one well of a six-well plate and placed in a 37°C, 5% CO2 cell culture incubator for 5-7 days to obtain a higher purity hES target cell line.

[0139] The piggybac-hnCD16-P2A-mbIL-15 plasmid and the piggybac transposase plasmid were co-transfected into hPSCs by electroporation. After electroporation, the cells were resuspended in E8 medium (Gibco, A1517001) and cultured for 5-7 days. Anti-human CD16 antibody and anti-human IL-15 antibody were used to select double positive cells co-expressing CD16 and IL-15. The cell sorting results are shown in Figure 3 The sorted cells were re-cultured for 5-7 days, and the cells were collected again and sorted again with anti-human CD16 antibody and anti-human IL-15 antibody to obtain CD16 and IL-15 double positive cells with higher purity. The cell sorting results are shown in Figure 4 shown.

[0140] Example 3 Differentiation of pluripotent stem cells (derived from embryonic stem cells) into lateral plate mesoderm

[0141] Figure 5 The process of obtaining lateral plate mesoderm cells by differentiation of stem cells is shown in the figure. The specific steps of differentiation are as follows:

[0142] The pluripotent stem cells (derived from embryonic stem cells) obtained in Example 2, which were cultured in a monolayer to a density of 80% adhesion, were digested with Accutase (Sigma-Aldrich, A6964) for 1 min, resuspended in E8 medium (Gibco, A1517001) to form a single cell suspension, and transferred to a cell culture dish covered with VTN (Animal-Free Human Vitronectin Matrix, PeproTech, AF-VMB-220) after adding 2 μM ROCK inhibitor thiazovivin (Selleck, S1459), and cultured in an incubator for 24 h (this time defined as Day 0 of the induction program). The ncTarget medium in the culture dish was aspirated, and medium I was added, and the culture was continued in the incubator for 24 h (this time defined as Day 1 of the induction program). The medium I was aspirated, and medium II was added, and the culture was continued for 24 h (this time defined as Day 2 of the induction program). The proportion of APLNR+ mesoderm cells was detected by flow cytometry, and the results are as follows: Figure 6 shown.

[0143] Medium I: Essential 6 medium (Gibco, A1516401 or STEMCELL Technologies, 05946) containing 40 ng / mL BMP4 (R&D Systems, 314-BP-010), 30 ng / mL ACTIVIN A (PeproTech, 120-14P), 20 ng / mL bFGF (PeproTech, 100-18B), 6 μM CHIR-99021 (Selleck, S1263) and 100 nM PIK-90 (Selleck, S1187).

[0144] Medium II: Essential 6 medium containing 30 ng / mL BMP4 (R&D Systems, 314-BP-010), 1 μM A-83-01 (Selleck, S7692) and 1 μM C59 (Selleck, S7037).

[0145] Example 4 Organoid Culture - Hematopoietic Stem / Progenitor Cell (Embryonic Stem Cell Source) Stage Induction

[0146] The lateral plate mesoderm cells obtained in Example 3 were digested and prepared into a single cell suspension, which was mixed with OP9 cells (mouse bone marrow stromal cells as feeder cells) at a mixing ratio of 1:20 and a total cell volume of 1 million to prepare organoids. Induction was continued for 10-18 days under medium III conditions, with the medium changed every 1-3 days. At the D8-D10 stage, a small amount of organoids was taken to detect the induction of hemogenic endothelial cells. Figure 7 The results showed that hemogenic endothelial cells can be obtained by using embryonic stem cells and OP9 as feeder cells to prepare organoids. On Day 16, a small amount of organoids were collected and flow cytometry was used to detect hematopoietic stem / progenitor cells (CD45 + CD34 + CD43 + ), the result is as follows Figure 8 As shown in the figure, it shows that both embryonic stem cells and OP9 as feeder cells can be used to prepare organoids to obtain CD45 + CD34 + CD43 + Phenotypic hematopoietic seed cells.

