Method for producing chimeric non-human mammal and method for producing heart or cardiac tissue

Abstract

To provide a complementing technique for regenerating a normally operating heart.SOLUTION: A method for producing a chimeric non-human mammal according to an aspect of the present invention includes a step of transplanting a stem cell derived from a mammal into a fertilized egg of a non-human mammal different from the mammal, in which the Mesp1 gene and the Mesp2 gene are knocked out. Stem cell-derived cardiomyocytes, endothelial cells, smooth muscle cells and fibroblasts complement the neonatal heart.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing a chimeric non-human mammal and said chimeric non-human mammal. The present invention also relates to a method for producing a heart or cardiac tissue and a heart or cardiac tissue obtained by said method. The present invention further relates to a kit for producing a heart or cardiac tissue. [Background technology]

[0002] More than 4,000 heart transplants are performed annually worldwide. For example, approximately 50 heart transplants are performed annually in Japan. However, the waiting period for heart transplant surgery is approximately three years, creating a need for a shorter waiting period. To shorten this waiting period, securing a large number of donor hearts is necessary, and research and development into donor heart production is underway. In this research and development, organ regeneration using blastocyst complementation has attracted attention. Blastocyst complementation is a method of transplanting stem cells into a genetically modified animal embryo lacking a specific organ, biological tissue, or cell group (hereinafter, the term "organ" is used to encompass biological tissue and cell group), thereby regenerating the stem cell-derived organ, biological tissue, or cell group in vivo. For example, Non-Patent Documents 1 and 2 describe organ regeneration using blastocyst complementation.

[0003] Non-Patent Documents 3 to 6 describe cardiac regeneration using the blastocyst complementation method. Specifically, Non-Patent Document 3 describes the results of verification when the Mesp1 gene and the Mesp2 gene are double knocked out. Non-Patent Document 4 describes the results of verification when the Mesp1 gene is knocked out. Non-Patent Document 5 describes the results of verification when the expression of the Nkx2.5 gene, which is located downstream of the Mesp1 gene and the Mesp2 gene, is manipulated.

[0004] Non-Patent Document 7 describes that abnormalities in the Mesp1 gene caused cardiac abnormalities. Non-Patent Document 8 describes that abnormalities in the Mesp2 gene did not cause cardiac abnormalities. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Cell 142, 787-799, September 3, 2010 [Non-patent document 2] Nature 2017 Feb 9;542(7640):191-196 [Non-patent document 3] Development 127, 3215-3226 (2000) [Non-patent document 4] Nat Cell Biol. 2014 Sep;16(9):829-40 [Non-patent document 5] Circulation 2015 Jul 14; 132(2):109-21 [Non-patent document 6] Cell 168, 473-486, January 26, 2017 [Non-Patent Document 7] Development 126, 3437-34447 (1999) [Non-patent document 8] Genes Dev. 1997 Jul 15;11(14):1827-39 Summary of the Invention [Problem to be solved by the invention]

[0006] However, the above-mentioned conventional techniques have not yet led to the regeneration of a normally functioning heart through organ complementation. In particular, there have been no reports of heart complementation using cardiomyocytes, endothelial cells, smooth muscle cells, and fibroblasts, which are the constituent cells of the heart. Further research and development into cardiac complementation is desired.

[0007] One aspect of the present invention aims to provide a complementary technique for regenerating a normally functioning heart. [Means for solving the problem]

[0008] To achieve the above object, the present inventors have conducted extensive research. As a result, they have discovered a technique for complementing cardiac myocytes, endothelial cells, smooth muscle cells, and fibroblasts by blastocyst complementation in mammalian embryos in which the Mesp1 and Mesp2 genes have been knocked out. They have then succeeded in regenerating a normally functioning heart using this complementation technique, leading to the present invention.

[0009] A method for producing a chimeric non-human mammal according to one embodiment of the present invention comprises a stem cell preparation step of preparing stem cells derived from a mammal; a transplantation step of transplanting the stem cells into fertilized eggs of a non-human mammal different from the mammal, the fertilized eggs having the Mesp1 gene and the Mesp2 gene knocked out; and a litter obtaining step of developing the fertilized eggs in the womb of the non-human mammal to obtain litter, wherein the heart of the litter is complemented by cardiomyocytes, endothelial cells, smooth muscle cells, and fibroblasts derived from the stem cells.

[0010] A chimeric non-human mammal according to one embodiment of the present invention is a chimeric non-human mammal in which the cardiomyocytes, endothelial cells, smooth muscle cells and fibroblasts that constitute the heart comprise cells derived from a mammal, and organs and tissues other than the heart comprise cells derived from the mammal and cells derived from a non-human mammal different from the mammal, the cells derived from the mammal have the Mesp1 gene and the Mesp2 gene, and the cells derived from the non-human mammal have the Mesp1 gene and the Mesp2 gene knocked out.

[0011] A method for producing a heart or cardiac tissue according to one embodiment of the present invention comprises a stem cell preparation step of preparing stem cells derived from a mammal; a transplantation step of transplanting the stem cells into fertilized eggs of a non-human mammal different from the mammal, the fertilized eggs having the Mesp1 gene and the Mesp2 gene knocked out; a litter obtaining step of developing the fertilized eggs in the womb of the non-human mammal to obtain offspring; and a heart obtaining step of obtaining a heart or cardiac tissue from the offspring, wherein the heart or cardiac tissue is complemented by cardiomyocytes, endothelial cells, smooth muscle cells, and fibroblasts differentiated from the stem cells.

[0012] One embodiment of the present invention provides a kit for producing a heart or cardiac tissue, which comprises stem cells derived from a mammal and a fertilized egg of a non-human mammal different from the mammal, in which the Mesp1 gene and the Mesp2 gene have been knocked out. [Effects of the Invention]

[0013] According to one aspect of the present invention, a complementary technique for regenerating a normally functioning heart can be provided. [Brief explanation of the drawings]

