Treatment of heart disorders

Abstract

The present invention relates to populations of cells with an increased proportion of left ventricular cardiomyocytes and their uses, for example, in treating left ventricular disorders and in screening for drugs that may be used in treating left ventricular disorders.

Description

[Technical Field]

[0001] The present invention relates to a population of cells with an increased proportion of left ventricular cardiomyocytes, and its use, for example, in the treatment of left ventricular disorders and in the screening of drugs that may be used to treat left ventricular disorders. [Background technology]

[0002] Cardiovascular disease is a leading cause of death in developed industrial nations. Myocardial infarction (MI), which causes a dramatic loss of contractile myocardium in the left ventricle (LV), is the most common cause of cardiac injury. When the infarction is large, or when a patient has multiple infarctions, it often leads to heart failure, for which the only effective treatment is a heart transplant. The life expectancy of patients with heart failure is up to 5 years, and as many as 60% of patients die within one year of diagnosis. Therefore, there is an urgent need for a safe and sustainable treatment that can replace the myocardium damaged in MI / heart failure. The key to improving patients' long-term outcomes is the regrowth of the LV by functional cardiomyocytes. [Overview of the Initiative]

[0003] The inventors have developed a novel method for providing a population of cardiomyocytes in which the number or percentage of cardiomyocytes expressing left ventricular cell-specific markers is remarkably high. This method results in the rapid generation (20 days) of left ventricular cardiomyocytes that can exhibit a more mature identity than can be achieved using previous differentiation methods and long-term culture (60 days or more).

[0004] Accordingly, the inventors provide, for the first time, a population of left ventricular cardiomyocytes with a high degree of homogeneity that may have a higher degree of maturity, and a method for generating such a population of cells in an effective and rapid manner. Such a population of cells may be used for various purposes, for example, as cell therapy to treat left ventricular dysfunction, to model left ventricular dysfunction, or for drug screening to identify drugs that may be used to treat left ventricular dysfunction, or for cardiotoxicity testing.

[0005] The present invention provides a population of cells containing at least about 60% of cells that are double-positive for the markers HAND1 and MLC2v. Such cells are left ventricular cardiomyocytes. The coexistence of the two markers HAND1 and MLC2v is beneficial as information about the left ventricular cardiomyocyte phenotype. This has not been recognized in the art to date.

[0006] The present invention enables for the first time the efficient production of such a population of left ventricular cardiomyocytes. This provides a significant contribution to the art, such as therapeutic uses, which are considered below. As described in this embodiment, evidence is provided that cells according to the present invention are more mature at day 20 than cells that have reached day 60 of long-term culture using monolayer differentiation methods known in the art. For example, immunofluorescence analysis for sarcomere markers is provided. Functional evidence is also provided from electrophysiology that the cells have ventricular action potential shapes (using three different assays). Furthermore, functional data from calcium imaging regarding calcium response are provided.

[0007] The inventors further provide evidence that these cells can successfully generate engineered heart tissue (EHT). Finally, the inventors provide evidence that EHT generated using left ventricular-like cardiomyocytes loses spontaneous pacemaker ability, consistent with what is expected in more mature ventricular cardiomyocytes.

[0008] In one embodiment, the present invention provides a method for treating left ventricular dysfunction in a subject, comprising administering to the subject a population of cells containing at least about 60% of cells that are double-positive for the markers HAND1 and MLC2v.

[0009] In one embodiment, the present invention provides a population of cells containing at least about 60% of cells that are double-positive for the markers HAND1 and MLC2v for use in the treatment or prevention of left ventricular disorders.

[0010] In one embodiment, the present invention provides a population of cells comprising at least about 60% of cells that are double-positive for the markers HAND1 and MLC2v for use in the manufacture of agents for use in the treatment or prevention of left ventricular disorders.

[0011] In one embodiment, the present invention provides the use of a population of cells containing at least about 60% of cells that are double-positive for the markers HAND1 and MLC2v for the treatment or prevention of left ventricular disorders.

[0012] In one embodiment, the present invention provides a method for preparing a population of cells containing at least about 60% of cells that are double-positive for the markers HAND1 and MLC2v, the method comprising the step of culturing pluripotent stem cells in a medium containing a retinoic acid receptor antagonist or an inverse agonist.

[0013] In one embodiment, the above method may include, for example, a step of culturing the cells or cell population in a medium lacking vitamin A from the 8th day of culture onward.

[0014] In a further embodiment, the present invention provides a method for screening for drugs suitable for treating or preventing left ventricular disorders, comprising contacting a population of cells described herein with a candidate drug.

[0015] In a further embodiment, the present invention provides a method for screening for cardiotoxicity with respect to a drug, comprising contacting a population of cells described herein with the drug. [Brief explanation of the drawing]

