JP7537726B2 - Extracellular matrix, method for producing mature cardiomyocytes, and cardiomyocyte maturation kit - Google Patents

Description

The present invention particularly relates to an extracellular matrix that matures immature cardiomyocytes induced to differentiate from pluripotent stem cells, a method for producing mature cardiomyocytes, a drug discovery support method, a treatment method, mature cardiomyocytes, and a cardiomyocyte maturation kit.

According to a statistical survey by the Japan Association for the Prevention of Lifestyle-Related Diseases (JPALD), the total number of patients with heart disease, particularly cardiomyopathy that develops after adulthood, is reported to be 1,729,000 (April 19, 2016), with the annual number of deaths from ischemic heart disease being 37,222 from "acute myocardial infarction" and 34,451 from "other ischemic heart disease," and the number of deaths due to heart disease being 196,113 per year (October 13, 2016). In addition, medical expenses for ischemic heart disease, which includes myocardial infarction, amount to 750.3 billion yen, of which 563 billion yen is for those aged 65 or older (November 10, 2015). As the aging population progresses, these figures are expected to increase further.

The use of regenerative medicine to treat such heart diseases is being considered. For example, it is believed that it may be possible to treat various heart diseases by inducing differentiation of pluripotent stem cells such as ES cells (embryonic stem cells) and iPS cells (induced pluripotent stem cells) to create cardiomyocytes and then transplanting these. Furthermore, it is expected that technology will be developed to analyze heart diseases and evaluate the toxicity of drugs by using cardiomyocytes created by such differentiation induction.

Now, referring to Patent Document 1, a method for differentiating cardiomyocyte progenitor cells and mature cardiomyocytes from pluripotent stem cells is described. In this method, pluripotent stem cells are differentiated on a substrate containing a mixture of (i) laminin 511 or 521 and (ii) laminin 221.

US Patent Application Publication No. 2016/0122717

However, cardiomyocytes created by inducing differentiation from pluripotent stem cells using the technology described in Patent Document 1 were of fetal to neonatal type and did not become similar to the cardiomyocytes of a fully mature adult heart.

The present invention was made in light of these circumstances, and aims to resolve the above-mentioned issues.

The extracellular matrix of the present invention is an extracellular matrix for maturing immature cardiomyocytes, and is characterized in that it contains a laminin fragment, and the laminin fragment is a laminin 511 E8 fragment and/or a laminin 521 E8 fragment.
The method for producing mature cardiomyocytes of the present invention is characterized in that immature cardiomyocytes are cultured in a medium containing the extracellular matrix to obtain mature cardiomyocytes .
The cardiomyocyte maturation kit of the present invention comprises an extracellular matrix for maturing immature cardiomyocytes, the extracellular matrix comprises a laminin fragment, and the laminin fragment is a laminin 511 E8 fragment and/or a laminin 521 E8 fragment.

According to the present invention, it is possible to provide an extracellular matrix containing laminin fragments that can obtain more mature cardiomyocytes than conventional methods from immature cardiomyocytes derived from pluripotent stem cells, etc.

1 is a photograph of cardiomyocytes according to an embodiment of the present invention. 1 is a photograph of cardiomyocytes according to an embodiment of the present invention. 1 is a heat map showing the results of clustering the expression of each extracellular matrix (ECM) at each stage according to an embodiment of the present invention. 1 is a graph showing the expression profile of each ECM according to an embodiment of the present invention. FIG. 1 is a schematic diagram of a test for evaluating the effect of each ECM on maturation according to an embodiment of the present invention. 1 is a graph showing the results of a test evaluating the effect of each ECM treatment on maturation according to an embodiment of the present invention. 1 is a graph summarizing the results of a test evaluating the effect of each ECM treatment on maturation according to an embodiment of the present invention. 1 shows photographs of morphological evaluation of cardiomyocytes by immunostaining after each ECM treatment according to an embodiment of the present invention. 1 is a graph showing the results of morphological evaluation of cardiomyocytes by immunostaining after each ECM treatment according to an embodiment of the present invention. 1 is a graph showing measurement of binucleation of cardiomyocytes by LN511/521 treatment according to an embodiment of the present invention. 1 is a photograph showing the formation of gap junctions by LN511/521 treatment according to an embodiment of the present invention. 1 is a graph of a representative trace of mitochondrial activity upon LN511/521 treatment according to an embodiment of the present invention. 1 is a graph showing the evaluation of mitochondrial activity by LN511/521 treatment according to an embodiment of the present invention. 1 is a graph showing representative traces of intracellular calcium transients following LN511/521 treatment according to an embodiment of the present invention. 1 is a graph showing an evaluation of intracellular calcium transient phenomenon caused by LN511/521 treatment according to an embodiment of the present invention. 1 shows photographs and graphs illustrating the results of a sarcomere shortening assay using LN511/521 treatment according to an embodiment of the present invention. 1 is a graph showing the overall results of transcriptome analysis by LN511/521 treatment according to an embodiment of the present invention. 1 is a graph showing the results of detailed analysis of transcriptome analysis by LN511/521 treatment according to an embodiment of the present invention. 1 is a graph showing maturity scores according to an embodiment of the present invention, the LN511/521 process.

<Embodiment>
FIG. 1 shows an example of cardiomyocytes (hereinafter referred to as "PSC-CMs") obtained by inducing differentiation from pluripotent stem cells. It is known that PSC-CMs become fetal (embryo) type to neonatal type, and do not become the same as cardiomyocytes of a fully mature heart. For example, FIG. 1(a) shows an example of PSC-CMs induced to differentiate from pluripotent stem cells such as mouse ES cells. FIG. 2(b) shows an example of actual mouse fetal cardiomyocytes. FIG. 2(a) shows PSC-CMs induced to differentiate from mouse ES cells on the 10th day of differentiation, FIG. 2(b) shows PSC-CMs on the 20th day of differentiation, and FIG. 2(c) shows PSC-CMs on the 30th day of differentiation. Thus, although PSC-CMs mature somewhat by culturing, they only reach the same level as cells isolated from newborn mice. In this state, there is a large morphological difference between them and adult mouse cells. Furthermore, the calcium transient, which shows the change in the amount of calcium in the cells, clearly shows that they are immature.
For this reason, a maturation method for further maturing PSC-CMs has been sought.

Here, the present inventors discovered that the extracellular matrix at each stage of development is expressed in a time-specific manner.
For this reason, we conducted repeated experiments to comprehensively investigate which extracellular matrices expressed specifically at this stage have a specific effect on the maturation of cardiomyocytes. In this study, we used a pluripotent stem cell line (reporter cell) in which a fluorescent protein was inserted into a gene locus whose expression increases as cardiomyocytes mature.
The present inventors then confirmed that laminin E8 fragment (E8 fragment) is effective, and further confirmed that laminin 511 cE8 fragment and/or laminin 521 E8 fragment increase the fluorescence intensity of reporter cells and mature their morphology, physiological activity, etc., thereby completing the present invention.

[Extracellular matrix]
Extracellular matrix (ECM) is an insoluble substance present outside cells in living organisms. The ECM serves as a physical scaffold for cells, and is also involved in biochemical and biomechanical signal cascades for tissue morphogenesis, differentiation, homeostasis, and the like. In animals, the ECM is composed of fibrous proteins, proteoglycans, glycosaminoglycans, and the like. Among these, fibrous proteins include collagen, elastin, fibronectin, and laminin. Among these, laminin is a giant protein that constitutes the basement membrane, which is the extracellular matrix.

