KR101535172B1 - A method of differentiating stem cells into IL-22-producing natural killer cells - Google Patents

Abstract

The present invention relates to a method of differentiating natural killer cells producing IL-22 from stem cells. More specifically, the present invention relates to a method for differentiating IL-15 from a bone marrow-derived hematopoietic stem cell, 22 induces differentiation into NK-22 cells producing IL-22 by mixing NK-22 and 23 (Interleukin-23). Therefore, NK-22 induced is useful for treating diseases such as inflammation, hepatitis and cancer Lt; / RTI >

Description

A method of differentiating stem cells into IL-22-producing natural killer cells}

The present invention relates to a method for differentiation from stem cells into NK-22 that produces IL-22 (interleukin-22), and more specifically, IL- 22 to produce NK-22.

Among the cells constituting the immune system, natural killer cells (hereinafter abbreviated as "NK cells") are known as cells capable of non-specifically killing cancer. The killing ability of these NK cells is used for solid tumor treatment or donor lymphocyte injection using lymphokine activated killer cells (LAK) and tumor infiltration lymphocytes (TIL). infusion) through immunotherapy (Tilden. AB et al., J. Immunol . , 136: 3910-3915, 1986; Bordignon C, et al., Hematologia 84: 1110-1149, 1999) Application is being attempted as a new cell therapy therapy to prevent rejection that occurs during organ transplantation. In addition, defects in the differentiation and activity of NK cells are related to breast cancer (Konjevic G, et al., Breast Cancer Res . Treat . , 66: 255-263, 2001), Melanoma cancer (Ryuke Y, et al., Melanoma Res ., 13: 349-356, 2003), lung cancer (Villegas FR , et al., Lung Cancer , 35: 23-28, 2002) has been reported to be associated with various cancer diseases, and NK cell therapy has emerged to treat these diseases.

_{In mice deficient in the expression of γ c} of the cytokine receptor, B cells and T cells are found, but NK cells are not _{, so it is known that receptors with γ c} play an important role in NK differentiation (Singer , B et al., Proc . Natl . Acad . Sci . USA 92, 377-381, 1995). _{The γ c} form of the receptor is a receptor for IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, of which IL-2 promotes the proliferation and activation of mature NK cells. It has been reported to have a function (Shibuya, A. et al., Blood 85, 3538-3546, 1995). It has been reported that the number of NK cells significantly decreases in humans and mice deficient in IL-2 (DiSanto, JP et al., J. Exp . Med . 171, 1697-1704, 1990), on the one hand, IL-2. And IL-2Ra deficiency indirectly affects the number and activation of NK cells. In addition, it is known that the IL-2R chain is involved in forming the receptor for IL-15.

IL-15 is involved in NK cell differentiation, which is deficient in NK cells in mice deficient in the transcription factor interferon (IFN)-regulatory factor 1 required for IL-15 production (Kouetsu et al., Nature). 391, 700-703, 1998), found that NK cells were not found in mice deficient in IL-15 or IL-15Ra. Accordingly, it has been reported that IL-15 directly enhances the growth and differentiation of NK cells through the IL-15 receptor expressed in NK cells (MrozekE et al., Blood). 87, 2632-2640, 1II996).

IL-23 is a member of the IL-12 family, consisting of p40 and p19, and is mainly secreted from APCs (antigen presenting cells) such as activated monocytes, macrophages, and dendritic cells, such as IL-12. IL-23 is known to activate APCs by applying them and induce the secretion of INF-γ and IL-12, as well as induce proliferation and secretion of INF-γ by acting on memory T cells. In particular, INF-γ induced by IL-12 and IL-23 increases the expression of APC's major histocomatibility complex (MHC) molecules and at the same time enhances the ability to present antigens, thereby imparting immunogenicity to cancer cells, thereby contributing to CTL and helper T. It induces the activity of lymphocytes and, in addition, increases the ability of NK cells to kill cancer cells.