[0014] [Figure 1] Schematic diagram of the targeting vector and targeting allele for generating Mesp − / − mice. [Figure 2] Photographs of Mesp+ / +, Mesp+ / -, and Mesp- / - mice at E9.0 are shown. [Figure 3] The results of qRT-PCR measurements of Mesp+ / +, Mesp+ / -, and Mesp- / - mice at E8.5 are shown. [Figure 4] The results of observation of Mesp+ / -, Mesp+ / -+GFP-blastomere, and Mesp- / -+GFP-blastomere mice at E9.5 are shown. [Figure 5]Immunostaining results of E13.5 hearts from Mesp+ / -, Mesp+ / -+GFP-blastomere, and Mesp- / -+GFP-blastomere mice are shown. [Figure 6] Immunostaining results of the heart of Mesp- / -+GFP-blastomere mice at E13.5 are shown. [Figure 7] This shows the results of observing GFP emission in organs other than the heart of Mesp- / -+GFP-blastomere mice at E13.5. [Figure 8] Eight-week-old Mesp+ / -+GFP-blastomere mice and Mesp- / -+GFP-blastomere mice are shown. [Figure 9] The results of immunostaining of the heart of an 8-week-old Mesp+ / -+GFP-blastomere mouse are shown. [Figure 10] The results of immunostaining of the heart of an 8-week-old Mesp- / -+GFP-blastomere mouse are shown. [Figure 11] The results of immunostaining of the heart of an 8-week-old Mesp+ / -+GFP-blastomere mouse are shown. [Figure 12] The results of immunostaining of the heart of an 8-week-old Mesp- / -+GFP-blastomere mouse are shown. [Figure 13] This shows the results of observing the heart of an 8-week-old Mesp- / -+GFP-blastomere mouse. [Figure 14] The results of measuring each cell that constitutes the heart of 8-week-old Mesp+ / -+GFP-blastomere mice and Mesp- / -+GFP-blastomere mice are shown. [Figure 15] The results of HE staining of the hearts of 8-week-old Mesp+ / +, Mesp+ / -+GFP-blastomere, and Mesp- / -+GFP-blastomere mice are shown. [Figure 16] The results of echocardiography measurements of 7- to 8-week-old Mesp+ / +, Mesp+ / -+GFP-blastomere, and Mesp- / -+GFP-blastomere mice are shown. [Figure 17]A graph summarizing the results of FIG. 16 is shown. [Figure 18] Photographs of the treadmill fatigue test are shown. [Figure 19] 1 shows a graph summarizing the results of the treadmill fatigue test. [Figure 20] The results of observation of Mesp+ / -, Mesp+ / -+GFP-mESCs, and Mesp- / -+GFP-mESCs mice at E9.5 are shown. [Figure 21] The results of observation of Mesp+ / -, Mesp+ / -+GFP-mESCs, and Mesp- / -+GFP-mESCs mice at E13.5 are shown. [Figure 22] Immunostaining results of E13.5 hearts from Mesp+ / -, Mesp+ / -+GFP-mESCs, and Mesp- / -+GFP-mESCs mice are shown. [Figure 23] This shows the results of observing GFP emission in organs other than the heart of Mesp- / -+GFP-mESCs mice at E13.5. [Figure 24] The results of observation of Mesp- / -+GFP-mESCs mice at P0 are shown. [Figure 25] This shows the results of evaluating chimerism in offspring when GRR mESCs were used as ES cells. [Figure 26] The results of a study into transplantation conditions that result in good chimerism are shown below. [Figure 27] The results of a study on the timing of ES cell transplantation (embryo maturity) are shown. [Figure 28] This shows the results of evaluating the chimerism of offspring when SGE2 mESCs were used as ES cells. [Figure 29] 8-week-old Mesp+ / -+GFP-mESCs mice and Mesp- / -+GFP-mESCs mice are shown. [Figure 30] The results of immunostaining of the hearts of 8-week-old Mesp- / -+GFP-mESCs mice and Mesp+ / -+GFP-mESCs mice are shown. [Figure 31] The results of immunostaining of the heart of an 8-week-old Mesp- / -+GFP-mESCs mouse are shown. [Figure 32] The results of immunostaining of the heart of an 8-week-old Mesp+ / -+GFP-mESCs mouse are shown. [Figure 33] The results of HE staining of the hearts of 8-week-old Mesp+ / +, Mesp+ / -+GFP-mESCs, and Mesp- / -+GFP-mESCs mice are shown. [Figure 34] The results of echocardiography measurements of 7-8 week-old Mesp+ / +, Mesp+ / -+GFP-mESCs, and Mesp- / -+GFP-mESCs mice are shown. [Figure 35] A graph summarizing the results of Figure 34 is shown. [Figure 36] Photographs of the treadmill fatigue test are shown. [Figure 37] 1 shows a graph summarizing the results of the treadmill fatigue test. [Figure 38] Electrocardiogram results of 7-8 week-old Mesp+ / +, Mesp+ / -+GFP-mESCs, and Mesp- / -+GFP-mESCs mice are shown. [Figure 39] 10 is a graph showing the ratio of heart weight to body weight in 7- to 8-week-old Mesp+ / -+GFP-mESCs and Mesp- / -+GFP-mESCs mice. [Figure 40] 10 is a graph showing the ratio of heart weight to tibia length in 7- to 8-week-old Mesp+ / -+GFP-mESCs and Mesp- / -+GFP-mESCs mice. [Figure 41] The results of chimerism evaluation of all surviving mice in the construction of heterologous chimeric mice are shown below. [Figure 42] This shows the results of evaluation of chimerism in surviving chimeric mice constructed from heterologous chimeric mice. [Figure 43] The observation results of wild-type, heterozygous, and homozygous individuals produced in the construction of heterologous chimeric mice are shown. [Figure 44] 1 shows the results of immunostaining of the heart of a homozygous individual produced in the construction of a heterologous chimeric mouse. [Figure 45]The results of immunostaining of the atria and ventricles of homozygous individuals produced in the construction of heterologous chimeric mice are shown. DETAILED DESCRIPTION OF THE INVENTION

[0015] [Method for producing a chimeric non-human mammal] A method for producing a chimeric non-human mammal according to one aspect of the present invention includes a stem cell preparation step, a transplantation step, and a litter obtaining step. Hereinafter, this method may be abbreviated as "a method for producing a chimeric non-human mammal according to this embodiment."

[0016] As used herein, the term "chimeric non-human mammal" refers to a non-human mammal composed of two or more types of cells with different genetic backgrounds. "Different genetic backgrounds" means that the cells are of different species (xenogeneic), or that the cells are of the same species but are allogeneic (allogeneic). Examples of non-human mammals include mice, rats, rabbits, pigs, cows, dogs, sheep, goats, monkeys, chimpanzees, gorillas, orangutans, marmosets, etc.

[0017] (Stem cell preparation process) In the stem cell preparation step, stem cells are prepared from mammals. The mammal is selected depending on the animal species of the target heart. Examples of such mammals include humans, mice, rats, rabbits, pigs, cows, dogs, sheep, goats, monkeys, chimpanzees, gorillas, orangutans, marmosets, etc.

[0018] Examples of the stem cells include embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells). ES cells or iPS cells may be derived from humans or non-humans. ES cells or iPS cells can be prepared by known techniques.

[0019] The stem cells may be prepared by known techniques so as to express a fluorescent protein such as GFP (green fluorescent protein). The expression of the fluorescent protein makes it easy to detect whether the heart in the chimeric non-human mammal is derived from the transplanted stem cells.

[0020] (Transplanting process) In the transplantation step, the stem cells prepared in the stem cell preparation step are transplanted into a fertilized egg in which the Mesp1 gene and the Mesp2 gene have been knocked out. This transplantation allows a chimeric cell mixture to be formed in the lumen of the fertilized egg from the internal cells derived from the fertilized egg and the transplanted stem cells. Transplantation of stem cells into the fertilized egg may be performed using known techniques.

[0021] Mesp1 gene is the mesoderm posterior 1 gene in mice and the mesoderm posterior bHLH transcription factor 1 gene in humans. Mesp2 gene is the mesoderm posterior 2 gene in mice and the mesoderm posterior bHLH transcription factor 2 gene in humans.

[0022] The Mesp1 gene and the Mesp2 gene can be knocked out by known techniques. For example, the Mesp1 gene and the Mesp2 gene can be knocked out by gene editing techniques such as the CRISPR-Cas9 system. The Mesp1 gene is described, for example, in Non-Patent Document 7. The Mesp2 gene is described, for example, in Non-Patent Document 8. When a fertilized egg in which the Mesp1 gene and the Mesp2 gene have been knocked out is developed, the heart does not develop and embryonic death is induced.