[0147] Medium III: Essential 6 medium containing 10 μM SB431542 (selleck, S1067), 10 μM hydrocortisone (selleck, S1696), 5 ng / mL Flt3L (PeproTech, 300-19), 5 ng / mL TPO (PeproTech, 300-18), 50 ng / mL SCF (PeproTech, 300-07), 50 ng / mL EGF (PeproTech, 100-47), 50 ng / mL VEGF (R&D System, 293-VE-010), 50 ng / mL bFGF (PeproTech, 100-18B), 50 ng / mL IGF-1 (PeproTech, 100-11), 50 μg / mL ascorbic acid and 2% SUPERGROW cell culture supplement (Dako, 6122011).

[0148] Example 5 Proliferation of mbIL-15-hnCD16-NK cells in the absence of IL-15 factor culture conditions

[0149] The hematopoietic seed cells obtained in Example 4 were further induced to differentiate under medium IV conditions in the absence of IL-15 factor for 10-14 days, with the medium changed every 1-3 days. At D27-D30, the organoids were digested to obtain mbIL-15-hnCD16-NK cells, and the cell output yield of each organoid was counted ( Fig. 9(B) Flow cytometry analysis of digested organoids. Fig. 9 As shown in A, it is shown that mbIL-15-hnCD16-NK cells were obtained by further inducing differentiation of hematopoietic seed cells.

[0150] Medium IV: Contains 15% SUPERGROW Cell Culture Supplement (Dakoway, 6122011), 2 mM GlutaMAX TM (Gibco, 35050061), 1 μM β-mercaptoethanol (Sigma-Aldrich, M3148), 5 ng / mL sodium selenite (Sigma-Aldrich, S5261), 50 μM ethanolamine (Sigma-Aldrich, 15014), 20 μg / mL ascorbic acid (Sigma-Aldrich, A-5960), 1% penicillin-streptomycin solution (Hyclone, SV30010), 5 ng / mL IL-3 (PeproTech, 200-03), 20 ng / mL SCF, 20 ng / mL IL-7 (PeproTech, 200-07) and 10 ng / mL Flt3L (PeproTech, 300-19) in DMEM medium.

[0151] Example 6 mbIL-15-hnCD16-NK cells combined with monoclonal antibodies enhance anti-tumor activity via ADCC

[0152] To explore the combined effects of mbIL-15-hnCD16-NK cells and monoclonal antibodies, the following experiments were performed:

[0153] The mbIL-15-hnCD16-NK cells obtained from the digestive organoids cultured at the D27-D30 stage in Example 5 were co-incubated with 1×10^4 CFSE-labeled human B lymphoma cell line Raji at a ratio of 1:1, 5:1 and 10:1, respectively. The control group did not add anti-CD20 antibody (Rituximab), and the experimental group added 20 μg / mL Rituximab. 7-AAD in CFSE+Raji cells was detected after 4 hours + Cell percentage (killing rate).

[0154] The results are as follows Fig.10 As shown, Rituximab is anti-CD20 rituximab, X axis: E:T ratio (NK cells: tumor cells), Y axis: percentage of tumor killing. The co-incubation time is 4 hours, n=4. Fig.10This indicates that mbIL-15-hnCD16-NK cells enhance the killing of B lymphoma cell line Raji through ADCC. In Example 5, the use of mbIL-15-hnCD16-NK cells can further improve the effect of NK cells combined with antibodies in killing tumor cells.

[0155] Example 7mbIL-15-hnCD16-NK cells significantly prolong survival time in animal models

[0156] Using the mbIL-15-hnCD16-NK cells obtained from the digestive organoids cultured at the D27-D30 stage in Example 5, 6-8 week old B-NDG mice were irradiated with a sublethal dose (1.5Gy). After an interval of 4 hours, NK cells / mbIL-15-hnCD16-NK cells were transplanted into B-NDG mice through the tail vein at a dose of 1×10^7 cells / mouse. Peripheral blood was collected from mice at Day 4, Day 7, and Day 14, and the proportion of donor NK cells in the peripheral blood was analyzed. The results are shown in Figure 2. Fig.11 As shown, it indicates that the survival time of mbIL-15-hnCD16-NK cells in animal models is significantly prolonged.

[0157] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", "some implementation schemes" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are contradictory.

