JP2020079329A - Composition for liver tissue regeneration - Google Patents

Abstract

To provide a composition for liver tissue regeneration, particularly, a composition for liver tissue regeneration for the treatment of liver disease.SOLUTION: A composition for liver tissue regeneration contains human dental pulp stem cells as an active ingredient.SELECTED DRAWING: None

Description

  The present invention relates to a composition for regenerating liver tissue, particularly a composition for regenerating liver tissue for the treatment of liver disease.

  Conventionally, cell transplantation has been performed for the purpose of repairing and regenerating various tissues and treating diseases. Cell transplantation is expected to have a direct therapeutic effect because it administers to an adult cells themselves constituting the tissue or cells that assist the construction of the tissue. For example, in the fields of regeneration of liver tissue and treatment of liver diseases, in the past preclinical studies and clinical studies, for example, transplantation of CD34-positive bone marrow stem cells into the liver or the like has been performed (for example, Non-Patent Document 1). . However, from the viewpoint of treatment of liver diseases, such transplantation of CD34-positive bone marrow stem cells shows only a slight improvement in liver disease, and is far from cure, waiting for life expectancy or cadaver liver transplant/living liver transplant. It only had the effect of lengthening the period.

  On the other hand, since dental pulp stem cells can be easily collected from deciduous or extracted teeth that have been conventionally discarded, such as milk teeth and wisdom teeth, they are expected as stem cells that can be used for tissue regeneration and disease treatment. However, up to now, a method for differentiating and maturing liver cells from dental pulp stem cells and producing (regenerating) a large amount of liver tissue in a mammalian body has not yet been established. In addition, at present, there is no established method for treating liver disease using dental pulp stem cells.

Nakamura et al., Journal of Gastroenterology and Hepatology, Vol. 29, 2014, pages 1830-1838

  As a result of earnest research, the present inventor has discovered that liver tissue can be effectively regenerated by using specific dental pulp stem cells. The present invention is based on such findings.

  Therefore, an object of the present invention is to provide a composition for regenerating mammalian liver tissue, which comprises human dental pulp stem cells as an active ingredient.

That is, the following are specifically exemplified as the present invention.
[1] A composition for regenerating mammalian liver tissue, which comprises human dental pulp stem cells as an active ingredient.
[2] The composition for regenerating liver tissue according to [1], wherein the human dental pulp stem cells are differentiated into hepatocytes.
[3] The composition for regenerating liver tissue according to [1] or [2], wherein the human dental pulp stem cells are human deciduous dental pulp stem cells.
[4] The liver tissue regeneration according to any one of [1] to [3], wherein the human dental pulp stem cells express CD29, CD73, CD90, CD105 and CD166 but do not express CD34, CD45 and CD133 Composition.
[5] The composition for regenerating liver tissue according to any one of [1] to [4], for treating human liver disease.
[6] The composition for regenerating liver tissue according to any one of [1] to [4], for treating liver disease in a non-human mammal.
[7] 5×10 ⁶ to 4×10 ¹⁰ human dental pulp stem cells per 15 kg body weight of the human or non-human mammal to be treated for liver disease are administered to the human or non-human mammal, The composition for regenerating liver tissue according to [5] or [6].
[8] The composition for regenerating liver tissue according to any one of [1] to [7], which is administered to the spleen of the human or non-human mammal.
[9] Needle treatment is required for the above-mentioned liver disease, liver cancer, liver failure, cirrhosis, hepatitis, non-alcoholic fatty liver disease and metastatic cancer, and side effects caused by cancer chemotherapy. The composition for regenerating liver tissue according to any one of [5] to [8], which is selected from the group consisting of:

  According to the present invention, it is possible to regenerate mammalian liver tissue using human dental pulp stem cells that can be easily collected. Further, according to the present invention, it becomes possible to effectively treat liver diseases such as non-alcoholic fatty liver disease (NAFLD).

A biopsy photograph of liver tissue of a liver disease (non-alcoholic fatty liver disease (NAFLD)) model animal (microminiature pig). The graph showing the blood albumin concentration before application of the composition of the present invention, at the start of application and 4 weeks after the start of application. The graph showing the blood total cholesterol level before application of the composition of the present invention, at the start of application and 4 weeks after the start of application. The graph showing the blood type IV collagen concentration before application of the composition of the present invention, at the start of application, and for 4 weeks after the start of application. The graph showing the blood hyaluronic acid concentration before application of the composition of the present invention, at the start of application, and for 4 weeks after the start of application. The graph showing the blood alanine aminotransferase (ALT) concentration before application of the composition of the present invention, at the start of application, and for 4 weeks after the start of application. 4 is a biopsy photograph of the spleen tissue of a liver disease model animal (microminiature pig) 4 weeks after the composition of the present invention was applied to the spleen.

