JP5230969B2 - Sheet containing cardiac myoblasts - Google Patents

Description

  The present invention relates to a sheet containing cardiac myoblasts, a method for obtaining a cardiac myoblast and a sheet containing cardiac myoblasts from adipose tissue-derived stem cells, and the like.

  In Japan, the incidence of lifestyle-related diseases and subsequent ischemic heart disease is increasing with the westernization of life. With recent advances in treatment technology, many ischemic heart diseases can be treated, but even with such treatment technology, it is difficult to treat severe heart failure, and there is an urgent need to establish a treatment method. Yes.

  In treating severe heart failure, regenerative medical techniques have attracted attention. To date, single cells such as bone marrow-derived mononuclear cells, skeletal muscle-derived cells, adipose tissue-derived stem cells, and cardiac myoblasts collected from the myocardium are transplanted into the myocardium by needle injection or via the coronary artery. Has been attempted (Non-Patent Document 1). However, this method has a problem that most of the transplanted cells do not engraft.

In recent years, it has been reported that cardiac function can be improved by transplanting a single cell sheet composed of adipose tissue-derived stem cells (ADSC) into the heart (Non-patent Document 2). However, the transplanted ADSC could not differentiate into cardiomyocytes and did not lead to myocardial recruitment. Therefore, it was necessary to obtain a large amount of cardioblasts in order to prepare a sheet of cardiac myoblasts that had been differentiated into cardiomyocytes, and such a sheet.
Suzuki K et al., Circulation. 2004 Sep 14; 110 (11 Suppl 1): II 225-30 Miyahara Y et al., Nat Med. 2006 Apr; 12 (4): 459-65

  An object of the present invention is to obtain a large amount of cardiac myoblasts and to provide a sheet containing such cardiac myoblasts.

  As a result of intensive studies in view of the above circumstances, the present inventors have found that when adipose tissue-derived stem cells are cultured in the presence of DMSO or OP9 culture supernatant, they can be differentiated into cardiac myoblasts. It came to be completed.

That is, the present invention
(1) a sheet containing cardiac myoblasts,
(2) A method for obtaining a cardiac myoblast characterized by culturing adipose tissue-derived stem cells;
(3) The method according to (2), wherein the culture is performed in the presence of DMSO or OP9 culture supernatant,
(4) The method according to (2) or (3), wherein the adipose tissue-derived stem cell is ADMPC,
(5) cardiomyocytes that can be obtained by the method according to any one of (2) to (4),
(6) A method for obtaining a sheet containing cardiac myoblasts,
The following process:
(A) differentiating adipose tissue-derived stem cells into cardiac myoblasts,
(B) forming a sheet containing cardiac myoblasts,
Including, method,
(7) The method according to (6), wherein the differentiation from adipose tissue-derived stem cells into cardiomyocytes is performed in the presence of DMSO or OP9 culture supernatant,
(8) The method according to (6) or (7), wherein the adipose tissue-derived stem cell is ADMPC,
(9) A sheet containing cardiomyocytes that can be obtained by the method according to any one of (6) to (8),
(10) A method for screening a substance that promotes differentiation into cardiac myoblasts, comprising the following steps:
(A) differentiating adipose tissue-derived stem cells into cardiac myoblasts in a medium containing a candidate substance;
(B) examining the differentiation of adipose tissue-derived stem cells into cardiac myoblasts,
A substance that promotes differentiation into cardiac myoblasts when differentiation is promoted compared to differentiation when adipose tissue-derived stem cells are cultured in a candidate substance-free medium. How to indicate that there is,
(11) A substance that can be obtained by the method according to (10), and a substance that promotes differentiation into cardiomyocytes and (12) a substance that promotes differentiation into cardiomyocytes used in the method according to (10) A kit for screening,
Is to provide.

  The present invention provides a sheet containing cardiac myoblasts, a method for obtaining a cardiac myoblast and a sheet containing cardiac myoblasts from adipose tissue-derived stem cells.

  The present invention provides a sufficient amount of cardiac myoblasts to constitute a sheet. Accordingly, in one aspect, the present invention provides a sheet containing cardiac myoblasts. In the present specification, cardioblasts are cells that are directed to differentiate into cardiomyocytes and that express α-cardiac actin (α-CA) and Myosin Light Chain (MLC). . The animal species from which the myocardial blast cells are derived is not particularly limited, and preferably, for example, mammals including humans, mice, rats, rabbits, dogs, cats, cows, horses, monkeys, etc., more preferably humans. Alternatively, the same type or the same as the target to which the sheet containing such cardiomyocytes is applied is preferable. For example, by transplanting a sheet containing cardiomyocytes derived from the same species or the same animal as the subject to the subject, it becomes possible to treat severe heart failure without fear of rejection.

  The sheet containing cardiomyocytes refers to a cell mass containing cardiomyocytes as an essential component. In the sheet, the cardiac myoblast may be contained in either a monolayer or a multilayer. By having a sheet form, handling when used for transplantation or the like is facilitated. The components other than the myocardial blasts of the sheet may be any one, and examples thereof include adipose tissue-derived stem cells, cardiomyocytes, cell scaffolds, vascular endothelium, and matrix. The size and thickness of the sheet can be appropriately selected according to various conditions such as the size of the applied injury area.

  The ratio of the myocardial blasts contained in the sheet is not particularly limited, and can be appropriately selected according to various conditions such as the state of the target to which the sheet is applied. The ratio is, for example, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, and the like. The percentage of cardiac myoblasts can be determined by quantifying the myocardial blast marker gene α-CA or MLC using means known to those skilled in the art, for example, quantitative RT-PCR.

  The function of the sheet of the present invention is a known method, for example, by transplanting a sheet into a subject, and the heart function of the transplanted subject is determined by echocardiography, or end diastolic diameter (LVDd), end systolic diameter (LVDs), left Evaluation can be made by measuring the chamber ejection rate (% EF) or the left chamber diameter shortening rate (% FS).

  In another aspect, the present invention provides a method for obtaining cardiac myoblasts, comprising culturing adipose tissue-derived stem cells. This method makes it possible to obtain a large amount of cardiac myoblasts from adipose tissue-derived stem cells. The method may include a step of obtaining adipose tissue-derived stem cells from adipose tissue-derived cells. Adipose tissue-derived stem cells refer to cells that can be differentiated into various cell lineages such as endoderm, mesoderm, ectoderm, etc., and adipose tissue-derived multilineage progenitors expressing Islet-1, which is an undifferentiated marker Contains cells (ADMPC). The animal species from which the adipose tissue-derived stem cells are derived is not particularly limited, and preferably, for example, mammals including humans, mice, rats, rabbits, dogs, cats, cows, horses, monkeys, etc., more preferably humans. Alternatively, the same or the same type of subject as that to be treated using cardiac myoblasts obtained from such adipose tissue-derived stem cells is preferable.

  Preferably, the method for obtaining cardioblasts of the present invention comprises a step of culturing adipose tissue-derived stem cells in the presence of DMSO or OP9 culture supernatant. By culturing adipose tissue-derived stem cells under such conditions, the cells can be differentiated and / or induced into cardiac myoblasts. The medium used for such culture can be appropriately selected. Such a medium may contain various factors such as retinoic acid, BMP2, BMP4, TGFβ2, HGF, bFGF, thyroxine, oxytocin or fatty acid concentrate. Differentiation into cardiac myoblasts can be examined, for example, by measuring the expression of a cardiac myoblast marker such as α-CA or MLC by RT-PCR.

  Accordingly, in a further aspect, the present invention provides a cardiomyocyte that can be obtained by the method for obtaining the cardiomyocyte. Such cardiomyocytes can be used for treatment of heart diseases such as myocardial infarction and cardiomyopathy, or can be used as a material for a sheet containing cardiomyocytes.

  In another aspect, the present invention provides a method for obtaining a sheet containing cardiac myoblasts, comprising the following steps: (a) differentiation of adipose tissue-derived stem cells into cardiac myoblasts, and (b) Forming a containing sheet. These steps may be performed continuously or in parallel. By such a method, a sheet containing cardiac myoblasts can be obtained easily and efficiently. The step of differentiating the adipose tissue-derived stem cells into cardiac myoblasts is as described above.