[0158] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. An expression vector, characterized in that: It comprises cDNA sequences encoding CD16 protein mutant and mbIL-15 protein, wherein the CD16 protein mutant has p.Phe158Val and p.Ser197Pro mutations relative to the wild-type CD16 protein.

2. The expression vector according to claim 1, characterized in that The nucleic acid sequence encoding the CD16 protein mutant is shown in SEQ ID NO: 1, and the nucleic acid sequence encoding the mbIL-15 protein is shown in SEQ ID NO:

2.

3. A host cell, characterized in that Comprising the expression vector according to claim 1 or 2.

4. The host cell according to claim 3, characterized in that The host cell is a human pluripotent stem cell; Optionally, the human pluripotent stem cells are embryonic stem cells within 14 days of fertilization that have not undergone in vivo development; Optionally, the host cell is a host cell into which the expression vector of claim 1 or 2 is introduced by piggybac transposon gene editing.

5. A lateral plate mesoderm cell, characterized in that: The lateral plate mesoderm cells are obtained by inducing differentiation of the host cells according to claim 3 or 4, wherein the host cells are human pluripotent stem cells, and the lateral plate mesoderm cells are capable of expressing the CD16 protein mutant and the mbIL-15 protein.

6. A method for obtaining lateral plate mesoderm cells, characterized in that: The expression vector of claim 1 or 2 is introduced into human pluripotent stem cells through piggyback transposon gene editing, and the human pluripotent stem cells are induced to differentiate, thereby obtaining the lateral plate mesoderm cells.

7. A hemogenic endothelial cell, characterized in that: The hemogenic endothelial cells are obtained by mixing the lateral plate mesoderm cells and feeder layer cells according to claim 5 to differentiate into the hemogenic endothelial cells, wherein the hemogenic endothelial cells are capable of expressing the CD16 protein mutant and the mbIL-15 protein; Optionally, the feeder layer cells include at least one of monocytes, macrophages, dendritic cells and B cells; Optionally, the feeder cells are macrophages and / or dendritic cells.

8. A method for obtaining hemogenic endothelial cells, characterized in that: The method comprises mixing and culturing the lateral plate mesoderm cells of claim 5 with feeder layer cells so as to differentiate and obtain the hemogenic endothelial cells; Optionally, the feeder layer cells include at least one of monocytes, macrophages, dendritic cells and B cells; Optionally, the feeder cells are macrophages and / or dendritic cells.

9. A hematopoietic stem / progenitor cell, characterized in that: The hematopoietic stem / progenitor cells are obtained by inducing differentiation of the hemogenic endothelial cells according to claim 7, wherein the hematopoietic stem / progenitor cells are capable of expressing the CD16 protein mutant and the mbIL-15 protein.

10. A method for obtaining hematopoietic stem / progenitor cells, characterized in that: The method comprises obtaining the hematopoietic stem / progenitor cells by inducing differentiation of the hemogenic endothelial cells according to claim 7.

11. A NK cell progenitor cell or a myeloid progenitor cell, characterized in that: The NK cell progenitor cells or myeloid progenitor cells are obtained by the induced differentiation of the hematopoietic stem / progenitor cells according to claim 9, wherein the NK cell progenitor cells or myeloid progenitor cells are both capable of expressing the CD16 protein mutant and the mbIL-15 protein.

12. A method for obtaining NK cell progenitor cells or myeloid progenitor cells, characterized in that: The method comprises obtaining the NK cell progenitor cells or myeloid progenitor cells by inducing differentiation of the hematopoietic stem / progenitor cells as described in claim 9.

13. Use of the NK cell progenitor cells or myeloid progenitor cells described in claim 11 or the NK cell progenitor cells or myeloid progenitor cells obtained by the method for obtaining NK cell progenitor cells or myeloid progenitor cells described in claim 12 in the preparation of CAR-related cell products.

14. The use according to claim 13, characterized in that The CAR-related cell products include CAR-iNK cells and CAR-T cells.

15. A CAR-iNK cell, characterized in that: It comprises iNK cells obtained by differentiating the NK cell progenitor cells or myeloid progenitor cells according to claim 11 and a chimeric antigen receptor.

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