Detailed explanation of the invention

  The composition for regenerating liver tissue of the present invention comprises human dental pulp stem cells as an active ingredient. The human dental pulp stem cells contained in the composition of the present invention may be derived from any of human deciduous teeth, permanent teeth or wisdom teeth (wisdom teeth), but are preferably dental pulp stem cells derived from deciduous deciduous teeth or wisdom teeth. ..

  The dental pulp stem cells used in the present invention may be prepared in advance before carrying out the present invention, or may be commercially available ones. When preparing dental pulp stem cells in the composition of the present invention, the individual from which the dental pulp stem cells are collected may be the same individual as the subject to which the composition of the present invention containing the dental pulp stem cells is applied or a different individual. Good. When the individual from which dental pulp stem cells are collected and the subject to which the composition of the present invention containing the dental pulp stem cells is applied are different, the human leukocyte antigen (HLA: Human Leucocyte Antigen) type of both individuals may be the same. It is essential, and it is more preferable that both individuals are closely related individuals with the same HLA type. Among these, the composition of the present invention is the same as the individual to which the composition of the present invention is applied, from the viewpoint of suppressing the occurrence of a rejection reaction when applying the composition and enhancing the engraftment efficiency and the regeneration efficiency. It is most preferable to use dental pulp stem cells collected from an individual, and then it is preferable to use dental pulp stem cells of an HLA-matched relative. On the other hand, as human dental pulp stem cells generally commercially available, for example, human dental pulp stem cells (CLI, product number: 300702-SF) and hDPSC-dental pulp stem cells (Lonza Japan, product number: PT-5025) and the like. Is mentioned.

  In the present invention, an example of a method for collecting and treating dental pulp stem cells when using previously prepared dental pulp stem cells is shown below. In this collection and treatment method, (1) collection of dental pulp, (2) enzyme treatment, (3) cell culture, and (4) cell collection are performed in order.

(1) Collection of dental pulp After sterilizing human milk teeth or permanent teeth such as wisdom tooth removed immediately before natural dislodgement with chlorhexidine or isodine solution, use a sterilized pulp extraction needle or spoon extractor to remove the root canal or pulp cavity. Pulp tissue is collected via.

(2) Enzyme treatment The collected pulp tissue is treated with collagenase I and dispase of 4 mg/ml each at 37°C for 1 hour.

(3) Cell Culture The cell suspension obtained by the enzyme treatment was seeded on a dish having an area of 25 cm ² , 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin and 0.25 μg. Incubated in Dulbecco's modified Eagle medium (DMEM) containing amphotericin B/ml at a CO ₂ concentration of 5%. The cells are cultivated until the cultured cells reach 85 to 90% confluence, and seeded in a new dish having an area of 25 cm ² at about 10 ⁵ cells/cm ² .

(4) Cell recovery After detaching the cultured cells from the dish by trypsin treatment or the like, centrifugation is performed to recover the cells, and the cells are recovered in Hanks' Balanced Salt Solution (HBSS: about 2 x 10). Resuspend at ⁶ cells/100 μl. The cells thus collected are used as dental pulp stem cells used in the present invention.

  Examples of the method for confirming that the prepared cells are dental pulp stem cells include a method for confirming the expression pattern of a marker specific to dental pulp stem cells as an index. Specifically, dental pulp stem cells are characterized by expressing CD29, CD73, CD90, CD105 and CD166, while not expressing CD34, CD45 and CD133. The expression of any of these markers can be detected and confirmed by, for example, an immunofluorescent staining method or FACS (Fluorescence activated cell sorting).

  The human dental pulp stem cells used in the present invention are preferably used after being differentiated into hepatocytes. The method for differentiating dental pulp stem cells into hepatocytes is not particularly limited, but, for example, a method known in the art for dental pulp stem cells (for example, Okada et al., “Hydrogen sulphide increases hepatic differentiation of human tooth pulp stem cells compared with human bone marrow stem cells, “method described in International Endodontic Journal)). Specifically, dental pulp stem cells were cultured in serum-free DMEM supplemented with 1% insulin-transferrin-sodium selenite and embryotrophic factor to about 70% confluency, and then 20 ng/ml hepatocytes were cultured. Growth factor (HGF) is added and treated for 5 days, followed by addition of Oncostatin M and dexamethasone for an additional 15 days.