[0023] The fertilized egg is a fertilized egg of a non-human mammal (recipient) different from the mammal (donor) from which the stem cells are derived. The stem cells and the fertilized egg are in a heterogeneous or allogeneic relationship. Examples of combinations of mammals (donors) and non-human mammals (recipients) are as follows: · Combination of mice (donor) and mice (recipient) -Mixture of mice (donors) and rats (recipients) · Combination of rats (donors) and mice (recipients) - Human (donor) and mouse (recipient) combination - Human (donor) and rat (recipient) combination - Human (donor) and pig (recipient) combination - Human (donor) and bovine (recipient) combination - Human (donor) and monkey (recipient) combination - Combination of monkeys (donor) and monkeys (recipient) - Combination of monkeys (donors) and mice (recipients) - Monkey (donor) and rat (recipient) combination -Combination of monkeys (donors) and pigs (recipients) -Combination of monkeys (donors) and cows (recipients)

[0024] The fertilized eggs are preferably fertilized eggs at the early embryo (embryo after fertilization to the 8-cell stage) to blastocyst stage, and more preferably fertilized eggs at the 8-cell to blastocyst stage, in that the chimerism of the offspring obtained in the offspring obtaining step described below will be 50% to 90%. As used herein, "offspring chimerism" refers to the proportion of cells with different genetic backgrounds within the offspring. Furthermore, "donor cell (recipient cell) chimerism" refers to the proportion of donor cells (recipient cells) within the body of a chimeric animal.

[0025] If the chimerism of the offspring is less than 50%, the heart may not be fully complemented, whereas if the chimerism of the offspring is more than 90%, there is a concern that the stem cells will be excessive and the entire offspring may be derived from stem cells.

[0026] An example of a method for evaluating chimerism in offspring is described below. Stem cells expressing a fluorescent protein such as GFP are transplanted into a fertilized egg, and the number of GFP-positive cells in the heart of the offspring obtained in the offspring acquisition process described below is counted. The chimerism of the offspring is then evaluated as the ratio of the number of GFP-positive cells to the total number of cells constituting the offspring.

[0027] It is preferable to select the number and type of stem cells to be transplanted into the fertilized eggs so that the chimerism of the offspring obtained in the offspring obtaining step described below is 50% or more and 90% or less.

[0028] The fertilized egg may be a fresh embryo or a frozen embryo. The fertilized egg is preferably a frozen embryo, since the chimerism of the offspring obtained in the offspring obtaining step described below is 50% or more and 90% or less.

[0029] (Lawn acquisition process) In the offspring obtaining step, the fertilized egg after the transplantation step is developed in the womb of the non-human mammal to obtain offspring. For example, the fertilized egg after the transplantation step can be developed in the womb of the non-human mammal by transplanting it into the uterus of a pseudopregnant or pregnant female animal of the same species as the non-human mammal from which the fertilized egg was derived. Then, the stem cell-derived heart transplanted in the transplantation step is complemented by the recipient non-human mammal to obtain offspring.

[0030] By the method for producing a chimeric non-human mammal of this embodiment, the heart of the offspring is complemented with cardiomyocytes, endothelial cells, smooth muscle cells, and fibroblasts derived from the stem cells. That is, the heart complemented by the method for producing a chimeric non-human mammal of this embodiment is a heart that functions normally.

[0031] [Chimeric non-human mammals] A chimeric non-human mammal produced by the method for producing a chimeric non-human mammal of this embodiment is also included in one aspect of the present invention. The chimeric non-human mammal has the following four characteristics:

[0032] (1) Cardiomyocytes, endothelial cells, smooth muscle cells, and fibroblasts that constitute the heart include cells of mammalian origin.

[0033] (2) Organs and tissues other than the heart include cells derived from the mammals described above and cells derived from non-human mammals different from the mammals described above.

[0034] (3) The mammal-derived cells contain the Mesp1 and Mesp2 genes. Therefore, the cardiomyocytes, endothelial cells, smooth muscle cells, and fibroblasts that constitute the heart of the chimeric non-human mammal have the Mesp1 and Mesp2 genes functioning normally.

[0035] (4) The Mesp1 gene and Mesp2 gene are knocked out in the cells derived from the non-human mammal.

[0036] The chimeric non-human mammal can be used as a heart disease model animal. For example, a chimeric non-human mammal in which a heart has been regenerated from human-derived stem cells can be used as a humanized heart disease model animal.

[0037] Furthermore, the above-mentioned chimeric non-human mammal can be used to screen or evaluate the efficacy of drugs for heart diseases.

[0038] [Method for producing heart or cardiac tissue] A method for producing a heart or cardiac tissue according to one aspect of the present invention includes a heart obtaining step in addition to the above-described stem cell preparation step, transplantation step, and offspring obtaining step. Hereinafter, this method may be abbreviated as "a method for producing a heart according to this embodiment." Examples of cardiac tissue include cardiac structures such as the right atrium, left atrium, right ventricle, left ventricle, and valve tissue, and cardiac cells such as cardiomyocytes or non-cardiomyocytes that constitute these structures.

[0039] (Heart acquisition process) In the heart obtaining step, a heart or cardiac tissue is obtained from the offspring obtained in the offspring obtaining step. The heart is a stem cell-derived heart and is substantially derived from the donor mammal. As used herein, "substantially derived from the donor mammal" means that the heart does not contain cells derived from the recipient non-human chimeric mammal, or contains only a very small amount of cells that does not induce a rejection reaction in transplantation, etc.

[0040] The obtained heart or heart tissue can be used as a heart transplant for a mammal with a heart disease.

[0041] Whether or not the obtained heart or cardiac tissue functions normally can be analyzed, for example, by visual observation, morphological analysis using tissue staining or fluorescence microscopy, gene expression analysis, or the like.

[0042] [Heart or cardiac tissue production kit] A kit for producing a heart or cardiac tissue according to one embodiment of the present invention comprises mammal-derived stem cells and a fertilized egg of a non-human mammal different from the mammal, in which the Mesp1 gene and the Mesp2 gene have been knocked out. The kit can be used to produce a heart for transplantation. The mammal-derived stem cells can be prepared in the same manner as in the stem cell preparation step of the method for producing a chimeric non-human mammal. The Mesp1 gene and the Mesp2 gene in the fertilized egg of the non-human mammal can be knocked out using known techniques.

[0043] 〔summary〕 The method for producing a chimeric non-human mammal according to aspect 1 of the present invention comprises: a stem cell preparation step of preparing stem cells derived from a mammal; a transplantation step of transplanting the stem cells into fertilized eggs of a non-human mammal different from the mammal, the fertilized eggs having the Mesp1 gene and the Mesp2 gene knocked out; and a litter obtaining step of developing the fertilized eggs in the womb of the non-human mammal to obtain litter, wherein the hearts of the litter are complemented by cardiomyocytes, endothelial cells, smooth muscle cells, and fibroblasts derived from the stem cells.

[0044] A method for producing a chimeric non-human mammal according to a second aspect of the present invention is the method of the first aspect above, wherein the fertilized egg of the non-human mammal is a fertilized egg at the early embryo to blastocyst stage.

[0045] A third aspect of the present invention relates to a method for producing a chimeric non-human mammal, wherein the chimerism of the offspring is 50% or more and 90% or less in the above-mentioned first or second aspect.

