METHOD FOR EFFICIENTLY PRODUCING MYOSIN HEAVY CHAIN IN CARDIAC MUSCLE CELLS DIFFERENTIATED FROM INDUCED PLURIPOTENT STEM CELLS DERIVED FROM HOMO SAPIENS

Abstract

The present invention provides a method for producing a .beta. myosin heavy chain in cardiac muscle cells differentiated from induced pluripotent stem cells derived from Homo sapiens. In the present method, first, a liquid culture medium containing the cardiac muscle cells is supplied onto a substrate comprising a first electrode, a second electrode and insulative fibers on the surface thereof. At least a part of the insulative fibers is located between the first electrode and the second electrode in a top view of the substrate. Then, the substrate is left at rest. Finally, the cardiac muscle cells are cultivated, while a pulse electric current is applied to the cardiac muscle cells through the first electrode and the second electrode.

Description

CROSS-REFERENCE OF RELATED APPLICATIONS

[0001] This application is a Divisional application of the patent application Ser. No. 15/848,020, filed on Dec. 20, 2017, which claims the benefit of Japanese Application No. 2017-039998, filed on Mar. 3, 2017, the entire disclosures of which applications are incorporated by reference herein.

INCORPORATION BY REFERENCE-SEQUENCE LISTING

[0002] The material contained in the ASCII text file named "P1006798US01_ST25.txt" created on Nov. 22, 2017, and having a file size of 18, 746 bytes is incorporated by reference herein.

BACKGROUND

1. Technical Field

[0003] The present invention relates to a method for efficiently producing a .beta. myosin heavy chain in cardiac muscle cells differentiated from induced pluripotent stem cells derived from Homo sapiens.

2. DESCRIPTION OF THE RELATED ART

[0004] Japanese patent application laid-open publication No. Sho 60-110287 discloses that cell proliferation is promoted by application of electric pulse to the cultivated cells.

[0005] Japanese patent application laid-open publication No. Hei 4-141087 discloses a method that cells are differentiated by application of electric voltage to the cells through a liquid culture medium.

[0006] U.S. Pat. No. 8,916,189 discloses a cell culture support for forming string-shaped cardiomyocyte aggregates.

[0007] Japanese patent application laid-open publication No. 2013-188173 discloses a method for creating cell tissue having function.

[0008] United States Patent Application Publication No. 2015/0017718 discloses a method for inducing cardiac differentiation of a pluripotent stem cell.

[0009] WO 2016/060260 discloses a method for producing a tissue fragment, particularly a myocardial tissue fragment which contains cultured cells having an oriented configuration. See FIG. 4B, FIG. 9A, and paragraphs 0055, 0131, 0141, 0142, and 0153 thereof.

SUMMARY

[0010] The present invention provides a method for producing a .beta. myosin heavy chain in cardiac muscle cells differentiated from induced pluripotent stem cells derived from Homo sapiens, the method comprising:

[0011] (a) supplying a liquid culture medium containing the cardiac muscle cells onto a substrate comprising a first electrode, a second electrode and insulative fibers on the surface thereof to coat a surface of the first electrode, a surface of the second electrode, and an region between the first electrode and the second electrode with the cardiac muscle cells;

[0012] wherein

[0013] at least a part of the insulative fibers is located between the first electrode and the second electrode in a top view of the substrate; and

[0014] an angle formed between each of not less than 90% of the insulative fibers and an imaginary straight line which passes through both the first electrode and the second electrode is not more than .+-.20 degrees in the top view;

[0015] (b) leaving the substrate at rest; and

[0016] (c) cultivating the cardiac muscle cells, while a pulse electric current is applied to the cardiac muscle cells through the first electrode and the second electrode.

[0017] The present invention provides a method for efficiently producing a .beta. myosin heavy chain in cardiac muscle cells differentiated from induced pluripotent stem cells derived from Homo sapiens.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 shows a top view of a substrate.

[0019] FIG. 2 shows an enlarged view of a region A included in FIG. 1.

[0020] FIG. 3 shows a graph showing an example of desirable pulse electric current.

[0021] FIG. 4 shows a top view of the substrate in one step included in a method for fabricating the substrate.

[0022] FIG. 5 shows an enlarged view of a region B included in FIG. 4.

[0023] FIG. 6A shows an enlarged top view of an end part of an electric wiring.

[0024] FIG. 6B shows a cross-sectional view taken along the line 6B-6B included in FIG. 6A.

[0025] FIG. 7A shows an enlarged top view of the end part of the electric wiring.

[0026] FIG. 7B shows a cross-sectional view taken along the line 7B-7B included in FIG. 7A.

[0027] FIG. 8A shows a cross-sectional view of the substrate on which a liquid culture medium has been supplied.

[0028] FIG. 8B shows a cross-sectional view of the substrate on which a liquid culture medium has been supplied.

[0029] FIG. 9A is a fluorescent microscope photograph of the cardiac muscle cells in the inventive example 1.

[0030] FIG. 9B is a fluorescent microscope photograph of the cardiac muscle cells in the comparative example 2.

[0031] FIG. 9C is a fluorescent microscope photograph of the cardiac muscle cells in the comparative example 4.

[0032] FIG. 9D is a fluorescent microscope photograph of the cardiac muscle cells in the comparative example 6.

[0033] FIG. 10A shows an enlarged top view of the end part of the electric wiring in the comparative examples 2 and 3.

[0034] FIG. 10B shows a cross-sectional view taken along the line 10B-10B included in FIG. 10A.

[0035] FIG. 11A shows an enlarged top view of the end part of the electric wiring in the comparative examples 4 and 5.

[0036] FIG. 11B shows a cross-sectional view taken along the line 11B-11B included in FIG. 11A.

[0037] FIG. 12A shows an enlarged top view of the end part of the electric wiring in the comparative examples 6 and 7.

[0038] FIG. 12B shows a cross-sectional view taken along the line 12B-12B included in FIG. 12A.

[0039] FIG. 13A is a microscope photograph of a first electrode, a second electrode, and an insulative fibers which have been formed on the thus-provided substrate in the inventive example 1.

[0040] FIG. 13B is another microscope photograph of the first electrode, the second electrode, and the insulative fibers which have been formed on the substrate in the inventive example 1.

[0041] FIG. 13C is a microscope photograph of the first electrode, the second electrode, and the insulative fibers which have been formed on the substrate 100 used in the comparative example 2 and the comparative example 3.

[0042] FIG. 13D is a microscope photograph of the first electrode, the second electrode, and the insulative fibers which have been formed on the provided substrate used in the comparative example 4 and the comparative example 5.

DETAILED DESCRIPTION OF THE EMBODIMENT

[0043] Hereinafter, an embodiment of the present invention will be described with reference to the drawings.

[0044] As disclosed in FIG. 2C of United States Patent Application Publication No. 2015/0017718, an amount of production of a .beta. myosin heavy chain (hereinafter, referred to as ".beta. MHC") is significantly smaller in cardiac muscle cells differentiated from induced pluripotent stem cells derived from Homo sapiens than in cardiac muscle cells included in a living body. The .beta. MHC is one kind of polypeptides providing support for a structure of the cell. For the maturation of the cardiac muscle cells differentiated from induced pluripotent stem cells derived from Homo sapiens, it is important to produce the .beta. MHC efficiently.

