KR101169835B1 - Methods for inducing the differentiation of CD34+ hematopoietic stem cells into megakaryocytes and platelets - Google Patents

Abstract

The present invention relates to a method for inducing differentiation of CD34 positive hematopoietic stem cells into megakaryocytes and platelets, and more particularly, co-culturing hematopoietic stem cells having CD34 positive surface markers with OP9 cells, and treating the compound of Formula 1 The present invention relates to a method for inducing differentiation of hematopoietic stem cells into megakaryocytes and platelets.

CD34, hematopoietic stem cells, megakaryocytes, platelets, differentiation

Description

Methods for inducing the differentiation of CD34 + hematopoietic stem cells into megakaryocytes and platelets}

The present invention relates to a method for inducing differentiation of CD34 positive hematopoietic stem cells into megakaryocytes and platelets, and more particularly, co-culturing hematopoietic stem cells having CD34 positive surface markers with OP9 cells, and treating the compound of Formula 1 The present invention relates to a method for inducing differentiation of hematopoietic stem cells into megakaryocytes and platelets.

Platelets are blood cell components that play an important role in the hemostatic apparatus of the living body, the diameter is 2 to 3㎛, and contains about 300,000 to 500,000 pieces in 1mm ² of blood. It is viscous and changes shape depending on conditions, sticks and aggregates to damaged tissues while releasing intracellular components, causing a series of coagulation reactions.

Platelet hematopoiesis refers to a process in which megakaryocyte progenitor cells derived from pluripotent hematopoietic stem cells are differentiated into megakaryocytes and platelets via megakaryocytes. The regulation of megakaryocytes (macrocytic cells) formation and platelet production was studied by Mazur (Exp. Hematol., 15: 248, 1987) and Hoffman (Blood, 74: 1196-1212, 1989). For example, myeloid pluripotent stem cells differentiate into megakaryocytes, red blood cells, and myeloid cell lines. The recognizable early constituent member cells of the megakaryocyte system are giant erythrocyte cells. These cells initially have a basophilic cytoplasm and one nucleus of slightly irregular shape, including loose and slightly reticular chromatin and several phosphorus, with a cell diameter of 20-30 μm. After a period of time, the megakaryocytes contain up to 32 nuclei (polyploids), but the cytoplasm remains sparse and immature. As the maturation progresses, the nucleus becomes more deciduous and enriched, the cytoplasm increases quantitatively, more eosinophilic and granulates. In the most mature cells of this line, you can see the platelet release from the edge of the cell. Typically, fewer than 10% of the megakaryocytes are in stage and more than 50% are mature. Morphological classifications commonly applied to the megakaryocyte system can be divided into early forms of megakaryocytes, intermediate forms of megakaryocytes or basophils, and terminal forms of mature (eosinophilic, granular or platelet producing) megakaryocytes. Mature megakaryocytes emit cytoplasmic filaments into the sinusoidal space where they fall apart and form individual platelets (Williams et al., Hematology, 1972).

Thrombocytopenia is a disease caused by the destruction of megakaryocyte colony-forming progenitor cells, platelet progenitor cells present in bone marrow cells, resulting in a lack of platelets. Currently, hereditary thrombocytopenia, idiopathic thrombocytopenic purpura, aplastic anemia, etc. are known, but clinically important thrombocytopenia is secondary thrombocytopenia caused by a drug that acts as a radiation systemic or hematopoietic inhibitory agent. Such secondary thrombocytopenia is caused by chemotherapy, radiation therapy, bone marrow transplantation therapy, etc., which is performed in cancer patients, and in many cases, the formation of myeloid megakaryocytes occurs and delays the recovery of the patient's prognosis. In some cases, it is a dangerous disease that causes death by bleeding.

The most frequently used treatment for thrombocytopenia is the transfusion of platelets so that platelet counts remain above 20,000 / μl. However, in addition to the problem of lack of a blood donor for platelet transfusion, such a method has side effects such as infection by an infectious agent derived from blood such as AIDS virus or hepatitis virus and induction of immune response due to transfusion of foreign platelets.

