WO2016056653A1 - Procédé de fabrication de cellules myocardiques, cellules myocardiques type ventricule ainsi que procédé de fabrication de celles-ci, et procédé de criblage - Google Patents

Procédé de fabrication de cellules myocardiques, cellules myocardiques type ventricule ainsi que procédé de fabrication de celles-ci, et procédé de criblage Download PDF

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WO2016056653A1
WO2016056653A1 PCT/JP2015/078784 JP2015078784W WO2016056653A1 WO 2016056653 A1 WO2016056653 A1 WO 2016056653A1 JP 2015078784 W JP2015078784 W JP 2015078784W WO 2016056653 A1 WO2016056653 A1 WO 2016056653A1
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cardiomyocytes
cardiomyocyte
ventricular
embryoid body
culture
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Japanese (ja)
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篤彦 内藤
小室　一成
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国立大学法人東京大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06—Quantitative determination

Definitions

  • the present invention relates to a method for producing cardiomyocytes, a ventricular cardiomyocyte and a method for producing the same, and a method for screening a preventive or therapeutic agent for at least one of hypertrophic cardiomyopathy, cardiac hypertrophy, and Noonan syndrome.
  • iPS cells induced pluripotent stem cells
  • cardiomyocytes differentiated from human iPS cells are expected to be applied to various uses such as regenerative medicine, drug discovery research, and drug safety tests.
  • regenerative medicine for example, it is expected to restore lost heart function by transplanting myocardial cells differentiated from self iPS cells or banked iPS cells into the heart.
  • drug discovery research for example, the development of new drugs for cardiovascular diseases and intractable diseases using iPS cells of healthy individuals and cardiomyocytes differentiated from disease-specific iPS cells prepared from patients with intractable diseases is used for analysis and screening. Is expected to be.
  • cardiomyocytes are expected to be used for screening for cardiotoxicity during development or for developed drugs.
  • a method for producing cardiomyocytes a method of inducing differentiation of cardiomyocytes using cytokines in the embryoid body formation method (see, for example, Non-Patent Document 1), cardiomyocytes without using cytokines in adhesion culture A method for inducing differentiation (for example, see Non-Patent Document 2), a method for inducing differentiation of cardiomyocytes without using cytokines (for example, see Non-Patent Document 3), etc. are proposed. ing.
  • the cardiomyocytes produced by these proposals have a problem that they cannot be said to be sufficient in quality in view of application to various uses such as regenerative medicine, drug discovery research, and drug safety testing.
  • the cardiomyocytes produced by the proposed technique have a problem that many non-cardiomyocytes are mixed and a problem that the cardiomyocytes are not homogeneous.
  • the heterogeneity of the cardiomyocytes means that pacemaker cardiomyocytes, atrial cardiomyocytes, and ventricular cardiomyocytes having different properties are mixed.
  • the cardiomyocytes are used for regenerative medicine due to the contamination of the non-cardiomyocytes and the inhomogeneity of the cardiomyocytes, the arrhythmogenicity and carcinogenicity become a problem. When used, there is a problem that an inaccurate result may be obtained.
  • the present invention relates to a method for producing cardiomyocytes capable of producing high-quality cardiomyocytes, mixing of non-cardiomyocytes and non-homogeneity of cardiomyocytes, and high quality ventricular myocardium.
  • An object of the present invention is to provide a method for screening a preventive or therapeutic agent for at least one of hypertrophic cardiomyopathy, cardiac hypertrophy, and Noonan syndrome using at least one of cardiomyocytes and ventricular cardiomyocytes And
  • Means for solving the problems are as follows. That is, ⁇ 1> An embryoid body forming step of culturing pluripotent stem cells to form an embryoid body, Culturing the embryoid body and inducing a mesoderm, Culturing the embryoid body after the mesoderm induction and inducing cardiomyocytes, An embryoid body dissociation step of dissociating the embryoid body after the cardiomyocyte induction, The method for producing cardiomyocytes, wherein the embryoid body dissociation step is performed from the start of the mesoderm induction step to the 40th day.
  • ⁇ 2> a fluorescent RNA probe introduction step of introducing a fluorescent RNA probe into cardiomyocytes produced by the method for producing cardiomyocytes according to ⁇ 1>, And a sorting step of sorting cells that emit fluorescence derived from the fluorescent RNA probe.
  • a ventricular cardiomyocyte having an action potential amplitude of 140 mV or more, a peak potassium current of 300 pA or more, a peak calcium current of 1 nA or more, and a peak sodium current of 6.5 nA or more.
  • a screening method for a preventive or therapeutic agent for at least one of hypertrophic cardiomyopathy, cardiac hypertrophy, and Noonan syndrome A screening method using at least one of cardiomyocytes and ventricular cardiomyocytes.
  • the present invention it is possible to solve the conventional problems, achieve the object, and to produce a high-quality cardiomyocyte, a method for producing a cardiomyocyte, mixing of non-cardiomyocytes, High-quality ventricular cardiomyocytes with reduced cell heterogeneity, a method for producing the same, and hypertrophic cardiomyopathy, cardiac hypertrophy, and Noonan syndrome using at least one of cardiomyocytes and ventricular cardiomyocytes It is possible to provide a method for screening a preventive or therapeutic agent for at least one of the above.
  • FIG. 1 is a diagram showing the results when no fluorescent RNA probe was introduced in Production Example 2.
  • FIG. 2 is a diagram showing the results when SF102 is introduced as a fluorescent RNA probe in Production Example 2.
  • FIG. 3 is a diagram showing the results when MYL2-1 was introduced as a fluorescent RNA probe in Production Example 2.
  • FIG. 4 is a diagram showing the results when MYL2-2 is introduced as a fluorescent RNA probe in Production Example 2.
  • FIG. 5 is a diagram showing the results when MYL3-1 was introduced as a fluorescent RNA probe in Production Example 2.
  • FIG. 6 is a diagram showing the results when MYL3-2 was introduced as a fluorescent RNA probe in Production Example 2.
  • FIG. 7 is a diagram showing the results when IRX4-1 was introduced as a fluorescent RNA probe in Production Example 2.
  • FIG. 8 is a diagram showing the results when IRX4-2 is introduced as a fluorescent RNA probe in Production Example 2.
  • FIG. 9 is a diagram showing the results when RPL3L-1 was introduced as a fluorescent RNA probe in Production Example 2.
  • FIG. 10 is a diagram showing the results when RPL3L-2 was introduced as a fluorescent RNA probe in Production Example 2.
  • FIG. 11 is a diagram illustrating a result of sorting by FACS in Test Example 1.
  • FIG. 12 is a diagram showing the results of sorting by FACS in Test Example 1.
  • FIG. 13 is a diagram showing the results of analyzing the gene expression level in Test Example 2.
  • FIG. 14 is a diagram showing a representative example of the action potential of cells in the MYL2-positive fraction in Test Example 3.
  • FIG. 15 is a diagram showing a representative example of the action potential of cells in the MYL2-negative fraction in Test Example 3.
  • FIG. 16 is a diagram showing the results of analyzing the gene expression level in Test Example 5.
  • FIG. 17 is a diagram showing the results of analyzing sodium current of ventricular cardiomyocytes purified by the method of the present invention in Test Example 7.
  • FIG. 18 is a diagram showing the results of analyzing sodium current of commercially available iCell Cardiomyocytes in Test Example 7.
  • FIG. 19 is a diagram showing the results of analyzing the sodium current of commercially available ReproCardio in Test Example 7.
  • FIG. 20 is a diagram showing the results of analyzing the potassium current of ventricular cardiomyocytes purified by the method of the present invention in Test Example 7.
  • FIG. 21 is a diagram showing the results of analyzing the potassium current of commercially available iCell Cardiomyocytes in Test Example 7.
  • FIG. 22 is a diagram showing the results of analyzing the potassium current of commercially available ReproCardio in Test Example 7.
  • FIG. 23 is a diagram showing the results of analyzing the calcium current of ventricular cardiomyocytes purified by the method of the present invention in Test Example 7.
  • FIG. 24 is a diagram showing the results of analyzing the calcium current of commercially available iCell Cardiomyocytes in Test Example 7.
  • FIG. 25 is a diagram showing the results of analyzing the calcium current of commercially available ReproCardio in Test Example 7.
  • FIG. 26 is a diagram showing a representative example of immunostaining cardiomyocytes produced using healthy person-derived iPS cells in Test Example 8.
  • FIG. 27 is a diagram showing a typical example of immunostaining cardiomyocytes produced using iPS cells derived from hypertrophic cardiomyopathy patients in Test Example 8.
  • FIG. 28 is a diagram showing the results of analyzing the cell area by the high content analysis device of Test Example 8.
  • FIG. 29 is a diagram showing the results of analyzing the roundness of cells by the high content analysis device of Test Example 8.
  • FIG. 30 is a diagram illustrating a result of analyzing the area of the nucleus by the high content analysis device of Test Example 8.
  • FIG. 31 is a diagram illustrating a result of analyzing the roundness rate of the nucleus by the high content analysis device of Test Example 8.
  • FIG. 32 is a diagram showing the results of analyzing the phalloidin staining intensity by the high content analyzer of Test Example 8.
  • FIG. 33 is a diagram illustrating a result of analyzing “Hole” of the texture analysis item attached to the high content analysis device of Test Example 8.
  • FIG. 34 is a diagram illustrating a result of analyzing “Edge” of the item of texture analysis attached to the high content analysis device of Test Example 8.
  • FIG. 35 is a diagram illustrating a result of analyzing “Ridge” of the item of texture analysis attached to the high content analysis device of Test Example 8.
  • FIG. 36 is a diagram illustrating a result of analyzing “Valley” of the texture analysis item attached to the high content analysis device of Test Example 8.
  • FIG. 37 is a diagram illustrating a result of analyzing “Bright” as an item of texture analysis attached to the high content analysis device of Test Example 8.
  • FIG. 38 is a diagram illustrating a result of analyzing “Dark” of the texture analysis item attached to the high content analysis device of Test Example 8.
  • FIG. 39 is a diagram illustrating a result of analyzing “Saddle” as an item of texture analysis attached to the high content analysis device of Test Example 8.
  • FIG. 40 is a diagram illustrating a result of analyzing “Spot” of the item of texture analysis attached to the high content analysis device of Test Example 8.
  • FIG. 41 is a diagram showing the results of analyzing the cell area with the high content analysis device of Test Example 9.
  • FIG. 42 is a diagram illustrating a result of analyzing the area of the nucleus by the high content analysis device of Test Example 9.
  • the method for producing cardiomyocytes of the present invention includes at least an embryoid body formation step, a mesoderm induction step, a cardiomyocyte induction step, and an embryoid body dissociation step, and further includes other steps as necessary.
  • the embryoid body formation step is a step of culturing pluripotent stem cells to form an embryoid body.
