JP2005224155A - Cell having ability of differentiating automatically pulsating cardiomyocyte isolated from tongue tissue and method for cell culture and differentiation induction - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain cells for creating a pulsating cardiomyocyte from self-tongue tissue, to provide a method for cell culture and differentiation induction and to greatly contribute to regeneration medicine. <P>SOLUTION: A pulsating cardiomyocyte is induced by collecting CD34 cells of SCA1 positive from tissue collectable even from human by a biopsy, especially from self-tongue stem cells, using a medium such as a growth factor such as a blood platelet-derived growth factor and an epithelial cell growth factor without using a demethylation agent and by a method for applying centrifugal loading as an extracellular environmental factor in culture to the cells. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a cell for creating beating cardiomyocytes from self-tongue tissue, and a method for isolating, purifying, culturing, and inducing differentiation of the cell. The present invention also relates to a method for growing cells capable of differentiating into cardiomyocytes and a method for controlling differentiation into cardiomyocytes using various cytokines, adhesion factors, and the like.

  Causes of heart failure include myocardial infarction, myocarditis, dilated cardiomyopathy, secondary cardiomyopathy, hypertension, and the like, and in recent years, the number of those due to myocardial infarction has increased due to westernization of lifestyle. Treatment of heart failure is mainly pharmacotherapy using cardiotonic drugs, angiotensin converting enzyme inhibitors, angiotensin receptor antagonists, β-blockers, aldosterone antagonists, diuretics, etc. In many cases, or when the degeneration of the myocardium itself is significant, there is only an effective treatment for heart transplantation. However, heart transplantation is difficult in particular in Japan, and it has been several years since heart transplantation has been performed, but there are still only a few dozen treatment examples. Therefore, embryonic stem cells and self-organized stem cells, especially stem cells in autologous bone marrow, have been tried to induce cardiomyocyte differentiation. However, the efficiency of induction into cardiomyocytes is extremely low, and reproducibility is poor. It does not form an electrical connection with its own cardiomyocytes, can cause arrhythmias, and there are ongoing ethical oppositions to use fetal and neonatal cardiomyocytes as a source of cells. Although a cell source is sought, an innovative cell source has not yet been found. In recent years, it has been reported that cells having the ability to differentiate into cardiomyocytes were obtained from adult mouse bone marrow (Non-patent Documents 1 and 2). Further, a patent has been filed regarding a cell having an ability to differentiate into at least cardiomyocytes isolated from living tissue, particularly bone marrow (Patent Document 1). However, since the above-described invention requires the use of a demethylating agent to obtain cells that differentiate into cardiomyocytes, there is a possibility that the chromosomal DNA is damaged, and this is a clinical application. It is considered an unbearable method. In addition, although a method has been reported in which tissue stem cells are isolated from the myocardium and myocardial infarction mice have been differentiated into myocardium (Non-patent Document 3), obtaining such cells from the heart is not possible in actual clinical practice. Is considered impossible. In addition, experiments have been conducted in which skeletal muscle progenitor cells are isolated from skeletal muscle and transplanted into the myocardium. However, cells from skeletal muscle cannot be electrically connected to the myocardium, which may cause arrhythmia. This method is also considered to have a limit (Non-Patent Document 4).

In addition, it has been found that cells derived from adult mouse muscle are differentiated into beating cardiac muscle-like cells by culturing without using a differentiation-inducing agent such as a demethylating agent.・ Identification and evaluation of genes involved in regeneration, (2) Cell transplantation of various diseases including heart failure such as heart failure and myocardial infarction, (3) Search and evaluation of compounds that promote myocardial differentiation, (4) Cell transplantation There are patents that claim to be used to search and evaluate drugs that increase efficiency (implantation of transplanted cells to living tissue and promote function), and (5) model animal development for developing therapeutic agents for heart failure ( Patent Document 2).
Japanese Patent Application 2001-548664 Japanese Patent Application No. 2003-59297 J. Clinical Investigation, 103, 10-18 (1999) Cell, 114: 763-776, (2003) PNAS, 100: 12313-12318,2003 Circulation, 107: 3088-3092, (2003)

  The present invention relates to a cell derived from a tongue tissue capable of differentiating into cardiomyocytes, its use and the like, and can be used for the treatment of human heart disease when using its own tongue tissue. In addition, transplanted animals (excluding humans) transplanted with pulsatile cardiomyocytes derived from tongue tissue capable of differentiating into cardiomyocytes can be used for screening to search for compounds that can be used as preventive / therapeutic agents for heart disease. Can also be used.