  The method for confirming that the dental pulp stem cells are differentiated into hepatocytes is not particularly limited, but a method for confirming the differentiation ability by actually transplanting into the liver of a living body, albumin, α-fetoprotein, or the like. It is carried out by a method of analyzing the expression level of a gene marker characteristic of hepatocyte differentiation using ELISA or real-time RT-PCR. In selecting hepatocytes differentiated from dental pulp stem cells, immunohistological expression of α-fetoprotein was observed, and the blood concentration of α-fetoprotein after transplantation was about 20 ng/ml or more. It is preferable to select young hepatocytes. When such a hepatocyte is used, regeneration of liver tissue including vascular and bile ducts accompanying the liver tissue is expected.

  Human dental pulp stem cells contained in the composition of the present invention may be dental pulp stem cells in a separated state, but in order to enhance the engraftment efficiency and regeneration efficiency of the dental pulp stem cells at the time of applying the composition, a sphere (sphere) is used. The formed pulp stem cells may be used. Further, the composition of the present invention may take any form of solid or liquid as long as it contains an effective amount of human dental pulp stem cells. Depending on the method of applying the composition, a pharmaceutically acceptable carrier or additive is added. It can also be prepared as a solid or liquid composition.

The amount of human dental pulp stem cells contained in the composition of the present invention is not particularly limited as long as it can regenerate liver tissue, but can be appropriately set according to the type and weight of the mammal to be applied, for example, application 5×10 ⁶ to 4×10 ¹⁰ dental pulp stem cells are contained for a target mammal body weight of 15 kg. Preferably, 1×10 ⁷ to 4×10 ⁷ dental pulp stem cells are contained with respect to a weight of a target mammal of 15 kg.

  The subject to which the composition of the present invention is applied is not particularly limited as long as it is a mammal, and may be a human or a non-human mammal. In addition, the composition of the present invention is particularly preferably used for higher mammals belonging to, for example, Primates, Artiodactyla, Artiodactyla or Perissodactyla.

  Further, the application site of the composition of the present invention is not particularly limited, but it is an organ such as liver, spleen and peritoneum. As a method for applying the composition of the present invention, a conventionally known method such as injection, embedding, filling or application is applied to an organ or tissue as an application site. By preparing the composition of the present invention in a gel form having an appropriate fluidity using a carrier such as an appropriate solvent, the composition can be applied by a simple technique such as filling, pouring or coating. If it is in the form of gel, it can be easily applied to the target organ or the like using an injection needle or the like. For example, when the composition of the present invention is applied to the spleen, it may be applied directly to the spleen, or may be injected into the sinus by a syringe and delivered to the spleen by the blood flow and applied. It has been confirmed that when the composition of the present invention is used to regenerate liver tissue in an organ other than the liver, the liver tissue can be regenerated without impairing the function of the organ.

  When the liver tissue is regenerated using the composition of the present invention, in order to suppress the immune reaction due to the application of the composition, it is possible to perform immunosuppression treatment on the animal to be applied before the application of the composition. preferable. The immunosuppressive treatment can be performed using a commercially available immunosuppressive agent. As a commercially available immunosuppressant, for example, tacrolimus (product name: Prograf (registered trademark), manufactured by Astellas Pharma Inc.) and the like can be used. The immunosuppressive treatment can be performed by a conventionally known method for each immunosuppressant used.

  An example of the application schedule of the prograph and the composition of the present invention when the composition of the present invention is applied using Prograf as an immunosuppressant is shown below.

