JP2006094827A - Method for identifying factor for adjusting platelet differentiation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for identifying a factor for deriving and/or promoting differentiation from megakaryocytes into proplatelets and/or platelets, by which many specimen treatments reflecting biological phenomena are simply possible, to provide a method for identifying a factor for inhibiting and/or suppressing differentiation from megakaryocytes into proplatelets and/or platelets, by which many specimen treatments reflecting biological phenomena are simply possible, and to provide factors identified by the methods and inducing, promoting, inhibiting and/or suppressing differentiations from the megakaryocytes into the platelets. <P>SOLUTION: The methods for identifying factors for deriving, promoting, inhibiting and/or suppressing differentiations from megakaryocytes into proplatelets and/or platelets use a cultured cell strain UT-7/TPO cell having properties as the megakaryocytes. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for identifying a factor that induces and / or promotes the differentiation of megakaryocytes into megakaryocytes and / or platelets. The present invention also relates to a method for identifying a factor that inhibits and / or suppresses the differentiation of megakaryocytes into megakaryocyte precursors and / or platelets. The present invention also relates to factors associated with the differentiation of megakaryocytes into megakaryocyte prosthetics and / or platelets identified using the methods described above.

  Platelets can be differentiated from hematopoietic stem cells to megakaryocyte progenitor cells, megakaryocytes (herein, megakaryocyte progenitor cells and megakaryocytes are collectively referred to as megakaryocyte cells), and further to megakaryocyte prosthetic processes. Go through the stages. The mechanism of megakaryocyte differentiation and / or maturation is gradually being elucidated by recent progress in cell biology and molecular biology. For example, thrombopoietin (TPO), identified in 1994, contributed greatly to elucidating this mechanism. TPO is a soluble protein essential for the production of megakaryocytes that acts on cells in the differentiation stage from megakaryocyte precursor cells to megakaryocytes (see Non-Patent Documents 1 and 2). However, TPO acts on the initial differentiation from megakaryocyte progenitor cells to megakaryocytes, but does not act on the stage of platelet formation from megakaryocytes, and the mechanism by which platelets are formed from megakaryocytes There are still many unknown parts.

  The main role of platelets is to form a thrombus around the wound and stop bleeding. If the platelet count decreases for some reason, the hemostatic mechanism breaks down and tends to bleed. Thrombocytopenia is an example of a disease associated with a decrease in the number of platelets. Causes of thrombocytopenia include suppression of platelet production in the bone marrow and peripheral platelet consumption. For example, a method for treating thrombocytopenia caused by suppression of platelet production in the bone marrow caused by cancer chemotherapy or radiation therapy includes platelet transfusion. However, platelets for blood transfusions prepared from blood supplied from healthy individuals have a short shelf life, and have problems such as infection by bacteria after preparation, virus infection from donors, and rejection after transfusion. Another method for treating thrombocytopenia is a method using TPO. TPO has been confirmed in preclinical studies and clinical studies to have an effect of increasing platelets and an effect of improving thrombocytopenia upon administration of a cancer chemotherapeutic agent, and is considered a candidate for a therapeutic agent for thrombocytopenia. However, since TPO does not show significant activity in the later stages of platelet production (differentiation stage from megakaryocytes to megakaryocyte processes and / or platelets), platelet increase by TPO is accompanied by an increase in the number of megakaryocytes. It does occur, and it is not possible to increase only platelets using TPO.

  On the other hand, if the number of platelets increases too much, a blood clot is easily formed. Thrombocytosis is an example of a disease associated with an increase in platelets. Examples of a method for treating thrombocytosis include treatment for suppressing thrombus formation. Thrombocytosis is a condition in which platelets in the blood are abnormally increased, and there are primary and secondary causes. In the former, myeloproliferative diseases (for example, essential thrombocythemia, chronic myelogenous leukemia, or idiopathic myelofibrosis) are the cause. The latter is caused by infections, malignant tumors and drugs. Therefore, the treatment of the primary disease is important for normal treatment, and antithrombotic treatment is performed as symptomatic treatment.

  For the development of therapeutic agents and / or therapeutic methods for diseases associated with platelets as described above (for example, thrombocytopenia or thrombocytosis), elucidation of the differentiation mechanism from megakaryocyte cells to platelets, in particular, megakaryocytes It is important to elucidate the mechanism of differentiation from nuclei to megakaryocytes and / or platelets. Currently, there are few reports on the mechanism of megakaryocyte-to-platelet differentiation and factors involved in megakaryocyte-to-platelet differentiation, and high density lipoprotein (HDL) and antithrombin III or C1 inhibitor Report that the complex promotes the formation of megakaryocyte processes from primary cultured cells derived from mouse bone marrow (Non-patent Document 3), and human fibronectin in combination with an activator of protein kinase C (human) There is only a report (Non-patent Document 4) that the induction of megakaryocyte structure is promoted for megakaryocyte strain CHRK-288 cells.

