WO2024153133A1 - Isclp cell, and preparation method therefor and use thereof - Google Patents

Abstract

Disclosed is an iSCLP cell or iSCLP cell population. The iSCLP cell or iSCLP cell population is prepared by means of the following method: inducing a lateral plate mesoderm cell to differentiate in a first culture medium for 13 to 23 days to obtain the iSCLP cell or iSCLP cell population. The prepared iSCLP cell or iSCLP cell population can be cryopreserved and transplanted in vivo to generate an NK cell. The regenerated NK cell has a long survival period, a high activity, a high proportion of CD16+ cells and a high anti-tumor activity. Therefore, the iSCLP cell or iSCLP cell population has the potential to be anti-tumor, anti-infection, anti-aging and to treat the defects of NK cells.

Description

iSCLP cell and its preparation method and application

This application is based on an application with CN application number 202310080661.9 and filing date January 19, 2023, and claims priority. The disclosed content of the CN application is hereby introduced as a whole into this application.

Technical Field

The present invention relates to the field of stem cell biology, and in particular to an iSCLP cell (stem cell-induced lymphoid progenitor cell) and a preparation method and application thereof.

Background technique

Pluripotent stem cells can differentiate into most cell types, including all derived cells of the endoderm, mesoderm, and ectoderm. It has been documented that hematopoietic stem cells isolated from mice, human bone marrow, human umbilical cord blood, or human peripheral blood can be used to induce lymphoid progenitor cells. However, the technology for differentiating pluripotent stem cells in vitro into lymphoid progenitor cells that can be cryopreserved and transplanted efficiently and regenerating NK cells in vivo is not yet mature.

Currently, the most widely used method for inducing NK cells from pluripotent stem cells is the embryoid body (EB) method. This method first prepares EB balls to induce hematopoietic progenitor cells, and then separates the hematopoietic progenitor cells for downstream NK cell differentiation. However, this method is time-consuming, complicated to operate, and costly. The regeneration efficiency of lymphoid hematopoietic progenitor cells is low during the hematopoiesis process, the induced NK cells are of low purity, and the expression of the cell surface protein CD16 is low, which is not conducive to antibody-dependent cell-mediated cytotoxicity (ADCC).

Moreover, the survival period of NK cells obtained through in vitro induction after transplantation into the body is short, and their activity and viability decrease after freezing, so it is still very challenging to achieve spot supply.

Summary of the invention

The present application provides a cryopreservable, spot-stock, high-transplantation-efficiency induced differentiation-derived lymphoid progenitor cell (iSCLP) from pluripotent stem cells PSC (including embryonic stem cells (ESC), haploid stem cells, induced pluripotent stem cells (iPSC), and extended pluripotent stem cells (EPSC)). After in vivo transplantation, iSCLP can obtain NK cells with high functional activity, strong amplification ability, long survival period, and high maturity, providing a new source for regenerative NK cells. The present application also provides a method for preparing iSCLP cells and their applications in the fields of anti-tumor, anti-infection, and anti-aging.

The iSCLP cells prepared in the present application were transplanted into mice at a rate of 0.2×10 ⁶ cells. After two weeks, NK cells could still be detected in the bone marrow, with a ratio of more than 40% in the lungs and more than 80% in the liver. The above data show that the iSCLP cells of the present invention still maintain a good transplantation efficiency after cryopreservation and resuscitation, have a good differentiation and amplification effect after transplantation, and have a long survival period in the body. The method of transplanting iSCLP after cryopreservation and resuscitation can overcome the disadvantage that the clinical treatment of NK cells is affected by cryopreservation and resuscitation.

Therefore, in a first aspect, the present application provides an iSCLP cell or an iSCLP cell population, which is prepared by the following method:

The lateral plate mesoderm cells are induced to differentiate in the first culture medium for 13-23 days to obtain the iSCLP cells or iSCLP cell population; wherein,

The first culture medium is a basal culture medium supplemented with one or more of the following substances: one or more serum replacements, one or more non-essential amino acids, glutamine or a stabilized dipeptide of L-alanyl-L-glutamine, a TGF-β inhibitor, hydrocortisone, ascorbic acid, and cell growth factors including IL-3 and IL-5.

In some embodiments, the lateral plate mesoderm cells are induced to differentiate in the presence of trophoblast cells.

In some embodiments, the lateral plate mesoderm cells are obtained by induced culture of stem cells.

In some embodiments, the stem cells are induced and cultured in a second culture medium and a third culture medium sequentially, wherein:

The second culture medium is a basal culture medium supplemented with the following substances: one or more serum replacements, one or more non-essential amino acids, glutamine or a stabilized dipeptide of L-alanyl-L-glutamine, and BMP4, ACTIVIN, bFGF, a Wnt agonist, and a PI3K inhibitor;

The third culture medium is a basal culture medium supplemented with the following substances: one or more serum replacements, one or more non-essential amino acids, glutamine or a stabilized dipeptide of L-alanyl-L-glutamine, and BMP4, a TGF-β inhibitor, and a Wnt inhibitor.

In some embodiments, the lateral plate mesoderm cells are obtained by culturing stem cells for 1-3 days.

In some embodiments, the lateral plate mesoderm cells are obtained by inducing and culturing in the second culture medium and the third culture medium for 1-2 days respectively, for example, inducing and culturing in the second culture medium for 1 day and inducing and culturing in the third culture medium for 1 day.

In some embodiments, more than 65% (e.g., more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 99%) of the lateral plate mesoderm cells are APLNR+ cells. In some embodiments, more than 65% (e.g., more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 99%) of the lateral plate mesoderm cells are APLNR+HAND1+ cells. In some embodiments, more than 65% (e.g., more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 99%) of the lateral plate mesoderm cells are APLNR+HAND1+MSGN1-OSR1- cells.

