WO2003047607A1

WO2003047607A1 - Medicinal compositions containing human amnion-origin cells

Info

Publication number: WO2003047607A1
Application number: PCT/JP2002/012761
Authority: WO
Inventors: Toshio Nikaido
Original assignee: Sankyo Company, Limited
Priority date: 2001-12-06
Filing date: 2002-12-05
Publication date: 2003-06-12
Also published as: AU2002354101A1; TW200301132A

Abstract

It is intended to provide medicinal compositions containing, as the active ingredient, epithelial cells or mesenchymal cells originating in human amnion which are usable as a substitute for human fetal stem cells because of having regeneration ability and can be relatively easily obtained by using usual surgical techniques from maternal humans giving informed consent. More specifically, medicinal compositions such as remedies for diabetes which contain the above epithelial cells as the active ingredient, or medicinal compositions such as remedies for bone metabolic errors or heart diseases which contain human amnion-origin mesenchymal cells as the active ingredient. Thus, it becomes possible to provide medicinal compositions such as remedies for diabetes by using regeneration therapy without causing any ethical problems.

Description

Specification

Human amniotic membrane-derived cell-containing pharmaceutical composition

[Technical field]

The present invention relates to a therapeutic agent for diabetes comprising human amniotic membrane-derived epithelial cells as an active ingredient and a method for treating diabetes comprising administering human amniotic membrane-derived epithelial cells to a diabetic patient. The present invention relates to a method for treating bone metabolism disorders, which comprises administering a therapeutic agent for bone metabolism disorder as an active ingredient and human amniotic membrane-derived mesenchymal cells to a patient with bone metabolism disorders. The present invention relates to a method for treating a heart disease, which comprises administering a therapeutic agent for a heart disease and a human amniotic membrane-derived mesenchymal cell to a patient with a heart disease.

[Background technology]

In recent years, the technology of regenerative medicine has come into the spotlight. In other words, the body of humans or other organisms loses part of their tissues or suffers severe damage due to trauma or disease during their lives. It varies from organization to organization. In order to regenerate organs and tissues that cannot be naturally regenerated and restore their functions, techniques have been developed to differentiate cells with biological functions from undifferentiated cells. Such a technique is generally called regenerative medicine. However, hitherto, stem cells derived from a human fetus have generally been used as cells subjected to such a technique, which has been regarded as a problem from an ethical aspect.

[Disclosure of the Invention]

In order to overcome such a problem, the present inventors have been searching for cells that can be substituted for stem cells derived from human fetuses.However, epithelial cells or mesenchymal cells derived from human amniotic membrane were administered to an organism under certain conditions. Then, they found that the pathology of living organisms could be improved, and completed the present invention. First, the present invention relates to a pharmaceutical composition containing human amniotic membrane-derived epithelial cells as an active ingredient. Second, the present invention relates to a therapeutic agent for diabetes containing human amniotic membrane-derived epithelial cells as an active ingredient. Third, the present invention relates to a method for treating diabetes mellitus, which comprises administering human amniotic membrane-derived epithelial cells to a diabetic patient. Fourth, the present invention relates to a pharmaceutical composition containing human amniotic membrane-derived mesenchymal cells as an active ingredient. Fifthly, the present invention relates to a therapeutic agent for bone metabolism disorder containing human amniotic membrane-derived mesenchymal cells as an active ingredient. Sixth, the present invention relates to a method for treating bone metabolism disorder, which comprises administering human amniotic membrane-derived mesenchymal cells to a patient with bone metabolism disorder. Seventh, the present invention relates to a therapeutic agent for heart disease containing human amniotic membrane-derived mesenchymal cells as an active ingredient. Eighth, the present invention relates to a method for treating heart disease, which comprises administering human amniotic membrane-derived mesenchymal cells to a heart disease patient. In the present invention, human amniotic cells can be obtained by cesarean section of a human pregnant woman who has obtained informal consent and by a usual surgical technique. Epithelial cells can be obtained from amniotic membrane by the following method. The obtained amniotic membrane is fragmented using sterilized scissors or the like, and is placed in a culture medium such as Dulbecco's modified Figur medium used for culturing normal animal cells, and shaken. In this case, to disperse the amniotic membrane fragments into individual cells, a protease such as trypsin is used. It is desirable that elements coexist. Suitable concentrations of proteolytic enzymes are between 0.1% and 0.5%. The temperature for shaking is preferably between 25 and 40 ° C. The shaking interval is preferably from 50 rpm to 200 rpm, and the shaking time is preferably from 20 minutes to 60 minutes. After collecting the cells by centrifugation and removing the supernatant, the cells are shaken again in the culture medium containing the protease under the same conditions as described above. However, the shaking interval is preferably from 400 rpm to 600 rpm, and the shaking time is preferably from 20 minutes to 60 minutes. Thereafter, the culture solution is passed through a mesh such as sterile gauze, and the epithelial cells are separated. Epithelial cells were collected by centrifugation, in a culture medium containing 10% serum, C0 ₂ incubator base - cultured in the evening. Culture temperature is 370C, C0 ₂ concentration is preferably 5%. The serum is preferably calf serum or fetal calf serum. Further, it is preferable that an antibiotic having a concentration of about 1% (for example, penicillin G, 100 units Zml: streptomycin, 100 g Zml: amphotericin B, 0.25 g / ml) is co-present in the culture solution. The amniotic membrane remaining on the gauze can be re-fragmented and further dispersed in cells in the same manner as described above. Epithelial cells obtained are collected by centrifugation, as above, in a culture medium containing 10% serum, cultured in C0 ₂ incubator. The cell concentration of the culture is preferably 1 × 10 ⁵ to 1 × 10 ⁶ Zml. Normally, 5 XK) ⁷ epithelial cells can be isolated from one amniotic membrane. Amniotic mesenchymal cells can be isolated from amniotic membrane from which epithelial cells have been completely removed. The amniotic membrane remaining on the mesh such as gauze is treated with a protease such as 0.1-1.0 mgZml of collagenase (manufactured by Wako Pure Chemical Industries, Ltd.) and a nuclease such as 0.01-0.1 mgZml of DNAase (Sigma). Shake and digest. The shaking temperature is preferably 20 to 40 ° C, and the shaking time is preferably 30 to 120 minutes. Preferably, the shaking interval is 100 to: LOOO rpm. The digested amniotic membrane can be passed through a mesh such as gauze to separate mesenchymal cells. Collected resulting mesenchymal cells by centrifugation, in a culture medium containing 10% serum, cultured in C0 ₂ incubator. Culture temperature 37QC, C0 ₂ concentration has preferably 5%. The serum is preferably calf serum or fetal calf serum. In addition, it is preferable that an antibiotic (as described above) having a concentration of about 1% coexist in the culture solution. The cell density is preferably from 10 ⁵ to 10 ⁶ cells Zml. Mesenchymal cells can usually be separated into 5 x 10 ⁶ cells from one amniotic membrane. The pharmaceutical composition of the present invention is characterized by containing human amniotic epithelial cells or mesenchymal cells obtained by the above-mentioned method. When epithelial cells or mesenchymal cells are administered to a patient, wash the cells carefully with saline to completely remove coexisting antibiotics and xenogenic serum. When administering cells, use cells suspended in physiological saline or culture solution. The cell concentration, 10 ⁶ ~: L0 ⁸ cells / ml are preferred. Growth factors and cell stabilizers may be mixed in to assist the cells. The administration site is not particularly limited. Depending on the disease, a site such as a spleen, a spleen, or a site of a rheumatic disease or a variety of blood vessels is selected. The total number of cells to be administered depends on the patient's condition, weight, and the like. The diseases that can be treated by the pharmaceutical composition or the therapeutic method of the present invention are as follows. Hyperlipidemia, hyperglycemia, impaired glucose tolerance (i-immediate ai red glucose tolerance: IGT), diabetic complications (eg retinopathy, nephropathy, cataract, coronary artery disease, etc.), arteriosclerosis, heart Vascular disease (eg, ischemic heart disease, etc.). Primary osteoporosis, endocrine osteoporosis (hyperthyroidism, hyperparathyroidism, Cushing's syndrome, and acromegaly), hereditary and congenital forms of osteoporosis (incomplete osteogenesis, homocystinuria, Menkes syndrome, and Riley) Osteoporosis such as osteoporosis due to di syndrome and limb fixation. Bone Paget's disease in adolescents and boys and girls (osteoarthritis). Hypercalcemia and hematological malignancies (solid myeloma, lymphoma and leukemia) from solid tumors (breast, lung and kidney), idiopathic hypercalcemia, and hyperthyroidism and renal function Hypercalcemia associated with insufficiency. Post-surgical steroid-induced osteopenia associated with small and large bowel disorders, and chronic hepatitis and kidney disease. Osteonecrosis or cell death associated with traumatic injury or with non-traumatic necrosis associated with Goge's disease, sickle cell anemia, systemic lupus erythematosus and other diseases. Bone loss due to chronic indirect rheumatism. Periodontal bone loss. Osteolytic metastases. Osteoarthritis, rheumatoid arthritis or spondyloarthropathy and indirect pain, shoulder and arm pain, low back pain and paralysis resulting therefrom. Ischemic heart disease (eg, angina, myocardial infarction, Kawasaki disease, etc.), valvular disease (eg, monk) Mitral stenosis, mitral regurgitation, mitral valve prolapse syndrome, aortic stenosis, aortic regurgitation, etc.), arrhythmia (eg, atrial fibrillation, atrial flutter, paroxysmal supraventricular tachycardia) , WPW syndrome, atrioventricular nodal recurrent tachycardia, ventricular extrasystole, ventricular tachycardia, ventricular fibrillation, bradyarrhythmia, sinus dysfunction syndrome, etc., aortic and vascular diseases (eg, ascending aortic aneurysm, arch Bilateral aortic aneurysm, thoracic aortic aneurysm, descending thoracic aortic aneurysm, thoracoabdominal aortic aneurysm, abdominal aortic aneurysm, obstructive atherosclerosis, venous thrombosis, pulmonary thromboembolism, etc.), congenital heart disease (eg, atrial septum) Deficiency, ventricular septal defect, Ei senmenger syndrome, pulmonary artery perfusion defect, patent arteriovascular patency, endocardial bed defect, complete macrovascular metastasis, modified macrovascular metastasis, pulmonary artery stenosis, Falot Tetralogy, aortic coarctation, Val salva sinus aneurysm, Ebstein malformation, etc.), cardiomyopathy (extended) Dilated cardiomyopathy, hypertrophic cardiomyopathy, restricted cardiomyopathy, etc.).

[Brief description of drawings]

FIG. 1 shows an amniotic membrane-derived epithelial cell and mesenchymal cell diagram.

FIG. 2 shows a blood glucose level fluctuation diagram in mice administered with amniotic epithelial cells and mesenchymal cells. In the figure, M represents the change in blood glucose level in mice administered with mesenchymal cells, and HAE represents changes in blood glucose levels in mice administered with epithelial cells.

FIG. 3 shows immunohistochemical staining showing the expression of human β2-microglobulin and human insulin in epithelial cell transplanted spleen.

Figure 4 shows the expression of human collagen type II gene in cultured amnion-derived mesenchymal cells. The left figure is a phase contrast micrograph, in which cells that have undergone morphological changes were observed. The right figure is an immunostaining diagram using an anti-human collagen type II antibody. Expression of human collagen type II was observed in cells that had undergone morphological changes.

Figure 5 shows that the isolated mesenchymal cells were cultured for about one month in the presence of 100 ng / ml FGF or 5 x M 5-azacitidine, and immunofluorescent staining was performed using a myocardial-specific antibody against NKX2.5. FIG.

Figure 6 shows that the isolated mesenchymal cells were cultured in the presence of 100 _ng / ml FGF or 5-azacitidine for about one month, and then RNA was extracted to determine whether the human NKX2.5 gene was expressed. FIG. 3 is a diagram in which RT was confirmed by the RT-PCR method. [Best Mode for Carrying Out the Invention]

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto. Example 1 Isolation of human epithelial cells and mesenchymal cells

A pregnant woman with informed consent was subjected to cesarean section to obtain an amniotic membrane. The resulting amniotic fragmented with surgical scissors sterile, cell culture medium containing 0.2% Toribushin (Dulbecco's modified Eagle's medium:. Hereinafter referred to as "DMEM") with placed in 50ml Falcon tubes, shaker of 37 ^Q C Shake at 100 rpm for 30 minutes. Removing the supernatant after centrifugation, fragmented with surgical scissors were sterilized again, shaking at 400-600rpm at 37 ^Q C Chez one force one 30 minutes, and digested with 0.2% trypsin. The solution was passed through sterile gauze to separate epithelial cells. Epithelial cells are collected by centrifugation, and the culture medium is 370C in DMEM containing 10% fetal calf serum and antibiotics (benicillin G, 100 units Zml: streptomycin, 100 g / ml: amphotericin B, 0.25 g / ml), 5% C0 ₂ and cultured under. The amniotic membrane remaining on the gauze was fragmented with re-sterilized surgical scissors and similarly digested with 0.2% trypsin for 30 minutes. The procedure of trypsin digestion and cell harvest was repeated three times, and the epithelial cells were collected by centrifugation at a cell density of 3 × 10 ⁵ cells / ml, 370C in DMEM containing 10% fetal calf serum and the same antibiotics as above, 5% C0 ₂ were cultured under. As for epithelial cells, usually 5 × 10 ⁷ cells can be separated from one amniotic membrane. Amniotic mesenchymal cells were isolated from amniotic membrane from which epithelial cells had been completely removed. The amniotic membrane left on the gauze was digested with collagenase (0.75 mgZml, manufactured by Wako Pure Chemical Industries, Ltd.) and DNAse (0.075 mgZml, manufactured by Sigma) at 37 ° C. for 60 minutes at 600 rpm for digestion. The digested amniotic membrane was passed through gauze to separate mesenchymal cells. Collected mesenchymal cells obtained al a centrifugal, 4 X 10 ⁵ cells / ml with 10% fetal calf serum at a density in including DMEM and 370C, 5% C0 ₂ under antibiotic (penicillin G, 100 units (Zml: streptomycin, 100 g / ml: amphotericin B, 0.25 Mg / ml). Normally, 5 × 10 ⁶ mesenchymal cells could be separated from one amniotic membrane. FIG. 1 shows photographs of the separated amniotic membrane-derived epithelial cells and mesenchymal cells. Test Example 1 Therapeutic effect of diabetes by human amniotic membrane-derived epithelial cells

(1) Creation of diabetic mice

Eight to 12 weeks of immunodeficient SCID (sever combined immunodeficient) mice can be treated with human amnion-derived epithelial cells (hereinafter simply referred to as “epithelial cells”) or human amniotic-derived mesenchymal cells (hereinafter simply referred to as “mesenchymal cells”). ). Intraperitoneal administration of 250mg / kg streptozotocin (STZ) to SCID mice at a concentration induces diabetes, resulting in a decrease in body weight and an increase in blood glucose (> 300mg / dl). ) It was confirmed. One week after STZ administration, under anesthesia, 1 × 10 ⁶ cells / 0.1 ml of epithelial cells or mesenchymal cells were transplanted into the mouse spleen. The blood glucose level of the transplanted mice was measured every 9 days between 9 and 11 am using the Aventis Pharma Co., Ltd. diasensor according to the experimental protocol of serum collected from the tail. Two to three weeks after the transplantation, after the blood glucose level recovered to the normal range <100 mg / dl), the mouse was laparotomized, and the spleen and the spleen were excised and subjected to the subsequent analysis.

(2) Immunostaining

The mouse spleen and tentum obtained in (1) were cut into 10% formalin-fixed paraffin slices, sliced to 4 to 6 microns, attached to a slide glass, and used. The following mouse-derived monoclonal antibodies were used as primary antibodies for immunostaining or fluorescence immunization. Anti-human insulin antibody (manufactured by Oncogene: Ab-lZl: 400 dilution), anti-human β2-microglobulin antibody (manufactured by Pharmaluminogen: Col ^ ZlOO dilution), and anti-human collagen type II antibody (manufactured by Sigma) : 1:99 dilution of Tu-99). A heron anti-mouse antibody was used as a secondary antibody, and a streptavidin-biotin method (SAB-PO kit: manufactured by Nichirei Co., Ltd.) was used as an antigen detection system. However, only for the detection of human collagen type II, FITC labeling as a secondary antibody ゥ A heron anti-mouse antibody was used. Heat treatment in a microwave oven for 15 minutes in 0.01M-pH6.0 citrate buffer, deparaffinization, and endogenous peroxidase inhibition (rest in methanol with 0.3% hydrogen peroxide for 30 minutes) . The primary antibody binding reaction is performed at room temperature for 1 hour, the biotin-labeled secondary antibody binding reaction is performed at room temperature for 30 minutes, and the peroxidase-labeled streptavidin (enzyme reagent) is allowed to bind at room temperature for 30 minutes, followed by DAB Aminobenzidine) The color was developed.

(3) Examination by PCR

In order to examine the presence of human cells in the mouse spleen and spleen obtained in (2), DNA was extracted from these tissues and the presence or absence of the human-specific β2-microglobulin gene was examined. Was confirmed by the PCR method. The details are described below.

DNA was extracted from the mouse spleen and spleen obtained in (2) using DNAzol from Molecular Research Senyuichi according to the company's experimental method. Using the obtained DNAlg as a substrate, a primer designed based on the human-specific nucleotide sequence in the human β2-microglobulin gene:

5, -gtgtctgggt ttcatcaatc -3 '(Rooster column number 1);

and

5'- ggcaggcata ctcatctttt -3 '(Toshimi system iJ number 2)

Using the Ready-To-Go PCR Beads flax one Siamese Inc. its experimental protocol - using accordance Le, 940C 1 minute, 56 ⁰ C 1 min, 720C2 minutes, PCR was performed in 30 cycles. The DNA strand obtained after PCR was subjected to electrophoresis at 100 V for 1 hour using a 2% agarose gel, and a 163 bp band corresponding to the human β2-microglobulin gene was identified under ultraviolet light.

(4) Result

On day 2 after STZ administration to 8- to 12-week immunodeficient SCID mice, blood glucose levels exceeded 300 mg / dl and diabetes was induced. One week after STZ administration, epithelial cells or mesenchymal cells were transplanted into the spleen. Blood glucose levels did not decrease to high levels in 3 of 3 mice treated with mesenchymal cells, while blood glucose levels remained low in 1 of 4 mice treated with epithelial cells Although they did not, they recovered to the normal range (KlOOmgZdl) in 3 cases (Fig. 2). Furthermore, in mice whose blood glucose levels had returned to the normal range, the expression of human insulin was identified immunohistologically in the spleen transplanted with epithelial cells (FIG. 3). Furthermore, it was confirmed immunohistologically that human β2-microglobulin was expressed in the spleen (FIG. 3). In addition, PCR confirmed the presence of the human β2-microglobulin gene in the spleen. From these results, it was confirmed that β cells were induced from human amniotic epithelial cells. Test Example 2. Differentiation of human amniotic membrane-derived mesenchymal cells into chondrocytes

The isolated mesenchymal cells were cultured in vitro in the presence of 100 ng / ml Bone Moropogenie Protein (BMP) for about 1 month with high molecular lactate-polyethylene glycol (molecular weight: about 9500) and 10% fetal calf serum. in containing DMEM 370C, 5% C0 ₂ with antibiotics (Bae two cylindrical G, 100 units ZML: streptomycin, 100 ^ g Zml: Anfoteri Singh B, 0.25 g / ml) after co-cultured in the presence of, cartilage cell-specific Analysis was performed by immunofluorescent staining with a collagen type II antibody as described below. The above cultured mesenchymal cells were washed with phosphate buffered saline (PBS), and fixed by treatment with 100% methanol at −20 for 10 minutes and 100% acetone at −20 ° C. for 1 minute. Then react with an anti-human collagen type II antibody (Sigma: Tu-99 / l: diluted 1000) for 1 hour at room temperature, wash with PBS, and use FITC-labeled heron anti-mouse antibody as a secondary antibody at room temperature for 30 hours. After reacting for 1 minute, the cells were washed with PBS and observed with a fluorescence microscope. As a result, the expression of the human collagen type II gene was specifically observed only in the cells whose morphology had changed (Fig. 4). The results showed that chondrocytes could be induced from human amniotic mesenchymal cells. Test Example 3. Differentiation of human amniotic membrane-derived mesenchymal cells into cardiomyocytes

(1) Isolated mesenchymal cells in vitro in the presence of 100 ng / ml FGF or 5-azacitidine (5-AZA) for about 1 month containing 10% fetal calf serum 370 ₅ % C0 _G under antibiotics (penicillin G, 100 units Roh ml: streptomycin, Loo g ml: amphotericin B, 0.25 g / ml) after culture in the presence, cardiomyocyte-specific translocation NKX2.5 (Komuro, I .; Izumo, S .: Csx: a murine

Nat. Acad. Sci. 90: 8145-8149, 1993.) homeobox-containing gene specifically expressed in the developing heart. The above cultured mesenchymal cells are washed with phosphate buffered saline (PBS) and then washed with 100% methanol at -200C for 10 minutes and with 100% acetone at -20. C, treated for 1 minute to fix cells. Next, using an anti-human NKX2.5 antibody (manufactured by SANTA CRUZ: sc-14033 diluted 1: 200), react for 1 hour in a room, wash with PBS, and use FITC-labeled antibody as a secondary antibody. After reacting with an anti-Peacock antibody at room temperature for 30 minutes, the plate was washed with PBS and observed with a fluorescence microscope. As a result, only amniotic mesenchymal cells treated with FGF

Expression of the NKX2.5 protein was observed (FIG. 5). The results showed that cardiomyocytes could be induced from human amniotic mesenchymal cells.

(2) Examination by RT-PCR

In order to examine whether the transcription of the NKX2.5 gene, a cardiomyocyte-specific transcription factor, is enhanced in the cells obtained in (1), total RNA was extracted from the above cells, and the NKX2.5 gene Expression was confirmed by RT-PCR. Total RNA was extracted from the amniotic cells obtained in (1) using RNAzol (Molecular Research Center) according to the attached protocol. Primers designed based on human-specific nucleotide sequence in human NKX2.5 gene using total RNA, l ^ g as substrate:

5'-cttcaagcca gaggcctacg -3 '(Rooster column number 3 in Rooster column);

and

5 ′ · ccgcctctgt cttcttcagc -3 ′ (SEQ ID NO: 4 in the sequence listing);

Was used to perform RT-PCR. The reaction is used according to the protocol attached RT-PCR kit (TAKARA Co.), a reverse transcriptase is reacted 420C, 30 minutes to prepare a cDNA, 30 seconds at 940C, 30 seconds at 55 ⁰ C, 30 seconds at 720C The temperature cycle was repeated 40 times to perform the PCR reaction. The obtained product was subjected to electrophoresis using a 2% agarose gel at 100 ports for 1 hour, and a 233 bp band corresponding to an amplification product from human NKX2.5 mRNA was identified under ultraviolet light. As a result, a human NKX2.5-specific amplification product was confirmed only in cells cultured in the presence of FGF (FIG. 6). [Possibility of industrial use]

By utilizing the present invention, it is possible to provide a remedy for diabetes or the like using regenerative medicine technology or to treat a diabetic patient or the like without causing ethical problems.

Claims

The scope of the claims

1. A pharmaceutical composition containing human amniotic membrane-derived epithelial cells as an active ingredient.

2. An antidiabetic agent comprising human amniotic membrane-derived epithelial cells as an active ingredient.

3. A method for treating diabetes, comprising administering human amniotic membrane-derived epithelial cells to a diabetic patient.

4. A pharmaceutical composition comprising human amniotic membrane-derived mesenchymal cells as an active ingredient.

5. An agent for treating bone metabolism disorders comprising human amniotic membrane-derived mesenchymal cells as an active ingredient.

6. A method for treating bone metabolism disorders, which comprises administering human amniotic membrane-derived mesenchymal cells to patients with bone metabolism disorders.

7. A therapeutic agent for heart disease comprising human amniotic membrane-derived mesenchymal cells as an active ingredient.

8. A method for treating heart disease, comprising administering human amniotic membrane-derived mesenchymal cells to a patient with heart disease.