Therefore, it can be said that it is important to develop a substance capable of directly promoting platelet hematopoiesis and to directly administer to a patient or to treat pluripotent hematopoietic stem cells to differentiate and grow platelets to secure platelet count.

On the other hand, stem cells are a kind of blast cells that grow into different cells or organs constituting the human body, also called hepatocytes (幹 細胞). These stem cells contain 'adult stem cells (multiple stem cells)' that can be made using human embryos, and 'adult stem cells (multifunctional stem cells)' such as bone marrow cells that constantly make blood cells. Adult stem cells (adult stem cells) are extracted from umbilical cord blood (umbilical cord blood) or mature adult bone marrow and blood, and are primitive cells just before they are differentiated into cells of specific organs such as bone, liver, and blood. These include hematopoietic stem cells, mesenchymal stem cells, and neural stem cells, which are in the spotlight of regenerative medicine. Umbilical cord blood (umbilical cord blood) contains a large amount of hematopoietic stem cells, bone marrow cells found in bone marrow in the bone has a variety of stem cells, including hematopoietic stem cells that can produce blood and lymphocytes, as well as mesenchymal stem cells. By developing materials that can differentiate these stem cells into megakaryocytes and platelets, and using these materials or platelets differentiated through them to treat thrombocytopenia, it can have the advantage of excluding the immune response or external contaminants of the patient. .

Accordingly, the present inventors have found that the compound of formula 1 of the present invention is an effective substance for inducing differentiation of CD34 positive hematopoietic stem cells into megakaryocytes and platelets during the study related to platelet hematopoiesis, and completed the present invention.

The object of the present invention is to (a) co-culture hematopoietic stem cells having a CD34 positive surface marker with OP9 cells; And (b) to provide a method and composition thereof for inducing differentiation of hematopoietic stem cells into megakaryocytes and platelets comprising the step of adding a compound of formula (1).

In one embodiment, the present invention provides a method of co-culturing hematopoietic stem cells having a CD34 positive surface marker with OP9 cells; And (b) inducing the differentiation of CD34 positive hematopoietic stem cells into megakaryocytes and platelets comprising the step of adding a compound of formula (1).

Hereinafter, a step-by-step method of inducing differentiation of CD34 positive hematopoietic stem cells of the present invention into megakaryocytes and platelets will be described.

In the present invention, the step (a) is a step of co-culture of hematopoietic stem cells having a CD34 positive surface marker with OP9 cells to co-culture CD34 + hematopoietic stem cells with OP cells to facilitate differentiation into megakaryocytes and platelets.

As used herein, the term "hematopoietic stem cell" refers to a cell having the ability to differentiate into blood cells such as red blood cells, white blood cells and platelets constituting blood. Hematopoietic stem cells have the capacity to differentiate into a variety of blood cells by specific differentiation-induced stimulation in addition to infinite self-renewal capacity in the undifferentiated state. The hematopoietic stem cells of the present invention are humans, monkeys, pigs, horses, cows, sheep, dogs, cats, mice It may be derived from all animals such as rabbits, rabbits, preferably human, more preferably may be isolated from human bone marrow, peripheral blood, umbilical cord blood and the like.

In addition, the hematopoietic stem cells of the present invention have a CD34 positive surface marker, where 'CD34 surface marker' is a marker for representative primitive hematopoietic progenitor cells, and having a CD34 surface marker means that the cell expresses a CD34 antigen protein. At the same time it means hematopoietic progenitor cells. The CD34 antigen is a glycophosphoprotein with a molecular weight of 116 kDa. The gene is present on chromosome 1 and is expressed in about 1 to 2% of normal bone marrow cells, especially in early hematopoietic progenitor cells. These CD34 positive (same meaning as 'CD34 +' notation) hematopoietic stem cells are usually capable of differentiating into various types of blood cells, such as red blood cells, white blood cells, platelets.