In some embodiments, the method is characterized by one or more of the following:

(i) the trophoblast cells are selected from one or more of AFT024 cells, OP9 cells, MS5 cells, HS-5 cells or other fibroblasts, and preferably express at least one, two, three, multiple or all of the following genes: DLL1, DLL4, IL7, IL15, IL3 and Flt3L. Further preferably, the trophoblast cells are OP9-DLL1, OP9-DLL4, or OP9-DLL1-DLL4;

(ii) the ratio of the lateral plate mesoderm cells to the trophoblast cells is 1:5-1:200, 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;

(iii) inducing differentiation of the lateral plate mesoderm cells in a first culture medium (eg, in the presence of trophoblast cells) for 13-14 days, 13-16 days, 13-18 days, 14-16 days, 14-19 days, 14-23 days, 16-18 days, or 16-23 days;

(iv) the serum substitute is a serum-free additive, for example, selected from KOSR, B27, UltroserTMG, SUPERGROW and any combination thereof; preferably, the serum substitute is SUPERGROW;

(v) the non-essential amino acids are selected from glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamine Acid, L-proline, L-serine and any combination thereof;

(vi) the TGF-β inhibitor is a small molecule compound A-83-01 or SB431542, preferably SB431542;

(vii) the cell growth factors further include 1, 2, 3, 4, 5, 6 or 7 of Flt3L, TPO, SCF, EGF, VEGF, bFGF and IGF-1;

(viii) the Wnt agonist is CHIR-99021;

(ix) the PI3K inhibitor is PIK-90 or LY294002;

(x) the Wnt inhibitor is a small molecule compound C59 or IWR-1-endo;

(xi) 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, preferably Essential 6 medium;

(xii) the total content of the one or more serum replacements 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);

(xiii) the one or more non-essential amino acids are each independently present in an amount of 0.1-0.5 mM, such as about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM or about 0.5 mM;

(xiv) the content of the stabilized dipeptide of glutamine or L-alanyl-L-glutamine is 1-5 mM, such as about 1 mM, about 2 mM, about 3 mM, about 4 mM, or about 5 mM;

(xv) the content of each factor of BMP4, ACTIVIN, bFGF, Flt3L, TPO, SCF, EGF, VEGF, bFGF, IGF-1, IL-3, and IL-5 is independently 1-100 ng/mL, such as 2-100 ng/mL, 2-50 ng/mL, 5-100 ng/mL, 5-50 ng/mL, or 5-20 ng/mL; such as about 1 ng/mL, about 2 ng/mL, about 3 ng/mL, about 5 ng/mL, about 8 ng/mL, or about 10 ng/mL; ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 45 ng/mL, about 50 ng/mL, about 55 ng/mL, about 60 ng/mL, about 65 ng/mL, about 70 ng/mL, about 75 ng/mL, about 80 ng/mL, about 85 ng/mL, about 90 ng/mL, about 95 ng/mL, or about 100 ng/mL;

(xvi) the content of each small molecule in the Wnt agonist, PI3K inhibitor, TGF-β inhibitor, Wnt inhibitor and hydrocortisone is 10nM-100μM, such as 10-30nM, 10-50nM, 10-80nM, 10-100nM, 100nM-1μM, 1μM-10μM, 10μM-30μM, 10μM-50μM, 10μM-80μM, 30μM-100μM;

(xvii) the ascorbic acid content is 1-100 μg/mL, such as 2-100 μg/mL, 2-50 μg/mL, 5-100 μg/mL, 5-50 μg/mL, or 5-20 μg/mL; such as about 1 μg/mL, about 2 μg/mL, about 3 μg/mL, about 5 μg/mL, about 8 μg/mL, about 10 μg/mL, about 15 μg/mL, about 20 μg/mL L, about 25 μg/mL, about 30 μg/mL, about 35 μg/mL, about 40 μg/mL, about 45 μg/mL, about 50 μg/mL, about 55 μg/mL, about 60 μg/mL, about 65 μg/mL, about 70 μg/mL, about 75 μg/mL, about 80 μg/mL, about 85 μg/mL, about 90 μg/mL, about 95 μg/mL, or about 100 μg/mL;

(xviii) the first culture medium is Essential 6 medium supplemented with the following substances: one or more serum replacements Substitutes, one or more non-essential amino acids, glutamine or stabilized dipeptide of L-alanyl-L-glutamine, SB431542, hydrocortisone, ascorbic acid, and cell growth factors including IL-3, IL-5, Flt3L, TPO, SCF, EGF, VEGF, bFGF and IGF-1; Essential 6 medium supplemented with the following substances is preferred: SB431542, hydrocortisone, ascorbic acid, IL-3, IL-5, Flt3L, TPO, SCF, EGF, VEGF, bFGF and IGF-1; preferably Essential 6 medium containing 10 μM SB431542, 10 μM hydrocortisone, 5 ng/mL Flt3L, 5 ng/mL TPO, 50 ng/mL SCF, 50 ng/mL EGF, 50 ng/mL VEGF, 50 ng/mL bFGF, 50 ng/mL IGF-1, 5 ng/mL IL-3, 10 ng/mL IL-15, 50 μg/mL ascorbic acid and 2% SUPERGROW Cell Culture Supplement;

(xix) the second culture medium is Essential 6 culture medium supplemented with the following substances: BMP4, ACTIVIN A, bFGF, CHIR-99021 and PIK-9; preferably, Essential 6 culture medium containing 40 ng/mL BMP4, 30 ng/mL ACTIVIN A, 20 ng/mL bFGF, 6 μM CHIR-99021 and 100 nM PIK-90;

(xx) the third culture medium is Essential 6 medium supplemented with the following substances: BMP4, A-83-01 and C59; preferably Essential 6 medium containing 30 ng/mL BMP4, 1 μM A-83-01 and 1 μM C59;

(xxi) The stem cells are totipotent stem cells or pluripotent stem cells, preferably embryonic stem cells (ESCs), haploid stem cells, induced pluripotent stem cells (iPSCs), or expanded pluripotent stem cells (EPSCs).