[0045] The .beta. MHC has a primary structure consisting of the amino acid sequence represented by the following SEQ ID NO: 1.

TABLE-US-00001 (SEQ ID NO: 1) MGDSEMAVFGAAAPYLRKSEKERLEAQTRPFDLKKDVFVPDDKQEFVKAK IVSREGGKVTAETEYGKTVTVKEDQVMQQNPPKFDKIEDMAMLTFLHEPA VLYNLKDRYGSWMIYTYSGLFCVTVNPYKWLPVYTPEVVAAYRGKKRSEA PPHIFSISDNAYQYMLTDRENQSILITGESGAGKTVNTKRVIQYFAVIAA IGDRSKKDQSPGKGTLEDQIIQANPALEAFGNAKTVRNDNSSRFGKFIRI HFGATGKLASADIETYLLEKSRVIFQLKAERDYHIFYQILSNKKPELLDM LLITNNPYDYAFISQGETTVASIDDAEELMATDNAFDVLGETSEEKNSMY KLTGAIMHFGNMKFKLKQREEQAEPDGTEEADKSAYLMGLNSADLLKGLC HPRVKVGNEYVTKGQNVQQVIYATGALAKAVYERMENWMVTRINATLETK QPRQYFIGVLDIAGFEIFDFNSFEQLCINFTNEKLQQFFNHHMFVLEQEE YKKEGIEWTFIDFGMDLQACIDLIEKPMGIMSILEEECMFPKATDMTFKA KLFDNHLGKSANFQKPRNIKGKPEAHFSLIHYAGIVDYNIIGWLQKNKDP LNETVVGLYQKSSLKLLSTLFANYAGADAPIEKGKGKAKKGSSFQTVSAL HRENLNKLMTNLRSTHPHFVRCIIPNETKSPGVMDNPLVMHQLRCNGVLE GIRICRKGFPNRILYGDFRQRYRILNPAAIPEGQFIDSRKGAEKLLSSLD IDHNQYKFGHTKVFFKAGLLGLLEEMRDERLSRIITRIQAQSRGVLARME YKKLLERRDSLLVIQWNIRAFMGVKNWPWMKLYFKIKPLLKSAEREKEMA SMKEEFTRLKEALEKSEARRKELEEKMVSLLQEKNDLQLQVQAEQDNLAD AEERCDQLIKNKIQLEAKVKEMNERLEDEEEMNAELTAKKRKLEDECSEL KRDIDDLELTLAKVEKEKHATENKVKNLTEEMAGLDEIIAKLTKEKKALQ EAHQQALDDLQAEEDKVNTLTKAKVKLEQQVDDLEGSLEQEKKVRMDLER AKRKLEGDLKLTQESIMDLENDKQQLDERLKKKDFELNALNARIEDEQAL GSQLQKKLKELQARIEELEEELESERTARAKVEKLRSDLSRELEEISERL EEAGGATSVQIEMNKKREAEFQKMRRDLEEATLQHEATAAALRKKHADSV AELGEQIDNLQRVKQKLEKEKSEFKLELDDVTSNMEQIIKAKANLEKMCR TLEDQMNEHRSKAEETQRSVNDLTSQRAKLQTENGELSRQLDEKEALISQ LTRGKLTYTQQLEDLKRQLEEEVKAKNALAHALQSARHDCDLLREQYEEE TEAKAELQRVLSKANSEVAQWRTKYETDAIQRTEELEEAKKKLAQRLQEA EEAVEAVNAKCSSLEKTKHRLQNEIEDLMVDVERSNAAAAALDKKQRNFD KILAEWKQKYEESQSELESSQKEARSLSTELFKLKNAYEESLEHLETFKR ENKNLQEEISDLTEQLGSSGKTIHELEKVRKQLEAEKMELQSALEEAEAS LEHEEGKILRAQLEFNQIKAEIERKLAEKDEEMEQAKRNHLRVVDSLQTS LDAETRSRNEALRVKKKMEGDLNEMEIQLSHANRMAAEAQKQVKSLQSLL KDTQIQLDDAVRANDDLKENIAIVERRNNLLQAELEELRAVVEQTERSRK LAEQELIETSERVQLLHSQNTSLINQKKKMDADLSQLQTEVEEAVQECRN AEEKAKKAITDAAMMAEELKKEQDTSAHLERMKKNMEQTIKDLQHRLDEA EQIALKGGKKQLQKLEARVRELENELEAEQKRNAESVKGMRKSERRIKEL TYQTEEDRKNLLRLQDLVDKLQLKVKAYKRQAEEAEEQANTNLSKFRKVQ HELDEAEERADIAESQVNKLRAKSRDIGTKGLNEE

[0046] For reference, myosin regulatory light chain 2 (hereinafter, referred to as "MYL2") is also produced in the cardiac muscle cells. The MYL2 has a primary structure consisting of the amino acid sequences represented by the following SEQ ID NO: 2.

TABLE-US-00002 (SEQ ID NO: 2) MAPKKAKKRAGGANSNVFSMFEQTQIQEFKEAFTIMDQNRDGFIDKNDLR DTFAALGRVNVKNEEIDEMIKEAPGPINFTVFLTMFGEKLKGADPEETIL NAFKVFDPEGKGVLKADYVREMLTTQAERFSKEEVDQMFAAFPPDVTGNL DYKNLVHIITHGEEKD

[0047] Hereinafter, the cardiac muscle cells differentiated from induced pluripotent stem cells derived from Homo sapiens are just referred to as "cardiac muscle cells". As well known, the induced pluripotent stem cells may be referred to as "iPS cells".

[0048] (Step (a))

[0049] First, a liquid culture medium containing cardiac muscle cells are supplied on a substrate 100 comprising a first electrode, a second electrode, and insulative fibers on the surface thereof.

[0050] FIG. 1 shows a top view of the substrate 100. FIG. 2 shows an enlarged view of a region A included in FIG. 1.

[0051] As shown in FIG. 1, the substrate 100 comprises a glass base 1 and an enclosure 10 located on the glass base 1. The surface of the glass base 1 is provided with electric contacts 2 and electric wirings 3. Each of the electric contacts 2 is connected to one end of one electric wiring 3. Within the enclosure 10, an insulative sheet 60 is disposed on the glass base 1. The electric wirings 3 are covered with the insulative sheet 60.

[0052] As shown in FIG. 2, other ends of the electric wirings 3 are exposed. The exposed parts function as a first electrode 31 and a second electrode 32. In FIG. 2, four electric wirings 3 are drawn. The first electrode 31 is formed of the exposed end part of the electric wiring 3 located on the left. Similarly, the second electrode 32 is formed of the exposed end part of the electric wiring 3 located on the right.

[0053] As shown in FIG. 1 and FIG. 2, insulative fibers 50 are disposed on the surface of substrate 100. The fibers 50 are required to be insulative. This is because a short circuit is prevented from being formed erroneously between the first electrode 31 and the second electrode 32. In case where the short circuit is formed erroneously, a pulse electric current which will be described later fails to be applied to the cardiac muscle cells.