  There are very few established identification methods as the cause of few in vivo factors or low molecular weight compounds having such functions that induce, promote, inhibit and / or suppress the differentiation of megakaryocytes into platelets. It is done. Currently, primary culture cells derived from mouse bone marrow are used as one of identification methods. This identification method requires a considerable period of time to acquire animal handling techniques, to prepare high-quality bone marrow cells, to examine the optimal conditions for culture, and to examine differentiation conditions for megakaryocytes for use in evaluation. In addition, a large amount of high-purity megakaryocytes are required for use in the identification method, but the number of cells obtained from primary cultured cells is much smaller than that of cultured cell lines. To secure it, a large number of animals are required. Thus, the above method using primary cultured cells derived from mouse bone marrow is unsuitable for conducting large-scale screening. In recent years, studies on megakaryocyte differentiation and platelet production have been conducted using blood cells derived from human umbilical cord blood. However, it is difficult for a general researcher to obtain human umbilical cord blood on a daily basis and / or continuously, and it is impossible to use it for a long-term screening.

  For large-scale screening, a method using a cultured cell line is convenient. The method using a cultured cell line is easier to obtain and handle than the method using the primary cultured cells. On the other hand, in the method using a cultured cell line, it is necessary to examine whether or not the cell line maintains the original properties of megakaryocytes and / or platelets. There are currently few known cell lines that show the transition from megakaryocytes to platelets, especially megakaryocytes to megakaryocytes. Recently, Jiang et al. Reported a cell line called CHRF-288, which forms a megakaryocyte structure by the simultaneous addition of human fibronectin and PMA (Phorbol 12-myristate 13-acetate) (Non-Patent Document 4). See However, the structural change of this cell line to megakaryocytic process has not been compared with the method using primary cultured cells derived from mouse bone marrow, so how much does it reflect the in vivo platelet production process? It is unknown. Further, the CHRF-288 cell line is like a megakaryocyte progenitor cell, requires an operation for maturation into a megakaryocyte, and the necessity and complexity of setting optimum conditions is a problem. In addition, it has been reported that the production of platelets having an original function can be induced using mouse ES cells (embryonic stem cells) (see Non-Patent Document 5). However, this method requires co-cultivation with a stromal cell line capable of supporting differentiation and is troublesome. Not all ES cells differentiate into megakaryocytes and / or platelets. Therefore, it is not suitable for use in screening.

  As described above, platelet production is currently being studied from various directions, but there is still no method for identifying a platelet differentiation-related factor capable of simple and multi-sample processing reflecting in vivo events.

de Sauvage et al. , Nature, vol. 369, 533-565 (1994) Bartley T. D. , Et al. , Cell, vol. 77, 11171-1124 (1994) Ishida Y. et al. , Thromb. Haemost. , Vol. 85, 349-55. (2001) Jiang F. et al. , Blood, vol. 99, 3579-3584. (2002) Fujimoto T. T.A. , Blood, vol. 102, 4044-4051, (2003)

  Therefore, an object of the present invention is to provide a method for identifying factors involved in differentiation from megakaryocytes to megakaryocyte processes and / or platelets, which is capable of multi-sample processing reflecting biological events and is simple, and To provide a factor involved in the differentiation of megakaryocytes identified by such an identification method into megakaryocyte processes and / or platelets.

  As a result of intensive studies to solve the above problems, the present inventors have determined that the cultured cell line UT-7 / TPO cells having properties as megakaryocytes differentiate from megakaryocytes to megakaryocyte processes, and the differentiation mechanism is as follows. It was found that it reflects in vivo events. Based on this finding, the present inventors constructed a method for identifying factors involved in the differentiation of megakaryocytes into megakaryocyte processes and / or platelets, and completed the present invention.

That is, this invention consists of the following.
[1] A method for identifying a factor that induces and / or promotes differentiation of megakaryocytes into platelets, comprising:
(1) adding a candidate factor to UT-7 / TPO cells, and (2) determining whether the UT-7 / TPO cells have differentiated into platelets,
[2] A factor that is identified by the method according to [1] and that induces and / or promotes differentiation of megakaryocytes into platelets,
[3] A method for identifying a factor that induces and / or promotes differentiation from a megakaryocyte to a megakaryocyte proplet, comprising:
(1) adding a candidate factor to a UT-7 / TPO cell; and (2) determining whether the UT-7 / TPO cell has differentiated into a megakaryocyte projection.
[4] A factor that induces and / or promotes differentiation from a megakaryocyte to a megakaryocyte proplatelet identified by the method according to [3],
[5] A method for identifying a factor that inhibits and / or suppresses differentiation of megakaryocytes into platelets, comprising:
(1) A step of adding fibronectin and a candidate factor into a culture solution of UT-7 / TPO cells,
(2) A method comprising the step of determining whether differentiation from UT-7 / TPO cells into platelets is inhibited and / or suppressed,
[6] A factor identified by the method according to [5], which inhibits and / or suppresses differentiation of megakaryocytes into platelets,
[7] A method for identifying a factor that inhibits and / or suppresses differentiation from a megakaryocyte to a megakaryocyte propellate, comprising:
(1) A step of adding fibronectin and a candidate factor into a culture solution of UT-7 / TPO cells,
(2) A method comprising the step of determining whether differentiation from UT-7 / TPO cells to megakaryocyte prosthetics has been inhibited and / or suppressed,
[8] A factor that is identified by the method according to [7], and that inhibits and / or suppresses differentiation from a megakaryocyte to a megakaryocyte precursor.

  The present invention provides a simple method for identifying factors involved in the differentiation of megakaryocytes into megakaryocyte processes and / or platelets, which allows multi-sample processing reflecting biological events. In addition, the identification method of the present invention makes it possible to provide factors involved in the differentiation of megakaryocytes into platelets.

