KR101738508B1 - Serum-free conditioned medium using perivascularcells and method for differentiating hematopoietic stem cell from pluripotent stem cell using the same - Google Patents

Abstract

According to the present invention, it is expected to contribute to commercialize cell treatment of malignant diseases such as immune defect disorder, aplastic anemia, hemoglobin disease, myelogenous leukemia and malignant lymphoma by promoting differentiation of hematopoietic stem cells from pluripotent stem cells using a serum-free conditioned medium using perivascular cells and treating with hematopoietic progenitor cells.

Description

TECHNICAL FIELD [0001] The present invention relates to a serum-free condition medium using vascular epithelial cells and a method for promoting differentiation of hematopoietic stem cells from pluripotent stem cells using the same. BACKGROUND ART [0002]

The present invention relates to a serum-free condition medium using vascular epithelial cells and a method for promoting differentiation of hematopoietic stem cells from the pluripotent stem cells using the same. The present invention relates to a method for the treatment of immune deficiency diseases, hemolytic anemia, hemoglobinopathies, hematopoietic diseases, and the like, which can be treated by hematopoietic stem cell transplantation by promoting the differentiation of pluripotent stem cells into hematopoietic progenitor cells by using serum- Leukemia and malignant lymphoma, and the like.

Leukemia is a disease caused by the abnormal proliferation of leukocyte cells. It is characterized by an increase in undifferentiated cells in the peripheral blood, a sharp decrease in normal red blood cells, white blood cells, and platelets, resulting in a decrease in various functions performed by normal cells It is caused by symptoms. Therefore, anemia due to reduction of red blood cells, many infectious symptoms due to reduction of leukocyte cells (infections caused by bacteria that are not routinely problematic, excessive frequent colds, pneumonia, etc.) and hemorrhagic symptoms due to reduction of platelet cells appear . In bone marrow tissues that produce blood, only abnormal cell proliferation occurs, and normal cell proliferation is difficult to see. There are various types of leukemia. Leukocyte cells can be divided into lymphoid cells and myeloid cells. The abnormal cells can be divided into lymph and myeloid, and they can be divided into chronic and acute according to how fast the symptoms of leukemia appear. do.

- Acute Lymphocytic Leukemia: Acute Lymphocytic Leukemia (ALL)

- Acute myelogenous leukemia: Acute Myelogenous Leukemia (AML)

- Chronic lymphocytic leukemia: Chronic lymphocytic leukemia (CLL)

- Chronic myelogenous leukemia: Chronic Myelogenous Leukemia (CML)

The treatment of leukemia is to remove abnormal white blood cells by chemotherapy through chemotherapy and radiotherapy. Through chemotherapy, abnormal white blood cells, myeloid cells and lymphatic cells which may be a problem are completely removed and the symptoms due to abnormal white blood cells are completely eliminated The hematopoietic stem cell transplantation is performed for the purpose of remission therapy. Hematopoietic stem cells are capable of differentiating into hematopoietic cells such as red blood cells, white blood cells, and platelets, and have self-replicating ability, multicellular cleavage ability, and differentiation-ability ability characteristic of stem cells (van der Kooy D., , pp 1439-1441, 2000). In the past, these hematopoietic stem cells were mainly obtained from bone marrow, but recently, hematopoietic stem cells can be obtained from cord blood or peripheral blood. However, even in normal peripheral blood, there is less than 0.1% hematopoietic stem cells. Therefore, in this condition, it is impossible to obtain enough hematopoietic stem cells for transplantation after the chemotherapy. Therefore, only hematopoietic stem cells Is attracting attention.

Currently, the transplantation of hematopoietic stem cells or progenitor cells using cord blood is clinically active, especially when the cancer cells after high-dose chemotherapy are malignant hematopoietic cells, when the cells of the self tissue are not suitable and the adult donor is absent, Progenitor cells are recognized as an important source of transplantation and various diseases are being treated through hematopoietic stem cell transplantation or progenitor cell transplantation. For example, it has been tried as a treatment for cure in mainly hematologic cancer related diseases such as acute / chronic leukemia, aplastic anemia, myelodysplastic syndrome, multiple myeloma and malignant lymphoma. Recently, treatment for breast cancer, ovarian cancer, kidney cancer, small cell lung cancer (Lennard, eral, BMJ, 321, pp 433-437, 200; Ikehare, Experimental Hematoligy, 29, pp661), which have been shown to have good therapeutic results for malignant autoimmune diseases such as solid tumors and refractory systemic lupus erythematosus and refractory rheumatoid arthritis -669, 2001).