The extracellular matrix of the present embodiment is a specific extracellular matrix for promoting the maturation of cardiomyocytes, and is characterized by containing laminin fragments.
Here, laminin is a very large protein composed of three chains (α, β, and γ chains). The inventors have found that using the entire laminin inhibits the maturation of cardiomyocytes and stably stops them at the stage of precursor cells. In fact, conventional recombinant laminin 521 is used as a laminin expressed in early human embryonic cells to maintain undifferentiated cells such as human pluripotent stem cells without causing them to differentiate (see, for example, URL = "https://www.veritastk.co.jp/sciencelibrary/pickup/laminin-521.html").

However, the present inventors have found through the above-mentioned comprehensive analysis of the extracellular matrix that, contrary to the conventional use of the entire laminin, the use of a fragment of laminin protein (laminin fragment) promotes the maturation of cardiomyocytes. In other words, the extracellular matrix of this embodiment is considered to function as a signaling molecule that promotes cell maturation and does not contain any other signaling molecule that suppresses cell maturation. In addition, the laminin fragment of this embodiment is considered to be useful for cardiac development because it provides a structural scaffolding necessary for cardiomyocyte maturation.
Here, as the laminin fragment of this embodiment, it is preferable to use the E8 fragment. The E8 fragment of laminin is a protein fragment (peptide chain) of the minimum constituent portion that binds to integrin on the cell surface. Among these, the use of the E8 fragment of laminin 511 and/or laminin 522 is particularly preferable for maturation of cardiomyocytes of vertebrates including humans and mice. It is preferable to use the E8 fragment of laminin 511 and/or laminin 522 having the sequence of each species. In addition, it may be chemically modified, may have a partially different peptide sequence, may be synthesized with other peptides, or may be chemically modified in other ways.

The animals in this embodiment are not particularly limited, and broadly include vertebrates and invertebrates that have a heart and laminin. Vertebrates include fish, amphibians, reptiles, birds, and mammals. Specifically, for example, mammals may be rodents such as mice, rats, ferrets, hamsters, guinea pigs, and rabbits, dogs, cats, sheep, pigs, cows, horses, or primates including rhesus monkeys, chimpanzees, orangutans, and humans. In addition to mammals, they also include fish, birds including poultry, and reptiles. In addition, invertebrates also broadly include animals with a heart, such as chordates, mollusks, annelids, and arthropods.

The cardiomyocytes of this embodiment may be PSC-CMs differentiated from pluripotent stem cells by a method using various mesodermal differentiation inducers. In addition, the cardiomyocytes of this embodiment may be induced cardiomyocytes (iCMs) induced by introducing a transcription factor into fibroblasts or blood cells of a patient or the like. Furthermore, the cardiomyocytes of this embodiment may be primary cell culture cardiomyocytes obtained from the heart of a patient or the like.
Furthermore, the cardiomyocytes of this embodiment may be cells that have been induced to differentiate into cardiomyocytes and selected in the form of colonies or the like using various markers or visual inspection. Furthermore, the cardiomyocytes of this embodiment may be a mixed cell population of various cells, tissues, organs, etc. (hereinafter referred to as "tissues, etc."). These cardiomyocytes may be mixed and contain cells in various states as indicated by the maturity score described below. That is, each cell may not be fully differentiated, may be immature, or may not be as mature as an adult cell. Furthermore, the cardiomyocytes of this embodiment may contain cells that have been induced to differentiate into reporter cells described below.
Furthermore, hereinafter, among these cardiomyocytes, mature cardiomyocytes will be simply referred to as "mature cardiomyocytes".

Here, the pluripotent stem cells of this embodiment include stem cells (stem cells) with pluripotency that can differentiate into various cells in organisms such as primates including humans, mammals other than primates, and other vertebrates. Here, the pluripotent stem cells of this embodiment are preferably passageable, maintain a state in which differentiation does not progress even when passaged, and have properties that the karyotype, etc., is unlikely to change, or the epigenetic phenotype is unlikely to change. In this regard, it is preferable that the pluripotent stem cells of this embodiment have sufficient proliferation ability in vitro or in vivo. Specific examples of such pluripotent stem cells of this embodiment include embryonic stem cells (hereinafter referred to as "ES cells"), induced pluripotent stem cells (hereinafter referred to as "iPS cells"), and other artificially generated or selected stem cells with pluripotency. These pluripotent stem cells of the present embodiment may be stem cells created by reprogramming somatic cells using various vectors such as retroviruses, adenoviruses, and plasmids containing specific genes, RNA, low molecular weight compounds, etc.
In addition, the pluripotent stem cells of this embodiment do not necessarily have to be cells with pluripotency close to totipotency, but it is also possible to use naive cells with higher pluripotency than normal. In addition, the pluripotent stem cells of this embodiment may be cells created from cells obtained from patients with a disease, cells that serve as models for other diseases, cells with reporter genes incorporated (reporter cells), cells that can be conditionally knocked out, other genetically modified cells, etc. This genetic modification includes the addition, modification, or deletion of genes in chromosomes, the addition of genes, etc. using various vectors or artificial chromosomes, changes in epigenetic control, the addition of artificial genetic materials such as PNA, and other genetic modifications.

[Method for producing mature cardiomyocytes, mature cardiomyocytes]
The method for producing mature cardiomyocytes of this embodiment is characterized in that immature cardiomyocytes are cultured in a medium containing the extracellular matrix of this embodiment to obtain mature cardiomyocytes (mature cardiomyocytes).
The mature cardiomyocytes of this embodiment are mature cardiomyocytes produced by the mature cardiomyocyte production method of this embodiment, and are characterized in that mature cardiomyocytes (mature cardiomyocytes) are obtained from immature cardiomyocytes using a mature cardiomyocyte marker whose expression increases with maturation of cardiomyocytes as an indicator.

Here, the mature cardiomyocyte markers of this embodiment are Acadvl, Acsl1, Acss1, Ankrd23, Aqp1, Atp1a2, Casq2, Cd36, Cep85l, Ckmt2, Clu, Cmya5, Coq10a, Cox7a1, Cox8b, Cryab, Dcn, Ech1, Ephx2, Fndc5, Gsn, Gstm1, Hfe2, Hrc, Hspb6, H spb8, Kcnip2, Klf9, Lpi1, Lrrc2, Lrtm1, Mb, Mgp, Myom2, Myoz2, Myzap, Nfix, Pdk2, Perm1, Pink1, Ptgds, Rgs5, Rpl3l, S100a1, Sgcg, Slc2a4, Sparcll, Tcap, Tmem182, Tuba4a, Txlnb, Xirp2, and Yipf7, or any combination thereof.
Of these, Myom2 is a gene encoding a protein that is expressed in adult cardiac muscle and stabilizes the three-dimensional arrangement of proteins including the M-band structure.

Specifically, the mature cardiomyocytes of this embodiment can be produced by creating pluripotent stem cells in which a reporter gene has been knocked into the gene locus of the above-mentioned mature cardiomyocyte marker, using this as a reporter cell, and maturing this reporter cell.
That is, it is possible to first prepare reporter cells as pluripotent stem cells with a reporter gene knocked in. Then, the reporter cells are induced to differentiate to produce PSC-CMs, and cells with an activated (expressed) reporter gene are selected and obtained by fluorescence activated cell sorting (hereinafter referred to as "FACS") using flow cytometry as an indicator, thereby making it possible to obtain purified mature cardiomyocytes.