  For the first time in the world, we succeeded in inducing cardiomyocytes that pulsate with growth factors and pressure without using a demethylating agent from autologous tongue tissue stem cells. In particular, the fact that it was derived from a tissue that can be collected by a biopsy called tongue tissue is very useful, and is considered to be an invention that can greatly contribute to regenerative medicine.

  In the present invention, differentiation can be induced without using a mutagenic drug, self-pulsating cardiomyocytes can be obtained with reproducibility, and an electrical junction is formed and beats in cooperation. We have filed a patent application for a novel cell source that can measure intracellular calcium transients and a method for inducing differentiation thereof.

  The ones that have been reported to be able to induce cardiomyocytes from their own tissue stem cells are pulsating cells that happened to appear while administering a demethylating agent to stem cells in the bone marrow and repeating frequent cultures. However, it is difficult to repetitively separate such cells from bone marrow. In addition, pulsating cardiomyocytes can be induced relatively easily from embryonic stem cells (ES cells), but this is not an autologous cell but has major immunological and ethical problems, and is currently used clinically. Can not. The present inventors have invented a method that can induce differentiation of beating cardiomyocytes in a first culture, at a high rate, with high reproducibility, and without using a mutagenic demethylating agent.

  The cells of the present invention are cells that have the ability to differentiate into autopulsating cardiomyocytes isolated from tongue tissue. In addition, this cell is a cell that can differentiate into cardiomyocytes that form electrical cell-cell communication, and is a cell that can differentiate into at least cardiomyocytes and skeletal muscle cells. These cells include cells that are positive for cell surface markers specific to stem cells (eg, SCA1), cells that are CD38 negative or positive, and cells that are CD31 negative or positive. Also included are myocardial progenitor cells that are specifically induced to differentiate into cardiomyocytes, cells that have the ability to differentiate into ventricular myocytes, and cells that have the ability to differentiate into sinus node cells. The tongue tissue is derived from mammals, and specifically can be selected from mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cows, goats, monkeys and humans.

  Cells that have the ability to differentiate into cardiomyocytes without the need for chromosomal DNA demethylating agents can increase the frequency of pulsating cells by pressure overload culture. Moreover, the cytokine used for culture is a group consisting of platelet-derived growth factor (PDGF), epithelial cell growth factor (EGF), leukemia inhibitory factor (LIF), and osteogenic factors 2, 4 (BMP-2, 4), Wnt. Is at least one selected from The adhesion molecule used for the culture is at least one selected from the group consisting of gelatin, laminin, collagen and fibronectin. Expression of a transcription factor that confirms differentiation into cardiomyocytes is at least one selected from the group consisting of Nkx2.5, GATA4, MEF-2C, ANP, BNP, Cx43, α-myosin heavy chain, MLC2a, MLC2v, and troponin I It is a seed. The extracellular environment factor that promotes differentiation into cardiomyocytes is at least one selected from factors dependent on extracellular calcium concentration and factors dependent on intracellular calcium concentration. Moreover, it is a cell via calcium-calmodulin kinase as a factor depending on intracellular calcium concentration as an extracellular environment factor that promotes differentiation into cardiomyocytes. Furthermore, it is a cell through a signal from Wnt as a factor that regulates intracellular calcium concentration as an extracellular environment factor that promotes differentiation into cardiomyocytes. Furthermore, it is a cell having the ability to differentiate into cardiomyocytes by transplanting into the heart.

  The cell culture method of the present invention includes a method of culturing under pressure as an extracellular environment factor that promotes differentiation into cardiomyocytes, and a method of culturing under a centrifugal load as an extracellular environment factor that promotes differentiation into cardiomyocytes. Use.

  The method of measuring intracellular calcium dynamics and calcium transients using a fluorescent dye such as fluo3 or Fura2 for the beating myocardium obtained by the present invention, the fluo3 fluorescent dye for the electrical coupling between beating cardiomyocytes The method of confirming using and the method of confirming the electrical connection between the beating cardiomyocytes using the immunostaining of Cx43 are also included in the present invention.

