JPH1094390A - Production of new peripheral blood stem cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtian the subject blood stem cell useful for treating disorders after a superhigh administration dose chemotherapy using a large dose of an anticancer medicine aiming a therapy for annihilating cancer cells by bringing peripheral blood stem cells with into contact with a factor capable of stimulating gp 130. SOLUTION: This method for producing new peripheral blood stem cells comprises bringing peripheral blood stem cells comprising peripheral blood- originated CD 34 positive cells into contact with interleukin-6 (IL-6) or interleukin-6 receptor (IL-6R) as a factor capable of stimulating gp 130 and further interleukin-3 (IL-3) and stem cell factor(SCF) in the wells of a 96 hole plate. When all of IL-6, IL-6R, IL-3 and SCF are added, the most amplified peripheral blood stem cells are obtained as understood from cell numbers expressed by void bars expressing 8-50 cells/well, black-colored bars expressing 51-500 cells/well and oblique line bars expressing >=501 cells/well.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for producing peripheral blood stem cells, which is suitable for performing peripheral blood stem cell transplantation, which has recently attracted attention. More specifically, production of peripheral blood stem cells, characterized by using a stimulating factor such as inter-leukin-6 (IL-6) or inter-leukin-6 receptor (IL-6R) capable of stimulating gp130. It is about the law.

[0002]

2. Description of the Related Art Peripheral blood stem cell transplantation aims to cure cancer cells by annihilation, and inevitably causes toxicity (degradation of bone marrow) in ultra-high dose chemotherapy using anticancer drugs several times higher than usual dose. This is a treatment method in which peripheral blood stem cells are collected and stored in advance from a patient in order to reconstruct), and then transfused into the patient after chemotherapy.

[0003] Peripheral blood stem cell transplantation has attracted attention as a third transplantation therapy after allogeneic bone marrow transplantation and autologous bone marrow transplantation.
Compared to allogeneic bone marrow transplantation or autologous bone marrow transplantation, this method has the advantages of not requiring general anesthesia, faster recovery of neutrophils and platelets after transplantation, and faster recovery of immune competence after transplantation. There is. In Japan, it was recognized as an insurance practice in April 1994, and the number of cases performed has dramatically increased. In contrast, the number of cases undergoing autologous bone marrow transplantation has decreased, and the number of cases undergoing allogeneic bone marrow transplantation has been flat. is there.

[0004]

In order to cause both rapid hematopoietic recovery and continuous hematopoietic recovery by peripheral blood stem cell transplantation, granulocyte / macrophage colony-forming cells must be included in the cells to be transfused. (CFU-GM), erythroid cell bar
Strike-forming cells (BFU-E), megakaryocyte colony-forming cells (CFU-Mk), mixed colony-forming cells (CFU-M)
It is desirable that a sufficient amount of CD34-positive cells such as Mix.

[0005] The amount of hematopoietic stem cells required for hematopoietic recovery is
In CFU-GM, 100,000-500,000 per kg of body weight,
CD containing colony-forming cells usually in an amount of about 30 to 40%
It is said that the number of 34 positive cells is 1 to 2 million per kg of body weight (Sonoda, peripheral blood stem cell transplantation-from basic to clinical, Nankodo, 5-15, 1995).

As described above, it is desirable to inject a sufficient amount of stem cells in order to promote hematopoietic recovery, but there is a limit in obtaining stem cells. For example, cyclophosphamide,
After anticancer chemotherapy using etoposide or an anticancer drug such as cytosine arabinosite or the like, granulocyte colony-stimulating factor (G-CSF) is administered to mobilize stem cells to peripheral blood,
With the method of obtaining CD34-positive cells by apheresis, it is difficult to secure a sufficient amount of stem cells in some patients such as acute myeloid leukemia.

[0007] A new method expected to secure a sufficient amount of stem cells is the production (expansion) of stem cells in vitro.
It is. If this becomes possible, C will greatly exceed the current amount
Not only can D34-positive cells be infused, but the burden on the patient can be greatly reduced by reducing the amount of blood subjected to apheresis. In fact, for CD34 + cells derived from bone marrow and umbilical cord blood, c-kit and gp130 are stimulated for in vitro production of stem cells, that is, for expansion of stem cells and induction of differentiation into red blood cells, white blood cells, and platelets. Has been reported to be effective, and for that purpose, CD34-positive cells may be cultured by adding stem cell factor (SCF), IL-6, IL-6R and the like (Sui et al., Proc. Natl. Aca
d. Sci. USA, 92, 2859-2863, 19
1995).

