A ric cell and a preparation method and application thereof

Abstract

The present application relates to a kind of RIC cells and its preparation method and application, especially it is related to the lineage-specific differentiation of pluripotent or multipotent stem cells, specifically related to a kind of RIC cell obtained from stem cell differentiation and its preparation method and application.

Description

[0001] This application is based on and claims priority to CN application No. 202110903886.0, filed on August 6, 2021, and PCT application No. PCT / CN2022 / 104873, filed on July 11, 2022, the disclosures of which are incorporated herein by reference in their entirety. Technical Field

[0002] This application relates to the field of stem cell biology, and more particularly to a recombinant intracellular column (RIC) cell, its preparation method, and its applications. Specifically, it relates to lineage-specific differentiation of pluripotent or multipotent stem cells, and more specifically to an RIC cell differentiated from stem cells, its preparation method, and its applications. Background Technology

[0003] Pluripotent stem cells can differentiate into the vast majority of cell types, including all cells derived from the ectoderm, endoderm, and mesoderm. Lymphogenesis, starting from hematopoietic cell populations isolated from mouse or human bone marrow, umbilical cord blood (UCB), or peripheral blood, is documented; however, little is known about the in vitro differentiation of pluripotent stem cells into lymphocytes, and the technology is not yet mature. Meanwhile, although innate immune cells derived from lymphoid lines have been used in cancer treatment in recent years, the efficient production of such cells remains a significant challenge. Summary of the Invention

[0004] Terminology Definition

[0005] In this document, unless otherwise stated, scientific and technical terms used have the meanings commonly understood by those skilled in the art. Furthermore, the procedures described herein, such as molecular genetics, nucleic acid chemistry, cell culture, biochemistry, and cell biology, are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0006] As used herein, the term "embryonic stem cell" can refer to pluripotent cells isolated from an embryo and maintained in an in vitro cell culture. These cells are cultured cells isolated from cultured embryos that are capable of rapid division and simultaneously maintain pluripotency. Embryonic stem cells can be cultured with or without feeder cells. Embryonic stem cells can be isolated from early embryos, including but not limited to blastocysts and preblastocysts. Embryonic stem cells may have a round cell morphology and can grow in round cell clumps on a feeder layer. Embryonic stem cells are well known to those skilled in the art. Their source can be human or non-human animals. For human embryonic stem cells, the use of stem cells isolated from human embryos within 14 days of fertilization that have not undergone in vivo development is limited to those obtained from such embryos.

[0007] As used herein, the term "in vitro" refers to an artificial environment and the processes and reactions within it. In vitro environments are exemplified by test tubes and cell cultures, but are not limited to these.

[0008] As used in this article, the term "in vivo" refers to the natural environment (i.e., the animal or cell) and the processes and reactions within it.

[0009] As used herein, the term "culture" refers to the product obtained by culturing cells (e.g., the RIC cell population of the present invention) in a culture medium.

[0010] As used herein, the term "culture supernatant" refers to a culture medium containing no cells themselves, obtained from culturing cells (e.g., the RIC cell population of the present invention). Thus, a culture supernatant suitable for use in the present invention can be obtained, for example, by separating and removing cellular components after culturing. This culture supernatant can also be subjected to other treatments, such as centrifugation, concentration, solvent replacement, dialysis, freezing, drying, freeze-drying, dilution, desalting, preservation, etc.

[0011] As used herein, the term "pharmaceutically acceptable carrier or excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, ionic strength enhancers, agents for maintaining osmotic pressure, agents for delaying absorption, diluents, adjuvants, preservatives, etc. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Agents for maintaining osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents for delaying absorption include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol), etc. Adjuvants include, but are not limited to, aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete Freund's adjuvant), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, p-hydroxybenzoate, chlorobutanol, phenol, sorbic acid, etc. In some embodiments, the pharmaceutically acceptable carrier or excipient is a sterile isotonic or non-aqueous solution (e.g., balanced salt solution or physiological saline), dispersion, suspension, or emulsion.

[0012] As used herein, the term “prevention” refers to methods implemented to prevent or delay the occurrence of a disease, condition, or symptom in a subject, or to minimize its effect if it occurs. The term “treatment” refers to methods implemented to achieve a beneficial or desired clinical outcome. Beneficial or desired clinical outcomes include, but are not limited to, reducing the rate of disease progression, improving or alleviating the disease state, and resolving or improving prognosis, whether detectable or undetectable. The amount of a therapeutic agent that effectively relieves symptoms of any particular disease can vary depending on factors such as the patient’s disease state, age, and weight, and the drug’s ability to elicit the desired response in the subject. Whether disease symptoms are relieved can be assessed by any clinical measurement typically used by a physician or other skilled healthcare provider to assess the severity or progression of the symptom.

[0013] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for disease prevention is an amount sufficient to prevent, stop, or delay the onset of disease; an effective amount for disease treatment is an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic purposes will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the manner of administration of the drug, and other concurrent treatments, etc.

[0014] As used herein, the term “subject” includes, but is not limited to, various animals, such as mammals, such as bovines, equines, sheep, suidae, canines, felines, rabbits, rodents (e.g., mice or rats), non-human primates (e.g., macaques or cynomolgus monkeys), or humans.

[0015] As used in this article, the terms “proliferation” or “expansion” refer to the ability of a cell or cell population to increase in number.

[0016] As used herein, a composition containing the terms "purified cell population" or "purified cell composition" means that at least 30%, 50%, 60%, generally at least 70%, and more preferably 80%, 90%, 95%, 98%, 99% or more of the cells in the composition are of an identified type.

[0017] As used in this article, the term "primary immune cells" refers to natural killer cells that are isolated directly from tissues (such as umbilical cord blood, peripheral blood, or bone marrow) taken from the body.

[0018] As used in this article, the term "CAR" refers to chimeric antigen receptor.

[0019] As used in this article, the term "expression rate" is the percentage of cells that express a particular protein. For example, an 80% CAR expression rate means that 80% of cells express CAR.

[0020] As used in this article, the term "RIC sac" refers to a sac-like structure that produces RIC cells.

[0021] As used herein, the term "RIC cell" refers to the immune cell or cell population described in this invention, the characteristics of which are set forth in the specification.

[0022] As used in this article, the term "seed cell" refers to an immature cell capable of forming an adult cell.

[0023] As used in this article, the term "X+ cell" refers to a cell that is X-positive or expresses the X gene. For example, a CD34+ cell is a cell that expresses CD34. X expression can be tested using existing techniques.

[0024] As used in this article, the term "X-cell" refers to an X-negative cell or a cell that does not express the X gene. For example, a CD34-cell is a cell that does not express CD34. X expression can be tested using existing techniques.

[0025] As used herein, the term "NKp30 / NCR3" belongs to the IgSF family and is a natural cytotoxic receptor. It is distributed in resting and activated NK cells and mediates NK cell killing by transducing activation signals with the ITAM-containing DAP12 and by cooperating with NKp46.

[0026] As used herein, the term "NKp44 / NCR2" belongs to the IgSF family and is a natural cytotoxic receptor. It is expressed on activated NK cells and participates in mediating NK cell killing activity by interacting with ITAM-containing DAP12.

[0027] As used herein, the term "NKG2C / KLRC2" binds to the non-classical HLA-I molecule HLA-E to deliver activation signals. NKG2C can recognize the UL40 peptide presented by HLA-E in HCMV-infected cells, promoting the release of IFNγ.

[0028] As used in this article, the term "NKG2D / KLRK1" is an activating receptor expressed on human natural killer cells, and DAP10 binds only to the intracellular region of NKG2D to pass on the activation signal. It plays an important role in innate immunity, participating in the recognition of virus-infected cells and the killing of tumor cells.

[0029] As used in this article, the term "SLAMF7 / CS1" refers to member 7 of the signaling lymphocyte activation molecule family, expressed on the surface of natural killer cells. There are two variants of the human CS1 molecule, long-form CS1-L and short-form CS1-S, both of which are expressed on NK cells. CS1-L is the NK cell activation receptor, while CS1-S cannot activate NK cells.

[0030] As used in this article, the term "KLRB1 / CD161" refers to member 1 of the cytotoxic lectin-like receptor subfamily B. It belongs to the NK cell inhibitory receptor family.

[0031] As used in this article, the term "KLRC1 / NKG2A" can bind to the non-classical HLA-I molecule HLA-E to transmit inhibitory signals. Uterine NK cells, however, highly express NKG2A. Under normal physiological conditions, placental trophoblast cells express HLA-E, which interacts with NKG2A to inhibit the killing effect of NK cells in the decidua and maintain placental immune tolerance. However, under pathological conditions, tumor cells upregulate HLA-E molecules to evade NK cell killing.

[0032] As used in this article, the term "LAIR1" refers to leukocyte-associated immunoglobulin-like receptor 1. It belongs to the NK cell inhibitory receptor family.

[0033] As used in this article, the term "SIGLEC7" refers to sialic acid-binding immunoglobulin-like lectin 7.

[0034] As used in this article, the term "CD96" refers to CD155 as the ligand for DNAM-1. The inhibitory receptors CD96 and TIGIT on NK cells can also bind to CD155 and CD112, forming the TIGIT-CD226-CD96 receptor family with DNAM-1.

[0035] As used in this article, the term “NCAM1 / CD56” refers to neural cell adhesion molecule 1, which is mainly expressed in NK or NKT cells in immune cells.

[0036] As used in this article, the term "KLRD1 / CD94" refers to human killer cell lectin-like receptor D1. The CD94 molecule can form a dimer with NKG2A, recognize HLA-E molecules, and transmit inhibitory signals.

[0037] As used in this article, the term "CD69" is a type II transmembrane protein that can be expressed on activated T cells and NK cells.

[0038] As used in this article, the term "TNFSF10 / TRAIL" refers to human tumor necrosis factor-associated apoptosis-inducing ligand, which is involved in mediating apoptosis.

[0039] As used in this article, the term "FASLG" refers to Fas ligand, which is involved in mediating apoptosis.

[0040] As used in this article, the term "PRF1 / Perforin" refers to perforin, which can disrupt the target cell membrane.

[0041] As used in this article, the term "GZMA / Granzyme A" refers to granzyme A, which enters target cells after being perforated by perforin, thereby inducing caspase-independent cell death.