IL-22 is a type II cytokine and showed sequence homology with IL-10. Its expression is upregulated in T cells by IL-9 or ConA (Dumoutier L. et al. (2000) Proc Natl. Acad Sci USA 97(18):10144-9). Further studies have shown that expression of IL-22 mRNA is induced in vivo in response to LPS administration and that IL-22 modulates parameters that are indicative of an acute phase response (Dumoutier L. et al. (2000); Pittman D. et al. (2001) Genes and Immunity 2:172). Additionally, IL-22 enhances the expression of antimicrobial peptides associated with host defense, including β-defensin, S100A7, S100A8, and S100A (Wolk et al., Immunity , 21:241-54 (2004); Boniface et al., J. Immunol . 174:3659-3702(2005); Liang et al., J. Exp . Med ., 203(10)2271-79(2006)). Therefore, it can be seen that IL-22 plays a specific role in inflammation (Kotenko SV (2002) Cytokine & Growth Factor Reviews 13(3):223-40).

It is known that NK cells are derived from hematopoietic stem cells (HSC) of the bone marrow. In As a method of in vitro culture, methods for differentiating hematopoietic stem cells into NK cells by treating and culturing appropriate cytokines have been reported (Immunity 3: 459-473, 1995; Blood 87:2632-2640, 1996; Eur J. Immunol. 33:3439-3447, 2003; Blood 108: 3824-3833, 2006). That is, Flt-3L, IL-7, SCF, IL-15, etc. can be added to HSC to differentiate into general NK (cNK) cells after culture. In vivo NK cells undergo a differentiation process in the bone marrow, and the microenvironment of the bone marrow is essential for the differentiation of NK cells. NK cells are characterized by sequentially obtaining various surface molecules according to the differentiation process. In the mouse model, NK cells sequentially express CD122, NK1.1, NKG2 family, Ly49 family, DX5 (CD49b), and CD43 during the differentiation process (Nat Immunol, 3: 523-528, 2008).

According to recent research results, NK cells (NK-22) that produce IL-22, an NK subset of different characteristics from cNK, have been identified (Nature 457:722-725, 2009; Nat Immunol. 10:75-82, 2009; Nat Immunol. 10:83-91, 2009). In particular, IL-22 plays a role in inducing an important immune response in the regulation of inflammation, infection, and cancer, while NK-22 cells are NK cells that secrete IL-22, and unlike cNK cells, a low amount of IFN-γ, It expresses granzyme and perforin and has low killing ability (Semin Immunopathol. 32:17-31, 2010; Nature 457:722-725, 2009; Nat Immunol. 10:83-91, 2009). In vivo, NK-22 cells are known to differentiate from lymphoid tissue inducer (LTi) cells present in the small intestine (J Exp Med. 207:281-290, 2010; Nat Immunol. 10:66-74, 2009). However, it is not yet known how NK-22 cells are differentiated from stem cells in particular in vitro. If NK-22 cells are obtained by these new methods, they can be actively applied to the treatment of diseases such as inflammation, infection, and cancer using the characteristics of these cells.

Accordingly, the present inventors were developing a method for more efficiently differentiating natural killer cells that produce IL-22 from stem cells, and hematopoietic stem cells by mixing low concentrations of IL-15 and IL-23 from hematopoietic stem cells. The present invention was completed by identifying NK-22 that produces IL-22 differentiated from.

An object of the present invention is to provide a method for differentiating natural killer cells that produce IL-22 (Interleukin-22) from hematopoietic stem cells.

To achieve the above object,

The present invention

1) adding a natural killer (NK) cell precursor inducing agent to hematopoietic stem cells to induce differentiation into NK precursor cells; And

2) It provides a method for differentiating hematopoietic stem cells into NK cells, comprising the step of differentiating into NK cells by mixing IL-15 (Interleukin-15) and IL-23 (Interleukin-23) into the NK precursor cells .