[0016] [Figure 1A-1]This figure shows the retinoic acid pathway gene expression phenotypes in different lumens of the mouse heart. A is a heatmap showing the expression patterns for each analyzed region and stage. Samples were analyzed from microscopically dissected hearts, as illustrated in the right figure. RV corresponds to the presumptive right ventricle, LV to the presumptive left ventricle, and A to the presumptive atrium. The analyzed stages (ST0-ST3) correspond to a series of cardiac developmental stages from the early duct (E8.25) to the end of the loop (E8.5), as shown in the right figure. The genes highlighted below, as indicated by the color codes, are those that are consistently and differentially expressed in the given regions across all analyzed stages. [Figure 1A-2] This figure shows the retinoic acid pathway gene expression phenotypes in different lumens of the mouse heart. A heatmap showing expression patterns for each analyzed region and stage. The analyzed stages (ST0-ST3) correspond to a series of cardiac developmental stages from the early cardiac duct (E8.25) to the end of the loop (E8.5), as shown in the figure on the right. The genes highlighted below, as indicated by the color codes, are those that are consistently and differentially expressed in the given regions across all analyzed stages. [Figure 1B] This figure shows the retinoic acid pathway gene expression phenotypes in different lumens of the mouse heart. B. Overview of pathway enrichment generated by the Reactome analysis tool when genes uniquely upregulated in atrial samples were used as the query dataset. The scale color bar shows the p-value of the enrichment. The arrows adjacent to the scale bar show the p-value of the pathway: retinoid metabolism and transport, and the enriched nodes of this pathway are highlighted in the color of the corresponding p-value. [Figure 1C-D]This figure shows the retinoic acid pathway gene expression phenotypes in different cavities of the mouse heart. Ci Reactome results for the pathway: Retinoid metabolism and transport were ranked 16th out of the top 25 most relevant pathways and were found to be enriched within a gene set consisting of genes specifically upregulated in atrial samples. Cii List of genes found to be upregulated within the retinoid metabolism and transport pathway, according to the Reactome analysis of genes specifically upregulated in atrial samples. D Gorilla GO-term analysis results for retinoic acid (RA) pathway-related GO terms enriched within the gene set (genes specifically upregulated in atrial samples). Hypergeometric p-values ​​(FDRs) are shown in parentheses. [Figure 1E] This figure shows the retinoic acid pathway gene expression phenotypes in different lumens of the mouse heart. E is a graph showing the normalized read count of RA direct target genes (i) or genes involved in the RA pathway (ii, highlighted by Reactome analysis in panel Cii). As highlighted in panel A, it shows expression for micro-dissected samples from mouse hearts, namely A (presumptive atrium), RV (presumptive right ventricle), and LV (presumptive left ventricle). * indicates a significant difference between group A and group LV (red for p<0.05, black for p<0.001), and # indicates a significant difference between group A and both groups LV and RV (red for p<0.05, black for p<0.001). [Figure 2] This figure shows hPSC proliferation and cardiac differentiation. A. Schematic diagram of the hPSC cardiomyocyte differentiation protocol showing different steps and components used in the protocol. B. Representative image of hPSCs seeded as single cells for cardiac differentiation and subsequently differentiated using the left ventricular cardiomyocyte protocol. [Figure 3A-D]This figure shows the determination of lumen identity in cardiomyocytes produced using the left ventricular cardiomyocyte protocol. A. Representative confocal micrograph showing immunostaining of MLC2V (green) in the upper panel and HAND1 (green) in the lower panel. Cells were stained on day 20 of the differentiation protocol. Cells were co-stained with TNNT2 to identify all cardiomyocytes and with DAPI to visualize all cells. Scale bar represents 100 μm. B. Representative flow cytometry analysis of the proportion of TNNT2+ cells in the day 20 population. Unstained control cells may appear blue. C. Magnified confocal micrograph showing immunostaining of MLC2V (green) and HAND1 (red) to highlight myofibril tissue at day 20 of differentiation. Cells were co-stained with DAPI to visualize all cells. Scale bar represents 35 μm. D. Representative flow cytometry analysis of the proportion of HAND1+ / MLC2V+ cells in the day 20 population. [Figure 3E] This figure shows the determination of lumen identity in cardiomyocytes produced using the left ventricular cardiomyocyte protocol. The graph shows the relative expression levels of TBX5, IRX4, and MYL2 on day 0 (D0), day 8 (D8), and day 20 (D20) as determined by qRT-PCR. Data were normalized to the housekeeping gene PBGD. T-tests were performed; * indicates a significant difference (p<0.01), and # indicates a significant difference (p<0.050). [Figure 4A-B] This figure shows the functional characterization of cardiomyocytes produced using a left ventricular cardiomyocyte protocol. The LEAP signal of typical 20-day cardiomyocytes acquired by the Axion Biosystems MEA system demonstrates at the individual cell level that cells exhibit sharp repolarization in the ventricular action potential shape, i.e., a plateau followed by a phase not seen in commercially available cardiomyocytes (gray line). The Bi 20000 electrode is used to measure the field potential of the entire well of differentiated 35-day cardiomyocytes by the Maxwell Biosystems MEA system. Principal component analysis of the electric field potentials shown in Bi 20000 indicates that only one cell population can be identified. [Figure 4C-J] This figure shows the functional characterization of cardiomyocytes produced using the left ventricular cardiomyocyte protocol. C The graph showing the average beat rate of cardiomyocytes at day 20, determined using di-4-ANEPPS and optical mapping (CellOptic), demonstrates that even at the early stage of differentiation at day 20, these cardiomyocytes have a low beat rate (slow). The beat rate of commercially available cardiomyocytes is shown by the gray line. D The graph showing the analysis of cardiomyocytes at day 20 using the DJ Axion Biosystems Maestro Pro MEA system. The average measured value of commercially available cardiomyocytes is shown by the gray line. D The beating cycle analysis demonstrates the regularity and uniform periodicity of the beating. E Conduction velocity analysis demonstrates that LV-like cardiomyocytes have a conduction velocity close to that of neonatal cardiomyocytes (0.3 mm / ms). F The average beating amplitude analysis demonstrates that LV-like cardiomyocytes have stronger contractility than commercially available cardiomyocytes. G Excitation-contraction delay analysis demonstrates that LV-like cardiomyocytes take longer to contract after action potential initiation than commercially available cardiomyocytes. This may be because LV-like cardiomyocytes exhibit a longer plateau associated with slow calcium channel opening and closing, typical of ventricular cardiomyocytes (see Figure 4A). H Field potential interval (FDP) analysis demonstrates that it is in the range of 300 ms to 500 ms. I Action potential rise time (Trise) analysis shows the rapid firing ability of LV-cardiomyocytes. Ji Action potential duration (ADP) analysis at 30% (ADP30), 50% (ADP50), and 90% (ADP90) of depolarization shows that ADP50 and ADP90 are not far apart, consistent with the presence of a plateau and subsequent rapid repolarization, which is consistent with the ventricular action potential shape. Action potential triangulation, determined by the ratio of Jii ADP50 to APD90, identifies LV-like cardiomyocytes (consistent with ventricular shape) that exhibit a triangular action potential shape, i.e., rapidly repolarizing after a plateau, in contrast to commercially available cardiomyocytes without a plateau. [Figure 4K-L]Figure showing the functional characterization of cardiomyocytes produced using the left ventricular cardiomyocyte protocol. Representative plot of the mean calcium transient (CaT) of day 20 cardiomyocytes determined using K Fura-4F. Data show a long CaT duration. Graph showing analysis of CaT obtained from day 20 cardiomyocytes described in L F. Li CaT rise time (time to peak, Tpeak), and Lii CaT duration at 50% (CaTD50), 75% (CaTD75), and 90% (CaTD90) decay. [Figure 5] Figure showing the intracellular characterization of day 20 cardiomyocytes produced using the left ventricular cardiomyocyte protocol. A Transmission electron microscope (TEM) image shows the ultrastructure of cardiomyocytes at day 20 of differentiation, highlighting (i) the nucleus, (ii) mitochondria, and (iii) sarcomeres. B Graph of sarcomere length at 20, 40, and 60 days after differentiation shows that the mean sarcomere length at day 20 is smaller than that of typical human adult cardiomyocytes (gray line), but over time the cells increase their sarcomere length and by day 60 have a length close to that of typical adult ventricular cardiomyocytes. C Graph of the mean mitochondrial DNA copy number at days 0, 10, 20, 40, and 60 of differentiation shows a sharp increase at day 20, suggesting an increase in mitochondrial activity thereafter. D Representative confocal micrograph of day 20 differentiated cardiomyocytes shows mitochondria stained with MitoTracker, endogenously GFP-tagged MLC2v, and nuclear stain Hoechst. These images highlight the extensive interconnected mitochondrial network typical of neonatal cardiomyocytes. [Figure 6A-C]This is a figure showing the characterization of the cell structure and maturity of day 20 cardiomyocytes produced using the left ventricular cardiomyocyte protocol. A Representative confocal micrographs showing immunostaining of connexin-43, a mature gap junction marker, and sarcomeric alpha-actinin demonstrate that, as in the case of rat neonatal cardiomyocytes (Ai), some cells already exhibit mature gap junctions on day 20 of differentiation. B Representative confocal micrographs showing immunostaining of telethonin and sarcomeric alpha-actinin, mature Z-disk markers, demonstrate that on day 20 of differentiation, cells exhibit Z-disk maturation similar to that seen in rat neonatal cardiomyocytes (Bi). C Representative confocal micrographs showing immunostaining of M-protein and myomesin, mature M-band markers, demonstrate that on day 20 of differentiation, cells exhibit M-band maturation similar to that seen in rat neonatal cardiomyocytes (Ci). [Figure 6D] This is a figure showing the characterization of the cell structure and maturity of day 20 cardiomyocytes produced using the left ventricular cardiomyocyte protocol. D Representative confocal micrographs showing immunostaining of desmin and MyBP-C, mature cardiomyocyte-related intermediate diameter filaments, demonstrate that, as in the case of rat neonatal cardiomyocytes (Di), desmin is seen concentrated around the Z-disks (and connecting them as expected in mature cardiomyocytes) in addition to filamentous signals. [Figure 7A-B] This is a figure showing the characterization of engineered heart tissue generated using day 40 cardiomyocytes produced using the left ventricular cardiomyocyte protocol. A Representative optical micrograph of an EHT showing the size of the EHT generated from pole to pole. B A graph showing the beating rate of the EHT over time in culture, demonstrating that beating began only on day 9 and the subsequent spontaneous beating rate was very low or absent. However, the EHT could be paced (red line), demonstrating that these cardiomyocytes exhibited a relatively mature ventricular beating phenotype. This is because adult ventricular cardiomyocytes beat only when stimulated. [[ID=,7]] [Figure 7C-F]This figure shows the characterization of designed cardiac tissue generated using 40-day cardiomyocytes produced using the left ventricular cardiomyocyte protocol. C / D / E / F Graphs comparing the EHT properties of EHT generated over time from cultured LV-like cardiomyocytes (LV-EHT, black) with EHT generated from other hPSC-derived cardiomyocytes (gray). C shows the beat rate of the EHT, confirming the inherently lower velocity of LV-EHT. D shows the force generated, confirming that LV-EHT can generate force. E shows the amount of time required to reach 20% of contraction, confirming that LV-EHT exhibits an average contraction time of 20% compared to other EHTs tested. F shows the amount of time required to reach 20% of relaxation, confirming that LV-EHT took the average amount of time to relax compared to other EHTs tested. [Modes for carrying out the invention]

[0017] Left ventricular cardiomyocytes As described herein, the present invention provides a population of cells comprising a substantial number or proportion of left ventricular cells, particularly left ventricular cardiomyocytes.