More specifically, the reporter cells of this embodiment can be created by knocking in a gene for a fluorescent protein, such as red fluorescent protein (hereinafter referred to as "RFP") or green fluorescent protein (hereinafter referred to as "GFP"), into the gene locus of a mature cardiomyocyte marker.

Furthermore, when using mouse ES cells, the reporter cells of this embodiment may be capable of producing chimeric mice. This allows the reporter cells to be widely used in experiments on the developmental stages of the animal's heart. Specifically, the reporter cells of this embodiment can be used as a tool for screening external stimuli related to the maturation of cardiomyocytes, such as extracellular matrix (ECM) and hormones, and optimizing maturation, in order to further promote the maturation of cardiomyocytes. More specifically, the reporter cells of this embodiment can be used for modeling heart disease and for screening candidate therapeutic drugs (candidate drugs) and candidate drugs for treating heart disease in the drug discovery support method of this embodiment described below.

The mature cardiomyocytes of this embodiment can be purified by obtaining the mature cardiomyocytes emitting fluorescence by maturing the reporter cells described above using FACS. In this case, the maturation of Cd36 can be evaluated using a fluorescent dye-bound antibody corresponding to the surface antigen as an indicator. This surface antigen is the extracellular portion of the transcription product of Cd36.
In the following Example, an example is shown in which a reporter cell in which the RFP gene is knocked in as a reporter gene into Myom2 is used.
In addition to fluorescent proteins, it is also possible to prepare cells transfected with drug resistance genes, induce their differentiation to produce PSC-CMs, and purify cardiomyocytes that have acquired drug resistance through maturation. In other words, drug resistance genes can be used as "reporter" cells in a broad sense.

As described above, the mature cardiac cells of this embodiment may be PSC-CMs obtained by maturing reporter cells. Alternatively, the mature cardiac cells of this embodiment may be PSC-CMs in which a reporter gene is not knocked in. That is, the mature cardiac cells produced by the mature cardiac cell production method of this embodiment may be any cardiac cells obtained by inducing differentiation of pluripotent stem cells, including the reporter cells described above. That is, the mature cardiac cells of this embodiment may be reporter cells and PSC-CMs other than reporter cells. Furthermore, when inducing differentiation of pluripotent stem cells, it is also possible to use a cardiac muscle cell maturation agent at the same time. Alternatively, the cardiac muscle cell maturation agent may be used to induce differentiation of pluripotent stem cells. For example, suitable viral vectors that transfer into the nucleus, various plasmids, PNA, etc. may be used. This viral vector may be constructed using viruses that are common to those skilled in the art, such as lentivirus, adenovirus, adeno-associated virus, and retrovirus. In addition, the cardiomyocyte maturation agent can be introduced into cells or nuclei using an appropriate DDS (Drug Delivery System), electroporation, or other methods used by those skilled in the art.

PSC-CMs (mature cardiomyocytes) produced by the mature cardiomyocyte production method of this embodiment are capable of forming a fibrous sarcomere structure by aggregation. When a sarcomere structure is observed in a cell mass in which PSC-CMs matured by the maturation method of this embodiment are cultured, it is possible to determine that the cardiomyocytes constitute functional myocardium. Alternatively, it is also possible to obtain a portion of the cell mass of PSC-CMs matured by the maturation method of this embodiment and evaluate the maturity score by the maturity evaluation method of this embodiment.

Here, the mature cardiomyocytes produced by the mature cardiomyocyte production method of this embodiment are cardiomyocytes that are functionally and morphologically similar to adult cardiomyocytes present in the living body. That is, in the mature cardiomyocytes of this embodiment, intracellular calcium transients and sarcomere shortening are observed, and mitochondrial activity is high. Furthermore, the ion channel function is developed, and, for example, the resting membrane potential, peak voltage, amplitude, etc. become more similar to adult cardiomyocytes in the living body. Furthermore, as described below, when evaluated by the cardiomyocyte maturity evaluation method of this embodiment, the mouse mature cardiomyocytes of this embodiment have a high score. Therefore, it is possible to distinguish the product from cardiomyocytes induced to differentiate by conventional differentiation induction methods.

The actual maturity of the mature cardiomyocytes of this embodiment can be evaluated by quantitatively evaluating the maturity of other types of cardiomyocytes by referring to common genes in the transcriptome data. In other words, it is also possible to evaluate the maturity using a cardiomyocyte maturity evaluation method and obtain mature cardiomyocytes based on this result. This can be done, for example, by obtaining a portion of a living tissue or a colony of PSC-CMs, evaluating the maturity, and obtaining the remaining cardiomyocytes.

Here, in this embodiment, the transcriptome data of the heart of an animal in the developmental stage can be, for example, data obtained by analyzing large-scale RNA sequencing (hereinafter referred to as "RNA-seq") using a next-generation sequencer, an evaluation panel with multiple genes set (Targeted RNA-seq, or qPCR), a DNA chip, or other data obtained by measuring the expression of multiple genes. This data includes, for example, data on the expression level of each gene at each stage (stage) from fetus or embryo corresponding to the number of days since fertilization to newborn baby after birth to maturity. In the case of RNA-seq, the expression level is data on transcripts per million (hereinafter referred to as "TPM"), which is the amount of transcription product.

In this embodiment, for example, as shown in the examples described below, it is possible to evaluate the maturity from the RNA-seq result data 120 of PSC-CMs differentiated from human iPS cells by using genes common to mice and humans as references from transcriptome data obtained by RNA-seq of mouse hearts at each stage of development. In other words, it is possible to quantitatively evaluate the degree of maturity of human PSC-CMs in particular.

The mature cardiomyocytes produced by the method for producing mature cardiomyocytes of this embodiment can be used in the drug discovery support method and treatment method of this embodiment, as described below.
In addition, in the field of basic biological medicine, it is impossible to completely know the relationship between the expression of tens of thousands of genes and the material, chemical changes, and operations that are actually proceeding in living cells at a specific time. Therefore, it is very difficult for those skilled in the art to quantitatively measure the maturity of cardiomyocytes beyond the maturity score using gene expression levels (transcriptome) as in this embodiment, so there is a special circumstance that it is difficult to directly identify the mature cardiomyocytes of this embodiment by their structure or characteristics.

[Cardiomyocyte Maturation Kit]
The cardiomyocyte maturation kit of this embodiment includes an extracellular matrix for maturing immature cardiomyocytes, and the extracellular matrix is characterized by including laminin fragments.
The cardiomyocyte maturation kit of this embodiment includes various reagents necessary for the method for producing mature cardiac cells of this embodiment. These reagents include, for example, a culture medium, an extracellular matrix, a medium, a reagent for FACS, an antibody, a container, and the like. The extracellular matrix may include, for example, a peptide of laminin 511 E8 fragment and/or laminin 521 E8 fragment. Furthermore, the extracellular matrix may be provided as an expression vector or the like so as to be expressed in the cardiomyocytes themselves or in feeder cells, and the like. In addition, the above-mentioned reporter cells themselves, as well as special media and drugs containing cardiomyocyte maturation agents including various hormones, agonists, and transcription factors, and others, may also be added to the cardiomyocyte maturation kit of this embodiment. Furthermore, a special medium for storing and maintaining the reporter cells, drugs necessary for differentiation induction and maturation, and the like may also be included.

[Drug discovery support method]
The drug discovery support method of this embodiment is characterized in that mature cardiomyocytes matured by the above-mentioned mature cardiomyocyte manufacturing method are cultured, a toxic and/or disease-related drug for drug discovery is administered to the cultured mature cardiomyocytes, and the state of the mature cardiomyocytes is evaluated.

