TW201307556A - Establishment of patient- or person- specific cardiac myocyte cell lines from human induced pluripotent stem cells (iPSCs) - Google Patents

Abstract

A composition and method for generating patient-or person-specific proliferative and substantially pure cardiac myocyte cell lines from pluripotent stem cells (iPSCs) is described herein. The patient-specific cardiac myocyte cell lines of the present invention find applications in research, drug screening and autologous cell-based therapy. The method of the present invention is simple and reproducible and generates cardiac myocyte cells having high purities and proliferating capacities.

Description

Establishment of a patient- or individual-specific cardiomyocyte cell line from human induced pluripotent stem cells (iPSCs)

SUMMARY OF THE INVENTION The present invention relates to techniques for differentiating and proliferating pluripotent stem cells, and more particularly to novel techniques for producing patient-specific cardiomyocyte strains from pluripotent stem cells derived from human fibroblasts.

Cross-references to related applications

The present application claims priority to U.S. Provisional Application Serial No. 61/504,637, filed on Jan. 5, 2011, the entire disclosure of which is hereby incorporated by reference.

Statement of federally funded research

no

Reference sequence table

no

Without limiting the scope of the invention, in combination with in vitro culture of differentiated cells and other related methods to produce cardiomyocyte strains and tissues for research, drug screening, and autologous cell-based therapies, the present invention is illustrated. background.

One such method is taught in PCT Patent Application No. PCT/US2011/056329, filed by Singla et al., which is incorporated herein by reference. Briefly, the present application is said to disclose iPS cells derived from myocardium or ventricular-specific cell types that have the potential to repair and regenerate infarcted myocardium. These cells are transduced with four stem cell factors and reprogrammed into iPS cells. These myocardial iPS cells can It is enough to differentiate into beating cardiomyocytes, forming a myocardial specific structure, and the myocardial specific protein staining is positive. Transplanted cells also significantly inhibited apoptosis and fibrosis and improved myocardial function.

Another application is U.S. Patent Application Serial No. 2011/097799, filed by Stankewicz et al. Briefly, it discloses methods and compositions for making cardiomyocytes by differentiation of pluripotent stem cells. For example, methods are set forth in certain aspects, including differentiation of pluripotent stem cells in the presence of a Rho-related (ROCK) inhibitor in a large suspension culture. Furthermore, it is said that the method of differentiating stem cells into cardiomyocytes is provided to overcome the differences between different stem cell lines and different batches of medium.

Another such technique is disclosed in U.S. Patent Application Publication No. 2005/037,489 (Gepstein et al. The Gepstein invention comprises the steps of: (a) partially dispersing a population of confluent cultured human stem cells, thereby producing a population of cells comprising cell aggregates; (b) subjecting the cell aggregates to a culture suitable for producing embryoid bodies Conditions; (c) subjecting the embryoid bodies to culture conditions suitable for inducing differentiation of the myocardial lineage in at least a portion of the cells of the embryoid bodies, the culture conditions suitable for inducing differentiation of the myocardial lineage comprising including the embryos The body is adhered to a surface and cultured in a medium supplemented with serum, thereby producing cells that exhibit at least one characteristic associated with the myocardial phenotype.

U.S. Patent Application Publication No. 2005/0054092 (Xu and Gold, 2005) provides a method of obtaining a population of human cardiomyocyte lineage cells. Allegedly Equal cell lines are obtained by differentiating pluripotent stem cell cultures in vitro and then harvesting cells with certain phenotypic characteristics. Differentiated cells have cell surface and morphological markers characteristic of cardiomyocytes, and some of them undergo spontaneous cyclic contraction. Highly enriched cardiomyocyte populations and their replication precursors are available, and are suitable for a variety of applications, such as drug screening and therapy for myocardial diseases.

A procedure for the production of a cardiomyocyte lineage cell from an embryonic stem cell for regenerative medicine is described in U.S. Patent No. 7,452,718 (2008) to the entire disclosure of the entire disclosure of The '718 patent describes methods that do not require the formation of embryoid bodies or differentiation in serum. Instead, the stem cells are placed on a solid substrate and differentiated in the presence of selection factors and morphogens. After enrichment of cells with the appropriate phenotype, the cells are clustered in cardiac bodies ^(TM ), which are very uniform and suitable for treating heart disease.

U.S. Patent Application Publication No. 2009/0017465 (Xu, 2009) provides a population of human cardiomyocyte lineage cells. Such cell lines are obtained by differentiating pluripotent stem cell cultures in vitro and then harvesting cells having certain phenotypic characteristics. Differentiated cells have cell surface and morphological markers characteristic of cardiomyocytes, and some of them undergo spontaneous cyclic contraction. High enrichment cardiomyocyte populations and their replication precursors are available, which are suitable for a variety of applications, such as drug screening and therapy for myocardial diseases.

U.S. Patent No. 7,611,852 (2009) to Thomson et al. discloses that human embryonic stem cells form embryoid bodies containing differentiated human cells in culture. Some human cells in the embryoid body differentiate into cardiomyocytes. Here, reference is made to the use of human embryonic stem cell-derived cardiomyocytes in a drug screening protocol for the mechanism of myocardial toxicity. Biological and electrical properties of cells.

The present invention includes compositions and methods for producing a patient-specific cardiomyocyte strain from, for example, pluripotent stem cells derived from human fibroblasts, which are intended for use as a cardiovascular disease research and cell-based therapy Proliferative high purity source. The present invention allows the production of a patient-specific cardiomyocyte cell line which is proliferative and thus provides a large number of cells and a high purity population. This makes it highly desirable for multiple studies and individual cell-based therapies for individual patients.

In one embodiment, the invention provides a method of producing a cell line or cell composition from one or more induced pluripotent stem cells (iPSCs) comprising the steps of: (a) obtaining the one or more iPSCs; (b Inducing differentiation of the one or more iPSCs; (c) separating one or more cells or clusters of cells exhibiting spontaneous pulsation; (d) performing the first incubation of the differentiated cells or the cluster of cells for a specified period of time; (e) separating one or more cells or cell clusters that exhibit spontaneous pulsation from the first culture; (f) performing a second incubation on the differentiated cells or the cluster of cells from the first culture for a specified time Segment (g) removing one or more cells or clusters of cells exhibiting spontaneous pulsation from the second culture, leaving one or more differentiated cells in the second culture; (h) harvesting remains in the second The one or more differentiated cells in the culture; and (i) amplifying the harvested differentiated cells from the second culture to obtain the cell strain or the cell composition.