[0046] A fourth aspect of the present invention relates to a method for producing a chimeric non-human mammal in any one of the above-mentioned first to third aspects, wherein the stem cells are embryonic stem cells (ES cells) or induced pluripotent stem cells (iPS cells).

[0047] A fifth aspect of the present invention relates to a method for producing a chimeric non-human mammal, in which, in any one of the above-mentioned first to fourth aspects, the stem cells are stem cells derived from a human, a mouse, or a rat.

[0048] In a chimeric non-human mammal according to aspect 6 of the present invention, the cardiomyocytes, endothelial cells, smooth muscle cells and fibroblasts that constitute the heart comprise cells derived from a mammal, and the organs and tissues other than the heart comprise cells derived from the mammal and cells derived from a non-human mammal different from the mammal, the cells derived from the mammal have the Mesp1 gene and the Mesp2 gene, and the cells derived from the non-human mammal have the Mesp1 gene and the Mesp2 gene knocked out.

[0049] The method for producing a heart or cardiac tissue according to aspect 7 of the present invention comprises a stem cell preparation step of preparing stem cells derived from a mammal; a transplantation step of transplanting the stem cells into fertilized eggs of a non-human mammal different from the mammal, the fertilized eggs having the Mesp1 gene and the Mesp2 gene knocked out; a litter obtaining step of developing the fertilized eggs in the womb of the non-human mammal to obtain offspring; and a heart obtaining step of obtaining a heart or cardiac tissue from the offspring, wherein the heart or cardiac tissue is complemented by cardiomyocytes, endothelial cells, smooth muscle cells, and fibroblasts differentiated from the stem cells.

[0050] A method for producing a heart or cardiac tissue according to an eighth aspect of the present invention is the method according to the seventh aspect above, wherein the fertilized egg of the non-human mammal is a fertilized egg at the early embryo to blastocyst stage.

[0051] A ninth aspect of the present invention relates to the method for producing a heart or cardiac tissue, wherein in the seventh or eighth aspect, the chimerism of the offspring is 50% or more and 90% or less.

[0052] A tenth aspect of the present invention relates to a method for producing a heart or cardiac tissue according to any one of the above seventh to ninth aspects, wherein the stem cells are embryonic stem cells (ES cells) or induced pluripotent stem cells (iPS cells).

[0053] A method for producing a heart or cardiac tissue according to an eleventh aspect of the present invention is the method of any one of the above seventh to tenth aspects, wherein the stem cells are stem cells derived from a human, a mouse, or a rat.

[0054] A heart or cardiac tissue according to a twelfth aspect of the present invention is obtained by the method for producing a heart or cardiac tissue according to any one of the seventh to eleventh aspects above.

[0055] A kit for producing a heart or cardiac tissue according to aspect 13 of the present invention comprises stem cells derived from a mammal and a fertilized egg of a non-human mammal different from said mammal, in which the Mesp1 gene and the Mesp2 gene have been knocked out.

[0056] The following examples are provided to further explain the embodiments of the present invention. It goes without saying that the present invention is not limited to the following examples, and various modifications are possible in detail. Furthermore, the present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed herein are also included in the technical scope of the present invention. Furthermore, all of the documents cited in this specification are incorporated by reference. [Example]

[0057] All experiments described below were conducted with the approval of the Animal Experiment Ethics Committee of the University of Tsukuba.

[0058] 〔material and method〕 (mouse) Mesp1 and Mesp2 double knockout (MespDKO) xenogeneic mice (Mesp + / - ) was provided by the RIKEN BioResource Research Center through the MEXT / AMED National BioResource Project. - / - ) Mice are heterologous Mesp + / -The transgenic mice were obtained by intercrossing GFP (green fluorescent protein)-dependent GFP transgenic mice. Transgenic mice overexpressing GFP (green fluorescent protein) under the control of the CAG promoter at the ROSA26 locus were generated as described in Hasegawa, Y. et al. Exp Anim 62, 295-304 (2013). ICR (Crl:CD1(ICR)) mice were purchased from Jackson Laboratory Japan. ICR mice stand for Institute of Cancer Research mice. No immunodeficiency or other health problems were observed in the transgenic mice. All animals were drug-naive prior to use. All animals were group-housed and maintained in a dedicated specific pathogen-free (SPF) facility under a 14-hour / 10-hour light / dark cycle. Food and water were provided ad libitum and checked daily. The animals' health status was routinely monitored to maintain SPF status. Animals undergoing surgical procedures were transferred to a satellite SPF facility under the same conditions.

[0059] (mouse ES cells) Mouse embryonic stem cells (mESCs) were used as stem cells. Specifically, R26GRR mouse (hereafter abbreviated as GRR mice) embryonic stem cells (hereafter abbreviated as GRR mESCs) and SGE2 mouse embryonic stem cells (hereafter abbreviated as SGE2 mESCs) were used. The production of GRR mESCs was described in Hasegawa, Y. et al. Exp Anim 62, 295-304 (2013), and the production of SGE2 mESCs was described in Yamaguchi, T. et al. Nature 542, 191-196 (2017). Both GRR mESCs and SGE2 mESCs are EGFP-expressing mES cells.

[0060] Undifferentiated GRR and SGE2 mESCs were maintained in mitomycin C-treated mouse embryonic fibroblasts (MEFs) established from ICR mice. The medium for GRR mESCs, prepared using standard procedures, consisted of Dulbecco's modified Eagle's medium (DMEM, 11995, Thermo Fisher Scientific) supplemented with 10% KnockOut serum replacement (KSR; 10828, Thermo Fisher Scientific), 1% non-essential amino acids (1681049, MP Biomedicals), 0.1 mM 2-mercaptoethanol (M3148, Merck), 1000 U / mL LIF (ESG1107, Merck), 3 μM CHIR99021 (04-0004-02, Reprocell), and 1 μM PD0325901 (04-0006-02, Reprocell). Culture medium for SGE2 mESCs, prepared using standard procedures, consisted of NDiff 227 (Y40002, Takara) supplemented with 1000 U / mL LIF (ESG1107, Merck), 3 μM CHIR99021 (04-0004-02, Reprocell), and 1 μM PD0325901 (04-0006-02, Reprocell).

[0061] (MespDKO embryo complementation with GRR blastomeres) To generate aggregation chimeras, 8-cell embryos from GRR mice were aggregated with 4-cell embryos from mutant mice. Briefly, 4-cell and 8-cell embryos were subjected to in vitro fertilization (IVF) with heterologous Mesp. + / - These embryos were obtained from GRR and GRR mice. These embryos were cultured in KSOM medium (MR-121-D, Merck). These embryos (allogeneic Mesp + / + , Heterogeneous Mesp + / - , or wild-type Mesp + / +Mutant chimeras were generated by aggregating one of the blastocysts (containing nuclei containing nuclei) with one or two GRR blastomeres (blastomere-stage embryos obtained from GRR mice). Twenty successfully aggregated chimeric blastocysts were surgically transferred into the uterine horns of pseudopregnant ICR female mice (2.5 dpc) and allowed to develop to the desired stage.