The step of forming a sheet containing cardiac myoblasts from cardiac myoblasts can be achieved by means or methods known to those skilled in the art. Preferably, the step can be achieved by growing cardioblasts in an attached state to form a cell mass, and then detaching the formed cell mass. The number of cardiomyocytes used and the culture time can be appropriately selected according to various conditions such as the size of the sheet to be obtained and the number of cardiomyocytes contained in the sheet. For example, the sheet may be obtained by culturing 10 ⁵ to 10 ⁶ cardiomyocytes for 24 to 72 hours. Separation of the cell mass can be performed by various means, for example, physical stimulation. Alternatively, the cell mass may be detached by culturing cardioblasts in a temperature-sensitive culture dish and incubating at, for example, 20 ° C. or lower.

  Therefore, in a further aspect, the present invention relates to a sheet containing cardiomyocytes that can be obtained by the method for obtaining a sheet containing cardiomyocytes. Such a sheet containing myoblasts can be used for the treatment of heart diseases such as myocardial infarction and cardiomyopathy.

  In another aspect, the present invention relates to a method for treating and / or preventing a disease caused by impaired myocardial function, comprising transplanting a sheet containing cardiomyocytes or cardiomyocytes into a subject. Examples of diseases caused by a decrease in myocardial function include acute myocardial infarction, old myocardial infarction, ischemic myocardial infarction, dilated cardiomyopathy, congenital heart disease, and the like. Transplantation into a subject can be performed by methods known to those skilled in the art. For example, it can be performed by transplanting a sheet containing myocardial blasts to a region where the function of the myocardium is reduced, or by transplanting myocardial blasts via the coronary artery. The size and thickness of the sheet to be transplanted, the number of cardiac myoblasts contained in the sheet, and the number of cardiac myoblasts can be appropriately selected according to various conditions such as the state of the subject and the size of the damaged area.

  Accordingly, the present invention provides, in a further aspect, a sheet containing cardiomyocytes or a composition containing cardiomyocytes for treating and / or preventing a disease caused by reduced cardiac function. In addition to the sheet or cardiac myoblast, such a composition may contain, for example, PBS, a medium, a substance that promotes engraftment, a cardiac function improving agent, a growth factor, a growth factor, and the like.

In another aspect, the present invention provides a method for screening a substance that promotes differentiation into cardiomyocytes, comprising the following steps:
(A) differentiating adipose tissue-derived stem cells into cardiac myoblasts in a medium containing a candidate substance;
(B) examining the differentiation of adipose tissue-derived stem cells into cardiac myoblasts,
A substance that promotes differentiation into cardiac myoblasts when differentiation is promoted compared to differentiation when adipose tissue-derived stem cells are cultured in a candidate substance-free medium. It relates to a method of indicating something. There are various candidate substances, and examples thereof include, but are not limited to, retinoic acid, BMP2, BMP4, TGFβ2, HGF, bFGF, thyroxine, oxyton analogs and derivatives, and the like. Differentiation of adipose tissue-derived stem cells into cardiac myoblasts is as described above. Differentiation into cardiac myoblast can be examined by various methods, for example, by measuring the expression of a cardiac myoblast marker such as α-CA or MLC by RT-PCR.

  Therefore, in a further aspect, the present invention relates to a substance that promotes differentiation into cardiac myoblasts, which can be obtained by the method for screening a substance that promotes differentiation into cardiac myoblasts. You may use this substance for the manufacturing method of the sheet | seat containing the above-mentioned myocardial blasts and myocardial blasts, and may increase the number of obtained myocardial blasts and sheets. Alternatively, such a substance may be used for treating or preventing a disease caused by a decrease in cardiac function.

In another aspect, the present invention provides a method for screening a substance that suppresses differentiation into cardiac myoblasts, which comprises the following steps:
(A) differentiating adipose tissue-derived stem cells into cardiac myoblasts in a medium containing a candidate substance;
(B) examining the differentiation of adipose tissue-derived stem cells into cardiac myoblasts,
A substance that suppresses differentiation into cardiomyocytes when the differentiation is suppressed as compared to differentiation when adipose tissue-derived stem cells are cultured in a candidate substance-free medium. It relates to a method that indicates that there is. There are various candidate substances, and examples thereof include, but are not limited to, noggin analogs and derivatives.

  Therefore, in a further aspect, the present invention relates to a substance that suppresses differentiation into cardiac myoblasts, which can be obtained by the method for screening a substance that suppresses differentiation into cardiac myoblasts. The substance may be used for the treatment or prevention of diseases caused by increased cardiac function.

  Furthermore, the present invention relates to a kit for screening for a substance that promotes or suppresses differentiation into cardiac myoblasts. The kit of the present invention may contain adipose tissue-derived stem cells, a medium, a culture container, means for examining differentiation into cardiomyocytes, and the like. Usually, an instruction manual is attached to the kit. The screening can be performed quickly and easily using such a kit.

In another aspect, the present invention provides a method for screening for a substance that promotes or suppresses the formation of a sheet containing cardiomyocytes, comprising the following steps:
(A) differentiating adipose tissue-derived stem cells into cardiac myoblasts in a medium containing a candidate substance; or (b) forming a sheet containing myocardial blasts in the presence of the candidate substance;
(C) examining the formation of a sheet containing cardiac myoblasts,
A substance that promotes or suppresses formation of a sheet containing cardiomyocytes when the formation is promoted or suppressed as compared to that formed in a candidate substance-free system. It relates to a method of indicating something. The method for examining the formation of the sheet is as described above. The addition of the candidate substance may be performed in both of the above steps (a) and (b), or may be performed on one side.

  Therefore, in a further aspect, the present invention relates to a substance that promotes or suppresses the formation of a sheet containing cardiac myoblasts, which can be obtained by the method for screening a substance that promotes or suppresses the formation of a sheet containing cardiac myoblasts. It is.

  Furthermore, this invention relates to the kit for screening the substance which accelerates | stimulates or suppresses formation of the sheet | seat containing the said cardiac myoblast.

  In one aspect, the present invention relates to a cell population comprising adipose tissue-derived multilineage progenitor cells. Adipose tissue-derived multilineage progenitor cells are cells that can differentiate into various cell lineages such as endoderm, mesoderm, ectoderm, etc., and express cells that are undifferentiated marker Islet-1. Adipose tissue-derived multilineage progenitor cells can be obtained by differentiation from adipose tissue, embryonic stem cells, and the like. The animal species from which the adipose tissue-derived multilineage progenitor cells are derived is not particularly limited, and preferably, for example, mammals including humans, mice, rats, rabbits, dogs, cats, cows, horses, monkeys, etc., more preferably humans It is. Alternatively, the same or the same subject as that to be treated by regenerative medicine using such a cell population is preferable.

  The cell population of the present invention has advantages such as ease of culture and high differentiation efficiency because the proportion of cells other than undesired contaminants such as red blood cells and vascular endothelial cells other than adipose tissue-derived multilineage progenitor cells is low. Have. As means for removing such contaminants, means / methods utilizing the difference from the specific gravity of the adipose tissue-derived multilineage progenitor cells, for example, the specific gravity method, utilizing the difference between the adherence of the adipose tissue-derived multilineage progenitor cells Means / methods, for example, chelating agents such as EDTA, methods using an enzyme such as trypsin, antigen-antibody methods such as sorting and MACS, morphological sorting methods, single cell cloning, hemolysis, etc. It is done. Reduction of contaminants in a cell population is confirmed visually by a microscope, or by flow cytometry or immunohistochemical staining, for example, by quantifying the markers of the contaminants using a method such as RT-PCR or ELISA. Can be done.

  The cell population of the present invention is preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 93%, 96%, or 99% Adipose tissue-derived multilineage progenitor cells. By including such a proportion of adipose tissue-derived multilineage progenitor cells, the cell population of the present invention has advantages such as easy maintenance of adipose tissue-derived multilineage progenitor cells and high efficiency when differentiated. Have. In addition to adipose tissue-derived multilineage progenitor cells, the cell population of the present invention includes cells effective for the maintenance or differentiation of adipose tissue-derived multilineage progenitor cells such as feeder cells, vascular endothelial cells, fibroblasts, and the like. May be. By including such cells, the above advantages can be enhanced.

