JP2015213441A - Production method of myocardium-like cells and medium used for same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method and medium which can efficiently produce beating myocardium-like cells.SOLUTION: The above subject is solved by culturing fibroblasts, into which only Mef2c and Tbx5 are introduced, with a serum-free medium containing VEGF, FGF2, and FGF10. The serum-free medium is preferred to contain further IWP4. After two weeks of introducing Mef2c and Tbx5 fibroblast, the fibroblasts into which only Mef2c and Tbx5 are introduced are preferred to be cultured with the serum-free medium containing VEGF, FGF2, and FGF10 for two weeks.

Description

  The present invention relates to a method and a medium capable of efficiently producing cardiomyocyte-like cells.

  A method has been proposed in which at least three factors (Gata4, Mef2c and Tbx5) are introduced into fibroblasts to induce differentiation directly into myocardial cells without going through iPS cells, thereby producing myocardial cells. (See Patent Document 1 and Non-Patent Documents 1 and 2).

Special table 2013-524837 gazette

M.M. Ieda et al. , “Direct Programming of Fibroblasts into Functional Cardiomyocytes by Defined Factors”, Cell, 142 (3), 375-386, 2010. R. Wada et al. "Induction of human cardiomyte-like cells from fibroblasts by defined factors", Proc Natl Acad Sci USA, 110 (31), 12667-12672, 2013.

  The techniques proposed in Patent Document 1 and Non-Patent Documents 1 and 2 are inefficient because the number of differentiation induction from fibroblasts into which at least three factors have been introduced into beating cardiac muscle-like cells is small. On the other hand, in order to reduce the risk of canceration of myocardial cells, it is necessary to develop a technology that can efficiently produce beating myocardial cells by reducing the number of factors introduced into fibroblasts. It has been.

  The present invention has been made to solve the above problems, and an object of the present invention is to provide a method and a medium capable of efficiently producing pulsating myocardial cells.

  As a result of intensive studies by the present inventors, a culture of fibroblasts into which three factors (Gata4, Mef2c, and Tbx5) have been introduced using a serum-free medium containing VEGF has been obtained by using conventional DMEM / M199. It has been found that pulsating myocardial cells can be produced more efficiently than those cultured using a medium. Further, it was found that by using a serum-free medium containing VEGF, FGF2 and FGF10, it is possible to efficiently produce pulsating myocardial cells even if the number of factors for gene transfer into fibroblasts is reduced from three to two. . Thus, the present inventors completed the present invention.

  (1) A method according to the present invention for solving the above-mentioned problem is a method for producing cardiomyocyte-like cells, wherein fibroblasts into which only Mef2c and Tbx5 have been introduced are treated with serum-free VEGF, FGF2 and FGF10 It includes a step of culturing in a medium.

  In the method according to the present invention, the serum-free medium can further comprise IWP4.

  In the method according to the present invention, the fibroblasts can be cultured in the serum-free medium for 2 weeks after the introduction of the Mef2c and Tbx5 genes.

  (2) A method according to the present invention for solving the above-mentioned problems is a method for producing cardiomyocyte-like cells, and fibroblasts into which Mef2c, Tbx5 and Gata4 have been introduced are cultured in a serum-free medium containing VEGF. Including the step of:

  In the method according to the present invention, the serum-free medium can be configured to contain FGF2 and / or FGF10.

  In the method according to the present invention, the serum-free medium can further comprise IWP4.

  In the method according to the present invention, the fibroblasts can be cultured in the serum-free medium for 2 weeks after the introduction of the Mef2c, Tbx5 and Gata4 genes.

  (3) A medium according to the present invention for solving the above-mentioned problems is a medium used for producing cardiomyocyte-like cells, characterized in that it contains VEGF and is serum-free.

  (4) A medium according to the present invention for solving the above-mentioned problems is characterized by containing VEGF, FGF2 and FGF10 and being serum-free.

  In the culture medium which concerns on this invention, it can comprise so that it may be a culture medium used in order to produce a myocardial cell.

  ADVANTAGE OF THE INVENTION According to this invention, the method and culture medium which can produce the beating myocardial-like cell efficiently can be provided.