  • pluripotent stem cells There is no restriction
  • the method for preparing the pluripotent stem cells is not particularly limited, and a known method can be appropriately selected.
  • pluripotent stem cells that are maintained in an undifferentiated state by a feeder cell culture step described below are dissociated. What was done can be used for the said embryoid body formation process.
  • embryoid body forming step medium As a medium in the embryoid body forming step (hereinafter, sometimes referred to as “embryoid body forming step medium” or “embryoid body forming step culture medium”), an embryoid body can be formed. There is no particular limitation, and it can be appropriately selected according to the purpose.
  • the embryoid body formation process medium include a medium in which Y27632 is added to a medium capable of maintaining pluripotent stem cells in an undifferentiated state at a final concentration of 5 ⁇ M to 10 ⁇ M, and StemPro34 (manufactured by Invitrogen) such as BMP4.
  • Examples include media supplemented with cytokines.
  • the medium capable of maintaining the pluripotent stem cells in an undifferentiated state include ReproFF (manufactured by Reprocell), ReproFF2 (manufactured by Reprocell), ReproXF (manufactured by Reprocell), Essential 6 (manufactured by Invitrogen), Essential 8 (manufactured by Invitrogen), mTeSR1 (manufactured by STEMCELL Technologies), TeSR2 (manufactured by STEMCELL Technologies), and the like.
  • the medium for the embryoid body formation process is described in Yang L, et al. Nature. 2008 May 22; 453 (7194): 524-8, Lian X, et al. Proc Natl Acad Sci US A. 2012 July 3; 109 (27): E1848-57, Minami I, et al. Cell Rep. , 2012 Nov 29; 2 (5): 1448-60 and the like.
  • medium-1 capable of maintaining the following pluripotent stem cells in an undifferentiated state
  • medium capable of maintaining pluripotent stem cells in an undifferentiated state- 2 can also be used.
  • DMEM / F12 manufactured by Nacalai Tesque Co., Ltd.
  • first differentiation-inducing medium a medium in which the components described in Table 1-1 and Table 1-2 below were added to the basal medium
  • an Iskov modified Dulbecco medium (hereinafter sometimes referred to as “IMDM”) is used, and a medium (hereinafter referred to as “hereinafter referred to as“ IMDM ”) to which the components described in Table 2-1 and Table 2-2 are added.
  • IMDM Iskov modified Dulbecco medium
  • a medium in which Y27632 is added at a final concentration of 5 ⁇ M to 10 ⁇ M to medium-1 capable of maintaining the pluripotent stem cells in an undifferentiated state, and the pluripotent stem cells in an undifferentiated state a medium in which Y27632 is added to Sustainable Medium-2 at a final concentration of 5 ⁇ M to 10 ⁇ M is preferable.
  • the culture in the embryoid body formation step is preferably suspension culture.
  • the suspension culture method is not particularly limited, and a known method can be appropriately selected.
  • the pluripotency can be selected using a culture dish that has not been subjected to adhesion processing or a culture dish that has been subjected to low adhesion processing. Examples thereof include a method for culturing stem cells and a method for culturing the pluripotent stem cells using a spinner flask.
  • Commercially available products can be used for the culture dish that has not been subjected to the adhesion process, the culture dish that has undergone the low adhesion process, and the spinner flask.
  • a well-known culture condition can be selected suitably.
  • the culture may be performed under low oxygen conditions or atmospheric conditions, but low oxygen conditions are preferable in terms of excellent differentiation efficiency.
  • Low oxygen conditions refer to oxygen concentration conditions below the normal oxygen concentration (21%).
  • the oxygen concentration in the low oxygen condition is not particularly limited as long as it is less than 21%, and can be appropriately selected according to the purpose, but it is preferably 0.1% or more and less than 21%, preferably 1% or more and 10%. The following is more preferable, and 4% or more and 6% or less are particularly preferable.
  • the mesoderm induction step is a step of culturing the embryoid body to induce mesoderm.
  • the medium in the mesoderm induction process (hereinafter, sometimes referred to as “medium mesoderm induction process medium” or “medium mesoderm induction process culture medium”) is not particularly limited as long as it can induce mesoderm. And can be appropriately selected according to the purpose. For example, Yang L, et al. Nature. 2008 May 22; 453 (7194): 524-8, Lian X, et al. Proc Natl Acad Sci US A. 2012 July 3; 109 (27): E1848-57, Minami I, et al. Cell Rep. 2012 Nov 29; 2 (5): 1448-60, and the like.
  • the medium for the mesoderm induction process include the following second differentiation induction medium, the one-part differentiation induction medium, StemPro34 (manufactured by Invitrogen), etc., Wnt signal activator, Activin A and BMP4. And a medium supplemented with cytokines.
  • a medium in which a Wnt signal activator is added to the following second differentiation induction medium, and a medium in which a Wnt signal activator is added to the one-part differentiation induction medium are preferable.
  • Wnt signal activator there is no restriction
  • the Wnt signal activator may be used alone or in combination of two or more.
  • the content of CHIR99021 in the medium for mesoderm induction process is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 2 ⁇ M to 6 ⁇ M, and more preferably 3 ⁇ M to 5 ⁇ M. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • -Second differentiation induction medium- A medium obtained by using IMDM (manufactured by Nacalai Tesque Co., Ltd.) as a basal medium and adding the components shown in Table 3 below to the basal medium.
  • IMDM manufactured by Nacalai Tesque Co., Ltd.
  • the culture in the mesoderm induction step is preferably suspension culture.
  • the suspension culture method is not particularly limited, and a known method can be appropriately selected. Examples thereof include the same methods as described in the item of culture in the embryoid body formation step.
  • the timing of performing the mesoderm induction step is not particularly limited and may be appropriately selected depending on the purpose.
  • the embryoid body formation step may be performed on the first day to the third day of the embryoid body formation step. Examples include day 1 to day 4 from the start, day 2 to day 4 from the start of the embryoid body formation step. The period is counted from the start date of the embryoid body formation process as day 0.
  • the culture conditions in the mesoderm induction step are not particularly limited, and known culture conditions can be appropriately selected.
  • the culture conditions are the same as those described in the item of culture conditions in the embryoid body formation step. Can do.
  • the cardiomyocyte inducing step is a step of culturing the embryoid body after the mesoderm induction to induce cardiomyocytes.
  • the cardiomyocyte induction step may be a mode consisting of a single cardiomyocyte induction process or a mode consisting of a plurality of cardiomyocyte induction processes. Embodiments are preferred. There is no restriction
  • the cardiomyocyte induction step may include a washing process.
  • the timing of the cardiomyocyte induction step is not particularly limited and may be appropriately selected depending on the purpose.
  • the embryoid body formation step is started on the 3rd to 20th day from the start of the embryoid body formation step. Examples include the 4th to 20th day. The period is counted from the start date of the embryoid body formation process as day 0.
  • first cardiomyocyte induction treatment medium the embryoid body after the mesoderm induction. It is the process which culture
  • the first cardiomyocyte induction treatment medium is not particularly limited as long as it can induce cardiomyocytes, and can be appropriately selected according to the purpose.
  • Yang L, et al. Nature. 2008 May 22; 453 (7194): 524-8 Lian X, et al. Proc Natl Acad Sci US A. 2012 July 3; 109 (27): E1848-57, Minami I, et al. Cell Rep. 2012 Nov 29; 2 (5): 1448-60, and the like.
  • the first cardiomyocyte induction treatment medium include a medium such as the second differentiation induction medium, the one-part differentiation induction medium, StemPro34 (manufactured by Invitrogen), a Wnt signal inhibitor, TGF- Examples thereof include a ⁇ signal inhibitor, a substance having an estrogen-like action, or a medium supplemented with cytokines.
  • a medium in which a Wnt signal suppressor, a TGF- ⁇ signal suppressor, and an estrogen-like agent are added to the second differentiation induction medium, a Wnt signal suppressor, TGF- ⁇ in the one-part differentiation induction medium A medium supplemented with a signal inhibitor and an estrogen-like substance is preferred.
  • the Wnt signal inhibitor is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include recombinant proteins such as Dkk-1 and Frizzled8-Fc, compounds such as IWP2, IWR1, XAV939, and KY02111. Can be mentioned. Among these, IWP2 is preferable in that it exhibits excellent differentiation induction efficiency from a low concentration.
  • the Wnt signal inhibitor may be used alone or in combination of two or more.
  • the content of IWP2 in the first cardiomyocyte induction treatment medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 1 ⁇ M to 10 ⁇ M, more preferably 2 ⁇ M to 9 ⁇ M, and more preferably 4 ⁇ M to 7 ⁇ M is particularly preferred. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the TGF- ⁇ signal inhibitor is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include SB431542, SB505124, A-83-01 and the like. Among these, SB431542 is preferable because it is excellent in differentiation induction efficiency.
  • the TGF- ⁇ signal inhibitor may be used alone or in combination of two or more.
  • the content of SB431542 in the first cardiomyocyte induction treatment medium is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 1 ⁇ M to 10 ⁇ M, more preferably 2 ⁇ M to 9 ⁇ M, and more preferably 3 ⁇ M to 8 ⁇ M is particularly preferred. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • the estrogen-like substance refers to hormones and low molecular compounds that exhibit estrogen action.
  • the estrogen-like substance is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include estradiol, estrone, estriol, and genistein. Among these, estradiol is preferable because it is excellent in differentiation induction efficiency.
  • the substances having an estrogen-like action may be used alone or in combination of two or more.
  • the content of the estrogen-like substance in the first cardiomyocyte induction treatment medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 1 nM to 10,000 nM, 10 nM to 1, 000 nM is more preferable. Within the above preferred range, it is advantageous in that the differentiation induction efficiency into cardiomyocytes is more excellent.
  • cytokine there is no restriction
  • the said cytokine may be used individually by 1 type, and may use 2 or more types together.
  • the cytokine content in the first cardiomyocyte induction treatment medium is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include 1 ng / mL to 100 ng / mL.
  • the culture in the first cardiomyocyte induction treatment is preferably suspension culture.
  • the suspension culture method is not particularly limited, and a known method can be appropriately selected. Examples thereof include the same methods as described in the item of culture in the embryoid body formation step.
  • the period of the first cardiomyocyte induction treatment is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include 48 hours to 72 hours.
  • the timing for performing the first cardiomyocyte induction treatment is not particularly limited and may be appropriately selected depending on the intended purpose. For example, on the 3rd to 6th day from the start of the embryoid body formation process, Examples include the 3rd to 5th day from the start of the process, and the 4th to 6th day from the start of the embryoid body formation process. The period is counted from the start date of the embryoid body formation process as day 0.
  • the culture conditions in the first cardiomyocyte induction treatment are not particularly limited, and known culture conditions can be appropriately selected.