An adult tongue tissue is treated with collagenase or the like to form isolated cells, labeled with an antibody recognizing a stem cell marker, and labeled with magnetic beads as a secondary antibody to separate positive cells. LIF, EGF, PDGFbb and the like are added to DMEM medium supplemented with 10% FCS, and cultured at 37 ° C. under 5% CO ₂ . At this time, a special incubator is used. That is, it culture | cultivates in the pressure | voltage resistant incubator which can apply a high pressure in an incubator. After culturing, rod-like and spherical cell masses appear, these cells fuse together, cause a certain automatic pulsation without electrical stimulation, and striates are observed, confirming that they differentiate specifically into cardiomyocytes. It was. Furthermore, these cells were confirmed to express the myocardium-specific genes NKX2.5, ANP, and connexin 43 by immunohistological studies and gene expression by RT-PCR. In addition, immunostaining with myosin, Cx 34, and troponin I confirmed that the cells had differentiated into myocardium, and these cells bound via gap junctions and confirmed that each cell can beat in synchronization. confirmed. Furthermore, intracellular calcium transients were loaded with the calcium-sensitive fluorescent dye Fluo3 and observed with a laser confocal microscope. As a result, intracellular calcium transients similar to those seen in cardiomyocytes were observed. When these cells were cultured for 2 weeks, they became sheet-like, and the whole sheet was observed to beat in synchronization. From the above, it was proved that the tongue-derived stem cells specifically differentiate into cardiomyocytes, have the ability to self-pulsate, and cause pulsation and electrical phenomena synchronously through intercellular junctions. Furthermore, it was proved that this cardiomyocyte differentiation can be induced with reproducibility without using harmful drugs having mutagenicity. Such self-pulsating cells can be obtained, and a pulsating myocardial sheet can be produced from these cells, which is considered to be very useful as a stem cell source that can be used for transplantation.

  We succeeded in inducing cardiomyocytes pulsating from self-organizing stem cells by growth factors and pressure loading without using a demethylating agent. In particular, the fact that it was derived from a tissue that can be collected from humans by a biopsy called tongue tissue is very useful and is considered to be an invention that can greatly contribute to regenerative medicine.

  Embodiments of the invention will be described with reference to the drawings based on examples.

The mouse tongue tissue is treated with 0.2% collagenase for 30 minutes to form isolated cells, labeled with a monoclonal antibody against SCA-1, a well-known stem cell marker, and labeled with magnetic beads as a secondary antibody. Then, positive cells were separated (using a commercial kit manufactured by Miltenyi). Next, LIF, EGF and PDGFbb were added to DMEM medium supplemented with 10% FCS, and the separated SCA-1 positive cells were cultured at 37 ° C. under 5% CO ₂ . At this time, a special incubator was used, and the incubator was cultured under a pressure of 110 mmHg. Seven days later, many rod-shaped cells and spherical cardiospheres appeared, these fused, developed a certain automatic pulsation without electrical stimulation, striated pattern was observed, and differentiated specifically into cardiomyocytes. Confirmed to do. Furthermore, these cells express myosin, Cx 43, and troponin I by immunohistochemical examination, and the genes of NKX2.5, ANP, and connexin 43, which are myocardial specific genes, were analyzed by RT-PCR. The expression was confirmed. These cells were connected through gap junctions, and it was confirmed that each cell beats synchronously. Further, intracellular calcium transients were loaded with calcium-sensitive fluorescent dye Fluo3 and observed with a laser confocal microscope. As a result, intracellular calcium transients consistent with cardiomyocytes were observed. It was observed that these cells became a sheet when cultured for 2 weeks, and the entire sheet was beaten synchronously. From the above, it was proved that the tongue-derived stem cells have the ability to specifically differentiate into cardiomyocytes. Moreover, it was proved that differentiation can be induced into cardiomyocytes with reproducibility without using harmful drugs.

An example of immunostaining of myocardial cells differentiated from tongue tissue-derived stem cells (TDS) (Myosin: red, connexin 43: yellow, DAPI: blue). It is a figure which shows that connexin43 is expressing in the fused myosin positive cell. An example of immunostaining of myocardial cells differentiated from tongue tissue-derived stem cells (TDS) (Myosin: red, connexin 43: yellow, DAPI: blue). It is a figure which shows that connexin43 is expressing in the fused myosin positive cell. The results are obtained by loading calcium-sensitive fluorescent dye Fluo3 in the second week of culturing tongue tissue-derived stem cells (TDS) and observing calcium transients with a laser confocal microscope. The figure on the left shows the measurement site, and the figure on the right shows It is a figure which shows a calcium transient. It is the result of loading calcium sensitive fluorescent dye Fluo3 in the 2nd week of culturing tongue tissue-derived stem cells (TDS) and observing calcium transients with a laser confocal microscope, and measuring the calcium transients of two adjacent cells, In order to show that these are synchronized, the left figure shows the measurement site and the right figure shows the calcium transient. Fig. A and Fig. B show the transients of each cell, and Fig. C shows the transients recorded by combining the two cells. If not synchronized, a waveform similar to that in FIGS. A and B should not be observed in FIG. C, indicating that the two cells are synchronized. It is a figure which shows the result of having examined the gene expression of the beating cell (1 week, 2 weeks) differentiation-induced from the tongue tissue origin stem cell (TDS) by RT-PCR, and using adult heart, water, and a thigh muscle as a control FIG. 2 shows that NKX2.5, ANP, connexin 43 and other gene expression found only in cardiomyocytes were observed in both TDS cells cultured for 1 week and 2 weeks (on the other hand, These gene expression is not observed). Cells isolated from tongue tissue by collagenase are labeled with SCA1 monoclonal antibody, a secondary antibody bound with magnetic beads is bound, and the cells after separation with a magnetic separation column twice (TDSC) are treated with PE conjugated SCA. -1 is a diagram showing the results of analysis of cell size and SCA-1 positive rate by Fluorescent activated cell sorter (FACS). A. side scatter (vertical axis), forward scatter (vertical axis ) Shows the cell distribution. B. Histogram of SCA-1 antigen. Black indicates TDSC and gray indicates negative control. C. Negative control distribution for SCA-1. D. Shows the distribution of SCA-1 positive cells in TDSC (SCA-1 positive rate was enriched to 86%). When TDS cells were cultured for 5 days, a self-pulsating spherical cell mass was observed, which was named Cardiosphere. Cardiosphere confirms self-pulsing and also confirms the presence of intracellular calcium transients due to Fluo3 loading. These cells also fuse with rod-shaped cells (myotube) and self-pulsate. It is the figure which showed that. Cardiosphere and myotubet undergo fusion (Figs. A, B, and C), and cardiosphere and myotube that have completely fused become wide spindle-shaped cells and self-pulsate (Fig. D).