In one embodiment of the present invention, mouse fetal fibroblasts transfected with Gata4, Mef2c and Tbx5 are cultured for 4 weeks in a DMEM / M199 medium or a serum-free medium containing a JAK inhibitor, FGF2, FGF10, or VEGF. FIG. 4 is a diagram showing the results of examining the number of pulsatile cardiomyocyte-like cells induced to differentiate. In one embodiment of the present invention, mouse fetal fibroblasts transfected with Gata4, Mef2c and Tbx5 were treated with DMEM / M199 medium, or FGF2, FGF10, VEGF, FGF2 and VEGF, FGF10 and VEGF, FGF2 and FGF10, or It is a figure which shows the result of having culture | cultivated for 4 weeks in the serum-free medium containing FGF2, FGF10, and VEGF (FFV), and having investigated the number of the pulsatile cardiomyocyte induced differentiation. In one embodiment of the present invention, mouse embryonic fibroblasts transfected with Gata4, Mef2c and Tbx5 are cultured for a predetermined period using a DMEM / M199 medium and / or a serum-free medium containing FFV, and then differentiated. It is a figure which shows the result of having investigated the number of induced beating myocardial cells and performing the same experiment three times and totaling the number. In one embodiment of the present invention, mouse fetal fibroblasts transfected with Gata4, Mef2c, and Tbx5, or mouse fetal fibroblasts transfected with Gata4, Mef2c, Tbx5, and Hand2 were treated with DMEM / M199 medium or FFV. It is a figure which shows the result of having investigated the number of the pulsating myocardial-like cell which induced differentiation after culture | cultivating for 4 weeks using the serum-free medium containing, performing the same experiment 3 times, and totaling the number. In one embodiment of the present invention, a mouse neonatal tail end-derived fibroblast transfected with Gata4, Mef2c and Tbx5, a mouse neonatal tail end-derived fibroblast transfected with Gata4, Mef2c, Tbx5 and Hand2, Alternatively, mouse neonatal tail-end fibroblasts transfected with Gata4, Mef2c, Tbx5, Mesp1 and Myocd were cultured for 8 weeks in serum-free medium containing DMEM / M199 medium or FFV, and then induced to differentiate. It is a figure which shows the result of having investigated the number of beating myocardial-like cells, performing the same experiment twice, and totaling the number. In one embodiment of the present invention, mouse fetal fibroblasts, mouse fetal fibroblasts transfected with Gata4, Mef2c and Tbx5, or mouse fetal fibroblasts transfected with Mef2c and Tbx5, serum-free containing FFV It is a figure which shows the result of having investigated the number of the pulsatile cardiomyocytes induced | guided | derived of differentiation after culture | cultivating using a culture medium for 4 weeks, performing the same experiment twice, and totaling the number.

  Hereinafter, a method for producing myocardial cells and a culture medium according to the present invention will be described with reference to the drawings. In addition, this invention is not limited to the following embodiment and an experiment example, It can implement in various deformation | transformation within the range of the summary.

[Method for producing myocardial cells]
The method for producing cardiomyocyte-like cells of the present invention includes a step of culturing fibroblasts into which at least two factors (Mef2c and Tbx5) have been introduced in a serum-free medium containing VEGF.

  According to such a method for producing myocardial cells, it is possible to efficiently produce beating myocardial cells from fibroblasts into which genes have been introduced. More specifically, fibroblasts transfected with three factors (Gata4, Mef2c and Tbx5) cultured in a serum-free medium containing VEGF are compared with those cultured in a conventional DMEM / M199 medium, It is possible to efficiently produce beating myocardial cells. Further, by culturing using a serum-free medium containing VEGF, FGF2 and FGF10, the three factors (Gata4, Mef2c and Tbx5) to be introduced into the fibroblasts can be reduced to two factors (Mef2c and Tbx5). Since differentiation can be induced into beating cardiac muscle-like cells, pulsating cardiac muscle-like cells can be efficiently produced.

  Hereinafter, a method for producing cardiac muscle-like cells will be described in detail.