In addition, the present invention

1) NK (stem cell factor), Flt3L (fms-like tyrosine kinase 3 ligand) and IL-7 (interleukin-7) composed of CD127 ⁺ CD117 ⁺ NK (natural killer) cell precursor inducer added to hematopoietic stem cells Inducing differentiation into precursor cells; And

2) Hematopoietic stem cells comprising the step of differentiating into NK cells by mixing IL-15 (Interleukin-15) and IL-23 (Interleukin-23) to the NK precursor cells were converted to IL-22 (Interleukin-22). It provides a method of differentiating into producing NK cells.

The present invention relates to a method for differentiating from hematopoietic stem cells into NK-22 cells that produce IL-22 (Interleukin-22), wherein a low concentration of IL-15 (Interleukin- 15) and IL-23 (Interleukin-23) can be mixed to induce differentiation into IL-22-producing NK-22 cells. Therefore, the induced NK-22 can be used to prevent inflammation, hepatitis, and cancer. It can be usefully used in the treatment of diseases.

1 is a diagram showing the results of differentiation from mouse hematopoietic stem cells into NK-22 cells;
(A) NK cells (cNK) differentiated with 100 ng/ml IL-15; And
^{(B) CD3-} CD127 ⁺ NKp46 ⁺ NK1.1 ^low/neg cells differentiated with 5 ng/ml IL-15 and 20 ng/ml IL-23.
2 is a diagram showing the results of measuring RORγt and IL-22 expression by RT-PCR after purification ^{of cNK and CD3-} CD127 ⁺ NKp46 ⁺ NK1.1 ^{low / neg cells.}
3 is a diagram showing the results of measuring RORγt expression ^{in cNK and CD3-} CD127 ⁺ NKp46 ⁺ NK1.1 ^{low / neg cells with a fluorescence cytometer.}
4 is a diagram showing the results of measuring RORγt and IL-22 expression of NK-22 cells differentiated from mouse hematopoietic stem cells;
(A) is purified ^{CD127 + CD117 +, CD127 + CD117} -, CD127 - CD117 +, CD127 - CD117 - a measure of the RORγt expression of cells with a fluorescent cell analyzer results;
(B) is purified ^{CD127 + CD117 +, CD127 + CD117} -, CD127 - CD117 +, CD127 - CD117 - a measure of the RORγt and IL-22 expression in cells of the RT-PCR results; And
(C) RT-PCR of RORγt and IL-22 expression of cNK, LTi cells isolated from mouse small intestine (LP CD127 ⁺ CD117 ^{+ ) and CD127 +} CD117 ⁺ cells (7d-CD127 ⁺ CD117 ⁺ ) differentiated from stem cells. The result measured with.
5 is a diagram showing the results of measuring differentiation of NK-22 cells cultured from ^{CD127 +} CD117 ^{+ cells;}
(A) is purified after differentiation from the stem cells, ^{CD127 + CD117 +, CD127 + CD117} -, CD127 - CD117 +, CD127 - CD117 - a result of the differentiation the cells with 100 ng / ml IL-15;
(B) is a CD127 purified after differentiation from stem cells ^{+ CD117 +, CD127 + CD117 -} , CD127 - CD117 + and CD127 ^- CD117 ^- differentiate the cells with 5 ng / ml IL-15 and 20 ng / ml IL-23 The result of analysis with a fluorescence cytometer after; And
(C) is a result of analyzing RORγt expression of NK-22 cells differentiated from ^{CD127 +} CD117 ^{+ cells.}
6 is a diagram showing the results of measuring differentiation of NK-22 cells generated after administration ^{of CD127 +} CD117 ^{+ cells to mice.}

Hereinafter, the present invention will be described in detail.

The present invention

1) adding a natural killer (NK) cell precursor inducing agent to hematopoietic stem cells to induce differentiation into NK precursor cells; And

2) It provides a method for differentiating hematopoietic stem cells into NK cells, comprising the step of differentiating into NK cells by mixing IL-15 (Interleukin-15) and IL-23 (Interleukin-23) into the NK precursor cells .