[0054] As shown in FIG. 2, at least a part of the insulative fibers 50 is located between the first electrode 31 and the second electrode 32. In case where the insulative fibers 50 are not located between the first electrode 31 and the second electrode 32 (including a case where no insulative fibers 50 are provided on the substrate 100), the .beta. MHC is not produced efficiently, as demonstrated in the comparative example 6 which will be described later.

[0055] The insulative fibers 50 are exposed on the surface of the substrate 100. The first electrode 31 and the second electrode 32 are also exposed on the surface of substrate 100.

[0056] The insulative fibers 50 have orientation such that an angle formed between each of not less than 90% of the insulative fibers 50 and an imaginary straight line which passes through both the first electrode 31 and the second electrode 32 is not more than .+-.20 degrees in the top view of substrate 100. In other words, each of the not less than 90% of the insulative fibers 50 forms an angle of not more than 20 degrees with regard to the imaginary straight line. Therefore, not less than 90% of the insulative fibers 50 are substantially parallel to a direction of an electric field generated when an electric current (e.g., pulse electric current) is caused to flow between the first electrode 31 and the second electrode 32. Needless to say, the imaginary straight line does not exist actually on the substrate 100. Desirably, the angle is not more than .+-.5 degrees. See the paragraph 0023 of U.S. patent application Ser. No. 15/519,341, which is incorporated herein by reference.

[0057] In case where less than 90% of the insulative fibers 50 are substantially parallel to the imaginary straight line which passes through both the first electrode 31 and the second electrode 32, the .beta. MHC is not produced efficiently. See the comparative examples 3-6 which will be described later. In the comparative examples 2-3, almost all of the insulative fibers 50 are substantially perpendicular to the imaginary straight line which passes through both the first electrode 31 and the second electrode 32. In other words, in the comparative examples 2-3, each of the almost all of the insulative fibers 50 forms an angle of approximately 90 degrees with regard to the imaginary straight line. In the comparative examples 4-5, a roughly half of the insulative fibers 50 are perpendicular to the imaginary straight line which passes through both the first electrode 31 and the second electrode 32, and the other roughly half of the insulative fibers 50 are parallel to the imaginary straight line.

[0058] Desirably, each of the insulative fibers 50 has a diameter of not less than 1 micrometer and not more than 5 micrometers. It is desirable that the material of the insulative fibers 50 is selected from the group consisting of polystyrene, polycarbonate, polymethylmethacrylate, polyvinyl chloride, polyethylene terephthalate, polyamide, polymethylglutarimide, or polylactic acid. It is desirable that the distance between the first electrode 31 and the second electrode 32 is not less than 150 micrometers and not more than 5,000 micrometers.

[0059] One example of a fabrication method of the substrate 100 will be described in more detail in the examples which will be described later. A skilled person who has read the examples which will be described later would understand easily the fabrication method of the substrate 100.

[0060] As shown in FIG. 8A, a liquid culture medium 182 containing cardiac muscle cells 180 is supplied to the surface of the above-mentioned substrate 100. The liquid culture medium 182 is spread onto the surface of the substrate 100 within the enclosure 10. In this way, the surface of the first electrode 31, the surface of the second electrode 32, and a region C between the first electrode 31 and the second electrode 32 are coated with the cardiac muscle cells. In case where at least one of the surface of the first electrode 31, the surface of the second electrode 32, and the region C fails to be coated with the cardiac muscle cells, the pulse electric current fails to be applied to the cardiac muscle cells 180 in the step (b) which will be described later. As a result, the .beta. MHC fails to be produced efficiently. As just described, in the step (a), the liquid culture medium 182 containing the cardiac muscle cells 180 having an amount sufficient to coat the surface of the first electrode 31, the surface of the second electrode 32, and the region C is supplied to the surface of substrate 100.

[0061] (Step (b))

[0062] The Step (b) is conducted out after the step (a). In the Step (b), the substrate 100 is left at rest. In this way, the cardiac muscle cells adhere on the insulative fibers 50 or the surface of substrate 100. Desirably, the substrate 100 is left at rest over 24 hours.

[0063] (Step (c))

[0064] The Step (c) is conducted after the step (b). In the step (c), while a pulse electric current is applied to the cardiac muscle cells 180 through the first electrode 31 and the second electrode 32, the cardiac muscle cells 180 are cultivated. The same pulse electric current may be applied to the first electrode 31 and the second electrode 32. When the pulse electric current is applied to the first electrode 31 and the second electrode 32, a reference electrode 4 may be used. The reference electrode 4 is grounded. As shown in FIG. 8A, the reference electrode 4 may be provided on the surface of the substrate 100. However, as shown in FIG. 8B, the reference electrode 4 is not necessary to be provided on the surface of the substrate 100. In FIG. 8B, the reference electrode 4 is included in the inside of the liquid culture medium 182. Anyway, it is desirable that the reference electrode 4 is in contact with the liquid culture medium 182.

[0065] FIG. 3 is a graph showing an example of a desirable pulse electric current. As shown in FIG. 3, the desirable pulse electric current has a period of 333 milliseconds to 2 seconds (1 second in FIG. 3). One pulse is either positive or negative. In FIG. 3, first, a negative pulse is applied, and then a positive pulse is applied. While the negative pulse is applied, an electric current flows from the cardiac muscle cells to the first electrode 31 (or the second electrode 32). While the positive pulse is applied, an electric current flows from the first electrode 31 (or the second electrode 32) to the cardiac muscle cells.

[0066] One pulse has a time length of 0.05 milliseconds to 4 milliseconds (0.4 milliseconds in FIG. 3) and a height (namely, an electric current value) of 1 microampere-20 microamperes (3-12 microamperes, in FIG. 3). It is desirable that the size of the pulse (namely, an area of one pulse in FIG. 3) is not less than 0.1 nano coulomb and not more than 1.0 nano coulomb. More desirably, the rate of the size of the pulse to the area of the first electrode 31 (or the second electrode 32) is not less than 0.04 coulombs/square meter and not more than 0.4 coulombs/square meter. It is desirable that the size of the negative pulse (namely, the area of the negative pulse in FIG. 3) is the same as the size of the positive pulse (namely, the area of the positive pulse in FIG. 3).

[0067] As demonstrated in the inventive example 1, the thus-cultivated cardiac muscle cells 180 contain a lot of .beta. MHC. In other words, the .beta. MHC is produced efficiently in the thus-cultivated cardiac muscle cells 180. In case where the pulse electric current fails to be applied, the .beta. MHC fails to be produced efficiently. Seethe comparative examples 1, 3, 5, and 7 which will be described later.

EXAMPLES

[0068] Hereinafter, the present invention will be described in more detail with reference to the following examples.

[0069] (Fabrication of Substrate 100)

[0070] The substrate 100 shown in FIG. 1 was fabricated as below. First, the glass base 1 having a shape of a square was prepared. The glass base 1 had a thickness of 0.7 millimeters and an area of approximately 2500 square millimeters (i.e., 50 millimeters.times.50 millimeters). Then, as shown in FIG. 4, the electric contacts 2 and the electric wirings 3 were formed on the glass base 1. The electric wirings 3 were formed by etching an indium tin oxide film having a thickness of 150 nanometers using a photoresist. The number of the electric contacts 2 and the electric wirings 3 was sixty-eight.