[0018] The cell population according to the present invention has a considerable degree of left ventricular cell homogeneity. Such a cell population has not been generated previously. As described in the examples herein, the inventors have developed a method for providing such a cell population.

[0019] As used herein, “cell population” or “population of cells” means more than one cell. The cells according to the present invention are cardiomyocytes, in particular left ventricular myocytes.

[0020] Left ventricular cells can be characterized by the presence of specific markers. One such marker may be TBX5. Tbx5 is a gene located on the long arm of chromosome 12. Tbx5 produces a protein called T-box 5, which acts as a transcription factor. The Tbx5 gene is involved in the development of the forelimbs and heart. This gene influences early forelimb development by inducing fibroblast growth factor FGF10. TBX5 is expressed only in the primary cardiac region, and therefore in the progenitor cells of the LV and atrial. In adult humans, TBX5 expression is highest in the atrial appendages, followed by the lungs, left ventricle, and esophagus.

[0021] A further marker may be IRX4 (also known as Iroquois class homeodomain protein IRX-4 or Iroquois homeobox protein 4), a protein encoded by the IRX4 gene in humans. IRX4 is a member of the Iroquois homeobox gene family, which appears to play multiple roles in pattern formation in vertebrate embryos. Among its related pathways are cardiac development and cardiac progenitor cell differentiation. While IRX4 is not sufficient for ventricular cavity formation in mice, it is required for the establishment of several components of the ventricular-specific gene expression program. In the absence of genes under the control of IRX4, ventricular function deteriorates and cardiomyopathy develops.

[0022] A further marker could be HAND1 (Heart- and neural crest derivatives-expressed protein 1), a protein encoded by the HAND1 gene in humans.

[0023] The HAND1 gene, a member of the HAND subclass of basic helix-loop-helix (bHLH) transcription factors, is essential for the development and differentiation of three distinct embryological lineages, including cardiac cardiomyocytes, placental trophoblasts, and yolk sac angiogenesis. Most closely related to the twisted bHLH gene in amino acid identity and embryonic expression, HAND1 can form homodimers and heterodimer combinations with multiple bHLH partners, mediating transcriptional activity in the nucleus.

[0024] HAND1 plays a role in cardiac morphogenesis. At 3 weeks of fetal development, the initial heart (bilateral symmetrical duct) undergoes characteristic right-sided looping, forming an asymmetrical structure with bulges representing the initial ventricles and atria of the heart. Developing from cells originating from the primary cardiac region of the cardiac primordium, HAND1 is expressed from both sides of the duct toward the ventral surface of the caudal cardiac segment and the aortic sac, and is then restricted to the lateral curvature of the left ventricle of the looped heart.

[0025] Along with HAND2 (a member of the bHLH transcription factor family), the complementary and overlapping expression patterns of HAND1 are thought to play a role in interpreting asymmetric signaling in the developing heart, resulting in characteristic looping. These two genes are involved in embryonic cardiac development based on a crucial HAND gene dosage system. Overexpression or underexpression of HAND1 can lead to morphological abnormalities, most notably cleft lip and palate. Expression has been modeled by phosphorylation knock-in, which turns on and off gene expression that induces craniofacial abnormalities. HAND1 is associated with hypoplastic left heart syndrome.

[0026] A further marker may be ventricular myosin light chain-2 (MLC-2v), which refers to ventricular myocardial morphology 2 (MYL2) of myosin light chains. MLC2v is strongly expressed in ventricular myocardium. MLC-2v plays an essential role in early embryonic cardiac development and function and represents one of the earliest markers of ventricular specificity. During early development (E7.5–8.0), MLC-2v is expressed within the cardiac primordium. The expression pattern of MLC-2v becomes restricted to the ventricular segment of the linear duct at E8.0 and remains restricted to the ventricles until adulthood.

[0027] In one aspect of the present invention, the cell population comprises at least about 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of cells that are double-positive for the markers HAND1 and MLC2v.

[0028] In one embodiment of the present invention, the cell population comprises at least about 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of cells that are positive for the marker TBX5, as well as HAND1 and MLC2v.

[0029] In one embodiment of the present invention, the cell population comprises at least about 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of cells that are positive for the markers TBX5, HAND1, MLC2v, and IRX4.

[0030] In one aspect of the present invention, the cell population comprises at least about 65, 70, 75, 80, 85, 90, 95, or 100% of cells that are double-positive for the markers HAND1 and MLC2v.

[0031] In one aspect of the present invention, the cell population comprises at least about 65, 70, 75, 80, 85, 90, 95, or 100% of cells that are positive for the marker TBX5, as well as for HAND1 and MLC2v.

[0032] In one aspect of the present invention, the cell population comprises at least about 65, 70, 75, 80, 85, 90, 95, or 100% of cells that are positive for the markers TBX5, HAND1, MLC2v, and IRX4.

[0033] In one embodiment, a population of cells containing at least approximately 85% of cells that are double-positive for the markers HAND1 and MLC2v.

[0034] In one embodiment, a population of cells containing at least about 90% of cells that are double-positive for the markers HAND1 and MLC2v.

[0035] In one embodiment, a population of cells containing at least about 95% of cells that are double-positive for the markers HAND1 and MLC2v.

[0036] In one embodiment, a population of cells containing at least about 85% of cells that are positive for the marker TBX5, as well as for HAND1 and MLC2v.

[0037] In one embodiment, a population of cells containing at least about 90% of cells that are positive for the marker TBX5, as well as for HAND1 and MLC2v.

[0038] In one embodiment, a population of cells comprising at least about 95% of cells that are positive for the marker TBX5, as well as for HAND1 and MLC2v.

[0039] In one embodiment, a population of cells containing at least approximately 85% of cells that are positive for the markers TBX5, HAND1, MLC2v, and IRX4.

[0040] In one embodiment, a population of cells containing at least about 90% of cells that are positive for the markers TBX5, HAND1, MLC2v, and IRX4.

[0041] In one embodiment, a population of cells containing at least about 95% of cells that are positive for the markers TBX5, HAND1, MLC2v, and IRX4.

[0042] In one aspect of the present invention, cells may be positive for HAND1 and / or MLC2v.

[0043] Methods for testing for the presence of such markers are commonplace for those skilled in the art. For example, FACS analysis or immunofluorescence can be used as described in this embodiment.

[0044] Left ventricular cells are different from right ventricular cells. Left ventricular cells and right ventricular cells originate from separate and distinct cell lineages.

[0045] In one aspect of the present invention as described herein, a population of cells may be further purified to increase the percentage of left ventricular cells in the population. Left ventricular cells are selected from the population using methods known in the art, for example, by selecting a subset of cells from a genome-edited cell line based on cell surface markers or other methods, such as reporter tags, or by killing unwanted cells based on killer gene selection.