[0042] As used in this article, the term "GZMB / Granzyme B" refers to granzyme B, which is the most important effector molecule of granzymes. After entering the target cell, it can activate the Caspase cascade reaction, thereby rapidly inducing apoptosis of the target cell.

[0043] As used herein, the term "therapeuticly effective" refers to the amount of RIC cells sufficient to treat or improve, or in some way alleviate, the symptoms associated with a disease such as cancer or a symptom such as an infection. When referred to in a method, the method is sufficient to effectively treat or improve, or in some way alleviate, the symptoms associated with a disease or symptom. For example, an effective amount for a disease is an amount sufficient to stop or prevent its onset; or, if the pathology of a disease has begun, to reduce, improve, stabilize, reverse, or slow the progression of the disease, or otherwise mitigate the pathological consequences of the disease. In any case, an effective amount may be administered as a single dose or in multiple doses.

[0044] As used herein, the term "pharmaceutical composition" refers to a pharmaceutically acceptable composition comprising RIC cells, and in some embodiments further comprising a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition may be a combination.

[0045] As used herein, the term "combination" refers to a fixed combination in the form of a dose unit, or to a kit for combined administration of portions thereof, wherein RIC cells and combination chaperones (e.g., another drug explained below, also referred to as "therapeutic agents" or "co-reagents") may be administered simultaneously or separately at time intervals. In some cases, combination chaperones exhibit synergistic effects, such as a combination effect.

[0046] As used herein, the terms “co-administration” or “combined administration” are intended to cover the administration of a selected combination of partners to a single subject in need of it, and are intended to include treatment regimens in which they are not necessarily administered via the same route of administration or at the same time.

[0047] As used herein, this is based on genome editing tools such as the regularly spaced short palindromic repeat (CRISPR) system, the TALEN editing tool, and others, which can be used for clustering in a variety of organisms (e.g., for adding, disrupting, or altering the sequence of specific genes). As used herein, the term "epitope" includes any protein determinant capable of specifically binding to an antigen of an antibody or T-cell receptor. Epitope determinants typically consist of chemically active molecular surface groups, such as amino acid or sugar side chains, and often possess specific three-dimensional structural features and specific charge characteristics.

[0048] As used herein, the term "antibody" includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the desired biological activity of binding to a target antigenic site and its homologue of interest. The term "antibody fragment" includes a portion of a full-length antibody, typically its antigen-binding region or its variable or constant region. In this document, the term "antibody" includes any antibody derived from any species and resource, including but not limited to human antibodies, rat antibodies, mouse antibodies, rabbit antibodies, etc., and which may be synthesized or naturally occurring.

[0049] As used herein, the term “differentiation” is a process by which less specialized cells become more specialized cell types in order to form offspring of at least one new cell type in culture or in vivo, rather than without differentiation under the same conditions. Under certain conditions, the proportion of offspring with characteristics of the new cell type may be at least about 1%, 5%, 25% or more, to increase priority.

[0050] RIC cell population

[0051] In a first aspect, the present invention provides a population of recurrent immunoglobulin (RIC) cells, wherein the average expression level of the NKG2-E gene in the RIC cells (e.g., without genetic modification) is at least about 10 times that of primary immune cells (e.g., at least about 20 times, at least about 30 times, at least about 50 times, at least about 60 times, at least about 80 times, at least about 90 times, at least about 100 times, at least about 150 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times).

[0052] In some embodiments, the expression levels of SELL and CD2 in the RIC cell population are at least about 10 times less than those of primary immune cells (e.g., at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, at least about 100 times, at least about 150 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times).

[0053] The RIC cell population shall contain RIC cells of not less than 60%, not less than 70%, not less than 80%, not less than 90%, and not less than 95%.

[0054] In some embodiments, the RIC cell population has any three or more, four or all of the activating receptors NCR1, NCR2, NCR3, KLRC2, KLRK1, and SLAMF7, and is not significantly different from an equivalent number of primary immune cells. In some embodiments, the RIC cell population has any three or more, four or all of the inhibitory receptors KLRB1, KLRC1, LAIR1, SIGLEC7, and CD96, and is not significantly different from an equivalent number of primary immune cells.

[0055] In some embodiments, the RIC cell population has any three or more, or four or all of the activating receptors NCR1, NCR2, CD96, KLRC2, and KLRK1, and is not significantly different from an equivalent number of primary immune cells. In some embodiments, the RIC cell population has any three or more, or four or all of the inhibitory receptors KLRB1, KLRC1, LAIR1, and SIGLEC7, and is not significantly different from an equivalent number of primary immune cells.

[0056] In some embodiments, the RIC cell population contains any three or more, four or all of the receptors NCR1, NCR2, CD96, KLRC2, and KLRK1, and is not significantly different from an equivalent number of primary immune cells. In some embodiments, the RIC cell population contains any three or more, four or all of the receptors KLRB1, KLRC1, LAIR1, and SIGLEC7, and is not significantly different from an equivalent number of primary immune cells.

[0057] In some embodiments, the RIC cell population is infected with a target CAR via a gene presentation vector or expresses the target CAR persistently or conditionally via gene editing, with the CAR expression rate being 60%-100%, preferably at least 70%, at least 80%, at least 90%, or at least 95%. Optionally, the expression rate of the CD19-targeting CAR is 60%-100%, preferably at least 70%, at least 80%, at least 90%, or at least 95%.

[0058] In this document, the expression can be monitored by measuring the levels of full-length mRNA, mRNA fragments, full-length protein, or protein fragments of the gene. Therefore, in some embodiments, the expression level is either the mRNA level or the protein level.

[0059] In some implementations, the expression is assessed by analyzing the expression of the mRNA transcripts of the genes. For example, the expression of the aforementioned genes in the cell population is determined by measuring the presence or abundance of mRNA of TBXT, APLNR, HAND1, CD2, SELL, NKG2-E, SLAMF7, NCR2, NCR3, CD96, KLRC2, KLRK1, KLRB1, KLRC1, LAIR1, or SIGLEC7 in the cell population using RT-PCR.

[0060] In other embodiments, the expression is assessed by analyzing the expression of the protein products of the gene. For example, the expression of the aforementioned genes in the cell population can be determined by immunological assays to measure the expression of proteins of BRACHYURY, APLNR, CD45, CD43, CD34, or CD56 on the cell membrane.

[0061] In some embodiments, the RIC cells possess one or more of the gene expression characteristics described above without genetic modification. The term "without genetic modification" means that the cell has not undergone a process of adding exogenous genetic material in the form of DNA or RNA to its total genetic material. Here, "exogenous genetic material" can refer to artificially introduced nucleotide sequences that are foreign to the unmodified cells. It is readily understood that the term "without genetic modification" is only used to describe the condition that the RIC cell population of the present invention possesses one or more of the gene expression characteristics described above, and is not intended to limit the RIC cell population of the present invention from containing genetic modification. Therefore, in some embodiments, the RIC cells of the present invention may contain one or more genetic modifications.

[0062] In some embodiments, the RIC cell population is derived from stem cells. Preferably, the stem cells are totipotent or pluripotent stem cells, more preferably pluripotent stem cells, and even more preferably pluripotent stem cells are selected from embryonic stem cells, haploid stem cells, and induced pluripotent stem cells.

[0063] In some implementations, the RIC cell population is generated in vitro.

[0064] In some embodiments, ≥80% (e.g., ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%) of the cells in the RIC cell population simultaneously express CD45 and CD56.

[0065] The RIC cell populations of the present invention can be formulated and administered as pharmaceutical compositions. Such pharmaceutical compositions can be in any form known in the medical field, preferably injectable formulations (including solutions, lyophilized powders). In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable sterile isotonic or non-aqueous solution (e.g., balanced salt solution or physiological saline), dispersion, suspension, or emulsion. General principles for the formulation of such pharmaceutical compositions can be found in *Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy*, edited by G. Morstyn and W. Sheridan, Cambridge University Press, 1996; and *Hematopoietic Stem Cell Therapy*, EDBall, J. Lister & P. ​​Law, Churchill Livingstone, 2000.

[0066] Methods for preparing RIC cell populations

[0067] In a second aspect, the present invention provides a method for generating the RIC cell population described in the first aspect, comprising the following steps:

[0068] (1) Culture stem cells to form lateral plate mesoderm seed cells;

[0069] (2) Inducing the differentiation of the mesodermal seed cells of the lateral plate into hematopoietic seed cells;

[0070] (3) Inducing the hematopoietic seed cells to differentiate into RIC cells.

[0071] More than 65% of the mesodermal seed cells in the lateral plate are APLNR+ cells; for example, more than 70% of the cells are APLNR+ cells; for example, more than 75% of the cells are APLNR+ cells; for example, more than 80% of the cells are APLNR+ cells; for example, more than 85% of the cells are APLNR+ cells; for example, more than 90% of the cells are APLNR+ cells; for example, more than 95% of the cells are APLNR+ cells.

[0072] More than 65% of the mesodermal seed cells in the lateral plate are APLNR+HAND1+ cells; for example, more than 70% of the cells are APLNR+HAND1+ cells; for example, more than 75% of the cells are APLNR+HAND1+ cells; for example, more than 80% of the cells are APLNR+HAND1+ cells; for example, more than 85% of the cells are APLNR+HAND1+ cells; for example, more than 90% of the cells are APLNR+HAND1+ cells; for example, more than 95% of the cells are APLNR+HAND1+ cells; for example, more than 99% of the cells are APLNR+HAND1+ cells.

[0073] More than 65% of the mesodermal seed cells in the lateral plate are APLNR+HAND1+MSGN1-OSR1- cells; for example, more than 70% of the cells are APLNR+HAND1+MSGN1-OSR1- cells; for example, more than 75% of the cells are APLNR+HAND1+MSGN1-OSR1- cells; for example, more than 80% of the cells are APLNR+HAND1+MSGN1-OSR1- cells; for example, more than 85% of the cells are APLNR+HAND1+MSGN1-OSR1- cells; for example, more than 90% of the cells are APLNR+HAND1+MSGN1-OSR1- cells; for example, more than 95% of the cells are APLNR+HAND1+MSGN1-OSR1- cells; for example, more than 99% of the cells are APLNR+HAND1+MSGN1-OSR1- cells.