First, prograf granules 1 mg packet (granule 500 mg) and 0.2 mg packet (granule 100 mg) were orally administered at an amount of 0.075 mg granules/kg body weight in the morning and the night before the application of the composition of the present invention. Administer (total daily dose of 0.15 mg/kg body weight). On the day of application, a prograf physiological saline solution prepared by dissolving 0.15 mg/kg body weight of Prograf injection solution (5 mg/ml) in 200 ml of physiological saline was intravenously administered by continuous infusion of 100 ml each in the early morning and the evening for 2 hours. Administered internally (total daily dose 0.15 mg/kg body weight).
From the day after the application to the 28th day after the application, a prograf physiological saline solution prepared by dissolving a prograf injection solution (5 mg/ml) in an amount of 0.10 mg/kg body weight in 200 ml of physiological saline, 100 ml each in the early morning and the evening, Intravenous administration by continuous infusion for 2 hours (total daily dose of 0.10 mg/kg body weight). When administered to an individual having a body weight of 15 kg, 1125 mg of Prograf (2 capsules of 1 mg of Prograf granules and 1 and 1/4 capsules of 0.2 mg) are administered in the morning and the evening before the application of the composition of the present invention, respectively. Orally administer. On the day of application, 2.25 mg (450 μl of prograf injection solution (5 mg/ml)) was dissolved in 200 ml of physiological saline, and 100 ml of prograf physiological saline solution was infused intravenously by continuous infusion of 100 ml each in the early morning and at night. Administer. From the day after the application until 28 days after the application, 1.5 mg (300 μl of prograf injection (5 mg/ml)) was dissolved in 200 ml of physiological saline, and 100 ml of prograf physiological saline solution was added to each in the early morning and the evening. Intravenous administration by continuous infusion of time. In addition, the day after the application of the composition of the present invention, the blood concentration of Prograf is measured daily, and 1.5 mg (Prograf injection solution (5 mg/ml) is used so that the blood concentration does not become 0.01 mg/ml or less. It is preferable to appropriately intravenously administer a Prograf physiological saline solution prepared by dissolving 300 μ) in 200 ml of physiological saline.

  4 to 10 weeks after the application of the composition of the present invention, the tissue (cell group) containing the human dental pulp stem cells contained in the applied composition of the present invention and the cells that have been divided/proliferated is collected. To confirm whether the liver tissue is regenerated, the collected tissue (cell group) and the liver tissue of a normal individual are histologically analyzed by histological staining, immunostaining for various liver markers, in situ hybridization. , ELISA, RT-PCR or real-time RT-PCR analysis, electron microscope analysis, etc. are used for comparison. Various liver markers include, but are not limited to, albumin, α-fetoprotein and CPS (carbamoyl phosphate synthase), and mitochondria. When these liver markers are expressed at the same level as the expression level in the liver of a normal individual, and when applied to a subject with defective liver function, the function of the defective liver is improved and/or Alternatively, when complemented, it is determined that the collected cell group has differentiated into liver tissue, that is, liver tissue has regenerated.

  The compositions of the present invention can be used to treat liver disease in mammals. Therefore, according to another aspect of the present invention, there is provided a pharmaceutical composition comprising the composition of the present invention. Also according to another aspect of the invention there is provided the use of the composition of the invention in the manufacture of a pharmaceutical composition. Further, according to a preferred embodiment of the present invention, the above-mentioned pharmaceutical composition is used for treating liver disease in mammals. In the above-mentioned pharmaceutical composition, the composition of the present invention may be used as it is, or a pharmaceutically acceptable carrier may be appropriately added to the composition of the present invention.

  The liver disease to be treated by the composition of the present invention is not particularly limited, but liver cancer, liver failure, cirrhosis, hepatitis, non-alcoholic fatty liver disease and metastatic cancer, and It is a condition requiring adjuvant therapy for side effects caused by cancer chemotherapy. The condition requiring adjuvant therapy for side effects caused by chemotherapy of cancer specifically means a condition such as liver failure or liver metastatic cancer. In addition, the mammal to which the composition of the present invention is applied may have a complication of lifestyle-related diseases such as obesity, diabetes, hyperlipidemia or hypertension in addition to the liver disease.

  Confirmation of the therapeutic effect on liver disease in the composition of the present invention is performed by applying the composition of the present invention, and then examining the pathological findings by histological staining of the whole organ or tissue to which the composition of the present invention is applied, It can be performed based on a blood test that is usually performed. Here, "the whole organ or tissue to which the composition of the present invention is applied" refers to an organ containing a human dental pulp stem cell contained in the applied composition of the present invention and a cell group consisting of cells that have been divided/proliferated therefrom. Or means an organization.