[0046] In one embodiment, purification may be carried out, for example, via metabolic selection. Metabolic selection is a method that relies on removing glucose from the culture medium and replacing it with lactate, so that very few cell types, including cardiomyocytes, can survive under these conditions. Metabolic selection can be used for extended periods to enrich left ventricular cardiomyocytes. In one example, cells may be cultured in metabolic selection medium between days 10 and 12 of the culture protocol as described herein.

[0047] In a further embodiment, purification can be performed via the use of beads or by flow cytometry, based on a surface marker gene for detecting the population according to the present invention. Purification may also require the use of a reporter cell line, for example, in which the HAND1 and MLC2V loci are tagged with fluorescent gene sequences, and the cells can be purified based on being double-positive to a reporter gene tagged to the HAND1 and MLC2V loci.

[0048] In one aspect of the present invention as described herein, a population of cells can be further matured by adding a supplement to the culture medium that may include, but is not limited to, triiodothyronine (T3) (16028, Cayman), insulin-like growth factor 1 (IGF-1) (I1271, Sigma-Aldrich), dexamethasone (Dex) (D4902, Sigma-Aldrich), and fatty acids, such as palmitic acid (810105P, Sigma-Aldrich), oleic acid (O3008, Sigma-Aldrich), and linoleic acid (L9530, Sigma-Aldrich).

[0049] In one embodiment, maturation can also be promoted by growing cells on an alternative substrate, such as fibronectin (PHE0023, Thermo Fisher), laminin 511 (LN511, BioLamina), laminin 521 (LN521, BioLamina), or a combination thereof.

[0050] In one aspect of the present invention as described herein, a high proportion of the population, for example, at least about 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the population exhibits various characteristics of maturity, for example, the cells present a well-developed myofibrils arrangement including a distinct Z-disk, conduction velocity close to that of neonatal cardiomyocytes, sarcomere length close to that of fully mature ventricular cardiomyocytes, an extensive interconnected mitochondrial network typical of neonatal cardiomyocytes, and expression of various maturity sarcomere markers, such as sarcomere alpha-actinin, M-protein, and telesonin.

[0051] Left ventricular dysfunction The present invention as described herein can be used for the treatment or prevention of left ventricular disorders.

[0052] The left ventricle is one of the four chambers of the heart. It is located in the lower left part of the heart, below the left atrium, and is separated by the mitral valve. When the heart contracts, blood eventually returns to the left atrium, and then passes through the mitral valve into the left ventricle. From there, the blood is pumped through the aortic valve to the aortic arch and then to the rest of the body. The left ventricle is the thickest of the heart chambers and is responsible for pumping oxygenated blood to all the tissues of the body. In contrast, the right ventricle only pumps blood to the lungs.

[0053] Various conditions can affect the left ventricle and interfere with its normal function. The most common is left ventricular hypertrophy, which causes hypertrophy and hardening of the muscular tissue that makes up the left ventricular wall, usually due to uncontrolled blood pressure. Another condition that can affect this area is left ventricular noncompaction cardiomyopathy, in which the muscular tissue surrounding the left ventricle becomes cavernous or "impaired noncompaction."

[0054] In one embodiment, the cells according to the present invention may be used to treat or prevent damage to the left ventricle. In particular, the present invention can be used to help identify cardiotoxic drugs or as a treatment for hearts damaged by these drugs. For example, left ventricular damage may be involved in or result of drug-induced damage to the heart, such as drug-induced heart failure. Some drugs or treatments may cause damage to the heart, for example, the left ventricle, as a side effect of their intended therapeutic use. This may apply, for example, to some cancer treatments or drugs.

[0055] In one aspect of the present invention, the left ventricular disorder may be selected from myocardial infarction, heart failure, left ventricular hypertrophy, hypoplastic left heart syndrome, and left ventricular noncompaction cardiomyopathy (LVNC).

[0056] In one aspect of the present invention, the left ventricular disorder is myocardial infarction.

[0057] Myocardial infarction (MI), commonly known as a heart attack, occurs when a portion of the heart is deprived of oxygen due to an obstruction of the coronary arteries. The coronary arteries supply oxygenated blood to the heart muscle (myocardium). Without oxygen, the muscle cells supplied by the obstructed artery begin to die (infarction). The causes of myocardial infarction are often atherosclerosis, the accumulation of fatty plaque, and other substances in the arteries. Plaque is covered with an inner lining of fibrous material. When this inner lining ruptures, the plaque is released and a blood clot forms. Myocardial infarction is practically synonymous with left ventricular infarction, as almost all myocardial infarctions affect the left ventricle. The heart has three coronary arteries, two of which always nourish the left ventricle, and a portion of the right coronary artery can exclusively nourish the right ventricle. Only 25% of heart attacks affect the right coronary artery, and of these, 1 / 12 affect only the right ventricle.

[0058] In one aspect of the present invention, the left ventricular disorder is left ventricular hypertrophy.

[0059] Left ventricular hypertrophy is the enlargement and thickening (increase) of the left ventricular wall. Left ventricular hypertrophy can occur, for example, in response to high blood pressure or cardiac conditions that impair the function of the left ventricle.

[0060] In one aspect of the present invention, left ventricular dysfunction is heart failure.

[0061] Heart failure, sometimes called congestive heart failure, occurs when the heart muscle is not pumping blood as effectively as it should. Certain conditions, such as narrowed arteries in the heart (coronary artery disease) or high blood pressure, can cause the heart to gradually weaken or become too stiff to fill and pump efficiently. Heart failure often occurs after other conditions have damaged or weakened the heart. However, heart failure can occur even if the heart is not weakened, or if the heart becomes too stiff.

[0062] In heart failure, the heart's main pump chambers (ventricles) can become stiff and unable to fill properly during each heartbeat. In some cases of heart failure, the heart muscle is damaged and weakened, causing the ventricles to stretch (dilate) to such an extent that the heart cannot efficiently pump blood throughout the body.

[0063] As time passes, the heart becomes unable to keep up with the normal demands placed on it to pump blood to the rest of the body.

[0064] Ejection fraction is an important measure of how well the heart is pumping and is used to help classify and treat heart failure. In a healthy heart, the ejection fraction is 50% or higher, meaning that more than half of the blood that fills the ventricles is pumped out with a single heartbeat. Heart failure can occur even when the ejection fraction is normal. This happens when the heart muscle hardens due to conditions such as high blood pressure. Heart failure can occur on the left side of the heart (left ventricle), the right side (right ventricle), or both sides. Generally, heart failure begins on the left side, especially in the left ventricle.

[0065] In one aspect of the present invention, the left ventricular disorder is left ventricular hypertrophy.

[0066] In left ventricular noncompaction cardiomyopathy (LVNC), the left inferior space of the heart, called the left ventricle, contains bundles or fragments of muscle extending into the space. These muscle fragments are called trabeculae. During development, the myocardium is a spongy network of muscle fibers.

[0067] As normal development progresses, the trabeculae become compact, transforming the myocardium from spongy to smooth and solid. LVNCs occur when compaction does not occur. These trabecular meshworks typically develop at the base of the heart, called the apex, but can be found anywhere in the left ventricle. Individuals with LVNCs may also have other types of myocardial disease (hypertrophic cardiomyopathy, dilated cardiomyopathy, or restrictive cardiomyopathy).

[0068] In one aspect of the present invention, the left ventricular disorder is hypoplastic left heart syndrome.

[0069] Hypoplastic left heart syndrome (HLHS) is a congenital anomaly that affects the normal blood flow through the heart. When it occurs in a fetus during pregnancy, the left side of the heart does not develop properly. Hypoplastic left heart syndrome is a type of congenital heart disease. In HLHS, the left ventricle of the heart does not develop properly and is much smaller than normal. The mitral valve between the left ventricle and the left superior filling chamber (left atrium) is often closed or very small.