(Fibroblast)
The fibroblast is not particularly limited as long as at least two factors (Mef2c and Tbx5) are transfected, but Gata4 may be further transfected. In addition, Hand2, or Mesp1 and Myocd may be introduced with a gene. Gene introduction of these factors, that is, introduction of a polynucleotide encoding these polypeptides, or a polynucleotide having a base sequence complementary to the base sequence thereof is, for example, a virus vector such as a retrovirus vector or an adenovirus vector. Can be carried out by a known method such as a method using a lipofection method, an electroporation method, or a microinjection method. Note that the number of factors for gene transfer is preferably small in order to reduce the risk of canceration of myocardial cells. In addition, the introduced fibroblast may be one that transiently expresses the introduced gene or one that stably expresses.

  The fibroblast may be one in which a polypeptide of some or all factors is introduced into the fibroblast. The polypeptide can be introduced by a known method such as a method using a fusion peptide in which a cell membrane-permeable peptide is fused to the above polypeptide. As the cell membrane permeable peptide, for example, HIV-1 TAT peptide, protein transduction domain (PTD) of the TAT peptide, peptide in which the PTD is all substituted with arginine, and the like can be used.

  The fibroblast into which the gene and / or polypeptide of each factor is introduced may be a subcultured cell, even if it is isolated from a human or a non-human vertebrate such as a mouse, rat, or pig. There may be. As fibroblasts, for example, fetal fibroblasts, tail tip-derived fibroblasts, cardiac fibroblasts, foreskin fibroblasts, skin fibroblasts, lung fibroblasts and the like can be used.

(Culture medium)
The medium is a serum-free medium, that is, a serum-free medium. As the serum-free medium, for example, StemPro34 serum-free medium (manufactured by Gibco) can be used, but is not limited thereto.

  The serum-free medium is not particularly limited as long as it contains VEGF (vascular endothelial growth factor), but besides VEGF, FGF2 (bFGF: basic fibroblast growth factor), FGF10 (fibroblast) Those further comprising one or two substances selected from growth factor-10), JAK inhibitor (JAK inhibitor), and IWP4 (Wnt inhibitor) are preferred. When the fibroblast is a fibroblast in which only two factors (Mef2c and Tbx5) are introduced, it is preferable to use a serum-free medium containing VEGF, FGF2 and FGF10, and further includes IWP4. Those are more preferred. When the fibroblast is a fibroblast into which at least three factors (Mef2c, Tbx5, and Gata4) have been introduced, any serum-free medium containing VEGF may be used, and FGF2 and / or FGF10 may be further added. It preferably contains, and more preferably further contains IWP4.

(Fibroblast culture)
The culture of fibroblasts into which genes and / or polypeptides of at least two factors (Mef2c and Tbx5) have been introduced is a step of inducing differentiation directly from the fibroblasts into cardiomyocyte-like cells. That is, fibroblasts into which genes and / or polypeptides of only two factors (Mef2c and Tbx5) have been introduced, or fibers into which genes and / or polypeptides of at least three factors (Mef2c, Tbx5 and Gata4) have been introduced. It is a step of culturing blast cells to produce myocardial cells.

The culture of fibroblasts into which genes and / or polypeptides of at least two factors (Mef2c and Tbx5) have been introduced is not particularly limited as long as the conditions are suitable, and is usually within the range of 25 ° C to 37 ° C. And under 5% CO ₂ conditions.

  The culture of fibroblasts into which genes and / or polypeptides of at least two factors (Mef2c and Tbx5) were introduced using a serum-free medium containing VEGF was performed two weeks after the introduction of the genes and / or polypeptides. Preferably it is done. The culture using the serum-free medium containing VEGF is preferably performed from 2 weeks to 4 weeks after the gene and / or polypeptide is introduced (for 2 weeks). By culturing in a serum-free medium containing VEGF during this period, differentiation induction into myocardial cells is efficiently performed, and beating myocardial cells can be efficiently produced. In addition, it is preferable to culture in a medium containing serum for 2 weeks after gene introduction. The serum is not particularly limited, and for example, fetal bovine serum (FBS), calf serum (NCS), horse serum and the like can be used. Examples of the medium to which serum is added include, but are not limited to, a DMEM / M199 medium, a DMEM medium, and an IMDM medium.