In some embodiments, in the process of inducing differentiation to obtain iSCLP cells or iSCLP cell groups, lateral plate mesoderm cells and trophoblast cells form cell capsules, which are referred to herein as organoids. In some embodiments, each organoid contains 2×10 ⁵ -1×10 ⁷ cells, such as 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, 9 million, etc. In certain embodiments, 1 organoid can be induced to obtain 1×10 ⁵ , 2×10 ⁵ , 3×10 ⁵ , 4×10 ⁵ , 5×10 ⁵ , 6×10 ⁵ , 7×10 ⁵ , 8×10 ⁵ , 9×10 ⁵ or 1×10 ⁶ iSCLP cells. Each organoid culture occupies an area ranging from 1-10 cm ² . Organoids can be cultured continuously for 13-23 days.

In another aspect, the present application provides an iSCLP cell (stem cell induced lymphoid progenitor cell) population or the iSCLP cell population described in any one of the first aspects, wherein the cells simultaneously expressing CD45, CD34, CD38 and CD7 account for ≥50%, ≥60%, ≥70%, ≥80%, ≥90%, ≥95% or ≥99%.

The main mechanisms by which NK cells exert their killing effects include (1) cell degranulation, such as secretion of cytotoxic granules such as perforin and granzyme; (2) expression of tumor necrosis factor (such as FasL, TRAIL, etc.) to activate cell apoptosis; (3) by expressing FcγRIII (CD16) and binding to the Fc segment of tumor antigen-specific antibodies, mediating antibody-dependent cellular cytotoxicity (ADCC). The NK cells obtained by transplanting and differentiating the iSCLP cells of the present invention in vivo have a high CD16+ ratio, which is more conducive to the ADCC process and can be used for anti-tumor, anti-viral infection, anti-aging, and treatment of diseases such as NK cell deficiency. In some embodiments, the CD16 positive ratio of NK cells obtained by in vivo regeneration of the iSCLP cells of the present invention is above 95%.

Therefore, in another aspect, the present invention provides a lymphoid immune cell obtained by the following method:

The iSCLP cells or iSCLP cell population described in any one of the first aspects are cultured in a bioreactor and separated to obtain the lymphoid immune cells.

In some embodiments, the lymphoid immune cells are NK cells.

In some embodiments, the bioreactor is a mammal, such as a mouse, rat, pig, or non-human primate.

In another aspect, the present application provides a culture comprising the iSCLP cell or iSCLP cell population described in any one of the first aspect, and a culture medium.

In another aspect, the present application provides a culture supernatant, which is the culture supernatant produced by culturing the iSCLP cells or iSCLP cell population described in any one of the first aspects.

In another aspect, the present application provides a pharmaceutical composition comprising the iSCLP cell or iSCLP cell population described in any one of the first aspect, the lymphoid immune cell described in any one of the second aspect, the culture described in any one of the third aspect or the culture supernatant described in any one of the fourth aspect, and a carrier or excipient.

In some embodiments, the pharmaceutical composition is an injection, a microneedle, a mucosal patch, an enema, a suppository, a gel, an oral agent, an aerosol, a drop, an ointment, an implant, a capsule, or an aerosol.

In some embodiments, the pharmaceutical composition is an injection.

In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solution, dispersion, suspension or emulsion.

In another aspect, the present application provides a culture medium having the characteristics of the first culture medium as defined in any one of the first aspects.

In another aspect, the present application provides a kit comprising the culture medium described above (particularly the first culture medium), and optionally the second culture medium and/or the third culture medium defined in any one of the first aspects.

In another aspect, the present application provides use of the iSCLP cell or iSCLP cell population described in any one of the first aspect, the lymphoid immune cell described in any one of the second aspect, the culture described in any one of the third aspect, the culture supernatant described in any one of the fourth aspect, or the pharmaceutical composition described in any one of the fifth aspect in the preparation of a drug, wherein the drug is used for:

(1) Anti-infection (including infections caused by viruses, bacteria, fungi and parasites). Preferably, the infections include infections caused by various pathogens (such as viruses, bacteria, fungi or parasites) after radiotherapy and chemotherapy, and early infections after bone marrow transplantation (such as bone marrow transplantation after radiotherapy or chemotherapy);

(2) Anti-tumor, preferably, the tumor is a blood tumor or a solid tumor, for example, leukemia, lymphoma, breast cancer, ovarian cancer, liver cancer, glioma or pancreatic cancer;

(3) Antiviral (e.g., coronaviruses such as SARS-CoV-2, human HIV, rhinovirus, cytomegalovirus CMV, hepatitis B virus HBV, herpes simplex virus HSV, herpes zoster virus) infections, for example, COVID-19, viral pneumonia, AIDS, acute respiratory disease caused by rhinovirus, genital and urinary tract infections, central nervous system infections or liver diseases caused by CMV, hepatitis B, blistering dermatitis, gingivostomatitis, keratoconjunctivitis, encephalitis, reproductive system infections or neonatal infections caused by HSV, or chickenpox, encephalomyelitis or conjunctivitis caused by herpes zoster virus;

(4) anti-aging; or

(5) Treatment of NK cell-related immunodeficiency diseases, such as primary NK cell deficiency, Chédiak-Higashi syndrome, X-linked lymphoproliferative syndrome (XLP), leukocyte adhesion deficiency (CD11/CD18 deficiency) or severe combined immunodeficiency (SCID).