In the present invention, hematopoietic stem cells having a CD34 positive surface marker are preferably used by directly separating CD34 + cells from bone marrow or umbilical cord blood, and commercially available human CD34 + stem cells may be used. The method of separating CD34 + hematopoietic stem cells from bone marrow or umbilical cord blood is a FACS method using a flow cytometer with sorting function, which is a known method used to obtain pluripotent stem cells expressing the surface antigen of interest from stem cell fluid (Int). Immunol., 10 (3): 275, 1998), method using magnetic beads, panning method using antibodies that specifically recognize pluripotent stem cells (J. Immunol., 141 (8): 2797, 1998 ), And the like, but may be separated using, for example, a MACS magnetobead separation system (Miltenyi Biotecl Bergisch-Gladbach, Germany), but is not limited thereto.

In the present invention, 'OP9 cells' are stromal cells, and macrophage-Colony stimulating factor (M-CSF) and CSF-1 (Colony) are required for the embryonic stem cells to form cells of various lineages. Stimulating factor-1) is absent, but refers to cells expressing differentiation factors into stem cell factor (SCF) and hematopoietic cells of IL-6. Cell lines derived from the neo-cranial canal of F2 (C57BL / 6xC3H) -op / op mice are examples of OP9 cells.

The term 'co-culture' in the present invention means that the different types of cells are cultured together in one culture vessel. In coculture with the CD34 positive hematopoietic stem cells and OP9 cells of the present invention, after culturing OP9 cells in a single membrane in a culture medium containing FBS, hematopoietic stem cells having CD34 positive surface markers are co-cultured on OP9 cells. It is preferable. In this case, the OP9 cells are preferably evenly distributed and cultured in a culture dish so that the cultured in a single membrane state, for example, 1 × 10 ³ to 1 × 10 ⁷ cells per well in a 40 mm diameter well, Preferably, 1 × 10 ⁴ to 1 × 10 ⁶ can be dispensed into 6-well culture dishes per well. Incubation time may be incubated for 18 to 36 hours, preferably 20 to 28 hours so that OP9 cells are evenly distributed at the bottom of the culture dish and become a single membrane.

In the present invention, the medium used for culturing CD34 + hematopoietic stem cells may use a culture medium known in the art that can support the growth and survival of cells in vitro, for example, DMEM (Dulbecco's Modified). Eagle's Medium, Minimal Essential Medium (MEM), Basic Medium Eagle (BME), RPMI, F-10, F-12, α Minimal Essential Medium (αMEM), Glass's Minimal Essential Medium (GMEM), Cell culture minimum medium ), Iscove's Modified Dulbecco's Medium and Stemspan ^™ H3000 medium, but are not limited thereto, and media and culture conditions may be selected according to the type of cells.

In a specific embodiment of the present invention, the OP9 bone marrow lipid cell line was adjusted to 1 × 10 ⁵ cells per well and dispensed into a 6-well culture dish, and cultured in a-MEM medium fed with FBS, penicillin / streptomycin and sodium bicarbonate for 24 hours. After that, the cells were uniformly distributed in a single membrane state, and CD34 + hematopoietic stem cells were adjusted to be 3 × 10 ⁵ cells per well and co-cultured onto OP cells in each well.

On the other hand, step (b) in the present invention is a step of inducing the differentiation of CD34 positive hematopoietic stem cells into megakaryocytes and platelets by culturing by adding the compound of formula 1 to the CD34 positive hematopoietic stem cells of step (a).

The compound of formula 1 of the present invention is N-steroyl-O-phosphocholine-D-serine methyl ester, the preparation method thereof is disclosed in detail in Korean Patent No. 10-0398892. In the present invention, the compound of Formula 1 is a concept including not only the compound itself, but also derivatives of the compound within a range capable of imparting differentiation ability of CD34 positive hematopoietic stem cells to megakaryocytes.

The term "differentiation" refers to the process by which undifferentiated cells acquire a specialized fate through additional developmental pathways, and in the present invention, the process by which undifferentiated CD34 positive hematopoietic stem cells are characterized as megakaryocytes with platelet production.