(How to check myocardial cells)
Cardiac-like cells can be confirmed by, for example, confirming whether or not the pulsation occurs under a microscope, or expressing a myocardial specific marker gene by a known method such as FACS method, RT-PCR method, immunostaining method, microarray assay method, etc. It can be performed by checking whether or not it is done, but is not limited to these methods.

  Examples of the myocardial specific marker gene include, but are not limited to, Myh6, cTnT, RyR2, HCN4, Actc1, Gja1, Col1a2, and the like.

[Culture medium]
The medium of the present invention is useful for producing cardiomyocyte-like cells. In particular, fibroblasts transfected with at least two factors (Mef2c and Tbx5), more specifically fibroblasts transfected with only two factors (Mef2c and Tbx5), or at least three factors (Gata4) , Mef2c and Tbx5) are useful for producing cardiomyocyte-like cells by inducing differentiation from fibroblasts into which the gene has been introduced into cardiomyocyte-like cells. In addition, differentiation from embryonic stem cells (ES cells) or induced pluripotent stem cells (iPS cells) to myocardial cells is also useful for producing myocardial cells. In addition, since each component of the culture medium of the present invention is as described in detail in the explanation section of the “method for producing myocardial cells” described above, the description thereof is omitted here.

  The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples.

[Experiment 1]
(Isolation of mouse embryo fibroblast (MEF))
Seven- to ten-week-old female ICR mice (CLEA Japan, Inc.) and α-myosin heavy chain-green fluorescent protein (αMHC-GFP) transgenic mice (male) were mated. The day when fertilization was confirmed was defined as day 0 of pregnancy, and fetuses were removed from ICR mice that became pregnant 12 days after the confirmation of pregnancy.

  The heart was extracted from the fetus, and the heart was projected with fluorescence using an inverted microscope (Olympus, IX71) to select a fetus that emits GFP fluorescence. From the selected fetus, limbs and heads 1/2 to 2/3, parenchymal organs such as lung, liver, kidney, intestine, etc. were excised. The remaining tissue of the trunk was washed with PBS (-) (manufactured by WAKO, 045-29795) to sufficiently remove blood cell components. The tissue was then sheared as finely as possible with sterile surgical scissors. A solution prepared by mixing 1: 1 0.25% trypsin-EDTA (Gibco, 25200-072) and PBS (−) was added to the sheared tissue pieces (15 mL / 6-7 fetuses). And it incubated at 37 degreeC for 15 minutes, shaking with a water bath.

After the incubation, 15 mL of FBS stock solution (HiClone Fetal Bovine Serum; Thermo Scientific, SV30014.03) was added and suspended sufficiently, and the suspension was centrifuged at 1500 ° C. (1500 rpm × 5 minutes). Was removed. The cell precipitate was resuspended in 30 mL of a medium for MEF shown in Table 1 and injected into a 10 cm tissue culture dish (Thermo Scientific, 172958) (one 10 cm dish for 2 to 3 fetuses). Thereafter, 37 ° C., and cultured under 5% CO _2. The next day, the medium was changed to a new medium for MEF, and thereafter, the medium was changed every 3 to 4 days.

  Migrated cells were collected with 0.25% trypsin-EDTA (Gibco, 25200-072), and filtered using a 40 μm cell strainer (BD, REF352340) to obtain mouse embryonic fibroblasts (MEF). Was isolated.

[Experiment 2]
(Isolation of mouse neonatal tail end-derived fibroblasts (TTF))
Seven- to ten-week-old female ICR mice and αMHC-GFP transgenic mice (male) were mated. The day when fertilization was confirmed was defined as day 0 of pregnancy, and newborns were obtained from ICR mice that became pregnant about 20 days after the confirmation of pregnancy.

The heart was removed from the newborn, and the heart was projected with fluorescence using an inverted microscope, and a newborn that emits GFP fluorescence was selected. From the selected neonate, the tail end in the range of 1/2 to 2/3 from the tip of the tail was cut with sterilized surgical scissors. Thereafter, the cut tail ends were collected and sheared into pieces of 2-3 mm ³ . The sheared tail end tissue was placed in a gelatin-coated 10 cm tissue culture dish at regular intervals and allowed to air dry at room temperature for 15 minutes (one 10 cm dish for 5 to 7 newborn tail tips). And in TTF for medium shown in Table 2, 37 ° C., and cultured under 5% CO _2. Thereafter, the medium was replaced with a new TTF medium every 5 to 7 days and cultured for 7 to 14 days.