[0008] Therefore, if stem cells can be produced in vitro from CD34-positive cells derived from peripheral blood as described above, it is effective for peripheral blood stem cell transplantation. However, various properties are different between cells derived from bone marrow or cord blood and cells derived from peripheral blood. For example, peripheral blood-derived CD mobilized by G-CSF after anticancer chemotherapy
34 positive cells, compared to CD34 positive cells in bone marrow,
It has been reported that the expression rate of HLA-DR antigen, CD33 antigen, and CD13 antigen is high, while the expression rate of c-kit is low (Sonoda, Peripheral Blood Stem Cell Transplantation-from basic to clinical, Nankodo, 5-15 , 1995). For this reason,
A method for producing stem cells from bone marrow-derived CD34-positive cells or cord blood-derived CD34-positive cells is directed to peripheral blood-derived CD3.
There was no guarantee that it would be effective in producing stem cells from 4 positive cells. For expansion and differentiation of stem cells contained in peripheral blood-derived CD34-positive cells, SCF, IL-3, granulocyte macrophage colony-stimulating factor (GM-CSF),
It has only been reported that G-CSF, erythropoietin and the like are effective (Sonoda et al., Blood
d, 81, 624-630, 1993, Sonoda.
Et al., Blood, 84, 4099-4106, 1994.
Year).

[0009]

The present inventors examined the expansion and differentiation of stem cells contained in CD34-positive cells derived from peripheral blood. As a result, the cells expressed gp130, and IL- 6, IL-6R, IL-3, SC
F and others have found that they are effective. That is, the present invention is a method for producing expanded peripheral blood stem cells, which comprises contacting peripheral blood stem cells with a factor capable of stimulating gp130. The present invention is also a composition for expanding peripheral blood stem cells, comprising a factor capable of stimulating gp130. The present invention further provides a peripheral blood-derived CD3, which comprises obtaining an interleukin-6 receptor-expressing cell.
A method for preparing a differentiated cell among 4 positive cells, and a method for preparing a peripheral blood stem cell-containing fraction in peripheral blood-derived CD34 positive cells, which comprises obtaining interleukin-6 receptor non-expressing cells. Hereinafter, the present invention will be described in detail.

Gp130 is obtained by gel electrophoresis (SDS-P
AGE) is a glycoprotein having an apparent molecular weight of 130 kDa, and has a role of transmitting stimulation from a complex of IL-6 and IL-6R to cells. In the present invention, gp
This is applied to peripheral blood-derived stem cells expressing 130 (peripheral blood stem cells). Peripheral blood stem cells
For example, those prepared as a cell (CD34-positive cell) fraction expressing the CD34 antigen can be used. By applying the present invention to such cells, the number of the cells can be increased, or CFU-GM-BF can be amplified.
It is possible to induce differentiation into EE, CFU-Mk, CFU-Mix and the like.

[0011] Factors capable of stimulating gp130 that can be used in the present invention are, for example, IL-6, IL-6R, inter-leukin-11 (IL-11), and oncostatin M. The stimulation of gp130 can be, for example, I
Triggered by a complex of L-6 and IL-6R,
Peripheral blood stem cells to be expanded have I other than gp130.
When L-6R is also expressed, only IL-6 is gp1
The effect of the present invention can be achieved by using 30 as a stimulable factor. However, since normal hematopoietic stem cells such as CD34-positive cells do not express IL-6R, other factors such as IL-11 that can stimulate gp130 alone or in combination with IL-6 and IL-6R alone may be used. It is preferred to use.