  In another aspect, the present invention provides a method for obtaining adipose tissue-derived multilineage progenitor cells from adipose tissue, comprising: (a) removing red blood cells from the adipose tissue-derived cell population, Next, (b) a method comprising a step of removing cells other than adipose tissue-derived multilineage progenitor cells from the pre-adipose tissue-derived multilineage progenitor cell population to obtain adipose tissue-derived multilineage progenitor cells. It is. According to the present invention, cells other than adipose tissue-derived multilineage progenitor cells can be reduced, and adipose tissue-derived multilineage progenitor cells can be obtained with high yield and high purity. In the present invention, the above steps may be performed continuously or in parallel. The adipose tissue used in this aspect of the present invention may be either subcutaneous adipose tissue or visceral adipose tissue in vivo. The animal species from which the adipose tissue is derived is not particularly limited, and is preferably a mammal including, for example, humans, mice, rats, rabbits, dogs, cats, cows, horses, monkeys, etc., more preferably humans. Alternatively, the same type or the same as the subject to be treated by regenerative medicine using adipose tissue-derived multilineage progenitor cells obtainable by the method of the present invention is preferable.

  As used herein, the adipose tissue-derived cell population refers to a cell population containing at least adipose tissue-derived multilineage progenitor cells. The adipose tissue-derived cell population may contain erythrocytes, vascular endothelial cells, fibroblasts and the like in addition to adipose tissue-derived multilineage progenitor cells. The adipose tissue-derived cell population can be obtained, for example, by treating adipose tissue with an enzyme such as collagenase or by physical means / method, and / or removing lipids by, for example, centrifugation or filtering.

  Erythrocytes have the property of adsorbing adipose tissue-derived multilineage progenitor cells, thereby reducing the yield of adipose tissue-derived multilineage progenitor cells. Therefore, it is necessary to remove red blood cells from the adipose tissue-derived cell population. Removal of erythrocytes from the adipose tissue-derived cell population may be performed by any means / method, for example, by means / methods other than those based on the difference in adhesion between erythrocytes and other cells. There may be. Preferably, such removal is performed by a specific gravity method, a hemolysis method, a filter method, more preferably by a specific gravity method. The specific gravity method can be performed using a specific gravity liquid having an appropriate specific gravity, for example, a commercially available specific gravity liquid such as Lymphoprep. The specific gravity of the specific gravity solution used may be between the specific gravity of red blood cells and other cells, preferably 1.063 to 1.119, more preferably 1.070 to 1.110, and most preferably. Is 1.077.

  As used herein, a pre-adipose tissue-derived multilineage progenitor cell population refers to a cell population containing at least adipose tissue-derived multilineage progenitor cells. The pre-adipose tissue-derived multilineage progenitor cell population may include vascular endothelial cells, fibroblasts, and the like in addition to the adipose tissue-derived multilineage progenitor cells. As described above, the pre-adipose tissue-derived multilineage progenitor cell population is substantially free of erythrocytes. By removing red blood cells to form a pre-adipose tissue-derived multilineage progenitor cell population, cells other than the subsequent adipose tissue-derived multilineage progenitor cells can be easily and efficiently removed.

  As used herein, cells other than adipose tissue-derived multilineage progenitor cells refer to adherent cells such as vascular endothelial cells and fibroblasts. Removal of cells other than the adipose tissue-derived multilineage progenitor cells from the pre-adipose tissue-derived multilineage progenitor cell population may be performed by any means / method, preferably a substance other than trypsin, more preferably It is carried out using a chelating agent such as EDTA or EGTA, most preferably EDTA. Preferably, such removal is performed based on the difference in adhesion between the adipose tissue-derived multilineage progenitor cells and other cells. In addition to the above, cells other than adipose tissue-derived multilineage progenitor cells can be removed by, for example, filter filtration. By removing these cells, the purity and yield of the resulting adipose tissue-derived multilineage progenitor cell population are increased.

  In another aspect, the present invention relates to an adipose tissue-derived multilineage progenitor cell that can be obtained by a method for obtaining an adipose tissue-derived multilineage progenitor cell from the adipose tissue. Such adipose tissue-derived multilineage progenitor cells express Islet-1 as described above.

  In a further aspect, the present invention relates to a cell population containing adipose tissue-derived multilineage progenitor cells obtainable by the method for obtaining adipose tissue-derived multilineage progenitor cells from the adipose tissue. Such a cell population is substantially free of cells derived from undesired adipose tissue such as erythrocytes and vascular endothelial cells, but contains cells effective for maintenance and differentiation of adipose tissue-derived multilineage progenitor cells such as feeder cells. May be.

  In one aspect, the present invention relates to a method for obtaining a hepatic lobule-like cell mass from adipose tissue-derived cells, which comprises culturing adipose tissue-derived cells. The present invention is excellent in that it can form a cell mass similar to a liver lobule, which is the smallest functional unit of the liver. Adipose tissue-derived cells are cells or cell populations obtained from visceral adipose tissue or subcutaneous adipose tissue, or cells contained in adipose tissue in vivo derived from stem cells such as mesenchymal hepatocytes and ES cells. Refers to a similar cell or cell population. Usually, adipose tissue-derived cells are adipose tissue-derived stem cells, adipose tissue-derived stromal cells, the above-mentioned adipose tissue-derived multilineage progenitor cells, adipose precursor cells or cells similar thereto, or all or A cell population containing a mixture of parts. Adipose tissue-derived cells can be obtained from adipose tissue and the like by means and methods known to those skilled in the art. Further, the obtained adipose tissue-derived cells may be grown using means / methods known to those skilled in the art, for example, to stabilize the character. Adipose tissue-derived cells can be transformed into a medium containing dexamethasone and ascorbic acid, such as ITS (10.0 mg / L insulin, 5.5 mg / L transferrin, 6.7 ng sodium selenite), 1 nM dexamethasone, 0.1 mM ascorbic acid, Adipose tissue-derived cells may be grown in 60% DMEM (low glucose) and 40% MCDB201 medium supplemented with 10 ng / mL rhEGF and 40% MCDB201 in a culture vessel such as a fibronectin-coated dish. .

  The animal species from which the adipose tissue-derived cells are derived is not particularly limited, and preferably, for example, mammals including mice, rats, rabbits, dogs, cats, cows, horses, monkeys, etc., more preferably humans. More preferably, the obtained hepatic lobule-like cell cluster is used for the same animal species as that for which the disease is to be prevented or treated, or adipose tissue-derived cells of the same individual. Since a sufficient amount of adipose tissue is present in the living body and can be obtained relatively easily, the present invention is very superior to, for example, a method for obtaining liver lobule from a limited material such as a cadaver. For example, the above-described method of the present invention is performed using self-derived adipose tissue-derived cells, and the obtained hepatic lobule-like cell cluster is self-transplanted, thereby reducing liver function such as cirrhosis without fear of rejection. It is possible to treat the resulting disease.

  The hepatic lobule-like cell mass refers to a hepatic lobule in a living body and a cell mass having a function / morphology similar thereto, and includes, for example, hepatocytes, bile duct epithelial cells, endothelial cells, Kupffer cells, hepatic stellate cells and the like. The hepatic lobule-like cell cluster of the present invention is very superior to individual hepatocytes in that it can produce a sufficient amount of secreted protein, has high metabolic ability, has high detoxification ability, and the like. Further, by forming a cell mass, there is an advantage that it is easy to use for the purpose of, for example, transplantation.

  The method for obtaining a hepatic lobule-like cell cluster from adipose tissue-derived cells of the present invention preferably includes a step of forming a hepatic lobule-like cell cluster from undifferentiated cells as an important step. The method for obtaining a hepatic lobule-like cell cluster from an adipose tissue-derived cell of the present invention preferably further includes a step of obtaining undifferentiated cells from the adipose tissue-derived cell as an important step. These steps may be performed continuously or in parallel.