  Migrated cells were collected with 0.25% trypsin-EDTA, and filtered using a 40 μm cell strainer to isolate mouse neonatal tail end-derived fibroblasts (TTF).

[Experiment 3]
(Production of transgenic cells)
Plate-E packaging cells (3.6 × 10 ⁶ cells) are seeded on a 10 cm tissue culture dish coated with gelatin, and the plate-E medium shown in Table 3 is used at 37 ° C. under 5% CO ₂ condition. The culture was stationary overnight.

Meanwhile, 300 μL Opti-MEM (manufactured by Gibco, 31985-070) and 27 μL FuGENE ^™ 6 Transfection Reagent (manufactured by Promega, E2691) were mixed and allowed to stand for 5 minutes, pMx-Gata4, pMx-Mef2c, pMx- 9000 ng of Tbx5, pMx-Hand2, pMx-Mesp1, or pMx-Myocd retroviral plasmid was mixed, tapped strongly, and allowed to stand at room temperature for 15 minutes. Subsequently, various retroviral plasmids were mixed into the overnight-plated Plat-E packaging cells, and each gene was transfected (transfected).

Twenty-four hours after transfection, the plate-E medium shown in Table 3 was replaced, and the plate was further allowed to stand at 37 ° C. and 5% CO ₂ for 24 hours. Thereafter, the supernatant was collected, filtered through a 0.45 μm pore size Minisart filter (Sartorius Stedim Biotech, 17598), and collected in a 50 mL tube (Corning, 430829). 4 μL of 10 mg / mL Polybrene Transfection Reagent (Millipore, # TR-1003-G) was injected into 10 mL of the collected supernatant, and each gene (Gata4, Mef2c, Tbx5, Hand2, Mesp1, Myocd, etc.) retro A virus solution was prepared.

  Gata4, Mef2c and Tbx5 retroviral solutions, Gata4, Mef2c, Tbx5 and Hand2 retroviral solutions, Gata4, Mef2c, Tbx5, Mesp1 and Myocd retroviral solutions, or Mef2c and Tbx5 retroviral solutions. Various mixed virus solutions were prepared by mixing.

The MEF isolated in Experiment 1 or the TTF isolated in Experiment 2 is spread on a multiwell plate for 12-well cell culture (manufactured by FALCON, 353043) (0.5 × 10 ⁵ cells / well) and allowed to stand overnight. I put it. Thereafter, the medium in the well was removed by aspiration, and 1 mL of various mixed virus solutions were injected (the day of infection was defined as day 0), and the culture was allowed to stand overnight at 37 ° C. under 5% CO ₂ conditions. MEF or TTF into which Gata4, Mef2c and Tbx5 have been introduced (respectively referred to as “GMT-MEF” and “GMT-TTF”), and MEF or TTF into which Gata4, Mef2c, Tbx5 and Hand2 have been introduced (each represented as “GHMT− MEF ”and“ GHMT-TTF ”), TTF into which Gata4, Mef2c, Tbx5, Mesp1 and Myocd have been introduced (hereinafter referred to as“ GMTMM-TTF ”), or MEF into which Mef2c and Tbx5 have been introduced (hereinafter referred to as“ METF ”). , "MT-MEF").

[Evaluation 1]
(Induction of differentiation into myocardial cells using serum-free medium supplemented with various substances)
The GMT-MEF produced in Experiment 3 was cultured under the conditions of 37 ° C. and 5% CO ₂ using each medium shown in Table 4 or Table 5. In addition, culture | cultivation was performed by changing a culture medium every 3 to 4 days. In addition, culture in a serum-free medium was performed at a final concentration of 0.5 μM JAK inhibitor (CALBOCHEM, # 4200999), 10 ng / mL FGF2 (recombinant human FGF basic146aa; R & D Systems, 233-FB-025) ), 25 ng / mL FGF10 (recombinant human FGF10; manufactured by R & D Systems, 345-FG-025), or 5 ng / mL VEGF (recombinant human VEGF165; manufactured by R & D Systems, 293-VE-050) It was performed using what was added to.