Drugs related to toxicity and/or disease for drug discovery in this embodiment can be candidate drugs for drug screening that require investigation of cardiotoxicity, candidate drugs for treating heart disease, etc. Candidate drugs in this embodiment can be, for example, low molecular weight compounds, peptides, proteins, cell extracts, supernatants, fermentation products, other synthetic compounds, natural compounds, etc. These candidate drugs may have any purity or degree of purification. In addition, diseases targeted by drug screening in this embodiment include any disease other than heart disease.

As the cardiotoxicity of this embodiment, drug-induced (acquired) long QT syndrome, which is a disease that causes serious arrhythmia in patients, may be evaluated by analyzing physiological characteristics described below. Drug-induced long QT syndrome is a serious disease in which the QT interval on the electrocardiogram is prolonged after administration of a drug, and TdP (torsades de pointes, nonsustained polymorphic ventricular tachycardia) often leads to ventricular fibrillation, resulting in fainting or sudden death.

The state of the mature cardiomyocytes of this embodiment can be evaluated by performing an analysis of physiological characteristics, an analysis of the expression of toxic marker genes, and the like. Among these, the analysis of physiological characteristics can be performed, for example, by treating the obtained PSC-CMs as a single cell or cell mass, and analyzing the electrophysiological characteristics by a patch clamp test, or measuring the field potential (extracellular action potential), etc. This extracellular action potential can be measured, for example, by placing a sample on a dish having a large number of electrodes. By measuring the extracellular action potential, it is possible to perform QT interval, beat (BPM) analysis, Na peak analysis, and the like. The QT interval is the time from the contraction of the ventricular muscle to the beginning of the diastole, and refers to the time from depolarization by the Na channel to repolarization by the K channel to the resting membrane potential. If QT prolongation, which is a lengthening of this QT interval, is observed, it may cause drug-induced arrhythmia due to inhibition of the K channel, so it is possible to perform an evaluation such that it is excluded from candidate drugs as being unsuitable in screening.

In addition, cardiotoxicity can be evaluated by analyzing the expression of various toxicity marker genes. Furthermore, screening may be performed according to a clinical trial protocol or by any method known to those skilled in the art.
In these analyses, if normal function is maintained in mature cardiomyocytes to which a candidate drug has been administered, it can be assumed that the candidate drug has little cardiotoxicity.

[Method of treating heart disease]
The method for treating a heart disease of this embodiment is characterized by culturing mature cardiomyocytes produced by the method for producing mature cardiomyocytes of this embodiment, and transplanting the cultured mature cardiomyocytes.

Specifically, in the treatment method of the present embodiment, the mature cardiomyocytes of the present embodiment can be used as cardiac regenerative medicine to treat cardiac diseases (heart disease, heart disease) in animals, including humans, including myocardial infarction, ischemic heart disease, myocarditis, and other heart failures.
For example, the mature cardiomyocytes of this embodiment are obtained from a library of pluripotent stem cells obtained and created or generated from the patient, or pluripotent stem cells with similar types such as HLA, induced to differentiate, and then cultured for a specific period of time with the addition of a cardiomyocyte maturation agent to mature. The matured cardiomyocytes can be injected into the heart of a patient suffering from a disease such as heart disease in the form of isolated cells or cell masses, and can be used for treatment such as transplantation by forming a myocardial sheet, myocardial tissue, or cardiac organ (hereinafter referred to as "myocardial sheet, etc."). This myocardial sheet, etc. may be prepared as a single-layer or multi-layer sheet using culture equipment used by those skilled in the art and transplanted into the heart of a patient with heart disease. Furthermore, the mature cardiomyocytes of this embodiment may be transplanted into the heart of a patient with heart disease in the state of cardiac muscle fibers or tissues having a sarcomere structure. Furthermore, it is also possible to culture the cells using an appropriate carrier or layer them using a 3D printer or the like to transplant a more organized culture into the heart of a patient with heart disease. The extracellular matrix of this embodiment can be included in these carriers, culture substrates, and/or culture media.
The mature cardiomyocytes of this embodiment can be used for various purposes other than regenerative medicine, such as the manufacture of bioreactors and artificial organs, and the creation of cloned individuals.
Furthermore, when the present invention is practiced in Japan, since treatment after provision of the culture is performed by a doctor, the "animal" in the treatment method of the present invention does not include humans (Homo sapiens). On the other hand, in other countries, the definitions of "animal" and "treatment method" are not limited.

When using mature cardiomyocytes according to an embodiment of the present invention for the above-mentioned treatment, the administration (introduction) interval and dosage can be appropriately selected and changed depending on various conditions such as the state of the disease and the condition of the subject.
The single dose and frequency of administration of mature cardiomyocytes according to an embodiment of the present invention can be appropriately selected and changed depending on the purpose of administration, as well as various conditions such as the patient's age and weight, symptoms, and severity of the disease.
The number of administrations and the duration of administration may be just once, or administration may be once to several times a day for several weeks, and the condition of the disease may be monitored, and administration may be repeated or repeated depending on the condition.

Additionally, the mature cardiomyocytes of the present invention can be used in combination with other compositions, drugs, etc. in addition to the extracellular matrix of this embodiment. The composition of the present invention may be administered simultaneously with the other compositions, or may be administered at intervals, with no particular restriction on the order of administration.
In addition, in an embodiment of the present invention, the period during which the disease is improved or alleviated is not particularly limited, but may be temporary improvement or alleviation, or improvement or alleviation for a certain period of time.

Furthermore, the extracellular matrix of this embodiment can be used as it is as a medicine for cardiomyocyte maturation. This medicine can be used, for example, for diseases caused by the presence of immature cardiomyocytes, for final maturation when a myocardial sheet or the like is transplanted, etc. Specifically, it is possible to add the extracellular matrix of this embodiment to various carriers such as gel-like substances at the time of transplantation.

The pharmaceutical for cardiomyocyte maturation according to the embodiment of the present invention may also contain any pharma- ceutical acceptable carrier. This carrier may be, for example, a liposome carrier, colloidal gold particles, a polypeptide, a lipopolysaccharide, a polysaccharide, a lipid membrane, etc. Among these, it is preferable to use a carrier that improves the expression regulating effect of the functional activity regulator.

Furthermore, the pharma- ceutically acceptable carrier may include, for example, physiological saline, an isotonic solution containing glucose or other auxiliary drugs, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, etc. Furthermore, it is possible to administer the drug together with a suitable solubilizing agent, such as alcohol, specifically ethanol, polyalcohol, such as propylene glycol, polyethylene glycol, non-ionic surfactant, such as polysorbate 80(TM), HCO-50, etc. Furthermore, it may further include a suitable excipient, etc.

The pharmaceutical for cardiomyocyte maturation of this embodiment may also contain a suitable pharma- ceutical acceptable carrier to prepare a pharma-ceutical acceptable carrier. The carrier may contain a biocompatible material such as silicone, collagen, gelatin, etc. The carrier may also be provided as an emulsion. Furthermore, it may contain any one or any combination of formulation additives such as diluents, flavorings, preservatives, excipients, disintegrants, lubricants, binders, emulsifiers, plasticizers, etc.

The administration route for cardiomyocyte maturation in this embodiment is not particularly limited, and administration can be parenteral or oral. Parenteral administration can be, for example, intravenous, intraarterial, subcutaneous, intradermal, intramuscular, or intraperitoneal administration, or direct administration to the kidney or the like.
The pharmaceutical agent for cardiomyocyte maturation according to an embodiment of the present invention can be formulated using pharma- ceutically acceptable carriers well known in the art in an administration form suitable for parenteral or oral administration.

