JPH07135969A - Precursor cell of natural killer cell and its differentiation and proliferation method - Google Patents

Abstract

PURPOSE:To provide a precursor cell of NK(natural killer) cell, its production process, a differentiation and proliferation agent for the precursor NK cell and a method for the differentiation and proliferation of the precursor NK cell. CONSTITUTION:A precursor cell of NK cell obtained from cells originated from human marrow. For example, a precursor NK cell negative to anti-human CD33 antibody and positive to anti-human CD34 antibody or a precursor NK cell positive to both anti-human CD33 antibody and anti-human CD34 antibody. A process for the preparation of the precursor NK cell by separating the precursor NK cell from cells originated from human marrow using positiveness to anti-human CD34 antibody as an index. A differentiation and proliferation agent for the precursor NK cell containing interleukin 2 and a stem cell proliferation factor. A differentiation and proliferation of the precursor NK cell containing interleukin 3 and a stem cell proliferation factor. A differentiation and proliferation of the precursor NK cell containing interleukin 2, interleukin 3 and a stem cell proliferation factor. A process for the differentiation and proliferation of the precursor NK cell using the above differentiation and proliferation agent.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a natural killer cell (hereinafter sometimes referred to as NK cell) precursor cell, NK.
The present invention relates to a method for producing cell progenitor cells, an agent for differentiating and proliferating NK cell progenitor cells, and a method for differentiating and proliferating NK cell progenitor cells. More specifically, the differentiation-proliferating agent and differentiation-proliferation method of the present invention utilize interleukin 2, interleukin 3, and stem cell growth factor (hereinafter sometimes referred to as SCF).

[0002]

Background Art and Problems to be Solved by the Invention NK cells are a type of immunocompetent cells and are known to play an important role in the early anticancer action. Therefore, if NK cells can be separated and expanded from the peripheral blood of a cancer patient and returned to the original patient, it can be a powerful method for cancer treatment. However, as described later, it is also known that it is extremely difficult to separate and proliferate specific blood cells such as NK cells.

In particular, NK cells have heretofore been unable to proliferate, and can only be separated from blood.
Separation of NK cells is carried out by mixing an antibody against a surface antigen specific to NK cells such as anti-human NK1.11 antibody to which an appropriate fluorescent dye is bound with leukocytes and allowing them to bind in ice water, followed by automatic fluorescence cell separation. It is carried out by sorting the antibody-bound cells on the device [Shibuya A. et.al. "I
nhibitory effect of granurocyte-macrophage colony-s
timulating factor therapy on the generation of nat
ural killer cells "Blood 12 (1991) p3124-3247 or Takao Hoshino Cell sorting-Principle and practice of cell separation-FACS-See Kazuo Ohta," Flow Cytometry "Kani Shobo 147-155]. However, the number of cells obtained by this method is very small. Moreover, from blood cells to N
Extracting a specific type of cells such as K cells requires a high degree of technical skill. Therefore, from blood cells to N
In the present situation, K cells can be separated only in several places in Japan.

As described above, in the conventional technique, although NK cells can be separated, the number of obtained cells is limited. On the other hand, when utilizing NK cells, more NK cells than the number of separated cells are required. Therefore, it is necessary to grow NK cells by some method. It is only necessary to separate cells at an immature stage and differentiate and proliferate them, but it is not known at all from which precursor cells NK cells differentiate and mature. Similarly, a method for differentiating and proliferating NK cell progenitor cells in a tester was also unknown.

Therefore, an object of the present invention is to provide a progenitor cell for NK cells and a method for producing the same.

A further object of the present invention is to provide an agent for differentiating and proliferating NK cell progenitor cells and a method for differentiating and proliferating NK cell progenitor cells.

[0007]

Means for Solving the Problems As a result of intensive studies by the present inventors, the presence of NK cell progenitor cells in bone marrow-derived cells, and the progenitor cells being interleukin 2 and SCF or interleukin 2, interleukin 3
The present invention has been completed based on the finding that they can be differentiated and propagated in the presence of SCF and SCF.