  28 days after infection, the whole of each well was photographed at a bright field of 4 times using an HS all-in-one fluorescence microscope (manufactured by Keyence Corporation, BIOREVO BZ9000). In addition, the number of myocardial cells pulsating with a phase difference of 20 times was measured using the same microscope, and the total of 12 wells was calculated. The result is shown in FIG.

  As shown in FIG. 1, GMT-MEF was cultured using a serum-free medium supplemented with a JAK inhibitor, FGF2, FGF10 or VEGF, compared to a culture using a conventional DMEM / M199 medium, It was confirmed that beating myocardial cells could be efficiently produced.

[Evaluation 2]
(Induction of differentiation into myocardial cells by serum-free medium containing various substances in combination)
Next, in order to examine whether or not pulsating myocardial cells can be efficiently produced by adding FGF2, FGF10, and VEGF in combination to a serum-free medium, FGF2, VEGF, FGF10 are added to the serum-free medium. And VEGF, FGF2, and FGF10, or FGF2, FGF10, and VEGF (FFV) were added, and the number of cardiac muscle-like cells that were cultured for 4 weeks was calculated by the same method as in Evaluation 1, to calculate the number of myocardial cells. The result is shown in FIG.

  As shown in FIG. 2, it was confirmed that beating myocardial cells could be produced more efficiently by adding VEGF and FGF2 to a serum-free medium and culturing GMT-MEF. In addition, a synergistic effect was observed when FGF10 was further added to a serum-free medium containing VEGF and FGF2.

[Evaluation 3]
(Induction of differentiation into myocardial cells by culture conditions of serum-free medium containing FFV)
Next, in order to examine in which period the culture of GMT-MEF in a serum-free medium containing FFV is effective, as shown in FIG. 3, a serum-free medium containing DMEM / M199 medium and / or FFV is used. Then, the number of beating myocardial cells was calculated by culturing in the same manner as in Evaluation 1 except that GMT-MEF was cultured for a predetermined period. The same experiment was performed three times, and the total of them was shown in FIG.

  As shown in FIG. 3, two weeks after infection (gene transfer), GMT-MEF was cultured for 2 weeks in a serum-free medium containing FFV, thereby efficiently inducing differentiation into myocardial cells. It was confirmed that cells could be produced efficiently. In addition, total RNA was isolated from beating cardiomyocyte-like cells obtained by culturing GMT-MEF in a serum-free medium containing FFV for 4 weeks, and ABI7900HT (Applied Biosystems) and TaqMan probe (Applied Biosystems) Quantitative RT-PCR was performed using a myocardial specific marker gene (for example, Myh6) in the same manner as total RNA isolated from beating myocardial cells obtained by culturing in DMEM / M199 medium. , CTnT, RyR2, HCN4, etc.) were observed (not shown).

[Evaluation 4]
(Induction of differentiation from GHMT-MEF into myocardial cells by serum-free medium containing FFV)
Next, in order to investigate whether GHMT-MEF can be efficiently produced by culturing in a serum-free medium containing FFV in GHMT-MEF, GHMT-MEF or GMT-MEF is added to DMEM / M199 medium or FFV. The number of beating myocardial cells was calculated by culturing in the same manner as in Evaluation 1 except that culture was performed using a serum-free medium containing. The same experiment was performed three times, and the total of them was shown in FIG.

  As shown in FIG. 4, culturing GHMT-MEF using a serum-free medium containing FFV efficiently pulsates myocardial cells as compared to those cultured using conventional DMEM / M199 medium. It was confirmed that it could be produced. In addition, it was confirmed that GHMT-MEF was able to efficiently produce beating myocardial cells by efficiently inducing differentiation into myocardial cells using a serum-free medium containing FFV compared to GMT-MEF.