[0062] (MespDKO embryo complementation with ES cells) Embryos were frozen two hours after IVF and used for blastocyst complementation with ES cells. Host embryos were prepared by thawing frozen embryos and incubating them in M16 medium (M7292, Sigma-Aldrich, Merck) for 24 hours. Three days before embryo manipulation, frozen ES cells were thawed and placed on a feeder layer. The expanded ES cells were dissociated with 0.25% trypsin immediately before injection. For the injection method, a hole was pierced in the zona pellucida and trophectoderm using a piezo-actuated micromanipulator (PMM-150FU, PrimeTech) under a microscope, and 10–20 ES cells were implanted into the blastocyst cavity near the inner cell mass. The manipulated chimeric embryos were then implanted at the blastocyst stage into the uterine horns of pseudopregnant recipient ICR female mice (2.5 dpc).

[0063] (Mouse fibroblasts for genotyping) MEFs and tail fibroblasts (TTFs) were isolated using the following methods: Kurotsu, S. et al., STAR Protoc 3, 101122 (2022); Muraoka, N. et al. Nat Commun 10, 674 (2019); Khan, M. et al., J Vis Exp (2016). Briefly, for MEF isolation, embryos isolated from pregnant mice were washed with phosphate-buffered saline (PBS). Each tissue of the embryo was washed with fresh PBS and minced using scissors. Each minced tissue of the embryo was then immersed in 0.25% trypsin / ethylenediaminetetraacetic acid (EDTA) solution (25200-072, Gibco). TMThe embryos were transferred to a 100-well plate (ThermoFisher) and incubated at 37°C for 5 minutes. After trypsin digestion, two volumes of MEF medium (DMEM containing 20% ​​fetal bovine serum (FBS) per embryo) were added, and the tissue was dissociated by pipetting several times. The supernatant was transferred to a fresh tube, and the cells were collected by centrifugation, resuspended in DMEM / 20% FBS (CCP-FBS-BR-500; COSMO Bio), and cultured at 37°C under 5% CO2. For TTF isolation, 1 cm of the tail from a 4-week-old mouse was minced to a size of less than 3 mm. The minced tail was transferred to an enzymatic digestion solution (0.4 mL of PBS containing 1 mg of collagenase D (11088866001, Merck)) and incubated at 37°C for 90 minutes. The digested tail tissue was transferred to a 70 μm cell strainer placed in a 10 cm cell culture dish. The medium in a 10 cm cell culture dish was 10 ml of TTF medium (Iscove's modified Dulbecco's medium (IMDM; 12440053, Gibco) containing 20% ​​FBS). TM A centrifuge was used (Thermo Fischer Scientific). The tissue was then forcibly crushed using a syringe plunger. The supernatant was transferred to a fresh tube, and cells were collected by centrifugation, resuspended in TTF medium, and cultured at 37°C under 5% CO.

[0064] (Genotyping of chimeric mice) Genotyping of chimeric mice was performed using genomic DNA extracted from GFP-negative fibroblasts isolated by fluorescence-activated cell sorting (using a cell sorter; Beckman Coulter, MoFlo XDP). MEFs were obtained from whole fetal tissues from embryonic day 9 (E9) to embryonic day 9.5 (E9.5) and head tissues from embryonic day 10.5 (E10.5). Adult TTFs were obtained from tail tissues after 4 weeks of age. Genotyping of Mesp by genomic PCR was performed. - / - Detection of the mutant allele was performed using the following four primer sets to amplify WT (439 and 829 bp PCR products) and KO (1.8 kbp PCR product). F1: 5'-GCCACCTGGCCCTTGCTACTCC-3' (SEQ ID NO: 1) F2: 5'-GAGCTGCACCTGGCCTGCAGTG-3' (SEQ ID NO: 2) R1: 5'-CTGAGTTCATTGACGTGGACCC-3 (SEQ ID NO: 3) R2: 5'-GCTTTGGTCTTGGGCTTCCAGTG-3' (SEQ ID NO: 4)

[0065] (Fluorescence-activated cell sorting) Analysis and cell sorting were performed using a MoFlo XDP. Fibroblasts were stained with an APC-conjugated anti-CD45 antibody (102410, Biolegend) to exclude GFP-silenced blood cells. All acquired data were analyzed using FlowJo software (Tomy Digital Biology).

[0066] (immunohistochemistry) Embryos were fixed in 4% paraformaldehyde (PFA) overnight at 4°C, followed by 30% sucrose overnight at 4°C. They were then embedded in optimal cutting temperature compound (OCT) for freezing in liquid nitrogen. Adult mouse hearts, intestines, kidneys, livers, and lungs were fixed overnight in 0.4% PFA and embedded in OCT compound for freezing in liquid nitrogen. These samples were cut into 7-μm sections using a cryostat microtome (CM3050S, Leica). Sections were stained with primary antibodies against α-actinin (A7811, Sigma, 1:800), α-SMA (A2547, Sigma, 1:400), CD31 (ab28364, Abcam, 1:10), cTnT (MS-295-P1, Thermo Fisher Scientific, 1:400), GFP (598, MBL, 1:500 and D153-3, MBL, 1:200), and vimentin (GP53, Progen, 1:100 and ab45939, Abcam, 1:100), followed by Alexa488- or 546-conjugated secondary antibodies and DAPI (D1306, Invitrogen, 1:100, Thermo Fisher Scientific). Images were acquired using an all-in-one fluorescence microscope (BZX810, Keyence) or a confocal microscope (LSM800, Carl Zeiss). GFP was measured in 10 randomly selected, non-overlapping areas per mouse section. + The percentage of cells was calculated and more than 1000 cells were counted in each mouse. Measurements and calculations were performed in a blinded manner.

[0067] (qRT-PCR) Total RNA was isolated from whole embryos using standard protocols. qRT-PCR was performed using the StepOnePlus Real-Time PCR system (Applied Biosystems, Thermo Fischer Scientific) with TaqMan probes. mRNA levels were normalized to the levels of Gapbh. Table 1 shows the details of the TaqMan gene expression assay (Applied Biosystems, Thermo Fischer Scientific), and Table 2 shows the details of the Universal Probe Library System (Roche).

[0068] [Table 1]

[0069] [Table 2]

[0070] (echocardiography) Cardiac function was analyzed in 7- to 8-week-old mice using transthoracic echocardiography (Visual Sonics, Vevo 2100). Mice were anesthetized with low doses of isoflurane for echocardiographic studies. Two-dimensional targeted M-mode tracings were obtained at the papillary muscle level. Using the Tischholz equation, ejection fraction (EF) was calculated according to the following formula: EF= (LVEDV - LVESV) / ​​LVEDV ×100 (%) (Formula 1) In addition, LVEDV (left ventricular end-diastolic volume) and LVESV (left ventricular end-systolic volume) were calculated using the following formulas 2 and 3, respectively. LVEDV = [{7.0 / (2.4+LVDd)} ×(LVDd) 3 ] ...(Formula 2) LVESV = [{7.0 / (2.4+LVDs)} ×(LVDs) 3] ...(Formula 3)

[0071] (Treadmill fatigue test in mice) After three consecutive days of treadmill exercise, a treadmill fatigue test was performed. As a warm-up exercise, mice ran at 10 m / min for 5 minutes up a 15° treadmill. Subsequently, the speed was increased by 2 m / min every 2 minutes according to the protocol. Mice were considered fatigued when they remained on the shock grid for 10 consecutive seconds. The time to fatigue was then measured, and the running distance was calculated.