When confirming the therapeutic effect on the liver disease of the composition of the present invention based on a blood test, it is performed by comparing at least one parameter selected from the following (1) to (6) with a normal individual.
(1) Blood total cholesterol concentration (2) Blood albumin concentration (3) Hepaplastin test (HPT) value (4) Blood type IV collagen concentration (5) Blood alanine aminotransferase (ALT) concentration (6) ) Blood hyaluronic acid concentration Specifically, in a mammal individual suffering from liver disease, when the composition of the present invention is applied, does at least one of the above-mentioned parameters have a value similar to that of a normal individual, Alternatively, it is judged that the composition of the present invention has a therapeutic effect on liver disease when it tends to approach the value of a normal individual.

  Also, according to another aspect of the present invention, there is provided a method of treating the above liver disease in a mammal, which comprises administering to the mammal an effective amount of the composition of the present invention.

  The effective amount of the composition of the present invention is not particularly limited, but is appropriately determined by a doctor according to the type, age, sex, condition, etc. of the target mammal.

  The administration method of the composition of the present invention is not particularly limited, but for example, it can be performed by the same procedure as the above-mentioned regeneration of liver tissue.

  Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to these Examples.

Preparation of dental pulp stem cells Milk teeth were collected from healthy humans aged 6 to 12 according to a conventional method. The collection of milk teeth and the preparation and use of the pulp stem cells of the collected milk teeth were explained to the subject or his/her guardian in advance and consent was given. From the collected milk teeth, pulp tissue was collected through a root canal using a sterilized pulp extraction needle. The collected dental pulp tissue was treated with 4 mg/ml dispase II (GIBCO, product number: 1728484) and 4 mg/ml collagenase I (Wako Pure Chemical Industries, Ltd., product number: CTH2297) at 37°C. Digested for hours. The cell suspension after digestion was used in a Dulbecco's modified Eagle medium (DMEM) (Invitrogen, 100 U/ml) containing 10% fetal bovine serum (FBS) using a 25 cm ² dish (manufactured by TPP). Penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B were contained), and the cells were cultured at 37° C. in an atmosphere with a CO ₂ concentration of 5%. After culturing until the cultured cells reached 85 to 90% confluence, the cells were subcultured to a new dish at 1×10 ⁵ cells/cm ² .

After three passages in DMEM medium containing 10% FBS, the cells were detached from the dish using trypsin. The detached cells were subjected to Manual MACS (registered trademark) Cell Separation protocol to isolate CD117 ⁺ dental pulp stem cells. CD117 ⁺ dental pulp stem cells were cultured in serum-free DMEM containing 20 ng/ml hepatocyte growth factor (HGF) for 5 days, and then 10 ng/ml oncostatin M (R&D) and 10 nM dexamethasone ( Wako Pure Chemical Industries, Ltd.) was added to the medium and the cells were further cultured for 15 days. After the completion of the culture, the cells were suspended in Hanks' balanced salt solution (manufactured by Gibco) to give about 1×10 ⁷ cells/ml, which was used as the composition of the present invention.

Preparation of animal model affected by liver disease Using microminiature pigs, an animal model affected by liver disease (nonalcoholic fatty liver disease (NAFLD)) was prepared by the following procedure.

  About 7 months old (body weight: about 10 kg), a male micro mini pig (strain: Fuji Micromini Pig) was purchased from Fuji Micra Co., Ltd., and natural ventilation, temperature: 20±5° C., relative humidity: 55±25% for 12 individuals. , Lighting cycle: 7:00 to 19:00 is light, and 19:00 to 7:00 is dark in a breeding environment, solid feed MP-A (manufactured by Oriental Yeast Co., containing choline: 0. (29%, crude protein content: 15.3%) was used as a feed, and 300 g was fed daily for acclimation. The feeding was performed around 8:00 each day.

A blood test was performed on 12 normal individuals (normal individuals) after the acclimation period of about one week. Specifically, venous blood is collected from each of 12 normal individuals to obtain serum, and the obtained serum sample is used to measure the following items (1) to (6), and the measurement is performed. The value was set to "normal value". The items (1) to (6) and their normal values were as follows.
(1) Blood total cholesterol concentration: 214 mg/dl
(2) Blood albumin concentration: 4.192 mg/dl
(3) Hepaplastin test (HPT) value: 21.9%
(4) Blood type IV collagen concentration: 0.095 μg/ml
(5) Blood alanine aminotransferase (ALT) concentration: 32 U/l
(6) Blood hyaluronic acid concentration: 32 ng/ml

The measurement of each item of the above (1) to (6) was performed using the following methods and kits.
(1) Blood total cholesterol concentration was measured using the cholesterol dehydrogenase (UV) method.
(2) Blood albumin concentration was measured using nephelometry (BCP improvement method).
(3) Hepaplastin test (HPT) values were measured using coagulation time measurement.
(4) Blood type IV collagen concentration was measured using Type IV collagen ELISA kit, ACB (manufactured by Funakoshi Co., Ltd.).
(5) Blood alanine aminotransferase (ALT) concentration was measured using the JSCC standardized method.
(6) Blood hyaluronic acid concentration was measured using the latex agglutination immunoturbidimetric method.