The present invention will be described below in order. Progenitor cells present invention of NK cells is related progenitor cells of NK cells obtained from cells derived from human bone marrow. Specific examples of the progenitor cells of the present invention include anti-human CD33 antibody negative (hereinafter sometimes abbreviated as CD33 ⁻ ) and anti-human CD34 positive (hereinafter, C
D34 ⁺ may be abbreviated) progenitor cells of NK cells (hereinafter sometimes abbreviated as CD33 ⁻ CD34 ⁺ ).

As another specific example, anti-human CD33
NK that is antibody-positive (hereinafter sometimes abbreviated as CD33 ⁺ ) and anti-human CD34 antibody-positive (CD34 ⁺ ).
Examples thereof include cell progenitor cells (hereinafter sometimes abbreviated as CD33 ⁺ CD34 ⁺ ).

The CD33 ^- CD34 ⁺ progenitor cells of NK cells are CD34 as a bone marrow cell surface antigen represented by a in FIG. 1 (vertical axis: CD34-APC, horizontal axis: CD34-FITC) in FIG. Among the cell groups having
Is a cell group that does not have the CD33 antigen. In addition, the progenitor cells of NK cells that are CD33 ⁺ CD34 ⁺ are the CD33 shown in c in the figure in the cell group having CD34 as the surface antigen in the bone marrow cells shown in a in FIG. 1A. It is a group of cells having an antigen.

As shown in FIG. 2, CD33 ^- CD3
4 progenitors NK cells ⁺ are immature stages of cells, progenitor cells NK cells CD33 ⁺ CD34 ⁺ is considered to be a cell slightly differentiated (Ema H. et.
al. "target cells for granulocyte colony-stimulatin
g factor, Interleukin-3, and Interleukin-5 in diff
erentiation pathways of neutrophils and eosinophil
s "Blood 76 (1990) 1956-1961).

The NK cell progenitor cells of the present invention can be produced by separating cells derived from human bone marrow using anti-human CD34 antibody positivity as an index. Especially CD3
3 ^- CD34 ⁺ NK cell progenitor cells are prepared by separating anti-human CD34 antibody-positive progenitor cells from human bone marrow-derived cells using anti-human CD34 antibody-positive as an index, and further separating the progenitor cells with anti-human CD33 antibody. It can be manufactured by separating using negative as an index. Also, CD33 ⁺ CD34
⁺ Progenitor cells of NK cells are anti-human CD34 cells derived from human bone marrow-derived cells using anti-human CD34 antibody positivity as an index.
It can be produced by separating antibody-positive progenitor cells and separating the separated progenitor cells using anti-human CD33 antibody-positive as an index.

The progenitor cells of the NK cells of the present invention include human bone marrow-derived cells such as bone marrow cells or placental blood containing peripheral blood, which is obtained by a conventional method (Shibuya A. et.al. "Inhibitory effect").
ofgranurocyte-macrophage colony-stimulating facto
r therapy on the generation of natural killer cell
s "Blood 12 (1991) p3124-3247), followed by flow cytometry (Shibuya A. et.al." Inh
ibitory effect of granurocyte-macrophage colony-st
imulating factor therapy on the generation of natu
ral killer cells "Blood 12 (1991) p3124-3247 or Takao Hoshino Cell Sorting-Principle and practice of cell separation-
FACS-Kazuo Ohta "Flow Cytometry" Crab Shobo 147-155), Immunomagnetic Bead Cell Separation Method (GaudernackG et.al. "Isolation of pure functional
ly active CD8 + T Cells. Positive selection w
ith monoclonal antibodies directly conjugated to m
onosized magnetic microspheres "J. Immunol. meetho
It can be fractionated by using ds 90 (1986) 179-87). For example, CD obtained from human bone marrow cells
Fractions that are 34 ⁺ and CD33 ⁻ (CD33 ⁻ CD3
4 ⁺ ) can be fractionated by collecting human bone marrow cells by a conventional method and then using a flow cytometry method, immunomagnetic bead cell separation method, or the like. Similarly, a fraction containing CD33 ⁺ CD34 ⁺ can be obtained from human bone marrow cells. In general, CD33 ^- CD34 ⁺
The cell group has a higher proliferative capacity in the tester than the CD33 ⁺ CD34 ⁺ cell group.