In the above method, the hematopoietic stem cells of step 1) are preferably derived from bone marrow, but are not limited thereto.

In the above method, the NK cell precursor inducing agent of step 1) is preferably a stem cell factor (SCF), a fms-like tyrosine kinase 3 ligand (Flt3L), and interleukin-7 (IL-7), but is not limited thereto.

In the above method, the NK cell precursor ^{is preferably a CD127 +} CD117 ⁺ precursor cell, but is not limited thereto.

In the above method, IL-15 in step 2) is preferably treated with 1 ng/ml-20 ng/ml, more preferably 5 ng/ml-10 ng/ml, and 5 ng/ml It is most preferable to treat it with, but is not limited thereto.

In the above method, IL-23 in step 2) is preferably 10 ng/ml-30 ng/ml, more preferably 20 ng/ml-25 ng/ml, and 20 ng/ml It is most preferable to do, but is not limited thereto.

In the above method, it is preferable that the NK cells of step 2) produce IL-22 (Interleukin-22), but are not limited thereto.

In one aspect of the present invention, in order to isolate hematopoietic stem cells from bone marrow, cells extracted from bone marrow isolated from mice are treated with an ACK solution to remove red blood cells, and then B220, CD2, NK1.1, CD11b, Gr-1, And TER-119 antibody is treated to attach to the corresponding B cells, T cells, NK/NKT cells, monocytes, granulocytes, and red blood cells, respectively, and these are attached to a magnetic-activated cell sorting (MACS) Cell Separation Kit ( Miltenyi Biotec, Bergisch Gladbach, Germany) was removed by negative selection. After removing the differentiated immune cells, hematopoietic stem cells were isolated by positive selection using microbeads and MACS for CD117 (c-kit), which is a specific surface molecule of hematopoietic stem cells.

In one aspect of the present invention, in order to induce differentiation of NK-22 producing IL-22 from hematopoietic stem cells, after inducing differentiation into NK precursor cells by adding SCF, Flt3L and IL-7 to hematopoietic stem cells , After mixed treatment with various concentrations of IL-15 and IL-23, the degree of NK cell differentiation was confirmed. When cNK cells were differentiated from hematopoietic stem cells, about 75% were differentiated into cNK (see FIG. 1A), and hematopoietic stem cells. Cells differentiated with 5 ng/ml IL-15 and 20 ng/ml IL-23 CD3 ^- CD127 ⁺ NKp46 ⁺ NK1.1 ^{low / neg} It was confirmed that the cells were about 37% (see Fig. 1B).

In one aspect of the invention, CD3 ^- CD127 ⁺ NKp46 ⁺ NK1.1 ^{low / neg} For cell analysis, RORγt and IL-22 expressions were measured by RT-PCR and fluorescence cytometry. As a result, RORγt and IL-22 gene expression in ^CD3- CD127 ⁺ NKp46 ⁺ NK1.1 ^{low/neg cells were compared with cNK cells.} In comparison, it was confirmed that it was significantly increased (see FIG. 2), and RORγt expression was confirmed ^{that about 2% of cells had in CD3- CD127 +} NKp46 ⁺ NK1.1 ^low/neg cells (see FIG. 3). Therefore, it was confirmed that ^{CD3- CD127 +} NKp46 ⁺ NK1.1 ^{low / neg} RORγt ⁺ cells were NK-22 cells differentiated from hematopoietic stem cells.

In one aspect of the present invention, in order to determine the precursor cells of NK-22 cells, as a result of analyzing precursor cells differentiated for 7 days from stem cells through a fluorescence cytometer and RT-PCR, ^{only CD127 +} CD117 ⁺ cells are RORγt. And IL-22 gene (see Fig. 4).