[Evaluation 5]
(Differentiation induction from various gene-transferred cells to myocardial cells by serum-free medium containing FFV)
Next, in GMT-TTF, GHMT-TTF, and GMTMM-TTF, in order to examine whether these cells can be efficiently produced by culturing in a serum-free medium containing FFV, The number of beating cardiomyocytes was calculated by culturing in the same manner as in Evaluation 1 except that the culture was performed for 8 weeks using a serum-free medium containing DMEM / M199 medium or FFV. Similar experiments were performed twice, and the sum of them was shown in FIG.

  As shown in FIG. 5, GHMT-TTF cultured in a serum-free medium containing FFV efficiently beats myocardial cells as compared to those cultured in a conventional DMEM / M199 medium. It was confirmed that it could be produced. In addition, those in which GMTMM-TTF was cultured using a serum-free medium containing FFV showed more efficient pulsating cardiomyocyte-like cells than those cultured in GHMT-TTF using a serum-free medium containing FFV. It was confirmed that it could be produced.

[Evaluation 6]
(Effects of IWP4)
Next, except that GMT-MEF was cultured in DMEM / M199 medium and replaced with serum-free medium containing IWP4, FGF2, FGF10 and VEGF (IFFV) 2 weeks after infection (gene transfer), and cultured for 2 weeks The number of cardiac muscle-like cells that were cultured for 4 weeks and pulsated was calculated in the same manner as in Evaluation 1. The serum-free medium containing IFFV was prepared by adding 4.8 μM IWP4 (manufactured by STEMGENT, 04-0036) to the serum-free medium containing FFV at a final concentration. Moreover, the number of beating myocardial cells was calculated by culturing for 4 weeks by the same method as in Evaluation 1, except that GMT-MEF was cultured in a DMEM / M199 medium or a serum-free medium containing FFV. These experiments were performed twice and the results are shown in Table 6.

  As shown in Table 6, GMT-MEF was cultured in a serum-free medium containing DMEM / M199 and IFFV, that is, cultured in a medium containing serum for 2 weeks after gene transfer, and then 2 It can be confirmed that the number of beating myocardial cells increased 100 to 200 times in those cultured in serum-free medium containing IFFV for a week compared to those cultured in conventional DMEM / M199 medium. It was.

[Evaluation 7]
(Induction from MT to myocardial cells)
Next, except for culturing MEF alone, GMT-MEF, and MT-MEF in a serum-free medium containing FFV, the number of beating myocardial cells was calculated by culturing for 4 weeks in the same manner as in Evaluation 1. did. Similar experiments were performed twice, and the sum of them was shown in FIG.

As shown in FIG. 6, even if the three factors (Gata4, Mef2c and Tbx5) introduced into fibroblasts are reduced to two factors (Mef2c and Tbx5) by using a serum-free medium containing FFV, pulsation is achieved. It was confirmed that the cardiomyocyte can be induced to differentiate into the cardiomyocyte, and the beating cardiomyocyte can be efficiently produced.

Claims (10)

A method of producing cardiomyocyte-like cells,
A method comprising a step of culturing fibroblasts into which only Mef2c and Tbx5 have been introduced in a serum-free medium containing VEGF, FGF2 and FGF10.   The method of claim 1, wherein the serum-free medium further comprises IWP4.   The method according to claim 1 or 2, wherein the fibroblasts are cultured in the serum-free medium for 2 weeks 2 weeks after the introduction of the Mef2c and Tbx5 genes. A method of producing cardiomyocyte-like cells,
A method comprising a step of culturing fibroblasts into which Mef2c, Tbx5 and Gata4 have been introduced in a serum-free medium containing VEGF.   The method according to claim 4, wherein the serum-free medium comprises FGF2 and / or FGF10.   The method according to claim 4 or 5, wherein the serum-free medium further comprises IWP4.   The method according to any one of claims 4 to 6, wherein the fibroblasts are cultured in the serum-free medium for 2 weeks 2 weeks after the introduction of the Mef2c, Tbx5 and Gata4. A medium used for producing cardiomyocyte-like cells,
A medium comprising VEGF and serum-free.   A medium containing VEGF, FGF2 and FGF10 and being serum-free. The medium according to claim 9, which is a medium used for producing cardiomyocyte-like cells.

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