With the above configuration, the following effects can be obtained.
Traditionally, primary cultures of cardiomyocytes obtained from patients have been difficult to maintain in a dish. For this reason, a protocol has been developed to induce cardiac differentiation of pluripotent stem cells by sequentially adding growth factors or small molecules. The pluripotent stem cell-derived cardiomyocytes (PSC-CMs) generated in this way are expected to be used for disease modeling and drug screening.

Here, conventional PSC-CMs are in an immature state with respect to their structure and function, similar to fetal or neonatal cardiomyocytes, and are different from typical in vivo mature cardiomyocytes after birth. Therefore, they are insufficient for purposes such as reproducing and analyzing the pathology of cardiac diseases such as cardiomyopathy that develop in adulthood, and evaluating the toxicity of drugs. This limitation has diminished the usefulness of PSC-CMs, particularly for in vitro models and medical purposes for drug discovery. Therefore, a method for maturing PSC-CMs to a level close to that of adult cells has been demanded.

In contrast, by maturing PSC-CMs using the laminin fragment of this embodiment, mature cardiomyocytes that were clearly more mature than those obtained by conventional techniques were obtained.
By applying the mature cardiomyocytes of this embodiment to iPS cells as a disease model, it may be possible to reproduce pathological conditions that could not be reproduced until now. Specifically, it becomes possible to select candidate drugs for idiopathic heart disease caused by ischemic heart disease such as myocardial infarction. In addition, it becomes possible to screen candidate drugs in response to a condition in which QT prolongation is asymptomatic in children but symptomatic in adults. This can contribute to the application of regenerative medicine to the treatment of cardiomyopathy.

Meanwhile, as described in paragraph [0057] of Patent Document 1, full-length laminin-511 and laminin-521 have been used to maintain pluripotent stem cells for a long period of time without the risk of spontaneous differentiation or genetic change, because they can serve as a scaffold to which pluripotent stem cells can adhere. Meanwhile, laminin-211 and laminin-221 are highly specific to cardiac and skeletal muscle fibers (cells), and as described in paragraph [0060] of Patent Document 1, pluripotent stem cells have been differentiated into PSC-CMs by culturing them on a substrate containing this laminin-221.
That is, there has been no prior knowledge that laminin-511 and laminin-521 are effective in maturing pluripotent stem cells into cardiomyocytes.
In contrast, the laminin 511 E8 fragment and/or laminin 521 E8 fragment of the present embodiment, contrary to conventional wisdom, serves as an extracellular matrix that is capable of maturing immature cardiomyocytes and producing mature cardiomyocytes by itself.

Furthermore, in this embodiment, reporter cells, which are pluripotent stem cells in which a fluorescent protein gene is knocked in as a reporter gene at the gene locus of a mature cardiomyocyte marker whose expression increases with maturation, are used. Then, by separating the mature cardiomyocyte (group) by FACS or the like using the reporter in the reporter cells or the Cd36 surface antigen as an indicator, highly pure mature cardiomyocytes can be obtained.
The mature cardiomyocytes produced from the reporter cells of this embodiment will enable the creation of pathological models, and may be useful for the application of regenerative medicine to the treatment of cardiomyopathy. For example, this may be useful for the development of a treatment for cardiomyopathy that develops in adults.

More specifically, in this embodiment, as shown in the examples, a pluripotent stem cell line in which RFP was knocked into the Myom2 locus was treated with laminin 511 E8 fragment and/or laminin 521 E8 fragment, and RFP intensity was measured to obtain mature cardiomyocytes. It is known that Myom2 is highly conserved among chickens, mice, and humans. For this reason, it can be more appropriately used as a reporter cell for the maturation of cardiomyocytes in pathological analysis, etc.

In addition to Myom2, the inventors have already created knock-in cells for Ckmt2, Hspb8, and Tcap, and have shown similar results in preliminary experiments. These also make it possible to create chimeric mice, and obtain the hearts of fetal and mature mice to obtain mature cells. It is also believed that similar results will be seen when other genes for mature cardiomyocyte markers are knocked in at each gene locus.

In addition, although the above embodiment describes an example in which FACS is used for cell sorting, cell sorting other than FACS can also be used.

The extracellular matrix, the method for producing mature cardiomyocytes, the drug discovery support method, the method for treating heart disease, the mature cardiomyocytes, and the cardiomyocyte maturation kit according to the embodiments of the present invention will be described in more detail below as examples based on specific experiments. However, this example is merely an example and is not intended to be limiting.

Materials and Methods
(Establishment of knock-in ES cell line (reporter cell))
Cardiomyocyte maturity reporter cells were established by performing genome editing on mouse ES cells, syNP4 cells. Since Myom2 is also expressed in skeletal muscle cells, it is possible to reliably purify cardiomyocytes in vitro by using cells of the syNP4 strain containing a puromycin resistance cassette driven by the sodium-calcium exchanger 1 promoter. Genome editing was also performed by cleaving the genome with CRISPR/Cas9 and inserting a knock-in vector into the target site. A guide RNA was designed to be directly above the stop codon of Myom2 at the guide RNA cloning position of px330, which expresses Cas9 protein and single guide RNA from a single vector, and inserted to create px330-Myom2. The knock-in vector has a homology arm of about 1000 bp from the gRNA target site, and TagRFP is inserted in the same frame as the Myom2 gene. In addition, downstream of TagRFP, there is a cassette called FRT-Blastcidin resistance gene-FRT (Myom2-TagRFP-Blast). Px330-Myom2 and Myom2-TagRFP-Blast were introduced into Synp4 by lipofection, and knock-in cells were obtained by performing blastcidin treatment. The knock-in cells were cultured from clonal density to obtain colonies derived from single cells. For each colony, it was confirmed by PCR and sequencing that the knock-in had been performed correctly, and it was also confirmed that the cardiomyocyte differentiation ability was maintained. The Myom2-RFP knock-in mouse ES cells obtained in this way were named SMM18. Furthermore, another Myom2-RFP strain was established by introducing the pCAG-Flpe-IRES-puro plasmid by lipofection and performing short-term puromycyn selection, which had lost the FRT-Blast-FRT cassette and was named SMM-B2. Loss of the cassette was confirmed by drug sensitivity testing and PCR. In this study, experiments were performed using SMM18 or SMM-B2 (hereinafter, these are simply referred to as "Myom2-RFP strains").

(Culture of mouse ES cells, differentiation induction, and maturation treatment with ECM)
Transgenic mouse ES cells (syNP4) expressing a Puromycin resistance gene under the control of an Ncx promoter were cultured in the presence of LIF, CHIR00021, PD0325901, and serum to maintain the undifferentiated state, and were passaged three times a week.
For differentiation induction, embryoid bodies were formed in suspension culture in the presence of B27 supplement (without Vitamin A) and N2 supplement instead of serum. From the second day of differentiation, Activin A, BMP4, and VEGF were added to continue differentiation. On the fourth day of differentiation, embryoid bodies were isolated by Tryple, and high-density flat culture (about 250k cells/ ^cm2 ) was performed in the presence of bFGF, FGF10, and VEGF. From about the seventh day of differentiation, pulsating cardiomyocytes were observed. By adding Puromycin, cells that did not differentiate into cardiomyocytes were killed, and highly pure cardiomyocytes were obtained. This method was also similar to the knock-in cells described below.
On the 10th day of differentiation, cells were isolated using Tryple and reseeded at 50k/ ^cm2 . When seeding, a culture plate coated with 0.1% gelatin was used, and 10% serum was added to the above differentiation medium (until the 11th day of differentiation). Viral infection was performed from the 11th day to the 12th day of differentiation. Purification of cardiomyocytes with Puromycin was continued from the 12th day to the 14th day of differentiation. For agonist treatment, Puromycin treatment was performed from the 11th day to the 14th day of differentiation, and then agonist treatment was performed from the 14th day of differentiation for up to 4 weeks.
Maturation treatment with ECM, in which culture was performed with ECM, was performed at concentrations of 0.125, 0.5, and 1 μg/cm ² for culture periods of 1, 2, and 4 weeks, respectively.

(RNA sample preparation and RNA-sequence (RNA-seq))
Mouse hearts were removed from embryonic day 11 to 10 months of age, separated into atria and ventricles, and dissolved using TRIzol. PSC-CMs were seeded on 0.1% gelatin on the 10th day of differentiation, agonist treatment was performed from the 14th day of differentiation, and dissolved in TRIzol two weeks later. RNA was extracted from mouse hearts by phenol-chloroform extraction or Direct-zol RNA kit (Zymo Research), and from PSC-CMs by Direct-zol RNA kit. RNA was quantified using Nanodrop (Thermo Fisher Scientific) or Qubit Fluorometer (Invitrogen). It was also confirmed that RNA was not decomposed using a Bioanalyzer. 500ng of RNA was prepared at 100ng/uL, and a library for RNA-seq was created using QUANT-seq 3'mRNA-Seq Library Prep Kit (Lexogen). The resulting library size was confirmed using Bioanalyzer (Agilent Technologies), and the concentration was quantified using Bioanalyzer results or Qubit Fluorometer. The samples were mixed in equal amounts, and massively parallel sequencing was performed using Next-seq high output 75SE (Illumina). 48 samples of heart samples and 96 samples of PSC-CMs were mixed. Additional heart samples were sequenced at 10 days, 30 weeks, and 10 months of age, with 96 samples each.

The Fastq files for each sample obtained by RNA-seq were trimmed using BBDuk from BBtools to remove poly A sequences, sequences at the sequence ends, adapter sequences, sequences with low sequence quality, etc. Then, mapping was performed to the mouse genome (GRC mm10) using STAR RNA-seq aligner. Finally, count data for each gene was obtained using feature counts included in the Subread package. The count data was further analyzed using a proprietary cardiomyocyte maturity evaluation program and the statistical analysis program "R". First, samples with counts of 1 million or less were excluded, and the transcript per million (TPM) was calculated for each sample and each gene by dividing the count for each gene by the total count. Using all experimental data (mice) that had been tested as of the end of August 2019, genes with an average of 1 TPM or more were defined as expressed genes, and the following analysis was performed using only expressed genes.

(Quantitative maturation assessment method (mouse))
Principal component analysis was performed on the data obtained by performing RNA-seq on mouse heart samples from each stage (Stages) from fetal (E) 11 to postnatal (P) 56 up to 10 months, and the eigenvector (coefficient) of each gene, which serves as the standard (hereinafter referred to as "reference") for calculating the maturation score, was calculated. In this case, the number obtained by adding 1 to TPM was converted to a logarithm with a base of 10, and then principal component analysis was performed. The maturity score was obtained by using the sum of the expression of each gene multiplied by the coefficient corresponding to the first principal component, adding a certain offset value (32), and multiplying by a coefficient (1.5). This calculation resulted in a maturity score ranging from approximately 0 to 100.

(Quantitative Maturity Assessment Method (Human))
Using biomaRt (a package that uses BioMart from R), we obtained a list of genes that correspond between humans and mice. In this case, two datasets, hsapiens_gene_ensembl and mmusculus_gene_ensembl, were used. In addition to those with the same gene name, genes with a single gene registered as an orthologue were set as the correspondence list.
Quantitative maturity evaluation of human cardiomyocytes was performed by projecting the RNA-seq data of these genes onto mouse data. RNA-seq was performed on samples 18, 32, and 46 days after differentiation induction using human fetal heart RNA samples, human adult heart RNA samples, and human PSC-CMs that had been induced to differentiate in house, and the results were projected onto mouse data. In addition, human PSC-CMs were also treated with Hydrocortisone (HC) and the thyroid hormone T3 as hormone treatments to promote maturation.
Specifically, using only the genes included in the correspondence list, a principal component analysis was performed on the RNA-seq data for each sample described above, and the eigenvector (coefficient) of each gene was calculated as a basis for calculating the maturity score. Similarly, an offset value (30) and a coefficient (1.5) were calculated for the first principal component score so that the range would be approximately 0 to 100.

When RNA-seq was performed using human fetal heart RNA samples and human adult heart RNA samples and projected onto mouse data, the fetuses obtained maturity scores equivalent to fetuses and the adults obtained maturity scores equivalent to adults. Furthermore, the maturity scores of samples taken 18, 32, and 46 days after induction of human PSC-CMs differentiation increased with the culture period.

(Immunostaining)
For hearts, tissues were embedded and directly frozen in optimal cutting temperature (OCT) compound (Tissue-Tek, Sakura) and cut on a Leica Cryostat (4 μm). Sections were washed with PBS and fixed in 4% paraformaldehyde (PFA, Wako) for 30 min at 4°C. Sections were then washed and cleared in 0.1% Triton X-100 (Amersham Biosciences) in PBS for 15 min at room temperature. After clearing, the cells were blocked with 3% bovine serum albumin, and then reacted with anti-MyoM2 polyclonal antibody (LS-B9842, 1:100, LifeSpan Biosciences) and anti-α-actinin monoclonal antibody (EA-53, 1:100, Sigma-Aldrich) at 4°C overnight. The cells were then washed with PBS containing 0.2% Tween-20 (Nacalai Tesque), and stained with secondary antibodies, anti-rabbit IgG (1:500, Thermo Fisher Scientific), and anti-mouse IgG (1:500, Thermo Fisher Scientific), and polymerized with Alexa Fluor Plus 555 and 488, respectively. DAPI (Dojindo Laboratories) was used for nuclear staining. Slides were mounted in VECTASHIELD antifade mounting medium (Vector Laboratories).

For PSC-CMs, PSC-CMs cultured in CellCarrier 96-well black polystyrene microplates (PerkinElmer) were fixed overnight with 4% paraformaldehyde. Cells were then washed with PBS and permeabilized for 15 min at room temperature using 0.2% Triton X-10 in PBS. After permeabilization, cells were blocked with 2% FBS in PBS and then treated overnight with anti-α-actinin antibody (1:500, Sigma-Aldrich) or anti-cardiac troponin T antibody (13-11, 1:500, Thermo Fisher Scientific), and anti-tRFP antibody (AAB233, 1:500, Evrogen). Cells were washed and stained with secondary antibodies, anti-mouse IgG (1:500, Thermo Fisher Scientific) and anti-rabbit IgG (1:500, Thermo Fisher Scientific), polymerized with Alexa Fluor 488 and 555, respectively. Nuclei were stained with DAPI solution. Immunofluorescence images were collected by confocal laser scanning microscope (Olympus, FluoView FV1200) or inverted fluorescence microscope (Olympus, IX83). Sarcomere length, cell size, circularity, circumference, and cell shape were analyzed by ImageJ software.