In another aspect of the invention, the method comprises the step of repeating one or more of steps (c) through (h). In one feature of the invention, the step of obtaining the one or more iPSCs comprises the steps of: (i) providing one or more Primary culture of non-multifunctional cells obtained from a mammalian individual and (ii) transfection of one or more transcription factors or stem cell-associated genes into one or more non-multifunctional cells, wherein the transfection results in a non-multiple Functional cells form iPSCs. In another aspect, the non-multifunctional cells comprise fibroblasts, blast cells, corneal cells, amniocytes, gastric cells, neural stem cells, or any combination thereof. In yet another aspect, the one or more transcription factors are selected from the group consisting of Sox2, Oct3/4, Klf4, c-Myc, Lin28, Nanog, or any combination thereof. In one aspect, a mammalian system of a human individual, wherein the system is at least one of a healthy individual, an individual having one or more of the following diseases: metabolic disorders, cardiovascular disease, cardiac dysfunction, myocardial infarction, myocardial deficiency Blood, myocardial reperfusion, subendocardial ischemia, high-angstrom arteritis, atrial fibrillation, hemorrhagic stroke, ischemia/reperfusion, reperfusion injury, congenital heart disease, or cardiac surgery.

In a particular aspect associated with the method, the metabolic disorder is Fabry's disease and the cell line comprises a cardiomyocyte strain. In another aspect, the cell strain or cell composition comprises cardiomyocytes. In yet another aspect, the step of inducing differentiation of one or more iPSCs comprises forming one or more embryoid bodies, or by exposing iPSCs to factors such as BMP4 or activin A. In one aspect, one or more cells or clusters of cells exhibiting spontaneous pulsation are separated by one or more mechanical methods. In another aspect, the isolated differentiated cells or clusters of cells comprise one or more beating cells. In yet another aspect, harvesting of one or more differentiated cells is performed by trypsinization or by mechanical separation using a cell scraper followed by repeated pipetting. In a particular feature, the cell strain or cell composition obtained by the method described above is pure Degree >90%. Finally, the cell lines or cell compositions obtained by the methods of the invention are suitable for cell/tissue transplantation, pharmaceutically active and toxic screening studies, in vitro disease mechanism models, cell-based therapies, optimized treatment regimens, or any combination thereof.

Another embodiment of the present invention provides a method of producing a cell composition of a cardiomyocyte cell line or a cardiomyocyte from one or more induced pluripotent stem cells (iPSCs), comprising the steps of: (i) obtaining the one or more iPSCs (ii) initiating differentiation of the one or more iPSCs; (iii) isolating one or more cells or clusters of cells exhibiting differentiation, wherein the one or more cells exhibiting differentiation or the clusters of cells comprise beating cardiomyocytes; (iv) performing the first incubation of the differentiated cardiomyocytes or the cluster of cells for a specified period of time; (v) isolating the one or more cardiomyocytes or clusters of cells exhibiting spontaneous pulsation from the first culture; Performing a second incubation of the isolated and differentiated cardiomyocytes or the cell cluster from the first culture for a specified period of time; (vii) removing one or more of the spontaneous beats from the second culture removal Cardiomyocytes or clusters of cells leaving one or more differentiated cardiomyocytes in the second culture; (viii) harvesting the one or more cell-differentiated cardiomyocytes remaining in the second culture; and (ix) Making the harvested differentiated myocardium from the second culture The cells are expanded to obtain a cell composition of the cardiomyocyte strain or cardiomyocytes. The method as described above further comprises the step of repeating one or more of steps (iii) through (viii). In one aspect of the methods described above, the step of obtaining one or more iPSCs comprises the steps of providing a primary culture comprising one or more dermal fibroblasts obtained from a mammalian individual and one or more Select transcription factor from a group consisting of Sox2, Oct3/4, Klf4, c-Myc or any combination thereof Dyeing into the one or more skin fibroblasts, wherein the transfection results in the formation of iPSCs from the dermal fibroblasts. In one aspect, a mammalian system of a human individual, wherein the system is at least one of a healthy individual, an individual having one or more of the following diseases: metabolic disorders, cardiovascular disease, cardiac dysfunction, myocardial infarction, myocardial deficiency Blood, myocardial reperfusion, subendocardial ischemia, high-angstrom arteritis, atrial fibrillation, hemorrhagic stroke, ischemia/reperfusion, reperfusion injury, congenital heart disease, or cardiac surgery. In a particular aspect, the metabolic disorder is Fabry disease. In yet another aspect, the purity of the cardiomyocyte strain or cardiomyocyte is about 100%. In a related aspect, the cardiomyocyte cell line or cardiomyocyte composition is suitable for cell/tissue transplantation, drug screening studies, in vitro disease mechanism models, cell-based therapies, optimized treatment regimens, or any combination thereof. Myocardial cell line or cardiomyocyte composition obtained by the method of the present invention exhibits myotube formation, showing continuous proliferation up to at least 12 passages, and exhibits sarcomeric α-actinin, transcription factor GATA-4, sarcomeric myosin heavy Chain (MF20), cardiac troponin T, human NKx2.5, and myosin light chain 2v, or any combination thereof.

In yet another embodiment, the invention provides an in vitro system or model for disease mechanism studies; for diagnosing one or more cardiovascular diseases, one or more metabolic disorders of cardiac complications; screening for one or more cardiovascular drugs Activity, toxicity, or both; an optimized cardiac treatment regimen, or any combination thereof, comprising: providing one or more cardiomyocyte strains or a composition comprising one or more cardiomyocytes; administering the candidate drug to a cardiomyocyte-containing strain or An in vitro system combination comprising a composition of one or more cardiomyocyte populations, wherein the cell strain or at least a portion of the cardiomyocytes are capable of proliferating, forming a myotube, expressing a myocardium a marker or any combination thereof; monitoring changes in activity, expression, morphology, or any combination thereof of the cardiomyocyte strain obtained by the combination with the candidate drug; or the cardiomyocyte cell or the cardiomyocyte The activity, the performance, the change in the morphology is associated with the activity, effectiveness, or both of the candidate drugs.

The cardiomyocyte strain used above or a composition comprising one or more cardiomyocytes is produced using a method comprising the steps of: (a) obtaining the one or more iPSCs; (b) initiating differentiation of the one or more iPSCs. (c) isolating one or more cells or clusters of cells exhibiting differentiation, wherein the one or more cells exhibiting differentiation or the clusters of cells comprise beating cardiomyocytes; (d) the differentiated cardiomyocytes or the cluster of cells Performing a first incubation for a specified period of time; (e) separating the one or more cardiomyocytes or cell clusters exhibiting spontaneous pulsation from the first culture; (f) separating the isolated cultures from the first culture And the differentiated cardiomyocytes or the cluster of cells are subjected to a second culture for a specified period of time; (g) removing one or more cardiomyocytes or clusters of cells exhibiting spontaneous pulsation from the second culture, in the second culture Leaving one or more differentiated cardiomyocytes; (h) harvesting the one or more cell-differentiated cardiomyocytes remaining in the second culture; and (i) subjecting the harvested differentiated myocardium from the second culture Cell expansion to obtain the fineness of the cardiomyocyte or cardiomyocyte combination. The system as described herein further comprises the step of repeating one or more of steps (c) through (h). In one aspect of the model, the step of obtaining one or more iPSCs comprises the steps of providing a primary culture comprising one or more dermal fibroblasts obtained from a mammalian individual and one or more selected from the group consisting of Sox2 a transcription factor of a group consisting of Oct3/4, Klf4, c-Myc, or any combination thereof Dyeing into the one or more skin fibroblasts, wherein the transfection results in the formation of iPSCs from the dermal fibroblasts. In one aspect, a mammalian system of a human individual, wherein the system is at least one of a healthy individual, an individual having one or more of the following diseases: metabolic disorders, cardiovascular disease, cardiac dysfunction, myocardial infarction, myocardial deficiency Blood, myocardial reperfusion, subendocardial ischemia, high-angstrom arteritis, atrial fibrillation, hemorrhagic stroke, ischemia/reperfusion, reperfusion injury, congenital heart disease, or cardiac surgery. In a particular aspect, the metabolic disorder is Fabry disease and the purity of the cardiomyocyte strain or cardiomyocyte is about 100%. The invention also provides a composition for treating cardiovascular complications of one or more cardiovascular diseases or Fabry disease in a human or animal subject, comprising a cardiomyocyte strain or a composition comprising one or more cardiomyocytes, Wherein the cardiomyocyte cell line or composition comprising one or more cardiomyocytes is produced by the methods previously described above.