[0071] Then, the surface of the glass base 1 was coated with an insulation film 40 consisting of a photosensitive acrylic acid resin. The electric contacts 2 were not coated with the insulation film 40. Each one end of the electric wirings 3 was not coated with the insulation film 40, since the one end of the electric wiring 3 was used as the first electrode 31, the second electrode 32, or the reference electrode 4. Subsequently, the glass base 1 was subjected to plasma surface treatment at an RF power of 18 W for two minutes with a plasma treatment apparatus (available from Harrick Plasma Company, trade name: "PDC-32G").

[0072] FIG. 5 shows an enlarged view of a region B included in FIG. 4. One electrode set 6 consisted of the ends of the four electric wirings 3, as shown in FIG. 5. The number of the electrode set 6 was 16 sets. The ends of remaining four electric wirings 3 were used for the reference electrode 4. FIG. 6A shows an enlarged top view of the end part of the electric wiring 3. FIG. 6B shows a cross-sectional view taken along the line 6B-6B included in FIG. 6A.

[0073] The end of the electric wiring 3 exposed on the surface (i.e., the first electrode 31 and the second electrode 32) had a size of approximately 15 micrometers.times.approximately 170 micrometers. The reference electrode 4 had an area of approximately 200 square micrometers. The distance between the ends of adjacent two electric wirings 3 was approximately 400 micrometers. The distance of adjacent two electrode sets 6 was approximately 4 millimeters.

[0074] Meanwhile, insulative fibers made of polymethyl glutaric imide were formed on the surface of an aluminum tape (available from Hitachi Maxell. Ltd., trade name: SLIONTEC) by an electrospinning method in accordance with the process disclosed in the paragraph 0122 of U.S. patent application Ser. No. 15/519,341. Unlike the process disclosed in the paragraph 0122 of U.S. patent application Ser. No. 15/519,341, an ejection time of polymethyl glutaric imide in the electrospinning method was 30 minutes in the inventive example 1. The insulative fibers had a surface coverage of 30%.

[0075] Then, the aluminum tape having the insulative fibers was disposed on the surface of the glass base 1 so that the insulative fibers were sandwiched between the aluminum tape and the electric wiring 3. The aluminum tape having the insulative fibers was impressed onto the surface of the insulation film 40 and the exposed ends of the electric wirings 3. Then, the aluminum tape was removed. FIG. 7A shows an enlarged top view of the end part of the electric wiring 3. FIG. 7B shows a cross-sectional view taken along the line 7B--7B included in FIG. 7A. As shown in FIG. 7A and FIG. 7B, the insulative fibers 50 were transcribed on the surface of the insulation film 40 and the exposed ends of the electric wirings 3. As shown in FIG. 2 and FIG. 7A, not less than 90% of the insulative fibers 50 were disposed in a direction parallel to the imaginary straight line which passes through the first electrode 31 and the second electrode 32 (namely, in a horizontal direction in the figures).

[0076] Then, as shown in FIG. 2, a silicone resin sheet 60 (available from Toray Dow Corning company, trade name: SYLGARD 184) was adhered on the insulation film 40 with a silicone adhesive. The silicone resin sheet 60 had a thickness of approximately 1 millimeter. The ends of the electric wirings 3 and their peripheries were not coated with the silicone resin sheet 60. Furthermore, the enclosure 10 was adhered with the silicone adhesive so as to include the silicone resin sheet 60 in the inside thereof. The enclosure 10 was formed of glass. The enclosure 10 had an internal diameter of approximately 22 millimeters, an external diameter of approximately 25 millimeters, and a height of approximately 10 millimeters.

[0077] The exposed ends of the electric wirings 3 were plated with platinum black 5. Specifically, the parts were plated at a current density of 20 mA/cm.sup.2 for two minutes using a plating solution. During the plating, the electric wirings 3 were used as cathodes. The plating solution had the composition shown in Table 1. The first electrode 31 or the second electrode 32 was formed through such plating on the surface of the end of the electric wiring 3. In other words, the first electrode 31 and the second electrode 32 were formed of platinum black.

TABLE-US-00003 TABLE 1 Composition Chemical formula Concentration Hexachloroplatinic H.sub.2PtCl.sub.6 6H.sub.2O 1% (IV) acid Lead acetate (CH.sub.3COO)2Pb 3H.sub.2O 0.01% Hydrochloric acid HCl 0.0025%

[0078] In this way, the substrate 100 was provided. FIG. 13A is a microscope photograph of the first electrode 31, the second electrode 32, and the insulative fibers 50 which have been formed on the thus-provided substrate 100. FIG. 13B is also a microscope photograph of the first electrode 31, the second electrode 32, and the insulative fibers 50 which have been formed on the substrate 100 provided similarly. As shown in FIG. 13B, a small amount of non-oriented fibers are included in the insulative fibers 50 due to the problem in the fabrication process by the electrospinning method. The amount of the non-oriented fibers is less than 10%.

[0079] (Cultivation of Cardiac Muscle Cells)

[0080] Using the substrate 100, cardiac muscle cells differentiated by induced pluripotent stem cells derived from Homo sapiens were cultivated. And then, production ratio of the .beta. MHC was measured. Specifically, cardiac muscle cells differentiated by induced pluripotent stem cells derived from Homo sapiens (available from iPS Academia Japan, Inc., trade name: iCell Cardiomycytes) were used. Pursuant to the protocol described in the manual attached to iCell Cardiomycytes, a liquid culture medium containing cardiac muscle cells differentiated by induced pluripotent stem cells derived from Homo sapiens was prepared.

[0081] Then, as shown in FIG. 8A, the liquid culture medium 182 was supplied onto the substrate 100. The density of the cardiac muscle cells 180 on the substrate 100 was 1.5.times.10.sup.4/square millimeter. In this way, the surface of the first electrode 31, the surface of the second electrode 32, and the region C were coated with the cardiac muscle cells 180. The cardiac muscle cells 180 was cultivated pursuant to the protocol described in the manual attached to iCell Cardiomycytes.

[0082] Two days after the supply of the liquid culture medium 182, the pulse electric current shown in FIG. 3 is applied with the reference electrode 4 to the cardiac muscle cells 180 through the first electrode 31 and the second electrode 32 shown in FIG. 2 to stimulate the cardiac muscle cells 180. For the application of the pulse electric current, a pulse electric current generator 200 was electrically connected to the first electrode 31 and the second electrode 32 through the electric contacts 2. The electric potential of the liquid culture medium 182 was maintained at standard electric potential (i.e., GND) through the reference electrode 4.

[0083] The pulse electric current was applied to the cardiac muscle cells 180 for 12 days, except in time of a change of a culture medium. In this way, the cardiac muscle cells 180 were cultivated.

[0084] (Measurement of Production Ratio of .beta. MHC)

[0085] The production ratio of the .beta. MHC contained in the thus-cultivated cardiac muscle cells 180 was measured as below.

[0086] The cardiac muscle cells were fixed with 4% paraformaldehyde and were permeabilized in phosphate buffered saline (PBS) plus 0.5% Triton X-100 for 0.5 hours. After blocking in a 5% normal donkey serum, 3% BSA, and 0.1% Tween 20 in PBS for 16 hours at 4 degrees Celsius, the cells were incubated for 16 hours at 4 degrees Celsius with mouse MYH7 monoclonal IgM primary antibodies (available from Santa Cruz Biotechnology, trade name: SC-53089) diluted at 1:100 with a blocking buffer. In this way, the primary antibodies were bound to the cardiac muscle cells. The antigen capable of binding to the primary antibody was .beta. MHC (GenBank: AAA51837.1).

[0087] Then, the cardiac muscle cells to which the primary antibodies were bound were washed with PBS. Subsequently, the cardiac muscle cells were incubated for 1 hour at 25 degrees Celsius with fluorescently-labelled anti-mouse IgM secondary antibodies (available from Jackson Immunoresearch labs., trade name: DyLight-594-Donkey anti-mouse IgM) diluted at 1:1,000 with the blocking buffer. In this way, the fluorescently-labelled secondary antibodies were bound to the primary antibodies. In this way, the cardiac muscle cells were fluorescently labelled.

[0088] The fluorescently-labelled cardiac muscle cells were observed using a fluorescent microscope. FIG. 9A is a fluorescent microscope photograph of the cardiac muscle cells in the inventive example 1. The brightness of the observed fluorescence was converted into 256 gradation digital brightness level. Digital brightness level 0 means that brightness is lowest. Digital brightness level 255 means that brightness is highest.

[0089] Hereinafter, the .beta. MHC production ratio is defined as a rate of the sum of the areas of the regions each having a digital brightness level of not less than 65 to the area of the whole of the observation region. In other words, the .beta.MHC production ratio is calculated according to the following mathematical formula.

(.beta. MHC Production Ratio)=(Sum of Areas of the regions each having a digital brightness level of not less than 65)/(Area of the whole of the observation region)

[0090] In the inventive example 1, the .beta. MHC production ratio was 57.9%.

[0091] For reference, production ratio of myosin regulatory light chain 2 (hereinafter, referred to as "MYL2") contained in the cultivated cardiac muscle cells was measured similarly. In particular, the MYL2 production ratio was calculated similarly to the case of the .beta. MHC production ratio, except for the following two matters.

[0092] (I) In place of the mouse MYH7 monoclonal IgM antibodies, rabbit MYL2 polyclonal IgG antibodies (dilution ratio: 1/200, available from Proteintech Company, trade name: 109060-1-AP) was used as the primary antibodies.

[0093] (II) In place of the anti-mouse IgM fluorescently-labelled secondary antibodies, anti-rabbit IgG fluorescently-labelled antibodies (available from Jackson Immunoresearch labs., trade name: Alexa Fluor 488 Donkey anti-rabbit IgG) was used as the secondary antibodies.

[0094] As a result, the MYL2 production ratio was 36.7% in the inventive example 1.

Comparative Example 1

[0095] An experiment similar to the inventive example 1 was conducted, except that no pulse electric current was applied.

Comparative Example 2

[0096] An experiment similar to the inventive example 1 was conducted, except that almost all of the insulative fibers 50 were disposed substantially perpendicularly (namely, in a vertical direction in FIG. 10A) to the imaginary straight line which passes through the first electrode 31 and the second electrode 32, as shown in FIG. 10A and FIG. 10B. FIG. 9B is a fluorescent microscope photograph of the cardiac muscle cells in the comparative example 2. FIG. 13C is a microscope photograph of the first electrode 31, the second electrode 32, and the insulative fibers 50 which have been formed on the thus-obtained substrate 100 used in the comparative example 2 and the comparative example 3 which will be described later. As shown in FIG. 13C, in the comparative examples 2-3, the insulative fibers 50 were disposed in a direction perpendicular to the imaginary straight line which passes through the first electrode 31 and the second electrode 32 (namely, in the vertical direction in the figure).

Comparative Example 3

[0097] An experiment similar to the inventive example 1 was conducted, except that almost all of the insulative fibers 50 were disposed substantially perpendicularly (namely, in a vertical direction in FIG. 10A) to the imaginary straight line which passes through the first electrode 31 and the second electrode 32, as shown in FIG. 10A and FIG. 10B, and except that no pulse electric current was applied.

Comparative Example 4

[0098] An experiment similar to the inventive example 1 was conducted, except that roughly half of the insulative fibers 50 were disposed parallel (namely, in the horizontal direction in FIG. 11A) to the imaginary straight line which passes through the first electrode 31 and the second electrode 32 and the other roughly half of the insulative fibers 50 were disposed perpendicularly (namely, in a vertical direction in FIG. 11A) to the imaginary straight line, as shown in FIG. 11A and FIG. 11B. FIG. 9C is a fluorescent microscope photograph of the cardiac muscle cells in the comparative example 4. FIG. 13D is a microscope photograph of the first electrode 31, the second electrode 32, and the insulative fibers 50 which have been formed on the thus-obtained substrate 100 used in the comparative example 4 and the comparative example 5 which will be described later. As shown in FIG. 13D, in the comparative examples 4-5, roughly half of the insulative fibers 50 (ejection time: 15 minutes) were disposed in a direction parallel to the imaginary straight line which passes through the first electrode 31 and the second electrode 32 (namely, in the horizontal direction in the figure), whereas the other roughly half of the insulative fibers 50 (ejection time: 15 minutes) were disposed in a direction perpendicular to the imaginary straight line (namely, in the vertical direction in the figure).

Comparative Example 5

[0099] An experiment similar to the inventive example 1 was conducted, except that some of the insulative fibers 50 were disposed parallel (namely, in the horizontal direction in FIG. 11A) to the imaginary straight line which passes through the first electrode 31 and the second electrode 32 and the other insulative fibers 50 were disposed perpendicularly (namely, in a vertical direction in FIG. 11A) to the imaginary straight line, as shown in FIG. 11A and FIG. 11B, and except that no pulse electric current was applied.

Comparative Example 6

[0100] An experiment similar to the inventive example 1 was conducted, except that no insulative fibers 50 were disposed, as shown in FIG. 12A and FIG. 12B. FIG. 9D is a fluorescent microscope photograph of the cardiac muscle cells in the comparative example 6.

Comparative Example 7

[0101] An experiment similar to the inventive example 1 was conducted, except that no insulative fibers 50 were disposed, as shown in FIG. 12A and FIG. 12B, and except that no pulse electric current was applied.

[0102] The following Table 2 shows the .beta. MHC production rate measured in the inventive example 1 and the comparative examples 1-7.

TABLE-US-00004 TABLE 2 Relation Between Direction of Insulative Pulse .beta. MHC fibers and Direction of electric production Electric Field current rate (%) I. E. 1 FIG. 13A or FIG. 13B Applied 57.9 C. E. 1 FIG. 13A or FIG. 13B No 14.5 C. E. 2 FIG. 13C Applied 31.9 C. E. 3 FIG. 13C No 10.3 C. E. 4 FIG. 13D Applied 36.5 C. E. 5 FIG. 13D No 15.8 C. E. 6 No insulative fibers Applied 15.4 C. E. 7 No insulative fibers No 9.8 "I. E." means "Inventive Example". "C. E." means "Comparative Example". "Electric Field" means the electric field generated between the first electrode 31 and the second electrode 32 by the electric current pulse.

[0103] The following Table 3 shows the MYL2 production rate measured in the inventive example 1 and the comparative examples 1-7.

TABLE-US-00005 TABLE 3 Relation Between Direction of Insulative Pulse MYL2 fibers and Direction of electric production Electric Field current rate (%) I. E. 1 FIG. 13A or FIG. 13B Applied 36.7 C. E. 1 FIG. 13A or FIG. 13B No 25.1 C. E. 2 FIG. 13C Applied 30.0 C. E. 3 FIG. 13C No 19.0 C. E. 4 FIG. 13D Applied 32.5 C. E. 5 FIG. 13D No 24.0 C. E. 6 No insulative fibers Applied 16.2 C. E. 7 No insulative fibers No 10.1

[0104] As is clear from Table 2, when both of the following requirements (I) and (II) are satisfied, the .beta. MHC production rate is a significantly high value of 57.9%. See the inventive example 1.

[0105] Requirement (I): The insulative fibers 50 have orientation such that an angle formed between each of not less than 90% of the insulative fibers 50 and an imaginary straight line which passes through both the first electrode 31 and the second electrode 32 is not more than .+-.20 degrees in the top view.

[0106] Requirement (II): The cardiac muscle cells 180 are cultivated, while the pulse electric current is applied thereto.

[0107] On the other hand, in case where at least one of the requirements (I) and (II) fails to be satisfied, the .beta. MHC production rate is a low value of less than 36.5%. See the comparative examples 1-7.

[0108] As is clear from Table 3, regardless to the direction of the insulative fibers, the MYL2 production rate is a constant value of approximately 32%-37%. On the other hand, as is clear from Table 1, the .beta. MHC production rate is significantly increased, when both of the requirements (I) and (II) are satisfied. In other words, the use of the insulative fibers increases the production amount of polypeptide (including protein) in the cardiac muscle cells. Among the polypeptide produced in the cardiac muscle cells, when both of the requirements (I) and (II) are satisfied, the .beta. MHC is produced at the significantly high production rate, unlike other polypeptide such as MYL2.

INDUSTRIAL APPLICABILITY

[0109] The present invention provides a method for efficiently producing 0 myosin heavy chain in cardiac muscle cells differentiated from induced pluripotent stem cells derived from Homo sapiens.

REFERENTIAL SIGNS LIST

[0110] 100 Substrate

[0111] 1 Glass plate

[0112] 2 Electric contact

[0113] 3 Electric wiring

[0114] 4 Reference electrode

[0115] 5 Platinum black

[0116] 6 Electrode set

[0117] 10 Enclosure

[0118] 31 First electrode

[0119] 32 Second electrode

[0120] 40 Insulation film

[0121] 50 Insulative fiber

[0122] 60 Insulative sheet

[0123] A Region

[0124] B Region

[0125] C Region

[0126] 180 Cardiac muscle cells

[0127] 182 Liquid culture medium

[0128] 200 Pulse electric current generator

Sequence CWU 1

1

211935PRTHomo sapiens 1Met Gly Asp Ser Glu Met Ala Val Phe Gly Ala Ala Ala Pro Tyr Leu1 5 10 15Arg Lys Ser Glu Lys Glu Arg Leu Glu Ala Gln Thr Arg Pro Phe Asp 20 25 30Leu Lys Lys Asp Val Phe Val Pro Asp Asp Lys Gln Glu Phe Val Lys 35 40 45Ala Lys Ile Val Ser Arg Glu Gly Gly Lys Val Thr Ala Glu Thr Glu 50 55 60Tyr Gly Lys Thr Val Thr Val Lys Glu Asp Gln Val Met Gln Gln Asn65 70 75 80Pro Pro Lys Phe Asp Lys Ile Glu Asp Met Ala Met Leu Thr Phe Leu 85 90 95His Glu Pro Ala Val Leu Tyr Asn Leu Lys Asp Arg Tyr Gly Ser Trp 100 105 110Met Ile Tyr Thr Tyr Ser Gly Leu Phe Cys Val Thr Val Asn Pro Tyr 115 120 125Lys Trp Leu Pro Val Tyr Thr Pro Glu Val Val Ala Ala Tyr Arg Gly 130 135 140Lys Lys Arg Ser Glu Ala Pro Pro His Ile Phe Ser Ile Ser Asp Asn145 150 155 160Ala Tyr Gln Tyr Met Leu Thr Asp Arg Glu Asn Gln Ser Ile Leu Ile 165 170 175Thr Gly Glu Ser Gly Ala Gly Lys Thr Val Asn Thr Lys Arg Val Ile 180 185 190Gln Tyr Phe Ala Val Ile Ala Ala Ile Gly Asp Arg Ser Lys Lys Asp 195 200 205Gln Ser Pro Gly Lys Gly Thr Leu Glu Asp Gln Ile Ile Gln Ala Asn 210 215 220Pro Ala Leu Glu Ala Phe Gly Asn Ala Lys Thr Val Arg Asn Asp Asn225 230 235 240Ser Ser Arg Phe Gly Lys Phe Ile Arg Ile His Phe Gly Ala Thr Gly 245 250 255Lys Leu Ala Ser Ala Asp Ile Glu Thr Tyr Leu Leu Glu Lys Ser Arg 260 265 270Val Ile Phe Gln Leu Lys Ala Glu Arg Asp Tyr His Ile Phe Tyr Gln 275 280 285Ile Leu Ser Asn Lys Lys Pro Glu Leu Leu Asp Met Leu Leu Ile Thr 290 295 300Asn Asn Pro Tyr Asp Tyr Ala Phe Ile Ser Gln Gly Glu Thr Thr Val305 310 315 320Ala Ser Ile Asp Asp Ala Glu Glu Leu Met Ala Thr Asp Asn Ala Phe 325 330 335Asp Val Leu Gly Phe Thr Ser Glu Glu Lys Asn Ser Met Tyr Lys Leu 340 345 350Thr Gly Ala Ile Met His Phe Gly Asn Met Lys Phe Lys Leu Lys Gln 355 360 365Arg Glu Glu Gln Ala Glu Pro Asp Gly Thr Glu Glu Ala Asp Lys Ser 370 375 380Ala Tyr Leu Met Gly Leu Asn Ser Ala Asp Leu Leu Lys Gly Leu Cys385 390 395 400His Pro Arg Val Lys Val Gly Asn Glu Tyr Val Thr Lys Gly Gln Asn 405 410 415Val Gln Gln Val Ile Tyr Ala Thr Gly Ala Leu Ala Lys Ala Val Tyr 420 425 430Glu Arg Met Phe Asn Trp Met Val Thr Arg Ile Asn Ala Thr Leu Glu 435 440 445Thr Lys Gln Pro Arg Gln Tyr Phe Ile Gly Val Leu Asp Ile Ala Gly 450 455 460Phe Glu Ile Phe Asp Phe Asn Ser Phe Glu Gln Leu Cys Ile Asn Phe465 470 475 480Thr Asn Glu Lys Leu Gln Gln Phe Phe Asn His His Met Phe Val Leu 485 490 495Glu Gln Glu Glu Tyr Lys Lys Glu Gly Ile Glu Trp Thr Phe Ile Asp 500 505 510Phe Gly Met Asp Leu Gln Ala Cys Ile Asp Leu Ile Glu Lys Pro Met 515 520 525Gly Ile Met Ser Ile Leu Glu Glu Glu Cys Met Phe Pro Lys Ala Thr 530 535 540Asp Met Thr Phe Lys Ala Lys Leu Phe Asp Asn His Leu Gly Lys Ser545 550 555 560Ala Asn Phe Gln Lys Pro Arg Asn Ile Lys Gly Lys Pro Glu Ala His 565 570 575Phe Ser Leu Ile His Tyr Ala Gly Ile Val Asp Tyr Asn Ile Ile Gly 580 585 590Trp Leu Gln Lys Asn Lys Asp Pro Leu Asn Glu Thr Val Val Gly Leu 595 600 605Tyr Gln Lys Ser Ser Leu Lys Leu Leu Ser Thr Leu Phe Ala Asn Tyr 610 615 620Ala Gly Ala Asp Ala Pro Ile Glu Lys Gly Lys Gly Lys Ala Lys Lys625 630 635 640Gly Ser Ser Phe Gln Thr Val Ser Ala Leu His Arg Glu Asn Leu Asn 645 650 655Lys Leu Met Thr Asn Leu Arg Ser Thr His Pro His Phe Val Arg Cys 660 665 670Ile Ile Pro Asn Glu Thr Lys Ser Pro Gly Val Met Asp Asn Pro Leu 675 680 685Val Met His Gln Leu Arg Cys Asn Gly Val Leu Glu Gly Ile Arg Ile 690 695 700Cys Arg Lys Gly Phe Pro Asn Arg Ile Leu Tyr Gly Asp Phe Arg Gln705 710 715 720Arg Tyr Arg Ile Leu Asn Pro Ala Ala Ile Pro Glu Gly Gln Phe Ile 725 730 735Asp Ser Arg Lys Gly Ala Glu Lys Leu Leu Ser Ser Leu Asp Ile Asp 740 745 750His Asn Gln Tyr Lys Phe Gly His Thr Lys Val Phe Phe Lys Ala Gly 755 760 765Leu Leu Gly Leu Leu Glu Glu Met Arg Asp Glu Arg Leu Ser Arg Ile 770 775 780Ile Thr Arg Ile Gln Ala Gln Ser Arg Gly Val Leu Ala Arg Met Glu785 790 795 800Tyr Lys Lys Leu Leu Glu Arg Arg Asp Ser Leu Leu Val Ile Gln Trp 805 810 815Asn Ile Arg Ala Phe Met Gly Val Lys Asn Trp Pro Trp Met Lys Leu 820 825 830Tyr Phe Lys Ile Lys Pro Leu Leu Lys Ser Ala Glu Arg Glu Lys Glu 835 840 845Met Ala Ser Met Lys Glu Glu Phe Thr Arg Leu Lys Glu Ala Leu Glu 850 855 860Lys Ser Glu Ala Arg Arg Lys Glu Leu Glu Glu Lys Met Val Ser Leu865 870 875 880Leu Gln Glu Lys Asn Asp Leu Gln Leu Gln Val Gln Ala Glu Gln Asp 885 890 895Asn Leu Ala Asp Ala Glu Glu Arg Cys Asp Gln Leu Ile Lys Asn Lys 900 905 910Ile Gln Leu Glu Ala Lys Val Lys Glu Met Asn Glu Arg Leu Glu Asp 915 920 925Glu Glu Glu Met Asn Ala Glu Leu Thr Ala Lys Lys Arg Lys Leu Glu 930 935 940Asp Glu Cys Ser Glu Leu Lys Arg Asp Ile Asp Asp Leu Glu Leu Thr945 950 955 960Leu Ala Lys Val Glu Lys Glu Lys His Ala Thr Glu Asn Lys Val Lys 965 970 975Asn Leu Thr Glu Glu Met Ala Gly Leu Asp Glu Ile Ile Ala Lys Leu 980 985 990Thr Lys Glu Lys Lys Ala Leu Gln Glu Ala His Gln Gln Ala Leu Asp 995 1000 1005Asp Leu Gln Ala Glu Glu Asp Lys Val Asn Thr Leu Thr Lys Ala 1010 1015 1020Lys Val Lys Leu Glu Gln Gln Val Asp Asp Leu Glu Gly Ser Leu 1025 1030 1035Glu Gln Glu Lys Lys Val Arg Met Asp Leu Glu Arg Ala Lys Arg 1040 1045 1050Lys Leu Glu Gly Asp Leu Lys Leu Thr Gln Glu Ser Ile Met Asp 1055 1060 1065Leu Glu Asn Asp Lys Gln Gln Leu Asp Glu Arg Leu Lys Lys Lys 1070 1075 1080Asp