  In order to solve the above-mentioned problems, the present inventors use a UT-7 / TPO cell, which is a cultured cell line having properties as a megakaryocyte, to identify a factor involved in the differentiation of megakaryocytes into platelets. Built. UT-7 / TPO cells are sub-lines of UT-7 cells isolated from patients with megakaryoblastic leukemia and have TPO-dependent proliferation ability and differentiation ability into megakaryocytes. . Previous detailed biochemical and / or molecular biological studies have revealed that UT-7 / TPO cells maintain megakaryocyte progenitor and megakaryocyte properties (Komatsu N. et al.). , Et al., Blood, vol. 87, 4552-4560, (1996)). However, whether or not the UT-7 / TPO cells differentiate and / or mature from megakaryocytes into platelets has not been investigated.

  The present inventors have found that UT-7 / TPO cells do not undergo spontaneous megakaryocyte formation but show a morphological change from megakaryocyte to megakaryocyte projection in certain culture conditions. . Specifically, UT-7 / TPO cells do not form any spontaneous megakaryocyte processes (ie, they do not change from megakaryocytes to megakaryocyte processes in normal passage and culture procedures). FIG. 4 shows differentiation from megakaryocytes to megakaryocyte processes in culture conditions supplemented with fibronectin.

  Furthermore, the present inventors have found that this morphological change reflects an in vivo event. Specifically, in a method using primary cultured cells derived from mouse bone marrow cells, which is a method established in the identification of factors related to differentiation from megakaryocytes to megakaryocyte processes, it is set in UT-7 / TPO cells. It was found that morphological changes from megakaryocytes to megakaryocyte spores are promoted under conditions similar to those described above (ie, addition of fibronectin). Accordingly, the present inventors have identified a method for identifying a factor related to differentiation from a megakaryocyte to a megakaryocyte vesicle process using UT-7 / TPO cells. Confirmed that the method reflects the stage. Since the method of the present invention using UT-7 / TPO cells does not require time and labor for culturing UT-7 / TPO cells, it is possible to carry out multiple samples conveniently and in a short time. In general, it is known that even when megakaryocytes are induced to differentiate into platelets in vitro, only megakaryocyte processes are differentiated. Since the cells of megakaryocyte vesicle processes are cells immediately before platelets, and differentiation to platelets is sufficiently directed if they can be induced to megakaryocyte processes, the present inventors have made UT-7 / If TPO cells are used, it is considered that not only factors related to differentiation from megakaryocytes to megakaryocyte processes but also factors related to differentiation from megakaryocytes to platelets can be identified.

  In the present specification, the “megakaryocyte progenitor cell” is a cell differentiated from a hematopoietic stem cell through a myeloid progenitor cell and further into a megakaryocyte cell. When this megakaryocyte progenitor cell is further differentiated, it becomes a megakaryocyte.

  In the present specification, the “megakaryocyte” is a further differentiated megakaryocyte progenitor cell, and the amount of DNA in the nucleus is 8 to 32 N due to DNA synthesis (endomitosis) without cytoplasm division. ing. The cytoplasm is wide, pink, fine azurophilic granules are observed, and some large cells exceed 100 μm. As a molecular marker, it is CD41 positive.

  In the present specification, the “megakaryocytes” means a cell immediately before platelets. Meganuclear vesicle process is a cell form characterized by a string-like structure in which mature megakaryocytes change without increasing the number of nuclei, and it is thought that this string-like structure is finely cut into platelets .

In the present specification, “platelet” is a megakaryocyte formed by undergoing a morphological change called megakaryocyte prosthesis and fragmenting the cytoplasm of the string-like structure. Platelet is a small blood cell component having a diameter of 2 to 4 μm without a nucleus that plays an important role in the hemostatic mechanism, and the molecular marker is CD41 positive.
UT-7 / TPO cells are a sub-line of UT-7 cells isolated from patients with megakaryoblastic leukemia. UT-7 / TPO cells can be obtained by allowing TPO to act on UT-7 cells according to a method described in the literature (Komatsu N., et al., Blood, vol. 87, 4552-4560, (1996)). It is. Previous detailed biochemical and / or molecular biological studies have revealed that UT-7 / TPO cells maintain megakaryocyte progenitor and megakaryocyte properties (Komatsu N. et al.). , Et al., Blood, vol. 87, 4552-4560, (1996)). However, it has not been investigated whether or not these UT-7 / TPO cells differentiate or mature from megakaryocytes into platelets.

In the method of the present invention, cells suspended in a serum-free medium are seeded on a culture plate such as a 96-well plate, a test sample is added, and the cells are cultured at 37 ° C. in a CO ₂ incubator. The addition of a candidate factor to test whether it is involved in differentiation from megakaryocytes to platelets may be before cell seeding, at the same time as seeding, or after seeding, Preferably, it is simultaneously with the seeding of cells. The culture plate may be a collagen-coated or non-collagen-coated plate, but a culture-coated plate is preferred. Preferably, after culturing for about 1 to 6 hours, more preferably about 1 to 3 hours, the activity of the candidate factor is evaluated by observing cell shape change.