Claims (26)

A cell isolated from the tongue tissue and capable of differentiating into a beating cardiomyocyte. The cell according to claim 1, which is a cell capable of differentiating into a cardiomyocyte that forms electrical cell-cell communication. The cell according to any one of claims 1 to 2, wherein the cell differentiates into at least cardiomyocytes and skeletal muscle cells. The cell according to any one of claims 1 to 3, wherein the cell is SCA1 positive. The cell of claim 4, wherein the cell is CD34 negative. The cell according to claim 5, wherein the cell is CD38 negative or positive. The cell according to claim 6, wherein the cell is further CD31 negative or positive. A myocardial progenitor cell specifically induced to differentiate into a cardiomyocyte derived from the cell according to any one of claims 1 to 7. The cell according to any one of claims 1 to 8, which has an ability to differentiate into ventricular myocytes. The cell according to any one of claims 1 to 8, which has an ability to differentiate into a sinus node cell. The cell according to any one of claims 1 to 10, wherein the tongue tissue is derived from a mammal. The cell according to claim 11, wherein the mammal is one selected from a mouse, rat, guinea pig, hamster, rabbit, cat, dog, sheep, pig, cow, goat, monkey and human. The cell according to any one of claims 1 to 12, which has an ability to differentiate into cardiomyocytes without requiring a chromosomal DNA demethylating agent. The cell of any one of Claims 1-13 which can increase the frequency of the cell which beats by pressure-load culture | cultivation. The cytokine used for culture is at least one selected from the group consisting of platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and leukemia inhibitory factor (LIF). A cell according to 1. 16. The cell according to claim 15, wherein the adhesion molecule used for culture is at least one selected from the group consisting of gelatin, laminin, collagen and fibronectin. Expression of a transcription factor that confirms differentiation into cardiomyocytes is at least selected from the group consisting of Nkx2.5, GATA4, MEF-2C, ANP, BNP, Cx43, α-myosin heavy chain, MLC2a, MLC2v, and troponin I The cell according to claim 11, which is one type. The cell according to claim 11, wherein the cell is at least one selected from factors dependent on extracellular calcium concentration and factors dependent on intracellular calcium concentration as an extracellular environment factor that promotes differentiation into cardiomyocytes. 19. The cell according to claim 18, wherein calcium-calmodulin kinase is mediated as a factor that depends on intracellular calcium concentration as an extracellular environment factor that promotes differentiation into cardiomyocytes. The cell according to claim 19, through a signal from Wnt as a factor that regulates intracellular calcium concentration as an extracellular environment factor that promotes differentiation into cardiomyocytes. 21. The cell according to any one of claims 1 to 20, which has the ability to differentiate into cardiomyocytes by transplantation into the heart. A method of culturing under pressure as an extracellular environmental factor that promotes differentiation into cardiomyocytes. A culture method in which centrifugal loading is applied as an extracellular environmental factor that promotes differentiation into cardiomyocytes. A method for measuring intracellular calcium dynamics and calcium transients in beating myocardium using fluorescent dyes such as fluo3 and Fura2. A method of confirming electrical connection between beating cardiomyocytes using fluo3 fluorescent dye. A method for confirming electrical connection between beating cardiomyocytes using Cx43 immunostaining.