[0074] The hematopoietic seed cells are CD45+CD43+CD34+CD235a- cells, for example, more than 70% of the cells are CD45+CD34+CD43+CD235a- cells; for example, more than 75% of the cells are CD45+CD43+CD34+CD235a- cells; for example, more than 80% of the cells are CD45+CD43+CD34+CD235a- cells; for example, more than 85% of the cells are CD45+CD43+CD34+CD235a- cells; for example, more than 90% of the cells are CD45+CD43+CD34+CD235a- cells; for example, more than 95% of the cells are CD45+CD43+CD34+CD235a- cells.

[0075] Step (3) can induce RIC cells. This monolayer induction protocol can induce RIC cells with high purity and high yield, with RIC cell content of not less than 60%, not less than 70%, not less than 80%, not less than 90%, and not less than 95%.

[0076] In the RIC cell population preparation process of the present invention, spin polymerization is not required, and spin embryos or EBs (embryomorphs) are not generated.

[0077] In summary, step (1) of this invention involves differentiating pluripotent stem cells into lateral mesoderm seed cells using a monolayer induction method. Optionally, two serum-free culture media, I and II, are used sequentially under matrix or matrix-free conditions, requiring 2-3 days. Spin polymerization is not required in step (1), and embryoid bodies (EBs) are not generated. Step (2) involves directly mixing the generated lateral mesoderm seed cells with a matrix trophoblast expressing a specific gene combination to prepare RIC sacs, without sorting or enrichment. The RIC sacs undergo stepwise culture and induction in two different culture media (III, IV), differentiating from LM to hematopoietic seed cells, with the LM to hematopoietic seed cell stage requiring 10-18 days; and step (3) the induction stage from hematopoietic seed cells to RIC (regenerative immune cells, or RIC for short), with the RIC development stage requiring 7-14 days. Optionally, RICs can also be obtained by monolayer induction after removing matrix trophoblast cells at the hematopoietic seed cell stage. The entire RIC cyst culture phase lasts 17-32 days.

[0078] In some implementations, the mesodermal seed cells of the lateral plate mentioned in step (1) are APLNR+HAND1+ cells obtained by monolayer induction of pluripotent stem cells.

[0079] The above steps can be implemented using the following scheme:

[0080] (1) Stem cells were cultured using culture media I and II to form lateral plate mesoderm seed cells; wherein, culture media I is a basal culture medium supplemented with the following substances: one or more serum substitutes, one or more non-essential amino acids, a stable dipeptide of glutamine or L-alanyl-L-glutamine, and BMP4, ACTIVIN A, bFGF, Wnt agonists (such as small molecule compound CHIR-99021) and PI3K inhibitors (such as small molecule compounds PIK-90, LY294002); culture media II is a basal culture medium supplemented with the following substances: one or more serum substitutes, one or more non-essential amino acids, a stable dipeptide of glutamine or L-alanyl-L-glutamine, and BMP4, TGF-β inhibitors (such as small molecule compounds A-83-01, SB431542) and Wnt inhibitors (such as small molecule compounds C59, IWR-1-endo);

[0081] In some embodiments, culture medium I is a basal culture medium supplemented with one or more serum substitutes, one or more non-essential amino acids, a stabilized dipeptide of glutamine or L-alanyl-L-glutamine, and BMP4, ACTIVIN, bFGF, CHIR-99021, and PIK-90; culture medium II is a basal culture medium supplemented with one or more serum substitutes, one or more non-essential amino acids, a stabilized dipeptide of glutamine or L-alanyl-L-glutamine, and BMP4, A-83-01, and C59.

[0082] (2) The lateral plate mesoderm seed cells are induced to differentiate into hematopoietic seed cells using culture medium III; wherein, culture medium III is a basal culture medium supplemented with the following substances: one or more serum substitutes, one or more non-essential amino acids, a stable dipeptide of glutamine or L-alanyl-L-glutamine, a TGF-β inhibitor (such as small molecule compounds A-83-01, SB431542), hydrocortisone, ascorbic acid, and one, two or more of cell growth factors such as Flt3L, TPO, SCF, EGF, VEGF, bFGF and IGF-1; in some embodiments, culture medium III is a basal culture medium supplemented with the following substances: one or more serum substitutes, one or more non-essential amino acids, a stable dipeptide of glutamine or L-alanyl-L-glutamine, and one, two or more of cell growth factors such as Flt3L, TPO, SCF, EGF, VEGF, bFGF and IGF-1;

[0083] (3) The hematopoietic seed cells are induced to differentiate into RIC cells using culture medium IV; wherein the culture medium IV is a basal culture medium supplemented with one or more of the following substances: one or more serum substitutes, one or more non-essential amino acids, a stabilized dipeptide of glutamine or L-alanyl-L-glutamine, sodium selenite, ethanolamine, ascorbic acid, and cell growth factors such as IL-3, SCF, IL-7, IL-15, and Flt3L, or two or more. In some embodiments, the culture medium IV further comprises β-mercaptoethanol. In some embodiments, the content of β-mercaptoethanol is 0.1-0.5% (v / v), for example, about 0.1% (v / v), about 0.2% (v / v), about 0.3% (v / v), about 0.4% (v / v), or about 0.5% (v / v);

[0084] In some embodiments, the culture medium IV is a basal culture medium supplemented with one or more serum substitutes, one or more non-essential amino acids, a stabilized dipeptide of glutamine or L-alanyl-L-glutamine, and one, two or more, of cell growth factors such as IL-3, SCF, IL-7, IL-15 and Flt3L; in some embodiments, the culture medium IV further comprises β-mercaptoethanol; in some embodiments, the β-mercaptoethanol content is 0.1-0.5% (v / v), for example about 0.1% (v / v), about 0.2% (v / v), about 0.3% (v / v), about 0.4% (v / v) or about 0.5% (v / v).

[0085] In this article, the term "basal medium" refers to any culture medium capable of supporting cell growth, typically containing inorganic salts, vitamins, glucose, buffer systems, and essential amino acids, and usually having an osmotic pressure of approximately 280–330 mOsmol. Examples include Essential 6, DMEM-high glucose, DMEM / F12, α-MEM, F-12, EBM2, MEM, BME, RPMI 1640, G-MEM, and any combination thereof.

[0086] In some embodiments, the stem cells in step (1) are totipotent stem cells or pluripotent stem cells, and more preferably the stem cells are pluripotent stem cells, and more preferably the pluripotent stem cells are selected from embryonic stem cells, haploid stem cells, and induced pluripotent stem cells.

[0087] In some embodiments, the culture in step (1) is performed in a matrix. In some embodiments, the matrix comprises an extracellular matrix. In some embodiments, the matrix comprises one or more of the following: Growth Factor Reduced Matrigel (GR-MTG material), glycoproteins (such as, but not limited to, vitrein (VTN), fibronectin), hyaluronic acid, laminin, fibronectin, collagen, elastin, heparan sulfate, dextran, dextran sulfate, and chondroitin sulfate. Preferably, the matrix is ​​a glycoprotein. Preferably, the matrix is ​​vitrein (VTN) and / or fibronectin. Preferably, the matrix is ​​vitrein (VTN).

[0088] In some embodiments, the cells generated in step (2) include: lateral plate mesoderm seed cells, hematopoietic endothelial cells, hematopoietic stem seed cells, and immune seed cells; step (2) includes: culturing the RIC capsule using culture medium III or IV, preferably culture medium III. Preferably, the RIC capsule is attached to a culture vessel.

[0089] In some embodiments, the culture media I, II, III, and IV possess one or more of the following characteristics:

[0090] (i) The total content of one or more serum substitutes is 2-30% (v / v), such as 3-30% (v / v), about 2% (v / v), about 3% (v / v), about 5% (v / v), about 8% (v / v), about 10% (v / v), about 12% (v / v), about 15% (v / v), about 18% (v / v), about 20% (v / v), about 22% (v / v), about 25% (v / v), about 28% (v / v), or about 30% (v / v);

[0091] (ii) The content of each of the one or more non-essential amino acids is 0.1-0.5 mM, for example, about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM or about 0.5 mM;

[0092] (iii) The content of the stabilized dipeptide of glutamine or L-alanyl-L-glutamine is 1-5 mM, for example about 1 mM, about 2 mM, about 3 mM, about 4 mM, or about 5 mM;

[0093] (iv) The concentration of each factor, including BMP4, ACTIVIN, bFGF, Flt3L, TPO, SCF, EGF, VEGF, bFGF, IGF-1, IL-3, IL-7, and IL-15, is independently 1–100 ng / mL, for example, 2–100 ng / mL, 2–50 ng / mL, 5–100 ng / mL, 5–50 ng / mL, or 5–20 ng / mL; for example, approximately 1 ng / mL, approximately 2 ng / mL, approximately 3 ng / mL, approximately 5 ng / mL. Approximately 8 ng / mL, approximately 10 ng / mL, approximately 15 ng / mL, approximately 20 ng / mL, approximately 25 ng / mL, approximately 30 ng / mL, approximately 35 ng / mL, approximately 40 ng / mL, approximately 45 ng / mL, approximately 50 ng / mL, approximately 55 ng / mL, approximately 60 ng / mL, approximately 65 ng / mL, approximately 70 ng / mL, approximately 75 ng / mL, approximately 80 ng / mL, approximately 85 ng / mL, approximately 90 ng / mL, approximately 95 ng / mL, or approximately 100 ng / mL.

[0094] (v) The concentration of each small molecule of Wnt agonists, PI3K inhibitors, TGF-β inhibitors and Wnt inhibitors can be 10 nM-100 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.

[0095] In some embodiments, the culture media I, II, III, and IV possess one or more of the following characteristics:

[0096] (a) The serum substitute is a serum-free additive, specifically selected from KOSR, B27, and Ultroser. TM G, SUPERGROW and any combination thereof; preferably, the serum substitute is SUPERGROW (Dacco, 6122011) (hereinafter referred to as SUPERGROW) or B27 (Gibco17504-044);

[0097] (b) 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;

[0098] (c) The basal culture medium is selected from Essential 6, DMEM-high glucose, DMEM / F12, α-MEM, F-12, EBM2, MEM, BME, RPMI 1640, G-MEM and any combination thereof.

[0099] In some embodiments, the culture media I, II, III, and IV possess one or more of the following characteristics:

[0100] Culture medium I: Essential 6 medium containing BMP4, ACTIVIN A, bFGF, CHIR-99021 and PIK-90.

[0101] Culture medium II: Essential 6 medium containing BMP4, A-83-01 and C59.

[0102] Culture medium III: Essential 6 medium containing SB431542, hydrocortisone, Flt3L, TPO, SCF, EGF, VEGF, bFGF, IGF-1, ascorbic acid and SUPERGROW cell culture supplement (Dakewei, 6122011).

[0103] Culture medium IV: DMEM-high glucose, DMEM / F12, SUPERGROW cell culture additive (Dakewei, 6122011), GlutaMAX TM β-mercaptoethanol, sodium selenite, ethanolamine, ascorbic acid, penicillin-streptomycin solution, IL-3, SCF, IL-7, IL-15 and Flt3L.

[0104] In some 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.

[0105] In some embodiments, medium II is Essential 6 medium containing 30 ng / mL BMP4, 1 μM A-83-01 and 1 μM C59.

[0106] In some embodiments, culture medium III is Essential 6 medium containing 10 μM SB431542, 10 μM hydrocortisone, 5 ng / mL LFlt3L, 5 ng / mL TPO, 50 ng / mL SCF, 50 ng / mL EGF, 50 ng / mL VEGF, 50 ng / mL bFGF, 50 ng / mL LIFG-1, 50 μg / mL ascorbic acid and 2% SUPERGROW cell culture additive (Dakeway, 6122011).

[0107] In some embodiments, culture medium IV contains: 55% DMEM-high glucose, 30% DMEM / F12, 15% SUPERGROW cell culture additive (Dakewei, 6122011), and 2 mM GlutaMAX. TM 1 μM β-mercaptoethanol, 5 ng / mL sodium selenite, 50 μM ethanolamine, 20 μg / mL ascorbic acid, 1% penicillin-streptomycin solution, 5 ng / mL IL-3, 20 ng / mL LSCF, 20 ng / mL IL-7, 10 ng / mL IL-15 and 10 ng / mL Flt3L.

[0108] In this document, the term "cell culture dish" refers to a culture dish with a coating that prevents proteins from adsorbing onto the surface of the dish, thereby minimizing the adhesion of a monolayer of cells to the culture vessel. Such cell culture dishes are well known to those skilled in the art, including, but not limited to, Corning's low-adhesion culture dish (catalog number 3262).

[0109] In some implementations, the duration of the culture in step (1) is 1-3 days, for example, about 1 day, about 1.5 days, about 2 days, about 2.5 days, or about 3 days.

[0110] In some implementations, the duration of the culture in step (2) is 10-18 days, for example, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, or about 17 days.

[0111] In some implementations, the duration of the culture in step (3) is 7-14 days, for example, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, or about 14 days.

[0112] In some implementations, step (2) includes using approximately one RIC capsule / 1-10cm 2The planar density of the RIC sac, composed of lateral plate mesoderm seed cells and trophoblast cells obtained in step (1), is seeded into the culture vessel. The lateral plate mesoderm seed cells do not require sorting or enrichment. In some embodiments, the total cell count of a single RIC sac ranges from 200,000 to 10,000,000 cells. In some embodiments, the mixing ratio of lateral plate mesoderm seed cells to trophoblast cells ranges from 1:5 to 1:200. In some embodiments, the trophoblast cells of the RIC sac are selected from one or more of AFT024, OP9 cells, MS5 cells, HS-5, or other fibroblasts, preferably expressing at least one, two, three, multiple, or all of the following genes: DLL1, DLL4, IL7, IL15, IL3, and Flt3L. More preferably, the trophoblast cells of the RIC sac are OP9-DLL1, OP9-DLL4, or OP9-DLL1-DLL4.

[0113] In some implementations, the duration of the incubation in steps (2) and (3) is 17-32 days, for example, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, about 30 days, about 31 days, or about 32 days.

[0114] In some implementations, steps (2) and (3) include replacing the culture medium with fresh medium daily or every 1 to 7 days (e.g., every 1, 2, 3, 4, 5, 6, or 7 days).

[0115] The entire process of culturing stem cells into RIC cells takes 19-34 days, for example, approximately 19 days, approximately 20 days, approximately 21 days, approximately 22 days, approximately 23 days, approximately 24 days, approximately 25 days, approximately 26 days, approximately 27 days, approximately 28 days, approximately 29 days, approximately 30 days, approximately 31 days, approximately 32 days, approximately 33 days, or approximately 34 days.

[0116] In some embodiments, the culture conditions in steps (1-3) are 37°C and 5% CO2. In some embodiments, the culture in steps (1-3) is carried out in an incubator at 37°C and 5% CO2.

[0117] In a third aspect, the present invention also relates to a population of RIC cells generated by the method described in the second aspect.

[0118] In some implementations, the RIC cell population is as defined in the first aspect.

[0119] In a fourth aspect, the present invention also relates to an intermediate cell assembly comprising lateral mesodermal seed cells formed by culturing stem cells, such as lateral mesodermal seed cells produced by step (1) of the aforementioned RIC cell population preparation method. In some embodiments, more than 65% of the lateral mesodermal seed cells are APLNR+ cells, preferably more than 65% are APLNR+HAND1+ cells.

[0120] In a fifth aspect, the present invention also relates to an intermediate cell assembly comprising hematopoietic seed cells and other immune seed cells (e.g., myeloid cells) produced by step (2) of the aforementioned RIC cell population preparation method. In some preferred embodiments, more than 65% of the hematopoietic seed cells are CD45+CD43+CD34+CD235a- cells.

[0121] RIC capsules

[0122] In a fifth aspect, the present invention also relates to a RIC sac, which is composed of lateral plate mesodermal seed cells and trophoblasts derived in step (1) above. In some embodiments, the ratio of the lateral plate mesodermal seed cells to trophoblasts may be in the range of 1:5 to 1:200, for example 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, etc. The trophoblasts are preferably AFT024, OP9 cells, MS5 cells, HS-5 or other fibroblasts, and preferably express at least one, two, three, more or all of the following genes: DLL1, DLL4, IL7, IL15, IL3 and Flt3L. The synergistic effect of the components of the RIC sac greatly promotes the directed differentiation of mesodermal seed cells from the lateral plate. The RIC sac exhibits dynamic changes over time, gradually shifting from a smooth, uniformly dense state to a dispersed state, expanding from the center towards the periphery. See details... Figure 6 As shown. In some implementations, one RIC capsule can induce 1×10 6 2×10 6 3×10 6 4×10 6 5×10 6 6×10 6 7×10 6 8×10 6 9×10 6 Or 1×10 7 One RIC cell.

[0123] The total cell count of a single RIC capsule can range from 200,000 to 10 million cells, for example, 250,000, 300,000, 400,000, 600,000, 800,000, 1 million, 1.5 million, 1.8 million, 2 million, 3 million, 4 million, 5 million, 6 million, 7 million, 8 million, and 9 million. The culture area occupied by each RIC capsule ranges from 1 to 10 cm². 2 RIC capsules can be cultured continuously for 10-18 days under medium III, followed by continuous culture for 7-14 days under medium IV.

[0124] Reagent test kit

[0125] In a sixth aspect, the present invention provides a kit comprising one, two, three, or four of separately provided culture media I, II, III, and IV.

[0126] The cultures of the present invention can be formulated and administered as pharmaceutical compositions. Such pharmaceutical compositions can be in any form known in the medical field, preferably injectable (including solutions, lyophilized powders). In some preferred embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable sterile isotonic or non-aqueous solution (e.g., balanced salt solution or physiological saline), dispersion, suspension, or emulsion. General principles for the formulation of such pharmaceutical compositions can be found in *Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy*, edited by G. Morstyn and W. Sheridan, Cambridge University Press, 1996; and *Hematopoietic Stem Cell Therapy*, EDBall, J. Lister & P. ​​Law, Churchill Livingstone, 2000.

[0127] In this invention, any culture medium and culture conditions known in the art for culturing immune cells can be used to culture the RIC cell population of this invention.

[0128] Used for the prevention and / or treatment of disease

[0129] In a seventh aspect, the present invention relates to the use of RIC cell populations and / or modified RIC cell-derived cell populations or pharmaceutical compositions containing RIC cell populations and their derivatives as described herein in the prevention and / or treatment of a disease in a subject, or in the preparation of a medicament for the prevention and / or treatment of a disease in a subject, and a method for the prevention and / or treatment of a disease in a subject comprising administering to a subject in need of the present invention the RIC cell populations and / or modified RIC cell-derived cell populations or pharmaceutical compositions containing RIC cell populations and their derivatives.

[0130] In some implementations, the subject is a mammal, such as a human.

[0131] In some implementations, the diseases prevented and / or treated in the subjects include cancer, infectious diseases, viral pneumonia, HIV / AIDS, immunodeficiency, immunodeficiency, or autoimmune diseases.

[0132] In embodiments of various aspects of this invention, examples of cancer include, but are not limited to, lymphoma, hematologic malignancies, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia), multiple myeloma, lymphocytic lymphoma, breast cancer, ovarian cancer, liver cancer, glioma, pancreatic cancer, lung cancer, colon cancer, rectal cancer, melanoma, kidney cancer, bladder cancer, head and neck cancer, stomach cancer, nasopharyngeal carcinoma, laryngeal cancer, cervical cancer, endometrial tumors, osteosarcoma, bone cancer, skin cancer, prostate cancer, uterine cancer, anal cancer, and testicular cancer. Testicular cancer, fallopian tube cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small bowel cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumors, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermal carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, including asbestos-induced cancers, and combinations of the aforementioned cancers.

[0133] In embodiments of various aspects of the present invention, the infectious disease may be caused by pathogens selected from viruses, bacteria, fungi, parasites, or protozoa, including but not limited to human HIV, immunodeficiency virus, poliovirus, hepatitis A virus, enterovirus, rhinovirus, cytomegalovirus (CMV), hepatitis B virus (HBV), herpes simplex virus (HSV), varicella-zoster virus, coronavirus, rabies virus, Ebola virus, parainfluenza virus, mumps virus, measles virus, influenza virus, papillomavirus, polyomavirus; Pasteurella, Staphylococcus, Streptococcus, Escherichia coli *C. coli*, *Pseudomonas*, *Salmonella*, *Helicobacter pylori*, *Mycobacterium*, *Neisseria gonorrhoeae*, *Neisseria meningitidis*, *Streptococcus*, *Erysipelothrix rhusiopathiae*, *Clostridium perfringens*, *Clostridium tetani*, *Enterobacter* aerogenes, Klebsiella pneumoniae, Pasteurella multocida, Leptospira and Rickettsia; Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis and Coccidioides; Babeosis bovis, Plasmodium, Leishmania spp., Toxoplasma gondii and Trypanosoma cruzi.

[0134] In some embodiments, the RIC cell is a RIC cell containing a chimeric antigen receptor (CAR) (CAR-RIC cell).

[0135] In some embodiments, the CAR includes an antigen-binding domain, such as an extracellular antigen-binding domain. In some embodiments, the antigen-binding domain is specific to antigens of pathogens causing infectious diseases. In some embodiments, the antigen-binding domain is specific to tumor-associated antigens.

[0136] The tumor-associated antigens include, but are not limited to, disialotetrahexosylganglioside GD2, epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, BCMA (CD269, TNFRSF17), NY-ESO-1, CD16, CD64, CD78, CD96, CLL1, CD116, CD117, CD71, CD45, CD71, CD123, CD138, ErbB2 (HER2 / neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), CD30, CD40, ductal epithelial mucin, gp36, TAG-72, glycosphingolipids, glioma-associated antigens, β-human chorionic gonadotropin, alpha fetal globulin (AFP), exogenous lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, and mucin. hsp70-2, M-CSF, prostase, prostate enzyme-specific antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53, Prostein, PSMA, survival and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, liver glycoside B2, CD22, insulin-like growth factor (IGF1)-I, IGF-II, IGFI receptor, mesothelin, major histocompatibility complex (MHC) molecule presenting tumor-specific peptide epitopes, 5T4, ROR1, Nkp30, tumor matrix antigen, extra domain A (EDA) and extra domain B (EDB) of fibronectin, A1 domain (TnC) of tendinin-C A1), fibroblast-associated protein (fap), CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, Foxp3, B7-1 (CD80), B7-2 (CD86), GM-CSF, cytokine receptors, endothelial factors, major histocompatibility complex (MHC) molecules, TNFRSF17 (UNIPROT Q02223), SLAMF7 (UNIPROT Q9NQ25), GPRC5D (UNIPROT Q9NZD1), FKBP11 (UNIPROT Q9NYL4), KAMP3, ITGA8 (UNIPROT P53708), and FCRL5 (UNIPROT Q68SN8).

[0137] In various embodiments of the present invention, the antigen-binding domain may be, for example, a monoclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a single-domain antibody, a nanobody, an antibody single-chain variable region (scFV), and its antigen-binding fragment. In some preferred embodiments, the antigen-binding domain is an scFV.

[0138] In this invention, RIC cell populations and / or modified RIC cell-derived cells, or pharmaceutical compositions containing RIC cell populations and their derivatives, or containing said RIC cell populations or cultures, can be administered to subjects via various suitable methods. In some embodiments, RIC cell populations and / or modified RIC cell-derived cells or pharmaceutical compositions as described herein are administered to subjects via local injection transplantation (e.g., stereotactic intracerebral injection transplantation or spinal cord local injection transplantation, intratumoral injection), blood circulation transplantation (e.g., intravenous injection transplantation or intra-arterial injection transplantation), or cerebrospinal fluid transplantation (e.g., lumbar puncture subarachnoid injection transplantation). Those skilled in the art know how to select an appropriate cell transplantation route based on the location and nature of the lesion.

[0139] In some embodiments, the drug is in unit dose form, and the unit dose contains not less than 1 × 10⁻⁶. 4 cells / ml (e.g., not less than 1×10⁻⁶) 4 cells / ml, not less than 3 × 10 4 cells / ml, not less than 5 × 10 4 cells / ml, not less than 7 × 10 4 cells / ml, not less than 1×10 5 cells / ml, not less than 3 × 10 5 cells / ml, not less than 5 × 10 5 cells / ml, not less than 7 × 10 5 cells / ml, not less than 1×10 6 cells / ml, not less than 3 × 10 6 cells / ml, not less than 5 × 10 6 cells / ml, not less than 7 × 10 6 cells / ml, not less than 1×10 7 cells / ml, not less than 3 × 10 7 cells / ml, not less than 5 × 10 7 cells / ml, not less than 7 × 10 7 cells / ml, not less than 1×10 8 cells / ml, not less than 3 × 10 8 cells / ml, not less than 5 × 10 8 cells / ml, not less than 7 × 10 8cells / ml, not less than 1×10 9 cells / ml, not less than 3 × 10 9 cells / ml, not less than 5 × 10 9 cells / ml, not less than 7 × 10 9 cells / ml, not less than 1×10 10 cells / ml, not less than 3 × 10 10 cells / ml, not less than 5 × 10 10 1 / ml or not less than 7×10 10 The RIC cells and / or modified RIC cell-derived cells (per cell / ml) are described. In some embodiments, the unit dose of the drug contains 1 × 10⁻⁶ cells. 4 ~1×10 10 1×10 6 ~1×10 8 1×10 6 ~1×10 7 One, or 1×10 6 ~5×10 6 The RIC cell population and / or modified RIC cell-derived cell populations are described in (1). Attached Figure Description

[0140] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and, together with their description, serve to explain the invention and do not constitute an undue limitation thereof. In the drawings:

[0141] Figure 1 The flowchart illustrates the process of inducing PSC differentiation into RIC, where PSC includes ESC and iPSC; PS, original streak; LM, lateral mesodermal layer; hematopoietic seed cells, hematopoietic progenitor cells; and RIC, induced RIC cells.

[0142] Figure 2 Cell morphology at different stages of embryonic stem cell differentiation in Example 1;

[0143] Figure 3 Embryonic stem cells from Example 1 differentiated into primary streak (TBXT+) and lateral mesoderm cells (APLNR+, HAND1+). The Y-axis value represents the result of qPCR analysis of the differential mRNA expression of relevant genes. The internal control gene was ACTIN, and the target genes were TBXT, APLNR, HAND1, MSGN1, and OSR1. TBXT is a primary streak-specific marker gene, APLNR and HAND1 are lateral mesoderm-specific marker genes, MSGN1 is a paraaxial mesoderm-specific marker gene, and OSR1 is an intercalary mesoderm-specific marker gene. The analytical method was 2... -ΔCt The method refers to the relative expression level of the target gene relative to the internal reference gene;

[0144] Figure 4 These are marker proteins for the flow cytometry analysis of the original strip (BRACHYURY+) and lateral plate mesoderm (APLNR+) in Example 1. BRACHYURY is a protein encoded by the TBXT gene and is a marker for the original strip. APLNR is a marker for the lateral plate mesoderm.

[0145] Figure 5 The flow cytometry analysis in Example 1 shows that, compared with GR-MTG material, the expression intensity of APLNR molecules from the lateral mesoderm derived from ESC was higher when differentiated using VTN material. VTN stands for vitronectin, and GR-MTG stands for growth factor reduced matrigel.

[0146] Figure 6 Stereoscopic images show the morphology of RIC cysts at different stages of pluripotent stem cell differentiation in Example 2.

[0147] Figure 7 RIC capsules can effectively produce CD45+CD34+CD43+CD235a- hematopoietic seed cells;

[0148] Figure 8 The changes in RIC maturity on Day 20, Day 24, and Day 27 of Example 3;

[0149] Figure 9 Changes in RIC cell numbers on Day 20, Day 24, and Day 27;

[0150] Figure 10 D27 GFP+ stromal cells disappeared, and RIC cells showed high purity;

[0151] Figure 11 Statistics on the yield of RIC cells (D27) obtained by monolayer induction of hematopoietic seed cells from isolated RIC sacs (D20);

[0152] Figure 12 RIC cyst hematopoietic seed cells were transplanted into recipient mice to induce regeneration and obtain mature RIC cells. In the figure, BM refers to bone marrow, PB refers to peripheral blood, and SP refers to spleen.

[0153] Figure 13 For ESC-RIC to express activating receptors, the NCR2, NCR3, KLRC2, KLRK1 and SLAMF7 genes encode NKp44, NKp30, NKG2C, NKG2D and CS1 activating receptor proteins, respectively.

[0154] Figure 14 The KLRB1, KLRC1, LAIR1, SIGLEC7, and CD96 genes encode inhibitory receptor proteins CD161, NKG2A, LAIR-1, SIGLEC7, and CD96, respectively, which are expressed as inhibitory receptors in RIC cells.

[0155] Figure 15 The NCAM1 and KLRD1 genes encode CD56 and CD94 proteins, respectively, which are mainly related to the maturation of RIC cells; the CD69, TNFSF10, FASLG, PRF1, GZMA and GZMB genes encode CD69, TRAIL, FasL, Perforin, Granzyme A and Granzyme B, respectively, which are related to the activation and natural killer function of RIC cells.

[0156] Figure 16 This is a unique gene expression pattern of RICs (derived from ESCs) compared to primary immune cells. RICs highly express NKG2-E, with expression levels more than 400 times higher than those of primary immune cells; they express SELL and CD2 at low levels, less than 1 / 50th of those of primary immune cells.

[0157] Figure 17 RIC cells derived from pluripotent stem cells (ESCs) were used in vitro to kill tumor cells derived from the Nalm-6 lymphoma cell line. ESC-1, -2, -3, -4, and -5 represent five ESC cell clones from the China National Stem Cell Resource Bank. X-axis: E:T ratio (RIC effector cells: tumor cells). Y-axis: percentage of residual tumor cells. Absolute viable cell count was performed after a co-incubation period of 6 hours.

[0158] Figure 18 This study demonstrated the high efficiency of ESC-RIC cells in killing AML cells THP-1 and Molm13 in vitro. X-axis: E:T ratio (RIC effector cells: tumor cells). Y-axis: tumor kill percentage. Co-incubation time was 4 hours.

[0159] Figure 19 RIC cells derived from pluripotent stem cells (ESCs) were used in vitro to kill tumor cells derived from the A1847 ovarian cancer cell line. ESC-1, -2, -3, -4, -5: five ESC cell clones from the China National Stem Cell Resource Bank. X-axis: E:T ratio (RIC cells: tumor cells). Y-axis: percentage of residual tumor cells relative to the initial infused tumor. Co-incubation time was 4 hours.

[0160] Figure 20Pluripotent stem cell (ESC-derived) RIC cells can enhance the killing of the Raji B-lymphoma cell line through ADCC. Rituximab is an anti-CD20 rituximab. X-axis, E:T ratio (RIC cells:tumor cells). Y-axis, percentage of tumor cells killed. Co-incubation time was 4 hours.