  Judgment that the cells to be evaluated are megakaryocytes, megakaryocyte processes, or platelets is made based on cell shape change. CD41 and von Willebrand factor, commonly used as megakaryocytes, megakaryocyte processes, or platelet differentiation markers, are expressed in common among the three, and using only differentiation markers, megakaryocytes, megakaryocyte processes, In addition, platelets cannot be identified, and visual observation with a microscope or a camera is essential. The morphology may be observed without staining the cells or may be observed after staining the cells, but it is preferable to observe after staining the cells. Cell morphology can be further clarified by staining based on enzyme activity or immunostaining. Examples of staining based on enzyme activity include cholinesterase staining. Examples of antibodies that can be used for immunostaining include anti-CD41 antibodies and anti-von Willebrand factor antibodies. As described in the following examples, CD41 (integrin aII, GPIIb) and von Willebrand factor were used as membrane protein-specific markers for megakaryocytes, megakaryocytes, or platelets, and the localization of these markers was determined. Analysis was performed by immunostaining. These markers are localized both on the megakaryocyte surface and on the platelet membrane surface. If these markers are also present in the protrusions of megakaryocytes that have undergone morphological changes, this protrusion structure is considered to appear during the process of platelet formation.

  The present inventors have found that UT-7 / TPO cells undergo morphological changes to megakaryocyte spores with good reproducibility by the addition of fibronectin. Examples of fibronectin include mammal-derived fibronectin. Fibronectin derived from human, monkey, mouse, rat, rabbit, cat, dog, cow, or horse is preferred, fibronectin derived from human is more preferred, and fibronectin derived from human serum is even more preferred. The concentration of fibronectin added to the cells is preferably 100 ng / mL to 10 μg / mL, more preferably 250 ng / mL to 8 μg / mL.

  According to the present invention, it is possible to identify factors that induce and / or promote differentiation from megakaryocytes to megakaryocyte processes and / or platelets. Specifically, (1) when a candidate factor is added to a UT-7 / TPO cell, and (2) when the UT-7 / TPO cell to which the candidate factor has been added has undergone a morphologic change to megakaryocyte processes and / or platelets. The candidate factor can be identified as a factor that induces and / or promotes the differentiation of megakaryocytes into megakaryocyte processes and / or platelets.

  In addition, according to the present invention, it is possible to identify factors that induce and / or promote differentiation from megakaryocytes to megakaryocyte processes and / or platelets using fibronectin as a positive control. Specifically, (1) fibronectin is added to one UT-7 / TPO cell, a candidate factor is added to the other UT-7 / TPO cell, and (2) UT-7 / TPO to which the candidate factor is added. When cells change shape to megakaryocyte processes and / or platelets as compared to cells supplemented with fibronectin, the candidate factor induces differentiation from megakaryocytes to megakaryocyte processes and / or platelets and And / or can be identified as a facilitating factor.

  Alternatively, fibronectin can be used to identify factors that inhibit and / or suppress the differentiation of megakaryocytes into megakaryocyte processes and / or platelets. Specifically, (1) a concentration of fibronectin and a candidate factor that induces and / or promotes the differentiation of megakaryocytes into megakaryocyte processes and / or platelets is added to UT-7 / TPO cells, and (2) UT When the -7 / TPO cells remain megakaryocytes, the candidate factor can be identified as a factor that inhibits and / or suppresses the differentiation of megakaryocytes to megakaryocyte processes and / or platelets. The concentration of fibronectin added to the cells is preferably 100 ng / mL to 10 μg / mL, more preferably 250 ng / mL to 8 μg / mL.

  In addition, fibronectin can be used to identify factors that induce and / or promote differentiation from megakaryocytes to megakaryocyte processes and / or platelets in conjunction with fibronectin. Specifically, (1) (A) fibronectin at a concentration that does not induce differentiation from megakaryocytes to megakaryocyte processes and / or platelets but directs differentiation from megakaryocytes to megakaryocyte processes and / or platelets. (B) A candidate factor is added to UT-7 / TPO cells, and (2) when the UT-7 / TPO cells differentiate into platelets and / or megakaryocyte processes, To platelets and / or megakaryocyte processes can be identified as factors that induce and / or promote in conjunction with fibronectin. In the present specification, “the concentration that does not induce differentiation from megakaryocytes to megakaryocyte processes and / or platelets but directs differentiation from megakaryocytes to megakaryocyte processes and / or platelets” means fibronectin at that concentration. Alone does not induce or promote the differentiation of megakaryocytes into megakaryocyte processes and / or platelets, but induces differentiation from megakaryocytes to megakaryocyte processes and / or platelets in the presence of co-acting factors And / or the concentration of fibronectin that promotes. The concentration of fibronectin that does not induce differentiation of megakaryocytes into megakaryocyte processes and / or platelets but directs differentiation from megakaryocytes to megakaryocyte processes and / or platelets is preferably 10 ng / mL to 125 ng / mL More preferably, it is the range of 10 ng / mL-62.5 ng / mL.

  Hereinafter, the present invention will be described in more detail and specifically with reference to Examples, but the present invention is not limited to the following Examples.

(Example 1. Induction of differentiation into platelets using UT-7 / TPO cells)
(1.1. Induction of morphological change using UT-7 / TPO cells)
Subculture medium (Iscove's Modified Dulbecco's Medium (IMDM) (manufactured by LIFE TECHNOLOGIES)) + 10% fetal bovine serum (FBS) (manufactured by MOREGATE) + 6 ng / mL TPO (PEPRO TECT) in a 5% CO ₂ incubator A suspension of UT-7 / TPO cells (10 mL) cultured in a 25 cm ² flask using a 25 cm ² flask was placed in a 15 mL centrifuge tube and allowed to spontaneously settle for about 2 hours. After removing 7 mL of the supernatant, 7 mL of the subculture medium was newly added, suspended, and the number of cells was measured. About 2 mL of the cell suspension was centrifuged at 1,000 rpm for 5 minutes at room temperature. Dullbecco including morphological changes induced medium to be 2.0 × 10 ⁵ cells / mL supernatant discarded (300 [mu] g / mL transferrin (LIFE TECHNOLOGIES Co.)'s Modified Eagle's Medium (DMEM ) (SIGMA Co. )) To suspend the cells. A 16 μg / mL fibronectin (manufactured by CHEMICON) solution was prepared in a morphological change induction medium.

A 2-fold serial dilution of fibronectin solution was prepared in the wells of Collagen Coated Microplate 96-well (Asahi Techno Glass). The liquid volume was 20 μL / well, and 11 stages of dilution were performed with the maximum concentration of 16 μg / mL. 20 μL of the cell suspension prepared above was added to each well and mixed, followed by incubation at 37 ° C. for about 1 hour in a 5% CO ₂ incubator. The volume of each well was 40 μL, and the final concentration of fibronectin was 7.8 ng / mL to 8 μg / mL. In addition, a control in which a 20 μL / well morphological change induction medium and a cell suspension were mixed was also prepared. The rate of morphological change was evaluated by microscopic observation.

  The observed morphological changes had several characteristics (FIG. 1). Protrusions appeared relatively frequently from large cells that were thought to have matured, but morphological changes were induced even in small cells. The protrusion structure appeared in a form extending almost linearly from the cell, and in many cases, 4 to 5 appeared from one cell (the cell indicated by the arrow in FIG. 1). Usually, only a straight protrusion structure was observed, but sometimes a node-like circular structure was observed near the middle of the protrusion. No branching from a single protrusion structure was observed. Morphological changes are characterized by the appearance of protrusions and usually do not undergo major changes in the cells themselves, but in particular in the case of small cells, the cell side also rarely changes, showing a spindle-like morphology as a whole. In some cases (FIG. 1, cells indicated by the upper arrow).

  Induction conditions, where cells having two or more protrusions formed are present on average at 5 or more per microscopic examination (100 times magnification), + is 1 or more and less than 5 and ± is less than 1 Was examined. As a result, it was found that morphological changes were induced at a fibronectin concentration of 250 ng / mL or more on a collagen-coated plate, and that the highest efficiency was obtained at a fibronectin concentration of 4-8 μg / mL (Table 1). The maximum number of cells that changed their shape was about 5%. An example of cells whose morphology has been changed by stimulation with 8 μg / mL fibronectin on a collagen-coated plate is shown in FIG.

(1.2. Immunostaining of anti-CD41 antibody of UT-7 / TPO cells in which morphological change was induced)
A suspension of UT-7 / TPO cells (10 mL) cultured in a 25 cm ² flask using a subculture medium (IMDM + 10% FBS + 6 ng / mL TPO) in a 5% CO ₂ incubator was placed in a 15 mL centrifuge tube. Allowed to settle for hours. After removing 7 mL of the supernatant, 7 mL of subculture medium (IMDM + 10% FBS + 6 ng / mL TPO) was newly added, suspended, and the number of cells was measured. About 2 mL of the cell suspension was centrifuged at 1,000 rpm for 5 minutes at room temperature. The supernatant was discarded and prepared in a morphological change induction medium (DMEM containing 300 μg / mL transferrin) so as to be 3.8 × 10 ⁵ cells / mL. A 16 μg / mL fibronectin solution was prepared in a morphological change induction medium. The prepared cell suspension (40 μL) was mixed with the prepared fibronectin solution (40 μL). Collagen I Cellware 8-well CultureSlide (manufactured by Becton Dickinson) was prepared, and the above mixture was spotted on 4 wells. Thereafter, the cells were kept at 37 ° C. for about 1 hour in a 5% CO ₂ incubator to induce morphological changes.

  100 μL of a 10% formalin solution was dropped onto the cell solution spot on the CultureSlide containing the cells subjected to the morphological change induction treatment. The cells were fixed by leaving at room temperature for 10 minutes. After fixing, the cells were washed three times with Phosphate Buffered Salts (PBS) (manufactured by TAKARA BIO INC) (0.1 mL). Cells were blocked for about 1 hour at room temperature with PBS (100 μL) containing 0.75% bovine serum albumin (BSA) (LIFE TECHNOLOGIES). After blocking, the cells were washed 3 times with PBS (0.1 mL), and a primary antibody (anti-CD41 antibody (manufactured by Funakoshi) or negative control antibody (normal mouse IgG: manufactured by Santa Cruz)) was added. The anti-CD41 antibody was added at 4 drops / well, and the negative control antibody was diluted 250-fold and added at 50 μL / well. It was left at room temperature for about 1 to 1.5 hours. The cells were washed three times with PBS (0.1 mL), and a secondary antibody (FITC-goat anti-mouse IgG: manufactured by Santa Cruz) diluted 100 times was added at 50 μL / well. This CultureSlide was shielded from light with aluminum foil and left at room temperature for about 1 to 1.5 hours. After washing 3 times with PBS (0.1 mL), the CultureSlide chamber was removed. 1 drop / well of VECTASHIELD (manufactured by VECTOR) was added. A cover glass was attached and sealed with a top coat, and then examined with a fluorescence microscope. After completion of the microscopic examination, the prepared slide was shielded from light with aluminum foil and stored refrigerated. After preparation of the preparation, it was microscopically examined with a confocal laser microscope within one week.

  When the prepared preparation was examined with a fluorescence microscope, only the cells to which the anti-CD41 antibody was added were stained, and the cells to which the negative control antibody was added were not stained. Further, when microscopically examined with a confocal laser microscope, the peripheral edge and the protrusion of the cell were markedly stained in the cells to which the anti-CD41 antibody was added, and the staining was not confirmed in the cells to which the negative control antibody was added. (FIG. 2).

(1.3. Immunostaining of UT-7 / TPO cells induced morphological changes using anti-von Willebrand factor antibody)
A suspension of UT-7 / TPO cells (10 mL) cultured in a 25 cm ² flask using a subculture medium (IMDM + 10% FBS + 6 ng / mL TPO) in a 5% CO ₂ incubator was placed in a 15 mL centrifuge tube, and about It was allowed to settle for 2 hours. After removing 7 mL of the supernatant, 7 mL of a subculture medium (IMDM + 10% FBS + 6 ng / mL TPO) was newly added, suspended, and the number of cells was measured. About 2 mL of the cell suspension was centrifuged at 1,000 rpm for 5 minutes at room temperature. The supernatant was discarded and prepared in a morphological change induction medium (DMEM containing 300 mg / mL transferrin) so as to be 3.8 × 10 ⁵ cells / mL. A 16 μg / mL fibronectin solution was prepared in a morphological change induction medium. The prepared cell suspension (40 μL) was mixed with the prepared fibronectin solution (40 μL). Collagen I Cellware 8-well CultureSlide (manufactured by Becton Dickinson) was prepared, and the above mixture was spotted on 4 wells. Thereafter, the cells were kept at 37 ° C. for about 1 hour in a 5% CO ₂ incubator to induce morphological changes.

  100 μL of a 10% formalin solution was dropped onto the cell solution spot on the CultureSlide containing the cells subjected to the morphological change induction treatment. The cells were fixed by leaving at room temperature for 10 minutes. After fixation, the plate was washed 3 times with PBS (0.1 mL). Blocking was performed with PBS containing 0.75% BSA (100 μL) at room temperature for about 1 hour. After blocking, the cells were washed 3 times with PBS (0.1 mL) and added with a primary antibody {anti-von Willebrand factor antibody (Santa Cruz) or negative control antibody (Normal goat IgG: Santa Cruz)} did. Anti-von Willebrand factor antibody was diluted 100-fold with 50 μL / well, and negative control antibody was diluted 250-fold with 50 μL / well. It was left at room temperature for about 1 to 1.5 hours. The cells were washed three times with PBS (0.1 mL), and a secondary antibody (FITC-donkey anti-goat IgG: manufactured by Santa Cruz) diluted 100 times was added at 50 μL / well. This CultureSlide was shielded from light with aluminum foil and left at room temperature for about 1 to 1.5 hours. The cells were washed 3 times with PBS (0.1 mL) and the CultureSlide chamber was removed. 1 drop / well of VECTASHIELD (manufactured by VECTOR) was added. A cover glass was attached and sealed with a top coat, and then examined with a fluorescence microscope. After completion of the microscopic examination, the prepared slide was shielded from light with aluminum foil and stored refrigerated. After preparation of the preparation, it was microscopically examined with a confocal laser microscope within one week.

  When the prepared preparation was examined with a fluorescence microscope, only the cells to which the anti-von Willebrand factor antibody was added were stained, and the cells to which the negative control antibody was added were not stained. Furthermore, when examined with a confocal laser microscope, the cells added with the anti-von Willebrand factor antibody were markedly stained at the periphery and protrusions, and the cells added with the negative control antibody were not stained ( FIG. 3).

  An experiment in which the immunostained sample was observed with a confocal laser microscope revealed that the anti-CD41 antibody and the anti-von Willebrand factor antibody were specifically bound to the cell surface and the protrusion (FIGS. 2 and 3). This indicates that CD41 and von Willebrand factor, which are platelet marker proteins, are expressed on the cell surface and protrusions of UT-7 / TPO cells whose differentiation was induced by fibronectin. On the other hand, almost no signal was detected with the control antibody. In addition, it has been reported that von Willebrand factor does not exist in the filamentous pseudopods that occur when normal megakaryocytes adhere to polylysine plates. It is unlikely. Therefore, the results of this example suggest that the morphological change (projection formation) of UT-7 / TPO cells is due to differentiation from human megakaryocytes to platelet formation.

(Example 2. Induction of differentiation into platelets using mouse bone marrow derived megakaryocyte cells)
In order to examine whether or not the method using UT-7 / TPO cells of Example 1 is appropriate, mouse bone marrow-derived megakaryocytes established in studies of differentiation and / or induction from megakaryocytes to platelets were used. The same experiment as in Example 1 was conducted and comparatively examined.