In another aspect, the present application provides a method for anti-infection, anti-tumor, anti-viral infection, anti-aging or treatment of NK cell-related immunodeficiency diseases, comprising the step of administering or transplanting a therapeutically effective amount of the iSCLP cells or iSCLP cell populations described in any one of the first aspect, the lymphoid immune cells described in any one of the second aspect, the culture described in any one of the third aspect, the culture supernatant described in any one of the fourth aspect, or the pharmaceutical composition described in any one of the fifth aspect to a subject in need thereof.

In some embodiments, the infection is caused by a virus, bacteria, fungus or parasite. In some embodiments, the infection is infection by various pathogens (e.g., viruses, bacteria, fungi or parasites) after radiotherapy or chemotherapy, or infection after bone marrow transplantation. In some embodiments, the bone marrow transplantation is bone marrow transplantation after radiotherapy or chemotherapy.

In some embodiments, the tumor is a hematological tumor or a solid tumor, for example, leukemia, lymphoma, breast cancer, ovarian cancer, liver cancer, glioma, or pancreatic cancer.

In some embodiments, the virus is a coronavirus (e.g., SARS-CoV-2), human HIV virus, rhinovirus, cytomegalovirus CMV, hepatitis B virus HBV, herpes simplex virus HSV, or herpes zoster virus. In some embodiments, the viral infection is COVID-19, viral pneumonia, AIDS, acute respiratory disease caused by rhinovirus, genital urinary system infection, central nervous system infection or liver disease caused by CMV, hepatitis B, blistering dermatitis, gingivostomatitis, keratoconjunctivitis, encephalitis, reproductive system infection or neonatal infection caused by HSV, or chickenpox, encephalomyelitis or conjunctivitis caused by herpes zoster virus.

In some embodiments, the NK cell-related immunodeficiency disease is a primary NK cell deficiency disease, Chédiak-Higashi syndrome, X-linked lymphoproliferative syndrome (XLP), leukocyte adhesion deficiency (CD11/CD18 deficiency), or severe combined immunodeficiency (SCID).

In some embodiments, the therapeutically effective amount is 1×10^5 cells/kg to 1×10^9 cells/kg.

In another aspect, the present application provides the culture medium or kit described above for preparing any one of the first aspect Use of iSCLP cells or iSCLP cell populations, the lymphoid immune cells according to any one of the second aspect, the culture according to any one of the third aspect, the culture supernatant according to any one of the fourth aspect, or the pharmaceutical composition according to any one of the fifth aspect.

In another aspect, the present invention provides a method for preparing the iSCLP cells or iSCLP cell population as described above, comprising the steps defined in any one of the first aspect.

Definition of Terms

In this document, unless otherwise specified, the scientific and technical terms used have the meanings commonly understood by those skilled in the art. In addition, the molecular genetics, nucleic acid chemistry, cell culture, biochemistry, cell biology and other operating procedures used in this document are conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

As used herein, the term "embryonic stem cell" may refer to a pluripotent cell isolated from an embryo, which is maintained in an in vitro cell culture. This cell is a cultured cell that is separated from the embryo of the culture and can divide rapidly, and simultaneously maintains pluripotency. Embryonic stem cells may be cultivated with or without feeder cells. Embryonic stem cells may be separated from early embryos, including but not limited to blastocyst stage embryos and pre-blastocyst stage embryos. Embryonic stem cells may have a round cell morphology, and may grow with round cell masses on feeder layers. Embryonic stem cells are well known to those of ordinary skill in the art. Its source may be a human or non-human animal. For human embryonic stem cells, it is limited to utilizing the human embryo separation and acquisition of fertilization within 14 days without in vivo development.

As used herein, the term "in vitro" refers to an artificial environment, and the processes and reactions therein. In vitro environments are exemplified by test tubes and cell cultures, but are not limited thereto.

As used herein, the term "in vivo" refers to the natural environment (ie, an animal or a cell) and the processes and reactions therein.

As used herein, the term "culture" refers to a product obtained after culturing cells in a culture medium.

As used herein, the term "culture supernatant" refers to a culture fluid obtained by culturing cells that does not contain the cells themselves. Thus, for example, the culture supernatant that can be used in the present invention can be obtained by separating and removing cell components after culturing. The culture supernatant can also be subjected to other treatments, such as centrifugation, concentration, solvent replacement, dialysis, freezing, drying, freeze drying, dilution, desalting, storage, etc.

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

As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease or disorder or symptom in a subject or to minimize its effect if it occurs. The term "treatment" refers to a method implemented to obtain a beneficial or desired clinical result. Beneficial or desired clinical results include, but are not limited to, reducing the rate of disease progression, improving or alleviating the disease state, and regressing or improving the prognosis, whether detectable or undetectable. The amount of the therapeutic agent that effectively alleviates any particular disease symptom can vary according to factors such as the patient's disease state, age and weight, and the ability of the drug to cause a desired response in the subject. Whether the disease symptoms are alleviated can be assessed by any clinical measurement, which is usually used by a doctor or other skilled health care provider to assess the severity or progression of the symptom.

As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, a disease prevention effective amount refers to an amount sufficient to prevent, prevent, or delay the occurrence of a disease; a disease treatment effective amount refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient who already has the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use 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 condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

As used herein, the term "subject" includes, but is not limited to, various animals, such as mammals, e.g., bovines, equines, ovines, porcines, canines, felines, lagomorphs, rodents (e.g., mice or rats), non-human primates (e.g., macaques or cynomolgus monkeys), or humans.

As used herein, the terms "proliferation" or "expansion" refer to the ability of a cell or a population of cells to increase in number.

As used herein, the term "pharmaceutical composition" refers to a pharmaceutically acceptable composition, wherein the composition comprises iSCLP cells, and in some embodiments further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition may be a combination.

As used herein, the term "combination" refers to a fixed combination in the form of a dosage unit, or to a kit of parts for joint administration, wherein iSCLP cells and a combination partner (e.g., another drug explained below, also referred to as a "therapeutic agent" or "co-agent") can be administered separately at the same time or within a time interval. In some cases, the combination partner exhibits a cooperative effect, such as a synergistic effect.