[0161] Figure 21 To demonstrate the in vivo killing effect of RIC (ESC-derived) on A1847 ovarian cancer cells. a. Treatment process and detection diagram. bc. Dynamic in vivo imaging to detect tumor burden in the RIC treatment group and the tumor burden control group. n=5, statistical methods: one-way ANOVA and Kruskal-Wallis test. NS, not significant. d. Survival curve analysis. n=5, statistical method: Log-rank test. e. Body weight of tumor-bearing mice in the treatment group and the control group at Day 0 and Day 35. n=5, statistical method: two-sided independent t-test.

[0162] Figure 22 To demonstrate the in vivo killing of HL60 acute myeloid leukemia cells by RIC (ESC-derived). a. Treatment process and detection diagram. bc. Dynamic in vivo imaging to detect tumor burden in the RIC treatment group and the tumor burden control group. n=5, statistical method: two-sided independent t-test. d. Survival curve analysis. n=5, statistical method: Log-rank test. e. Body weight of tumor-bearing mice in the treatment group and the control group on Day 0 and Day 28. n=5, statistical method: two-sided independent t-test. NS, not significant. *: p<0.05, **: p<0.01, ***: p<0.001.

[0163] Figure 23 The infection rate of CD19-CAR in RIC cells is shown. Compared with primary immune cells, the expression rate of CD19-CAR virus in RIC cells after infection (MOI=20) is as high as 82.4%, while the infection rate of innate immune cells (MOI=35) is only 14.9%.

[0164] Figure 24 The CD19-CAR-ESC-RIC method was used to efficiently kill primary tumor cells from ALL (acute lymphoblastic leukemia) patients. CD19-CAR-carrying RIC cells were co-incubated with primary tumor cells (CD19+B cells) from two ALL patients at a 1:1 ratio in vitro for 6 hours, after which the number of CD19+B cells was detected. The Y-axis represents the percentage of residual tumor cells relative to the initial infused tumor.

[0165] Figure 25 To determine the cell morphology at different stages of pluripotent stem cell differentiation;

[0166] Figure 26 This study aimed to detect specific cell markers for inducing pluripotent stem cell differentiation into primary streak (TBXT+) and lateral mesodermal cells (APLNR+HAND1+). The Y-axis value represents the result of qPCR analysis of differential mRNA expression of relevant genes. The internal control gene was ACTIN, and the target genes were TBXT, APLNR, HAND1, MSGN1, and OSR1. TBXT is a primary streak-specific marker gene, APLNR and HAND1 are lateral mesodermal cell-specific marker genes, MSGN1 is a paraaxial mesodermal-specific marker gene, and OSR1 is a mesenchymal-specific marker gene. The analytical method was 2... -ΔCt The method refers to the relative expression level of the target gene relative to the internal reference gene;

[0167] Figure 27 To identify marker proteins in the protostome (BRACHYURY+) and lateral mesoderm (APLNR+) by flow cytometry. BRACHYURY is a protein encoded by the TBXT gene and is a marker for protostome identification. APLNR is a marker for lateral mesoderm identification.

[0168] Figure 28 Flow cytometry analysis showed that, compared with GR-MTG materials, iPSC-derived lateral mesoderm APLNR molecules were expressed at higher intensity. VTN stands for vitronectin, and GR-MTG stands for growth factor-reduced matrigel.

[0169] Figure 29 The changes in RIC maturity on Day 20, Day 24, and Day 27;

[0170] Figure 30 Changes in RIC cell numbers on Day 20, Day 24, and Day 27;

[0171] Figure 31 The D27 GFP+ stromal cells disappeared;

[0172] Figure 32 Yield of RIC cells (D27) obtained by monolayer induction of hematopoietic seed cells isolated from RCI cysts (D20);

[0173] Figure 33For iPSC-RIC to express activating receptors, the NCR2, NCR3, KLRC2, KLRK1 and SLAMF7 genes encode NKp44, NKp30, NKG2C, NKG2D and CS1 activating receptor proteins, respectively.

[0174] Figure 34 For iPSC-RIC cells to express inhibitory receptors, the genes KLRB1, KLRC1, LAIR1, SIGLEC7 and CD96 encode CD161, NKG2A, LAIR-1, SIGLEC7 and CD96 inhibitory receptor proteins, respectively.

[0175] Figure 35 The NCAM1 and KLRD1 genes encode CD56 and CD94 proteins, respectively; the CD69, TNFSF10, FASLG, PRF1, GZMA, and GZMB genes encode CD69, TRAIL, FasL, Perforin, Granzyme A, and Granzyme B, respectively.

[0176] Figure 36 This is a unique gene expression pattern of RICs (iPSC-derived cells) compared to primary immune cells. RICs highly express NKG2-E, with expression levels more than 400 times higher than those of primary immune cells; they express SELL and CD2 at low levels, less than 1 / 50th of those of primary immune cells.

[0177] Figure 37 In vitro killing of Nalm-6 lymphoma cell line tumor cells by pluripotent stem cell (iPSC-derived) RIC cells. X-axis, E:T ratio (RIC effector cells: tumor cells). Y-axis, percentage of residual tumor cells. Absolute viable cell count was performed after a co-incubation period of 6 hours.

[0178] Figure 38 RIC cells derived from pluripotent stem cells (iPSCs) killed tumor cells derived from the A1847 ovarian cancer cell line in vitro. X-axis: E:T ratio (RIC cells: tumor cells). Y-axis: percentage of residual tumor cells relative to the initial infused tumor. Co-incubation time: 4 hours.

[0179] Figure 39 RIC cells, derived from pluripotent stem cells (iPSCs), can enhance the killing of the Raji B-cell lymphoma cell line through ADCC. Rituximab is an anti-CD20 rituximab. X-axis: E:T ratio (RIC cells:tumor cells). Y-axis: percentage of tumor cells killed. Co-incubation time: 4 hours.

[0180] Figure 40RIC (iPSC-derived) in vivo killing of A1847 ovarian cancer cells. a. Treatment process and detection diagram. bc. Dynamic in vivo imaging detection of tumor burden in the RIC treatment group and the tumor burden control group. n=5, statistical methods: one-way ANOVA and Kruskal-Wallis test. NS, not significant. d. Survival curve analysis. n=5, statistical method: Log-rank test. e. Body weight of tumor-bearing mice in the treatment group and the control group at Day 0 and Day 35. n=5, statistical method: two-sided independent t-test.

[0181] Figure 41 Infection rate of CD19-CAR in RIC cells. Compared with primary immune cells, the expression rate of CD19-CAR virus in RIC cells after infection (MOI=20) was as high as 83.0%, while the expression rate of innate immune cells (MOI=35) after infection was only 33.4%.

[0182] Figure 42 CD19-CAR-iPSC-RIC efficiently kills primary tumor cells from ALL (acute lymphoblastic leukemia) patients. CD19-CAR-carrying RIC cells and primary tumor cells (CD19+B cells) from ALL patients were co-incubated in vitro at a 1:1 ratio for 6 hours, and the number of CD19+B cells was then detected. The Y-axis represents the percentage of residual tumor cells relative to the initial infused tumor. Detailed Implementation

[0183] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention or its application or use. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0184] Experimental methods in the following examples that do not specify specific conditions should be performed according to conventional methods and conditions, or according to the product instructions. All reagents and raw materials used in this invention are commercially available.

[0185] For reference Figure 1 PSCs were induced to differentiate into RICs, where... Figure 1 The meanings of the symbols and the composition of the culture medium are shown below:

[0186] PSC includes ESC and iPSC; PS refers to the original streak; LM refers to the lateral mesoderm; hematopoietic seed cells, i.e., hematopoietic progenitor cells; RIC refers to induced RIC cells.

[0187] Culture 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).

[0188] Culture 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).

[0189] Culture 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 LSCF (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 (Dakewei, 6122011).

[0190] Culture medium IV: 55% DMEM-high glucose (Hyclone, SH30022.01B), 30% DMEM / F12 (Hyclone, SH30023.01B), 15% SUPERGROW cell culture supplement (Dakewei, 6122011), 2mM 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), 10 ng / mL IL-15 (PeproTech, 200-15), and 10 ng / mL Flt3L (PeproTech, 300-19).

[0191] The specific implementation plan is as follows:

[0192] Example 1: Pluripotent stem cells (ESC-derived) differentiate into lateral mesoderm.

[0193] Pluripotent stem cells (ESC-derived) cultured to 80% adherence density were digested with Accutase (Sigma-Aldrich, A6964) for 1 min, resuspended in ncTarget medium (Zhong Sheng Su Yuan, RP01020) to form a single-cell suspension, and then transferred to cell culture dishes lined with VTN (Animal-Free Human Vitronectin Matrix, PeproTech, AF-VMB-220) after adding 2 μM ROCK inhibitor thiazovivin (Selleck, S1459). The cells were incubated for 24 h (Day 0 of the induction program). The ncTarget medium was removed from the culture dishes, and medium I was added. The cells were incubated for another 24 h (Day 1 of the induction program). Medium I was removed, and medium II was added. The cells were incubated for another 24 h (Day 2 of the induction program). Cell images were taken at D0, D1, and D2. Figure 2 ), qPCR was used to detect the identity markers of the original strip (BRACHYURY+, encoding the TBXT gene) and the lateral mesoderm (APLNR+HAND1+MSGN1-OSR1-). Figure 3 ) and flow cytometry analysis of the induction efficiency of the original strip and lateral plate mesoderm ( Figure 4 The reason for choosing VTN is that, compared to GR-MTG (Growth Factor Reduced Matrigel), VTN material achieves a higher efficiency in obtaining side plate mesoderms under otherwise identical conditions. Figure 5 ).

[0194] Example 2: RIC capsule culture - hematopoietic seed cell (ESC-derived) stage induction

[0195] The specific steps are as follows:

[0196] Mesodermal cells from the lateral plate were digested and prepared into a single-cell suspension, which was then mixed with OP9-DLL1 / DLL4 feeder cells. The mixing ratio of the two cell types could range from 1:5 to 1:200. The total cell count was adjusted from 200,000 to 10,000,000,000 depending on the size of the culture dish wells, to prepare RIC capsules. Each RIC capsule occupied 1-10 cm² of culture surface area. 2 Under medium III conditions, induction continued for 10-18 days, with the medium changed every 1-3 days. Morphological changes in the RIC capsule units were photographed and recorded on Day 9 and Day 16. Figure 6 Single-cell flow cytometry analysis was performed on RIC capsules from Day 16 to detect hematopoietic seed cells (CD45+CD34+CD43+CD235a-). This method is more efficient than directly inducing monolayer cells from LM to obtain CD34+ phenotype hematopoietic seed cells. Figure 7 ).