  After measuring the normal value, CDAA feed (RESERCH DIETS, product number: A15022101), which does not substantially contain protein, was used as a feed, and 300 g/day was fed to the feed under the same feeding environment for 16 weeks. It was bred by food. The feeding was performed around 8:00 each day. Twelve individuals after being bred for free for 16 weeks were used as liver disease model. Blood tests were carried out on 12 individual liver disease models in the same manner as in normal individuals.

  The liver tissue of one of the obtained models of liver disease was taken out to obtain a biopsy photograph. The biopsy picture is shown in FIG. In the biopsy photograph of FIG. 1, the gray portion indicates hepatocytes, and the small white granular portion indicates fat (lipid). In the liver tissue of the microminiature pig obtained by the method of the present invention, a large amount of fat (lipid) is deposited on hepatocytes, which is not observed in normal liver tissue. Findings of liver disease were noted.

The results of blood tests on 12 individuals with liver disease models were as follows. These measured values were defined as "morbidity value".
(1) Blood total cholesterol concentration: 87 mg/dl
(2) Blood albumin concentration: 1.768 mg/dl
(3) Hepaplastin test (HPT) value: 6.9%
(4) Blood type IV collagen concentration: 0.998 μg/ml
(5) Blood alanine aminotransferase (ALT) concentration: 246 U/l
(6) Blood hyaluronic acid concentration: 51 ng/ml

  From the results of the above blood test, the findings (markers) of liver disease are: (1) decrease in total blood cholesterol concentration, (2) decrease in blood albumin concentration, (3) decrease in HPT value, (4) blood An increase in medium type IV collagen concentration, (5) increase in blood ALT concentration, and (6) increase in blood hyaluronic acid concentration were observed.

  Thus, the obtained liver disease model was a liver disease model that can be extrapolated to liver disease (NAFLD) from the findings of biopsy photographs and blood tests.

<Example: Examination of therapeutic effect on liver disease by application of composition containing dental pulp stem cells>
The composition of the present invention containing dental pulp stem cells prepared by the above-described method was applied to the spleen of the liver disease-affected model prepared by the above-mentioned method, and the therapeutic effect on liver disease of the composition of the present invention was examined. .

  The 12 models of liver disease affected by the above-described method were divided into an application group of 6 individuals to which the composition of the present invention was applied and a control group of 6 individuals to which the composition of the present invention was not applied.

To the application group, the composition of the present invention containing dental pulp stem cells was applied to the spleen of a liver disease model by the following method. First, tacrolimus (product name: Prograf (registered trademark) granules, manufactured by Astellas Pharma Inc.) was administered to 6 individuals in the application group on the day before the start of application of the composition of the present invention at a single dose of Prograf. Immunosuppression was carried out by oral administration twice a day in the early morning and at night so as to obtain 0.075 mg/kg body weight (the total daily dose was 0.15 mg/kg body weight). Next, the composition of the present invention (containing about 1×10 ⁷ dental pulp stem cells/ml) produced by the above-mentioned method is applied using a 1 ml syringe (manufactured by Terumo, product number: SS-01T). Six individuals in the group were injected into the splenic sinuses. Four weeks after the start of application, venous blood was collected from each individual to obtain serum, and a blood test was performed using the obtained serum sample. On the day of application of the composition of the present invention, Prograf (registered trademark) injection solution (5 mg/ml) was added to physiological saline (Otsuka Pharmaceutical Co., Ltd.) so that a single dose of Prograf was 0.075 mg/kg body weight. (Manufactured by Co., Ltd., product number: D05352) was dissolved in 100 ml and administered intravenously twice a day in the early morning and in the evening for immunosuppression (total daily dose: 0.15 mg/kg body weight). On and after the day after the application of the composition of the present invention, Prograf (registered trademark) injection solution (5 mg/ml) was dissolved in 100 ml of physiological saline so that the dose of Prograf at one time was 0.05 mg/kg body weight. Immunosuppression was performed by intravenous administration twice a day in the early morning and at night (total daily dose was 0.10 mg/kg body weight). From the day after the application of the composition of the present invention, the blood concentration of prograph in each individual was measured daily, and 1.5 mg (prograph injection was performed so that the prograf concentration in venous blood was about 0.013 mg/ml). The immunosuppression was maintained by appropriately intravenously administering a Prograf physiological saline solution prepared by dissolving Prograph of the liquid (5 mg/ml) (300 μl) in 200 ml of physiological saline. In the control group, the same amount of physiological saline was administered instead of Prograf.