  • the culture conditions are the same as those described in the item of culture conditions in the embryoid body formation step. It can be.
  • Second cardiomyocyte induction process the embryoid body after the first cardiomyocyte induction treatment is referred to as a second cardiomyocyte induction treatment medium (hereinafter, “second cardiomyocyte induction treatment medium”). In some cases).
  • the second cardiomyocyte induction treatment medium is not particularly limited as long as it can induce cardiomyocytes, and can be appropriately selected according to the purpose.
  • Yang L, et al. Nature. 2008 May 22; 453 (7194): 524-8 Lian X, et al. Proc Natl Acad Sci US A. 2012 July 3; 109 (27): E1848-57, Minami I, et al. Cell Rep. 2012 Nov 29; 2 (5): 1448-60, and the like.
  • the second cardiomyocyte induction treatment medium include a medium such as the second differentiation induction medium, the one-part differentiation induction medium, StemPro34 (manufactured by Invitrogen), a Wnt signal inhibitor, TGF- Examples thereof include a ⁇ signal inhibitor, a substance having an estrogen-like action, or a medium supplemented with cytokines.
  • a medium in which a Wnt signal inhibitory substance and an estrogen-like substance are added to the second differentiation-inducing medium and a medium in which a Wnt signal inhibitory substance and an estrogen-like substance are added to the one-part differentiation-inducing medium. preferable.
  • the Wnt signal suppressing substance and the content thereof are the same as those described in the item of the first medium for cardiomyocyte induction treatment.
  • the TGF- ⁇ signal inhibitor and the content thereof are the same as those described in the item of the first cardiomyocyte induction treatment medium.
  • the estrogen-like substance and the content thereof are the same as those described in the item of the first cardiomyocyte induction treatment medium.
  • the cytokine and the content thereof are the same as those described in the item of the first cardiomyocyte induction treatment medium.
  • the culture in the second cardiomyocyte induction treatment is preferably suspension culture.
  • the suspension culture method is not particularly limited, and a known method can be appropriately selected. Examples thereof include the same methods as described in the item of culture in the embryoid body formation step.
  • the period of the second cardiomyocyte induction treatment is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include 48 hours to 72 hours.
  • the timing for performing the second cardiomyocyte induction treatment is not particularly limited and can be appropriately selected according to the purpose.
  • the embryoid body formation process is started on the 6th to 8th days. Examples include the 5th to 8th days from the start of the process. The period is counted from the start date of the embryoid body formation process as day 0.
  • the culture conditions in the second cardiomyocyte induction treatment are not particularly limited, and known culture conditions can be appropriately selected. For example, the same as described in the item of culture conditions in the embryoid body formation step. It can be.
  • third cardiomyocyte induction treatment culture medium the embryoid body after the second cardiomyocyte induction treatment.
  • third cardiomyocyte induction treatment culture medium the embryoid body after the second cardiomyocyte induction treatment. In some cases).
  • the third cardiomyocyte induction treatment medium is not particularly limited as long as it can induce cardiomyocytes, and can be appropriately selected according to the purpose.
  • Yang L, et al. Nature. 2008 May 22; 453 (7194): 524-8 Lian X, et al. Proc Natl Acad Sci US A. 2012 July 3; 109 (27): E1848-57, Minami I, et al. Cell Rep. 2012 Nov 29; 2 (5): 1448-60, and the like.
  • the third cardiomyocyte induction treatment medium include a medium such as the second differentiation induction medium, the one-part differentiation induction medium, StemPro34 (manufactured by Invitrogen), a Wnt signal inhibitor, TGF- Examples thereof include a ⁇ signal inhibitor, a substance having an estrogen-like action, or a medium supplemented with cytokines.
  • a medium in which an estrogen-like substance is added to the second differentiation-inducing medium, and a medium in which an estrogen-like substance is added to the one-part differentiation-inducing medium are preferable.
  • the Wnt signal suppressing substance and the content thereof are the same as those described in the item of the first medium for cardiomyocyte induction treatment.
  • the TGF- ⁇ signal inhibitor and the content thereof are the same as those described in the item of the first cardiomyocyte induction treatment medium.
  • the estrogen-like substance and the content thereof are the same as those described in the item of the first cardiomyocyte induction treatment medium.
  • the cytokine and the content thereof are the same as those described in the item of the first cardiomyocyte induction treatment medium.
  • the culture in the third cardiomyocyte induction treatment is preferably suspension culture.
  • the suspension culture method is not particularly limited, and a known method can be appropriately selected. Examples thereof include the same methods as described in the item of culture in the embryoid body formation step.
  • the period of the third cardiomyocyte induction treatment is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include 4 to 20 days. Among these, 8 days to 12 days are preferable.
  • the time for performing the third cardiomyocyte induction treatment is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include the 8th to 20th day from the start of the embryoid body formation process. The period is counted from the start date of the embryoid body formation process as day 0.
  • the culture conditions in the third cardiomyocyte induction treatment are not particularly limited, and known culture conditions can be appropriately selected. For example, the same as described in the item of culture conditions in the embryoid body formation step. It can be.
  • the washing process is a process of washing the embryoid body after at least one of the first cardiomyocyte induction process, the second cardiomyocyte induction process, and the third cardiomyocyte induction process, It can be performed in the same manner as the cleaning step described later.
  • the embryoid body dissociation step is a step of dissociating the embryoid body after the cardiomyocyte induction.
  • the timing for performing the embryoid body dissociation step is not particularly limited as long as it is from the start of the mesoderm induction step to the 40th day, and can be appropriately selected according to the purpose.
  • the period from the 16th day to the 30th day is preferable.
  • the time for continuously contacting the embryoid body and the proteolytic enzyme is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 1 minute to 30 minutes. Within the preferable range, cell death caused by continuous exposure of the embryoid body to a proteolytic enzyme for a long time can be prevented, and the cells can be efficiently recovered.
  • a preferred embodiment of the method for dissociating the embryoid body is as follows. (1) To prevent the embryoid body from adhering to the inner surface of the pipette or the collection tube, a buffer solution containing 0.1% by mass to 1% by mass of the protein component is brought into contact with the inner surface of the pipette or the collection tube. (2) The embryoid body is collected in a collection tube and precipitated, and then the supernatant is removed. Subsequently, the operation of adding a buffer solution and reprecipitating the supernatant and then removing the supernatant is repeated twice. (3) Add a dissociation solution containing proteolytic enzyme and nucleolytic enzyme, hold at 37 ° C.
  • the embryoid body dissociation step preferably includes a process of culturing cells after the dissociation process.
  • cultivates the cell after performing the said dissociation process According to the objective, it can select suitably, For example, the culture medium etc. which added the serum to DMEM etc. are mentioned. As content of the said serum, 20 mass% etc. are mentioned, for example.
  • the culture in the treatment for culturing the cells after the dissociation treatment is preferably an adhesion culture.
  • adhesion culture method There is no restriction
  • the treatment period for culturing cells after the dissociation treatment is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include 1 to 3 days.
  • timing for performing the treatment for culturing the cells after the dissociation treatment as long as it is from the start of the mesoderm induction step to the 40th day, and may be appropriately selected according to the purpose.
  • the period from the start of the mesoderm induction step to the 30th day is preferable. The period is counted from the start date of the mesoderm induction step as day 0.
  • the culture conditions in the process of culturing the cells after the dissociation treatment are not particularly limited, and known culture conditions can be appropriately selected.
  • the culture conditions in the embryoid body formation step It can be the same as described.
  • the seeding density of the cells in the adhesion culture is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 2.5 ⁇ 10 4 cells / cm 2 to 2 ⁇ 10 5 cells / cm 2 , 8 ⁇ 10 4 cells / cm 2 to 1.2 ⁇ 10 5 cells / cm 2 are more preferable.
  • the culture vessel in the treatment of culturing the cells after the dissociation treatment there is no particular limitation on the culture vessel in the treatment of culturing the cells after the dissociation treatment, and it can be appropriately selected according to the purpose, but the culture vessel for performing adhesion culture is coated with gelatin. Is preferred.
  • feeder cell culture step washing step
  • cardiomyocyte maturation step non-myocardial
  • non-myocardial examples thereof include a cell growth suppression step.
  • the feeder cell culturing step is a step of culturing pluripotent stem cells and feeder cells together to maintain the pluripotent stem cells in an undifferentiated state.
  • the feeder cell culturing step is a step of culturing pluripotent stem cells and feeder cells together to maintain the pluripotent stem cells in an undifferentiated state.
  • limiting in particular as said feeder cell A well-known thing can be selected suitably.
  • the washing step is a step of washing the embryoid body after at least one of the aforementioned embryoid body forming step, mesoderm induction step, and cardiomyocyte induction step.
  • the washing method is not particularly limited and can be appropriately selected depending on the purpose.
  • the cultured embryoid bodies are collected in a container together with the culture solution and allowed to stand to precipitate the embryoid bodies. After the treatment, the supernatant is removed, and then a culture solution or a buffer solution is added, and the mixture is allowed to stand again to precipitate the embryoid body, and then the supernatant is removed.
  • cleaning Although it can select suitably according to the objective, It is preferable to perform in multiple times.
  • the non-cardiomyocyte growth suppressing step is a step of suppressing non-cardiomyocyte growth.
  • the timing for performing the non-cardiomyocyte growth suppression step is not particularly limited and may be appropriately selected depending on the purpose. However, the period between the cardiomyocyte induction step and the embryoid body dissociation step (hereinafter referred to as “No. 1 non-cardiomyocyte growth inhibition step ”or“ non-cardiomyocyte growth inhibition step-1 ”), and between the embryoid body dissociation step and the cardiomyocyte maturation step (hereinafter referred to as“ the first cardiomyocyte growth inhibition step-1 ”). 2 ”(sometimes referred to as“ non-cardiomyocyte growth inhibition step 2 ”or“ non-cardiomyocyte growth inhibition step-2 ”).
  • first non-cardiomyocyte growth inhibition step culture medium a first non-cardiomyocyte growth inhibition step culture medium. It may be referred to as “)”.
  • the medium for the first non-cardiomyocyte growth inhibition step is not particularly limited as long as the growth of non-cardiomyocytes can be suppressed, and can be appropriately selected according to the purpose.
  • culture without glucose examples thereof include a medium in which lactic acid is added to the liquid. By using the medium, it is possible to suppress the proliferation of non-cardiomyocytes and increase the proportion of cardiomyocytes contained in the embryoid body.
  • a medium in which lactic acid is added to a culture solution not containing glucose is described in, for example, International Publication No. 2007/088884, Toyama et al. It can be prepared with reference to descriptions such as Cell Stem cell 2013.
  • Specific examples include a medium in which bovine serum albumin and L-lactic acid are added to DMEM. Examples of the bovine serum albumin content include 5,000 mg / L. Examples of the content of L-lactic acid include 360 mg / L.