  An undifferentiated cell refers to a cell that can differentiate into a wide variety of cells, for example, hepatic progenitor cells, pancreatic progenitor cells, myocardial progenitor cells, vascular endothelial progenitor cells, osteoblasts, chondroblasts, and the like. The step of obtaining undifferentiated cells may further include a step of growing the obtained undifferentiated cells. By proliferating undifferentiated cells, it is possible to increase the formation efficiency of hepatic lobule-like cell clusters, increase the number of hepatic lobule-like cell clusters obtained, and the like. The step of obtaining undifferentiated cells is performed using a known method such as a method based on an antigen-antibody reaction such as sorting, MACS, or rosetta formation, a density gradient method, a morphological sorting method, or single cell cloning. Alternatively, it may be performed by culturing adipose tissue-derived cells in a suspended state to form adipospheres. Since already differentiated cells can be killed and undifferentiated cells can survive and proliferate, it is preferable to culture adipose tissue-derived cells in a suspended state to form adipospheres. The culture in the suspended state will be described later. Here, an adiposphere is defined as a spherical body containing undifferentiated cells as a main component. The formation of adiposphere and the subsequent differentiation into hepatic lobule-like cell mass can occur sequentially or simultaneously, so that adipospheres are not only undifferentiated cells but also hepatocytes, bile duct epithelial cells, endothelial cells, Kupffer cells, hepatic stellate It may contain cells and the like.

  The step of obtaining a hepatic lobule-like cell mass is performed by culturing undifferentiated cells in a medium containing, for example, fibroblast growth factor, hepatocyte growth factor, oncostatin M, epidermal growth factor, and dimethyl sulfoxide, Preferably, this step is performed by culturing undifferentiated cells in a suspended state. By suspending and culturing, a cell mass having a form similar to the liver lobule in a living body can be obtained more easily. Cultivation of undifferentiated cells in a suspended state means culturing by placing the cells in a released state by preventing or suppressing adhesion to the culture vessel. The suspension of the cells can be performed by various known means / methods. For example, the cells may be in a suspended state using a culture vessel or apparatus that has been treated to prevent or inhibit cell adhesion, or made of a material that prevents or inhibits cell adhesion. Examples of the culture container or apparatus include a low-adhesion culture container such as a silicon-treated culture container (for example, a siliconized flask) or a low-adhesion culture dish (for example, a HydroCell (CellSeed)). Alternatively, the cells may be cultured in a suspended state using a hanging drop culture method. In addition, known means and methods may be used in combination as appropriate at the start of floating or in order to continue floating. Examples of such means / methods include releasing cells with an enzyme or chelating agent such as trypsin / EDTA, collagenase, Cell Dissociation Buffer (GIBCO Invitrogen), physically scraping cells using a scraper, or the like. There is a method in which cells are cultured with a temperature-responsive culture equipment for cell recovery (for example, CellSeed), and then incubated at 20 ° C. for 30 minutes to detach the cells. By culturing undifferentiated cells in a suspended state, the hepatic lobule-like cell mass is formed.

  In another aspect, the present invention relates to a hepatic lobule-like cell cluster obtainable by the above method. As described above, the hepatic lobule-like cell cluster of the present invention includes hepatocytes. For example, the above method is performed using a subject collected from a disease or a predisposition to a disease caused by a decrease in liver function such as cirrhosis or the like, or adipose tissue of the same type as the subject, and the obtained hepatic lobule-like cell cluster is used as the subject It is possible to treat or prevent diseases caused by decreased liver function such as cirrhosis.

  In a further aspect, the present invention relates to a hepatocyte contained in a hepatic lobule-like cell cluster obtainable by the above method.

  In a still further aspect, the present invention prevents or treats a disease caused by a decrease in liver function, including a hepatic lobule-like cell cluster obtainable by the above method and / or a hepatocyte contained in the hepatic lobule-like cell cluster. The present invention relates to a pharmaceutical composition. Diseases caused by decreased liver function include diseases caused by dysfunction as well as decreased liver function, such as hepatitis, cirrhosis, liver cancer, liver failure, drug-induced liver injury, alcoholic liver injury, inborn metabolism Abnormalities, cholestatic liver disorders, etc. In the pharmaceutical composition of the present invention, the hepatic lobule-like cell cluster or hepatocyte may be suspended in a suitable solution such as PBS. Further, the pharmaceutical composition of the present invention contains, in addition to hepatic lobule-like cell clusters or hepatocytes, substances that promote their engraftment in the liver, liver function improving agents, appropriate additives, excipients and the like. May be.

  In another aspect, the present invention provides a liver lobule-like cell cluster obtainable by the above culture method and / or hepatocytes contained in the liver lobule-like cell cluster, which are administered to a subject. The present invention relates to a method for treating or preventing a disease caused by a decrease. From the viewpoint of rejection and the like, preferably, hepatic lobule-like cell clusters or hepatocytes that can be obtained from the same species or from adipose tissue-derived cells are used in the present invention. The hepatic lobule-like cell cluster or hepatocyte may be transplanted or injected into the liver, omentum, intraperitoneal cavity, intraspleen, subcutaneous, etc., for example, under the renal capsule and portal vein. The subject may be any, a human subject, or a non-human subject, for example, a mammal such as a mouse or monkey. The dose and the number of administrations of the hepatic lobule-like cell cluster or hepatocyte are appropriately selected according to various factors such as the condition of the subject and the severity of the disease.

  The present invention further relates to a method for reducing blood bilirubin concentration, comprising administering hepatic lobule cell mass and / or hepatocytes contained in such hepatic lobule-like cell mass. Such a method may be performed either in vivo or in vitro.

  In yet another embodiment, the present invention is characterized in that when a fatty tissue is cultured to obtain a hepatic lobule-like cell mass, a candidate substance is added to the medium. The present invention relates to a method for screening a substance that promotes the formation of hepatic lobule, which indicates that the candidate substance is a substance that promotes the formation of hepatic lobule when it is promoted as compared with the formation in the system. As described above, since the hepatic lobule-like cell cluster contains hepatocytes, this method also includes a screening method for a substance that promotes differentiation into hepatocytes. There are various candidate substances, such as, but not limited to, basic fibroblast growth factor, hepatocyte growth factor, or oncostatin M analog or derivative. The addition of the candidate substance to the medium when obtaining the hepatic lobule-like cell mass from the adipose tissue is one of the step of obtaining undifferentiated cells from the adipose tissue-derived cells and the step of obtaining hepatic lobule-like cell masses from the undifferentiated cells, Alternatively, both may be performed once or a plurality of times.

  The formation of hepatic lobule-like cell mass can be achieved, for example, by measuring the number of hepatic lobule-like cell masses formed by microscopic observation, α-fetoprotein, albumin, etc. secreted into the culture supernatant using, for example, ELISA. Quantifying, measuring the expression of genes such as α-fetoprotein, albumin, CYP1B1, glutamine synthetase, keratin 18, keratin 19, etc. by quantitative PCR, or expression or phenomenon increases with differentiation or formation into liver lobule It can be examined by measuring a marker substance known to do, for example, transthyretin, α1-antitrypsin, tyrosine aminotransferase, glucose-6-phosphatase, etc. by quantitative PCR or ELISA.

  Therefore, in a further aspect, the present invention relates to a substance that promotes the formation of hepatic lobule, which can be obtained by the above screening method. Such a substance may be used in the method for obtaining a hepatic lobule-like cell mass from the adipose tissue-derived cell of the present invention to increase the number of hepatic lobule-like cell mass obtained or increase the formation rate of the hepatic lobule-like cell mass. You may let them. Alternatively, such a substance may be used for treating or preventing a disease caused by a decrease in liver function.

  Still another embodiment of the present invention is characterized in that when a fatty tissue-derived cell is cultured to obtain a hepatic lobule-like cell mass, a candidate substance is added to the medium, and the formation of a hepatic lobule-like cell mass is a candidate substance The present invention relates to a method for screening a substance that suppresses the formation of hepatic lobule, which indicates that the candidate substance is a substance that suppresses the formation of hepatic lobule, when the candidate substance is suppressed as compared with the formation in a non-containing system. . As described above, since the hepatic lobule-like cell cluster contains hepatocytes, this method also includes a screening method for a substance that suppresses differentiation into hepatocytes. There are various candidate substances, and examples thereof include, but are not limited to, analogs or derivatives of drugs having hepatotoxicity such as carbon tetrachloride and phenobarbital. Substances that can be obtained by such screening methods are considered to be suitable for the treatment or prevention of diseases caused by enhanced liver function. Means and methods for examining the addition of candidate substances to the medium and the formation of hepatic lobule-like cell clusters are as described above.

  Therefore, this invention relates to the substance which suppresses formation of a liver lobule which can be obtained with the said screening method in the further aspect.

  In still another embodiment, the present invention relates to a kit used in the above method for screening a substance that promotes or suppresses the formation of hepatic lobule. The kit of the present invention may include a means for obtaining cells from adipose tissue, a medium, a culture vessel, a means for examining the formation of hepatic lobule-like cell clusters, and the like. Usually, an instruction manual is attached to the kit. The screening can be performed quickly and easily using such a kit.

  In another aspect of the present invention, the hepatic lobule-like cell mass obtained by culturing adipose tissue-derived cells is cultured in a medium containing a candidate substance, and the activity of the hepatic lobule-like cell mass does not contain a candidate substance. The present invention relates to a method for screening for a substance that increases the activity of liver lobule, which indicates that the candidate substance is a substance that increases the activity of liver lobule when it is increased compared to the activity in the system. The activity of liver lobule refers to the detoxification action, protein synthesis ability, metabolic action, etc. of liver lobule.

  The increase in the activity of the hepatic lobule-like cell mass is determined by, for example, quantifying α-fetoprotein, albumin, etc. secreted into the culture supernatant using ELISA, etc., and by increasing / decreasing the amount of such protein, or α-fetoprotein, albumin, The expression of genes such as CYP1B1, glutamine synthetase, keratin 18, keratin 19 and the like can be measured by quantitative PCR and examined by increasing or decreasing the expression of such genes. There are various candidate substances, such as, but not limited to, basic fibroblast growth factor, hepatocyte growth factor, or oncostatin M analog or derivative. The addition of the candidate substance to the medium may be performed once or a plurality of times.

  Therefore, in a further aspect, the present invention relates to a substance that increases the activity of liver lobule, which can be obtained by the above screening method. Such a substance may be used in the method for obtaining a hepatic lobule-like cell mass from the adipose tissue-derived cell of the present invention to increase the activity of the obtained hepatic lobule-like cell mass. Alternatively, such a substance may be used for treating or preventing a disease caused by a decrease in liver function.

  In another aspect of the present invention, a hepatic lobule-like cell mass obtained by culturing adipose tissue-derived cells is cultured in a medium containing a candidate substance, and the activity of the hepatic lobule-like cell mass is a candidate substance-free system. The present invention relates to a method of screening for a substance that reduces the activity of liver lobule, which indicates that the candidate substance is a substance that reduces the activity of liver lobule when the activity is reduced compared to There are various candidate substances, and examples thereof include, but are not limited to, carbon tetrachloride and phenobarbital analogs or derivatives. Means and methods for adding the candidate substance to the medium and examining the activity of the hepatic lobule-like cell cluster are as described above.

  Therefore, this invention relates to the substance which reduces the activity of a liver lobule which can be obtained by the said screening method in the further aspect.

  The present invention will be described in detail and specifically with reference to examples, but the examples are not intended to limit the present invention.

Preparation of ADMPC Excess adipose tissue was removed during surgery for gastric cancer from 10 subjects (4 men and 6 women) who received informed consent. The protocol was similar to the Osaka University Graduate School of Medicine Review Boards for Human Research. All subjects were fasted for at least 10 hours. Subjects' age was 55 ± 5 years (mean ± SE; range 40-60 years). None of the subjects were taking steroids or TZD. From the subject, 1-10 g of abdominal subcutaneous (outside the fascial surface) adipose tissue and omental adipose tissue were obtained. Adipose tissue was minced and then digested in Hanks buffered saline (HBSS) containing 0.075% collagenase (Sigma Chemical Co.) for 1 hour with shaking in a 37 ° C. water bath. The digested product was filtered through Cell Strainer (BD Bioscience) and centrifuged at 800 g for 10 minutes. Using Lymphoprep (d = 1.077) (Nycomed), erythrocytes were removed by a specific gravity method, and the resulting preadipose tissue-derived multilineage progenitor cell population was placed in DMEM containing 10% fetal calf serum (Hyclone). Sowing. Cells were allowed to attach by culturing for 24 hours, washed, and then treated with EDTA to obtain ADMPC. ADMPC was then added to human fibronectin at a density of 10,000 cells / cm ² in medium I: 60% DMEM-low glucose, 40% MCDB201, 10 μg / mL EGF, 1 nM dexamethasone, 100 μM ascorbic acid, and 5% FBS. They were seeded on a coat dish, passaged 3 to 5, and used for the experiment (FIG. 1).

Characterization of ADMPC Gene expression in ADMPC was examined by RT-PCR. As a control, adipose tissue-derived stem cells (hereinafter referred to as “ADSC”) obtained by the method described in JP-T-2005-502352 were used. RT-PCR was performed as follows. Total RNA was isolated from the obtained cells using a Mag-Extractor kit (TOYOBO) according to the manufacturer's recommended protocol. 500 ng of total RNA was treated with DNase, and cDNA was synthesized using Superscript III reverse transcriptase RNase H (−) (Invitrogen). RT-PCR was performed for Islet-1, Nkx2.5, GATA-4, α-CA, MLC, MHC, and GAPDH using KOD-plus (TOYOBO) under the following conditions: 94 ° C. for 2 minutes Denaturation, followed by denaturation at 94 ° C. for 15 seconds, annealing at a predetermined temperature for 30 seconds, and extension at 68 ° C. for 30 seconds for 40 cycles. The annealing temperature and primer sequence for each gene are shown in Table 1. The obtained amplification product was electrophoresed on a 2% agarose gel. The results are shown in FIGS. It was confirmed that ADMPC expresses Islet-1, which is an undifferentiated marker, and does not express α-CA and MLC, which are markers of cardiomyocytes.

  Furthermore, real-time PCR (details will be described later) was performed on the ADMPC using an Applied Biosystems 7900 Fast Real-Time PCR system, and the expression of undifferentiated markers Sca-1 and ABCG2 was examined. The TaqMan probe used is shown in Table 2. The results are shown in FIGS. It was confirmed that ADMPC expresses Sca-1 and ABCG2.

Five ADMPCs (2 × 10 ⁵ ) were fixed with a 0.5% formalin-PBS solution at 4 ° C. for 30 minutes. A primary antibody (anti-SSEA-4 antibody (Chemicon)) diluted 50 times with PBS was added and reacted at 4 ° C. for 45 minutes. After the reaction, a secondary antibody (anti-mouse IgG antibody-PE (Becton Dickinson)) was added, reacted at 4 ° C. for 45 minutes, and FACS analysis was performed. As a control, a mouse IgG antibody (Becton Dickinson) was used as a primary antibody. The results are shown in FIG. It was confirmed that ADMPC expresses SSEA-4.

Differentiation / Induction from ADMPC into Cardioblasts ADMPC was cultured for 14 days in medium I containing DMSO (group 1) or OP9 culture supernatant (group 2), and gene expression in the resulting cells was as described above. Investigated by RT-PCR. As a control, ADMPC cultured in medium I was used. The results are shown in FIG. It was confirmed that α-CA and MLC, which are myocardial blast markers, were expressed in the cells of groups 1 and 2, that is, ADMPC was differentiated and induced into cardiomyocytes.

Examination of differentiation / induction period into cardiomyocytes Next, ADMPC was cultured in the presence of DMSO for 1, 2, 3, 4, 5, 7, 10, 14 days to examine differentiation into cardiomyocytes. ADMPC and cardiomyocytes were used as controls. The results are shown in FIGS. It was found that cardiomyocytes can be obtained by culturing in the presence of DMSO.

Preparation of sheet containing cardiac myoblasts The obtained cardiac myoblasts were cultured at 37 ° C. in a temperature-sensitive culture dish (provided by Cellseed Co., Ltd.) in medium I containing DMSO to form cell clusters. By incubating at 20 ° C. or lower for 30 minutes, the cell mass was detached, and a sheet containing cardiomyocytes was obtained (see FIG. 13). The obtained sheet was used for the following transplantation experiments.

Transplantation of sheet into myocardial infarction model rat A myocardial infarction model rat was prepared by ligating the coronary artery of a nude rat. Next, a sheet containing myocardial blasts was transplanted into the injured area. Cardiac function at 2 weeks and 10 weeks after transplantation was measured with echo to measure end diastolic diameter (LVDd), end systolic diameter (LVDs), left ventricular ejection fraction (% EF), and left ventricular diameter shortening ratio (% FS) And evaluated. As a control, a sheet containing ADMPC formed as described above was used. The results are shown in FIGS. Wall motion was confirmed in rats 2 and 10 weeks after transplantation of the sheet containing cardiac myoblasts, and it was found that cardiac function was remarkably improved. In contrast, in a rat transplanted with a sheet containing ADMPC, wall motion was confirmed after 2 weeks, but not after 10 weeks.

Tissue analysis of heart transplanted with sheet Rats were sacrificed 12 weeks after transplantation, and the heart was removed. The excised heart was fixed with 4% paraformaldehyde solution and then replaced with 70% ethanol. The fixed heart was cut out to a width of several millimeters and hardened with paraffin to prepare a block. The obtained paraffin block was sliced into 2 μm using a microtome, attached to a slide glass, and dried. The obtained thin sections were stained with hematoxylin and eosin and immunohistochemically as follows.

1. Hematoxylin and eosin staining Thin sections were deparaffinized and washed with water. The plate was stained with hematoxylin solution for 10 minutes, and colored with lukewarm water for 3 minutes. After washing with water, it was stained with eosin for 5 minutes. Fractionated with alcohol and dehydrated. The sample was sealed with xylene, sealed, and observed with a microscope. The results are shown in FIGS. It was confirmed that the transplanted sheet was engrafted.

2. Immunohistochemical staining Thin sections were deparaffinized and washed with water. Immunostimulation treatment was performed, and the cells were immersed in TBS-T supplemented with 10% normal goat serum and blocked at 4 ° C. for 24 hours. After washing with TBS-T, a primary antibody diluted 100-fold with TBS-T supplemented with 10% normal goat serum was added dropwise to the sample and reacted at 37 ° C. for 1 hour. As primary antibodies, anti-human α-cA antibody (American Research Products, Inc), anti-human MHC antibody (Upstate cell signaling solutions) and anti-human HLA-ABC antibody (Hokudo Co., Ltd.) were used. After washing with TBS-T, simple stain rat MAX-PO (Nichirei Bioscience Co., Ltd.) was added dropwise and reacted at room temperature for 30 minutes. After washing with TBS-T, a simple stain AEC solution (Nichirei Bioscience Co., Ltd.) was added dropwise, and color was developed with a microscope. After washing with water, nuclear staining was performed by staining with hematoxylin solution for 3 minutes. After washing with water, a water-insoluble mounting agent (Nichirei Bioscience Co., Ltd.) was dropped, sealed with a cover glass, and observed with a microscope. The results are shown in FIGS. It was confirmed that the site where the sheet containing the myocardial blasts was transplanted was HLA-ABC positive, that is, human-derived, and expressed α-CA and MHC. Therefore, it was found that the transplanted cardiomyocytes were transdifferentiated into cardiomyocytes.

Confirmation of pluripotency of ADMPC Differentiation ability into pancreatic cells ADMPC was differentiated into pancreatic endocrine cells by the method described in International Publication WO2007 / 039986. The obtained pancreatic endocrine cells are shown in FIG. It was confirmed that ADMPC can differentiate into pancreatic endocrine cells, that is, has a function as a pancreatic progenitor cell. In addition, the efficiency of differentiation from such ADMPC to pancreatic endocrine cells was higher than that from ADSC (data not shown).

2. Differentiation ability into hepatocytes DMSO (0.1%), HGF (10 ng / mL), bFGF (10 ng / mL), and onco known to differentiate hepatic progenitor cells into hepatoblasts and hepatocytes Statin M (10 ng / mL) was added to Medium I to prepare Medium II. ADMPC was cultured in medium II for 14 days to obtain hepatocytes. The obtained hepatocytes are shown in FIG. It was confirmed that ADMPC can differentiate into hepatocytes, that is, has a function as a hepatic progenitor cell. In addition, the efficiency of differentiation from ADMPC to hepatocytes was higher than that from ADSC (data not shown).

  Furthermore, the obtained hepatocytes were subjected to real-time PCR (details will be described later) using an Applied Biosystems 7900 Fast Real-Time PCR system, and α-fetoprotein (AFP), a marker indicating differentiation into hepatocytes, albumin, The expression of CYP1B1 and glutamine synthase was examined. As a control, ADMPC was used. The results are shown in FIGS. It was confirmed that the expression of these genes was elevated in the obtained hepatocytes.

3. Ability to differentiate into adipocytes ADMPC was cultured using a PPAR-γ agonist, which is an adipocyte differentiation agent, and differentiated into adipocytes. The obtained fat cells were stained with oil red O, and the lipid content was measured. ADSC was used as a control. The results are shown in FIG. It was found that ADMPC can differentiate into adipocytes, that is, has a function as a preadipocyte. Moreover, the efficiency of such differentiation was higher than that of ADSC. From the above, it was confirmed that ADMPC is a cell having the ability to differentiate into multilineage cells.

Isolation and culture of adipose tissue-derived cells Human adipose tissue was minced into fragments of 2-3 mm ² in size and digested with collagenase I. The digest was cultured in DMEM containing 10% FBS and antibiotics for 24-36 hours and treated with 0.02% EDTA to obtain adipose tissue-derived cells. The obtained adipose tissue-derived cells were treated with 60% DMEM (low glucose), 40% MCDB201, 1 × ITS (10.0 mg / L insulin, 5.5 mg / L transferrin, 6.7 ng sodium selenite), 10 ng / mL. The culture was expanded by subculturing 3 to 5 in a fibronectin-coated dish in a medium containing rhEGF, 1 nM dexamethasone, 0.1 mM ascorbic acid, and 5% FCS (Hyclone). A microscopic image of adipose tissue-derived cells is shown in FIG.

Acquisition of undifferentiated cells from adipose tissue-derived cells Adipose tissue-derived cells were dissociated by treatment with 0.25% trypsin / EDTA (Nacalai Tesque), and the singulated cells were treated with 20% FCS, 1 mM glutamine (GIBCO Invitrogen ) And 1% non-essential amino acid (GIBCO Invitrogen) in a knockout DMEM (GIBCO Invitrogen), the cells were cultured in a low adhesion culture dish (Hydrocell; cell seed) for 2 to 3 days. The cells self-aggregated and formed adipospheres. A microscope image of the adiposphere is shown in FIG.

Formation of hepatic lobule-like cell mass from undifferentiated cells The obtained adipocytes were washed 2 to 3 times with PBS (centrifuged at 1000 to 1200 rpm), 60% DMEM (low glucose), 40% MCDB201, 1 XITS, 1 nM dexamethasone, 100 μM ascorbic acid, 10 ng / mL rhEGF, bFGF, HGF, and OSM (Oncostatin M) were cultured in a low adhesion culture dish (Hydrocell; cell seed) for 3 to 4 weeks. From the 10th day after the start of the culture, 0.1% DMSO was added to obtain a hepatic lobule-like cell cluster. A microscopic image of the obtained hepatic lobule-like cell cluster is shown in FIG.

Hepatocyte gene expression characteristics of hepatic lobule-like cell cluster Extraction of RNA RNA was extracted from the hepatic lobule-like cell cluster using RNeasy Protect Mini Kit (QIAGEN) as follows. The hepatic lobule-like cell mass was collected, and buffer RLT containing 10 μl / ml 2-mercaptoethanol (Naacalai Tesqu) was added at a rate of 600 μl / 10 ⁷ cells. The cells were homogenized by pipetting with a 20G needle, and then 600 μl of 70% ethanol was added. 700 μl of the obtained mixed solution was transferred to an RNeasy Mini column in a 2 ml collection tube, and centrifuged at 1000 rpm for 15 seconds. Next, 350 μl of buffer RW1 was added to the column and centrifuged at 1000 rpm for 15 seconds. 10 μl of DNase I stock solution (QIAGEN) was added to 70 μl of buffer RDD, mixed by inversion, added to the RNeasy silica gel membrane in the RNeasy Mini column, and incubated at room temperature for 15 minutes. 350 μl of buffer RW1 was added and centrifuged at 1000 rpm for 15 seconds. The 2 ml collection tube was replaced with a new one. 500 μl of buffer RPE was added to the column and centrifuged at 1000 rpm for 15 seconds. Furthermore, 500 μl of buffer RPE was added, centrifuged at 1000 rpm for 2 minutes, and then centrifuged at 1500 rpm for 1 minute. The column was transferred to a 1.5 ml collection tube, RNase free water 30-50 μl was added to the RNeasy silica gel membrane, and centrifuged at 1000 rpm for 1 minute to extract RNA.

2.1 Preparation of single-stranded cDNA To 11.5 μl of the extracted RNA solution, 0.5 μl of 0.5 mM Random Primer (Invitorogen) and 1 μl of 10 mM dNTPmix (Invitorogen) were added and reacted at 65 ° C. for 5 minutes, and then And cooled on ice. 4 μl of 5 × First-Strand buffer (Invitorogen), 1 μl of 0.1 M DTT (Invitorogen), 1 μl of RNaseOUT (Invitorogen), and 1 μl of SuperScript III RT (Invitorogen) are added to the resulting mixture at 25 ° C. for 5 minutes. Reaction was performed at 60 ° C. for 60 minutes and at 70 ° C. for 15 minutes to prepare single-stranded cDNA. The obtained cDNA was stored at 4 ° C. until use.

3. Real-time PCR
To 9 μl of the prepared cDNA, 10 μl of TaqMan Universal PCR Master Mix (Applied Biosystems) and 1 μl of TaqMan Gene Expression Assays (Applied Biosystems) were added. Real-time PCR was performed on an Applied Biosystems 7900 Fast Real-Time PCR system under the following conditions: denaturation at 95 ° C. for 10 minutes, 95 ° C. for 15 seconds and 60 ° C. for 1 minute for 40 cycles. Table 3 below shows TaqMan probes used for α-fetoprotein, albumin, CYP1B1, CYP2B6, glutamine synthetase, keratin 18 and keratin 19. Moreover, GAPDH (Applied Biosystems) was used as an internal standard. As control samples, hepatocytes obtained by the method described in Seo MJ. Et al., 2005 Mar 4; 328 (1): 258-64 (referred to as “conventional method”), fibroblasts, HepG2, and adipose tissue Derived cells (denoted “ADSC”) were used. The results are shown in FIGS. 38 to 43 (the vertical axis in the figure indicates the fluorescence intensity). The hepatic lobule-like cell cluster obtained by the method of the present invention is higher in α-fetoprotein, albumin, keratin 18, keratin 18, keratin 19, CYP1B1 and glutamine synthase than the hepatocytes obtained by the conventional method. It was found to express. Moreover, the expression of these genes except albumin was significantly higher than that in HepG2.

Production of hepatic proteins by hepatic lobule-like cell clusters Western blot analysis Sample preparation The hepatic lobule-like cell mass was washed three times with PBS (Nacalai Tesque), and then M-PER (PIERCE) was added. Cells were lysed by sonication and centrifuged at 14000 g for 15 minutes to remove undissolved cell components. A sample buffer (Nacalai Tesque) equivalent to the sample was added, boiled at 100 ° C. for 5 minutes, and ice-cooled. The protein concentration of the obtained sample was measured using BCA Protein Assay Reagent (PIERCE).

2. SDS-PAGE
SDS-PAGE was performed using a gel miniplate for electrophoresis (PAG mini “first”; first chemical) and running buffer. The amount of protein used was 5 μg. The electrophoresis conditions were 10 mA in the stacking gel and 40 mA in the running gel.

3. Western blot The electrophoresed gel was washed with blotting buffer for 10 minutes. Next, the protein in the gel was copied onto a nitrocellulose membrane by wet blotting (100 mA, overnight).

4). Immunostaining The membrane was washed with PBS containing 0.1% Tween 20 for 10 minutes. It was made to react at room temperature for 1 hour using blocking one (Nacalai Tesque). As a primary antibody, it was reacted with a 500-fold dilution of Human Albumin antibody (BETHYL) or Alpha Fetoprotein Ab-2 (LAB Vision) for 1 hour. Washed with PBS containing 0.1% Tween 20 for 15 minutes (3 times). As a secondary antibody, it was reacted for 1 hour with a 1000-fold dilution of polyclonal porcine anti-rabbit immunoglobulin / HRP or polyclonal rabbit anti-goat immunoglobulin / HRP. Washed with PBS containing 0.1% Tween 20 for 15 minutes (3 times). Bands were detected using ECL Plus Western Blotting Detection Reagents. The results are shown in FIGS. It was found that the hepatic lobule-like cell cluster obtained by the method of the present invention produced a sufficient amount of α-fetoprotein and albumin.

B. Immunohistochemical staining Preparation of section The hepatic lobule-like cell cluster was washed three times with PBS (Nacali Tesque) and centrifuged. The obtained pellet was embedded in Tissue-Tek OCT-compound (Sakura Fineteck Inc.) and stored at −30 ° C. A 7 μm section was prepared using a cryostat, attached to a slide glass, and stored at −30 ° C.

2. Immunohistochemical staining The above sections were dried with a dryer and fixed with 4% paraformaldehyde for 30 minutes. Washed 3 times with PBS for 5 minutes. It was made to react at room temperature for 1 hour using blocking one (Nacalai Tesque). Washed 3 times for 5 minutes with PBS. The mixture was reacted with a 400-fold diluted primary antibody polyclonal rabbit anti-human albumin antibody (DAKO) or a 300-fold diluted polyclonal rabbit anti-human α-1-fetoprotein antibody (DAKO) for 1 hour. Washed 3 times with PBS for 10 minutes. The secondary antibody AlexaFluor [trademark] 466 goat anti-rabbit IgG antibody (Molecular Probes) was diluted with a 500-fold diluted solution for 1 hour. Washed 3 times with PBS for 10 minutes. The sample was embedded using Perma Fluor (Nihon Turner) and observed with a microscope. The results are shown in FIGS. 46 and 47. The presence of α-fetoprotein and albumin in the hepatic lobule cell mass was confirmed. This revealed that the hepatic lobule cell mass actually produces these proteins.

1. Production of urea by hepatic lobule-like cell mass Urea production Hepatic lobule-like cell clusters were incubated in 5 ml of Hank's buffered salt solution (Gibco) containing 5 mM NH ₄ Cl for 2 hours. The urea concentration in 0.5 ml of the supernatant was measured using a Quantum Chure Urea Assay Kit (Bioassay Systems). The total amount of produced urea was calculated by multiplying the resulting concentration by the total volume of the supernatant. HepG2 was used as a control.

2. Measurement of DNA amount Hank's buffer salt solution containing NH ₄ Cl was removed, and the hepatic lobule-like cell mass was washed with PBS. Buffer was added, sonicated and the cells were homogenized. 1 ml of buffer solution was added to 50 μl of cell homogenate, and 50 μl of coloring solution was further added and stirred. The fluorescence value of the obtained solution was measured at excitation: 356 nm and emission: 458 nm to determine the DNA concentration. Total DNA was calculated by multiplying the DNA concentration by the volume of buffer solution added.

3. Amount of product urea The total amount of product urea obtained was divided by the amount of total DNA to determine the amount of product urea produced. The results are shown in FIG. It was found that the hepatic lobule-like cell mass produced a sufficient amount of urea compared to HepG2. This confirmed that the hepatic lobule-like cell cluster obtained by the present invention has a sufficient detoxifying action.