Differentiation and Proliferation of Progenitor Cells In addition, as shown in FIG. 2, the progenitor cells of the above-mentioned NK cells of the present invention are interleukin-3 depending on the type of progenitor cells.
(IL-3), interleukin 2 (IL-2) and SCF in an appropriate combination in a medium added in an appropriate amount,
By culturing for an appropriate period, NK cells can be differentiated and proliferated.

Culture for proliferating precursor cells is a general culture condition for animal cells, eg, 37 ° C., 5%
It can be carried out with carbon dioxide gas and 100% humidity. The culture period is, for example, 1 day to 50 days, and can be appropriately determined while measuring the degree of differentiation.

The interleukin 3, interleukin 2 and SCF used for differentiation and proliferation can be natural or produced by gene recombination technology. In addition, the production method and cells and hosts used in the production are not particularly limited. Furthermore, these interleukin 3, interleukin 2 and SCF are
Naturally occurring or genetically engineered variants having these activities are also included.

For differentiation and proliferation, a medium generally used for culturing bone marrow-derived cells can be used as it is. For example, RPMI1640 medium [Morton HJIn
virto 6 (1970) 89 Moore GE et.al. J. Natl. Cancer
Insitute 36 (1966) 519 (Dainippon Pharmaceutical RPMI1640 Part Number
110-601-20)], DMEM medium, complete medium and the like. The concentrations of interleukin 3, interleukin 2 and SCF contained in these media are, for example, in the range of 5 to 500 U / ml for interleukin 3, 50 to 5000 U / ml for interleukin 2, and SCF. Is preferably in the range of 5 to 500 ng / ml.

More specifically, for example, CD33 ⁺ CD3
In 4 ⁺ fractionated cells, interleukin 2 and S
A medium containing CF, for example, 10% fetal bovine serum, 500
U / ml interleukin 2 and 50 mg / ml S
By culturing in RPMI1640 medium containing CF under conditions of 37 ° C., 5% carbon dioxide and 100% humidity for 20 to 40 days, NK cells characterized by anti-CD56 antibody positive (CD56 ⁺ ) can be obtained.

In addition, for example, in CD33 ^- CD34 ⁺ fractionated cells, a medium containing interleukin 3, interleukin 2 and SCF, such as 10% fetal calf serum,
In RPMI1640 medium containing 500 U / ml interleukin 3, 500 U / ml interleukin 2, and 50 mg / ml SCF, 37 ° C., 5% carbon dioxide 10
By culturing for 20-40 days under the condition of 0% humidity,
NK cells characterized by anti-CD56 antibody positivity are obtained. Alternatively, R containing interleukin 3 and SCF
Similarly, by culturing in PMI1640 medium under similar conditions for 5 to 10 days, and then culturing in RPMI1640 medium containing interleukin 2, interleukin 3 and SCF under similar conditions for 15 to 30 days, NK cells are similarly obtained. Can be obtained.

Differentiation / Proliferation Agent Further, the present invention relates to a differentiation / proliferation agent for progenitor cells of natural killer cells, which comprises at least one selected from interleukin 3, interleukin 2 and SCF.
More specifically, the first aspect is a differentiation and growth agent for natural killer cell progenitor cells containing interleukin 2 and stem cell growth factor. The proliferative agent is useful when the natural killer cell progenitor cells are CD33 ⁻ CD34 ⁺ progenitor cells or CD33 ⁺ CD34 ⁺ progenitor cells.