  • the culture in the first non-cardiomyocyte growth inhibition step is preferably suspension culture.
  • the suspension culture method is not particularly limited, and a known method can be appropriately selected. Examples thereof include the same methods as described in the item of culture in the embryoid body formation step.
  • the period of the first non-cardiomyocyte growth suppression step is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include 6 to 10 days.
  • the timing for performing the first non-cardiomyocyte growth inhibition step is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include the 20th to 28th days from the start of the embryoid body formation step. It is done. The period is counted from the start date of the embryoid body formation process as day 0.
  • the culture conditions in the first non-cardiomyocyte growth inhibition step are not particularly limited, and known culture conditions can be appropriately selected. For example, those described in the culture condition item in the embryoid body formation step And can be similar.
  • the culture vessel used in the first non-cardiomyocyte growth inhibition step is not particularly limited and may be appropriately selected depending on the intended purpose.
  • protein components such as serum or bovine serum albumin are contained in an amount of 0.1% by mass to 1%.
  • a treatment in which a buffer solution such as PBS or HBSS containing about mass% is brought into contact with the inner surface of the container in advance.
  • the medium in which lactic acid is added to the culture solution not containing glucose contains almost no protein component, and the embryoid body in culture is easily adhered to the inner surface of a plastic container such as a pipette or a collection tube.
  • a plastic container such as a pipette or a collection tube.
  • second non-cardiomyocyte growth inhibition step culture medium a second non-cardiomyocyte growth inhibition step medium
  • second non-cardiomyocyte growth inhibition step culture medium a second non-cardiomyocyte growth inhibition step medium
  • the medium for the second non-cardiomyocyte growth inhibition step is not particularly limited as long as the growth of non-cardiomyocytes can be suppressed, and can be appropriately selected according to the purpose. The thing similar to what was described in the item of the culture medium of a cardiomyocyte growth suppression process is mentioned.
  • the culture in the second non-cardiomyocyte growth inhibition step is preferably adhesion culture.
  • adhesion culture method A well-known method can be selected suitably.
  • the period of the second non-cardiomyocyte growth suppression step is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include 3 to 7 days. It is preferable that the cardiomyocytes after the embryoid body dissociation step have a shorter period than the first non-cardiomyocyte growth suppression step because of their various susceptibility to culture in a medium not containing glucose.
  • the culture conditions in the second non-cardiomyocyte growth inhibition step are not particularly limited, and known culture conditions can be appropriately selected. For example, those described in the culture condition item in the embryoid body formation step And can be similar.
  • the seeding density of the cells in the adhesion culture is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 2.5 ⁇ 10 4 cells / cm 2 to 2 ⁇ 10 5 cells / cm 2 , 8 ⁇ 10 4 cells / cm 2 to 1.2 ⁇ 10 5 cells / cm 2 are more preferable.
  • the culture vessel in the second non-cardiomyocyte growth inhibition step is not particularly limited and may be appropriately selected depending on the intended purpose. However, a culture vessel coated with gelatin is preferably used for adhesion culture.
  • the cardiomyocyte maturation step is a step of culturing and maturing the cardiomyocytes after the embryoid body dissociation step.
  • cardiomyocyte maturation step medium As the medium used for the cardiomyocyte maturation step (hereinafter, sometimes referred to as “cardiomyocyte maturation step medium” or “cardiomyocyte maturation step culture medium”), the cardiomyocytes can be matured. As long as there is no particular limitation, it can be appropriately selected depending on the purpose. For example, Knockout Serum Replacement (manufactured by Invitrogen), Fetal Bovine Serum (manufactured by Biowest), Newborn Calf Serum (manufactured by Hyclone) , Bovine Calf Serum (manufactured by Hyclone), or culture medium containing 1% by mass to 20% by mass of Adult Bovine Serum (manufactured by Hyclone).
  • Knockout Serum Replacement manufactured by Invitrogen
  • Fetal Bovine Serum manufactured by Biowest
  • Newborn Calf Serum manufactured by Hyclone
  • Bovine Calf Serum manufactured by Hyclone
  • the culture in the cardiomyocyte maturation step is preferably an adhesion culture.
  • adhesion culture method A well-known method can be selected suitably.
  • the culture conditions in the cardiomyocyte maturation step are not particularly limited, and known culture conditions can be appropriately selected.
  • the culture conditions are the same as those described in the item of culture conditions in the embryoid body formation step. be able to.
  • the seeding density of the cells in the adhesion culture is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 2.5 ⁇ 10 4 cells / cm 2 to 2 ⁇ 10 5 cells / cm 2 , 8 ⁇ 10 4 cells / cm 2 to 1.2 ⁇ 10 5 cells / cm 2 are more preferable.
  • the culture vessel in the cardiomyocyte maturation step is not particularly limited and can be appropriately selected according to the purpose.
  • cardiomyocyte maturation By performing the cardiomyocyte maturation step, cardiomyocyte maturation can be promoted. Maturation of cardiomyocytes, for example, ventricular cardiomyocytes will be more highly expressed in the ventricular cardiomyocyte-specific gene, and ventricular cardiomyocytes using a technique for labeling the ventricular cardiomyocyte-specific gene will be applied. Is easily isolated. In addition, by maturating cardiomyocytes, changes in cardiomyocytes that occur in the postnatal heart (increase in cell area, loss of division ability, increased expression of calcium handling-related genes, etc.) can be reproduced, and regenerative medicine and drug screening More suitable cardiomyocytes can be obtained.
  • the method for confirming that cardiomyocytes were obtained from pluripotent stem cells by the above method is not particularly limited and can be appropriately selected according to the purpose.
  • expression of a gene specific for cardiomyocytes And a method for confirming the expression of a gene specific to cardiomyocytes by a PCR method.
  • the cardiomyocytes produced by the method for producing cardiomyocytes of the present invention can be suitably used as a material for a method for producing a ventricular cardiomyocyte of the present invention described later, a material for a screening method, and the like.
  • the ventricular cardiomyocytes of the present invention can be preferably produced by the method for producing ventricular cardiomyocytes of the present invention.
  • the ventricular cardiomyocytes of the present invention will be described below together with the description of the method for producing ventricular cardiomyocytes of the present invention.
  • the method for producing ventricular cardiomyocytes of the present invention includes at least a fluorescent RNA probe introduction step and a sorting step, and further includes other steps as necessary.
  • the fluorescent RNA probe introduction step is a step of introducing a fluorescent RNA probe into cardiomyocytes produced by the method for producing cardiomyocytes of the present invention.
  • the cardiomyocytes are cardiomyocytes induced to differentiate from pluripotent stem cells, and are a cell population in which pacemaker cardiomyocytes, atrial cardiomyocytes, ventricular cardiomyocytes and the like are mixed.
  • the fluorescent RNA probe is used to detect a target gene in the cardiomyocytes. By detecting the presence or absence of the target gene in the cardiomyocytes, it becomes possible to specifically detect ventricular cardiomyocytes.
  • the said fluorescent RNA probe may be used individually by 1 type, and may use 2 or more types together.
  • the target gene is not particularly limited and may be appropriately selected depending on the intended purpose. However, at least one of the MYL2 gene, the IRX4 gene, the MYL3 gene, and the RPL3L gene is preferable, and ventricular cardiomyocytes are more specific. The MYL2 gene is more preferable because it can be detected easily.
  • the target genes may be used alone or in combination of two or more.
  • the target sequence in the target gene is not particularly limited as long as the target gene can be detected, and can be appropriately selected according to the purpose, but covers all isoforms in each target gene. It is preferable that it is a sequence which can be performed.
  • the length of the target sequence is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 20 mer to 30 mer.
  • the said target sequence may be used individually by 1 type, and may use 2 or more types together.
  • MYL2 gene- The official name of the MYL2 gene is Myosin Light Chain 2, Regulatory, Cardiac, Slow, and its base sequence can be easily obtained through a public database such as GenBank (NCBI).
  • NCBI GenBank
  • the base sequence of the human MYL2 gene can be obtained under NCBI accession number NM_000432.3.
  • the target sequence for detecting the MYL2 gene is not particularly limited and may be appropriately selected depending on the intended purpose.
  • the sequence represented by SEQ ID NO: 1 the sequence represented by SEQ ID NO: 2
  • the sequence represented by SEQ ID NO: 1 is more preferable.
  • the official name of the IRX4 gene is Iroquois Homeobox 4, and its base sequence can be easily obtained through a public database such as GenBank (NCBI).
  • NCBI GenBank
  • the base sequence of the human IRX4 gene can be obtained under NCBI accession number NM_016358.2.
  • the target sequence for detecting the IRX4 gene is not particularly limited and may be appropriately selected depending on the intended purpose.
  • the sequence represented by SEQ ID NO: 3 and the sequence represented by SEQ ID NO: 4 are preferable.
  • MYL3 gene- The official name of the MYL3 gene is Myosin Light Chain 3, Alkali; Venticular, Skeletal, Slow, and its base sequence can be easily obtained through a public database such as GenBank (NCBI).
  • NCBI GenBank
  • the base sequence of the human MYL3 gene can be obtained under NCBI accession number NM — 0258.2.
  • the target sequence for detecting the MYL3 gene is not particularly limited and may be appropriately selected depending on the intended purpose.
  • the sequence represented by SEQ ID NO: 5 and the sequence represented by SEQ ID NO: 6 are preferable.
  • RPL3L gene- The official name of the RPL3L gene is Ribosomal Protein L3-Like, and its base sequence can be easily obtained through a public database such as GenBank (NCBI).
  • NCBI GenBank
  • the base sequence of the human RPL3L gene can be obtained by NCBI accession number NM_005061.2.
  • the target sequence for detecting the RPL3L gene is not particularly limited and may be appropriately selected depending on the intended purpose.
  • the sequence represented by SEQ ID NO: 7 and the sequence represented by SEQ ID NO: 8 are preferable.
  • a well-known structure can be selected suitably, for example, the structure which couple
  • the fluorescent RNA probe having a structure in which a fluorescently labeled double-stranded oligonucleotide is bound to the gold nanoparticle, one strand has a fluorescent dye molecule (hereinafter sometimes referred to as “reporter strand”), and the other The chain (hereinafter sometimes referred to as “capture chain”) is bonded to the gold nanoparticles.
  • reporter strand a fluorescent dye molecule
  • capture chain is bonded to the gold nanoparticles.
  • the fluorescent dye molecule of the reporter chain is quenched by the gold nanoparticles.
  • the fluorescent RNA probe when the fluorescent RNA probe approaches a target gene (hereinafter sometimes referred to as “target RNA”), the target RNA binds to the capture strand, the reporter strand is released, and fluorescence is emitted. By detecting the fluorescence, the presence or absence of expression of the target gene in the cardiomyocytes can be confirmed, and ventricular cardiomyocytes can be sorted in the sorting step described below.