In the present invention, the compound of Formula 1 is treated on CD34 positive hematopoietic stem cells in co-culture with OP9 cells, and the concentration of the compound in the culture medium is within the range where the CD34 positive hematopoietic stem cells do not exhibit cytotoxicity by the compound. It can be added to the extent that can be differentiated, Preferably it is about 10-60 microgram / ml, More preferably, it is 40 microgram / ml, but it is not limited to this.

In addition, in step (b) of the present invention, a known substance effective for differentiating hematopoietic stem cells into megakaryocytes and platelets may be further added. For example, cytokines such as interleukin-3 (IL-3), IL-6 and IL-11, leukemia inhibitors (LIF), erythropoietin (EPO), stem cell factor (SCF), and thrombopoie Tin (TPO), megacult-C, and the like can be added.

On the other hand, cells induced to differentiate from CD34 positive hematopoietic stem cells to megakaryocytes by the method of steps (a) and (b) of the present invention have physiological or immunological characteristics of megakaryocytes, preferably a megakaryocyte-specific labeling factor. Their gene expression may be increased. For example, the expression rate of CD41 or CD61, which is a receptor specifically expressed in megakaryocytes, may be increased.

In addition, cells induced to differentiate into megakaryocytes by the method of the present invention have platelet secretion ability, for example, the degree of ploidy through mitosis may be increased, multinuclearized, and the size of the cells may be increased. In addition, these cells may have a demarcation channel through which platelets are released. The megakaryocytes with this characteristic are then further matured, the nucleus becomes more lobular and enriched, the cytoplasm increases quantitatively, more eosinophilized and granulates, and finally platelets are released from the mature cell edges.

In a specific embodiment of the present invention, CD34 + hematopoietic stem cells were treated with CC100, CC200, IL-3, IL-6, TPO and SCF, which are known as hematopoietic differentiation inducers, to induce differentiation. Cells treated with compounds not only showed morphological characteristics similar to megakaryocytes due to the increased cytoplasmic volume and increased cell size, but also the degree of ploidization through mitosis, which is characteristic of megakaryocytes with platelet secretion ability. As a result, the number of nuclei was increased.

In addition, CD34 + hematopoietic stem cells were treated with a compound of Formula 1 and CC200 and Megacult-C, known as hematopoietic differentiation inducers, alone or in combination to induce differentiation. When the compound of Formula 1 was treated and differentiated to cells, it was confirmed that the expression rate of CD41 or CD61, which is a megakaryocyte specific marker, was high. These results show that the compound of formula 1 effectively induces differentiation into megakaryocytes and platelets by enhancing the megakaryocyte differentiation capacity of CD34 + hematopoietic stem cells.

In still another aspect, the present invention relates to a composition for inducing differentiation of hematopoietic stem cells having a CD34 positive surface marker into megakaryocytes and platelets containing the compound of Formula 1 as an active ingredient.

Since the composition for inducing differentiation into megakaryocytes and platelets of the present invention allows CD34-positive hematopoietic stem cells to have differentiation ability to megakaryocytes and platelets, the composition is directly administered to patients whose platelet formation ability is reduced by chemotherapy or radiation therapy. By improving the platelet forming ability of hematopoietic cells, it can be used as a pharmaceutical composition for the treatment of thrombocytopenia.

In another aspect, the present invention relates to a thrombocytopenia treating agent comprising megakaryocytes and platelets obtained through the differentiation induction method comprising the steps (a) and (b).

Megakaryocytes having excellent platelet production capacity obtained by the method of the present invention and platelets produced therefrom can be used as cell therapeutics for the treatment of thrombocytopenia. The cell therapeutic agent herein is meant to encompass pharmaceutical compositions comprising megakaryocytes and platelets obtained by the method of the present invention, and may be provided in the form of pharmaceutical preparations containing such compositions.