[0070] The cell population according to the present invention can be used as a cell therapy for treating or preventing left ventricular disorders, as described herein. Cell therapy is a treatment in which cell material is injected, transplanted, or implanted into a patient, which generally refers to intact living cells.

[0071] In this case, the cell population according to the present invention can be administered to subjects with left ventricular dysfunction. The present invention includes the use of the cell population according to the present invention as cell therapy.

[0072] In one embodiment, the present invention provides a cell population for use as a cell therapy for treating or preventing left ventricular disorders.

[0073] The present invention also provides the use of a cell population according to the present invention as a cell therapy for treating or preventing left ventricular disorders.

[0074] Methods for producing cell populations In one aspect of the present invention, a method for producing a population of cells as described herein is provided.

[0075] The present invention provides an improved method for generating a population of cells that include an increased number or proportion of left ventricular cardiomyocytes, i.e., levels that may be practically and therapeutically useful. The advantages of the present invention over protocols known in the art are the rapid achievement of a high-quality cell population and the acquisition of more than 85% LV cardiomyocytes. Cells produced according to the present invention are more mature than cells produced under equivalent monolayer conditions using methods known in the art.

[0076] The method according to the present invention comprises the step of culturing pluripotent stem cells in a medium containing a retinoic acid receptor antagonist or an inverse agonist. It was not evident from previous studies in the art that antagonism of the retinoic acid receptor increases left ventricular cardiomyocyte differentiation. This was a surprising discovery by the inventors.

[0077] Thus, the present invention provides a method for producing a population of cells of the present invention, comprising the step of culturing pluripotent stem cells in a medium containing a retinoic acid receptor antagonist or an inverse agonist.

[0078] "Pluripotent stem cells" refer to stem cells that have the potential to differentiate into any of the three layers of the lung: the endoderm (endoderm, gastrointestinal tract, lungs), the mesoderm (muscle, bone, blood, urogenital organs), or the ectoderm (epidermal tissue and nervous system).

[0079] In a preferred embodiment, the pluripotent stem cells are embryonic stem cells, most preferably human embryonic stem cells.

[0080] As is well known in this art, embryonic stem cells are pluripotent stem cells derived from early embryos. Embryonic stem cell lines (ES cell lines) are cultures of cells derived from epiblast cells of the inner cell mass (ICM) of a blastocyst, or from early morula-stage embryos. A blastocyst is an early embryo in humans, approximately 5-7 days old, and composed of 100-300 cells. ES cells are pluripotent and, during development, give rise to derivatives of all three primary germ layers: ectoderm, endoderm, and mesoderm. In other words, they can differentiate into each of the adult cell types.

[0081] In alternative embodiments, the pluripotent stem cells are induced pluripotent stem cells, most preferably human induced pluripotent stem cells.

[0082] Induced pluripotent stem cells are a type of pluripotent stem cell that is artificially prepared from non-pluripotent cells, typically adult somatic cells, or terminally differentiated cells, such as fibroblasts, hematopoietic cells, muscle cells, nerve cells, and epidermal cells, by inserting certain genes called reprogramming factors or by using non-integrated mRNA or chemical substances.

[0083] Cells can be transduced, transfected, electroporated, or nucleofected by the presence of any one or a combination of the following: the transcription factors SOX2 (SRY-related HMG-box 2), OCT4 (octamer-binding transcription factor 4), KLF4 (Kruppel-like factor 4), and inhibitors of c-MYC (V-myc tricohyloma virus oncogene homolog), L-MYC, N-MYC, NANOG, LIN28, SALL4, UTF1, TBX3, p53, and / or p21, and / or epigenetic modifiers, such as 5'-azacitidine and RG108. Those skilled in the art will understand that this list is not exhaustive and is merely an example of some of the factors or combinations of factors used to generate induced pluripotent stem (iPS) cells similar to hES cells. These factors influence the conversion of non-pluripotent cells to iPS cells. It is known in the art that adult mice can be derived from iPS cells. These reprogrammed cells acquire ES cell-like characteristics and therefore have the potential to generate any tissue (Boland et al., (2009) Nature 461: pp. 91-94; Quinlan et al., (2011) Cell Stem Cell 9: pp. 366-373).

[0084] Those skilled in the art are familiar with methods for culturing, isolating, or producing embryonic stem cells.

[0085] For example, hES cells can be obtained from blastocysts using the methods described in, for example, Thomson et al., (1995) Proc. Natl. Acad. Sci. USA Vol. 92: pp. 7844-7848; Thomson et al., (1998) Science Vol. 282: p. 1145; Thomson & Marshall (1998) Curr. Top. Dev. Biol. Vol. 38: pp. 133-165; Reubinoff et al., (2000) Nat. Biotechnol. Vol. 18: pp. 399-404; Chen and Egli et al., Cell Stem Cell, February 2009, Vol. 6; p. 4.

[0086] Established ES cell lines are also available. Various hES cell lines are known, and their growth and reproduction conditions are defined, for example, in hES cell lines Shef6, WA01, WA07, WA09, WA13, and WA14. Any ES cell or ES cell line is suitable for use according to the present invention.

[0087] ES cells can be obtained from blastocysts, by culturing the inner cell mass of blastocysts, or from cultures of established cell lines. Therefore, as used herein, the term “ES cells” may refer to ES cells obtained from the inner cell mass of blastocysts, ES cells obtained from cultures of cells from the inner cell mass, and ES cells obtained from cultures of ES cell lines.

[0088] iPS cells can be obtained by various methods. For example, see the method described by Takahashi et al. (2007) in Cell, Vol. 126 (No. 4): pp. 663-666. iPS cells are morphologically similar to hES cells and express various hES cell markers.

[0089] Human embryonic stem cells can be defined by the presence of several transcription factors and cell surface proteins, as determined by immunohistochemistry and / or flow cytometry. Suitable transcription factor markers include OCT4, NANOG, and SOX2, while suitable antigen markers include glycolipids SSEA-1 (in its absence), SSEA3, and SSEA4, as well as keratan sulfate antigens TRA-1-60 and TRA-1-81. Such methods are commonplace in the art.

[0090] iPS cells can be defined by the presence of several transcription factors and cell surface proteins, as determined by immunohistochemistry and / or flow cytometry. Suitable transcription factor markers include OCT4, NANOG, and SOX2, while suitable antigen markers include glycolipids SSEA-1 (in its absence), SSEA3, and SSEA4, as well as keratan sulfate antigens TRA-1-60 and TRA-1-81. Such methods are commonplace in the art.

[0091] The pluripotency of embryonic stem cells is determined by spontaneous or directional differentiation in vitro, or by the development of approximately 0.5–10 × 10¹⁶ embryonic stem cells in the hindlimb muscles of 8–12-week-old male SCID mice. 6 This can be confirmed by injecting cells, which generates a teratoma that exhibits at least one cell type from each of the three germ layers.

[0092] Suitable cells are known to those skilled in the art. For example, WA09 cells, WA01 cells, AICS cells (iPSCs), or disease cell lines, such as Progeria cells (iPSCs), can be used.

[0093] Suitable retinoic acid receptor antagonists or inverse agonists are known in the art.