In another embodiment, the present invention discloses a method of treating or optimizing a cardiovascular complication of one or more cardiovascular diseases or Fabry disease in a human or animal subject, comprising the steps of: determining the need for resistance or a human or animal subject for treatment of a plurality of cardiovascular diseases or cardiovascular complications of Fabry disease; and transplanting a therapeutically effective amount of a composition comprising a cardiomyocyte strain or a composition comprising one or more cardiomyocytes to the In human or animal individuals. The cardiomyocyte strains used in the methods herein or compositions comprising one or more cardiomyocytes are produced by the methods previously described.

In one embodiment, the invention provides for assessing the effectiveness of a candidate drug against a cardiovascular complication of one or more cardiovascular diseases or Fabry disease in a human or animal subject, screening for activity, toxicity, or both, or both Conduct an assessment A method for screening, comprising: providing a drug candidate for cardiovascular complications against one or more cardiovascular diseases or Fabry disease; and the candidate drug comprising a cardiomyocyte cell strain or comprising one or more cardiomyocyte populations An in vitro system combination of a composition, wherein the cell strain or at least a portion of the cardiomyocyte population is capable of proliferating, forming a myotube, expressing a myocardial marker, or any combination thereof; monitoring the cardiomyocyte cell obtained by combination with the candidate drug Or a change in the activity, expression, morphology or any combination thereof of the cardiomyocyte population; and the activity, the manifestation, the change in the morphology, the activity of the candidate drug, and the activity of the cardiomyocyte population or the cardiomyocyte population; Sex or both. In one aspect, a cardiomyocyte cell strain or a composition comprising one or more cardiomyocytes is produced by the methods previously described herein.

In another embodiment, the invention relates to an in vitro disease model for detecting one or more cardiovascular diseases, one or more cardiac complications of Fabry disease, or both, comprising a cardiomyocyte strain or inclusion a composition of one or more cardiomyocyte populations, wherein at least a portion of the cell strain or the cardiomyocyte population is capable of proliferating, forming a myotube, expressing a myocardial marker, or any combination thereof, wherein the cardiomyocyte strain or the one or more The composition of the cardiomyocyte population is produced by one or more induced pluripotent stem cells (iPSCs) obtained from a healthy individual, an individual suffering from one or more cardiovascular diseases or metabolic disorders, or both. In one aspect, a cardiomyocyte strain obtained from iPSC or a composition comprising one or more cardiomyocytes is produced by the methods previously described.

In yet another embodiment, the present invention discloses methods of detecting, investigating, assessing a risk, or any combination thereof, of one or more cardiovascular diseases, one or more cardiac complications of Fabry disease, or both, including The following steps: (i) Providing an in vitro disease model comprising a cardiomyocyte strain or a composition comprising one or more cardiomyocytes, wherein the cell strain or at least a portion of the cardiomyocytes are capable of proliferating, forming a myotube, expressing a myocardial marker, or any combination thereof, Wherein the cardiomyocyte cell or the composition comprising the cardiomyocytes is obtained from one or more induced pluripotent stem cells (iPSCs) from a healthy individual, an individual having one or more cardiovascular diseases or metabolic disorders, or both. Producing; (ii) monitoring the activity, performance, morphology, or any combination thereof of the cardiomyocyte cell or the cardiomyocytes under normal or stress physiological conditions after exposure to one or more drugs or cardiac agents or any combination thereof. And (iii) causing the activity, manifestation, and morphology of the cardiomyocyte strain or the cardiomyocytes to be associated with one or more cardiovascular diseases, one or more cardiac complications of Fabry disease, or both , the risk of non-existence or occurrence is associated. In one aspect, a cardiomyocyte cell strain obtained from iPSC or a composition comprising one or more cardiomyocytes is produced by the methods previously described herein.

For a fuller understanding of the features and advantages of the present invention, reference should be

Although the preparation and use of the various embodiments of the present invention are discussed in detail herein below, it is understood that The specific embodiments discussed herein are merely illustrative of specific ways of making and using the invention, and not limiting the scope of the invention.

To facilitate an understanding of the invention, a number of terms are defined below. The terms defined herein have the ordinary meaning as understood by one of ordinary skill in the art to which the invention pertains. Terms such as "a (a, an)" and "the" are not exclusive To refer to a singular entity, including a general category that can be described using a particular instance. The terminology herein is used to describe a particular embodiment of the invention, and is not intended to limit the invention unless otherwise indicated.

The term "muscle cell" as used herein refers to a muscle cell characterized by the inclusion of myosin. The term "cardiomyocyte" refers to a cell containing myosin (isolated or stored in culture) that is isolated from or derived from the muscle (myocardium) or conductive tissue of the heart and that is capable of eliciting a flow.

The term "cell line" refers to a pure line of immortalized mammalian cells. The terms "cell strain" and "established cell strain" as used herein are consistent with the definitions published by Federoff in the Tissue Culture Association Manual, Vol. 1, No. 1, pp. 53-57 (1975), which is incorporated by reference. The manner is incorporated herein.

The term "fibroblasts" as used herein refers to the primary cells in the dermis that produce connective tissue components (collagen and the like) and form tissue by combining with such components.

The term "pluripotent stem cell" as used herein denotes a cell which is capable of self-replication indefinitely and which, under appropriate conditions, can produce a plurality of cell types, in particular from all three embryonic germ layer-mesoderm, endoderm and ectoderm cell types.

The term "embryonic body" as used herein is an industry term synonymous with "aggregate", which refers to differentiated and undifferentiated cells that occur when pluripotent stem cells are overgrowth in monolayer cultures or remain in suspension culture. Aggregate. "Original embryos" are usually mixtures of different cell types from several germ layers that can be immunologically cellified by morphological criteria. Learn to identify the cell markers to distinguish.