As used herein, the terms "co-administration" or "administered in conjunction with" and the like are meant to encompass administration of the selected combination partners to a single subject in need thereof, and are intended to include treatment regimens in which administration is not necessarily by the same route of administration or at the same time.

As used herein, based on genome editing tools, such as clustered regularly spaced short palindromic repeats (CRISPR) systems, TALEN editing tools, etc., which can be used in a variety of organisms (e.g., for adding, destroying or changing the sequence of a specific gene). As used herein, the term "epitope" includes any protein determinant clusters of antigens that can specifically bind to antibodies or T cell receptors. Epitope determinants are generally composed of chemically active molecular surface groups, such as amino acids or sugar side chains, and generally have specific three-dimensional structural features and specific charge characteristics.

As used herein, the term "differentiation" is the process by which less specialized cells become A more specialized cell type that forms progeny 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 progeny with characteristics of the new cell type can be at least about 1%, 5%, 25% or more to increase preference.

iSCLP: induced Stem Cell-derived Lymphocyte progenitors

NK：Natural killer cells

ESC:Embryonic stem cells

iPSC: induced Pluripotent stem cells

EPSC: Extended Pluripotent stem cells

EB: Embryoid body

ADCC: Antibody-dependent cell cytotoxicity

AML

PMA/Ionomycin: Phorbol ester/Ionomycin

TNF-α：Tumor necrosis factor-α

Advantageous Effects of the Invention

The present invention provides a lymphoid progenitor cell iSCLP derived from a pluripotent stem cell that can be cryopreserved and transplanted in vivo to produce regenerative NK cells. The NK cells regenerated in vivo by iSCLP cells after transplantation have a long survival period, high activity, a high CD16+ ratio, and strong anti-tumor activity. The iSCLP cells can be used for anti-tumor, anti-viral infection, anti-aging, and treatment of diseases such as NK cell deficiency.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a flow chart of the induction and differentiation of pluripotent stem cells into iSCLP cells according to the present invention.

FIG. 2 shows the morphology and flow cytometry phenotype of lateral plate mesoderm cells obtained by differentiation of pluripotent stem cells (derived from embryonic stem cells) in Example 1. APLN is an identity marker of lateral plate mesoderm.

FIG. 3 shows the immunophenotype of iSCLP cells obtained by differentiation induction in Example 2 (CD45+CD34+CD7+CD38+).

FIG. 4 is a flow cytometric analysis of the distribution of NK cells generated by transplantation of iSCLP cells (derived from embryonic stem cells) in recipient mice in Example 3.

FIG. 5 shows that iSCLP-derived NK cells (derived from embryonic stem cells) express tumor necrosis factor TNF-α.

Figure 6 shows that the iSCLP (embryonic stem cell-derived) in vivo model effectively inhibits human AML tumor cells: a, treatment process and detection schematic diagram; b, dynamic in vivo imaging on Day 0 and Day 7 to detect the tumor burden of the iSCLP treatment group and the tumor control group; c, statistical results of the Total Flux values of in vivo imaging of mice in the iSCLP treatment group and the tumor control group on Day 0 and Day 7.

Detailed ways

The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

The process of inducing pluripotent stem cells to differentiate into iSCLP cells is shown in Figure 1, where the meaning of each symbol and the composition of the culture medium are as follows:

PSC refers to pluripotent stem cells, which may include embryonic stem cells (ESC), haploid stem cells, induced pluripotent stem cells (iPSC), and extended pluripotent stem cells (EPSC); PS refers to primitive streak; LM refers to lateral plate mesoderm; iSCLP refers to stem cell-induced lymphoid progenitor cells; and iNK refers to induced NK cells.

Medium I: 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.

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

Medium III: Essential 6 medium containing 10 μM SB431542, 10 μM hydrocortisone, 5 ng/mL Flt3L, 5 ng/mL TPO, 50 ng/mL SCF, 50 ng/mL EGF, 50 ng/mL VEGF, 50 ng/mL bFGF, 50 ng/mL IGF-1, 5 ng/mL IL-3, 10 ng/mL IL-15, 50 μg/mL ascorbic acid, and 2% SUPERGROW Cell Culture Supplement.

Example 1 Pluripotent stem cells (derived from embryonic stem cells) were differentiated into lateral plate mesoderm.

The pluripotent stem cells (derived from embryonic stem cells) cultured in a monolayer to 80% adherent density were digested with Accutase for 1 min, resuspended in nc Target medium to form a single cell suspension, and transferred to a cell culture dish covered with vitronectin after adding 2 μM Thiazovivin. Cultured in the incubator for 24 h (this time is defined as Day 0 of the induction program). The nc Target medium in the culture dish was aspirated, and medium I was added, and cultured in the incubator for another 24 h (this time is defined as Day 1 of the induction program). Medium I was aspirated, and medium II was added, and cultured for another 24 h (this time is defined as Day 2 of the induction program). Highly pure lateral plate mesoderm cells can be obtained on Day 2, and the cell morphology and flow immunohistotype (lateral plate mesoderm cell characteristic protein: APLNR+) are shown in Figure 2.

Example 2 Organoid Culture-iSCLP Cell (Embryonic Stem Cell Derived) Stage Induction

The specific steps are:

Digest the lateral plate mesoderm cells to prepare a single cell suspension, and mix with OP9-DLL4 trophoblasts. The mixing ratio of the two cells can range from 1:5-1:200. The total number of cells is adjusted from 200,000 to 1000,000 according to the pore size of the culture dish to prepare organoids. Each organoid occupies a culture surface area of 1-10 ^cm2 . Under the conditions of medium III, continue to induce for 13-23 days, and change the medium every 1-3 days. The organoids on Day 18 were selected for flow cytometry and iSCLP cells (CD45+CD34+CD7+CD38+) were sorted. The characteristic immunophenotype of iSCLP cells is shown in Figure 3.