[0094] Antagonists are a type of receptor ligand or drug that, unlike agonists which activate receptors, binds to receptors and blocks or attenuates biological responses by blocking them. These are sometimes called blockers, and examples include alpha-blockers, beta-blockers, and calcium channel blockers. Pharmacologically, antagonists have affinity but no efficacy towards their congener receptors; binding interferes with interaction and inhibits the function of agonists or inverse agonists towards the receptor. Antagonists may mediate their actions by binding to the active or allosteric site of the receptor, or they may interact at intrinsic binding sites not normally involved in the biological regulation of receptor activity. Antagonist activity is reversible or irreversible depending on the lifespan of the antagonist-receptor complex, which depends on the nature of the antagonist-receptor binding. Most drug antagonists achieve their efficacy by competing with endogenous ligands or substrates at structurally defined binding sites on the receptor.

[0095] Antagonists are different from inverse agonists. Inverse agonists are drugs that bind to the same receptor as agonists but induce a pharmacological response opposite to that of the agonist. Neutral antagonists are inactive in the absence of either an agonist or an inverse agonist, but can block the activity of either. Inverse agonists exert the opposite effect to agonists, but the effects of both are blocked by antagonists. Agonists increase receptor activity above the basal level, while inverse agonists decrease activity below the basal level.

[0096] In one aspect of the present invention, AGN193109 (sc-210768, Santa Cruz Biotechnology), which is marketed as a high-affinity panretinoic acid receptor (RAR) antagonist, can be used.

[0097] In one aspect of the present invention, BMS493 (available, for example, from StemCell Technologies, Tocris, Merck, R&D Systems) can be used. BMS493 is a pan-RA inverse agonist.

[0098] The basal media that can be used in accordance with the present invention as described herein include, but are not limited to, StemPro-34, Dulbecco's Modified Eagle Medium (DMEM), Ham's F10 Medium, Ham's F12 Medium, Advanced DMEM, Advanced DMEM / F12, Minimum Essential Medium, DMEM / F-12, DMEM / F-15, Liebovitz L-15, RPMI 1640, Iscove Modified Dulbecco's Medium (IMDM), OPTI-MEM SFM (Invitrogen Inc.), N2B27, MEF-CM, and specified basal ESC media, ExVivo 10, ESGrow, or combinations thereof.

[0099] In one embodiment, the culture medium is RPMI1640 medium, which is available, for example, from LifeTech.

[0100] In one embodiment, the culture medium includes, for example, an insulin-free B27 supplement available from Gibco (ThermoFisher Scientific MA, USA).

[0101] In one embodiment, the culture medium may contain Wnt. The Wnt signaling pathway is a group of signal transduction pathways that begin with proteins that transmit signals to cells via cell surface receptors. The Wnt signaling pathway uses either nearby intercellular communication (paracrine) or same-cell intercellular communication (autocrine).

[0102] In one embodiment, the culture medium may contain a Wnt agonist.

[0103] In one embodiment, the culture medium may contain a glycogen synthase kinase-3 (Gsk3) inhibitor. For example, the low molecular weight Chiron, CHIR99021 (a Gsk3 inhibitor), may be present in the culture medium. A suitable source of Chiron for use according to the present invention is commercially available, for example, from Selleck Chem (S2924).

[0104] In one embodiment, the culture medium may contain approximately 1 to 12 μM / ml of Chiron, for example, approximately 2 to 5 or 2 to 3 μM / ml of Chiron.

[0105] In one embodiment, the culture medium may also contain BMPs. Originally discovered for their ability to induce bone and cartilage formation, BMPs are now considered important in regulating tissue structure throughout the body. Seven BMPs were originally discovered. Six of these (BMP2 to BMP7) belong to the transforming growth factor beta superfamily of proteins. BMP1 is a metalloproteinase. Subsequently, 13 more BMPs were discovered, bringing the total to 20.

[0106] In one embodiment, the culture medium contains BMP4. A suitable source of BMP4 for use according to the present invention is commercially available, for example, from R&D Systems (314-BP-010).

[0107] In one embodiment, the culture medium may contain approximately 1 to 10 ng / ml of BMP, for example, BMP4, for example, approximately 1 to approximately 6 ng / ml, or approximately 3 to approximately 5 ng / ml of BMP, for example, BMP4.

[0108] In one embodiment, the culture medium may contain activin, Nodal, or TGFβ. For example, exogenous activins, such as activin A, activin AB, and / or activin B, may be present in the cell culture medium. Suitable sources of activin for use according to the present invention are commercially available, for example, from R&D Systems (catalog no. 338-AC / CF) or Peprotech (catalog no. 120-14).

[0109] In one embodiment, the culture medium contains activin A.

[0110] In one embodiment, the culture medium may contain about 1 to 10 ng / ml of activin, for example, activin A, or about 3 to about 10 ng / ml of activin. In another embodiment, the culture medium may contain about 5 ng / ml of activin, for example, activin A.

[0111] Nodal is a secreted protein encoded in humans by the NODAL gene located on chromosome 10. It belongs to the transforming growth factor beta (TGF-β) superfamily. In one embodiment, the culture medium can contain Nodal. For example, exogenous Nodal may be present in the cell culture medium. A suitable source of Nodal for use according to the present invention is commercially available, for example, from R&D Systems (catalog no. 3218-ND / CF). Those skilled in the art can determine an appropriate amount of Nodal that may be included in the culture medium.

[0112] Transforming growth factor beta (TGF-β) is a multifunctional cytokine belonging to the transforming growth factor superfamily. In one embodiment, the culture medium can contain TGF-β. For example, exogenous TGF-β, such as TGF-β1, TGF-β2, and / or TGF-β3, may be present in the cell culture medium. A suitable source of TGF-β for use according to the present invention is commercially available, for example, from R&D Systems (catalog no. 7754-BH / CF). Those skilled in the art can determine an appropriate amount of TGF-β that may be included in the culture medium.

[0113] In one embodiment, the culture medium may also contain FGF. FGF is a family of growth factors whose members are involved in angiogenesis, wound healing, embryogenesis, and various endocrine signaling pathways. As used herein, the term “FGF” is intended to encompass any member of the FGF family, e.g., FGF1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23. FGF is commercially available.

[0114] In one embodiment, FGF is FGF2. A suitable source of FGF2 for use according to the present invention is commercially available, for example, from R&D Systems (233-FB-025).

[0115] In one embodiment, the culture medium may contain about 1 to 20 ng / ml of FGF, for example, FGF2, for example, about 3 to about 10 ng / ml. In another embodiment, the culture medium may contain about 5 ng / ml of FGF, for example, FGF2.

[0116] In one embodiment, the culture medium may also contain a Wnt inhibitor. Such inhibitors are known to those skilled in the art. In one embodiment, such inhibitors may be, for example, IWR1, IWP2, C59, or DKK.

[0117] In one embodiment, the culture medium may also contain L-ascorbic acid (also known herein as L-AA).

[0118] In one embodiment, the culture medium contains vitamin A. In another embodiment, the culture medium does not contain vitamin A.

[0119] Those skilled in the art can determine the appropriate amount of such factors to be added to the culture medium.

[0120] The present invention encompasses cell populations obtained or obtainable by the methods described herein. Such cell populations can be used in any of the methods or uses described herein. In one embodiment, the present invention encompasses producing a cell population described herein according to the method of the present invention, and then using the cells to treat or prevent a left ventricular disorder described herein.

[0121] In one embodiment, the method includes culturing pluripotent stem cells in the medium described herein from day 0 of pluripotent stem cell differentiation. Day 0 can be defined as the first day on which pluripotent stem cell differentiation begins.

[0122] As described herein, cells can be plated at a predetermined density and then grown in pluripotency maintenance medium until a specific confluence is reached. Those skilled in the art can determine the appropriate quantity of cells, as well as the time required to achieve the appropriate density and compression.

[0123] A suitable method for providing a cell population according to the present invention is as described in this embodiment.