The pharmaceutical compositions of the present invention may contain one or more pharmaceutically acceptable excipients, such as fillers, binders, disintegrants, flow regulators, lubricants, sugars, sweeteners, flavors, preservatives, stabilizers, wetting agents, emulsifiers, solubilizers and buffers. It may further include.

In addition, such pharmaceutical compositions can be used as pharmaceutical preparations, including conventionally acceptable and excipients, wherein their proportions and properties are determined by the pharmaceutical practice of the selected route and standard of administration. Pharmaceutical formulations prepared from the pharmaceutical compositions of the present invention can be administered to a patient in any form or manner that formulates in a bioavailable effective amount. In addition, those skilled in the art of preparing formulations may readily select the appropriate form and mode of administration depending upon the condition of the disease being treated, the stage of the disease, the age, sex and weight of the patient and other related situations.

Diseases associated with platelet reduction in the present invention are caused by leukemia, metastatic cancer, blood and bone marrow diseases (eg, aplitic anemia, primary myelofibrosis, myelodysplasia, etc.), vitamin 12 or folate deficiency, or bone marrow damage. When platelet production is reduced, infectious diseases and drugs (eg, penicillin, cephalosporin), such as sepsis, heart valve surgery, systemic lupus erythematosus (SLE), lymphoma, and infectious mononucleosis And thrombocytopenia, such as when the platelets are destroyed by thiazide, etc., or when the platelet distribution is abnormal due to enlargement of the spleen due to tumor or portal hypertension.

As described above, differentiation of CD34-positive hematopoietic stem cells into megakaryocytes and platelets can be effectively induced by co-culture of OP9 cells and CD34-positive hematopoietic stem cells.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. However, these Examples are only for illustrating the present invention in detail, and the scope of the present invention is not limited by these Examples.

Manufacturing example  1. Preparation of Compound of Formula 1

1-1. D-serine methyl  Synthesis of Ester Hydrochloride

47.7 mmol of D-serine was dissolved in 476 ml of methanol, saturated with hydrochloric acid gas, and reacted at room temperature for 2 hours. The solvent was evaporated and then recrystallized with methanol and ether to give the target compound L-serine methyl ester hydrochloride (yield: 99%, melting point: 163-164 DEG C, [α] ²⁵ D = -4.3 (c 1.8, EtOH)). It was. The structure of the synthesized compound was confirmed by FTIR, ¹ H-NMR and ¹³ C-NMR.

FTIR (KBr, cm-1): 3349 O-H peak, 2943 sp3 C-H peak, 1749 ester carbonyl peak

¹ H NMR (CD3 OD): δ 4.07-4.10 (1H, t, J = 3.9 Hz), 3.38-3.93 (2H, m), 3.79 (3H, s) methoxy carbon protons (s: singlet, d: Doublet, t: triplet, m: multiplet)

¹³ C NMR (CD3OD): δ 52.69, 55.10, 59.67, 168.37 carbonyl peak

1-2. N- Steroyl -D-serine methyl  Synthesis of Ester

Compound (1eq) described in 1-1 above was dissolved in 257 ml of dichloromethane and the temperature was lowered to 0 ° C. N-methyl morpholine (2.1eq), stearic acid (1.1eq), 1-hydroxybenzotriazole (1.1eq), and 1,3-dicyclohexylcarbodiimide (1.1eq) were sequentially added thereto. After the reaction was carried out for 3 hours at room temperature. After the reaction, the byproduct dicyclourea was filtered off under reduced pressure, and the remaining solution was concentrated. Purification by column chromatography (dichloromethane: acetone = 9: 1 to 7: 1) synthesized the target compound N-Styroyl-D-serine methyl ester (yield: 88%, melting point: 82-83 ° C). , [a] ²⁵ D = -15.7 (c 2.0, CHCl 3)). The structure of the synthesized compound was confirmed by FTIR, ¹ H-NMR and ¹³ C-NMR.