As described above, IL-6 is a protein that forms a complex with IL-6R expressed on the cell membrane or soluble IL-6R to transmit stimulus into cells through gp130 expressed on the cell membrane. . In the present invention, both recombinant and natural purified IL-6 can be used, but from the viewpoint of homogeneity, recombinant IL-6 is used.
Is preferred. As the recombinant IL-6, for example,
Recombinant IL-6 derived from Escherichia coli (Yasukawa
a et al., Biotech. Lett. , 12, 419-4
24, 1990). As IL-6R used in combination with IL-6, either IL-6R of recombinant or purified from nature can be used, but from the viewpoint of homogeneity, recombinant IL-6R is preferable. In particular, the present invention is applied to peripheral blood stem cells that do not express IL-6R, because IL-6R lacking a portion corresponding to the extracellular region is soluble and thus easy to manufacture and handle. Is particularly preferred. Such soluble IL
As -6R, a recombinant CHO cell-derived IL-6R composed of 344 amino acids in the extracellular region can be exemplified (Yasukawa, J. Biotech., 10).
8, 673-676, 1990).

For example, when IL-6 and IL-6R are used as factors capable of stimulating gp130, IL-3 and IL-6R are further added.
Alternatively, it is preferable to use IL-3 and SCF together.
Although IL-3 or IL-3 and SCF are not considered to be factors that can stimulate gp130, their combined use can enhance the effect of expanding and differentiating peripheral blood stem cells.
As for IL-3 and SCF, either recombinant or purified from nature can be used, but from the viewpoint of homogeneity, etc., recombinant IL-3 and recombinant SC
F is preferred (Martin et al., CELL, 63, 20).
3-212, 1990). Furthermore, gp13
0 (for example, a combination of IL-6 and IL-6R), in addition to the above-mentioned IL-3, etc., for example, FLK2 ligand, thrombopoietin (TPO) or G-CSF
, Etc. can also be used in combination.

In the present invention, the peripheral blood stem cells are gp1
30 is contacted with an agent capable of stimulating, for example, IL-6 and IL-6R, and for further purposes, eg, I
Peripheral blood stem cells may be cultured in a medium containing L-3 or the like. Culture conditions (medium, temperature, pH, etc.) can also be performed according to a conventional method.

The peripheral blood stem cells to be expanded and differentiated to which the present invention is applied can be collected according to a conventional method. Here, as shown in Examples described later, peripheral blood-derived CD3
For 4 positive cells, IL-6R
Is expressed, and IL-6R is not expressed in cells that have not yet differentiated. Thus, CD3 containing peripheral blood stem cells
By examining the expression of IL-6R for the 4 positive cells, differentiated cells can be prepared, or a peripheral blood stem cell-containing fraction can be prepared (secured).

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

Embodiment 1 FIG. Analysis of surface antigen of peripheral blood-derived CD34-positive cells After peripheral blood is immunostained with various monoclonal antibodies, it is subjected to flow cytometry (manufactured by Becton Dickinson) according to a conventional method, and IL-6R, gp of CD34-positive cells is analyzed.
The expression rates of 130, c-kit, and IL-3R (α chain) were analyzed. The results are shown in FIG. As is clear from FIG.
CD34-positive cells have an IL-6R expression rate of about 80%,
The expression rate of gp130 is almost 100%, the expression rate of c-kit is about 20%, and the expression rate of IL-3R (α chain) is about 10%.
It was 0%.

Next, CD3 was analyzed by flow cytometry.
4-positive IL-6R-positive cells and CD34-positive IL-6R-negative cells were fractionated and examined for their properties. As a result, the CD34-positive IL-6R-positive cells are differentiated,
Most were found to be CFU-GM. Therefore,
Undifferentiated stem cells conventionally contained in CD34 positivity are CD3
It was included in the 4 positive IL-6R negative fraction, and this fraction was used in the following experiments.

Embodiment 2 FIG. Peripheral blood-derived CD34-positive IL-
Granulocyte / macrophage / erythroid bar of 6R negative cells
Colony formation of strike Clonal culture using the methylcellulose method
da et al. (Blood, 84, 4099-4106, 19)
1994). C fractionated in Example 1
Α-medium containing 200 D34-positive IL-6R-negative cells (1% bovine serum albumin, respectively, in the absence of 30% fetal bovine serum (A in Table 1) or in the absence of fetal bovine serum (B in Table 1)
5 × 10 ⁻⁵ M 2-mercaptoethanol and various factors described in Table 1. The concentrations of the various factors are as follows: I
L-3; 10 ng / mL, GM-CSF; 10 ng / m
L, G-CSF; 20 ng / mL, Epo; 2 U / m
L, SCF; 20 ng / mL, IL-6: 20 ng / m
L, soluble IL-6R (sIL-6R); 1 μg / m
L) 1 mL was placed in a 35 mm diameter petri dish and cultured for 2 weeks.

After the cultivation, various colonies were calculated by a conventional method. Table 1 shows the results. In the table, CSFs are SCF,
3 shows a mixture of IL-3, GM-CSF, G-CSF and Epo.

[0021]

[Table 1]

As is apparent from Table 1, SCF, IL-3, GM-CSF, and GC, which are well known as factors that form granulocyte, macrophage, and erythroid bursts, are known.
When SF and erythropoietin (Epo) were added (Table 1A or B, row of CSFs), the colony number was 100.
%, SCF, IL-3, IL-6, IL-6
Combination of R (SCF + IL-3 + I in Table 1A or B)
L-6 + soluble IL-6R (sIL-6R) row) gave approximately the same (77.6% in Table 1A, 101% in Table 1B) colony numbers. In Table 1, G is the number of granulocyte colonies, M is the number of macrophage colonies, GM is the number of granulocyte macrophages, B is the number of erythrocyte burst colonies, Eo is the number of eosinophil colonies, and E-Mix is the number of eosinophil colonies. The number of mixed colonies is shown. The numbers in Table 1 are all mean ± SD values.

This result indicates that GC, which has been said to be essential for forming granulocytes and macrophages, has been used.
In the absence of SF, Epo, which was said to be essential for the formation of erythroid bursts, SCF, IL-3, I
This shows that granulocytes, macrophages, erythroid blasts and the like are formed by the combination of L-6 and IL-6R. In other words, it is shown that the stimulation of gp130 can induce the expansion of peripheral blood stem cells (CD34-positive cells) and the differentiation into granulocytes and the like, similarly to bone marrow stem cells and the like.

Embodiment 3 FIG. Peripheral blood-derived CD34-positive IL-
Megakaryocyte formation of 6R-negative cells CD34-positive IL-6R-negative cells 2 fractionated in Example 1
Α-medium containing 100 cells (10% human plasma (A in Table 2)
Alternatively, 1% bovine serum albumin, 5 × 10 ⁻⁵ M 2-mercaptoethanol and various factors described in Table 2 are contained in the absence of human plasma (B in Table 2). The concentrations of various factors are as follows. Thrombopoietin (TPO); 100
ng / mL, otherwise as in Example 2) 20 mL
It was placed in 20 Petri dishes having a diameter of 35 mm and cultured for 2 weeks.

After the cultivation, various colonies were counted by a conventional method. Table 2 shows the results.

[0026]

[Table 2]

As is clear from Table 2, when TPO, which is well known as a factor for forming megakaryocytes (megakaryosites), was added (Table 2A, B, TPO rows), the colony of megakaryocytes was changed. Appearance (203 in A, 126 in B
SCF, IL-3, IL-6, IL
-6R combination (SCF + IL-3 + IL- in Table 2)
6+ soluble IL-6R (sIL-6R) row), in the presence of human plasma, a colony of approximately the same number of megakaryocytes in total.
(24) and colonies including megakaryocytes (162),
Mixed colonies containing half of megakaryocytes even in the absence of human plasma
Numbers (60) were obtained. In Table 2, Meg indicates the number of megakaryocyte colonies, and M-Mix indicates the number of mixed colonies containing megakaryocytes. The numbers in Table 2 are all mean ±
SD value.

This result indicates that SCF, IL-3, I-C, in the absence of TPO, which is important for the formation of megakaryocyte colonies.
It shows that megakaryocytes are formed by the combination of L-6 and IL-6R. That is, by the stimulation of gp130,
It shows that peripheral blood stem cells (CD34-positive cells) can be induced to expand and differentiate into megakaryocytes, similarly to bone marrow stem cells and the like.

Embodiment 4 FIG. Effect of Various Neutralizing Antibodies on the Formation of Granulocyte, Macrophage, and Erythroid Burst CD34 + IL-6R + cells 2 fractionated in Example 1
Α-medium containing 30 cells (30% fetal bovine serum, 1% bovine serum albumin, 5 × 10 ⁻⁵ M 2-mercaptoethanol-
, IL-3: 10 ng / mL, SCF: 20 ng / m
L, IL-6; 20 ng / mL) and control serum; 1/100 of a mixed serum of normal mouse serum and normal rabbit serum.
Diluted, anti-G-CSF serum; 1/100 dilution, rabbit-derived, anti-SCF antibody; 20 μg / mL, mouse-derived, anti-IL-3 serum; 1/100 dilution, rabbit-derived, anti-IL-6R antibody; 5 μg / ML mouse origin, anti-gp1
30 antibody; anti-gp130 monoclonal antibody mixture (G
Mixture of PX-7, GPX-22 and GPZ-35, mouse-derived, see JP-A-5-304986, total 1 μg
/ ML) or a mixed antibody of the above-mentioned anti-SCF antibody, anti-IL-3 serum, anti-IL-6R antibody and anti-gp130 antibody, and 1 mL thereof was spread on two 35 mm diameter Petri dishes and cultured for 2 weeks.

After completion of the culture, various colonies were counted by a conventional method. The results are shown in Table 3A.

On the other hand, CD34-positive I fractionated in Example 1
Α-medium containing 200 L-6R negative cells (30% fetal bovine serum, 1% bovine serum albumin, 5 × 10 ⁻⁵ M 2-
Mercaptoethanol, IL-3; 10 ng / mL, S
CF: 20 ng / mL, IL-6: 20 ng / mL, soluble IL-6R (sIL-6R); 1 μg / mL)
Further, control serum; a mixed serum of normal mouse serum and normal rabbit serum diluted 1/100, anti-Epo antibody; 5 μg
/ ML mouse-derived, anti-SCF antibody; 20 μg / mL
Mouse, anti-IL-3 serum; 1/100 dilution, rabbit, anti-IL-6R antibody; 5 μg / mL, mouse,
Anti-gp130 antibody; anti-gp130 monoclonal antibody mixture (mixture of GPX-7, GPX-22 and GPZ-35, mouse-derived, see JP-A-5-304986, total 1 μg / mL) or the above-mentioned anti-SCF antibody, anti-IL-3
A mixed antibody of serum, anti-IL-6R antibody and anti-gp130 antibody was added, and 1 mL thereof was spread on two 35 mm diameter Petri dishes and cultured for 2 weeks. After completion of the culture, various colonies were counted by a conventional method. The results are shown in Table 3B.

[0032]

[Table 3]

In the system shown in Table 3A, 45 granulocyte macrophage colonies were formed ("non" in Table 3A).
e), GM column), the anti-G-CSF antibody had no inhibitory effect on this colony formation. On the other hand, anti-SCF antibody, anti-IL-3 serum, anti-IL-6R antibody or anti-gp13
Antibodies 0 each inhibited the formation of this granulocyte macrophage colony. In particular, when an anti-SCF antibody, anti-IL-3 serum, anti-IL-6R antibody and anti-gp130 antibody were mixed, colonies of granulocyte macrophages were not formed. (Comparison of GM columns in Table 3A).

In the system shown in Table 3B, 69 erythroid burst colonies were formed ("non" in Table 3B).
e), column B), anti-Epo antibody showed no inhibitory effect on this colony formation. On the other hand, anti-SCF antibody, anti-IL-3 serum, anti-IL-6R antibody, anti-gp130 antibody or anti-SCF antibody, or anti-IL-3 serum, anti-IL-6R
When the antibody and the anti-gp130 antibody were mixed, no erythroid burst colonies were formed (comparison of row B in Table 3B). In Table 3, GM indicates the number of colonies of granulocyte colony-stimulating factor, B indicates the number of erythroid burst colonies, and E-Mix indicates the number of mixed colonies containing erythroblasts. The numbers in Table 3 are all mean ± SD values. * Indicates P <for the row of “none” and the column of “B”.
** indicates that there is a significant difference under the condition of 0.01.
The row “e” and the column “B” indicate that there is a significant difference under the condition of P <0.001, and NS indicates that there is no significant difference.

The above results indicate that peripheral blood stem cells were
Granulocytes, macrophages supported by culturing with the addition of a combination of L-3, IL-6 and IL-6R
This shows that the formation of erythroid blasts is not affected by G-CSF or Epo.

Embodiment 5 FIG. Effect of various neutralizing antibodies on the formation of colonies containing megakaryocytes CD34-positive IL-6R-negative cells 2 fractionated in Example 1
Α-medium containing 10 cells (10% human plasma, 1% bovine serum albumin, 5 × 10 ⁻⁵ M 2-mercaptoethanol-
And a control serum; a mixture of normal mouse serum and normal rabbit serum diluted 1/100; anti-TPO antibody; 5 μg / mL mouse-derived anti-TPO receptor
(C-Mpl) antibody; 20 μg / mL mouse, anti-SCF antibody; 20 μg / mL mouse, anti-IL-3
Serum; 1/100 dilution, rabbit-derived, anti-IL-6R antibody; 5 μg / mL, mouse-derived, anti-gp130 antibody; anti-gp130 monoclonal antibody mixture (GPX-7, G
A mixture of PX-22 and GPZ-35, mouse-derived, see JP-A-5-304986, total 1 μg / mL) or a mixed antibody of the above-mentioned anti-SCF antibody, anti-IL-3 serum, anti-IL-6R antibody and anti-gp130 antibody 1m
L was spread on two 35 mm diameter Petri dishes and cultured for 2 weeks. After completion of the culture, various colonies were counted by a conventional method.
Table 4 shows the results.

[0037]

[Table 4]

As is evident from Table 4, 25 colonies containing megakaryocytes were formed (see the row of "none" in Table 4;
M-Mix column), anti-TPO antibody or anti-c-Mpl antibody showed no inhibitory effect on this colony formation. On the other hand, anti-SCF antibody, anti-IL-3 serum, anti-IL-6R antibody or anti-gp130 antibody, or anti-IL-3 serum, anti-IL
When the -6R antibody and the anti-gp130 antibody were mixed, the formation of the megakaryocyte-containing colony was inhibited (comparison of the M-mix column in Table 4). In Table 4, Meg indicates the number of megakaryocyte colonies, and M-Mix indicates the number of mixed colonies containing megakaryocytes. In addition, the numbers in Table 4 are all mean ± SD values, and * indicates a row of “none” and “MM”.
** indicates that there is a significant difference under the condition of P <0.05, and ** indicates a significant difference under the condition of P <0.005 with respect to the row of “none” and the column of “M-Mix”. That there is a difference
NS indicates that there is no significant difference.

The above results indicate that peripheral blood stem cells were
It is shown that megakaryocyte formation by adding and culturing a combination of L-3, IL-6, and IL-6R is not affected by TPO.

Embodiment 6 FIG. Effect of various factors on single CD34-positive IL-6R-negative cell 1 CD34-positive IL-6R-negative cell 1 fractionated in Example 1
Medium containing 30% fetal bovine serum, 1% bovine serum albumin, 5 × 10 ⁻⁵ M 2-mercaptoethanol and the various factors described in Table 5. 0.2 ml) was placed in each well of a 96-well plate and cultured. The number and type of colonies formed on day 14 were counted. Table 5 shows the results.

[0041]

[Table 5]

As apparent from Table 5, SCF, IL-
6, 3.5% when used in combination with IL-6R
Colonies were formed in the wells. In particular, SCF, I
By using a combination of L-3, IL-6 and IL-6R, the frequency of colony-formed wells was 3
It increased to 8.1%, and colony formation of all types of hematopoietic cells was observed.

The above results indicate that the combination of SCF, IL-6 and IL-6R has an effect on the induction of proliferation and differentiation of peripheral blood stem cells, and in particular, SCF, IL-3, IL-6 and I-6
This shows that the combination of L-6R is effective.

Embodiment 7 FIG. Effect of various factors on single CD34-positive IL-6R-negative cell 2 CD34-positive IL-6R-negative cell 1 fractionated in Example 1
Medium containing 30% fetal bovine serum, 1% bovine serum albumin, 5 × 10 ⁻⁵ M 2-mercaptoethanol and the various factors described in FIG. 0.2 ml) was placed in each well of a 96-well plate and cultured. On the fifth, tenth, and fourteenth days after the start of the culture, the number of wells in which cell expansion was observed and the number of expanded cells were counted. The results are shown in FIG.

As is apparent from FIG. 2, especially SCF, I
By using the combination of L-3, IL-6, and IL-6R, the frequency of appearance of wells in which cell expansion was observed was increased, and wells in which cells were amplified to 500 cells or more accounted for about half. Was.

The above results indicate that, similarly to Example 6, the induction of expansion and differentiation of peripheral blood stem cells, particularly SCF, IL-3, I
It strongly suggests that the combination of L-6 and IL-6R is effective.

[0047]

According to the method for producing peripheral blood stem cells characterized by stimulating gp130, CFU-GM (granulocyte / macrophage colony-forming cells), BFU-E
It is possible to effectively produce in vitro peripheral blood stem cells including (erythroid burst-forming cells), CFU-Mk (megakaryocytic colony-forming cells), CFU-Mix (mixed colony-forming cells) and the like. Will be possible. This makes it possible to easily produce stem cells required for peripheral blood stem cell transplantation, which has undergone a dramatic increase in the number of cases performed in recent years. While reconstructing, it can be expected to improve treatment results. In addition, various possibilities can be provided in a wide range of fields such as research and development relating to the production of artificial blood.

[Brief description of the drawings]

FIG. 1 is a two-dimensional scattergram showing the results of Example 1 and analyzed by flow cytometry after immunostaining peripheral blood with various monoclonal antibodies. (A) FI
Control IgG conjugated with TC and control IgG conjugated with PE (phycoerythrin), (b) shows binding of anti-CD34 antibody (CD34-FITC) conjugated with FITC and PE IL-6
R stained with an antibody against R, (c) shows CD34-F
Those stained with an antibody against gp130 bound to ITC and PE, (d) those stained with an antibody against c-kit bound to CD34-FITC and PE,
(E) IL bound to CD34-FITC and PE
3 shows the fluorescence pattern of a sample stained with an antibody against -3R (α chain).

FIG. 2 shows the results of Example 7, wherein peripheral blood stem cells (CD34-positive IL-6R-negative cells) were cultured in a 96-well plate, one cell per well, and cultured in the presence of various factors. In each case, the results are shown on day 5 (left column), day 10 (middle column), and day 14 (right column).
The numbers on the axis indicate the number (well) of wells in which cell amplification was observed on the measurement date, and each bar indicates white (8 to 50 cells were observed per well) and black (well). And cells with 51 to 500 cells per well) and oblique lines (cells with 501 or more cells per well) are shown.

Claims (12)

[Claims] 1. A method for producing expanded peripheral blood stem cells, comprising contacting peripheral blood stem cells with a factor capable of stimulating gp130. 2. The method for producing peripheral blood stem cells according to claim 1, wherein inter-leukin-6 and inter-leukin-6 receptor are used as stimulating factors capable of stimulating gp130. 3. In addition to a stimulating factor capable of stimulating gp130,
3. The method according to claim 1, further comprising using interleukin-3.
Method for producing peripheral blood stem cells. 4. In addition to a stimulator capable of stimulating gp130,
The method for producing peripheral blood stem cells according to claim 1 or 2, further comprising using interleukin-3 and stem cell factor. 5. The method for producing peripheral blood stem cells according to claim 1, wherein the peripheral blood stem cells are CD34-positive cells derived from peripheral blood. 6. A composition for expanding peripheral blood stem cells, comprising a factor capable of stimulating gp130. 7. The composition according to claim 6, comprising interleukin-6 and an interleukin-6 receptor as factors capable of stimulating gp130. 8. The composition according to claim 6, further comprising inter-leukin-3. 9. The composition according to claim 6, further comprising interleukin-3 and stem cell factor. 10. The composition according to claim 6, which is used for peripheral blood-derived CD34-positive cells. 11. A method for preparing differentiated cells from peripheral blood-derived CD34-positive cells, which comprises obtaining interleukin-6 receptor-expressing cells. 12. Peripheral blood-derived CD34, characterized by obtaining cells not expressing interleukin-6 receptor.
A method for preparing a fraction containing peripheral blood stem cells in positive cells.