[0124] In one embodiment, the present invention provides a method for culturing left ventricular cardiomyocytes, the method comprising culturing embryonic stem cells with Wnt / Wnt agonist / GSK3b inhibitor (preferably Chiron), BMP (preferably BMP4), activin (preferably activin A), and FGF (preferably FGF2). In one embodiment, the culture may last for about 1 or 2 days, preferably about 1 day. The amounts of each component may be as described above.

[0125] In one embodiment, cells may be cultured with B27 (preferably without insulin) and a retinoic acid inhibitor (preferably AGN193109) or an inverse agonist. Such a medium is referred to herein as “cardiac medium 1”. L-ascorbic acid may be added as needed. The Wnt inhibitor IWR is added on day 2.

[0126] In one embodiment, cells are incubated in cardiac medium 1 for approximately 8 days. The first day of incubation in cardiac medium 1 is called day 0. Cells may be pluripotent on day 0. Cells are incubated with the above factors (Wnt / Wnt agonist / GSK3b inhibitor (preferably Chiron), BMP (preferably BMP4), activin (preferably activin A), and FGF (preferably FGF2) on day 0, day 1, and optionally on day 2.

[0127] In one embodiment, the culture medium is changed on approximately day 8 to a medium containing vitamin B27, which does not contain vitamin A but may contain insulin. Optionally, the medium may contain a retinoic acid inhibitor (preferably AGN193109) or an inverse agonist. Such a medium is referred to herein as “cardiac medium 2”. Optionally, cardiac medium 2 may contain L-ascorbic acid.

[0128] In one embodiment, the cells are incubated in cardiac medium 2 for approximately 2 days, or until day 10-12.

[0129] In one embodiment, the culture medium is changed between days 10 and 12 to a medium lacking glucose, containing B27 (vitamin A-free but potentially insulin), L-lactic acid, and additionally containing or not containing a positive or negative retinoic acid inhibitor (preferably AGN193109) or an inverse agonist, and optionally containing L-ascorbic acid. Such a medium is referred to herein as “cardiac medium 3”. In one embodiment, cells may be cultured in cardiac medium 3 for a period of 2 to 4 days, for example, between days 10 and 12.

[0130] In one embodiment, the culture medium is changed to "cardiac medium 2" on approximately day 12-14. L-ascorbic acid may be added if necessary. A retinoic acid inhibitor (preferably AGN193109) or an inverse agonist may be added if necessary. In one embodiment, the cells are incubated in cardiac medium 2 and then incubated until the experiment is completed.

[0131] In one embodiment, the basal medium may be RPMI medium.

[0132] In one embodiment, the culture medium may be as follows: Cardiac culture medium 1: 1. RPMI 1640 medium containing glucose and L-glutamine. 2. B27 supplement - (negative) insulin (preferably 1 ml per 50 ml of RPMI). 3. AGN193109 (preferably with a final concentration of 20-200 nM). Cardiac culture medium 2: 1. RPMI 1640 medium containing glucose and L-glutamine. 2. B27 supplement - Vitamin A (preferably 1 ml per 50 ml of RPMI). 3. In some cases, AGN193109 (preferably with a final concentration of 20-200 nM). Cardiac culture medium 3: 1. RPMI 1640 medium containing L-glutamine but without glucose. 2. B27 supplement - Vitamin A (preferably 1 ml per 50 ml of RPMI). 3. Depending on the case, AGN (preferably a final concentration of 20-200 nM). 4. L-lactic acid (preferably 4 mM)

[0133] This method can include culturing for approximately 20 days or more after day 0.

[0134] In one embodiment of the present invention, the protocol shown in Table 1 below may be followed.

[0135] [Table 1]

[0136] In one embodiment, cells may be cultured in cardiac medium 2 containing L-ascorbic acid for a period of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 days after day 20 of the protocol. Cardiac medium 2 + L-ascorbic acid may be administered every other day.

[0137] Cellular functional characteristics As described herein, cells generated according to the present invention may have functional features or properties related to ventricular cardiomyocytes, particularly left ventricular cardiomyocytes.

[0138] As supported by this embodiment, in one aspect, a cell may exhibit one or more of the following features: 1. Ventricular action potential shape. 2. Low beating rate and / or beating periodicity are already observed by day 20 and become more pronounced over time during culture, especially when the cells are used to produce EHT. The lower the beating rate, the more mature the cells are, i.e., a lower beating rate or lack of spontaneous beating is desirable. Mature ventricular cardiomyocytes do not bend spontaneously, but only when stimulated. The present invention facilitates the production of cells with lower and therefore improved beating rates, but importantly, the cells can still bend when stimulated. 3. Conduction velocity characteristic of neonatal ventricular cells. While a conduction velocity close to that of adult cardiomyocytes is desirable, having a conduction velocity of neonatal cardiomyocytes (0.3 mm / ms) is already very good. The present invention provides cells with a conduction velocity of approximately 0.3 mm / ms, which is an improvement over conventional methods disclosed in the art that yielded a conduction velocity of approximately 0.04 mm / ms. Therefore, the present invention provides a method for improving the conduction velocity of a cell population. 4. The average pulse amplitude is higher than that of commercially available cardiomyocytes. Therefore, the present invention provides a method for improving the strength of a cell population. 5. The excitation-contraction delay is longer than that of commercially available cardiomyocytes, because LV-like cardiomyocytes exhibit a long plateau associated with slow opening and closing of calcium channels. This plateau is typical of ventricular cardiomyocytes. 6. The longer duration of the action potential of ventricular cells results in a longer field potential interval (approximately 400 ms). 7. The action potential rise time (Trise) is fast, and the action potential duration and / or action potential triangulation are maintained to those expected of ventricular cells. 8. Calcium transients (CaT) are similar to those found in adult human ventricular cardiomyocytes. 9. The CaT rise time (time to peak, Tpeak) and CaT duration are slower than those of mature ventricular cells, but are improved compared to conventional methods disclosed in the art.

[0139] In one embodiment, the cells according to the present invention can be paced. This feature is particularly useful in the context of engineered cardiac tissue, as described herein. This embodiment demonstrates that cardiomyocytes can be used to generate EHT exhibiting a relatively mature ventricular beating phenotype (i.e., low or no beating). This is because adult ventricular cardiomyocytes bend only when stimulated. In one embodiment, for example, in the context of engineered cardiac tissue, the force generated by the cells according to the present invention during spontaneous beating is consistent with that of other cardiomyocytes produced commercially or by methods already disclosed in the art. Similarly, for example, in the context of engineered cardiac tissue, the contraction and relaxation times of the cells according to the present invention during spontaneous beating are consistent with those of other cardiomyocytes produced commercially or by methods already disclosed in the art.

[0140] The method according to the present invention can provide a population of cells having one or more of these characteristics.

[0141] The present invention also provides a method for improving the maturity of a cell population in a short period (20 days). Maturity markers can be improved, for example, with respect to sarcomere organization, length, and function. The present invention facilitates the production of cells having sarcomere lengths characteristic of adult cardiomyocytes.

[0142] The cells according to the present invention may also exhibit connexin-43, a maturation gap junction marker; sarcomere alpha-actinin; telesonin, a maturation z-disk marker; M-protein, a maturation M-band marker; and / or desmin, a mature cardiomyocyte-associated intermediate filament.

[0143] The cells according to the present invention can also exhibit a broad, interconnected mitochondrial network typical of neonatal cardiomyocytes. The present invention also facilitates the rapid activation of mitochondrial function by increasing mitochondrial DNA activation observed on day 20, which is characteristic of activated metabolism.

[0144] subject In preferred embodiments of the present invention, the subject is a mammal, preferably a cat, dog, horse, donkey, sheep, pig, goat, cow, mouse, rat, rabbit, or guinea pig, but most preferably a human.

[0145] Where defined herein, “treatment” means reducing, alleviating, or eliminating one or more symptoms of the disease being treated compared to the symptoms before treatment.