Phe Glu Leu Asn Ala Leu Asn Ala Arg Ile Glu Asp Glu Gln 1085 1090 1095Ala Leu Gly Ser Gln Leu Gln Lys Lys Leu Lys Glu Leu Gln Ala 1100 1105 1110Arg Ile Glu Glu Leu Glu Glu Glu Leu Glu Ser Glu Arg Thr Ala 1115 1120 1125Arg Ala Lys Val Glu Lys Leu Arg Ser Asp Leu Ser Arg Glu Leu 1130 1135 1140Glu Glu Ile Ser Glu Arg Leu Glu Glu Ala Gly Gly Ala Thr Ser 1145 1150 1155Val Gln Ile Glu Met Asn Lys Lys Arg Glu Ala Glu Phe Gln Lys 1160 1165 1170Met Arg Arg Asp Leu Glu Glu Ala Thr Leu Gln His Glu Ala Thr 1175 1180 1185Ala Ala Ala Leu Arg Lys Lys His Ala Asp Ser Val Ala Glu Leu 1190 1195 1200Gly Glu Gln Ile Asp Asn Leu Gln Arg Val Lys Gln Lys Leu Glu 1205 1210 1215Lys Glu Lys Ser Glu Phe Lys Leu Glu Leu Asp Asp Val Thr Ser 1220 1225 1230Asn Met Glu Gln Ile Ile Lys Ala Lys Ala Asn Leu Glu Lys Met 1235 1240 1245Cys Arg Thr Leu Glu Asp Gln Met Asn Glu His Arg Ser Lys Ala 1250 1255 1260Glu Glu Thr Gln Arg Ser Val Asn Asp Leu Thr Ser Gln Arg Ala 1265 1270 1275Lys Leu Gln Thr Glu Asn Gly Glu Leu Ser Arg Gln Leu Asp Glu 1280 1285 1290Lys Glu Ala Leu Ile Ser Gln Leu Thr Arg Gly Lys Leu Thr Tyr 1295 1300 1305Thr Gln Gln Leu Glu Asp Leu Lys Arg Gln Leu Glu Glu Glu Val 1310 1315 1320Lys Ala Lys Asn Ala Leu Ala His Ala Leu Gln Ser Ala Arg His 1325 1330 1335Asp Cys Asp Leu Leu Arg Glu Gln Tyr Glu Glu Glu Thr Glu Ala 1340 1345 1350Lys Ala Glu Leu Gln Arg Val Leu Ser Lys Ala Asn Ser Glu Val 1355 1360 1365Ala Gln Trp Arg Thr Lys Tyr Glu Thr Asp Ala Ile Gln Arg Thr 1370 1375 1380Glu Glu Leu Glu Glu Ala Lys Lys Lys Leu Ala Gln Arg Leu Gln 1385 1390 1395Glu Ala Glu Glu Ala Val Glu Ala Val Asn Ala Lys Cys Ser Ser 1400 1405 1410Leu Glu Lys Thr Lys His Arg Leu Gln Asn Glu Ile Glu Asp Leu 1415 1420 1425Met Val Asp Val Glu Arg Ser Asn Ala Ala Ala Ala Ala Leu Asp 1430 1435 1440Lys Lys Gln Arg Asn Phe Asp Lys Ile Leu Ala Glu Trp Lys Gln 1445 1450 1455Lys Tyr Glu Glu Ser Gln Ser Glu Leu Glu Ser Ser Gln Lys Glu 1460 1465 1470Ala Arg Ser Leu Ser Thr Glu Leu Phe Lys Leu Lys Asn Ala Tyr 1475 1480 1485Glu Glu Ser Leu Glu His Leu Glu Thr Phe Lys Arg Glu Asn Lys 1490 1495 1500Asn Leu Gln Glu Glu Ile Ser Asp Leu Thr Glu Gln Leu Gly Ser 1505 1510 1515Ser Gly Lys Thr Ile His Glu Leu Glu Lys Val Arg Lys Gln Leu 1520 1525 1530Glu Ala Glu Lys Met Glu Leu Gln Ser Ala Leu Glu Glu Ala Glu 1535 1540 1545Ala Ser Leu Glu His Glu Glu Gly Lys Ile Leu Arg Ala Gln Leu 1550 1555 1560Glu Phe Asn Gln Ile Lys Ala Glu Ile Glu Arg Lys Leu Ala Glu 1565 1570 1575Lys Asp Glu Glu Met Glu Gln Ala Lys Arg Asn His Leu Arg Val 1580 1585 1590Val Asp Ser Leu Gln Thr Ser Leu Asp Ala Glu Thr Arg Ser Arg 1595 1600 1605Asn Glu Ala Leu Arg Val Lys Lys Lys Met Glu Gly Asp Leu Asn 1610 1615 1620Glu Met Glu Ile Gln Leu Ser His Ala Asn Arg Met Ala Ala Glu 1625 1630 1635Ala Gln Lys Gln Val Lys Ser Leu Gln Ser Leu Leu Lys Asp Thr 1640 1645 1650Gln Ile Gln Leu Asp Asp Ala Val Arg Ala Asn Asp Asp Leu Lys 1655 1660 1665Glu Asn Ile Ala Ile Val Glu Arg Arg Asn Asn Leu Leu Gln Ala 1670 1675 1680Glu Leu Glu Glu Leu Arg Ala Val Val Glu Gln Thr Glu Arg Ser 1685 1690 1695Arg Lys Leu Ala Glu Gln Glu Leu Ile Glu Thr Ser Glu Arg Val 1700 1705 1710Gln Leu Leu His Ser Gln Asn Thr Ser Leu Ile Asn Gln Lys Lys 1715 1720 1725Lys Met Asp Ala Asp Leu Ser Gln Leu Gln Thr Glu Val Glu Glu 1730 1735 1740Ala Val Gln Glu Cys Arg Asn Ala Glu Glu Lys Ala Lys Lys Ala 1745 1750 1755Ile Thr Asp Ala Ala Met Met Ala Glu Glu Leu Lys Lys Glu Gln 1760 1765 1770Asp Thr Ser Ala His Leu Glu Arg Met Lys Lys Asn Met Glu Gln 1775 1780 1785Thr Ile Lys Asp Leu Gln His Arg Leu Asp Glu Ala Glu Gln Ile 1790 1795 1800Ala Leu Lys Gly Gly Lys Lys Gln Leu Gln Lys Leu Glu Ala Arg 1805 1810 1815Val Arg Glu Leu Glu Asn Glu Leu Glu Ala Glu Gln Lys Arg Asn 1820 1825 1830Ala Glu Ser Val Lys Gly Met Arg Lys Ser Glu Arg Arg Ile Lys 1835 1840 1845Glu Leu Thr Tyr Gln Thr Glu Glu Asp Arg Lys Asn Leu Leu Arg 1850 1855 1860Leu Gln Asp Leu Val Asp Lys Leu Gln Leu Lys Val Lys Ala Tyr 1865 1870 1875Lys Arg Gln Ala Glu Glu Ala Glu Glu Gln Ala Asn Thr Asn Leu 1880 1885 1890Ser Lys Phe Arg Lys Val Gln His Glu Leu Asp Glu Ala Glu Glu 1895 1900 1905Arg Ala Asp Ile Ala Glu Ser Gln Val Asn Lys Leu Arg Ala Lys 1910 1915 1920Ser Arg Asp Ile Gly Thr Lys Gly Leu Asn Glu Glu 1925 1930 19352166PRTHomo sapiens 2Met Ala Pro Lys Lys Ala Lys Lys Arg Ala Gly Gly Ala Asn Ser Asn1 5 10 15Val Phe Ser Met Phe Glu Gln Thr Gln Ile Gln Glu Phe Lys Glu Ala 20 25 30Phe Thr Ile Met Asp Gln Asn Arg Asp Gly Phe Ile Asp Lys Asn Asp 35 40 45Leu Arg Asp Thr Phe Ala Ala Leu Gly Arg Val Asn Val Lys Asn Glu 50 55 60Glu Ile Asp Glu Met Ile Lys Glu Ala Pro Gly Pro Ile Asn Phe Thr65 70 75 80Val Phe Leu Thr Met Phe Gly Glu Lys Leu Lys Gly Ala Asp Pro Glu 85 90 95Glu Thr Ile Leu Asn Ala Phe Lys Val Phe Asp Pro Glu Gly Lys Gly 100 105 110Val Leu Lys Ala Asp Tyr Val Arg Glu Met Leu Thr Thr Gln Ala Glu 115 120 125Arg Phe Ser Lys Glu Glu Val Asp Gln Met Phe Ala Ala Phe Pro Pro 130 135 140Asp Val Thr Gly Asn Leu Asp Tyr Lys Asn Leu Val His Ile Ile Thr145 150 155 160His Gly Glu Glu Lys Asp 165

Claims

1. A substrate comprising: a first electrode; a second electrode; and insulative fibers, wherein the first electrode, the second electrode, and the insulative fibers are provided on a surface of the substrate; at least a part of the insulative fibers is located between the first electrode and the second electrode in a top view of the substrate; and an angle formed between each of not less than 90% of the insulative fibers and an imaginary straight line which passes through both the first electrode and the second electrode is not more than .+-.20 degrees in the top view.

2. The substrate according to claim 1, further comprising a reference electrode on the surface thereof.