Transplantation effect of hepatic lobule-like cell cluster in mouse hepatitis model Creating NOD-SCID mice murine hepatitis model, the four-week twice carbon chloride (CCl ₄₎ at 300 [mu] l / kg, was injected 12 weeks intraperitoneally, was created mouse hepatitis model.

2. Transplantation of hepatic lobule-like cell mass The hepatic lobule-like cell mass was washed with Hanks buffered salt solution and centrifuged to give a pellet. The mouse was anesthetized with sevofluene. The left paramedian incision was restored, the left kidney was exposed, the kidney capsule was peeled off, and a pocket was created. The pelleted cell mass was injected into the created pocket and transplanted. The abdominal wall was closed in two layers.

3. Analysis On day 10 of transplantation, blood was collected from the mice, and the blood bilirubin concentration was measured. The results are shown in FIG. It was found that the blood bilirubin concentration was reduced in the group transplanted with hepatic lobule-like cell mass compared to the group not transplanted. As a result, it was found that the hepatic lobule-like cell cluster obtainable by the method of the present invention is effective in treating diseases caused by a decrease in liver function.

  According to the present invention, a sheet containing cardiac myoblasts, a method for obtaining myocardial blasts and a sheet containing cardiac myoblasts, and the like can be obtained. This is useful in screening for substances to be improved.

FIG. 1 is a microscopic image of ADMPC obtained from adipose tissue. FIG. 2 shows the results of RT-PCR when ADMPC and ADSC are passaged three times. FIG. 3 shows the results of RT-PCR when ADMPC was passaged 6 times. FIG. 4 is a graph showing the expression level of Sca-1 in ADMPC. FIG. 5 is a graph showing the expression level of ABCG2 in ADMPC. FIG. 6 shows the results of FACS analysis showing the expression of SSEA-4 in ADMPC. FIG. 7 shows the results of RT-PCR in cells cultured in the presence of DMSO or OP9 culture supernatant. FIG. 8 is a graph showing the expression of Nkx2.5 in cells cultured in the presence of DMSO. FIG. 9 is a graph showing the expression of GATA-4 in cells cultured in the presence of DMSO. FIG. 10 is a graph showing the expression of α-CA in cells cultured in the presence of DMSO. FIG. 11 is a graph showing the expression of MLC in cells cultured in the presence of DMSO. FIG. 12 is a graph showing the expression of MHC in cells cultured in the presence of DMSO. FIG. 13 is a strongly magnified image of a sheet containing cardiac myoblasts. FIG. 14 is echocardiograms at 2 weeks and 10 weeks after transplantation of a sheet containing cardiac myoblasts. The longer arrow represents diastole and the shorter arrow represents systole. FIG. 15 is an echocardiogram at 2 weeks and 10 weeks after transplantation of a sheet containing ADMPC. The longer arrow represents diastole and the shorter arrow represents systole. FIG. 16 is a graph showing an improvement in LVDd of a heart transplanted with a sheet containing cardiomyocytes (circle) and a sheet containing ADMPC (square). FIG. 17 is a graph showing an improvement in LVDs of a heart transplanted with a sheet containing cardiomyocytes (circle) and a sheet containing ADMPC (square). FIG. 18 is a graph showing improvement in% EF of a heart transplanted with a sheet containing cardiomyocytes (circle) and a sheet containing ADMPC (square). FIG. 19 is a graph showing the improvement in% FS of a heart transplanted with a sheet containing cardiomyocytes (circle) and a sheet containing ADMPC (square). FIG. 20 is an HE-stained image (100 ×) of a heart transplanted with a sheet containing cardiac myoblasts. FIG. 21 is an HE-stained image (100 times) of a heart transplanted with a sheet containing ADMPC. FIG. 22 is a microscopic image (100 ×) when a heart transplanted with a sheet containing cardiac myoblasts is immunostained with an anti-human α-CA antibody. FIG. 23 is a microscopic image (100 ×) when a heart transplanted with a sheet containing ADMPC is immunostained with an anti-human α-CA antibody. FIG. 24 is a microscopic image (100 ×) when a heart transplanted with a sheet containing cardiac myoblasts is immunostained using an anti-human MHC antibody. FIG. 25 is a microscopic image (100 ×) when a heart transplanted with a sheet containing ADMPC is immunostained with an anti-human MHC antibody. FIG. 26 is a microscopic image (100 times) when a heart transplanted with a sheet containing cardiomyocytes is immunostained using an anti-human HLA-ABC antibody. FIG. 27 is a microscopic image (100 ×) when a heart transplanted with a sheet containing ADMPC is immunostained with an anti-human HLA-ABC antibody. FIG. 28 is a microscopic image of pancreatic endocrine cells obtained by differentiating ADMPC. FIG. 29 is a microscopic image of hepatocytes obtained by differentiating ADMPC. FIG. 30 is a graph showing the expression level of α-fetoprotein in hepatocytes obtained by differentiating ADMPC. FIG. 31 is a graph showing the expression level of albumin in hepatocytes obtained by differentiating ADMPC. FIG. 32 is a graph showing the expression level of CYP1B1 in hepatocytes obtained by differentiating ADMPC. FIG. 33 is a graph showing the expression level of glutamine synthetase in hepatocytes obtained by differentiating ADMPC. FIG. 34 is a graph showing the lipid content contained in adipocytes obtained by differentiating ADMPC. FIG. 35 is a microscopic image of the obtained adipose tissue-derived cells. FIG. 36 is a microscopic image of the obtained adiposphere. FIG. 37 is a microscopic image of the obtained hepatic lobule-like cell cluster. FIG. 38 is a graph showing the results of quantitative RT-PCR for α-fetoprotein. FIG. 39 is a graph showing the results of quantitative RT-PCR for albumin. FIG. 40 is a graph showing the results of quantitative RT-PCR for keratin 18. FIG. 41 is a graph showing the results of quantitative RT-PCR for keratin 19. FIG. 42 is a graph showing the results of quantitative RT-PCR for CYP1B1. FIG. 43 is a graph showing the results of quantitative RT-PCR for glutamine synthetase. FIG. 44 shows the results of Western blot analysis for α-fetoprotein produced by the hepatic lobule-like cell cluster. FIG. 45 shows the results of Western blot analysis for albumin produced by the hepatic lobule-like cell cluster. FIG. 46 is a microscopic image showing the presence of α-fetoprotein in the hepatic lobule-like cell cluster by immunohistochemical staining. FIG. 47 is a microscopic image showing the presence of albumin in the hepatic lobule-like cell cluster by immunohistochemical staining. FIG. 48 is a graph showing the amount of urea produced by the hepatic lobule-like cell cluster. FIG. 49 is a graph showing the effect of transplanting hepatic lobule-like cell clusters in a hepatitis mouse model.

SEQ ID NO: 1: Islet-1 forward primer
SEQ ID NO: 2: Islet-1 reverse primer
SEQ ID NO: 3: Nkx2.5 forward primer
SEQ ID NO: 4: Nkx2.5 reverse primer
SEQ ID NO: 5: GATA-4 forward primer
SEQ ID NO: 6: GATA-4 reverse primer
SEQ ID NO: 7: α-CA forward primer
SEQ ID NO: 8: α-CA reverse primer
SEQ ID NO: 9: MLC2v forward primer
SEQ ID NO: 10: MLC2v reverse primer
SEQ ID NO: 11: MHC forward primer
SEQ ID NO: 12: MHC reverse primer
SEQ ID NO: 13: GAPDH forward primer
SEQ ID NO: 14: GAPDH reverse primer

Claims (4)

A method for producing a sheet containing cardiac myoblasts,
The following process:
(A) differentiating adipose tissue-derived stem cells into cardiac myoblasts in the presence of DMSO; and (b) forming a sheet containing cardiac myoblasts;
Including a method.   The method according to claim 1, wherein the adipose tissue-derived stem cell is ADMPC. Comprising a step of differentiating adipose tissue-derived stem cells into cardiac myoblasts in the presence of DMSO.
A method for producing myoblasts . The method according to claim 3, wherein the adipose tissue-derived stem cell is ADMPC.

Images