FTIR (KBr, cm-1): 3310 O-H peak, 2919 sp3 C-H peak, 1720 ester carbonyl peak, 1650 amide carbonyl peak

¹ H NMR (CDCl3): δ 0.83-0.88 (3H, m) stearic acid terminal carbon proton, 1.23 (28H, s) hydrocarbon proton, 1.60 ~ 1.63 (2H, m) carbonyl-β-carbon proton, 2.21-2.28 (2H, t, J = 7.6 Hz), 2.52 (1H, m) hydroxy peak, 3.78 (3H, s) methoxy carbon protons, 3.93-3.94 (2H, d, J = 3.4 Hz), 4.64-4.70 (1H , m), 6.36-6.39 (1H, d, J = 6.5 Hz) Amide nitrogen proton

¹³ C NMR (CDCl 3): δ 14.1 stearic acid terminal carbon, 22.7 hydrocarbon carbon, 25.5 carbonyl-β-carbon, 29.2, 29.3, 29.5, 29.7, 31.9

Hydrocarbon, 36.5, 52.8 methoxy carbon, 54.6, 63.7, 171.0 carbonyl peak, 173.8 carbonyl peak

1-3. N- Steroyl -O- Phosphocholine -D-serine methyl  Synthesis of Ester

Compound (1eq) described in 1-2 above was dissolved in 260 ml of tetrahydrofuran and the temperature was lowered to -10 ° C. N-diisopropylethylamine (4eq) and ethylene chlorophosphite (3eq) were added thereto, and it was made to react for 1 hour. Bromine (3eq) was added thereto, reacted for 15 minutes, and 86.6 ml of water was added thereto, followed by reaction at room temperature for 1 hour. After the reaction was completed, the separated organic layer was evaporated and recrystallized with dichloromethane and acetone. This was again dissolved in 87.5 ml of chloroform / isopropanol / acetonitrile (3: 5: 5, v / v / v) at 0 ° C., and trimethylamine (3eq) in 40% aqueous solution was added and reacted for 11 hours. Purification by column chromatography (dichloromethane: methanol: water = 3: 1: 0-> 2: 1: 0.1) synthesized the target compound N-Styroyl-O-phosphocholine-D-serine methyl ester (yield). : 12%, [a] ²⁵ D = +8.8 (c 2.0, MeOH). The structure of the synthesized compound was confirmed by ¹ H-NMR and ¹³ C-NMR.

¹ H NMR (CDCl 3): δ 0.90 to 0.93 (3H, m) stearic acid terminal carbon protons, 1.31 (28H, s) hydrocarbon protons, 1.63 to 1.65 (2H, m) carbonyl-β-carbon protons, 2.27 to 2.33 (2H, t, J = 7.2 Hz), 3.25 (9H, s) trimethylamine carbon proton, 3.65-3.67 (2H, m), 3.77 (3H, s) methoxy peak, 4.15-4.19 (1H, m), 4.21-4.28 (3H, m), 4.68 (1H, m);

¹³ C NMR (CDCl 3): δ 13.5 stearic acid terminal carbon, 22.8 hydrocarbon carbon, 25.9 carbonyl-β-carbon, 29.3, 29.5, 29.8, 32.1, 35.7, 51.9 methoxy carbon, 53.7, 59.5, 65.1, 66.4, 170.6 Carbonyl peak, 175.4 carbonyl peak

Example  One. OP  Cells and CD34 Of hematopoietic stem cells Coculture

OP9 bone marrow stromal cells were purchased from the American Type Culture Collection, Rockville, MD. Medium StemSpan ™ H3000 for umbilical cord blood-derived CD34 + hematopoietic stem and CD34 + cell cultures was purchased from STEMCELL Technologies Inc. (Vancouver, Canada).

The OP9 bone marrow lipid cell line was adjusted to 1 × 10 ⁵ per well and dispensed in a 6-well culture dish to a-MEM fed 20% heat-inactivated FBS, 0.5% penicillin / streptomycin and 1.5 g / L sodium bicarbonate. Cells were incubated for 24 hours to bring the cells into a single membrane. Confirm that the OP cells were evenly distributed in a single membrane at the bottom, change the medium to StemSpanTM H3000 for culture of CD34 + hematopoietic stem cells, and adjust the CD34 + hematopoietic stem cells to 3 × 10 ⁵ cells per well to control OP cells in each well. Busy on.