(2.1. Formation of megakaryocyte prosthesis using mouse bone marrow derived megakaryocyte cells)
First, the following operations were performed to collect and culture mouse bone marrow. For each mouse bone marrow collection, 10 ddy mice (Japan SLC, 5 weeks old or older) were prepared, and their body weights were measured. For each ddy mouse, two femurs were extracted and wet-stored in PBS (manufactured by TAKARA BIO INC) (20 mL petri dish (Asahi Techno Glass)) After collecting all 10 mouse femurs At this time, the petri dish containing 20 mL of PBS (-) was replaced with a new one as appropriate, and items other than the femur such as meat pieces and body hair were carried over to the next stage. Subsequently, a piece of meat attached to the femur was further trimmed using a sterilized Kimwipe etc. After cutting both ends of the femur with an animal dissection scissors with a diamond blade, HBSS (-) (Hanks' Balanced Salt Solution, manufactured by LIFE TECHNOLOGIES) (in a 20 mL petri dish, 25 Using a 1 mL syringe with 1 needle (both from Terumo), bone marrow was collected by suspending with HBSS (−), and 20 mL of the cell suspension was transferred to a 50 mL centrifuge tube after bone marrow collection. Was further washed with 5 mL of HBSS (−), and the cell suspension was transferred to the previous 50 mL centrifuge tube, and about 25 mL of the cell suspension in the 50 mL centrifuge tube was suspended lightly and uniformly. After 10 minutes, the supernatant in the 50 mL centrifuge tube was collected and the cells were counted, and after counting the cells, the supernatant was centrifuged at 1,000 rpm at room temperature for 5 minutes. After completion, the precipitate was suspended in 20 mL of megakaryocyte growth medium (IMDM + 10% FBS + 10 ng / mL TPO), and cultured in a 75 cm ² flask for floating cells (manufactured by Sumitomo Bakelite). 8) were prepared, and 2.5 mL of the previous cell suspension and 17.5 mL of megakaryocyte growth medium were mixed per 75 cm ² flask, and maintained at 37 ° C. in a 5% CO ₂ incubator. For 5 days.

Next, in order to selectively culture mouse bone marrow-derived megakaryocytes, the following operation was performed. As described above, 8 mouse bone marrow cells (20 mL) cultured in a 75 cm ² flask in a 5% CO ₂ incubator for 5 days were prepared, and the bone marrow cell culture solution was transferred to 4 50 mL centrifuge tubes (approximately 40 mL x 4). And it centrifuged at 1,000 rpm at room temperature for 5 minutes. After completion of the centrifugation, the precipitate was suspended in 40 mL of megakaryocyte induction medium (IMDM + 10 ng / mL TPO + 10 μg / mL insulin (Wako Pure Chemical Industries) +5 μg / mL transferrin + 0.1% BSA) (10 mL per centrifuge tube). ). For the culture, 25 cm ² flasks (manufactured by Sumitomo Bakelite Co., Ltd.) for floating cells were used, and eight were prepared. Then, 5 mL of the previous cell suspension was dispensed per 25 cm ² flask and cultured at 37 ° C. for 2 days in a 5% CO ₂ incubator.

Subsequently, the following operation was performed in order to prepare mouse bone marrow-derived megakaryocytes. As described above, 8 mouse bone marrow cells (5 mL) cultured in a 25 cm ² flask in a 5% CO ₂ incubator for 2 days were prepared, and the bone marrow cell culture solution was transferred to 4 15 mL centrifuge tubes (about 10 mL). X4) The cells were allowed to stand for 1 hour to allow the cells to settle naturally. After spontaneous sedimentation for 1 hour, the lower 2.0 mL of 15 mL centrifuge tube was collected (2.0 mL × 4 tubes). Two new 15mL centrifuge tubes are prepared, each one from the bottom 16% BSA / HBSS (-) 1.5mL, 4% BSA / HBSS (-) 3.0mL, 2% BSA / HBSS (-) 1.5 mL, and 4.0 mL of the sample that had spontaneously settled at the top, were layered gently, allowed to stand for 1 hour, and subjected to BSA / HBSS (−) density gradient sedimentation. After performing the BSA / HBSS (−) density gradient sedimentation method for 1 hour as described above, 2.0 mL of the lower part was collected from each 15 mL centrifuge tube. Subsequently, the cell solutions in the two centrifuge tubes were combined into one (2.0 mL × 2 to 4.0 mL). Further, 6.0 mL of IMDM was added, the suspension was gently suspended, and centrifuged at 1,000 rpm for 5 minutes at room temperature to collect selectively cultured mouse bone marrow-derived megakaryocytes. After centrifuging, the precipitate (mouse bone marrow-derived megakaryocyte) was treated with megakaryocyte projection medium (IMDM + 10 μg / mL insulin + 5 μg / mL transferrin + 10 ng / mL IL-6 (upstate) +20 μM zinc sulfate (Wako Pure Chemical Industries)) (+ 0.1% BSA) was suspended in 1.0 mL, and the cells were counted. After counting the cells, the cells were diluted with a megakaryocyte formation medium to a cell solution concentration according to the purpose of the experiment. The mouse bone marrow-derived megakaryocytes recovered in this manner were prepared at a cell solution concentration according to the purpose of the experiment.

Using the mouse bone marrow-derived megakaryocytes prepared as described above, the following differentiation into megakaryocyte blastoid cells was examined. The megakaryocytes prepared as described above were seeded in Collagen Coated Microplate 96-well (Asahi Techno Glass Co., Ltd.) at 700 cells / well. The liquid volume was 75 μL / well. A fibronectin solution was prepared using a megakaryocyte projection formation medium, and a 7-fold 2-fold serial dilution was prepared up to 8 μg / mL with 512 μg / mL as the maximum concentration. To each well seeded with the above cells, 25 μL of the fibronectin solution prepared above was added and mixed, and then culture was started at 37 ° C. in a 5% CO ₂ incubator. The volume of each well was 100 μL and the final concentration of fibronectin was 2 μg / mL to 128 μg / mL. As a control, a sample prepared by adding only the megakaryocyte formation medium and mixing them was also prepared.

  After culturing for one day, it was microscopically examined with an inverted microscope Leica DMIL, and the number of megakaryocyte projections per well was counted. FIG. 4 shows the morphology of the megakaryocyte sprout cells induced. FIG. 5 shows the relationship between the concentration of fibronectin and the number of megakaryocyte-like cells induced.

  When mouse bone marrow derived megakaryocytes were used, morphological changes to megakaryocyte spores were observed by fibronectin (FIGS. 4 and 5).

  In this example, the morphological change of UT-7 / TPO cells added with fibronectin (Example 1) was similar to the morphological change of mouse bone marrow-derived megakaryocytes added with fibronectin (Example 2). Has been demonstrated. As described above, mouse bone marrow-derived megakaryocyte cells are frequently used in studies on differentiation induction from megakaryocytes to platelets. Since the differentiation form similar to that using a reliable mouse bone marrow-derived megakaryocyte cell is shown, the method using a UT-7 / TPO cell can be said to be a method reflecting the differentiation process from megakaryocytes to platelets. Furthermore, since UT-7 / TPO cells do not cause spontaneous megakaryocyte formation, they are easy to use in experiments, and are suitable for identifying factors that induce, promote, inhibit or suppress differentiation from megakaryocytes to platelets. It can be said that there is.

FIG. 1 shows the prominent morphological changes observed in UT-7 / TPO cells upon fibronectin stimulation. A. No fibronectin added. B. Cell morphology upon addition of 8 μg / mL fibronectin. Arrows indicate cells that have undergone typical morphological changes. FIG. 2 shows the localization of CD41 in UT-7 / TPO cells whose morphology has been changed by fibronectin stimulation. FITC-goat anti-mouse IgG was used as the secondary antibody, and was examined with a confocal laser microscope. A: Anti-CD41 antibody was used as the primary antibody. B: As a primary antibody, a negative control antibody (normal mouse IgG) was used. FIG. 3 shows the localization of von Willebrand factor in UT-7 / TPO cells morphologically changed by fibronectin stimulation. FITC-donkey anti-goat IgG was used as the secondary antibody, and microscopically examined with a confocal laser microscope. A: An anti-von Willebrand factor antibody was used as the primary antibody. B: As a primary antibody, a negative control antibody (normal goat IgG) was used. FIG. 4 shows the morphology of megakaryocyte spores induced by fibronectin from mouse bone marrow cell-derived megakaryocytes. The morphology of megakaryocytes cultured overnight in fibronectin-containing medium was observed with a microscope. A string-like structure characteristic of megakaryocyte projections was observed. 1-3. Cells that have undergone megakaryocytic process changes due to the addition of fibronectin. 1-3 are cells under the same conditions. 4). Control cells (megakaryocytes) cultured in the absence of fibronectin. FIG. 5 shows the effect of fibronectin on the differentiation of mouse bone marrow cell-derived megakaryocytes into megakaryocyte progenitor cells. Megakaryocytes matured from bone marrow cells derived from ddy mice (700 cells / well / 96-well plate) were cultured overnight in a medium containing various concentrations of fibronectin using a 96-well plate, followed by megakaryocytes. The number of cells that caused a change in protrusion morphology was measured. Experiments were performed at each concentration in triplicate and average values were shown. Error bars indicate standard deviation. The vertical axis represents the number of megakaryocyte spores per well, and the horizontal axis represents the fibronectin concentration (μg / mL).

Claims (8)

A method for identifying a factor that induces and / or promotes differentiation of megakaryocytes into platelets, comprising:
(1) adding a candidate factor to UT-7 / TPO cells, and (2) determining whether the UT-7 / TPO cells have differentiated into platelets. A factor that induces and / or promotes differentiation of megakaryocytes into platelets, identified by the method of claim 1. A method for identifying a factor that induces and / or promotes differentiation of a megakaryocyte into a megakaryocyte proplet, comprising:
(1) adding a candidate factor to UT-7 / TPO cells, and (2) determining whether the UT-7 / TPO cells have differentiated into megakaryocyte projections. A factor that induces and / or promotes differentiation from a megakaryocyte to a megakaryocyte protelet identified by the method of claim 3. A method for identifying a factor that inhibits and / or suppresses differentiation of megakaryocytes into platelets, comprising:
(1) A step of adding fibronectin and a candidate factor into a culture solution of UT-7 / TPO cells,
(2) A method comprising a step of determining whether differentiation from UT-7 / TPO cells into platelets is inhibited and / or suppressed. A factor identified by the method according to claim 5, which inhibits and / or suppresses differentiation of megakaryocytes into platelets. A method for identifying a factor that inhibits and / or suppresses differentiation of a megakaryocyte to a megakaryocyte proplet, comprising:
(1) A step of adding fibronectin and a candidate factor into a culture solution of UT-7 / TPO cells,
(2) A method comprising the step of determining whether differentiation from UT-7 / TPO cells to megakaryocyte prosthetics is inhibited and / or suppressed. A factor that is identified by the method according to claim 7 and that inhibits and / or suppresses the differentiation of megakaryocytes into megakaryocyte prosthetics.

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