[0197] Example 3: RIC capsules generate RIC (ESC source)

[0198] Replace medium III with medium IV and continue culturing the RIC capsules for 7-14 days, changing the medium every 1-3 days. Photograph the RIC capsules on Day 20 and Day 27 for record-keeping. Figure 6 On Day 20, Day 24, and Day 27, single-cell suspensions were prepared from RIC capsules for flow cytometry analysis of ESC-RIC cells (CD45+CD56+), and cell yield was statistically analyzed by sampling. Figure 8 ,9). Furthermore, GFP+ stromal cells were no longer detectable at D27 ( Figure 10 ), and photographs were taken of the ESC-RIC cells harvested on Day 27. Figure 2 ).

[0199] Example 4: RIC (ESC-sourced) monolayer induction

[0200] Selectively, Day 20 RIC cells cultured in medium III were removed and prepared into a single-cell suspension. GFP+ stromal cells were sorted and removed. The remaining mixture of hematopoietic and immune seed cells was added to medium IV and transferred to a culture dish for monolayer culture. The medium was changed every 2 days, and cultured for 7-14 days to induce mature RIC cells. This monolayer induction protocol can also induce high-purity, high-yield RIC cells. Figure 11 ).

[0201] Example 5: In vivo induction of RIC (ESC source)

[0202] After isolating hematopoietic seed cells from the Day 20 RIC capsule and transplanting them into recipient mice, mature RIC cells were efficiently regenerated in bone marrow (BM), peripheral blood (PB), and spleen (SP) after 12 days. Figure 12 ).

[0203] Example 6: Single-cell sequencing transcriptome features of RIC (ESC-derived).

[0204] Deep single-cell RNA sequencing was performed on individual RICs and individual primary immune cells derived from umbilical cord blood (32 primary immune cells and 41 ESC-RIC cells, sequencing method: Smartseq2). Further comparative analysis of the sequencing data showed that RICs and primary immune cells are similar in expressing activating receptors NKp30, NKp44, NKG2C, NKG2D, CS1, etc. Figure 13 ), inhibitory receptors KLRB1, KLRC1, LAIR1, SIGLEC7, CD96, etc. Figure 14 ), surface markers and functional activity-related proteins such as CD56, CD94, CD69, TRAIL, FasL, Perforin, Granzyme A, and Granzyme B, etc. Figure 15 However, RICs differ significantly from natural primary immune cells, particularly in their unique gene expression characteristics. For example, RICs highly express NKG2-E and express SELL and CD2 lower than natural primary immune cells. Figure 16 ).

[0205] Example 7: RIC (ESC-derived) in vitro killing of Nalm-6 tumor cells

[0206] RIC (derived from ESC) cells were co-incubated with the Nalm-6 tumor cell line to test the natural killer activity of RIC cells against tumor cells. Figure 17 : Pluripotent stem cell (ESC-derived) RIC cells killed tumor cells derived from the Nalm-6 lymphoma cell line in vitro. ESC-1, -2, -3, -4, -5: Five ESC cell clones derived from the China National Stem Cell Resource Bank. X-axis, E:T ratio (RIC effector cells: tumor cells). Y-axis, percentage of residual tumor cells. Absolute viable cell count was performed after a co-incubation period of 6 hours.

[0207] Example 8: RIC (ESC-derived) in vitro highly efficient killing of acute myeloid leukemia (AML) cells

[0208] See details Figure 18Pluripotent stem cell (ESC-derived) RIC cells killed AML cell lines THP-1 and Molm13 in vitro. X-axis, E:T ratio (RIC effector cells: tumor cells). Y-axis, tumor killing percentage. Co-incubation time was 4 hours.

[0209] Example 9: RIC (ESC-derived) in vitro killing of A1847 ovarian cancer tumor cells

[0210] See details Figure 19 Pluripotent stem cell (ESC-derived) RIC cells killed tumor cells derived from the A1847 ovarian cancer cell line in vitro. ESC-1, -2, -3, -4, -5: five ESC cell clones from the China National Stem Cell Resource Bank. X-axis: E:T ratio (RIC cells: tumor cells). Y-axis: percentage of residual tumor cells relative to the initial infused tumor. Co-incubation time was 4 hours.

[0211] Example 10: RIC (ESC-derived) can be combined with monoclonal antibodies to enhance antitumor activity via ADCC.

[0212] See details Figure 20 RIC cells (ESC-derived) can enhance the killing of the Raji B-cell lymphoma cell line through ADCC. Rituximab is an anti-CD20 rituximab. X-axis, E:T ratio (RIC cells:tumor cells). Y-axis, percentage of tumor cells killed. Co-incubation time was 4 hours.

[0213] Example 11: RIC (ESC-derived) in vivo killing of A1847 ovarian cancer tumor cells

[0214] First, an A1847 ovarian cancer mouse model was established. Each 6-8 week old NSG mouse (an immunodeficient mouse lacking RIC, T, and B immune cells) was injected intraperitoneally with 2 × 10⁶ cells / mL. 5 A1847-luciferase+ (luciferase-positive) cells were used. One day after inoculation with tumor cells, in vivo imaging (IVIS Spectrum, PerkinElmer) was performed on the tumor model. Then, 1–1.5 × 10⁶ cells were transplanted into each mouse via intraperitoneal injection. 7 Mice were treated with 500 μL of ESC-RIC cells weekly for two weeks. IL-2 (10000 U / mouse) was injected every two days for 21 days post-treatment. In vivo imaging was performed on mice every 3-4 days to analyze changes in tumor burden. ESC-RIC effectively killed A1847 ovarian cancer cells in vivo. Figure 21 )

[0215] Example 12 RIC (ESC-derived) in vivo AML cell killing

[0216] First, an HL60 AML mouse model was established. Each 6-8 week old NSG mouse (an immunodeficient mouse lacking RIC, T, and B immune cells) was injected intraperitoneally with 1×102 6 HL60-luciferase+ (luciferase-positive) cells were used. Three days after tumor cell inoculation, in vivo imaging (IVIS Spectrum, PerkinElmer) was performed on the tumor model. Then, 1–1.5 × 10⁶ cells were transplanted into each mouse via tail vein injection. 7 Mice were treated with 200 μL of ESC-RIC cells three times a week for two to three weeks. In vivo imaging was performed on mice every 7 days to analyze changes in tumor burden. ESC-RIC effectively killed HL60 AML cells. Figure 22 )

[0217] Example 13: CD19-CAR-ESC-RIC (ESC source) kills primary tumor cells in patients with acute lymphoblastic leukemia (ALL).

[0218] First, primary immune cells derived from umbilical cord blood and ESC-RIC cells were infected using CD19-CAR lentivirus. 5 × 10⁶ cells of each were collected. 6 Primary immune cells and ESC-RIC cells were infected with CD19-CAR lentivirus via centrifugation (primary immune cells: MOI=35, ESC-RIC cells: MOI=20). Infection efficiency was assessed by flow cytometry 48 hours after infection. A small number of cells (2 × 10⁶ cells per cell type) were initially collected. 5 (Number of cells) were incubated with CD56-APC and Anti-Mouse FMC63 scFv Monoclonal Antibody, PE (CD19-CAR-PE) antibodies. Infection efficiency was detected by flow cytometry after incubation. Unstained primary immune cells (NC) and ESC-RIC (NC) served as negative controls to detect the positive rate of CD19-CAR. The infection efficiency of CD19-CAR-primary immune cells was 14.9%, while the infection rate of CD19-CAR-ESC-RIC was as high as 82.4%. Figure 23 RICs expressing CD19-CAR can efficiently kill B cells from patients with acute B-lymphoblastic leukemia in vitro. Figure 24 ). Figure 24CD19-CAR-ESC-RIC efficiently kills primary tumor cells from ALL (acute lymphoblastic leukemia) patients. RIC cells carrying CD19-CAR were co-incubated with primary tumor cells (CD19+B cells) from two ALL patients at a 1:1 ratio in vitro for 6 hours, after which the number of CD19+B cells was detected. The Y-axis represents the percentage of residual tumor cells relative to the initial infused tumor.

[0219] Example 14: Pluripotent stem cells (iPSC-derived) differentiate into lateral mesoderm.

[0220] Pluripotent stem cells (iPSC-derived) cultured to 80% adherence density were digested with Accutase (Sigma-Aldrich, A6964) for 1 min, resuspended in ncTarget medium (Zhong Sheng Su Yuan, RP01020) to form a single-cell suspension, and then transferred to cell culture dishes lined with VTN (Animal-Free Human Vitronectin Matrix, PeproTech, AF-VMB-220) after adding 2 μM ROCK inhibitor thiazovivin (Selleck, S1459). The cells were incubated for 24 h (Day 0 of the induction program). The ncTarget medium was removed from the culture dishes, and medium I was added. The cells were incubated for another 24 h (Day 1 of the induction program). Medium I was removed, and medium II was added. The cells were incubated for another 24 h (Day 2 of the induction program). Cell images were taken at D0, D1, and D2. Figure 25 ), qPCR was used to detect the identity markers of the original strip (BRACHYURY+, encoding the TBXT gene) and the lateral mesoderm (APLNR+HAND1+MSGN1-OSR1-). Figure 26 ) and flow cytometry analysis of the induction efficiency of the original strip and lateral plate mesoderm ( Figure 27 The reason for choosing VTN is that, compared to GR-MTG (Growth Factor Reduced Matrigel), VTN material achieves a higher efficiency in obtaining side plate mesoderms under otherwise identical conditions. Figure 28 ).

[0221] Example 15: RIC capsule generates RIC (iPSC source)

[0222] Following the methods described in Examples 1-4, iPSCs were induced to produce iPSC-derived RIC cells using culture media I, II, III, and IV. On Day 20, Day 24, and Day 27, single-cell suspensions of RIC cells were prepared for flow cytometry analysis of iPSC-RIC cells (CD45+CD56+), and cell yield was statistically analyzed by sampling. Figure 29 ,30). Furthermore, GFP+ stromal cells were undetectable on Day 27 ( Figure 31 ).