  • a SmartFlare TM RNA detection probe manufactured by Merck & Co., Inc.
  • the fluorescent RNA probe having a structure in which a fluorescently labeled double-stranded oligonucleotide is bound to the gold nanoparticles.
  • the fluorescent dye is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include Cy3 and Cy5. These may be used individually by 1 type and may use 2 or more types together. Among the fluorescent dyes, Cy5 is preferable because it can be detected by a general-purpose cell sorter.
  • the cardiomyocytes are preferably seeded at a seeding density of 8 ⁇ 10 4 cells / cm 2 to 1.2 ⁇ 10 5 cells / cm 2 for about 2 days before introduction of the fluorescent RNA probe, and are subjected to adhesion culture. .
  • a seeding density 8 ⁇ 10 4 cells / cm 2 to 1.2 ⁇ 10 5 cells / cm 2 for about 2 days before introduction of the fluorescent RNA probe.
  • adhesion culture There is no restriction
  • the method for introducing the fluorescent RNA probe into the cardiomyocytes is not particularly limited and may be appropriately selected depending on the intended purpose.
  • the SmartFlare TM RNA detection probe (Merck Co., Ltd.) Can be introduced.
  • the introduction of the fluorescent RNA probe it is preferable not to use Lipofectamine LTX Plus (manufactured by Invitrogen) from the viewpoint of cytotoxicity.
  • the sorting step is a step of sorting cells that emit fluorescence derived from the fluorescent RNA probe (hereinafter sometimes referred to as “fluorescently labeled cells”). By the sorting step, ventricular cardiomyocytes are isolated and purified.
  • the means for sorting the cells that emit fluorescence is not particularly limited, and a known means can be appropriately selected. Examples thereof include an automatic cell analysis / separation apparatus (Fluorescence Activated Cell Sorter (FACS)).
  • FACS Fluorescence Activated Cell Sorter
  • the timing of performing the sorting step is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably one day after the introduction of the fluorescent RNA probe.
  • the method for confirming that ventricular cardiomyocytes have been obtained by the above method is not particularly limited and can be appropriately selected according to the purpose.
  • expression of a gene specific to ventricular cardiomyocytes can be performed. Examples thereof include a method for confirming by immunostaining, a method for confirming expression of a gene specific to ventricular cardiomyocytes by a PCR method, a method for confirming an action potential peculiar to ventricular cardiomyocytes by a patch clamp method and the like.
  • the ventricular cardiomyocytes of the present invention have an action potential amplitude of 140 mV or higher, a peak potassium current of 300 pA or higher, a peak calcium current of 1 nA or higher, and a peak sodium current of 6.5 nA or higher.
  • ventricular cardiomyocytes can be sorted alive from a cell population in which pacemaker cardiomyocytes, atrial cardiomyocytes, ventricular cardiomyocytes and the like are mixed.
  • the ventricular cardiomyocytes are very useful for the realization of regenerative medicine because they are more specific for diseases that are the subject of regenerative medicine such as severe heart failure.
  • by using only ventricular myocardial cells for screening it becomes possible to develop new drugs that are more useful for diseases in which ventricular myocardial cells exhibit abnormalities such as heart failure, cardiomyopathy, and Noonan syndrome. .
  • the screening method of the present invention is a screening method for a prophylactic or therapeutic agent for at least one of hypertrophic cardiomyopathy, cardiac hypertrophy, and Noonan syndrome.
  • the cells used in the screening method are not particularly limited as long as they are at least one of cardiomyocytes and ventricular cardiomyocytes, and can be appropriately selected according to the purpose. There is no restriction
  • the cardiac muscle cell manufactured by the manufacturing method of the cardiac muscle cell of this invention is preferable.
  • the ventricular cardiomyocytes are not particularly limited and may be appropriately selected depending on the purpose, but the ventricular cardiomyocytes of the present invention are preferred.
  • the screening method is not particularly limited as long as at least one of cardiomyocytes and ventricular cardiomyocytes is used, and can be appropriately selected according to the purpose, a test substance administration step, a measurement step, A method including a determination step is preferable.
  • a test substance administration step e.g., a test substance administration step
  • a measurement step e.g., a measurement step
  • a method including a determination step is preferable.
  • the cardiomyocytes it is preferable to further include a step of distinguishing ventricular cardiomyocytes from atrial cardiomyocytes.
  • the test substance administration step is a step of administering a test substance to at least one of the cardiomyocytes and the ventricular cardiomyocytes.
  • the cardiomyocytes and the ventricular cardiomyocytes may be cells derived from healthy subjects, cells derived from patients with hypertrophic cardiomyopathy, or cells derived from patients with cardiac hypertrophy. Alternatively, cells derived from a Noonan syndrome patient may be used. These may be used individually by 1 type and may use 2 or more types together.
  • test substance There is no restriction
  • the culture method of at least one of the cardiomyocytes to which the test substance is administered and the ventricular cardiomyocytes is not particularly limited, and can be appropriately selected according to the method used in the measurement step described later.
  • the patch clamp method it is necessary to culture at least one of the cardiomyocytes and the ventricular cardiomyocytes at a low density so that the cells are cultured in a single cell state. / Cm 2 to 10,000 cells / cm 2 are preferred.
  • the seeding density is preferably 15,000 cells / cm 2 to 60,000 cells / cm 2, more preferably 15,000 cells / cm 2 to 35,000 cells / cm 2 .
  • the seeding density is preferably 15,000 cells / cm 2 to 60,000 cells / cm 2, more preferably 15,000 cells / cm 2 to 35,000 cells / cm 2 .
  • Is preferably 1 ⁇ 10 5 cells / cm 2 to 2 ⁇ 10 5 cells / cm 2 .
  • cultivation According to the objective, it can select suitably, For example, the culture medium which added serum to DMEM etc. are mentioned. Examples of the serum content include 5% by mass to 10% by mass.
  • the measurement step is a step of measuring at least one of the cardiomyocytes administered with the test substance and the ventricular cardiomyocytes.
  • the measurement method is not particularly limited and can be appropriately selected depending on the purpose.
  • a method for measuring cell changes a method for measuring cell morphology, protein expression and protein modification using a high content analysis instrument, a method for observing cell movement using a cell imaging system, and the like.
  • the measurement step includes staining the cardiomyocytes with a cytoplasmic staining compound, measuring the area of the cardiomyocytes, staining the cardiomyocytes with an actin fiber staining compound, Treatment for measuring the dyeing strength of the fiber dyeing compound.
  • cytoplasmic staining compound is not particularly limited and may be appropriately selected depending on the intended purpose, e.g., HCS CellMask TM Blue (Invitrogen) and the like.
  • HCS CellMask TM Blue Invitrogen
  • staining to the end of the cell is not performed or evenly stained in the staining with the actin fiber staining compound or in the immunostaining in the process of distinguishing the ventricular cardiomyocytes and the atrial cardiomyocytes described later. It is possible to solve the problem that it is difficult to define a cell due to the absence of a cell.
  • the actin fiber dyeing compound is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include phalloidin.
  • the cytoskeleton can be stained with the actin fiber staining compound.
  • the nuclear staining compound is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include Hoechst.
  • the determination step is a step of determining a preventive or therapeutic effect on at least one of hypertrophic cardiomyopathy, cardiac hypertrophy, and Noonan syndrome of the test substance using the result of the measurement step as an index.
  • the cell area of a cardiomyocyte or ventricular cardiomyocyte derived from a patient with hypertrophic cardiomyopathy, cardiac hypertrophy, or Noonan syndrome administered with a test substance the roundness of the cell
  • the test substance is It can be determined that it has a therapeutic effect on hypertrophic cardiomyopathy, cardiac hypertrophy, or Noonan syndrome.
  • the various parameters of the texture analysis are not particularly limited and can be appropriately selected according to the purpose.
  • Operata manufactured by PerkinElmer
  • the evaluation step uses at least one of an area of ventricular cardiomyocytes and an abnormal cytoskeleton of ventricular cardiomyocytes as an index.
  • an area of the ventricular cardiomyocytes By using the area of the ventricular cardiomyocytes as an index, the degree of hypertrophy of the ventricular cardiomyocytes can be evaluated.
  • the cytoskeletal abnormality can be evaluated based on the staining intensity with the actin fiber staining compound and various parameters of the texture analysis.
  • a method for distinguishing between the ventricular cardiomyocytes and the atrial cardiomyocytes is not particularly limited and may be appropriately selected depending on the intended purpose. However, immunostaining is preferable. Whether or not it is a ventricular cardiomyocyte can be determined by immunostaining a gene expressed specifically in the ventricular cardiomyocyte. In addition, genes that are expressed specifically in atrial cardiomyocytes may be immunostained. There is no restriction
  • the screening method of the present invention it becomes possible to efficiently screen for preventive or therapeutic agents for at least one of hypertrophic cardiomyopathy, cardiac hypertrophy, and Noonan syndrome.
  • DMEM / F12 manufactured by Nacalai Tesque Co., Ltd.
  • Y27632 manufactured by Sigma, 5 ⁇ M
  • IMDM Iskov modified Dulbecco medium
  • ⁇ Mesodermal induction process> After the washing step, the following medium (hereinafter sometimes referred to as “culture medium for mesoderm induction process”) was added, and suspension culture was resumed in the culture dish (embryoid body formation process starting day 1 to 3 Day).
  • culture medium for mesoderm induction process As a basal medium, IMDM (manufactured by Nacalai Tesque Co., Ltd.) was used.
  • CHIR99021 manufactured by Nacalai Tesque Co., Ltd.
  • CHIR99021 second differentiation induction medium
  • ⁇ Washing process> The embryoid body after the mesoderm induction was collected in a centrifuge tube together with the culture solution, allowed to stand to precipitate the embryoid body, and then the supernatant was removed. Next, IMDM was added and the mixture was allowed to stand again to precipitate the embryoid body, and then the supernatant was removed. This operation was repeated twice.
  • first culture medium for cardiomyocyte induction treatment A medium in which IWP2 (Sigma, 5 ⁇ M), SB431542 (Sigma, 5 ⁇ M), and estradiol (Sigma, 100 nM) are added to the second differentiation induction medium prepared in the culture medium for the mesoderm induction process.
  • IWP2 Sigma, 5 ⁇ M
  • SB431542 SB431542
  • estradiol Sigma, 100 nM
  • ⁇ Cleaning process The embryoid body after the first cardiomyocyte induction treatment was collected in a centrifuge tube together with the culture solution, allowed to stand to precipitate the embryoid body, and then the supernatant was removed. Next, IMDM was added and the mixture was allowed to stand again to precipitate the embryoid body, and then the supernatant was removed. This operation was repeated twice.
  • Second cardiomyocyte induction process After the washing step, the following medium (hereinafter sometimes referred to as “second culture medium for cardiomyocyte induction treatment”) was added, and suspension culture was resumed in the culture dish (embryoid body formation process start day 6 Eyes to 8th).