(Calcium Transients and Sarcomere Shortening)
To measure intracellular calcium transients, PSC-CMs were cultured in glass-bottom 24-well plates (MatTek Corporation) for 28 days. Cells were then washed with PBS and treated with Tyrode's solution (140 mM NaCl, 5.4 mM KCl, 0.5 mM MgCl2, 0.33 mM _NaH2PO4 , _{2 mM} CaCl2, 5 mM HEPES _, and 11 mM D-glucose, pH adjusted to 7.4 with NaOH) containing calcium indicator Calbryte 520-AM (AAT Bioquest) for 30 min. Cells were stimulated with a 1 Hz electric field (C-Pace, IonOptics). Intracellular calcium transients were recorded with an inverted fluorescence microscope (Olympus IX83, equipped with ORCA-Flash 4.0 V3) using a 40x objective lens, 10 ms exposure, and 20 ms intervals. Intracellular calcium transients were quantified using ImageJ. For sarcomere shortening, cell contraction was continuously recorded in time-lapse recordings for live cell imaging. Time-lapse recordings were analyzed by SarcOptiM on ImageJ.

Mitochondrial Activity Assay
Mitochondrial function was analyzed using a Seahorse XF96 extracellular flux analyzer. Seahorse XF96 microplates were coated with 0.1% gelatin (control) or each ECM. On day 10 after differentiation induction, PSC-CMs were seeded onto the plates at a density of 50,000 cells/well. Cells were cultured for up to day 38 before starting the assay. One hour before performing the assay and during the measurements, the culture medium was replaced with base medium (Seahorse XF RPMI medium supplemented with 1 mM pyruvate, 2 mM glutamine, and 25 mM glucose). Selective inhibitors were sequentially injected during the measurement, including 3 μM oligomycin (an inhibitor for complex V [ATPase] of the mitochondrial electron transport chain [METC]), 0.5 μM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, a mitochondrial oxidative phosphorylation uncoupler), 3 μM rotenone (an inhibitor of complex I of METC), and antimycin A (an inhibitor of complex III of METC). Basal respiration, a major index of mitochondrial respiration, was expressed as oxygen consumption rate (OCR) before applying oligomycin. ATP-linked respiration was expressed as the oligomycin-sensitive respiration rate. Proton leak was calculated by the difference between the ratio of oligomycin and rotenone/antimycin A. Maximal respiration was calculated relative to FCCP.

(Flow Cytometry)
PSC-CMs generated from the Myom2-RFP line were treated with TrypLE (Thermo Fisher Scientific) for 10 min at 37°C to dissociate into single cells. After washing with PBS, the cells were suspended in 2% FBS in PBS containing DAPI (1:2000). Measurement of the percentage of RFP positive (hereafter referred to as "RFP+") and RFP intensity and cell sorting were performed using SH800 (Sony).

(Statistical Analysis)
Data were presented as the mean ± standard deviation (SD) of at least three replicate samples. For experiments with fewer samples, such as transcriptomic data, all data points were shown. Student's t-test, Dunnett's test, chi-square test, or one-way ANOVA were used where appropriate. All statistical analyses were performed using the statistical program "R". A p-value of less than 0.05 (p<0.05) was considered significant.

〔result〕
To test whether the Myom2-RFP line could be used to screen for cardiomyocyte maturation, we investigated the effect of extracellular matrix (ECM) on the maturation of PSC-CMs. To identify candidate ECMs that promote cardiomyocyte maturation, we performed RNA-seq of wild-type mouse ventricles at stages E11 to P56.

Figure 3 shows the results of hierarchical clustering of the expression of each ECM when RNA-seq was performed. The concentration was calculated by dividing the normalized transcript (TPM) by the highest expression level, and low or high expression levels were indicated by darker colors.
This hierarchical clustering showed groupings of gene expression patterns in early embryonic, neonatal, and adult ventricles, from which representatives were selected.

Figure 4 shows the expression profile of ECM including laminin, collagen, and fibronectin in the developing heart (E11-P56). Fibronectin (Fn1), and collagen types II (Col2a1) and IV (Col4a6) were abundantly expressed in fetal hearts. Collagen types I (Col1a1 and Col1a2) and III (Col3a1) were also highly upregulated in neonatal hearts. On the other hand, laminin (α2, α5, and β2) and one member of collagen type IV (Col4a4) were expressed in mature mouse hearts. This led to the prediction that ECM expression is timed to coincide with the development and maturation of cardiomyocytes.

Next, an evaluation test of the effect of ECM on cardiomyocyte maturation will be described with reference to Figure 5. Specifically, 10 days after differentiation induction, various concentrations (range of 0.125 to 1 μg/ ^cm2 ) of laminin E8 fragments (isoforms 111, 121, 211, 221, 311, 321, 332, 411, 421, 511, and 521), collagen (types I, III, and IV), fibronectin, and 0.1% gelatin as a control were plated as each ECM. Thereafter, in order to quantitatively measure the RFP intensity of RFP+ PSC-CMs, the PSC-CMs were cultured for 7 days (1 week, 1w), 14 days (2 weeks, 2w), and 28 days (4 weeks, 4w), i.e., for a total of 17 to 38 days after differentiation induction (hereinafter, the addition of ECM to PSC-CMs after differentiation induction and culturing for a predetermined period of time is referred to as "ECM treatment". The same applies to "laminin 511/521 treatment" described below.). Then, flow cytometry was performed.

Figure 6 shows the results of the test evaluating the effect of each ECM. Specifically, for each ECM treatment, the proportion of RFP+ PSC-CMs and RFP intensity were measured 17 days (d17, 1w), 24 days (d24, 2w), and 38 days (d38, 4w) after differentiation induction. Data are shown as mean ± SD (n = 3), and RFP fluorescence intensity (RFP intensity) is shown as arbitrary units (a.u.).
As a result, it was found that both the proportion of RFP+ PSC-CMs and RFP intensity increased from days 17 to 38. In some ECM PSC-CMs, an increase in RFP intensity was observed in a dose-dependent manner.

Figure 7 is a graph summarizing the results of 1 μg/ ^cm2 ECM at day 38 of differentiation in Figure 6 above. Each ECM fraction was compared to the control (gelatin) using a Dunnett's test. "*" indicates P<0.05, "§" indicates P<0.01, "#" indicates P<0.001, and "†" indicates P<0.0001.
Among these ECMs, the proportion of RFP+ PSC-CMs remained the same; that is, it was considered that there was no effect of inducing maturation. In contrast, RFP intensity increased in several ECMs. In particular, laminin 511 E8 fragment and laminin 522 E8 fragment (hereinafter abbreviated as "laminin 511/521") showed the highest RFP intensity compared to the others. This result suggests that ECMs, especially laminin 511/521, promoted the maturation of cardiomyocytes in vitro, rather than inducing maturation.

Figure 8 shows the structure and morphology of the ECM on PSC-CMs to examine whether laminin-511 promotes cardiomyocyte maturation. Figure 8(a) shows staining with TagRFP, Figure 8(b) shows staining with α-actinin antibody, and Figure 8(c) shows staining with DAPI for nuclei. Scale bar is 20 μm.
Treatment with each ECM notably increased cell size and sarcomere length.

Figure 9 shows the results of statistical calculations of the measurements of structural and morphological features. The measurements are represented by violin plots: (a) sarcomere length (μm), (b) cell length (μm), (c) cell area ( ^μm2 ), and (d) cell width (μm). In the violin plots, the black line in the white box indicates the median, the box limits indicate the 25th and 75th percentiles, the whiskers indicate 1.5 times the interquartile range of the 25th and 75th percentiles, and the polygon indicates the density estimate of the data, drawn to the extremes.