[0072] (statistical analysis) Statistical parameters, including sample size (n), descriptive statistics (mean and standard error of the mean), and significance, were calculated. Generally, at least n = 3 was used for each time point in each study. Statistical significance of differences between groups was tested using Student's t-test or one-way analysis of variance (ANOVA) followed by Dunnett's post-hoc test. Differences were considered significant when p-values ​​were <0.05. Statistical analysis was performed using GraphPad Prism software.

[0073] 〔result〕 (Observation of MespDKO mice) MespDKO(Mesp - / - A schematic diagram of the targeting vector and target allele used in the generation of Mesp mice is shown in Figure 1. + / + , Mesp + / - , Mesp - / - A photograph of a mouse at E9.0 is shown in Figure 2. As shown in Figure 2, Mesp - / - The mice were confirmed to have died during the fetal stage due to impaired differentiation of the mesoderm, from which the heart is derived.

[0074] Also, Mesp + / + , Mesp + / - , Mesp - / - The results of qRT-PCR at E8.5 in mice are shown in Figure 3. + / + , Mesp1 / 2 + / - , Mesp1 / 2- / - are respectively Mesp + / + , Mesp + / - , Mesp - / - The results for mice are shown. "ns" indicates non-significant, "*" indicates P=0.01, and "**" indicates P<0.01.

[0075] As shown in Figure 3, Mesp - / - In mice, expression of the target genes Mesp1 and Mesp2 was defective, and expression of the cardiac-related genes Myh6 and Tnn2 was reduced.

[0076] (Mesp - / - +GFP-blastomere mouse construction) Heart-defective embryos (Mesp - / - ) and a normal embryo (GFP-positive embryo, GFP-blastomere) (hereafter referred to as Mesp - / - We constructed chimeric mice (called +GFP-blastomere mice) and examined whether cardiac regeneration was possible. The observation results of these chimeric mice are shown in Figures 4 to 19. GFP-blastomere (GFP-positive blastomere) is an embryo at the blastomere stage during the development of a fertilized egg from a mouse that has been genetically engineered to express Green Fluorescent Protein.

[0077] Figure 4 shows the Mesp + / - , Mesp + / - +GFP-blastome and Mesp - / - The results are from observations of +GFP-blastomere mice at E9.5. In Figure 4, BF indicates the results of observations in bright field, and GFP indicates the results of observations of GFP emission. The heart developed from a GFP-positive embryo emits green light due to GFP. As shown in Figure 4, Mesp + / - No GFP emission was observed in the hearts of +GFP-blastomere mice. - / - GFP emission was observed in the hearts of +GFP-blastomere mice, confirming that heart regeneration using GFP-positive embryos was successful and embryonic death was avoided.

[0078] Figure 5 shows the Mesp + / - , Mesp + / - +GFP-blastome, Mesp - / - Figure 6 shows the immunostaining results of the heart of an E13.5 +GFP-blastomere mouse. - / - Immunostaining of the heart of a +GFP-blastomere mouse at E13.5 is shown. RV, IVS, and LV indicate the right ventricle, interatrial septum, and left ventricle, respectively. α-actinin is a marker for cardiomyocytes.

[0079] As shown in Figures 5 and 6, Mesp - / - It was found that almost all cardiomyocytes in the hearts regenerated from +GFP-blastomere mice were derived from GFP-positive embryos.

[0080] Figure 7 shows the Mesp - / - The results of observing GFP emission in organs other than the heart (lungs, liver, intestines, and kidneys) of +GFP-blastomere mice at E13.5 are shown in Figure 7. As shown in Figure 7, cells in organs other than the heart were GFP- + / - It was found to be a chimera with mixed cells.

[0081] The left side of Figure 8 shows an 8-week-old Mesp + / - +GFP-blastomere mouse (control), on the right is an 8-week-old Mesp - / - +GFP-blastomere mice. - / - The dorsal side of the +GFP-blastomere mice is black, suggesting that it originates from GFP-positive embryonic cells. - / - The ventral side of the +GFP-blastomere mice is gray, and Mesp - / - It is thought that they originate from embryonic cells. - / - The tails of +GFP-blastomere mice were deformed. This deformity was due to the development of embryonic heart defects (Mesp - / - This is due to the mixture of GFP-blastomere and normal embryos (GFP-blastomere).

[0082] Figures 9 and 11 show the results of 8-week-old Mesp + / -10 and 12 show the results of immunostaining of the hearts of 8-week-old Mesp1+GFP-blastomere mice. - / - Immunostaining results of the hearts of +GFP-blastomere mice are shown in Figures 9 to 12. + / - +GFP-blastomere mouse heart is GFP + / - While cardiomyocytes derived from Mesp cells are mixed, - / - It was found that almost all of the cardiomyocytes in the hearts of +GFP-blastomere mice were regenerated from cells derived from GFP-positive embryos.

[0083] Figure 13 shows the results of 8-week-old Mesp - / - The heart of an 8-week-old Mesp1+GFP-blastomere mouse was immunostained for smooth muscle cell marker (α-SMA), endothelial cell marker (CD31), and fibroblast marker (vimentin). + / - +GFP-blastomere mice and Mesp - / - 13 and 14 are graphs showing the chimerism of each cell type constituting the heart of +GFP-blastomere mice. - / - It was found that the cells that make up the hearts of +GFP-blastomere mice, including not only cardiomyocytes but also representative non-cardiomyocytes such as smooth muscle cells, endothelial cells, and fibroblasts, were almost entirely regenerated from cells derived from GFP-positive embryos.

[0084] Figure 15 shows the Mesp + / + , Mesp + / - +GFP-blastome and Mesp - / - The results of hematoxylin-eosin staining (HE staining) of the hearts of +GFP-blastomere mice are shown. As shown in Figure 15, Mesp - / - No abnormalities were observed in the cardiac structure of +GFP-blastomere mice.

[0085] Figure 16 shows the Mesp + / + , Mesp + / -+GFP-blastome and Mesp - / - The results of echocardiography of +GFP-blastomere mice are shown in Figure 17. The vertical axis of Figure 17 represents LVEF, which stands for left ventricular ejection fraction. As shown in Figures 16 and 17, Mesp - / - The regenerated hearts in the +GFP-blastomere mice were found to function normally.

[0086] Figure 18 shows photographs of the treadmill fatigue test. The mice running in the two lanes in the center (the mice marked with * in Figure 18) were Mesp - / - +GFP-blastomere mice. Figure 19 is a graph summarizing the results of the treadmill fatigue test. The vertical axis of Figure 19 shows the running distance of the mice. As shown in Figure 19, Mesp - / - +GFP-blastomere mice showed normal exercise tolerance.

[0087] (Mesp using GRR mESC as stem cells - / - + GFP-mESCs mouse construction) Heart-defective embryos (Mesp - / - We investigated whether cardiac regeneration is possible in chimeric mice by transplanting GFP-expressing mouse ES cells (GFP-mESCs) into the peritoneal cavity of the ovary. GRR mESCs were used as mouse ES cells. The observation results of the chimeric mice are shown in Figures 20 to 24.

[0088] Mesp + / - , Mesp + / - +GFP-mESCs and Mesp - / - The observation results of +GFP-mESCs mice at E9.5 are shown in Figure 20, and the observation results at E13.5 are shown in Figure 21. The arrow in Figure 20 and the square frame in Figure 21 indicate the heart. As shown in Figures 20 and 21, Mesp - / - Overall GFP emission was observed in the hearts of +GFP-mESCs mice, confirming successful cardiac regeneration.

[0089] Figure 22 shows the Mesp+ / - , Mesp + / - +GFP-mESCs, Mesp - / - The results of immunostaining of the hearts of +GFP-mESC mice at E13.5 are shown in Figure 22. - / - It was found that the majority of cardiomyocytes in the hearts regenerated in +GFP-mESCs mice were derived from mouse ES cells expressing GFP.

[0090] Figure 23 shows the Mesp - / - 23 shows the results of observing GFP emission in organs other than the heart of +GFP-mESCs mice at E13.5. As shown in FIG. 23, cells in organs other than the heart were Mesp - / - The cells were a mixture of mouse ES cell-derived cells and mouse ES cell-derived cells.

[0091] Figure 24 shows the Mesp - / - The results of heart regeneration by blastocyst complementation are shown in Fig. 1. - / - +GFP-mESC mice were able to give birth. However, although some survived at birth, all died early. Furthermore, malformations were observed even in WT / heterozygous mice. - / - +GFP-blastomere mice survived to adulthood, indicating that Mesp - / - It was suggested that the early death of +GFP-mESCs mice may be due to the quality of the ES cells.

[0092] (Examination of chimerism in offspring) Mesp + / + , Mesp + / - +mESCs and Mesp - / - The chimerism of surviving +GFP-blastomere mice was evaluated. GRR was used as the GFP-blastomere mouse. The results of the evaluation at adulthood are shown in Figure 25. The chimerism (%) on the vertical axis of Figure 25 indicates the proportion of GFP-positive cells (GRR-mESCs) to the cells that make up the offspring.

[0093] As shown in Figure 25, the surviving Mesp - / -The chimerism of the +GFP-blastomere mice was around 60-90%. The other mice had chimerism of less than 50%, but Mesp - / - These results indicate that cardiac complementation achieved chimerism of 50-90%.

[0094] Next, we transplanted ES cells expressing GFP into wild-type mouse embryos and investigated the transplantation conditions that would result in good chimerism (50-90% GFP-positive cells). The results of this study at E13.5 are shown in Figure 26. The chimerism (%) on the vertical axis of Figure 26 represents the proportion of GFP-positive cells (GRR-mESCs) to the cells that make up the offspring.

[0095] As shown in Figure 26, it was found that ICR mice were less likely to form chimeras than B6 mice. B6 mice were purchased from CLEA Japan. In many studies, 5 to 10 ES cells were transplanted into embryos. Based on the results in Figure 26, we decided to transplant 20 ES cells into ICR mouse embryos to compare chimerism.

[0096] Twenty ES cells were transplanted into ICR mouse embryos at the 8-cell, morula, and blastocyst stages, and chimerism was compared. The results are shown in Figure 27.

[0097] As shown in Figure 27, high chimerism was achieved by transplanting 20 ES cells. We also found that the maturity of the transplanted embryos also affects chimerism. When 20 ES cells are transplanted, the highest chimerism was achieved when the transplanted embryos were at the morula stage, which was the condition for achieving a chimerism of around 50-90%.

[0098] Next, we changed the type of ES cells expressing GFP to SGE2 mESCs and used Mesp - / - The chimerism of surviving +mESC mice was evaluated. The results of the evaluation at E13.5 are shown in Figure 28. The vertical axis of Figure 28 shows the ratio of GFP-positive cells (SGE2 mESCs) to the cells constituting the offspring.

[0099] As shown in Figure 28, when 20 ES cells were transplanted into embryos, not only were fewer individuals with the optimal chimerism of 50-90%, but the number of surviving individuals was also low. Figure 28 also revealed that in SGE2, the optimal number of ES cells to be transplanted into embryos was 10. The chimerism of the three surviving chimeric mice (circled in Figure 28) when 10 ES cells were transplanted into embryos was approximately 50-90%. These results demonstrate that the number of transplanted cells that achieves optimal chimerism depending on the type of transplanted cells (stem cells) is necessary. Furthermore, regardless of the type of transplanted cells, the chimerism required for cardiac complementation was found to be approximately 50-90%.

[0100] We also used SGE2 mESCs as stem cells for blastocyst complementation, optimized chimerism to 50-90%, and verified cardiac regeneration by blastocyst complementation. Four mice derived from these SGE2 mESCs survived.

[0101] (Mesp using SGE2 mESCs as stem cells - / - + GFP-mESCs mouse construction) Heart-defective embryos (Mesp - / - We examined whether cardiac regeneration is possible in chimeric mice by transplanting GFP-expressing mouse ES cells (GFP-mESCs) into the oocytes. SGE2 mESCs were used as mouse ES cells. The observation results of the chimeric mice are shown in Figures 29 to 40.

[0102] The left side of Figure 29 shows an 8-week-old Mesp + / - +GFP-mESCs mouse (control), on the right is an 8-week-old Mesp - / - +GFP-mESCs mice. - / - +GFP-blastomere mice, as well as Mesp - / - The tails of the +GFP-mESCs mice were deformed.

[0103] The upper part of Figure 30 and Figure 31 show 8-week-old Mesp - / -The results of immunostaining of the hearts of 8-week-old Mesp1+GFP-mESC mice are shown in the lower part of Figure 30 and in Figure 32. + / - 31 and 32 show the results of immunostaining of the hearts of +GFP-mESCs mice. In Figures 31 and 32, SMCs represent smooth muscle cells, ECs represent endothelial cells, and CFs represent fibroblasts.

[0104] As shown in Figures 30-32, Mesp + / - The hearts of +GFP-mESC mice were analyzed using Mesp - / - The cardiomyocytes derived from Mesp and mouse ES cells were mixed. - / - It was found that almost all of the cardiomyocytes in the hearts of +GFP-mESCs mice were regenerated from mouse ES cells expressing GFP. - / - It was found that the cells that make up the hearts of +GFP-mESCs mice, including not only cardiomyocytes but also representative non-cardiomyocytes such as smooth muscle cells, endothelial cells, and fibroblasts, were almost entirely regenerated by mouse ES cells expressing GFP.

[0105] Figure 33 shows the Mesp + / + , Mesp + / - +GFP-mESCs and Mesp - / - The results of HE staining of the hearts of +GFP-mESC mice are shown in Figure 33. - / - No abnormalities were observed in the cardiac structure of +GFP-mESCs mice.

[0106] Figure 34 shows the Mesp + / + , Mesp + / - +GFP-mESCs and Mesp - / - The results of echocardiography of +GFP-mESC mice are shown in Figure 35. The results of Figure 34 are summarized in a graph. The vertical axis of Figure 35 indicates LVEF. As shown in Figures 34 and 35, Mesp - / - The function of the regenerated hearts in the +GFP-mESCs mice was found to be normal.

[0107] Figure 36 shows photographs of the treadmill fatigue test. The mice running in the two lanes in the center (the mice marked with *** in Figure 36) were Mesp - / - +GFP-mESCs mice. Figure 37 is a graph summarizing the results of the treadmill fatigue test. The vertical axis of Figure 37 shows the distance traveled by the mice. As shown in Figure 37, the vertical axis of Figure 37 shows the distance traveled by the mice. - / - +GFP-mESCs mice showed normal exercise tolerance.

[0108] Figure 38 shows the Mesp + / + , Mesp + / - +GFP-mESCs and Mesp - / - As shown in Figure 38, Mesp - / - The regenerated hearts of +GFP-mESC mice showed no arrhythmias and their hearts functioned normally.

[0109] Figure 39 shows the Mesp + / - +GFP-mESCs and Mesp - / - 39 and 40 show the ratio of heart weight (HW, mg) to body weight (BW, g) in the +GFP-mESCs mice. FIG. 40 shows the ratio of heart weight (HW, mg) to tibia length (TL, mm). From the results in FIGS. 39 and 40, Mesp - / - It was found that the +GFP-mESCs mice did not have heart failure and their hearts functioned normally.

[0110] (Construction of heterologous chimeric mice) Next, using rat ES cells, Mesp - / - We constructed +GFP-ratESC mice. We used SD-Tg(CAG-EGFP) rats, which express GFP, as rat ES cells. The production of EGFP-expressing rESCs (EGFP-expressing rES cells) was performed according to the methods described in Ping Li, et al. Cell. 2008 Dec 26;135(7):1299-310 and Kazushi Kawaharada, et al. World J Stem Cells.

[0111] Chimeric embryos were generated using mutant mouse embryos and rat ES cells. Briefly, host embryos used for blastocyst complementation with ES cells were frozen 2 hours after IVF. The frozen embryos were thawed and incubated in M16 medium (M7292, Sigma-Aldrich, Merck) for 24 hours. Three days before embryo manipulation, frozen ES cells were thawed and placed on a feeder layer. Proliferated ES cells were dissociated with 0.25% trypsin immediately before injection. For the injection method, a piezo-driven micromanipulator (PMM-150FU, PrimeTech) was used under a microscope to puncture the zona pellucida and trophectoderm, and 10 ES cells were implanted into the blastocyst cavity near the inner cell mass. The engineered chimeric embryos were surgically transferred at the blastocyst stage into the uterine horns of pseudopregnant recipient ICR female mice (2.5 dpc) and allowed to develop to the desired stage.

[0112] The evaluation results when fresh or frozen embryos were used as mouse embryos are summarized in Table 3. In Table 3, "WT / hetero / homo" indicates wild-type individuals (WT, Mesp) in the chimera. + / + ), hetero individuals (hetero, Mesp + / - ) and homozygous individuals (Mesp - / - ) indicates the number of

[0113] [Table 3]

[0114] As shown in Table 3, one of the 50 normal embryos (15 fresh and 35 frozen) obtained in the first experiment was a homozygous individual (Mesp - / - The embryos were confirmed to be homozygous (+GFP-rat ESCs mice) (E11.5). In Table 3, "+na1" indicates "not available." No homozygous individuals were confirmed among the 34 normal embryos obtained in the second round (16 fresh embryos, 18 frozen embryos) (E11.5).

[0115] Figure 41 shows the results of evaluating the chimerism of all surviving mice (E11.5). Figure 42 shows the results of evaluating the chimerism of surviving chimeric mice (E11.5). The chimerism (%) on the vertical axis in Figures 41 and 42 indicates the ratio of rat ES cells to the cells that make up the offspring. "Fresh" indicates fresh embryos, and "Frozen" indicates frozen embryos.

[0116] As shown in Figures 41 and 42, when frozen embryos were used, individuals with high chimerism similar to that of allogeneic chimeric mice were obtained. In addition, when frozen embryos were used, homozygous individuals (Mesp - / - The results showed that frozen embryos are suitable for generating heterologous chimeras.

[0117] Figure 43 shows the results of observing wild-type, heterozygous, and homozygous individuals at E11.5. The chimerism of the individuals observed in Figure 43 was 59.7% for wild-type individuals, 34.1% for heterozygous individuals, and 79.5% for homozygous individuals.

[0118] Figure 44 shows a homozygous individual (Mesp) at E11.5. - / - The figures show the results of immunostaining of the heart of a homozygous mouse (+GFP-ratESCs mouse). The upper panel of Figure 45 shows the results of immunostaining of the atrium of an E11.5 homozygous mouse, and the lower panel shows the results of immunostaining of the ventricle. Figure 44 shows that the heart of the homozygous mouse is regenerated by cells derived from GFP-positive rat ES cells. Furthermore, Figure 45 shows that the cardiomyocytes that make up the atrium and ventricle of the homozygous mouse are regenerated by cells derived from GFP-positive rat cells. [Industrial Applicability]

[0119] The present invention makes it possible to produce hearts of mammalian species of interest, and the produced hearts can be used in the clinical field as transplant hearts.

Claims

1. a stem cell preparation step for preparing mammalian-derived stem cells; a transplantation step of transplanting the stem cells into a fertilized egg of a non-human mammal different from the mammal, the fertilized egg having a knockout of the Mesp1 gene and the Mesp2 gene; and a litter obtaining step of developing the fertilized egg in a womb of the non-human mammal to obtain a litter, A method for producing a chimeric non-human mammal in which the heart of the offspring is complemented by cardiomyocytes, endothelial cells, smooth muscle cells and fibroblasts derived from the stem cells.

2. The method according to claim 1, wherein the fertilized egg of the non-human mammal is a fertilized egg at the early embryo to blastocyst stage.

3. The method of claim 1, wherein the chimerism of the offspring is between 50% and 90%.

4. The method of claim 1, wherein the stem cells are embryonic stem cells (ES cells) or induced pluripotent stem cells (iPS cells).

5. The method of claim 1 , wherein the stem cells are stem cells of human, mouse, or rat origin.

6. Cardiomyocytes, endothelial cells, smooth muscle cells, and fibroblasts that constitute the heart include cells derived from mammals, the organs and tissues other than the heart comprise cells derived from the mammal and cells derived from a non-human mammal different from the mammal, the mammalian cell has a Mesp1 gene and a Mesp2 gene; A chimeric non-human mammal, wherein the Mesp1 gene and the Mesp2 gene have been knocked out in the cells derived from the non-human mammal.

7. a stem cell preparation step for preparing mammalian-derived stem cells; a transplantation step of transplanting the stem cells into a fertilized egg of a non-human mammal different from the mammal, the fertilized egg having a knockout of the Mesp1 gene and the Mesp2 gene; a step of obtaining offspring by developing the fertilized eggs in the womb of a non-human mammal; and a heart obtaining step of obtaining a heart or cardiac tissue from the offspring; A method for producing a heart or cardiac tissue, wherein the heart or cardiac tissue is complemented by cardiomyocytes, endothelial cells, smooth muscle cells and fibroblasts differentiated from the stem cells.

8. The method according to claim 7, wherein the fertilized egg of the non-human mammal is a fertilized egg at the early embryo to blastocyst stage.

9. The method according to claim 7, wherein the chimerism of the offspring is between 50% and 90%.

10. The method of claim 7, wherein the stem cells are embryonic stem cells (ES cells) or induced pluripotent stem cells (iPS cells).

11. The method of claim 7 , wherein the stem cells are stem cells of human, mouse, or rat origin.

12. A heart or heart tissue obtained by the method according to any one of claims 8 to 11.

13. Mammalian-derived stem cells; A kit for producing a heart or cardiac tissue, comprising: a fertilized egg of a non-human mammal different from the mammal, in which the Mesp1 gene and the Mesp2 gene have been knocked out.

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