To In one aspect of the invention, to determine whether NK-22 cells are differentiated from CD127 ⁺ CD117 ⁺ precursor ^{cells, CD127 + CD117 +, CD127 +} CD117 -, CD127 - CD117 +, CD127 - CD117 - 5 ng / ml cells As a result of differentiation with IL-15 and 20 ng/ml IL-23, it was confirmed ^{that about 60% of CD127 +} CD117 ⁺ cells differentiated into NK-22 cells (see FIG. 5).

In one aspect of the invention, In ^{To confirm whether NK-22 cells differentiate from CD127 +} CD117 ⁺ precursor cells in the in vivo model, 14 days after administration of the precursor cells to mice, the phenotype and RORγt expression of NK-22 cells isolated from the small intestine were analyzed using a fluorescence cytometer. As a result of the analysis, about 10% of the cells of the small intestine were RORγt ⁺ cells, and CD127 ⁺ CD117 ⁺ RORγt ⁺ was measured to be about 0.6%. Thus, In It was confirmed that NK-22 cells were differentiated from stem cells in vivo (see FIG. 6).

Therefore, it was confirmed that the differentiation of IL-22-producing NK-22 cells was induced by mixing low concentrations of IL-15 and IL-23 to precursor cells differentiated from hematopoietic stem cells.

In addition, the present invention provides a pharmaceutical composition for preventing and treating cancer containing NK-22 cells prepared by the method according to the present invention as an active ingredient.

The cancer is preferably any one selected from the group consisting of breast cancer, melanoma cancer, gastric cancer, liver cancer, colon cancer and lung cancer, but is not limited thereto.

The present invention confirmed that the differentiation of IL-22-producing NK-22 cells is induced by mixing low concentrations of IL-15 and IL-23 to precursor cells differentiated from hematopoietic stem cells. It can be usefully used as a composition for preventing and treating cancer containing as an active ingredient.

The composition of the present invention may further contain one or more active ingredients exhibiting the same or similar function to the NK-22 cells. For administration, it may be prepared by including one or more pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers can be used by mixing saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components, and if necessary, antioxidants, buffers, Other conventional additives such as bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare injectable formulations such as aqueous solutions, suspensions, emulsions, powders, tablets, capsules, pills, granules, or injection solutions. Further, it may be preferably formulated according to each disease or component by an appropriate method in the art or by using a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

The composition of the present invention can be administered parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically), and the dosage is the patient's weight, age, sex, health status, diet, administration time, administration method, The range varies depending on the excretion rate and the severity of the disease. The daily dosage of the composition according to the present invention is 0.01 to 5000 mg/kg, preferably 0.01 to 10 mg/kg, and it is more preferable to administer once to several times a day.

In addition, the present invention provides an agent for inducing differentiation from hematopoietic stem cells into NK cells containing IL-15 and IL-23 as active ingredients.

The hematopoietic stem cells are preferably those derived from bone marrow, but are not limited thereto.

The NK cells preferably produce IL-22, but are not limited thereto.

The present invention confirmed that the differentiation of IL-22-producing NK-22 cells is induced by mixing IL-15 and IL-23 from hematopoietic stem cells, so differentiation containing IL-15 and IL-23 as active ingredients The inducer can be usefully used to induce differentiation from hematopoietic stem cells into NK cells.

Hereinafter, the present invention will be described in detail by examples.

However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

< Example 1> Isolation of hematopoietic stem cells from mouse bone marrow

The calf bone, thigh bone, and pelvic bone of an experimental mouse (Central Experimental Animal Co., Ltd., Korea) were isolated, and total bone marrow cells were extracted from this by a method known in the art. The cells extracted from the bone marrow were treated with ACK solution to remove red blood cells, and then B220, CD2, NK1.1, CD11b, Gr-1, and TER-119 antibodies were treated to the corresponding B cells, T cells, and NK/NKT. Cells, monocytes, granulocytes, and red blood cells were attached and removed by negative selection using a magnetic-activated cell sorting (MACS) Cell Separation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). After removing the differentiated immune cells, hematopoietic stem cells were isolated by positive selection using microbeads and MACS for CD117 (c-kit), which is a specific surface molecule of hematopoietic stem cells.