Example  2. Treated Compound of Formula 1 CD34 Morphological changes in hematopoietic stem cells Drainage

To determine whether cord blood-derived CD34 + hematopoietic stem cells differentiate into characteristic forms of megakaryocytes and platelets upon treatment of the compound of Formula 1, compounds of Formula 1 and CC100, CC200, IL-3, IL-6, known as inducers of hematopoietic differentiation, The cells were treated with TPO and SCF to examine the morphological changes and degree of ploidy of CD34 + hematopoietic stem cells.

Each material was treated on CD34 + hematopoietic stem cells co-cultured with OP9 cells through Example 1. Hematopoietic stem cell differentiation inducers were CC100 (rhFlt-3 ligand 100ng / ml, SCF 100ng / ml, IL-3, 20ng / ml, IL-6 20ng / ml), CC200 (rhTPO 50ng / ml, rhSCF 50ng / ml, IL -3 10ng / ml), Megacult-C (TPO 5ng / ml, IL-3 1ng / ml, IL-6 2ng / ml), IL-3 10ng / ml, IL-6 20ng / ml, SCF 50ng / ml, (R) -NALPCE was treated in culture medium at a concentration of 35 ug / ml.

After 8 days, the cells were photographed at 200 × magnification to observe the morphology of the cells (FIG. 1).

In addition, FACS (flow cytometry) analysis was performed to determine the degree of ploidy of cells. The cells were pooled and resuspended in 1 × PBS with 0.1% saponin and 10 mg / mL RNAse A and propidium iodine were added. Thereafter, the cells were incubated for 30 minutes at room temperature. Cells were analyzed by FACS calibur flow cytometer and Cell quest software. Results are expressed as mean ± SD (n = 3). Similar results were obtained from three independent experiments (FIG. 1).

As shown in FIG. 1, the cells treated with the compound of Formula 1 compared to other differentiation inducing substances not only show morphological characteristics similar to megakaryocytes such as increased cytoplasmic volume and increased cell size, but also have platelet secretion ability. This resulted in an increase in the number of nuclei through mitosis, a characteristic characteristic of megakaryocytes.

In addition, the compound of formula 1 was shown to further promote the differentiation of megakaryocytes by other differentiation inducing agents, in particular compared to the treatment of CC200 alone or megacult-C, a differentiation inducing agent using some stimulation alone. Treatment with the compounds resulted in further differentiation into megakaryocytes and platelets.

Example  3. The compound of formula 1 was treated CD34 + Confirmation of differentiation of hematopoietic stem cells into megakaryocytes and platelets

To determine whether cord blood-derived CD34 + hematopoietic stem cells differentiate into megakaryocytes and platelets by treatment with the compound of Formula 1, the compound of Formula 1 and CC200 and Megacult-C, known as hematopoietic differentiation inducers, may be treated alone or in combination. After inducing differentiation, it was confirmed whether the megakaryocyte specific marker CD41 or CD61 was expressed.

Each of the differentiation-inducing substances on CD34 + hematopoietic stem cells co-cultured with OP9 cells through Example 1 was CC200 (rhTPO 50ng / ml, rhSCF 50ng / ml, IL-3 10ng / ml), Megacult-C (TPO 5ng / ml , (R) -NALPCE was treated in culture medium at a concentration of 35 ug / ml.