As a result, ECM-treated cardiomyocytes showed significant increases in sarcomere length, cell area, cell length, and cell width compared to the control (gelatin), especially with treatment with laminin (LN) 511/521.
Each ECM treatment was also compared to the control (gelatin) using Dunnett's test. "*" indicates P<0.05, "§" indicates P<0.01, "#" indicates P<0.001, and "†" indicates P<0.0001. PSC-CMs cultured with laminin-511/521 showed a significant increase in cell size, cell length, and cell width. Furthermore, sarcomere length of PSC-CMs was longer in laminin-511/521 compared to the control.
These results demonstrated that laminin-511/521 potently promotes the structural maturation of cardiomyocytes.

Next, to further analyze whether laminin-511/521 enhanced the functional and transcriptional maturation of PSC-CMs, we first examined binucleation of the cells.

Figure 10 shows the percentage of mononuclear and binuclear cells in PSC-CMs cultured with laminin 511/521 compared to gelatin (n>100). The lighter shades indicate the percentage of mononuclear cells, and the darker shades indicate the percentage of binuclear cells. "†" indicates significance at P<0.0001.
As a result, laminin-511/521-treated PSC-CMs had a higher population of binucleated cells than control gelatin (7.77%), specifically, 38.68% for laminin-511 E8 fragment and 54.72% for laminin-521 E8 short side.

Next, we examined whether laminin 511/521 treatment results in the formation of gap junctions by measuring the localization of connexin-43 (Cx43), a gap junction protein that reflects cardiomyocyte maturation.
Here, in embryonic cardiomyocytes, Cx43 is diffusely expressed in the cytoplasm. Then, in neonatal and juvenile stages, it is localized to the lateral surface of cardiomyocytes. When cardiomyocytes are fully mature, Cx43 is mainly localized to the intercalation disks of cardiomyocytes. We observed whether such morphological changes were observed.

Figure 11 shows representative photographs of PSC-CMs stained for Cx43 at day 38 after differentiation. Scale bar is 20 μm.
Control PSC-CMs on gelatin expressed weak Cx43, whereas laminin 511/521-treated PSC-CMs expressed strong Cx43 on the lateral surface of the cells, indicating that gap junctions (Cx43) were formed upon incubation with laminin 511/521.
These results suggested that laminin 511/521 treatment caused PSC-CMs to become more mature, at least reaching the neonatal or juvenile stage.

Next, we measured the mitochondrial activity of PSC-CMs using oxygen consumption rate (OCR). After birth, cardiomyocytes oxidize carbohydrates to fatty acids to produce energy.

FIG. 12 shows representative traces corresponding to oligomycin, FCCP, and rotenone/antimycin A for laminin 511/521 treated PSC-CMs and gelatin.
Statistical analysis of OCR for basal respiration (a), ATP production-related respiration (b), proton leak (c), and maximum respiration (d) are shown in Fig. 13(a), 13(b), 13(c), and 14(d), respectively. Data are shown as mean ± SD (n = 3). Dunnett's test was performed to compare PSC-CMs on laminin and gelatin as controls. * indicates P < 0.05, § indicates P < 0.01, # indicates P < 0.001, and † indicates P < 0.0001.
As a result, laminin 511/521-treated PSC-CMs had higher basal and maximal respiration levels, suggesting higher mitochondrial activity than PSC-CMs on control gelatin.

Next, to further investigate physiological changes, we examined calcium transients in laminin 511/521-treated PSC-CMs.

Figure 14 shows representative traces of calcium transients stimulated at 1 Hz on day 38. PSC-CMs cultured in laminin-511/521 were compared to gelatin control.
Fig. 15(a) shows the average time to peak of calcium transients, Fig. 15(b) shows the peak amplitude, and Fig. 15(c) shows the decay time (n>80). In Dunnett's test, "*" indicates P<0.05, and "†" indicates P<0.0001.
As a result, laminin-521-treated PSC-CMs exhibited a significantly reduced time to peak, a significantly reduced time to decay, and a higher peak amplitude.

Next, we investigated sarcomere shortening in PSC-CMs treated with laminin 511/521.

Figure 16(a) shows a representative trace of sarcomere length during electrical pulse (1 Hz) stimulation. Figure 16(b) shows average data of sarcomere shortening. Data are shown as mean ± SD (n>40). "*" indicates P<0.05 and "§" indicates P<0.01 by Dunnett's test.
As a result, it was found that laminin 511/521 treatment significantly increased the rate of sarcomere shortening.
These results demonstrated that laminin 511/521 treatment improved the physiological properties of PSC-CM.

Next, we performed transcriptome analysis of laminin 511/521-treated PSC-CMs.

Figure 17 shows the total expression of genes of eight GO terms selected for biological processes as a transcriptome analysis. Compared to the observed morphological or physiological changes, the expression of certain GO terms did not change significantly.
Detailed transcriptome analysis is shown in Figure 18. In contrast to the total expression shown above, genes known to be associated with cardiomyocyte development were upregulated in laminin 511/521-treated PSC-CMs compared to gelatin, including cardiac marker genes (Tnnt2 and Actc1), a transcription factor gene involved in cardiac gene expression (Ankrd23), a calcium-related gene (Casq2), and sarcomere genes (Mybpc2, Mybpc3, Myh7, and Myl2).
These results suggest that sarcomere genes and maturation-related genes tended to be upregulated in laminin 511/521-treated PSC-CMs, suggesting that the effect of ECM was through changes in translation rather than transcription.

Next, we performed transcriptome-based cardiomyocyte maturity assessment on laminin 511/521-treated PSC-CMs to quantitatively measure their degree of maturity.

19 is a graph showing the results of maturation score analysis. PSC-CMs treated with laminin 511/521 for 2 weeks (2w) and 4 weeks (4w) starting 10 days after differentiation induction of the Myom2-RFP line and control PSC-CMs treated with gelatin only were obtained by cell sorting using flow cytometry, and the maturation score was measured.
As a result, laminin 511/521-treated PSC-CMs were more mature even at 2 w than those cultured on gelatin for 4 w.

In summary, RFP+ PSC-CMs differentiated from the Myom2-RFP line showed sarcomere shortening upon treatment with laminin 511/521, and were more morphologically, structurally, and functionally mature than RFP- PSC-CMs. Therefore, by obtaining PSC-CMs treated with laminin 511/521, it is possible to obtain mature cardiomyocytes.

Although not shown in the results above, in preliminary experiments, human PSC-CMs were also treated with laminin 511/521 and their maturity scores were evaluated. This confirmed that the maturity was actually increased compared to controls such as gelatin.

It goes without saying that the configurations and operations of the above-described embodiments are merely examples and can be modified as appropriate without departing from the spirit and scope of the present invention.

According to the present invention, the method for producing mature cardiomyocytes can provide mature cardiomyocytes that can be used in transplant medicine, drug discovery support, etc., and can be used industrially.

Claims (3)

An extracellular matrix for maturing immature cardiomyocytes, comprising:
Contains laminin fragments,
The laminin fragment is a laminin 511 E8 fragment and/or a laminin 521 E8 fragment. Cultivating immature cardiomyocytes in a medium containing the extracellular matrix of claim 1;
A method for producing mature cardiomyocytes, comprising obtaining mature cardiomyocytes. containing an extracellular matrix for maturing immature cardiomyocytes;
the extracellular matrix comprises laminin fragments;
The cardiomyocyte maturation kit, wherein the laminin fragment is a laminin 511 E8 fragment and/or a laminin 521 E8 fragment.

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