< Example 2> From hematopoietic stem cells isolated from bone marrow NK -22 Induction of differentiation of cells

The hematopoietic stem cells isolated in Example 1 were placed in a 24-well plate (Becton and Dickinson Bioscience, USA) at ^{a concentration of 1.5 x 10 6} cells/well at 10% bovine. Fetal bovine serum (FBS), 2 μg/ml indometacin (Sigma-Aldrich, St. USA), 20 μg/ml gentamicin (Sigma-Aldrich. USA), 30 ng/ml ml murine SCF (Stem cell factor) (PeproTech, Rocky Hill, USA), 50 ng/ml murine Flt3L (PeproTech, USA), 0.5 ng/ml murine IL-7 (PeproTech, USA) It was cultured for 7 days at 37°C and 5% CO _{2 using the RPMI 1640 medium.} After 3 days of initial culture, half of the culture supernatant was replaced with a new medium containing cytokines of the same composition. For the differentiation into NK cells, intermediate progenitor cells cultured for 7 days were recovered for an additional 7 days in a medium containing various concentrations of murine IL-15 and murine IL-23 (PeproTech, USA). Cultured. At intervals of 3 days, half of the medium was replaced with a new medium containing cytokines of the same composition. Using the 14th day, wherein -NK1.1 and wherein -CD3 antibody NK1.1 ⁺ CD3 ^- a flow cytometer the differentiation of NK cells using an antibody for the analysis of the purity of NK cells, and a variety of NK cell surface molecules It was analyzed by (flow cytometry).

As a result, as shown in FIG. 1, when cNK cells were differentiated from hematopoietic stem cells, about 75% were differentiated into cNK (FIG. 1A), and hematopoietic stem cells were 5 ng/ml IL-15 and 20 ng/ml IL- 23 CD3 ^- CD127 ⁺ NKp46 ⁺ NK1.1 ^{low / neg} It was confirmed that the cells were about 37% (FIG. 1B).

< Example 3> NK -22 Confirmation of cell differentiation

cNK and CD3 ^- CD127 ⁺ NKp46 ⁺ NK1.1 ^{low / neg} After the cells were isolated and purified, RORγt and IL-22 expression were measured by RT-PCR.

Specifically, NK cells during the differentiation process were recovered and total RNA was isolated using Trizol Reagent (Invitrogen, Carlsbad, USA). 1-3 μg of RNA was reacted with 2.5 U of Moloney murine leukemia virus (M-MLV) reverse transcriptase (Roche Diagnostics, Basel, Switzerland) at 37° C. for 1 hour to cDNA Was synthesized. The synthesized cDNA transcript was quantified using SYBR Premix Ex Taq (Takara Bio, Japan) and Real-time system TP 800 (Takara Bio, Japan), and GAPDH was used for standardization of the sample. Each primer was prepared using Primer3 software. The primers used were RORγt forward primer SEQ ID NO: 1(5'-CATCATCTCTGCAAGACTCATCGAC-3') and RORγt reverse primer SEQ ID NO: 2(5'-TTTCCACATGTTGGCTGCAC-3'), IFN-γ forward Primer SEQ ID NO: 3(5'-GATGCATTCATGAGTATTGCCAAGT-3') and IFN-γ reverse primer SEQ ID NO: 4(5'-GTGGACCACTCGGATGAGCTC-3'), IL-22 forward primer SEQ ID NO: 5(5'-GTGAGAAGCTAACGTCCATC-3 ') and IL-22 reverse primer SEQ ID NO: 6 (5'-GTCTACCTCTGGTCTCATGG-3'), Perforin forward primer SEQ ID NO: 7 (5'-TTCGGGAACCAAGCTACACCA-3') and Perforin reverse primer SEQ ID NO: 8 (5'-CAGGCTGTAGTCCACCAGACCA-3') and GranzymeA forward primer SEQ ID NO: 9 (5'-AAGAACTGGGTGTTGACTGCTG-3') GranzymeA reverse primer SEQ ID NO: 10 (5'-CACGTGTATATTCATCATAGCATGG-3') were used, respectively.