Example 3 In vivo transplantation of iSCLP cells (derived from embryonic stem cells) to produce NK cells

After thawing iSCLP cells, 200,000 iSCLP cells were transplanted into the tail vein of mice irradiated with a sublethal dose (1.5 Gy). B-NDG hIL15 (NOD.CB17-Prkdc ^scid Il2rg ^tm1 Il15 ^{tm1 (IL15)} /Bcgen background, Biocytogen Co., Ltd) mice are severely immunodeficient mice without NK, T and B cells. Two weeks later, various tissues and organs (peripheral blood, liver, lungs, spleen, bone marrow, peritoneal cavity) were taken for flow cytometry detection of iNK cells (CD45 ⁺ CD3 ^- CD56 ⁺ CD16 ^+/- ). The results showed that iSCLP-derived iNK cells could be detected in the peripheral blood, liver, lungs, spleen, bone marrow, and peritoneal cavity of the recipient mice. Among them, more iNK cells could be detected in the peripheral blood, liver, and lungs compared with the spleen, bone marrow, and peritoneal cavity. Detailed results are shown in Figure 4.

Example 4 iSCLP-derived NK cells (derived from embryonic stem cells) express tumor necrosis factor TNF-α

Under the stimulation of PMA/Ionomycin or K562 tumor cells, iSCLP-derived NK cells can be stimulated to secrete tumor necrosis factor TNF-α. The Medium group was PRMI 1640 medium containing 10% FBS, which was set as the negative control group. PMA/Ionomycin was added to the medium for stimulation, which was used as the positive control group. The experimental group was 2×10^5 K562 tumor cells added to the medium. Each group contained 1×10^5 iSCLP transplanted and resuscitated into B-NDG hIL15 mice, and NK cells isolated from the liver were detected by flow cytometry to secrete TNF-α by NK cells. The results showed that iNK can secrete the cytokine TNF-α under the stimulation of PMA/Ionomycin or K562 tumor cells. The results are shown in Figure 5.

Example 5 iSCLP in vivo regenerated NK cells effectively inhibit human acute myeloid leukemia (AML) tumor cells

First, the HL60 acute myeloid leukemia mouse model was established. Each 6-8 week old B-NDG hIL15 mouse was inoculated with 5×10^5 HL60-luciferase+ (luciferase positive) cells by tail vein transplantation. One day after the inoculation of tumor cells, the tumor model was imaged in vivo (IVIS Spectrum, PerkinElmer). The model mice were then irradiated with a sublethal dose (1.5Gy), and 4 hours later, iSCLP cells were transplanted into B-NDG hIL15 mice through the tail vein at a dose of 200,000 cells/mouse. The mice were imaged in vivo at Day 0 and Day 7 to analyze the changes in tumor burden. The results showed that after treatment, the tumor burden of mice transplanted with iSCLP cells was significantly lower than that of untreated control mice, indicating that transplanted iSCLP cells can effectively inhibit human AML tumor cells in vivo (Figure 6).

Although the specific embodiments of the present invention have been described in detail, those skilled in the art will appreciate that various modifications and substitutions may be made to those details based on all the teachings disclosed, and these changes are within the scope of protection of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims (14)