  • second culture medium for cardiomyocyte induction treatment A medium obtained by adding IWP2 (manufactured by Sigma, 5 ⁇ M) and estradiol (manufactured by Sigma, 100 nM) to the second differentiation-inducing medium prepared in the culture medium for the mesoderm induction process is used for the second cardiomyocyte induction treatment. A culture solution was obtained.
  • ⁇ Cleaning process The embryoid body after the second cardiomyocyte induction treatment was collected in a centrifuge tube together with the culture solution, allowed to stand to precipitate the embryoid body, and then the supernatant was removed. Next, IMDM was added and the mixture was allowed to stand again to precipitate the embryoid body, and then the supernatant was removed. This operation was repeated twice.
  • third culture medium for cardiomyocyte induction treatment After the washing step, the following medium (hereinafter sometimes referred to as “third culture medium for cardiomyocyte induction treatment”) was added, and suspension culture was resumed in the culture dish (8 days after the embryoid body formation step) Eyes to 20th).
  • third culture medium for cardiomyocyte induction treatment A medium obtained by adding estradiol (manufactured by Sigma, 100 nM) to the second differentiation induction medium prepared in the culture medium for the mesoderm induction process was used as the second culture medium for cardiomyocyte induction treatment.
  • ⁇ Cleaning process The embryoid body after the third cardiomyocyte induction treatment was collected in a centrifuge tube together with the culture solution, allowed to stand to precipitate the embryoid body, and then the supernatant was removed. Next, IMDM was added and the mixture was allowed to stand again to precipitate the embryoid body, and then the supernatant was removed. This operation was repeated twice.
  • Non-cardiomyocyte growth-inhibiting culture medium As a basal medium, DMEM (glucose-free, manufactured by Nacalai Tesque) was used, and 5,000 mg / L of bovine serum albumin (manufactured by Sigma) and L-lactic acid (manufactured by Wako Pure Chemical Industries, Ltd.) were used as the basal medium. ) was added as a culture solution for inhibiting non-cardiomyocyte growth.
  • embryoid body dissociation process The embryoid bodies after being cultured in the non-cardiomyocyte growth-suppressing culture medium were dissociated as follows (embryoid body formation process start day 28, mesoderm induction process start day 27).
  • -Dissociation solution- A solution obtained by adding 20 mg / L of DNase I (Millipore) to 2.5 mg / L-Trypsin / 1 mmol / L-EDTA solution (Nacalai Tesque) was used as a dissociation solution. (4) After pipetting, 3 mL of proteolytic reaction stop solution having the following composition was added and held at 37 ° C. for 5 minutes to stop the proteolytic reaction and leak into the solution due to cell destruction. Genomic DNA was degraded.
  • the cells precipitated as pellets after removing the supernatant in (11) above are suspended in a medium (a culture medium in which 20% by mass of serum (Biowest) is added to DMEM (Nacalai Tesque)). It became turbid and seeded on a gelatin-coated culture dish (made by Greiner) so that the seeding density was 100,000 cells / cm 2 , followed by adhesion culture (culture conditions: 37 ° C., 5% CO 2 , embryoid body) 28th to 30th day from the formation process).
  • the gelatin coating can be performed by bringing a solution obtained by adding 0.1% by mass of gelatin (manufactured by Sigma) into PBS and bringing it into contact with the surface of the culture dish for 30 minutes or more, and then removing the solution.
  • Non-cardiomyocyte growth inhibition step-2 After the embryoid body dissociation step, the medium was changed to the non-cardiomyocyte growth inhibition culture solution in the non-cardiomyocyte growth inhibition step-1 and the culture was continued (culture conditions: 37 ° C., 5% CO 2 , embryoid-like Day 30 to 35 of the body formation process).
  • ⁇ Myocardial cell maturation process> After the non-cardiomyocyte growth inhibition step, the medium was changed to the following culture medium for cardiomyocyte maturation step, and the culture was continued (culture conditions: 37 ° C., 5% CO 2 , embryoid body formation step 35th day- Day 42).
  • the cardiomyocytes were manufactured by the above process.
  • -Culture medium for cardiomyocyte maturation process- As a basal medium, DMEM (manufactured by Nacalai Tesque Co., Ltd.) was used, and a medium obtained by adding 5% by mass of Bovine Calf Serum (manufactured by Hyclone) to the basal medium was used as a culture solution for cardiomyocyte maturation process.
  • RNA probe introduction process Two days before the introduction of the fluorescent RNA probe, the cardiomyocytes obtained in Production Example 1 were seeded on a culture dish (made by Greiner) coated with gelatin so that the seeding density was 100,000 cells / cm 2 . Adhesion culture was performed (culture conditions: 37 ° C., 5% CO 2 ). As the medium, the culture solution for cardiomyocyte maturation step in the cardiomyocyte maturation step of Production Example 1 was used.
  • SmartFlare solution containing a SmartFlare TM RNA detection probe (Millipore Corp., refer to the section of fluorescent RNA probe below for the target sequence) is 1 mL of culture solution. The culture was continued by adding 1 ⁇ L per unit. Cy5 was used as the fluorescent dye molecule of the reporter chain.
  • RNA fluorescently labeled probe (sometimes referred to as "RNA fluorescently labeled probe")- (1)
  • a probe whose target gene is the MYL2 gene and whose target sequence is represented by the following SEQ ID NO: 1 (hereinafter sometimes referred to as “MYL2-1”) 5′-catggcacctaagaagaagcaagaagag-3 ′ (SEQ ID NO: 1)
  • the sequence represented by SEQ ID NO: 1 is the 71st to 97th region of the MYL2 gene.
  • SEQ ID NO: 2 A probe whose target gene is MYL2 gene and whose target sequence is represented by the following SEQ ID NO: 2 (hereinafter sometimes referred to as “MYL2-2”) 5'-catcacccacggagagagagaggacta-3 '(SEQ ID NO: 2)
  • the sequence represented by SEQ ID NO: 2 is the 545th to 571st region of the MYL2 gene.
  • a probe whose target gene is the IRX4 gene and whose target sequence is represented by the following SEQ ID NO: 3 (hereinafter sometimes referred to as “IRX4-1”) 5′-ctatggcaactacgtgacctacgggctc-3 ′ (SEQ ID NO: 3)
  • SEQ ID NO: 3 The sequence represented by SEQ ID NO: 3 is the 365th to 391st region of the IRX4 gene.
  • a probe whose target gene is an IRX4 gene and whose target sequence is represented by the following SEQ ID NO: 4 (hereinafter sometimes referred to as “IRX4-2”) 5′-agagctccagagaacgcagagccccgtgg-3 ′ (SEQ ID NO: 4)
  • the sequence represented by SEQ ID NO: 4 is the 838th to 864th region of the IRX4 gene.
  • Probe whose target gene is MYL3 gene and whose target sequence is represented by the following SEQ ID NO: 5 (hereinafter sometimes referred to as “MYL3-1”) 5'-ctaaggagggtcgagtttgatgcttccca-3 '(SEQ ID NO: 5)
  • the sequence represented by SEQ ID NO: 5 is the 188th to 214th region of the MYL3 gene.
  • the sequence represented by SEQ ID NO: 6 is the 675th to 701st region of the MYL3 gene.
  • a probe whose target gene is RPL3L gene and whose target sequence is represented by SEQ ID NO: 7 below (hereinafter sometimes referred to as “RPL3L-1”) 5′-tgcagaacacctcagtgatgagtgccg-3 ′ (SEQ ID NO: 7)
  • the sequence represented by SEQ ID NO: 7 is the 365th to 391st region of the RPL3L gene.
  • Probe whose target gene is RPL3L gene and whose target sequence is represented by the following SEQ ID NO: 8 (hereinafter sometimes referred to as “RPL3L-2”) 5'-attacgctgagaaaagtcccctccgtggtg-3 '(SEQ ID NO: 8)
  • the sequence represented by SEQ ID NO: 8 is the 1028th to 1054th region of the RPL3L gene.
  • Negative control (labeled scrambled oligo, hereinafter sometimes referred to as “SF102”)
  • FIGS. 12 Test Example 1: Evaluation of ventricular cardiomyocytes-1) Using MYL2-1 as a fluorescent RNA probe, ventricular cardiomyocytes were purified in the same manner as in Production Example 2. The results of sorting by FACS are shown in FIGS. In FIG. 12, P1 represents a MYL2-1 positive fraction (the fraction with the highest fluorescence intensity labeled by MYL2-1), and P2 represents a MYL2-1 negative fraction (labeled by MYL2-1). (Fraction with lower 10% fluorescence intensity).
  • -Immunostaining- A cell group before sorting by FACS (hereinafter, sometimes referred to as “unsorted”), a cell group with the highest 10% fluorescence intensity labeled with MYL2-1 (hereinafter, referred to as “MYL2-positive fraction”) And a cell group having a fluorescence intensity labeled with MYL2-1 of the lower 10% (hereinafter sometimes referred to as “MYL2-negative fraction”), the seeding density is 30,000 cells / cm 2.
  • the cells were seeded in a 96-well plate (Cell Carrier-96, manufactured by PerkinElmer) coated with gelatin as described above, and adhesion culture was started (culture conditions: 37 ° C., 5% CO 2 ).
  • a culture solution in which 10% by mass of serum (manufactured by Biowest) was added to DMEM (manufactured by Nacalai Tesque) was used. Two days later, the medium was removed, washed with PBS, and fixed by performing 4 mass% paraformaldehyde at room temperature for 10 minutes.
  • Immunostaining of cells fixed with antibodies against the ventricular myosin light chain and the atrial myosin light chain was performed as follows, and analyzed using a high-content analysis device (PerkinElmer, Operatta).
  • the fixed cells were washed twice with PBS, and then a penetration operation was performed by allowing PBS containing 0.1% by mass of Triton X-100 (manufactured by Sigma) to act at room temperature for 5 minutes.
  • the cells after penetration were washed once with PBS, followed by blocking with PBS containing 5% by mass of normal goat serum (manufactured by Sigma) at room temperature for 60 minutes.
  • the blocked cells were washed once with PBS, and then a rabbit polyclonal antibody against the ventricular myosin light chain (catalog number 10906-1-AP, manufactured by Proteintech) in PBS containing 5% by mass of normal goat serum (manufactured by Sigma). ) And a mouse monoclonal antibody against the atrial myosin light chain (catalog number 311011, manufactured by Synaptics) diluted 1: 200 was allowed to act at 4 ° C. overnight.