As a result, as shown in Fig. 2, CD3 ^- CD127 ⁺ NKp46 ⁺ NK1.1 ^{low / neg} It was confirmed that RORγt and IL-22 gene expression in cells was significantly increased compared to cNK cells (FIG. 2).

In addition, RORγt ^{expression in cNK and CD3-} CD127 ⁺ NKp46 ⁺ NK1.1 ^{low / neg} cells was measured with a fluorescence cytometer.

As a result, as shown in FIG. 3, it was confirmed that RORγt expression was found in about 2% of cells in ^CD3- CD127 ⁺ NKp46 ⁺ NK1.1 ^{low/neg cells (FIG. 3).} Therefore, it was confirmed that ^{CD3- CD127 +} NKp46 ⁺ NK1.1 ^{low / neg} RORγt ⁺ cells were NK-22 cells differentiated from hematopoietic stem cells.

< Example 4> NK -22 cell progenitor cell analysis

Using cell fluorescence cell analyzer ^- NK-22 is 7 days differentiated precursor cells, which are CD127 ⁺ CD117 ^+, CD127 ⁺ CD117 from stem cells to see if erupting from which the precursor cells ^-, CD127 ^- CD117 ⁺ and CD127 ^- CD117 after separation by, purified ^{CD127 + CD117 +, CD127 + CD117} - was measured RORγt expression of cells with a fluorescent cell analyzer ^-, CD127 ^- CD117 ⁺ and CD127 ^- CD117.

As a result, as shown in Fig. 4A, ^{only CD127 +} CD117 ⁺ cells expressed RORγt (Fig. 4A).

In addition, RORγt and IL-22 gene expression was confirmed by real-time PCR.

As a result, as a result, ^{it was confirmed that only CD127 +} CD117 ⁺ cells expressed a lot of RORγt and IL-22 genes as shown in FIG. 4B (FIG. 4B ).

In addition, RORγt and IL-22 expressions were compared and analyzed in LTi cells isolated from mouse small intestine (LP CD127 ⁺ CD117 ⁺ ) and CD127 ⁺ CD117 ⁺ cells differentiated from stem cells (7d-CD127 ⁺ CD117 ^{+ ).}

As a result, it was confirmed that the LTi cells expressed more RORγt and IL-22 as shown in FIG. 4C (FIG. 4C ).

< Example 5> from precursor NK -22 Confirmation of cell differentiation

CD127 ⁺ CD117 ⁺ precursor from NK-22 cells to differentiate that the CD127 ⁺ CD117 ^+, CD127 ⁺ CD117 purified to identify cells ^-, CD127 ^- CD117 ⁺ and ^{CD127 - CD117 - 100 ng / ml} IL-15 cells, respectively, and After differentiation into 5 ng/ml IL-15 and 20 ng/ml IL-23, they were analyzed with a fluorescence cytometer.

As a result, as shown in FIG. 5A, as a result of differentiating with 100 ng/ml IL-15, ^{about 20% of CD127 +} CD117 ⁺ cells were differentiated into cNK cells, and about 50% of CD127 ^- CD117 ^{+ cells became cNK cells.} It was confirmed that it was differentiated (Fig. 5A).

In addition, purified CD127 ⁺ CD117 ^+, CD127 ⁺ CD117 ^-, CD127 ^- CD117 ^+, CD127 ^- CD117 ^- After the cells were differentiated with 5 ng / ml IL-15, and 20 ng / ml IL-23, analyzed by fluorescent cell analyzer I did.

As a result, it was confirmed ^{that about 60% of CD127 +} CD117 ⁺ cells were differentiated into NK-22 cells, and CD127 ^- CD117 ⁺ cells hardly differentiated into NK-22 cells (FIG. 5B).

In addition, RORγt expression of NK-22 cells differentiated from ^{CD127 +} CD117 ^{+ cells was analyzed.}

As a result, it was confirmed that 13% were differentiated into final NK-22 cells as shown in FIG. 5C (FIG. 5C ).

< Example 6> ( in vivo ) In mice NK Confirmation of the differentiation of -22

In In an in vivo model as well as in Example 5 above, in order to confirm whether NK-22 cells were differentiated, CD127 ⁺ CD117 ⁺ cells were administered to mice, and then the differentiation of the generated NK-22 cells was measured.

Specifically, 2 x 10 ⁴ CD127 ⁺ CD117 ⁺ precursor cells ^{were administered intravenously to Rag2 -/-} IL2rg ^-/- mice, and then, in order to analyze NK-22 cells differentiated from the precursor cells, the precursor cells were used as mice. After 14 days of administration, the phenotype and RORγt expression of NK-22 cells isolated from the small intestine were analyzed using a fluorescence cytometer.

As a result, as shown in FIG. 6, about 10% of the cells of the small intestine were RORγt ⁺ cells, and CD127 ⁺ CD117 ⁺ RORγt ⁺ was measured to be about 0.6%. Therefore, it was confirmed that NK-22 cells were differentiated from stem cells in vivo (FIG. 6).

<110> Korea Research Institute of Bioscience and Biotechnology <120> A method of differentiating stem cells into IL-22-producing natural killer cells <130> 14p-12-051 <150> KR 10-2011-0037846 <151> 2011-04-22 <160> 10 <170> KopatentIn 1.71 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> ROR gamma t forward primer <400> 1 catcatctct gcaagactca tcgac 25 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ROR gamma t reverse primer <400> 2 tttccacatg ttggctgcac 20 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> IFN-gamma forward primer <400> 3 gatgcattca tgagtattgc caagt 25 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> IFN-gamma reverse primer <400> 4 gtggaccact cggatgagct c 21 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Interleukin-22 forward primer <400> 5 gtgagaagct aacgtccatc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Interleukin-22 reverse primer <400> 6 gtctacctct ggtctcatgg 20 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Perforin forward primer <400> 7 ttcgggaacc aagctacacc a 21 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Perforin reverse primer <400> 8 caggctgtag tccaccagac ca 22 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GranzymeA forward primer <400> 9 aagaactggg tgttgactgc tg 22 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> GranzymeA reverse primer <400> 10 cacgtgtata ttcatcatag catgg 25

Claims (6)

1) CD127 ⁺ CD117 ⁺ NK (natural killer) cell precursor directing agent consisting of SCF (stem cell factor), Flt3L (fms-like tyrosine kinase 3 ligand) and IL-7 (Interleukin- Inducing differentiation into precursor cells; And
2) Interleukin-22 (IL-22) was prepared by mixing hematopoietic stem cells with NK precursor cells by mixing IL-15 (Interleukin-15) and IL-23 (Interleukin- Lt; RTI ID = 0.0 &gt; NK &lt; / RTI &gt;
2. The method according to claim 1, wherein the hematopoietic stem cells of step 1) are derived from bone marrow.
The method according to claim 1, wherein the IL-15 of step 2) is treated at 1 to 20 ng / ml, wherein the hematopoietic stem cells are differentiated into NK cells.
The method according to claim 1, wherein the IL-15 of step 2) is treated at 5 to 10 ng / ml, wherein the hematopoietic stem cells are differentiated into NK cells.
The method according to claim 1, wherein the IL-23 of step 2) is treated at 10 to 30 ng / ml, wherein the hematopoietic stem cells are differentiated into NK cells.
The method according to claim 1, wherein the IL-23 of step 2) is treated at 20 to 25 ng / ml.

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