However, such hematopoietic stem cells or progenitor cells have only a small amount of blood to be collected from the umbilical cord blood of the umbilical cord, resulting in a disadvantage that only a small number of cells are obtained. Transplantation without amplification of hematopoietic stem cells is usually limited to children. Thus, over the past decade, there has been an increased interest in in vitro amplification of human hematopoietic or progenitor cells in the cord blood as well as in the cord blood (Cairo MS, et al., Blood, 90, pp 4665-4678, 1997; Mayani H , et al., Stem cells, 16, pp153-165, 1998). In addition, recent reports have shown that the umbilical cord blood, which was frozen in storage, had a significantly lower physiological survival rate when thawed by the thawing cells (Shim J S., et al., Br J Haematol, 135, pp 210-213, 2006).

Therefore, it is very useful to develop a technique for obtaining a large number of hematopoietic stem cells necessary for transplantation of hematopoietic stem cells. Therefore, in order to amplify and obtain such a large number of hematopoietic stem cells, various methods and development of optimal culture conditions have been attempted. As a representative culture method known so far, there have been methods of culturing in vitro various recombinant stimulatory cytokines (Metcalf D., et al., Biomed Pharmacother, 55, pp 75-78, 2001; Ian NcNiee, et al., Experimental Hematology, 29, pp3-11, 2001). Some studies have used antibodies against hematopoietic inhibitors and other studies have used other stromal cell lines to culture hematopoietic cells (Yamaguchi M., et al., Exp. Hematol., 29, pp 174- 182, 2001).

The cytokine used in culturing the hematopoietic stem cells has various problems in clinical use and there are concerns about the risk of infection or tumor due to its side effects. For example, the use of IL-2 in the extracted lymphocytes and re-infusion of the patient after mass culture has serious limitations such as serious side effects such as hypotension and capillary leak syndrome. The capillary vasoconstrictor syndrome refers to the accumulation of tissue fluid flowing from the capillaries of tissues (Rosenstein M., et al., J. Immunol., 137, pp 1735-1742, 1986) Studies are underway to improve the differentiation efficiency by supplying specific growth factors or co-culturing with specific cells to induce differentiation into specific cells. Among them, the method of co-culturing is limited in the clinical application due to the problem that the possibility of contamination by the cells used for co-culture can not be excluded and the solution thereof is required.

Therefore, the present inventors have made intensive efforts to discover new factors effective for differentiation into human hematopoietic stem cells from human pluripotent stem cells. As a result, in the case of using serum-free conditioned media using vascular epithelial cells, differentiation of stem cells into hematopoietic stem cells The present invention has been completed.

As used herein, the term " hematopoietic stem cells " refers to ancestral cells of undifferentiated myeloid hematopoietic cells that make red blood cells, white blood cells, platelets. Hematopoietic stem cells (hematopoietic stem cells) are present in about 1% of normal human bone marrow blood cells (CD34-positive cells) capable of producing all blood cells. It is a mother cell that makes blood. It is found in whole body, but it is produced in large quantity especially in bone marrow. From these cells, red blood cells, white blood cells and platelets corresponding to the cells constituting the blood are differentiated and made. In addition, it has a self-replicating ability to produce the same self, and accounts for about 0.05 ~ 0.25% of total bone marrow stem cells. Peripheral blood stem cells are hematopoietic stem cells originating from the bone marrow and circulating in the bloodstream, and have the property of self-reproduction and differentiation into mature cells.

As used herein, the term "differentiation " refers to a phenomenon in which the structure or function of a cell is specialized during cell division and proliferation and growth of the entire individual. In other words, it refers to a process in which cells, tissues, etc. of a living organism are changed into appropriate forms and functions to perform their respective roles. For example, differentiation of ectodermal, mesodermal, and endodermal cells into differentiable stem cells, such as embryonic stem cells, is also differentiation, and the process of transformation of hematopoietic stem cells into red blood cells, white blood cells, platelets, etc. is also differentiated.

As used herein, the term "embryonic stem cell" refers to a cell that has been isolated from the inner cell mass of a blastocyst, an early stage of development, and has pluripotency. it means.

As used herein, the term " pre-differentiating stem cell "refers to a stem having a differentiating ability to differentiate into all three germ layers constituting the living body, that is, endoderm, mesoderm and ectoderm Cells. Traditionally embryonic stem cells fall into this category.

As used herein, the term " dedifferentiated stem cell or induced pluripotent stem cell (iPS) "refers to a cell that has undergone artificial reprogramming (reprogramming) It has pluripotency.

According to one aspect,

A serum-free condition medium using vascular epithelial cells for promoting differentiation from pluripotent stem cells into hematopoietic stem cells is disclosed.

The term "conditioned medium" in the present invention refers to a medium in which a cell is subjected to liquid suspension culture and when it reaches the logarithmic growth phase, which is the highest stage of cell division, the dividing cells are removed by centrifugation or filtration and only the culture medium is collected and mixed with the culture medium Means a medium. This is due to the unknown growth factor extracted from the dividing cells in the medium, which is often used for low density cell plating or protoplast culture.

In the serum-free condition medium using the vascular epithelial cells of the present invention, the pluripotent stem cells may include human embryonic stem cells or inducible pluripotent stem cells (iPS).

In the serum-free condition medium using the vascular epithelial cells of the present invention, the serum-free condition medium may further include a growth factor or a cytokine. For example, the growth factor may include one or more selected from the group consisting of IGF, bFGF, TGF, HGF, EGF, VEGF, and PDGF. The cytokine may include at least one selected from the group consisting of IL-1, IL-4, IL-6, IFN-r, IL-10 and IL-17.

According to another embodiment,

(A) preparing a serum-free condition medium using vascular epithelial cells; And

(B) a step of differentiating the pluripotent stem cells into hematopoietic stem cells in the serum-free medium, and a method for promoting the differentiation of hematopoietic stem cells from the pluripotent stem cells.

In the method of promoting the differentiation of hematopoietic stem cells from the pluripotent stem cells of the present invention, the pluripotent stem cells may include human embryonic stem cells or inducible pluripotent stem cells (iPS).

In the method for promoting the differentiation of hematopoietic stem cells from the pluripotent stem cells of the present invention, the serum-free condition medium may further include growth factors or cytokines. For example, the growth factor may include one or more selected from the group consisting of IGF, bFGF, TGF, HGF, EGF, VEGF, and PDGF. The cytokine may include at least one selected from the group consisting of IL-1, IL-4, IL-6, IFN-r, IL-10 and IL-17.

In the method for promoting the differentiation of hematopoietic stem cells from the pluripotent stem cells of the present invention, the hematopoietic stem cells may be selected from the group differentiating into a common myeloid progenitor or a common lymphoid progenitor . The common myeloid origin cell may be, but is not limited to, differentiating into a group comprising at least one of erythroblast, megakaryoblast, myeloblast, and monoclast . The common lymphoid origin cell may be differentiated into a group including lymphoblasts, but is not limited thereto

The present invention relates to a method for the treatment of immune deficiency diseases, hemolytic anemia, hemoglobinopathies, hematopoietic diseases, and the like, which can be treated by hematopoietic stem cell transplantation by promoting the differentiation of pluripotent stem cells into hematopoietic progenitor cells by using serum- Leukemia and malignant lymphoma, and the like.

Fig. 1 shows the vascular epithelial cell proliferation according to Example 2 of the present invention.
2 shows the phenotype of vascular epithelial cells according to Experimental Example 1-1 of the present invention.
3 shows the differentiation of vascular epithelial cells according to Experimental Example 1-2 of the present invention.
4 shows a culture plan of the test group according to Experimental Example 2-1 of the present invention.
5 shows the proliferating stem cell proliferation according to Experimental Example 2-1 of the present invention.
6 shows the expression of hematopoietic stem cell markers according to Experimental Example 2-2 of the present invention.
7 shows the number of differentiated cells according to Experimental Example 2-3 of the present invention.
8 shows the proportion of differentiated cells according to Experimental Example 2-3 of the present invention.

Hereinafter, various embodiments are provided to facilitate understanding of the present invention. The following examples are provided to facilitate understanding of the invention and are not intended to limit the scope of the invention.

< Example >

Example  1. Isolation of arterial blood vessels from human umbilical cord

All maternal and fetal pregnancies over 37 weeks of gestation were collected by collecting the umbilical cord that was discarded after cesarean section in mothers with no internal / surgical disease or genetic disease. This course was approved by the ethics committee of Kangwon National University and was approved by the mother. The umbilical cord was collected at about 30 cm each and placed in a test tube containing sterile pH 7.2 phosphate buffered saline (PBS, Invitrogen). The umbilical cord was collected in sterile 100 U / mL penicillin (Sigma) and 100 After washing the umbilical cord blood as much as possible by washing three times with sterilized PBS supplemented with ug / mL streptomycin (Sigma), wharton's jelly, the umbilical cord connective tissue, was removed and two umbilical arteries were removed. .

Example  2. Of vascular epithelial cells (PVC)  Isolation and Culture

The umbilical artery blood vessels isolated according to Example 1 were suspended in an α-Minimum Essential Medium (FBS) supplemented with 20% fetal bovine serum (FBS, Invitrogen), 100 U / mL penicillin, 100 μg / mL streptomycin and 2 mM L- α-MEM; Invitrogen), and cultured for 14 days. Perivascular cell (PVC) clusters formed from arterial blood vessels were separated by treatment with 0.125% trypsin-EDTA 1 mM (Sigma). The isolated vascular epithelial cells were washed twice with PBS and then subcultured in the medium at a temperature of 37 ° C and a humidified environment of 5% CO ₂ . If the cells grew with confluence of about 80-90% of the culture dish, the cells were removed by adding 1 mM of 0.125% trypsin-EDTA, washed with PBS, and 2 × 10 ³ cells per cm ^{2 of} the culture dish , And replaced with fresh medium at intervals of 24 hours (Fig. 1).

Example  3. Vascular epithelial cells  Used Serum-free Condition badge  Produce

The vascular epithelial cells grown by subculturing in Example 2 were washed with PBS and stained with serum-free medium Stemline? II &lt; / RTI &gt; Hematopoietic Stem Cell Expansion Medium (Sigma). The incubation temperature was maintained at 37 캜 and cultured in a humidified environment of 5% CO ₂ . If the cells grew with confluence of about 80-90% of the culture dish, the cells were removed by adding 1 mM of 0.125% trypsin-EDTA, and the cells were washed with PBS and 2 x 10 ³ cells per 1 cm ^{2 of} the culture dish Subsequently, the culture medium was replaced with fresh medium at intervals of 24 hours, and the culture medium was recovered. The recovered culture was filtered using a 0.22 ㎛ filter, and the serum-free medium was completed and cryopreserved at -80 ° C.

<Experimental Example>

Experimental Example  One. Vascular epithelial cells Multiparameter  Confirm

1-1. Observation of vascular epithelial cell phenotype by flow cytometry

In order to confirm the phenotype of vascular epithelial cells isolated and cultured according to Examples 1 and 2, the expression pattern of the marker was observed using flow cytometry. Three times subcultured vascular epithelial cells were detached from the culture dish using cell dissociation buffer (Invitrogen), washed with sterile 1% FBS-PBS and suspended at a concentration of 1 x 10 ⁶ cells / mL. Antibodies used to identify phenotypes include anti-human CD31-phycoerythrin (PE) (BD Biosciences), anti-human CD34-fluorescein isothiocyanate (FITC) FITC (BD), anti-human CD146-FITC (BD) and anti-human CD90-APC (BD) ⁶ cell concentration and reacted at 4 ℃ for 1 hour. The stained cells were washed 2 times with 1% FBS-PBS, suspended, and 2 μl of 7-amino-actinomycin (7AAD; Immunotech) was added to separate living and dead cells. The fluorescence quantification and expression patterns of each marker were observed using AccuriC6 (BD) and analyzed using Accuri C6 Analysis software (BD). The results are shown in Fig.

1-2. Identification of adipocytes, osteocytes and cartilage cell differentiation ability

To confirm the ability of the vascular epithelial cells isolated and cultured according to Examples 1 and 2 to differentiate into adipocytes, osteocytes and cartilage cells, cells corresponding to 3 to 5 passages were seeded on a 12-well tissue culture plate containing PVC medium x 10 &lt; ³ &gt; cells / cm &lt; ² &gt; and cultured until the density of the plate became 90%. The differentiation of adipocytes was carried out in commercial StemPro adipogenic media (Gibco) for 14 days. Cells were fixed with 10% formalin at 4 ° C for 30 minutes in 0.2% Oil Red O (Sigma) (Gibco) for 21 days. To determine the degree of differentiation, 10% formalin (10% formalin) was added to the oocytes, And then stained with 2% Alizarin Red S (Sigma) solution at pH 4.2 for 5 min at room temperature to observe calcified nodules around the cells differentiated by microscopy. To differentiate into chondrocytes, commercial StemPro chondrogenic media (Gibco) was used, and stained with Alcian blue (Sigma) solution at room temperature for 5 minutes to observe the degree of differentiation. The results are shown in Fig.

Experimental Example  2. Vascular epithelial cells  Used Serum-free On the conditioned medium  Induction of differentiation by

2-1. Culturing of total differentiating stem cells in serum-free condition medium using vascular epithelial cells

Human pre-differentiating stem cells cultured in 15 to 35 passages were divided into three groups as shown in Fig. 4 and subcultured at 2 x 10 ³ per 1 cm ^{2 of} culture dish to induce differentiation for 18 days. The control group (CON) was prepared by using the serum-free condition medium using the vascular epithelial cells according to the present invention. (D0) to 6 days (d6) in the serum-free conditioned medium according to Experimental Example 3, and after 6 days from the start of culture Stemline? II hematopoietic Stem Cell Expansion Medium (Sigma). In the second experimental group (Group 2), Stemline? II hematopoietic Stem Cell Expansion Medium (Sigma) and cultured on a serum-free conditioned medium according to Experimental Example 3 after 6 days. The pre-differentiating stem cells used were purchased from cell lines registered with the CDC. This course was conducted after the approval of the ethics committee of Kangwon National University and the public IRB. The results are shown in Fig.

2-2. Observation of hematopoietic stem cell phenotype by flow cytometry

Expression patterns of markers were examined using flow cytometry in order to confirm the phenotype of hematopoietic stem cells cultured in Experimental Example 2-1. The hematopoietic stem cells were detached from the culture dish using cell dissociation buffer (Invitrogen), washed with sterile 1% FBS-PBS, and suspended at a concentration of 1 × 10 ⁶ cells / mL. Antibodies used to identify phenotypes include anti-human CD31-phycoerythrin (PE) (BD Biosciences), anti-human CD34-fluorescein isothiocyanate (FITC) FITC (BD), anti-human CD146-FITC (BD) and anti-human CD90-APC (BD) ⁶ cell concentration and reacted at 4 ℃ for 1 hour. The stained cells were washed 2 times with 1% FBS-PBS, suspended, and 2 μl of 7-amino-actinomycin (7AAD; Immunotech) was added to separate living and dead cells. The fluorescence quantification and expression patterns of each marker were observed using AccuriC6 (BD) and analyzed using Accuri C6 Analysis software (BD). The results are shown in Fig.

As can be seen from FIG. 6, the pluripotent stem cells cultured in serum-free conditioned medium (PVC-CM) using vascular epithelial cells showed more hematopoietic stem cell markers than the control cells.

2-3. Observation of differentiation ability by colony forming unit test

After the expression of the markers of hematopoietic stem cells was observed in Experimental Example 2-2, a colony forming unit assay was performed to observe the differentiation into hematopoietic stem cells. Respect to the colony, and 3 groups were (CON, Group1, Group2) of the vessel shell cells NH CFU-F one after temperature inoculated in Petri dishes containing medium (MiltenyBiotec) maintains 37 ℃ and, 5% CO ₂ in a humid environment For 14 days. After incubation, the formation of colonies was confirmed using a microscope, fixed with methanol, and dried. After staining with Sigma-Aldrich, incubation at room temperature for 5 minutes, washing and drying, colonies (20 cells or more) with diameters of 1-8 mm were counted. The results are shown in Fig. 7 and Fig.

As can be seen from FIG. 7, the pluripotent stem cells cultured in serum-free condition medium (PVC-CM) using vascular epithelial cells showed more differentiated cells than in the control group. 8, CFU-G in the granulocyte production step, CFU-M in the monocyte production step, CFU-E in the erythrocyte production step, CFU-G in the generation step, and CFU-GM and CFU- CFU-M, and CFU-E, which correspond to the precursor cell stage, are increased in the progenitor cell stage CFU-GEMM than in the CFU-GMEM stage and the CFU-GEMM stage And it was confirmed that the differentiation of hematopoietic stem cells was induced by the serum-free condition medium according to the present invention.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (10)

delete delete delete delete delete A method for promoting differentiation from pre-differentiating stem cells into hematopoietic stem cells, said method comprising:
(A) preparing a serum-free condition medium using vascular epithelial cells; And
(B) dividing the pluripotent stem cells into hematopoietic cells in the serum-free condition medium,
Wherein said pluripotent stem cells comprise human embryonic stem cells or inducible pluripotent stem cells (iPS).
delete The method according to claim 6,
Wherein the serum-free condition medium further comprises a growth factor or cytokine.
9. The method of claim 8,
Wherein the growth factor comprises at least one selected from the group consisting of IGF, bFGF, TGF, HGF, EGF, VEGF and PDGF.
9. The method of claim 8,
Wherein the cytokine comprises at least one selected from the group consisting of IL-1, IL-4, IL-6, IFN-r, IL-10 and IL-17. Way.

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