After 8 days, the culture was centrifuged (1,000 rpm, 5 min), the cell mass was washed three times with PBS buffer, and then 20 μl FITC-bound mouse anti-human was added to 200 μl PBS buffer containing 0.01% BSA. Fluorescein isothiocyanate (FITC) -conjugated mouse anti-human monoclonal CD41 antibody, DAKOCycomation, Denmark monoclonal CD61 antibody, DAKOCycomation, Denmark) were treated and rotated at 37 ° C. for 30 minutes to achieve even fluorescent staining. After staining, the fluorescent stained solution was centrifuged (1,000 rpm, 10 minutes), and then the cell mass was washed three times with PBS buffer added with 0.01% (BSA), and the flow cytometry was performed using the Cellquest program. Staining degree of fluorescence was measured by an analyzer (fluorescence-activated cell sorter, FACS Aria). Results are expressed as mean ± SD (n = 3). Similar results were obtained from three independent experiments (FIG. 2).

As shown in Figure 2, when the differentiation of the compound of formula (1) to cells compared to the CC200 or megacult-C material, the expression rate of the megakaryocyte-specific marker CD41 or CD61 was higher, and the CC200 or megacult-C material When treated with the compound of Formula 1 together compared to the single treatment, the expression rate of CD41 or CD61 was higher. This is a result showing that the compound of formula 1 is a useful substance to increase the differentiation ability of CD34 + hematopoietic stem cells into megakaryocytes and platelets.

The method of inducing the differentiation of CD34 positive hematopoietic stem cells into megakaryocytes and platelets using the compound of formula 1 of the present invention increases the differentiation ability of CD34 positive hematopoietic stem cells into megakaryocytes and platelets, thereby effectively inducing differentiation into megakaryocytes and platelets. The megakaryocytes and platelets differentiated by the method can be prepared in the form of a pharmaceutical preparation and can be effectively used for the treatment of thrombocytopenia.

Figure 1 shows the results of photography to observe the morphological changes of CD34 ⁺ cells and FACS analysis results to determine the degree of diploidization.

Figure 2 shows the results of analyzing the CD41 and CD61 expression level of CD34 + cells co-cultured with OP9 bone marrow stromal cells with a fluorescence-activated cell separator.

Claims (11)

(a) coculture with hematopoietic stem cells having a CD34 positive surface marker with OP9 cells; And (b) inducing the differentiation of CD34 positive hematopoietic stem cells into megakaryocytes and platelets.   [Formula 1]

The method of claim 1, wherein said hematopoietic stem cells are derived from umbilical cord blood, bone marrow or peripheral blood. The method of claim 1, wherein the co-culture in step (a) is characterized in that after culturing the OP9 cells in a single membrane in a culture medium containing FBS, hematopoietic stem cells having a CD34 positive surface marker is dispensed on the OP9 cells Way. The method of claim 1, wherein the CD34 positive hematopoietic stem cells are derived from humans. The method of claim 1 wherein the compound of formula 1 has a concentration of 10 to 60 μg / ml. The method of claim 1, wherein the megakaryocytes differentiated from CD34 positive hematopoietic stem cells have an increased expression rate of CD41 or CD61. The method of claim 1, wherein in step (b), interleukin-3 (IL-3), IL-6, IL-11, leukemia inhibitory factor (LIF), erythropoietin (EPO), stem cell factor (SCF), throm A method of further adding one or more selected from the group consisting of bopoietin (TPO), CC100, CC200 and megacult-C. A composition for inducing differentiation of hematopoietic stem cells having a CD34 positive surface marker into megakaryocytes and platelets containing the compound of Formula 1 as an active ingredient.  [Formula 1]

According to claim 8, wherein the composition is interleukin-3 (IL-3), IL-6, IL-11, leukemia inhibitory factor (LIF), erythropoietin (EPO), stem cell factor (SCF), thrombopoie The composition further comprises at least one selected from the group consisting of tin (TPO), CC100, CC200 and megacult-C (megacult-C). (a) inducing the differentiation of CD34 positive hematopoietic stem cells into megakaryocytes and platelets by the method of claim 1; And (b) preparing a thrombocytopenia treating agent comprising the megakaryocytes or platelets Comprising a method for producing a thrombocytopenia treatment. [Formula 1]

An agent for treating thrombocytopenia comprising the composition of claim 8.

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