The term "cardiovascular disease" as used herein is intended to mean all pathological conditions that result in stenosis and/or obstruction of systemic blood vessels. In particular, the term "cardiovascular disease" refers to conditions including atherosclerosis, thrombosis, and other related pathological conditions, particularly in the arteries of the heart and brain. Thus, the term "cardiovascular disease" encompasses, but is not limited to, various types of heart disease, as well as Alzheimer's disease and blood vessel size.

The term "metabolic disease" as used herein includes a group of defined disorders in which metabolic abnormalities, metabolic disorders, or poor metabolism occur. The term "metabolic disease" as used herein also encompasses a condition that can be modulated by metabolic therapy, but the disease may or may not be triggered by specific metabolic blockade.

The term "Fabbreviation" as used herein refers to an abnormality in the catabolism of X-chromosomal congenital glycosphingolipids caused by the lack of lysosomal α-galactosidase A activity. This deficiency results in the accumulation of spheroids, sphingosine (sphingosine triglucoside) and related glycosphingolipids in vascular endothelins of the heart, kidney, skin and other tissues.

The term "polymerase chain reaction" (PCR), as used herein, refers to the methods of U.S. Patent Nos. 4,683,195, 4,683,202 and 4,965,188, the disclosures of each of which are hereby incorporated by reference in A method of increasing the concentration of a fragment of a target sequence in a mixture of genomic DNA in the case. This method for amplifying a target sequence consists of introducing a large excess of two oligonucleotide primers into a DNA mixture containing the desired target sequence, followed by introduction of a precise sequence of thermal cycling in the presence of a DNA polymerase. The two primers are complementary to their respective strands of the double stranded target sequence. To effect amplification, the mixture is denatured and the primers are then annealed to their complementary sequences in the target molecule. After annealing, the primers are extended with a polymerase to form a new pair of complementary strands. The denaturation, primer annealing, and polymerase extension steps can be repeated multiple times (ie, denaturation, annealing, and extension to form a "cycle"; multiple "cycles" can be present) to obtain amplified fragments of the desired target sequence at high concentrations. It is expected that the length of the amplified fragment of the target sequence is determined by the relative positions of the primers relative to each other, and therefore, this length is a controllable parameter. This method is called "polymerase chain reaction" (PCR below) due to the repeated nature of the method. Since the desired amplified fragment of the target sequence becomes the main sequence (in terms of concentration) in the mixture, it is called "PCR amplification". Using PCR, it is possible to amplify a single copy-specific target sequence in genomic DNA to a number of different methods (eg, hybridization with a labeled probe; incorporating biotinylated primers followed by anti-biotinase conjugate detection; The concentration of ³² P-labeled deoxynucleotide triphosphate (eg, DCTP or DATP) is included in the amplified fragment). In addition to genomic DNA, any oligonucleotide sequence can be amplified with a suitable set of primer molecules. In particular, the amplified fragment produced by the PCR method itself is an effective template for subsequent PCR amplification.

The term "in vivo" as used herein refers to the interior of the body. The term "in vitro" as used in this application shall be taken to mean an operation carried out in a non-viable system.

As used herein, "pharmaceutically acceptable carrier" refers to a material that, when combined with an immunoglobulin (Ig) fusion protein of the invention, maintains Ig biological activity and generally does not react with the individual's immune system. Examples include, but are not limited to, standard pharmaceutical carriers such as phosphate buffered saline solutions, water, emulsions (eg, oil-in-water Type emulsion) and various types of wetting agents. Certain diluents can be used with the present invention, for example, for aerosol or parenteral administration, which can be phosphate buffered saline or physiological saline (0.85%).

The term "administration of or administration" is used herein to provide a compound of the invention in a form which is therapeutically usable and therapeutically usable in the form of a pharmaceutical composition, including, but not limited to, an oral dosage form, For example, lozenges, capsules, syrups, suspensions and the like; injectable formulations, such as intravenous, intramuscular or intraperitoneal, and the like; transdermal formulations, including creams, jellies, powders or patches; buccal dosage forms; Inhalation of powders, sprays, suspensions and the like; and rectal suppositories, the compounds.

The term "effective amount" or "therapeutically effective amount" as used herein shall be understood to mean the amount of individual compound that is sought by a researcher, veterinarian, physician or other clinician to cause a biological or medical response to a tissue, system, animal or human.

The term "treatment" or "treating" as used herein includes administration of any of the compounds of the invention and (1) inhibiting the disease of an animal that is undergoing or exhibiting the pathology or symptoms of the disease (ie, preventing the pathology and/or symptoms). Further development), or (2) improving the disease (ie, reversing the pathology and/or symptoms) of an animal that is undergoing or displaying the pathology or symptoms of the disease. Non-limiting examples of diseases that can be treated using the cells of the invention include cardiac dysfunction, myocardial infarction, myocardial ischemia, myocardial reperfusion, subendocardial ischemia, Takanysu's arteritis, atrial fibrillation, hemorrhagic Stroke, ischemia/reperfusion, reperfusion injury, cardiac surgery.

One current method for producing cardiomyocytes separates cardiomyocytes directly from human heart tissue. Cardiomyocytes produced by this method are commercially available from a number of companies including PromoCell (GmbH Heidelberg, Germany) and ScienCell (Carlsbad, CA). However, all of these cardiomyocytes were isolated from healthy individuals. Another method used is to differentiate human embryonic stem (ES) cells or induced pluripotent stem cells (iPSCs) into cardiomyocytes in vitro (1). However, the current method for differentiating pluripotent stem cells into cardiomyocytes in vitro can only provide relatively impure cardiomyocytes with a large amount of contaminants (about 20%) of other cell types and having low proliferative capacity, and therefore, the method is limited in providing Use in commercially available cardiomyocytes.

The present invention describes a novel method for producing a proliferative high purity human cardiomyocyte cell line from an induced pluripotent stem cell (iPSC). Since these cardiomyocyte strains are originally produced by dermal fibroblasts, the methods described herein allow for the production of a patient or a human-specific cardiomyocyte strain in vitro. This feature makes the method of the present invention highly suitable for basic research and drug screening, myocardial therapy optimization, and treatment of individual patients by, for example, autologous cell transplantation.

The method described below is simple and reproducible. In addition, it takes only a few months to obtain a high-purity cardiomyocyte cell line from skin fibroblasts. Unlike the currently available methods for obtaining cardiomyocytes from human iPSCs, cardiomyocytes produced by the method of the present invention have high proliferative capacity and high purity. These are superior to the significant advantages of current methods. Although the present inventors produced cardiomyocytes from iPSCs, cardiomyocyte strains can be produced from human ES cells or other types of stem cells using this method due to genetic and phenotypic similarity between iPSCs and ES cells.

The inventors have used the method described herein from one healthy individual and two not The same patient with, for example, Fabry disease establishes several cardiomyocyte strains. Cardiac complications are common and are the main manifestations of Fabry disease. Such Fabri myocardial cell lines (and cell lines to be produced) are invaluable tools for studying disease mechanisms (including the relationship between genotype and myocardial phenotype) and for evaluating new treatments for Fabry disease, and Therefore, researchers in the field of metabolic diseases and cardiovascular diseases are interested.

Department of cardiovascular heart disease leading causes of death, which results in the United States each year about 2.5 × 10 ⁶ deaths (2). Treatment of risk factors for acute myocardial infarction and cardiovascular disease reduces mortality in patients, but on the other hand increases the number of patients with heart failure after acute myocardial infarction. Cell-based therapies are excellent in the treatment of both acute myocardial infarction and chronic heart failure (3). Human cardiomyocyte cell lines are important materials for cell-based therapies and other important applications such as drug screening and disease mechanism studies (4, 5). The purity and proliferative potential of transplantable cardiomyocytes are two important factors for successful transplantation. There is currently no method for obtaining a proliferative pure cardiomyocyte strain from a patient. Such cells are highly desirable for autologous cell/tissue transplantation and patient customization studies.

Human cardiomyocytes can be obtained and cultured from cardiac tissue. Some biotech companies, including PromoCell and ScienCell, provide human cardiomyocytes obtained from normal individuals for research purposes. In theory, patient-specific cardiomyocytes can be obtained by cardiac biopsy and cultured by this method. However, the invasive procedure of cardiac tissue obtained by biopsy and a limited number limit the clinical application of this method.

A versatile stem cell line including ES cells and induced pluripotent stem cells (iPSC) is an alternative source of human cardiomyocytes, which is why stem cells have It has excellent proliferative ability and can be differentiated into cardiomyocytes in vitro (6). Since iPSCs can be produced by human skin fibroblasts, they are significantly superior to human ES cells with ethical and immunological rejection problems. Currently, human iPSCs are highly competitive in the production and purification of cardiomyocyte cell lines. Current methods for obtaining human cardiomyocytes use an embryoid body-based (EB)-based differentiation method (1) after separation of cardiomyocytes from a mixed-type cell bank by a Percoll gradient. However, the purity of the cells is about 80% and the cell proliferation ability is extremely limited. Transplantation experiments have shown that the purity and proliferative capacity of differentiated cardiomyocytes are critical for successful transplantation, as the higher the purity, the less chance of developing teratomas during transplantation and the need to establish a supply of blood vessels that determine graft survival. Proliferation ability. Researchers are developing methods such as enrichment of cardiomyocytes (7), co-culture with visceral endoderm-like cells (8), and lineage-directed cardiomyocyte differentiation (9, 10) to increase the purity and quantity of cardiomyocytes. However, none of these methods can obtain high-purity proliferative cardiomyocytes.

The present invention provides for obtaining pure or almost pure cardiomyocytes (eg, 90%, 95%, 96%, 97%, 98%, 99%, or even 100%) from human iPSCs that can proliferate continuously for more than 12 generations in vitro. A simple method of pure cardiomyocytes. This method enables the establishment of a source of patient-specific cardiomyocytes for research and therapeutic purposes. The passage algebra can be 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th or more generations. Figure 1A is a schematic comparison of the method of the present invention with the methods currently used, and Figure 1B compares the cardiomyocyte strain produced by the method of the present invention and the prior art.

Fig. 1A is a schematic diagram showing the technical difference between the prior art method of producing cardiomyocytes by induced pluripotent stem cells (iPSC) and the present invention. In short, Human iPSCs are prepared in a manner known in the art, including the formation of embryoid bodies (EB) in suspension cultures followed by attachment of EBs. In certain instances, the invention does not require, but may also benefit from, hanging drop suspension cultures, but is not required. Due to the attachment of EB, a certain percentage of cells further differentiate into pulsatile clusters. At this stage the invention differs from the prior art in which pulsatile clusters or cardiomyocytes are harvested from such clusters. The present invention divides individual beat clusters and allows them to adhere to allow cardiomyocytes to be expanded from clusters. Then, the beating cluster is discarded, and the cells proliferating and expanding from the self-pulsating cluster are proliferated. These cells are then further grown and the beater clusters are removed again and discarded. The remaining cells can then be harvested and used to allow them to continue to grow or to be made into cell lines, thereby allowing amplification or allowing a large number of homologous cells to be reinserted into the patient. Figure 1B is a comparison of the characteristics of cardiomyocytes obtained from iPSC using existing methods and methods as described in various embodiments of the present invention, wherein the methods disclosed herein yield cells with high proliferative capacity, high yield, and high purity, in some cases. More than 60%, 70%, 80%, 85%, 90% or even 97% of the cell line iPSC. The cell line of the invention can also be passaged and expanded multiple times, including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more times. repeatedly.

Production of cardiomyocyte strains: The present inventors generated human iPSCs by transducing Sox2, Oct3/4, Klf4, and c-Myc into human fibroblasts in the presence/absence of heart disease (Figs. 2A and 2B). (eg, human genes with the following accession numbers: NM_003106; NM_001173531; NM_004235; NM_002467). The present invention discloses a method of producing cardiomyocytes by such human iPSCs. EBs were first formed from iPSCs in suspension cultures and these EBs were attached to obtain pulsatile cardiomyocyte clusters (Figs. 2C and 2D). The pulsatile clusters were mechanically divided and attached to a 12-well plate at 1 cluster/well and cultured for 2 weeks (first culture, Figure 2E). In order to improve the purity of the cardiomyocyte strain, the pulsatile cluster in the first culture was again divided into new wells (1 cluster/well) to achieve the migration of pure cardiomyocytes from the cluster for 2 weeks (second culture, figure) 2F). Then, the pulsatile cluster in the second culture was removed (Fig. 2F, indicated by the arrow front) and the migrating cells were harvested by trypsinization (Fig. 2F, arrow). The cells from each cluster were expanded into a cardiomyocyte strain. Using the present invention, a number of cardiomyocyte strains can be obtained from a patient/person that are spontaneously produced during use of the methods of the invention.

Characterization of the resulting cardiomyocyte strain: (i) Cell morphology: The cardiomyocyte cell line was uniform in morphology and showed typical morphology of cardiomyocytes (Fig. 3). It grows in a stripe form at a lower confluence and grows on top of each other when grown to confluence and detaches in the form of a sheet during subculture.

(ii) Myotube formation: Myocardial cell lines form myotubes in culture (Fig. 3). The myotube system is formed by cell fusion, a functional feature of cell fusion cells. This further demonstrates the obtained cell line of cardiomyocytes.

(iii) Growth: The cardiomyocyte cell line is continuously proliferated in vitro for at least 12 passages. The doubling time of the fourth generation was 38.2 hours.

(iv) Performance of cardiomyocyte markers: Immunostaining showed that 100% of the cells exhibited cardiomyocyte-labeled sarcomeric α-actinin. About 30% were positive for the cardiomyocyte-specific transcription factor GATA-4 (Fig. 4).

Identification of the obtained cardiomyocyte strains was carried out by short tandem repeat (STR) analysis to confirm that the cell strain was derived from the patient fibroblasts (Table 1). The results showed that the parental fibroblast, iPSC and the obtained cardiomyocyte cell line had the same Genetic marker, which indicates that it is from the same individual.

The analysis also confirmed that there was no interspecies contamination.

The obtained cardiomyocyte strains were further characterized for the expression of myocardial lineage markers, ion channels, and calcium (Ca ²⁺ ) regulatory proteins. The cardiomyocyte cell line expresses ISL1 (marker of myocardial progenitor cell lineage, accession number NM_002202.2 (human), NM_021459.4 (mouse)) in a similar degree in the beating cardiomyocyte cluster. Myocardial cell lines also showed the expression of sodium (Na ⁺ ) channels, potassium (K ⁺ ) channels, and Ca ²⁺ channels and Ca ²⁺ regulatory proteins, triadin (Triadin) and adaptor proteins (Junctin). Figure 5 shows the expression of ISL1 mRNA in the obtained cardiomyocyte cell line and beating cardiomyocyte (CM) cluster.

Figure 6 shows quantitative mRNA analysis of ion channel subunits and Ca ²⁺ regulatory proteins in the obtained cardiomyocyte strains. (Na ⁺ channel: SLC8A1; K ⁺ channel: KCNH2, KCNN1 and KCNJ2; Ca ²⁺ channel and regulatory protein: CACNA1C, SERCA2a, triplet and adaptor). The values were normalized to 18S and expressed as a percentage in the cluster of pulsatile cardiomyocytes (CM).

Then, the expression of ion channel proteins and gap junction proteins was studied by immunocytochemistry. The expression of L-type Ca ²⁺ channel and connexin 43 was confirmed in the obtained cardiomyocyte strain. The expression and distribution of Ca ²⁺ channels and gap junction proteins indicate the morphology and functional integrity of cardiomyocyte strains, which are important for the production of cardiomyocyte properties such as electrical activity and electrical transmission. Figure 7 shows immunostaining of L-type Ca ²⁺ channel α-subunit (Cav1.2) and connexin 43 in the obtained cardiomyocyte strain by specific antibodies.

The effect of the drug that induces hypertrophy on the obtained cardiomyocyte strain was tested. The response to cardiotoxic agents is important in assessing the nature of the obtained cardiomyocyte strains and the future use of cells in testing for cardiotoxic agents and in the study of mechanisms of cardiomyopathy. Induction was performed using tumor necrosis factor (TNF)-α and cell size changes 24 hours after drug administration were compared. The size of the treated cardiomyocytes is increased. Figure 8 shows a relative image of two representative fields of view of a cardiomyocyte cell obtained before and after (24 hours) use of TNF-[alpha].

Additional data on myotube formation. Myotubes were stained with desmin for immunostaining to confirm cardiomyocyte identity. Myotubes are positive for desmin. Figure 9 shows immunostaining of myotubes using desmin.

To study the electrophysiological characterization of cardiomyocyte strains in vitro, it is more important to consider the use of these cell lines for the study of the mechanisms of cardiac physiology and pathology. However, in therapeutic studies, the transplanted cell line may follow an intrinsic ecological location for further differentiation and integration into the environment, with appropriate electrophysiology at the site of integration. In vivo transplantation can be designed for this purpose the study.

The present invention contemplates that any of the embodiments discussed in this specification can be practiced in accordance with any of the methods, kits, reagents or compositions of the present invention, and vice versa. Additionally, the compositions of the invention can be used to achieve the methods of the invention.

It is understood that the specific embodiments described herein are shown by way of illustration and not limitation. The main features of the invention can be used in various embodiments without departing from the scope of the invention. Those skilled in the art will be able to determine or determine various equivalents of the particular procedures described herein using only routine experimentation. Such equivalents are considered to be within the scope of the invention and are covered by the scope of the claims.

All publications and patent applications mentioned in the specification are intended to be <RTIgt; All publications and patent applications are hereby incorporated by reference in their entirety in the extent of the extent of the disclosure of the disclosures

In the context of the patent application and/or the description, the words "a" or "an" may mean "one" when used in conjunction with the term "comprising", but it is also associated with "one or more" or "at least one" The meaning of "one or more than one" is the same. The term "or" as used in the context of the disclosure is intended to mean only the meaning of "and/or", unless the term is used to mean that the term merely refers to the substitution or the substitution is mutually exclusive, the term "or" is used in the scope of the application. Is used to mean "and / or". Throughout this application, the term "about" is used to indicate that a value includes a variation of the intrinsic error of the device or method used to determine the value or a change in the subject.

The words "comprising" (and any form thereof, such as "comprise" and "comprises"), "having" (and any form thereof, such as "have" and "has"), "including" (and any form thereof, such as "includes" and "include") or "containing" (and any form thereof, such as "contains" and "contain") They are all inclusive or open and do not exclude additional elements or method steps not listed.

The term "or a combination thereof" as used herein refers to all permutations and combinations of items previously recited by the term. For example, "A, B, C, or a combination thereof" is intended to include at least one of the following: A, B, C, AB, AC, BC, or ABC, and if the order is important in a particular context, BA, CA, CB, CBA, BCA, ACB, BAC or CAB. Continuing with this example, it explicitly includes combinations of duplicates of one or more entries or terms, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, and the like. It will be understood by those skilled in the art that the number of items or terms is not limited in any combination, unless the context indicates otherwise.

All of the compositions and/or methods disclosed and claimed herein can be obtained and practiced without undue experimentation. Although the compositions and methods of the present invention have been described in terms of the preferred embodiments, it is apparent to those skilled in the art that the compositions and/or methods may be modified without departing from the spirit, scope and scope of the invention. And the steps or sequence of steps of the methods described herein. All such similar substitutes and modifications that are obvious to those skilled in the art are intended to be included within the spirit, scope and concept of the invention as defined by the appended claims.

references

PCT Patent Application No. PCT/US2011/056329: Cardiac Induced Pluripotent Stem Cells And Methods Of Use In Repair And Regeneration Of Myocardium .

US Patent Application No. 2011/097799: Cardiomyocyte Production .

US Patent Application Publication No. 20050037489: Methods of Generating Human Cardiac Cells and Tissues and Uses Thereof .

U.S. Patent Application Publication No. 20050054092: Process for Making Transplantable Cardiomyocytes from Human Embryonic Stem Cells .

US Patent No. 7,452,718: Direct Differentiation Method for Making Cardiomyocytes from Human Embryonic Stem Cells .

U.S. Patent Application Publication No. 20090017465: Compound Screening Using Cardiomyocytes .

U.S. Patent No. 7,611,852: Functional Cardiomyocytes from Human Embryonic Stem Cells .

1. Xu C et al. (2002) Characterization and enrichment of cardiomyocytes derived from human embryonic stem cells. Circ Res 91(6): 501-508.

2. Lloyd-Jones D et al. (2009) Heart disease and stroke statistics-2009 update: from American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Circulation. 119(3): Report of e21-181.

3. Taylor DA, Zenovich AG. (2008) Cardiovascular cell therapy and endogenous repair. Diabetes Obes Metab. 10, Supplement 4:5-15.

4. Yazawa M et al. (2011) Using induced pluripotent stem cells to investigate cardiac phenotypes in Timothy syndrome. Nature 471 (7337): 230-234.

5. Laflamme MA et al. (2007) Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts. Nat Biotechnol 25(9): 1015-1024.

6. Kehat I et al. (2001) Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. J Clin Invest 108(3): 407-414.

7. Zhang J et al. (2009) Cardiac bodies: a novel culture method for enrichment of cardiomyocytes derived from human embryonic stem cells. Functional cardiomyocytes derived from human induced pluripotent stem cells. Circ Res 104(4): e30-41.

8. Mummery C et al. (2003) Differentiation of human embryonic stem cells to cardiomyocytes: role of coculture with visceral endoderm-like cells. Circulation 107(21): 2733-2740.

9. Paige SL et al. (2010) Endogenous Wnt/beta-catenin signaling is required for cardiac differentiation in human embryonic stem Cells. PLoS One 5(6): e11134.

10. Yang L et al. (2008) Human cardiovascular progenitor cells develop from a KDR+embryonic-stem-cell-derived population. Nature 453 (7194): 524-528.

1A is a schematic diagram showing the technical difference between the prior art method of producing cardiomyocytes by induced pluripotent stem cells (iPSC) and the present invention; FIG. 1B is obtained from iPSC using existing methods and methods as described in various embodiments of the present invention. The characteristics of cardiomyocytes; Figures 2A to 2F show the different steps involved in the production of cardiomyocytes from iPSCs derived from patient fibroblasts; Figure 3 shows the cardiomyocytes of patients established according to the techniques described in the examples of the present invention. The morphology of the strain and myotube formation; and Figure 4 shows the expression of the cardiomyocyte marker of the established cardiomyocyte cell line; Figure 5 shows the expression of ISL1 mRNA in the acquired cardiomyocyte cell line and the pulsatile cluster of cardiomyocytes (CM); Quantitative mRNA analysis of ion channel subunits and Ca2+ regulatory proteins in the obtained cardiomyocyte strains is shown; Figure 7 shows the L-type Ca2+ channel α-subunits (Cav1.2) by specific antibodies in the obtained cardiomyocyte strains. And immunostaining of connexin 43; Figure 8 shows a relative ratio of two representative fields of view of the obtained cardiomyocyte strain before and after TNF-α (24 hours); Figure 9 shows the use of desmin The muscular tube immunostaining.

Claims (37)

A method of producing a cell strain or cell composition from one or more induced pluripotent stem cells (iPSCs), comprising the steps of: (a) obtaining the one or more iPSCs; (b) initiating the one or more iPSCs (c) separating one or more cells or clusters of cells exhibiting spontaneous pulsation; (d) performing the first incubation of the differentiated cells or the cluster of cells for a specified period of time; (e) from the first culture Separating the one or more cells or clusters of cells that exhibit spontaneous pulsation; (f) performing a second incubation on the differentiated cells or the cluster of cells from the first culture for a specified period of time; (g) from the first The second culture removes one or more cells or clusters of cells that exhibit spontaneous pulsation, leaving one or more differentiated cells in the second culture; (h) harvesting the one or more remaining in the second culture And differentiated cells; and (i) amplifying the differentiated cells harvested from the second culture to obtain the cell strain or the cell composition. The method of claim 1, further comprising the step of repeating one or more of steps (c) through (h). The method of claim 1, wherein the step of obtaining the one or more iPSCs comprises the step of providing a non-functional fine comprising one or more derived from a mammalian individual Primary culture of cells; and transfection of one or more transcription factors or stem cell-associated genes into the one or more non-multifunctional cells, wherein the transfection causes the non-multifunctional cells to form the iPSCs. The method of claim 3, wherein the non-multifunctional cells comprise fibroblasts, blast cells, corneal cells, amniocytes, gastric cells, neural stem cells, or any combination thereof. The method of claim 3, wherein the one or more transcription factors are selected from the group consisting of Sox2, Oct3/4, Klf4, c-Myc, Lin28, Nanog, or any combination thereof. The method of claim 3, wherein the mammalian system is a human subject, wherein the system is a healthy individual, at least one of the individuals having one or more of the following diseases: metabolic disorders, cardiovascular diseases, cardiac dysfunction, myocardial infarction , myocardial ischemia, myocardial reperfusion, subendocardial ischemia, Takanysu's arteritis, atrial fibrillation, hemorrhagic stroke, ischemia/reperfusion, reperfusion injury, congenital heart disease or cardiac surgery. The method of claim 6, wherein the metabolic disorder is Fabry's disease. The method of claim 1, wherein the cell strain comprises a cardiomyocyte cell line. The method of claim 1, wherein the cell strain or the cell composition comprises cardiomyocytes. The method of claim 1, wherein the step of inducing differentiation of the one or more iPSCs comprises forming one or more embryoid bodies, or being exposed by iPSCs, such as Factors such as BMP4 or activin A. The method of claim 1, wherein the one or more cells or cell clusters exhibiting spontaneous pulsation are separated by one or more mechanical methods. The method of claim 1, wherein the isolated cells or clusters exhibiting differentiation comprise one or more beating cells. The method of claim 1, wherein the one or more differentiated cells are harvested by trypsin digestion or mechanical separation using a cell scraper followed by pipetting. The method of claim 1, wherein the cell strain or the cell composition has a purity of >90%. The method of claim 1, wherein the cell strain or cell composition is suitable for cell/tissue transplantation, drug activity and toxicity screening studies, optimized treatment regimens, in vitro disease mechanism models, cell-based therapies, or any combination thereof. A method for producing a cell composition of a cardiomyocyte cell line or cardiomyocyte from one or more induced pluripotent stem cell iPSCs, comprising the steps of: (a) obtaining the one or more iPSCs; (b) initiating the one or more (i) differentiation exhibiting one or more cells or clusters of cells, wherein the one or more cells exhibiting differentiation or the clusters of cells comprise beating cardiomyocytes; (d) the differentiated cardiomyocytes Or the cell cluster is cultured for the first time for a specified period of time; (e) separating the one or more of the spontaneous beats from the first culture a cardiomyocyte or cluster of cells; (f) performing a second incubation of the isolated and differentiated cardiomyocytes or the cluster of cells from the first culture for a specified period of time; (g) removing from the second culture Showing one or more cardiomyocytes or clusters of spontaneous pulsations, leaving one or more differentiated cardiomyocytes in the second culture; (h) harvesting the one or more differentiations remaining in the second culture Cardiomyocytes; and (i) amplifying differentiated cardiomyocytes harvested from the second culture to obtain a cell composition of the cardiomyocyte strain or cardiomyocytes. The method of claim 16, further comprising the step of repeating one or more of steps (c) through (h). The method of claim 16, wherein the step of obtaining the one or more iPSCs comprises the steps of: providing a primary culture comprising one or more dermal fibroblasts obtained from a mammalian individual; and selecting one or more a transcription factor of a group consisting of free Sox2, Oct3/4, Klf4, c-Myc, or any combination thereof, transfected into the one or more dermal fibroblasts, wherein the transfection causes the dermal fibroblasts to form such iPSC. The method of claim 18, wherein the mammalian system is a human subject, wherein the system is a healthy individual, at least one of the individuals having one or more of the following diseases: metabolic disorders, cardiovascular diseases, cardiac dysfunction, myocardial infarction , myocardial ischemia, myocardial reperfusion, subendocardial ischemia, high Anilitis, atrial fibrillation, hemorrhagic stroke, ischemia/reperfusion, reperfusion injury, congenital heart disease or cardiac surgery. The method of claim 19, wherein the metabolic disorder is Fabry disease. The method of claim 16, wherein the cardiomyocyte strain or the cardiomyocytes have a purity of about 100%. The method of claim 16, wherein the cardiomyocyte cell line or the cardiomyocyte composition is suitable for cell/tissue transplantation, drug activity and toxicity screening studies, optimized treatment regimens, in vitro disease mechanism models, cell-based therapies, or any combination thereof . The method of claim 16, wherein the cardiomyocyte cell line or the cardiomyocyte composition exhibits myotube formation. The method of claim 16, wherein the cardiomyocyte strain or the cardiomyocyte composition is continuously proliferated for at least 12 passages. The method of claim 16, wherein the cardiomyocyte cell line or the cardiomyocyte composition exhibits sarcomeric α-actinin, transcription factor GATA-4, sarcomeric myosin heavy chain (MF20), cardiac troponin T Human NKx2.5 and myosin light chain 2v or any combination thereof. An in vitro system or model for disease mechanism studies; for diagnosing one or more cardiovascular diseases, one or more metabolic disorders of cardiac complications; screening for activity, toxicity, or both of one or more cardiovascular drugs; A cardiac treatment regimen; or any combination thereof, comprising: providing one or more cardiomyocyte strains or a composition comprising one or more cardiomyocytes; administering the candidate drug to a cardiomyocyte containing strain or comprising one or more myocardium An in vitro system combination of a composition of cells, wherein the cell strain or at least a portion of the cardiomyocytes are capable of proliferating, forming a myotube, expressing a myocardial marker, or any combination thereof; monitoring the cardiomyocyte cell obtained in combination with the candidate drug Or a change in the activity, expression, morphology or any combination thereof of the cardiomyocytes; and the activity, the manifestation, the change in the morphology of the cardiomyocyte cell or the cardiomyocytes and the activity, effectiveness or The two are related. The system of claim 26, wherein the cardiomyocyte cell line or a composition comprising one or more cardiomyocytes is produced using the method of any one of claims 16 to 21. A composition for treating or optimizing the cardiovascular complications of one or more cardiovascular diseases or Fabry disease in a human or animal subject, comprising a cardiomyocyte strain or a composition comprising one or more cardiomyocytes Wherein the cardiomyocyte cell line or a composition comprising one or more cardiomyocytes is produced by the method of claim 16. Use of a composition comprising a cardiomyocyte strain or a composition comprising one or more cardiomyocytes for the manufacture of cardiovascular concurrency for treating one or more cardiovascular diseases or Fabry disease in a human or animal individual Symptoms or agents that optimize their treatment. The use of claim 29, wherein the cardiomyocyte strain or a composition comprising one or more cardiomyocytes is produced by the method of claim 16. The use of claim 29, wherein the one or more cardiovascular diseases are cardiac dysfunction, myocardial infarction, myocardial ischemia, myocardial reperfusion, subendocardial Ischemia, high-amp; arteritis, atrial fibrillation, hemorrhagic stroke, ischemia/reperfusion, reperfusion injury, congenital heart disease, or cardiac surgery. A method for assessing the effectiveness of a candidate drug against a cardiovascular complication of one or more cardiovascular diseases or Fabry disease in a human or animal subject, screening for activity, toxicity, or both, or simultaneously evaluating and screening. Included: providing the candidate drug for the cardiovascular complications of the one or more cardiovascular diseases or Fabry disease; combining the candidate drug with a composition comprising a cardiomyocyte strain or comprising one or more cardiomyocyte populations An in vitro system combination, wherein the cell strain or at least a portion of the cardiomyocyte population is capable of proliferating, forming a myotube, expressing a myocardial marker, or any combination thereof; monitoring activity of the cardiomyocyte strain or cardiomyocyte population obtained in combination with the candidate drug a change in expression, morphology, or any combination thereof; and correlating the activity, the manifestation, the change in morphology of the cardiomyocyte cell or the cardiomyocyte population with the activity, effectiveness, or both of the candidate drug. The method of claim 32, wherein the cardiomyocyte cell line or a composition comprising one or more cardiomyocytes is produced by the method of any one of claims 16 to 21. An in vitro disease model for detecting one or more cardiovascular diseases, one or more cardiac complications of Fabry disease, or both, comprising a cardiomyocyte strain or a composition comprising one or more cardiomyocyte populations, Wherein the cell strain or at least a portion of the cardiomyocyte population is capable of proliferating and forming a muscle Tube, a myocardial marker, or any combination thereof, wherein the cardiomyocyte strain or the composition comprising the one or more cardiomyocyte populations is from one or more self-healthy individuals, suffering from one or more cardiovascular diseases or metabolic disorders Inducible pluripotent stem cell iPSC production by an individual or both. The in vitro disease model of claim 34, wherein the cardiomyocyte strain obtained from the iPSCs or the composition comprising one or more cardiomyocytes is produced by the method of claim 16. A method of detecting, studying, assessing a risk, or any combination thereof, of one or more cardiovascular diseases, one or more cardiac complications of Fabry disease, or both, comprising the steps of: providing a cardiomyocyte-containing strain or An in vitro disease model comprising a composition of one or more cardiomyocytes, wherein the cell strain or at least a portion of the cardiomyocytes are capable of proliferating, forming a myotube, expressing a myocardial marker, or any combination thereof, wherein the cardiomyocyte cell line or The composition comprising the cardiomyocytes is produced by one or more inducible pluripotent stem cell iPSCs obtained from a healthy individual, an individual having one or more cardiovascular diseases or metabolic disorders, or both; monitoring the cardiomyocyte cell line or Changes in the activity, manifestations, morphologies, or any combination thereof of the cardiomyocytes under normal or stress physiological conditions upon exposure to one or more drugs or cardiac agents, or any combination thereof; and the cardiomyocyte cell line Or the activity, the manifestation, the change in morphology of the cardiomyocytes and one or more cardiovascular diseases, one or more cardiac complications of Fabry disease Or the risk of the existence, non-existence or occurrence of the two. The method of claim 36, wherein the cardiomyocyte strain obtained from the iPSCs or a composition comprising one or more cardiomyocytes is produced by the method of claim 16.