[0223] Example 16 RIC (iPSC source) monolayer induction

[0224] Selectively, Day 20 RIC cells cultured in medium III were removed and prepared into a single-cell suspension. GFP+ stromal cells were sorted and removed. The remaining mixture of hematopoietic and immune seed cells was added to medium IV and transferred to a culture dish for monolayer culture. The medium was changed every 2 days, and cultured for 7-14 days to induce mature RIC cells. This monolayer induction protocol can also induce high-purity, high-yield RIC cells. Figure 32 ).

[0225] Example 17 Single-cell sequencing transcriptome features of RIC (iPSC source).

[0226] Deep single-cell RNA sequencing was performed on individual RICs and individual primary immune cells derived from umbilical cord blood (32 primary immune cells and 41 iPSC-RIC cells, sequencing method: Smartseq2). Further comparative analysis of the sequencing data showed that RICs and primary immune cells are similar in expressing activating receptors NKp30, NKp44, NKG2C, NKG2D, CS1, etc. Figure 33 ), inhibitory receptors KLRB1, KLRC1, LAIR1, SIGLEC7, CD96, etc. Figure 34 ), surface markers and functional activity-related proteins such as CD56, CD94, CD69, TRAIL, FasL, Perforin, Granzyme A, and Granzyme B, etc. Figure 35 However, RICs differ significantly from natural RICs, particularly in their unique gene expression characteristics. For example, RICs express NKG2-E more highly and SELL and CD2 less than natural primary immune cells. Figure 36 ).

[0227] Example 18: RIC (iPSC-derived) in vitro killing of Nalm-6 tumor cells

[0228] RICs (iPSC-derived) were co-incubated with the Nalm-6 tumor cell line to test the natural killer activity of RICs against tumor cells. Figure 37 : In vitro killing of Nalm-6 lymphoma cell line-derived tumor cells by pluripotent stem cell (iPSC-derived) RIC cells. X-axis, E:T ratio (RIC effector cells: tumor cells). Y-axis, percentage of residual tumor cells. Absolute viable cell count was performed after a co-incubation period of 6 hours.

[0229] Example 19: RIC (iPSC-derived) in vitro killing of A1847 ovarian cancer tumor cells

[0230] Figure 38 : In vitro killing of ovarian cancer cell line A1847-derived tumor cells by pluripotent stem cell (iPSC-derived) RIC cells. X-axis, E:T ratio (RIC cells: tumor cells). Y-axis, percentage of residual tumor cells relative to the initial infused tumor. Co-incubation time was 4 hours.

[0231] Example 20: RIC (iPSC-derived) can be combined with monoclonal antibodies to enhance antitumor activity via ADCC.

[0232] See details Figure 39 iPSC-RIC cells enhance the killing of the Raji B-lymphoma cell line through ADCC. Rituximab is an anti-CD20 rituximab. X-axis: E:T ratio (RIC cells:tumor cells). Y-axis: percentage of tumor cells killed. Co-incubation time: 4 hours.

[0233] Example 21: RIC (iPSC-derived) in vivo killing of A1847 ovarian cancer tumor cells

[0234] First, an A1847 ovarian cancer mouse model was established. Each 6-8 week old NSG mouse (an immunodeficient mouse lacking RIC, T, and B immune cells) was injected intraperitoneally with 2 × 10⁶ cells / mL. 5 A1847-luciferase+ (luciferase-positive) cells were used. One day after inoculation with tumor cells, in vivo imaging (IVIS Spectrum, PerkinElmer) was performed on the tumor model. Then, 1–1.5 × 10⁶ cells were transplanted into each mouse via intraperitoneal injection. 7 Mice were treated with 500 μL of iPSC-RIC cells weekly for two weeks. IL-2 (10000 U / mouse) was injected every two days within 21 days post-treatment. In vivo imaging was performed on mice every 3-4 days to analyze changes in tumor burden. iPSC-RIC effectively killed A1847 ovarian cancer cells in vivo. Figure 40 RIC (iPSC-derived) kills A1847 ovarian cancer cells in vivo. A, Treatment process and detection diagram. B, Dynamic in vivo imaging to detect tumor burden in the RIC treatment group and the tumor burden control group.

[0235] Example 22: CD19-CAR-iPSC-RIC (iPSC source) kills primary tumor cells in patients with acute lymphoblastic leukemia (ALL).

[0236] First, primary immune cells derived from umbilical cord blood and iPSC-RIC cells were infected using CD19-CAR lentivirus. 5 × 10⁶ cells of each were collected. 6 Primary immune cells and iPSC-RIC cells were infected with CD19-CAR lentivirus via centrifugation (primary immune cells: MOI=35, iPSC-RIC cells: MOI=20). Infection efficiency was assessed by flow cytometry 48 hours after infection. A small number of cells (2 × 10⁶ cells per cell type) were initially collected. 5 (1) cells were incubated with CD56-APC and Anti-Mouse FMC63 scFv Monoclonal Antibody, PE (CD19-CAR-PE) antibodies. After incubation, their infection efficiency was detected by flow cytometry. Unstained primary immune cells (NC) and iPSC-RIC (NC) were used as negative controls to detect the positive rate of CD19-CAR. Figure 41 The infection rate of CD19-CAR in RIC cells is shown. Compared with primary immune cells, the expression rate of CD19-CAR virus in RIC cells after infection (MOI=20) is as high as 83.0%, while the expression rate of innate immune cells (MOI=35) after infection is only 33.4%. Figure 42 This study aimed to efficiently kill primary tumor cells from ALL (acute lymphoblastic leukemia) patients using CD19-CAR-iPSC-RIC. CD19-CAR-carrying RIC cells were co-incubated with primary tumor cells (CD19+B cells) from ALL patients at a 1:1 ratio in vitro for 6 hours, followed by detection of CD19+B cell count. The Y-axis represents the percentage of residual tumor cells relative to the initial infused tumor.

[0237] In addition to those described herein, various modifications to the invention will be apparent to those skilled in the art based on the foregoing description, and such modifications are also intended to fall within the scope of the appended claims.

Claims

1. A method for producing a RIC cell population (regenerative immune cell population), comprising the following steps: (1) inducing culture of stem cells to form lateral plate mesoderm seed cells using medium I and medium II in sequence; (2) preparing RIC sacs and inducing culture in medium III to obtain hematopoietic seed cells, wherein the RIC sacs are composed of lateral plate mesoderm seed cells derived from step (1) and trophoblast cells; (3) inducing culture of the hematopoietic seed cells to differentiate into the RIC cells using medium IV; wherein: the medium I is Essential 6 medium added with 40 ng / mL BMP4, 30 ng / mL ACTIVIN A, 20 ng / mL bFGF, 6 µM CHIR-99021 and 100 nM PIK-90; the medium II is Essential 6 medium added with 30 ng / mL BMP4, 1 µM A-83-01 and 1 µM C59; the medium III is Essential 6 medium added with 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; the medium IV is composed of 55 % DMEM-high glucose, 30 % DMEM / F12, 15 % SUPERGROW cell culture supplement, 2 mM GlutaMAX™, 1 μM β-mercaptoethanol, 5 ng / mL sodium selenite, 50 uM ethanolamine, 20 μg / mL ascorbic acid, 1 % penicillin-streptomycin solution, 5 ng / mL IL-3, 20 ng / mL SCF, 20 ng / mL IL-7, 10 ng / mL IL-15 and 10 ng / mL Flt3L.

2. The method of claim 1, wherein, The mixing ratio of the lateral plate mesoderm seed cells to the trophoblast cells ranges from 1:5 to 1:

200.

3. The method of claim 1, wherein, The trophoblast cells of the RIC sacs are selected from one or more of AFT024, OP9 cells, MS5 cells, HS-5 or other fibroblasts.

4. The method of claim 3, wherein, The trophoblast cells of the RIC sacs express at least one, two or three of the following genes: DLL1, DLL4, IL7, IL15, IL3 and Flt3L.

5. The method of claim 1, wherein, The total cell amount of a single RIC capsule is in the range of 2-10 million cells, and the culture footprint of each RIC capsule is in the range of 1-10 cm2 2 .

6. The method according to any one of claims 1 to 5, characterized in that one or more of the following: the duration of the step (1) culture is 1-3 days; the duration of the step (2) culture is 10-18 days; the duration of the step (3) culture is 7-14 days.

7. The method of claim 6, wherein one or more of the following: the duration of the step (1) culture is 1 day, 1.5 days, 2 days, 2.5 days, or 3 days; The duration of the culturing in step (2) is 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, or 17 days. The duration of the culturing in step (3) is 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days.

8. The method of any one of claims 1-5, wherein, The duration of the culturing in steps (2) and (3) is 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, or 32 days.

9. The method of claim 1, wherein, The fresh medium is replaced every day or every 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days in steps (2) and (3).

10. The method of claim 1, wherein, The total duration of the culturing from the stem cells to the RIC cells is 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, or 34 days.

11. The method of claim 1, wherein, The culturing in steps (1)-(3) is performed at 37°C under 5% CO2.

12. The method of claim 1, wherein, The culturing in steps (1)-(3) is performed in an incubator at 37°C under 5% CO2.

13. The method of claim 1, wherein, The culturing in step (1) is performed in a matrix, which is one or more of a glycoprotein, hyaluronic acid, laminin, fibronectin, collagen, elastin, heparan sulfate, dextran, dextran sulfate, and chondroitin sulfate.

14. The method of claim 13, wherein the glycoprotein is vitronectin (VTN) or fibronectin.

15. The method of claim 1, wherein, The average NKG2-E expression level of the RIC cell population is at least 10 times that of the primary immune cells. The expression levels of SELL and CD2 of the RIC cell population are less than at least 10 times that of the primary immune cells.

16. The method of claim 1, wherein, The RIC cell population has one or more of the following characteristics: (1) the RIC cell population expresses a target CAR by means of a gene delivery vector or expresses a target CAR by means of gene editing, and the expression rate of the CAR is 60%-100%; (2) has any three or more of the activating receptors NCR1, NCR2, NCR3, KLRC2, KLRK1, and SLAMF7; (3) has any three or more of the inhibitory receptors KLRB1, KLRC1, LAIR1, SIGLEC7, and CD96; (4) comprises ≥80% of cells that express CD45 and CD56 simultaneously; (5) the RIC cell population is generated from stem cells; (6) the stem cells are totipotent stem cells or pluripotent stem cells, wherein the pluripotent stem cells are selected from embryonic stem cells, haploid stem cells, and induced pluripotent stem cells.

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