  The results of the blood test of each of the application group and the control group (average value of 6 individuals: “treatment value”), and graphs summarizing the normal value and the morbidity value by item are shown in FIGS. As is clear from the results of FIGS. 2 to 6, in the application group, each marker of liver disease in the blood test approaches a normal value 4 weeks after the application of the composition of the present invention (from the morbidity value to the treatment value). This means that the liver disease has been cured by applying the composition of the present invention. Although not shown in the figure, in the blood test 4 weeks after the application of the composition of the present invention, the value of the hepaplastin test (HPT) was 11.0%, and the blood type IV collagen concentration was It was 0.382 μg/ml. These liver disease markers also tend to approach normal values within 4 weeks after the application of the composition of the present invention, indicating that the liver disease has been cured by applying the composition of the present invention. is there.

  On the other hand, in the control group, each marker of liver disease showed almost no change between the morbidity value and the treatment value. Specifically, the treatment value tends to be slightly closer to the normal value, or the treatment value deviates from the normal value rather than the morbidity value. These results indicate that liver disease is poorly cured or rather advanced.

In addition, 4 weeks after the start of application of the composition of the present invention, one individual spleen tissue was taken out from the application group and stained with diaminobenzidine (DAB). The biopsy photograph after staining is shown in FIG.
In the biopsy photograph of FIG. 7, small black particles indicate liver cells. From this biopsy photograph, it can be seen that in the spleen to which the composition of the present invention has been applied, the liver tissue has been regenerated and a so-called second liver that complements the function of the liver has been created. Based on the pathological findings, the results of this biopsy photograph show a pathological image leading to the healing of the liver disease by further progressing the regeneration of liver tissue over time.

  From the above, based on the blood test and biopsy photograph, by applying the composition of the present invention containing human dental pulp stem cells, it is clear that the effect of regeneration of liver tissue, and the treatment of liver disease accompanying it are exhibited. became. Further, from this result, it can be said that the composition of the present invention containing human dental pulp stem cells can be used for regeneration of liver tissue and treatment of liver disease.

  INDUSTRIAL APPLICABILITY According to the present invention, it becomes possible to produce a composition for regeneration of liver tissue and treatment of liver disease by using human dental pulp stem cells that can be easily collected from deciduous teeth or extracted teeth.

Claims (9)

  A composition for regenerating mammalian liver tissue, which comprises human dental pulp stem cells as an active ingredient.   The liver tissue regeneration composition according to claim 1, wherein the human dental pulp stem cells are differentiated into hepatocytes.   The composition for regenerating liver tissue according to claim 1, wherein the human dental pulp stem cells are human deciduous dental pulp stem cells.   The composition for regenerating liver tissue according to any one of claims 1 to 3, wherein the human dental pulp stem cells express CD29, CD73, CD90, CD105 and CD166, but do not express CD34, CD45 and CD133.   The composition for regenerating liver tissue according to any one of claims 1 to 4, which is used for treating human liver disease.   The composition for regenerating liver tissue according to any one of claims 1 to 4, which is used for treating liver disease in a non-human mammal. 6. The human or non-human mammal is administered 5×10 ⁶ to 4×10 ¹⁰ human dental pulp stem cells per 15 kg body weight of the human or non-human mammal to be treated for liver disease. Or the composition for regenerating liver tissue according to 6 above.   The composition for regenerating liver tissue according to any one of claims 1 to 7, which is administered to the spleen of the human or non-human mammal.   The liver disease, liver cancer, liver failure, cirrhosis, hepatitis, non-alcoholic fatty liver disease and metastatic cancer, and from the pathology requiring adjuvant therapy for side effects caused by cancer chemotherapy The composition for liver tissue regeneration according to any one of claims 5 to 8, which is selected from the group consisting of: