JPH11180877A - Cell preparation containing megakaryocyte precursor cells and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide cell preparation that is effective for hematopoietic stem cell isotransplantation thrombocytopenia that shows effective recovering effect in the treatment of a variety of diseases with hemorrhagic diathesis causes by thrombocytopenia accompanied by chemotherapy and bone marrow transplantation. SOLUTION: In order to attain efficient in vitro proliferation of human megakaryocyte precursor cells, the hematopoietic undifferentiated cells are preliminarily cultured using the megakaryocyte growth factor and FLT3 ligand, and then by adding other hemopoietic factor, the objective cell preparation is more amplified in the megakaryocyte cells.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a cell preparation for transplantation of allogeneic hematopoietic stem cells containing megakaryocyte precursor cells generated by culturing hematopoietic undifferentiated cells in a culture system, and a method for producing the same.

[0002]

2. Description of the Related Art Blood cells, such as red blood cells, lymphocytes, and platelets, exist in blood which is essential for somatic cells constituting a living body. The blood cells share their respective functions,
Has a role to keep the living body constantly. In recent years, it has become clear that various blood cells differentiate and mature from hematopoietic undifferentiated cells in the bone marrow, and that various in vivo humoral factors are involved in the process of differentiation and maturation. It has become to.

[0003] Platelets have a diameter of 2-3 μm, which is present in blood.
m cells, which are a type of formed component in blood that plays an important role in hemostasis and thrombus formation in living organisms. It has been clarified that hematopoietic undifferentiated cells in bone marrow become megakaryocytes via megakaryocyte precursor cells, and the platelets generated by fragmentation of megakaryocyte cytoplasm are released into blood.

[0004] Recently, various research results on megakaryocyte and platelet systems have also been reported. For example, it has been reported that megakaryocyte growth factor has an action of promoting amplification of megakaryocytes producing platelets. [Shunichi Kato “Umbilical Cord Blood Stem Cell Transplantation” Daily Practice and Blood Vol. 7, p. 479 Special reprint, Pharmaceutical Journal). Cancer treatments, such as high-dose chemotherapy and high-dose radiation, destroy hematopoietic cells in the bone marrow, leaving the patient with a severely depleted platelet. After such treatment, thrombocytopenia occurs, resulting in reduced coagulation and bleeding disorders that require platelet transfusion.
Platelet deficiency is a major cause of morbidity and mortality following these cancer treatments, and has increased the cost of cancer treatment. The recovery of megakaryocytes and platelets generally requires more than 15 days, during which the patient's blood clotting capacity is lacking.

[0005] In recent years, cord blood banks and the like have been established, and allogeneic hematopoietic stem cell transplantation, in which collected cord blood is transplanted to a patient different from the individual from which the cord blood was collected, has begun to spread. In many cases of transplanted patients, delayed platelet recovery is a problem.

[0006]

In order to solve this problem, it has been desired to develop a method for producing a cell preparation in which undifferentiated cells derived from cord blood are cultured to efficiently expand megakaryocyte cells. . Such cell processing is expected to be performed outside hospitals by domestic and foreign companies.

[0007]

Means for Solving the Problems The present inventors have conducted intensive studies for the purpose of developing a culture method that acts on hematopoietic undifferentiated cells and promotes proliferation of megakaryocyte precursor cells.
The inventors succeeded in clarifying the culture method, and completed the present invention. The present invention relates to megakaryocyte growth factor and FLT.
Provided is a method for promoting differentiation of hematopoietic undifferentiated cells into megakaryocyte progenitor cells by a combination of three ligands, and further promoting proliferation of megakaryocyte progenitor cells by causing a stem cell factor to act. The present invention can provide a cell preparation for allogeneic hematopoietic stem cell transplantation that promotes recovery from thrombocytopenia in the treatment of a disease associated with thrombocytopenia or a disease associated with abnormal platelet function. The cell suspension obtained according to the present invention promotes the recovery of platelets immediately after cancer treatment.

That is, the present invention is characterized in that (1) it is produced by culturing hematopoietic undifferentiated cells in a medium containing megakaryocyte growth factor and FLT3 ligand in advance, and then culturing in a medium containing hematopoietic factors. (2) Production of the cell preparation according to (1), wherein the undifferentiated hematopoietic cells are derived from cord blood, (3) production of the cell preparation according to (1) or (2), About the method.

Hereinafter, the present invention will be described in detail. Hematopoietic factors (cytokines) include granulocyte macrophage colony stimulating factor (GM-CSF) and granulocyte colony stimulating factor (G
-CSF), interleukin 1 (IL-1), interleukin 3 (IL-3), interleukin 6 (IL
-6), megakaryocyte growth factor, erythropoietin (EP
O), FLT3 ligand, stem cell factor (SCF), GM
-CSF / IL-3 binding protein and the like are indicated. Megakaryocyte growth factor is known as thrombopoietin (TPO) and has been reported as a factor for growing megakaryocytes in vivo [Wendling, F. et al. , Et. al. ,
Nature, 369, 571 (1994), Masanao Teramura, Annual Review Blood, (199)
6)]. In addition, the megakaryocyte growth factor is Pepro Te.
ch (USA). FLT3 ligand is the same molecule as FLK2 ligand, its gene is cloned, and its effect on hematopoietic undifferentiated cells is reported [Lyman, S. et al. D. , Blood, 83, 27
95 (1994), Atsushi Iwama, Annual Review
w Blood, 12-32 (1995)). Stem cell factor
Z as Stem Cell Factor (SCF)
Reported by sebo et al. (Cell, 63, 195-20)
1 (1990)). This molecule is a ligand for the tyrosine kinase c-kit, KL (Huan, E .;
Cell 63, 225-233), mast cell growth factor M
GF (Mast cell Growth Facto)
r) (Williams, DE, Cell, 63,
167-174 (1990)], and its effect on hematopoietic undifferentiated cells is reported [Shimosaka, Special Issue on Experimental Medicine, “Cytokine and Signaling”, 185, (199)
2)]. These factors are usually 1 ng / ml to 1 ng / ml.
μg / ml, but preferably from 10 to 10
Can be used at 0 ng / ml.

[0010] The present invention provides a method for culturing megakaryocytes by culturing hematopoietic undifferentiated cells in advance in a medium containing megakaryocyte growth factor and FLT3 ligand as main stimulating factors for at least one day, and further culturing in a medium containing hematopoietic factors. Progenitor cells can be efficiently propagated. A number of cultures using combinations of hematopoietic factors have been reported (WO95 / 2446).
9). However, megakaryocyte precursor cells and megakaryocytes have not been efficiently generated. The present inventors have found a two-stage culture method, established a large number of megakaryocyte progenitor cells in the first stage, and established a method for inducing the differentiation of blood progenitor cells having a required composition in the second stage. In the present invention, pre-hematopoietic undifferentiated cells and megakaryocyte growth factor,
When culturing in a medium supplemented with FLT3 ligand as a main stimulating factor, some cytokines can be used in combination, but megakaryocyte growth factor and FL
It is desirable to use only T3 ligand. Hematopoietic factors added later are GM-CSF, G-CSF,
Although IL-1, IL-3, IL-6, megakaryocyte growth factor, erythropoietin (EPO), FLT3 ligand, stem cell factor (SCF), and GM-CSF / IL-3 binding protein can be used, stem cell factor is used. It is desirable to do. By combining these various kinds of cytokines, it is possible to set more appropriate stimulating conditions. However, culturing in a medium containing megakaryocyte growth factor and FLT3 ligand of the present invention, furthermore, By culturing the cells in the added medium, therapeutically useful megakaryocyte precursor cells can be efficiently generated. How to add hematopoietic factors
The hematopoietic factor can be added during the culturing, or the medium can be replaced with a centrifuge or the like.

According to the present invention, it is an industrially effective requirement that the amount of added cytokines be small and the timing of addition be adjusted to reduce the amount of addition to the minimum. And the possibility of providing a culture system and a cultured cell preparation at a particularly low price. The cell preparation refers to a suspension containing cells, a cell mass such as a pellet concentrated by centrifugation or the like, or a cryopreservation of the cell suspension. These cell preparations are not only available at medical institutions,
It can be manufactured in an external facility dedicated to cell processing.

The medium to be used may be supplemented with serum derived from humans or animals. More preferably, a serum-free medium may be used. For example, in addition to a basic medium for animal cells which can be generally used, as an additive composition for a serum-free medium, corticosteroid, transferrin, insulin, cholesterol, ethanolamine, human albumin and the like can be mentioned.

According to the present invention, a population of megakaryocyte precursor cells can be easily prepared in vitro. The cells originate from either bone marrow, cord blood or peripheral blood. Bone marrow fluid and peripheral blood can be obtained from normal donors. Hematopoietic undifferentiated cells as referred to herein are generically referred to as hematopoietic pluripotent stem cells having pluripotency obtained from bone marrow fluid, umbilical cord blood, peripheral blood, etc., and undifferentiated cells capable of forming hematopoietic colonies. Includes hematopoietic cells having cell surface antigens. In peripheral blood, the number of CD34 positive cells makes up only about 0.1% of total leukocytes. In cord blood, C
D34 positive cells make up about 0.1-1% of total leukocytes. Typically, normal bone marrow contains only 1-2% of CD34 positive cells. Leukocytes are separated from bone marrow or cord blood or peripheral blood by standard methods such as centrifugation by gradient. [Geigy Scientific
Tables, Vol 3, C.I. Lentner, e
d. Ciba-Geigy, Basel, Switzerland
rand, (1984)]. Analysis by Wright-Giemsa staining revealed that mature megakaryocytes in the bone marrow comprised only about 0.05% of the leukocyte population, while immunostaining specific for megakaryocytes showed about 0.2% Labeled up to. In healthy individuals, megakaryocyte progenitor cells are fully differentiated in the bone marrow, so that progenitor cells are found very rarely in normal blood. After separation, the leukocytes may be immediately cultured in a serum-free medium. Preferably, hematopoietic undifferentiated cells are isolated from the leukocyte population. The isolation of hematopoietic undifferentiated cells
34 may be based on their binding to antibodies specific for 34 positives, followed by separation of antibody-bound stem cells using magnetic beads [Hardwick, R .; A. , Et
al. , J. et al. Hematother. 1,379-38
6 (1992)]. The isolated hematopoietic undifferentiated cells are 5,
It is placed in culture at a density range of 000 to 500,000 cells / ml, preferably 10,000 cells / ml. Any tissue culture flask or bag or hollow fiber module or filter module can be used in either a static or perfusion culture system [Koller,
M. R. , Et al. , BIO / TECHNOLOG
Y, 11, 358-363, Emerson, S .; G,
et al. , PCT WO92 / 11355]. If a static culture system is used, the cells are fed with media at intervals of 5-7 days to replenish nutrients and remove waste. Cells are 7-14 days, more preferably 9
Cultured for ~ 12 days. At this time, the cell suspension contains a population of megakaryocyte progenitor cells suitable for use in treating thrombocytopenia.

[0014] Flow cytometer analysis was performed to determine cell phenotype based on labeling with cell surface antigens. CD34 positive cells indicate hematopoietic undifferentiated cells,
D61-positive cells indicate megakaryocytic cells [CD Classification Handbook, Cancer and Chemotherapy]. Mature megakaryocytes show increased cytoplasm,
Maturation can be easily analyzed by exhibiting multinucleation. The main trait of hematopoietic undifferentiated cells is CD34
Positive CD61 negative. Hematopoietic undifferentiated cells can be efficiently treated with CD34-positive CD61 by the treatment according to the present invention.
Promotes differentiation into positive megakaryocyte progenitor cells and further promotes CD3
4-negative CD61-positive immature megakaryocyte cells can be expanded. The differentiation into megakaryocyte precursor cells is promoted by FLT3 ligand and megakaryocyte growth factor, and the proliferation of the megakaryocyte precursor cells is promoted by stem cell factor and megakaryocyte growth factor, and immature megakaryocytes are amplified.

The cell suspension produced according to the present invention contains megakaryocyte progenitor cells and immature megakaryocytes as well as various types of blood cell precursor cells, and is further cultured with various types of hematopoietic factors. Thus, the required megakaryocyte differentiation into cells is promoted. Further, the cells in the megakaryocyte cell suspension according to the present invention are cell fragments PPF that cannot release platelets.
(Proplatelet Process Form
ation) is not generated, and unlike mature megakaryocytes, the degree of multinucleation is low. Therefore, there is an advantage that it is difficult to form a thrombus when transplanted into a living body.

The enriched cell suspension of megakaryocyte progenitor cells of the present invention allows for effective treatment of various types of thrombocytopenia in addition to those produced by cancer treatment.
Thus, the cell suspension provided herein also suggests the possibility of replacing platelet infusion in the treatment of thrombocytopenia. When the above-mentioned cell population is administered to a patient after chemotherapy or the like, the administered cells are transformed in vivo to produce megakaryocytes, which ultimately produce platelets.
It is expected to further differentiate in vo. Further, when placed in a fibrin clot assay, it is observed that the megakaryocyte progenitors form mature megakaryocytes and release platelets. The results from these assays suggest that cells from the serum-free culture can undergo further maturation even after they have been subjected to in vivo conditions.

[0017]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The following examples illustrate the invention but do not limit its scope.

[0018]

Example 1 Umbilical cord blood was fractionated using Ficoll Pack (Pharmacia), the mononuclear cell layer was collected, and washed with RPMI-1640 medium (Gibco) containing 5% fetal bovine serum.
The obtained cells were seeded on a plastic petri dish for culture, and cultured for 1 hour in a cell culture incubator (37 ° C., 5% carbon dioxide gas) to remove adherent cells. Dynabeads (Dynal) anti-CD34 antibody detachable beads were added to the obtained cell suspension, and reacted on ice for 30 minutes. The separation operation of the dynabeads was performed according to the attached manual. The obtained suspension of CD34-positive cells was added to fetal bovine serum to a final concentration of about 15% and dimethyl sulfoxide (DMSO) to a final concentration of 10%, divided into vials, and frozen. Was.

The percentage of isolated CD34-positive cells was measured with a flow cytometer. The method is as follows. A phycoerythrin (PE) -labeled anti-CD34 antibody (Becton) is added to a part of the cells, and the cells are stained on ice for about 30 minutes.
It was measured with a CS caliber (Becton). The positive rate of CD34 cells was about 80 to 90%.

[0020]

Example 2 The cells frozen in Example 1 were thawed and washed in a thawing solution (RPMI-1640 medium containing 2% dextran (Midori Cross) and 10% fetal bovine serum), and washed with 2 to 2.5 × 10 ^5. X-VIVO to a density of / ml
The cells were suspended in 10 media (Bio Whittaker). Transfer these to sterile test tubes (Falcon, product number 2)
No. 057). As a first experimental section, the cell suspension was subjected to two-stage culture. FLT3 ligand (100 ng) was used as a stimulator used in the first stage culture.
/ Ml, Genzyme) and megakaryocyte growth factor (5
0 ng / ml) and the cell suspension was cultured in a cell incubator (Tavai Co., 5% carbon dioxide, 37 ° C.) for 5 days.

Subsequently, the second stage of culture was performed. The method is described below. The cells after culture are collected, centrifuged, and an X-VIVO 10 medium (hereinafter referred to as X-VIV
2 to 2.5 × 10 ⁵ / ml in O10 modified medium)
Was prepared to have a density of. Additives were insulin (final concentration 10 μg / ml, Sigma I6634),
Transferrin (final concentration 10 μg / ml, Sigma T4778), low density lipoprotein (LDL, final concentration 5.5 μg / ml, Sigma L5402), 2-
Mercaptoethanol (2-ME, final concentration 4.3 × 1
0 ^-5 M, Merck). After the medium is changed, the second
SCF (50 ng / ml) and megakaryocyte growth factor (50 ng / ml) were added as stimulating factors for the steps. The cells were further cultured in a cell incubator for 5 days.

After collecting the cells, wash the cells with a centrifuge,
The cells were suspended in 50 μl of RPMI-1640 medium (Gibco) containing 5% fetal calf serum, fluorescein isothiocyanate (FITC) -labeled anti-CD61 antibody (Dako) was added in 2 μl portions, and staining was performed on ice for 30 minutes. Was. After washing twice with a centrifuge, the degree of staining of CD61 was measured with a FACS caliber (Becton). As a reference control, a system to which no stimulating factor was added, and as a stimulant control, a megakaryocyte growth factor (50 ng / ml), SCF as a stimulating factor in both the first and second stage cultures as a second experimental group
(50 ng / ml), and as a third experimental group, FLT3 ligand (100 ng / ml) and megakaryocyte growth factor (50 ng / ml) as stimulants in both the first and second stages of culture. / Ml).

As a result, the abundance of CD61-positive cells after culturing was 141.2 times in the first experimental group, 11.8 times in the second experimental group, and 6% in the third experimental group compared to the control without the stimulant.
It was 7.1 times. Therefore, it was shown that megakaryocyte cells were proliferated at high magnification by switching the stimulating factor in the middle.

[0024]

Example 3 The cells frozen in Example 1 were thawed with a thaw solution (RPMI-1640 medium containing 2% dextran (Midori Cross) and 10% fetal bovine serum), washed, and washed with 2 to 2.5 × 10 ^5. X-VIVO to a density of / ml
The cells were suspended in 10 media (Bio Whittaker). Transfer these to sterile test tubes (Falcon, product number 2)
No. 057). As a first experimental section, FLT3 ligand (100 ng / ml, Genzyme) and megakaryocyte growth factor (50 ng / ml) were added to the cell suspension as a first-step stimulating factor, and a cell culture vessel ( The cells were cultured for 5 days in Tabai, 5% carbon dioxide, at 37 ° C).

The cells after the culture were collected, and the second stage of the culture was performed as follows. After centrifugation, it was adjusted to a density of 2 to 2.5 × 10 ⁵ / ml with X-VIVO10 modified medium. After the medium was changed, S
CF (50 ng / ml), megakaryocyte growth factor (50 ng / ml)
ml) was added. Subsequently, the cells were cultured in a cell incubator for 5 days.

As a control, in the second experimental group, megakaryocyte growth factor (50 ng / m2) was used as a stimulator in both the first and second stages.
l), a system in which SCF (50 ng / ml) was continued to be added, and the third experimental group: FLT3 ligand (100 ng / ml), megakaryocyte growth factor (50 ng / ml) / Ml) was carried out in a system cultured with the addition. Each test was performed in three or more independent experiments.

After the culturing, a part of the cells was recovered, labeled with an antibody, and the cell type was measured. That is, the cells are washed with a centrifuge, and the cells are washed with RPMI containing 5% fetal bovine serum.
Suspended in 50 μl of -1640 medium (Gibco), 10 μl of PE-labeled anti-CD34 antibody (Becton), and FIT
2 μl of C-labeled anti-CD61 antibody (Dako) was added, and staining was performed on ice for 30 minutes. After washing with cold saline twice using a centrifuge, the degree of staining of CD34 and CD61 was measured with a FACS caliber (Becton Dickinson).

As a result, CD34 showing megakaryocyte precursor cells
The number of positive CD61-positive cells could hardly be detected before the culture, whereas the number of cells in the first experimental section was 7.4 × 10 ⁴ ,
3.2 × 10 ^{4 in} the second experimental section, 4.2 × 10 in the third experimental section
It was shown that amplification was carried out to ^four . CD61-positive cells, which are megakaryocytic cells, were 12.7 × 1 in the first experimental section.
0 ^{5, five} second experimental group 7.2x10, a third experiment District 9.
The number was 2 × 10 ⁵ . Therefore, it was shown that megakaryocyte precursor cells and megakaryocyte cells were proliferated at high magnification by switching the stimulating factor in the middle.

[0029]

Industrial Applicability The cell preparation of the present invention and the method for producing the same are effective in the treatment of thrombocytopenia associated with chemotherapy or bone marrow transplantation, and various diseases showing bleeding tendency considered to be caused by thrombocytopenia. It is useful for allogeneic hematopoietic stem cell transplantation.

Claims (3)

[Claims] 1. A megakaryocyte precursor cell produced by culturing hematopoietic undifferentiated cells in a medium containing megakaryocyte growth factor and FLT3 ligand in advance, and then culturing the medium in a medium containing hematopoietic factors. A cell preparation comprising: 2. The cell preparation according to claim 1, wherein the hematopoietic undifferentiated cells are derived from cord blood. 3. A method for producing the cell preparation according to claim 1 or 2.