  • the cells treated with the primary antibody solution were washed three times with PBS, and then goat anti-rabbit IgG antibody (Invitrogen) fluorescently labeled with AlexaFluor (registered trademark) 488 in PBS containing 5% by mass of normal goat serum (manufactured by Sigma). And a secondary antibody solution prepared by diluting goat anti-mouse IgG antibody (manufactured by Invitrogen) fluorescently labeled with AlexaFluor (registered trademark) 546 at 1: 500 was allowed to act at room temperature for 1 hour.
  • goat anti-rabbit IgG antibody Invitrogen
  • AlexaFluor registered trademark
  • Hoechst 33342 (10 mg / mL, manufactured by Invitrogen) was added 1: 10,000 in PBS containing 5% by mass of normal goat serum (manufactured by Sigma). Then, HCS CellMask TM Blue (manufactured by Invitrogen) dissolved in DMSO was diluted 1: 5,000, and phalloidin (manufactured by Biotium) fluorescently labeled with CF TM 647 dissolved in methanol was diluted 1:40. The counterstain solution was allowed to act for 30 minutes at room temperature. After the counterstained cells were washed twice with PBS, a post-fixing operation was performed by allowing 4% paraformaldehyde to act at room temperature for 10 minutes.
  • the ratio of ventricular muscle (MYL2 gene alone positive) cells was 64.1%, the percentage of atrial muscle (MYL7 gene alone positive) cells was 13.7%, and the other percentage was 22.2. 2%.
  • the ratio of ventricular cardiomyocytes was 94.3%, the ratio of atrial cardiomyocytes was 3.2%, and the other ratio was 2.6%.
  • the ratio of ventricular cardiomyocytes was 44.3%, the ratio of atrial cardiomyocytes was 24.6%, and the other ratio was 31.0%. Therefore, it was confirmed that ventricular cardiomyocytes can be purified by the method for producing ventricular cardiomyocytes of the present invention.
  • Ventricular cardiomyocyte-specific genes (MYL2 gene, MYL3 gene, IRX4 gene) in each cell of the MYL2-positive fraction and MYL2-negative fraction obtained in Test Example 1, and a gene related to calcium handling (PLN gene, RYR2 gene, ATP2A2 gene, CASQ1 gene) expression levels were analyzed by real-time PCR. The results are shown in FIG.
  • RNA extraction reagent Trizol, manufactured by Invitrogen
  • genomic DNA was removed using a kit (Quantitative Reverse Transcription Kit, manufactured by QIAGEN), and reverse transcription was performed to prepare cDNA.
  • Real-time PCR was performed using a universal probe and LightCycler 480 II (Roche). Primer sequences and universal probe numbers used for real-time PCR are as follows.
  • the horizontal axis indicates genes, the left side in each gene item indicates the result of analyzing gene expression in cells of the MYL2-negative fraction, and the right side indicates gene expression in cells of the MYL2-positive fraction.
  • the result of analyzing is shown. From the results of FIG. 13, in the MYL2-positive fraction, the expression of the ventricular cardiomyocyte-specific gene and the gene relating to calcium handling was high. The more ventricular cardiomyocytes that have more genes related to calcium handling are mature ventricular cardiomyocytes and are suitable for application. Therefore, the ventricular cardiomyocytes obtained by the production method of the present invention may be superior. Indicated.
  • Test Example 3 Evaluation of ventricular cardiomyocytes-3) Electrophysiological analysis using a patch clamp was performed on each cell of the MYL2-positive fraction and the MYL2-negative fraction obtained in Test Example 1. The results are shown in FIGS.
  • the cells are seeded on a gelatin-coated cover glass and cultured for 5 to 10 days. Then, spontaneously beating single cells are made into a whole cell state, and spontaneous activity is performed under current fixation (0 pA). The potential was measured.
  • the composition of the extracellular fluid was NaCl 150 mmol / L, KCl 4 mmol / L, CaCl 2 1.2 mmol / L, MgCl 2 1 mmol / L, D (+)-glucose 10 mmol / L, HEPES 10 mmol / L (using NaOH adjusted to pH 7.4).
  • the composition of the electrode internal solution used was KCl 140 mmol / L, MgCl 2 1 mmol / L, EGTA 5 mmol / L, MgATP 5 mmol / L, HEPES 10 mmol / L (adjusted to pH 7.2 using KOH).
  • FIG. 14 shows a typical example of the action potential of cells in the MYL2-positive fraction
  • FIG. 15 shows a typical example of the action potential of cells in the MYL2-negative fraction.
  • the MYL2-positive fraction action potentials were observed in all cells, and the proportion of cells in which ventricular muscle-like action potentials were observed was 92% (11 out of 12 cells).
  • the proportion of cells in which action potential was observed was 50%, of which the proportion of cells in which ventricular myogenic action potential was observed was 33% (2 out of 6 cells). . Therefore, the results of electrophysiological analysis showed that the ventricular cardiomyocytes obtained by the production method of the present invention were excellent.
  • the MYL2-positive fraction collected by FACS was immunostained in the same manner as in Test Example 1.
  • Test Example 5 Examination of purification time of ventricular cardiomyocytes -2) Ventricular cardiomyocyte-specific genes (MYL2 gene, MYL3 gene, IRX4 gene) in cells of each MYL2-positive fraction obtained in Test Example 4, and genes related to calcium handling (PLN gene, RYR2 gene, ATP2A2 gene, The expression level of CASQ1 gene) was measured in the same manner as in Test Example 2. The results are shown in FIG.
  • the horizontal axis represents genes, and the left side of each gene item is (1) genes in MYL2-positive fraction cells when using cardiomyocytes after the embryoid body dissociation step of Production Example 1
  • the results of the analysis of the expression of are shown in the middle, and (2) the expression of the gene in the MYL2-positive fraction cells when using the cardiomyocytes after the non-cardiomyocyte growth inhibition step-2 of Production Example 1 was analyzed.
  • a result is shown and the right side shows the result of having analyzed the gene expression in the cell of the MYL2 positive fraction at the time of using the cardiomyocyte after the cardiomyocyte maturation process of the said manufacture example 1 (3). From the results of FIG. 16, it was shown that the expression of the ventricular cardiomyocyte-specific gene and the gene involved in calcium handling is increased by performing the cardiomyocyte maturation step.
  • Test Example 6 Examination of purification time of ventricular cardiomyocytes-3) Using the results of immunostaining in Test Example 4, the areas of ventricular cardiomyocytes and atrial cardiomyocytes were measured. The results are shown in Table 6-1 to Table 6-3.
  • Test Example 7 Evaluation of ventricular cardiomyocytes-4) Electrophysiological analysis using patch clamp for cells of MYL2 positive fraction obtained in Test Example 1, commercially available cardiomyocytes, iCell Cardiomyocytes (Cellular Dynamics International), and ReproCardio (manufactured by Reprocell, Inc.) Analysis was performed.
  • the cells were seeded on a gelatin-coated cover glass and cultured for 5 to 10 days. Then, spontaneously beating single cells were put into a whole cell state and observed under potential fixation.
  • the composition of the extracellular fluid was NaCl 150 mmol / L, KCl 4 mmol / L, CaCl 2 1.2 mmol / L, MgCl 2 1 mmol / L, D (+)-glucose 10 mmol / L, HEPES 10 mmol / L (using NaOH adjusted to pH 7.4).
  • the composition of the electrode internal solution used was KCl 140 mmol / L, MgCl 2 1 mmol / L, EGTA 5 mmol / L, MgATP 5 mmol / L, HEPES 10 mmol / L (adjusted to pH 7.2 using KOH).
  • the following was used as the potential protocol for measuring the ion channel current.
  • Sodium current A depolarization stimulus of 20 milliseconds from a holding potential of ⁇ 90 mV to ⁇ 20 mV was applied at 1 second intervals.
  • Calcium current A depolarization stimulus of 100 milliseconds was applied at intervals of 10 seconds from a holding potential of ⁇ 40 mV to ⁇ 0 mV.
  • Potassium current After 50 milliseconds of depolarization from a holding potential of -90 mV to -40 mV, a stimulus of 20 mV, 2 seconds of depolarization, and further -40 mV, repolarization to 0.5 seconds was given at 15 second intervals.
  • FIG. 17 shows the result of analyzing the sodium current of cells of the MYL2-positive fraction obtained in Test Example 1
  • FIG. 18 shows the result of analyzing the sodium current of commercially available iCell Cardiomyocytes
  • FIG. The result of having analyzed the sodium current of ReproCardio of is shown.
  • FIG. 20 shows the result of analyzing the potassium current of cells of the MYL2-positive fraction obtained in Test Example 1
  • FIG. 21 shows the result of analyzing the potassium current of commercially available iCell Cardiomyocytes
  • FIG. The result of having analyzed the potassium current of ReproCardio of is shown.
  • FIG. 23 shows the result of analyzing the calcium current of cells of the MYL2-positive fraction obtained in Test Example 1
  • FIG. 24 shows the result of analyzing the calcium current of commercially available iCell Cardiomyocytes, and FIG. The result of having analyzed the calcium current of commercially available ReproCardio is shown.
  • ventricular cardiomyocytes obtained by the production method of the present invention have a large peak sodium current of 6.5 nA or more, a large peak potassium current of 300 pA or more, and a large peak calcium current of 1 nA or more. It was shown that it was very excellent compared with the commercial product.
  • the cardiomyocytes were seeded in 96-well plates at 15,000 cells / cm 2 .
  • a culture solution in which 10% by mass of serum (manufactured by Biowest) was added to DMEM (manufactured by Nacalai Tesque) was used.
  • DMEM manufactured by Nacalai Tesque
  • MYL2 gene ventricular myosin light chain
  • MYL7 gene atrial myosin light chain
  • cell nuclei and cytoplasm are stained, and a high content analysis device ( Observation and analysis were carried out using PerkinElmer (Operetta).
  • the immunostaining was performed as follows.
  • the medium was removed, washed with PBS, and fixed by performing 4 mass% paraformaldehyde for 10 minutes at room temperature.
  • the fixed cells were washed twice with PBS, and then a penetration operation was performed by allowing PBS containing 0.1% by mass of Triton X-100 (manufactured by Sigma) to act at room temperature for 5 minutes.
  • the cells after penetration were washed once with PBS, followed by blocking with PBS containing 5% by mass of normal goat serum (manufactured by Sigma) at room temperature for 60 minutes.
  • the blocked cells were washed once with PBS, and then a rabbit polyclonal antibody against the ventricular myosin light chain (catalog number 10906-1-AP, manufactured by Proteintech) in PBS containing 5% by mass of normal goat serum (manufactured by Sigma). ) And a mouse monoclonal antibody against the atrial myosin light chain (catalog number 311011, manufactured by Synaptics) diluted 1: 200 was allowed to act at 4 ° C. overnight.