An iSCLP cell (stem cell-induced lymphoid progenitor cell) or an iSCLP cell population is prepared by the following method: The lateral plate mesoderm cells are induced to differentiate in the first culture medium for 13-23 days to obtain the iSCLP cells or iSCLP cell population; wherein, The first culture medium is a basal culture medium supplemented with one or more of the following substances: one or more serum replacements, one or more non-essential amino acids, glutamine or a stabilized dipeptide of L-alanyl-L-glutamine, a TGF-β inhibitor, hydrocortisone, ascorbic acid, and cell growth factors including IL-3 and IL-5. The iSCLP cell or iSCLP cell population according to claim 1, wherein the lateral plate mesoderm cells are obtained by induced culture of stem cells; Preferably, the stem cells are induced and cultured in the second culture medium and the third culture medium in sequence, wherein: The second culture medium is a basal culture medium supplemented with the following substances: one or more serum replacements, one or more non-essential amino acids, glutamine or a stabilized dipeptide of L-alanyl-L-glutamine, and BMP4, ACTIVIN, bFGF, a Wnt agonist, and a PI3K inhibitor; The third culture medium is a basal culture medium supplemented with the following substances: one or more serum replacements, one or more non-essential amino acids, glutamine or a stabilized dipeptide of L-alanyl-L-glutamine, and BMP4, a TGF-β inhibitor, and a Wnt inhibitor; Preferably, the lateral plate mesoderm cells are obtained by inducing and culturing stem cells for 1-3 days; Preferably, the lateral plate mesoderm cells are obtained by inducing and culturing in the second culture medium and the third culture medium for 1-2 days respectively, for example, inducing and culturing in the second culture medium for 1 day and inducing and culturing in the third culture medium for 1 day; Preferably, more than 65% (for example, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 99%) of the lateral plate mesoderm cells are APLNR+ cells, further preferably, more than 65% (for example, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 99%) are APLNR+HAND1+ cells, and more preferably, more than 65% (for example, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 99%) are APLNR+HAND1+MSGN1-OSR1- cells. The iSCLP cell or iSCLP cell population according to claim 1 or 2, wherein the method is characterized by one or more of the following: (i) inducing differentiation of the lateral plate mesoderm cells in the presence of trophoblast cells, preferably, the trophoblast cells are selected from one or more of AFT024 cells, OP9 cells, MS5 cells, HS-5 cells or other fibroblasts, and preferably express at least one, two, three, multiple or all of the following genes: DLL1, DLL4, IL7, IL15, IL3 and Flt3L. Further preferably, the trophoblast cells are OP9-DLL1, OP9-DLL4, or OP9-DLL1-DLL4; (ii) the ratio of the lateral plate mesoderm cells to the trophoblast cells is 1:5-1:200, 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; (iii) inducing differentiation of the lateral plate mesoderm cells in the first culture medium for 13-14 days, 13-16 days, 13-18 days, 14-16 days, 14-19 days, 14-23 days, 16-18 days or 16-23 days; (iv) the serum replacement is a serum-free additive, for example, selected from KOSR, B27, Ultroser ^™ G, SUPERGROW and any combination thereof; preferably, the serum replacement is SUPERGROW; (v) the non-essential amino acids are selected from the group consisting of glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, and any combination thereof; (vi) the TGF-β inhibitor is a small molecule compound A-83-01 or SB431542, preferably SB431542; (vii) the cell growth factors further include 1, 2, 3, 4, 5, 6 or 7 of Flt3L, TPO, SCF, EGF, VEGF, bFGF and IGF-1; (viii) the Wnt agonist is CHIR-99021; (ix) the PI3K inhibitor is PIK-90 or LY294002; (x) the Wnt inhibitor is a small molecule compound C59 or IWR-1-endo; (xi) 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, preferably Essential 6 medium; (xii) the total content of the one or more serum replacements 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); (xiii) the one or more non-essential amino acids are each independently present in an amount of 0.1-0.5 mM, such as about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM or about 0.5 mM; (xiv) the content of the stabilized dipeptide of glutamine or L-alanyl-L-glutamine is 1-5 mM, such as about 1 mM, about 2 mM, about 3 mM, about 4 mM, or about 5 mM; (xv) the content of each factor of BMP4, ACTIVIN, bFGF, Flt3L, TPO, SCF, EGF, VEGF, bFGF, IGF-1, IL-3, and IL-5 is independently 1-100 ng/mL, such as 2-100 ng/mL, 2-50 ng/mL, 5-100 ng/mL, 5-50 ng/mL, or 5-20 ng/mL; such as about 1 ng/mL, about 2 ng/mL, about 3 ng/mL, about 5 ng/mL, about 8 ng/mL, or about 10 ng/mL; ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 45 ng/mL, about 50 ng/mL, about 55 ng/mL, about 60 ng/mL, about 65 ng/mL, about 70 ng/mL, about 75 ng/mL, about 80 ng/mL, about 85 ng/mL, about 90 ng/mL, about 95 ng/mL, or about 100 ng/mL; (xvi) the content of each small molecule in the Wnt agonist, PI3K inhibitor, TGF-β inhibitor, Wnt inhibitor and hydrocortisone is 10nM-100μM, such as 10-30nM, 10-50nM, 10-80nM, 10-100nM, 100nM-1μM, 1μM-10μM, 10μM-30μM, 10μM-50μM, 10μM-80μM, 30μM-100μM; (xvii) the ascorbic acid content is 1-100 μg/mL, such as 2-100 μg/mL, 2-50 μg/mL, 5-100 μg/mL, 5-50 μg/mL, or 5-20 μg/mL; such as about 1 μg/mL, about 2 μg/mL, about 3 μg/mL, about 5 μg/mL, about 8 μg/mL, about 10 μg/mL, about 15 μg/mL, about 20 μg/mL L, about 25 μg/mL, about 30 μg/mL, about 35 μg/mL, about 40 μg/mL, about 45 μg/mL, about 50 μg/mL, about 55 μg/mL, about 60 μg/mL, about 65 μg/mL, about 70 μg/mL, about 75 μg/mL, about 80 μg/mL, about 85 μg/mL, about 90 μg/mL, about 95 μg/mL, or about 100 μg/mL; (xviii) the first culture medium is Essential 6 medium supplemented with the following substances: one or more serum replacements, one or more non-essential amino acids, glutamine or stabilized dipeptide of L-alanyl-L-glutamine, SB431542, hydrocortisone, ascorbic acid, and cell growth factors including IL-3, IL-5, Flt3L, TPO, SCF, EGF, VEGF, bFGF and IGF-1; preferably, Essential 6 medium supplemented with the following substances: SB431542, hydrocortisone, ascorbic acid, IL-3, IL-5, Flt3L, TPO, SCF, EGF, VEGF, bFGF and IGF-1; preferably Essential 6 medium containing 10 μM SB431542, 10 μM hydrocortisone, 5 ng/mL Flt3L, 5 ng/mL TPO, 50 ng/mL SCF, 50 ng/mL EGF, 50 ng/mL VEGF, 50 ng/mL bFGF, 50 ng/mL IGF-1, 5 ng/mL IL-3, 10 ng/mL IL-15, 50 μg/mL ascorbic acid and 2% SUPERGROW Cell Culture Supplement; (xix) the second culture medium is Essential 6 culture medium supplemented with the following substances: BMP4, ACTIVIN A, bFGF, CHIR-99021 and PIK-9; preferably, Essential 6 culture medium containing 40 ng/mL BMP4, 30 ng/mL ACTIVIN A, 20 ng/mL bFGF, 6 μM CHIR-99021 and 100 nM PIK-90; (xx) the third culture medium is Essential 6 medium supplemented with the following substances: BMP4, A-83-01 and C59; preferably Essential 6 medium containing 30 ng/mL BMP4, 1 μM A-83-01 and 1 μM C59; (xxi) The stem cells are totipotent stem cells or pluripotent stem cells, preferably embryonic stem cells (ESCs), haploid stem cells, induced pluripotent stem cells (iPSCs), or expanded pluripotent stem cells (EPSCs). An iSCLP cell population (stem cell induced lymphoid progenitor cell) or the iSCLP cell population according to any one of claims 1 to 3, wherein the cells expressing CD45, CD34, CD38 and CD7 account for ≥50%, ≥60%, ≥70%, ≥80%, ≥90%, ≥95% or ≥99%. A lymphoid immune cell obtained by the following method: Cultivating the iSCLP cell or iSCLP cell population according to any one of claims 1 to 4 in a bioreactor, separating and obtaining the lymphoid immune cell; Preferably, the lymphoid immune cells are NK cells; Preferably, the bioreactor is a mammal, such as a mouse, rat, pig, or non-human primate. A culture comprising the iSCLP cell or iSCLP cell population according to any one of claims 1 to 4, and a culture medium. A culture supernatant, which is the culture supernatant produced by culturing the iSCLP cell or iSCLP cell group according to any one of claims 1 to 4. A pharmaceutical composition comprising the iSCLP cell or iSCLP cell population according to any one of claims 1 to 4 or the lymphoid immune cell according to claim 5, the culture according to claim 6 or the culture supernatant according to claim 7, and a carrier or an excipient; Preferably, the pharmaceutical composition is an injection, microneedle, mucosal patch, enema, suppository, gel, oral agent, aerosol, drop, ointment, implant, capsule or aerosol; Preferably, the pharmaceutical composition is an injection; Preferably, the pharmaceutical composition comprises a pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solution, dispersion, suspension or emulsion. A culture medium having the features of the first culture medium as claimed in claims 1 and 3. A kit comprising the culture medium according to claim 9, and optionally the second culture medium and/or the third culture medium according to any one of claims 1 to 3. Use of the iSCLP cell or iSCLP cell population according to any one of claims 1 to 4, the lymphoid immune cell according to claim 5, the culture according to claim 6, the culture supernatant according to claim 7 or the pharmaceutical composition according to claim 8 in the preparation of a drug, wherein the drug is used for: (1) Anti-infection (including infections caused by viruses, bacteria, fungi and parasites). Preferably, the infections include infections caused by various pathogens (such as viruses, bacteria, fungi or parasites) after radiotherapy and chemotherapy, and early infections after bone marrow transplantation (such as bone marrow transplantation after radiotherapy or chemotherapy); (2) Anti-tumor, preferably, the tumor is a blood tumor or a solid tumor, for example, leukemia, lymphoma, breast cancer, ovarian cancer, liver cancer, glioma or pancreatic cancer; (3) Antiviral (e.g., coronaviruses such as SARS-CoV-2, human immunodeficiency virus, rhinovirus, cytomegalovirus CMV, hepatitis B virus HBV, herpes simplex virus HSV, herpes zoster virus) infections, such as COVID-19, viral pneumonia, AIDS, acute respiratory diseases caused by rhinovirus, CMV-induced Genitourinary tract infection, central nervous system infection or liver disease, hepatitis B, vesicular dermatitis, gingivostomatitis, keratoconjunctivitis, encephalitis caused by HSV, reproductive system infection or neonatal infection, or chickenpox, encephalomyelitis or conjunctivitis caused by herpes zoster virus; (4) anti-aging; or (5) Treatment of NK cell-related immunodeficiency diseases, such as primary NK cell deficiency, Chédiak-Higashi syndrome, X-linked lymphoproliferative syndrome (XLP), leukocyte adhesion deficiency (CD11/CD18 deficiency) or severe combined immunodeficiency (SCID). A method for anti-infection, anti-tumor, anti-viral infection, anti-aging or treatment of NK cell-related immunodeficiency diseases, comprising the step of administering or transplanting a therapeutically effective amount of the iSCLP cell or iSCLP cell population according to any one of claims 1 to 4, the lymphoid immune cell according to claim 5, the culture according to claim 6, the culture supernatant according to claim 7 or the pharmaceutical composition according to claim 8 to a subject in need thereof; Preferably, the infection is caused by viruses, bacteria, fungi or parasites. Preferably, the infection is infection by various pathogens (such as viruses, bacteria, fungi or parasites) after radiotherapy or chemotherapy, or infection after bone marrow transplantation. Preferably, the bone marrow transplantation is bone marrow transplantation after radiotherapy or chemotherapy. Preferably, the tumor is a blood tumor or a solid tumor, for example, leukemia, lymphoma, breast cancer, ovarian cancer, liver cancer, glioma or pancreatic cancer; Preferably, the virus is a coronavirus such as SARS-CoV-2, human HIV virus, rhinovirus, cytomegalovirus CMV, hepatitis B virus HBV, herpes simplex virus HSV or herpes zoster virus, and further preferably, the viral infection is COVID-19, viral pneumonia, AIDS, acute respiratory disease caused by rhinovirus, genital urinary system infection, central nervous system infection or liver disease caused by CMV, hepatitis B, vesicular dermatitis, gingivostomatitis, keratoconjunctivitis, encephalitis, reproductive system infection or neonatal infection caused by HSV, or chickenpox, encephalomyelitis or conjunctivitis caused by herpes zoster virus; Preferably, the NK cell-related immunodeficiency disease is a primary NK cell deficiency disease, Chédiak-Higashi syndrome, X-linked lymphoproliferative syndrome (XLP), leukocyte adhesion deficiency (CD11/CD18 deficiency) or severe combined immunodeficiency (SCID); Preferably, the therapeutically effective amount is 1×10^5 cells/kg to 1×10^9 cells/kg. Use of the culture medium according to claim 9 or the kit according to claim 10 in preparing the iSCLP cells or iSCLP cell populations according to any one of claims 1 to 4, the lymphoid immune cells according to claim 5, the culture according to claim 6, the culture supernatant according to claim 7, or the pharmaceutical composition according to claim 8. The method for preparing the iSCLP cell or iSCLP cell population according to claim 4, comprising the steps defined in any one of claims 1 to 3.