The second aspect is a differentiation and growth agent for natural killer cell precursors containing interleukin 3 and stem cell growth factor. This proliferative agent is useful when the natural killer cell progenitor cells are progenitor cells that are CD33 ⁻ CD34 ⁺ .

The third embodiment is a differentiation and proliferation agent for natural killer cell progenitor cells containing interleukin 2, interleukin 3 and stem cell growth factor. In this differentiation-proliferating agent, natural killer cell precursor cells are CD33 ^- C
It is useful when the progenitor cells are D34 ⁺ .

The differentiation / proliferation agent of the present invention can be used, for example, by adding it to the culture medium of the above-mentioned cultured cells.
The amount of the differentiation / proliferation agent of the present invention added to the medium varies depending on the type of natural killer cell progenitor cells, culture conditions, and the like. For example, interleukin-3 is 5 to 500 U / ml,
It is appropriate that the SCF is in the range of 5 to 500 ng / ml and the interleukin 2 is in the range of 50 to 5000 U / ml.

Furthermore, the agent for differentiation and proliferation of the present invention promotes the differentiation and proliferation of NK cells in mammals by direct parenteral (eg, intravenous injection) and oral administration to mammals such as humans. You can also The dose of the differentiation / proliferation agent of the present invention is determined in consideration of the symptoms, weight, etc. of the patient.
0 kU, SCF is 5-500 μg, interleukin 2
Is a dose of 50 to 5000 kU, and can be administered, for example, in 1 to 3 days.

The differentiation-proliferating agent of the present invention may contain various components, if necessary, in addition to interleukin 2, interleukin 3 and / or stem cell growth factor.
For example, an excipient (eg physiological saline) for the active ingredients interleukin 2, interleukin 3 and / or stem cell growth factor, a stabilizer (eg albumin, saccharide) and the like can be further included.

[0026]

EXAMPLES The present invention will now be described in more detail with reference to examples. Example 1 Fractionation of CD33 ⁺ CD34 ⁺ cells 4 ml of bone marrow (BM) was collected from the sternum of a healthy human, and immediately heparin-containing HBSS (Hank's balanced salt sol) was collected.
ution; Hanks JH et.al. Proc.Soc.Exp.Biol.Med.71
(1949) 196-200 (Dainippon Pharmaceutical HBS product number 17-10
1)) diluted in 15 ml. The cell suspension obtained is 3
% Fetal bovine serum and 0.02% sodium azide phosphate buffered saline (staining medium; hereinafter referred to as SM) about 3
After doubling the dilution, the cells are overlaid on a 15 ml centrifuge tube containing 3 ml of Ficoll Conley. 18 ℃ 1
Centrifuge at 500 rpm for 30 minutes to separate the mononuclear cell layer, and
M mononuclear cells (MNC) were obtained.

The BM mononuclear cells thus obtained are
Allophycocyani
n; APC) -conjugated anti-human CD34 antibody (4A1 (1
6); IgG1), C bound with phycoerythrin (PE) as a lineage marker (surface antigen possessed by cells already differentiated into NK cells)
D3 (Leu4; IgG1), CD16 (Leu11
c; IgG1) and CD56 (NKH-1; IgG
1), and fluorescein
n; FITC) conjugated anti-human CD33 antibody (My9; Ig
G2b) incubated at 4 ° C for 30 minutes, then 2
Washed twice.

Then, the cells were sorted (cell sort) by using a fluorescence automatic cell separation device (FACStar ^PLUS ) equipped with a 488 nm argon laser and a 599 nm dye laser.
(Beckman Dickinson)). CD3
For the CD33 fraction of 4 ⁺ cells, the sort gate is A
-A (total CD34 ⁺ cells), Ab (negative 30)
%), (Blyest 50%). Percentages are approximate values obtained from the results of 3 independent experiments. In this way, phycoerythrin (phyco)
erythrin (PE) negative, fluorescein (F
Fluorescein (FITC) -negative, allophycocyan (APC) -positive cell population (Lin ⁻ (= CD3 ⁻ , CD16 ⁻ , CD
56 ^-) CD33 ⁺ CD34 ⁺ fraction) was obtained.

Example 2 Differentiation and Proliferation of CD33 ⁺ CD34 ⁺ Cells 500 U / ml of recombinant human IL-2, 50 ng
Complete medium containing 1 / ml of recombinant human SCF 200
μl was placed in a 96 well round bottom microplate. 500 Lin ⁻ CD33 ⁺ CD34 ⁺ fractionated cells obtained in Example 1 were added to each well and incubated at 37 ° C. 5% carbon dioxide 100%.
After culturing for 28 days under the condition of humidity, the number of CD56-positive cells that are surface antigens of NK cells that appeared was counted (Experiment 3).

For comparison, no IL-2 was included (Experiment 2), or added only for a part of the culture period (Experiments 6 and 7), and no SCF was included (Experiment 1), or When the cells were added to only a part of the culture period (Experiments 4 and 5), the cells were similarly cultured and the number of CD56-positive cells was counted. The results are shown in Table 1.

[0031]

[Table 1]

As shown in Table 1, CD56-positive cells were not obtained in the medium containing only one of IL-2 and SCF (Experiments 1 and 2), and CD33 ⁺ CD34 ⁺ cells were differentiated and proliferated. It can be seen that both IL-2 and SCF are required (experiment 3). Furthermore, SCF is present at a certain time after the start of culture, and CD33 ⁺ CD34
It is necessary to differentiate and proliferate ⁺ cells, and it can be seen that differentiation and proliferation are possible even in the latter stage of culture (Experiment 4).
And 5). Further, it can be seen that even if IL-2 does not exist for a certain period from the start of culture, the differentiation and proliferation of CD33 ⁺ CD34 ⁺ cells proceed, and rather the differentiation and proliferation do not proceed unless it is present in the latter stage of the culture (Experiment 6 And 7).

Example 3 NK cell activity assay The CD56-positive cells obtained in Example 2 were assayed for their properties as NK cells by the following method.

The activity of NK-like cells was assayed by the NK activity assay method targeting K562 cells. That is,
K562 cells were suspended in RPMI 1640 medium containing 5% FCS. K562 target cells were labeled with ⁵⁰ μCi of ⁵¹ Cr for 2 hours at 37 ° C. and washed thoroughly before use. The target cells (5
× 10 ³ ) and the above CD56 suspended in 10 μl of medium
Positive cells and various ratios (20: 1, 10: 1, 5:
1 or 2.5: 1) in microtiter wells. After culturing at 37 ° C. for 4 hours, the supernatant was collected and the radioactivity was measured with a gamma counter. Cytotoxicity was determined from the amount of ⁵¹ Cr released by the lysed target cells. ^The amount of ⁵¹ Cr released is calculated by the following formula: 100 ×
([Cpm in experimental well]-[cpm in well containing target cells alone]) / (c contained in target cells
pm).

One degradation unit (LU) was defined as the NK activity that resulted in 15% release of ⁵¹ Cr from 5 × 10 ³ target cells. The LU per 10 ⁵ cells was determined for each sample from the percent cell lysis regression curve. NK activity per well was calculated as follows. LU /
10 ⁵ cells x (1/10 ⁵ ) x total viable cell counts / well. Analysis of NK activity was performed after starting culture of sorted cells 14,
It was carried out on 21st and 28th. The result of NK activity is shown in FIG. From FIG. 3, it can be seen that the cells obtained in Experiment 3 of Example 2 show a corresponding NK activity.

Example 4 Fractionation of CD33 ^- CD34 ⁺ cells A phycoerythrin-negative, fluorescein-negative and allophycocyanin-positive cell population (Lin ^- CD33 ^- CD34 ⁺ fraction) was prepared in the same manner as in Example 1. Obtained.

Example 5 Differentiation and Proliferation of CD33 ^- CD34 ⁺ Cells 500 U / ml of Recombinant Human IL-2, 100 U
/ Ml recombinant human IL-3, 50 ng / ml recombinant human SCF in 200 μl of complete medium was placed in a 96-well round bottom microplate. 500 Lin ⁻ CD33 ⁻ CD34 ⁺ fractionated cells obtained in Example 4 were
The cells were added to each well and cultured for 28 days under the conditions of 37 ° C., 5% carbon dioxide gas and 100% humidity, and the number of CD56-positive cells that appeared as surface antigens of NK cells was counted (Experiment 6). The results are shown in Table 2.

For comparison, when IL-2 was not included (Experiments 2 and 3) or added only for a part of the culture period (Experiment 7), IL-3 was not included (Experiments 1, 2 and 3). 4) or when added only to a part of the culture period (experiments 6, 7 and 8) and without SCF (experiment 1,
3, 5 and 8) or when added to only a part of the culture period (Experiment 7), the same culture was performed, and CD5
The number of 6-positive cells was counted. The results are shown in Table 2.

[0039]

[Table 2]

As shown in Table 2, no CD56-positive cells were obtained in the medium containing only one or two of IL-2, IL-3 and SCF (Experiments 1-5 and 8), and C
IL-2, I is required for the differentiation and proliferation of D33 ^- CD34 ⁺ cells.
It turns out that three of L-3 and SCF are required (experiment 6). Furthermore, it is found that the presence of IL-3 at a certain time from the start of the culture is necessary for the differentiation and proliferation of CD33 ⁻ CD34 ⁺ cells, and the differentiation and proliferation are possible even if they are not always required at the latter stage of the culture ( Experiments 6 and 7). Also,
SCF is present at a certain time after the start of culture,
It is found that it is necessary for the differentiation and proliferation of D33 ^- CD34 ⁺ cells, and that the differentiation and proliferation is possible even if it is not always required in the latter stage of the culture (Experiments 6 and 7).

Example 6 Examination of morphological characteristics CD33 ⁺ CD34 ⁺ cells (200
0.5-1 × 10 ³ cells in 800 μl of medium at 800 rpm
Centrifuge for 3 minutes on a microscope slide. After centrifugation, it was immediately air-dried and stained with Wright. It is shown in the photograph of FIG. 4, which is a photograph of cells stained with Wright. C obtained in Example 4
D33 ^- CD34 ⁺ cells were also stained with Wright in the same manner as above, and shown in the photograph of FIG. From the photograph of FIG. 4, C
D33 ⁺ CD34 ⁺ cells are large and have fine cytoplasmic granules. On the other hand, from the photograph in Figure 5, CD33 ^- CD
34 ⁺ cells are small to medium sized basophilic cytoplasms (basoph
It was a lymphoblastoid cell with iliccytoplasm. These show that CD33 ^- CD34 ⁺ cells
⁺ Suggests that it is more primitive than CD34 ⁺ cells.

The cells obtained in Experiment 3 of Example 2 (0.5 to 1)
For even × 10 ⁵ cells), and photographed cells Wright's stain in the same manner as described above, shown in the photograph of FIG. The cells shown in the photograph of FIG. 6 showed a large-sized, granular-like lymphocyte-like structure. This morphology is similar to that of NK cells. Besides these, granulocytes, macrophages and mast cells were also present.

[0043]

INDUSTRIAL APPLICABILITY The present invention can provide NK cell progenitor cells and a method for producing the same. Furthermore, the present invention can provide an agent for differentiating and proliferating NK cell progenitor cells and a method for differentiating and proliferating NK cell progenitor cells, and it has become possible to obtain a large number of NK cells. Further, by using the method of the present invention, it has become possible to develop a method for treating cancer by separating NK progenitor cells from a cancer patient, proliferating them in a tester, and then transplanting them into the patient's body.

[Brief description of drawings]

FIG. 1 shows the results of cell sorting (cell classification) performed in Examples. Of the Lin ⁻ fractions (a), (b) in FIG.
D33 ^- CD34 ⁺ fraction, (c) is CD33 ⁺ C
D34 ⁺ fraction.

FIG. 2: Progenitor cells of NK cells of the present invention (CD33 ^- CD
34 ⁺ and CD33 ⁺ CD34 ⁺ ) NK cells (CD5
6 is a scheme showing the steps of differentiation into 6 ⁺ ).

FIG. 3 shows the results of assaying NK activity. Figure A is counted
Total cell count, Figure B shows CD56⁺Number of cells, Figure C shows NK
Each activity is shown. The solid line is CD33.⁺CD34
⁺Lin^-The results for the fractions of
33^-CD34⁺Lin ^-Results for the
It

FIG. 4 is a photograph instead of a drawing, which shows the morphology of cells (organisms) obtained by Wright-staining CD33 ⁺ CD34 ⁺ cells.

FIG. 5 is a photograph instead of a drawing, which shows the morphology of Wright-stained cells (organism) of CD33 ⁻ CD34 ⁺ cells.

FIG. 6 is a photograph instead of a drawing, which shows the morphology of cells (organisms) obtained by Wright-staining cells obtained by differentiating and growing CD33 ⁻ CD34 ⁺ cells.

Claims (15)

[Claims] 1. A precursor of natural killer cells obtained from human bone marrow-derived cells. 2. An anti-human CD33 antibody-negative and anti-human C
The progenitor cell according to claim 1, which is positive for D34 antibody. 3. Anti-human CD33 antibody-positive and anti-human C
The progenitor cell according to claim 1, which is positive for D34 antibody. 4. Anti-human CD34 derived from human bone marrow-derived cells
A method for producing natural killer cell progenitor cells, wherein natural killer cell progenitor cells are isolated using antibody positivity as an index. 5. Anti-human CD33 derived from cells derived from human bone marrow
The production method according to claim 4, wherein the precursor cells of natural killer cells are isolated using antibody negative as an index. 6. Anti-human CD33 derived from cells derived from human bone marrow
The production method according to claim 4, wherein the precursor cells of natural killer cells are separated using antibody positivity as an index. 7. A differentiation and growth agent for natural killer cell progenitor cells, which comprises interleukin 2 and stem cell growth factor. 8. The natural killer cell progenitor cell is a progenitor cell that is anti-human CD33 antibody-negative and anti-human CD34 antibody-positive, or a progenitor cell that is anti-human CD33 antibody-positive and anti-human CD34 antibody-positive. 7. The differentiation and proliferation agent according to 7. 9. A differentiation and proliferation agent for natural killer cell progenitor cells, which comprises interleukin 3 and stem cell growth factor. 10. The differentiation and proliferation agent according to claim 9, wherein the natural killer cell progenitor cells are anti-human CD33 antibody-negative and anti-human CD34 antibody-positive progenitor cells. 11. A differentiation and proliferation agent for natural killer cell progenitor cells, which comprises interleukin 2, interleukin 3, and stem cell growth factor. Breeding agent. 12. The differentiation / proliferation agent according to claim 11, wherein the natural killer cell progenitor cells are anti-human CD33 antibody-negative and anti-human CD34 antibody-positive progenitor cells. 13. A natural killer cell progenitor cell, which is anti-human CD33 antibody-positive and anti-human CD34 antibody-positive, is cultured in a medium containing interleukin-2 and stem cell growth factor. A method for differentiating and proliferating progenitor cells. 14. A progenitor cell of a natural killer cell, which is anti-human CD33 antibody negative and anti-human CD34 antibody positive, is cultured in a medium containing interleukin 2, interleukin 3 and stem cell growth factor. A method for differentiating and proliferating precursor cells of natural killer cells. 15. Precursor cells of natural killer cells that are anti-human CD33 antibody negative and anti-human CD34 antibody positive are cultured in a medium containing interleukin 3 and stem cell growth factor, and then interleukin 2 and interleukin 3 are cultured. And a method for differentiating and proliferating precursor cells of natural killer cells, which comprises culturing in a medium containing a stem cell growth factor.