  • the cells treated with the primary antibody solution were washed three times with PBS, and then goat anti-rabbit IgG antibody (Invitrogen) fluorescently labeled with AlexaFluor (registered trademark) 488 in PBS containing 5% by mass of normal goat serum (manufactured by Sigma). And a secondary antibody solution prepared by diluting goat anti-mouse IgG antibody (manufactured by Invitrogen) fluorescently labeled with AlexaFluor (registered trademark) 546 at 1: 500 was allowed to act at room temperature for 1 hour.
  • goat anti-rabbit IgG antibody Invitrogen
  • AlexaFluor registered trademark
  • Hoechst 33342 (10 mg / mL, manufactured by Invitrogen) was added at 1: 10,000 in PBS containing 5% by mass of normal goat serum (manufactured by Sigma). This was prepared by diluting HCS CellMask TM Blue (manufactured by Invitrogen) dissolved in DMSO at 1: 5,000, and phalloidin (manufactured by Biotium) fluorescently labeled with CF TM 647 dissolved in methanol at 1:40. The counterstain solution was allowed to act for 30 minutes at room temperature. After the counterstained cells were washed twice with PBS, a post-fixing operation was performed by allowing 4% paraformaldehyde to act at room temperature for 10 minutes.
  • FIG. 26 A representative example of immunostaining cardiomyocytes produced using iPS cells derived from healthy subjects is shown in FIG. 26, and a representative example of immunostaining cardiomyocytes produced using iPS cells derived from hypertrophic cardiomyopathy patients is shown. An example is shown in FIG.
  • FIGS. 28 to 40 show the results of analysis by the high content analysis device.
  • “healthy person” shows the results of ventricular cardiomyocytes in cardiomyocytes produced using iPS cells derived from healthy persons
  • “Noonan syndrome” shows iPS cells derived from Noonan syndrome patients. Shows the results of ventricular cardiomyocytes in the cardiomyocytes produced using “hypertrophic cardiomyopathy” is the result of ventricular cardiomyocytes in the cardiomyocytes produced using iPS cells derived from hypertrophic cardiomyopathy patients Show.
  • the cell area was compared with ventricular cardiomyocytes manufactured using healthy person-derived iPS cells, and ventricular cardiomyocytes and hypertrophy manufactured using Noonan syndrome patient-derived iPS cells. Ventricular cardiomyocytes produced using cardiomyopathy patient-derived iPS cells were larger.
  • the roundness of the cells is a ventricular type produced using iPS cells derived from patients with hypertrophic cardiomyopathy, compared to ventricular type cardiomyocytes produced using iPS cells derived from healthy subjects. Myocardial cells were smaller.
  • the area of the nucleus is a ventricular cardiomyocyte produced using a hypertrophic cardiomyopathy patient-derived iPS cell as compared with a ventricular cardiomyocyte produced using a healthy person-derived iPS cell.
  • a hypertrophic cardiomyopathy patient-derived iPS cell as compared with a ventricular cardiomyocyte produced using a healthy person-derived iPS cell.
  • the roundness rate of the nucleus is a ventricular type produced using iPS cells derived from patients with hypertrophic cardiomyopathy, compared to ventricular type cardiomyocytes produced using iPS cells derived from healthy subjects. Myocardial cells were smaller.
  • the intensity of phalloidin staining is higher for ventricular cardiomyocytes produced using Noonan syndrome patient-derived iPS cells than for ventricular cardiomyocytes produced using healthy person-derived iPS cells. It was getting smaller.
  • “Saddle (FIG. 39)” and “Spot (FIG. 40)” are iPS cells derived from healthy subjects. Compared with the ventricular cardiomyocytes manufactured using this, the ventricular cardiomyocytes manufactured using iPS cells derived from hypertrophic cardiomyopathy patients were smaller.
  • the cardiomyocytes and ventricular cardiomyocytes obtained by the method of the present invention can be applied to screening methods for preventive or therapeutic drugs for at least one of Noonan syndrome, hypertrophic cardiomyopathy, and cardiac hypertrophy. It was.
  • Cardiomyocytes were produced in the same manner as in Production Example 1 except that iPS cells derived from patients with hypertrophic cardiomyopathy established at the University of Tokyo Hospital as a pluripotent stem cell were used.
  • the cardiomyocytes were seeded in 96-well plates at 15,000 cells / cm 2 .
  • a culture solution in which 10% by mass of serum (manufactured by Biowest) was added to DMEM (manufactured by Nacalai Tesque) was used.
  • the medium was replaced with a culture solution in which 5% by mass of serum (manufactured by Hyclone) was added to DMEM (manufactured by Nacalai Tesque Co., Ltd.), and rapamycin, a drug that has been reported to have an effect of suppressing cardiac hypertrophy Nifedipine and carvedilol were added at a final concentration of 10 ⁇ M.
  • the cells expressing MYL2 gene (ventricular myosin light chain) and MYL7 gene (atrial myosin light chain) were expressed by immunostaining in the same manner as in Test Example 8. Cells, cell nuclei, and cytoplasm were stained, and observed and analyzed using a high-content analyzer (PerkinElmer, Operatta).
  • FIGS. 41 to 42 The results of analysis of ventricular myocardial cells using a high content analyzer are shown in FIGS.
  • carvedilol was added, as shown in FIGS. 41 to 42, both the cell area and the nucleus area were reduced compared to the control.
  • rapamycin was added and when nifedipine was added, the nuclear area decreased compared to the control, as shown in FIG. Therefore, it is confirmed that a compound having a preventive or therapeutic effect on cardiac hypertrophy can be appropriately detected by using at least one of the cardiomyocytes produced by the production method of the present invention and the ventricular cardiomyocytes of the present invention. It was done.
  • Examples of the aspect of the present invention include the following. ⁇ 1> An embryoid body forming step of culturing pluripotent stem cells to form an embryoid body, Culturing the embryoid body and inducing a mesoderm, Culturing the embryoid body after the mesoderm induction and inducing cardiomyocytes, An embryoid body dissociation step of dissociating the embryoid body after the cardiomyocyte induction, The method for producing cardiomyocytes, wherein the embryoid body dissociation step is performed from the start of the mesoderm induction step to the 40th day.
  • ⁇ 2> The method for producing cardiomyocytes according to ⁇ 1>, wherein the embryoid body dissociation step is performed from the 16th day to the 30th day from the start of the mesoderm induction step.
  • ⁇ 3> The cardiomyocyte according to any one of ⁇ 1> to ⁇ 2>, wherein the time during which the embryoid body is continuously contacted with the proteolytic enzyme in the embryoid body dissociation step is 1 to 30 minutes It is a manufacturing method.
  • ⁇ 4> The method for producing cardiomyocytes according to any one of ⁇ 1> to ⁇ 3>, wherein the embryoid body dissociation step is performed a plurality of times.
  • ⁇ 5> The method for producing cardiomyocytes according to any one of ⁇ 1> to ⁇ 4>, including a cardiomyocyte maturation step of culturing and maturing the cardiomyocytes after the embryoid body dissociation step.
  • ⁇ 6> The method for producing cardiomyocytes according to any one of ⁇ 1> to ⁇ 5>, further including a non-cardiomyocyte growth suppression step of suppressing non-cardiomyocyte growth.
  • ⁇ 7> The ⁇ 1>, wherein the non-cardiomyocyte growth suppression step is performed at least one of a cardiomyocyte induction step and an embryoid body dissociation step and an embryoid body dissociation step and a cardiomyocyte maturation step > To ⁇ 6>.
  • cardiomyocytes are used in at least one of a prophylactic or therapeutic drug screening method for at least one of hypertrophic cardiomyopathy, cardiac hypertrophy, and Noonan syndrome, and production of ventricular cardiomyocytes
  • ⁇ 7> A method for producing cardiomyocytes according to any one of the above.
  • ⁇ 9> A fluorescent RNA probe introduction step of introducing a fluorescent RNA probe into cardiomyocytes produced by the method for producing cardiomyocytes according to any one of ⁇ 1> to ⁇ 8>, And a sorting step of sorting cells that emit fluorescence derived from the fluorescent RNA probe.
  • ⁇ 10> The method for producing a ventricular cardiomyocyte according to ⁇ 9>, wherein the target gene of the fluorescent RNA probe is at least one of a MYL2 gene, an IRX4 gene, a MYL3 gene, and an RPL3L gene.
  • the target gene of the fluorescent RNA probe is the MYL2 gene.
  • the ventricular cardiomyocyte according to ⁇ 12> which is produced by the method for producing a ventricular cardiomyocyte according to any one of ⁇ 9> to ⁇ 11>.
  • a screening method for a preventive or therapeutic agent for at least one of hypertrophic cardiomyopathy, cardiac hypertrophy, and Noonan syndrome A screening method using at least one of cardiomyocytes and ventricular cardiomyocytes.
  • the cardiomyocyte is a cardiomyocyte produced by the method for producing a cardiomyocyte according to any one of ⁇ 1> to ⁇ 8>, and the ventricular cardiomyocyte is any of the above ⁇ 12> to ⁇ 13>
  • the screening method according to ⁇ 14> wherein the screening method is any one of ventricular cardiomyocytes.
  • the screening method according to any one of the above.
  • the screening method according to any one of ⁇ 15> to ⁇ 16> including a step of distinguishing ventricular cardiomyocytes from atrial cardiomyocytes by immunostaining.
  • the measurement step stains cardiomyocytes with a cytoplasmic staining compound, measures the area of the cardiomyocytes, stains the cardiomyocytes with an actin fiber staining compound, and measures the staining intensity with the actin fiber staining compound.
  • the present invention it is possible to solve the conventional problems, achieve the object, and to produce a high-quality cardiomyocyte, a method for producing a cardiomyocyte, mixing of non-cardiomyocytes, High-quality ventricular cardiomyocytes with reduced cell heterogeneity, a method for producing the same, and hypertrophic cardiomyopathy, cardiac hypertrophy, and Noonan syndrome using at least one of cardiomyocytes and ventricular cardiomyocytes It is possible to provide a method for screening a preventive or therapeutic agent for at least one of

Abstract

L'invention concerne un procédé de fabrication de cellules myocardiques qui permet de fabriquer des cellules myocardiques de haute qualité. En outre, l'invention concerne des cellules myocardiques type ventricule de haute qualité ainsi qu'un procédé de fabrication de celles-ci, lesquelles cellules myocardiques type ventricule présentent une réduction de la contamination par des cellules non myocardiques, et de la non homogénéité des cellules myocardiques. Enfin, l'invention concerne un procédé de criblage d'un médicament de prévention ou de traitement contre au moins la cardiomyopathie hypertrophique, la cardiomégalie ou le syndrome de Nooman qui impliquent les cellules myocardiques et/ou les cellules myocardiques type ventricule.
PCT/JP2015/078784 2014-10-09 2015-10-09 Procédé de fabrication de cellules myocardiques, cellules myocardiques type ventricule ainsi que procédé de fabrication de celles-ci, et procédé de criblage WO2016056653A1 (fr)

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