[0146] "Prevention" (or "prophylaxis") refers to delaying or preventing the onset of symptoms of a disease. Prevention may be absolute (preventing the disease from occurring) or it may be effective for only some individuals or for a limited period of time.

[0147] Combination therapy The present invention as described herein may also be used in combination with other appropriate therapeutic or surgical procedures.

[0148] The method and use of the present invention for treating left ventricular disorders can be used in combination with additional treatments, such as treatments or procedures used to treat or prevent left ventricular disorders, including heart failure.

[0149] Such treatments may include, for example, lifestyle factors such as weight management and smoking cessation.

[0150] Such treatments may also include two or more medications to treat or improve left ventricular function in patients suffering from left ventricular disease, such as heart failure. Such treatments may include blood pressure medications, such as angiotensin-converting enzyme inhibitors, such as benazepril, captopril, enalapril, hosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, trandolapril (Lotensin, Vasotec, Prinivil, Accupril, Mavik, and others), or angiotensin receptor blockers (ARBs), such as azilsartan, candesartan, eprosartan, irbesartan, losartan, olmesartan, telmisartan, valsartan (Atacand, Avapro, Benicar, Diovan, Edarbi, Micardis, Teveten, and others).

[0151] Additionally, blood pressure medications, such as Entresto (sacubitril / valsartan), which is typically used to treat heart failure, may also be included.

[0152] Such treatments may also include beta-blockers, such as carvedilol (Coreg), metoprolol (Lopressor), and bisoprolol (Zebeta), which are drugs that lower heart rate, reduce blood pressure, limit or reverse some of the damage to the heart, and reduce the risk of certain abnormal arrhythmias.

[0153] Such treatments also include calcium channel blockers, which are drugs that prevent calcium from entering the cells of the heart and blood vessel walls, resulting in a decrease in blood pressure; for example, they may include amlodipine (Norvasc) and diltiazem (Cardizem, Tiazac).

[0154] Such treatments may also include diuretics combined with potassium and magnesium supplements; diuretics, such as fluoremide (Lasix), may help reduce fluid in the lungs and facilitate breathing in patients with left ventricular dysfunction.

[0155] Such therapies also include potassium-sparing diuretics and aldosterone antagonists such as spironolactone (Aldactone) and eplerenone (Inspra), which have additional properties that may help extend the survival time of patients with severe systolic heart failure.

[0156] Such treatments may also include vasoactive agents, which are intravenous medications used in hospitals for patients with severe heart failure to improve cardiac output and maintain blood pressure. Such treatments may also include digoxin (also known as lanoxin or digitalis), a drug that tends to increase myocardial contractility and slow the heart rate. Such treatments may also include nitrates to relieve chest pain, statins to lower cholesterol, or blood-thinning medications.

[0157] Such treatments may also include surgical interventions to address the underlying problem that led to left ventricular disease, such as aortic valve stenting, coronary artery bypass surgery, or heart valve repair or replacement.

[0158] Such treatments may also include the implantation of medical devices, such as an implantable cardioverter-defibrillator (ICD) or biventricular pacemaker, to provide cardiac resynchronization therapy (CRT).

[0159] composition The population of cells according to the present invention as described herein can be provided in the form of a composition.

[0160] The composition may be a pharmaceutical composition further comprising a pharmaceutically acceptable carrier, diluent, or excipient. The pharmaceutical composition may optionally comprise one or more further pharmaceutically active polypeptides and / or compounds. Such formulations may be in a form suitable for intravenous infusion, for example.

[0161] The compositions according to the present invention are administered using any amount and any route of administration that is effective in preventing or treating a subject. An effective amount refers to an amount of the composition sufficient to beneficially prevent or improve the symptoms of a disease or condition.

[0162] The precise dosage is selected by the individual physician, taking into account the patient being treated. The dosage and administration are adjusted to provide a sufficient level of activator(s) or to maintain the desired effect in the patient. Additional factors that may be taken into consideration include the severity of the disease state, e.g., liver function, cancer progression, and / or the mid- or advanced stage of macular degeneration; age; weight; sex; diet and timing; frequency of administration; route of administration; drug combinations; response sensitivity; level of immunosuppression; and tolerance / response to treatment. Long-acting pharmaceutical compositions are administered, depending on the half-life and clearance rate of the specific composition, for example, every hour, twice hourly, every 3-4 hours, once daily, twice daily, every 3-4 days, once weekly, once every two weeks, twice a year, once a year, or even as a single dose.

[0163] The active agents of the pharmaceutical compositions of embodiments of the present invention are preferably formulated in dosing unit form for ease of administration and uniformity of dosage. As used herein, the term “dosing unit form” refers to a physically separated unit of the active agent suitable for the subject being treated. The total daily, weekly, monthly, yearly, or single-dose doses of the compositions of the present invention are determined by the attending physician within the bounds of normal medical judgment. For any active agent, the therapeutically effective dose is first estimated in a cell culture assay or in an animal model (potentially a mouse, pig, goat, rabbit, sheep, primate, monkey, dog, camel, or high-value animal). The cell-based models, animal models, and in vivo models provided herein are also used to achieve desired concentrations, total dosing ranges, and routes of administration. Such information is used to determine useful doses and routes of administration in humans.

[0164] A therapeutically effective dose refers to the amount of an activator that improves symptoms or conditions, or prevents the progression of a disease or condition. The therapeutic efficacy and toxicity of an activator are determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED. 50 (The dose that is effective in 50% of the population) and LD 50 It is determined by the lethal dose (50% of the population). The dose-to-toxicity ratio is the LD50. 50 / ED 50 This is a therapeutic index expressed as a ratio. Pharmaceutical compositions with a large therapeutic index are preferred. Data obtained from cell culture assays and animal studies are used to formulate the dosage range for human use.

[0165] The pharmaceutical compositions or methods provided herein are administered to humans and other mammals, for example surgically (e.g., via injection into the heart), for preventive or therapeutic purposes, and depending on the severity and nature of the disorder, so that they may be formulated with a suitable pharmaceutically acceptable carrier at a desired dosage.

[0166] Injections of pharmaceutical compositions include, for example, direct injection into the heart, intravenous injection, subcutaneous injection, intramuscular injection, or intraperitoneal injection.

[0167] Liquid drug formulations are, but are not limited to, intravenous, intraocular, mucosal, pharmaceutically acceptable emulsions, microemulsifies, solutions, suspensions, syrups, and elixirs. In addition to at least one active agent, liquid drug formulations may potentially contain an inert diluent commonly used in the art, such as water or other solvents. Besides the inert diluent, compositions delivered ocularly, orally, or otherwise systemically may also contain adjuvants, such as wetting agents, emulsifiers, and suspending agents.

[0168] The activator can be mixed with a pharmaceutically acceptable carrier under sterile conditions. A preservative or buffer may be required. Dosage may be in therapeutic or prophylactic forms. Certain embodiments of the present invention can be combined with implantable devices (e.g., pacemakers) and methods for manufacturing or using such devices or products.

[0169] In one embodiment, the pharmaceutical composition may be in the form of a patch, for example, a patch that can be applied directly to the left ventricle. Suitable patches are known to those skilled in the art.

[0170] In one embodiment, cells may be administered in combination with other elements, such as supporting cells, carriers, loaded vesicles, microRNAs, growth factors, and / or small molecules that may be advantageous for cell proliferation and development.

[0171] Patches offer the additional advantage of providing controlled delivery of the active ingredient. Such drug delivery forms can be prepared by dissolving or distributing the compound in a suitable culture medium. Absorption enhancers are used to increase the flow of the compound across cells, including thick epithelium such as the epicardium. The rate is controlled by providing a rate-controlled membrane or by dispersing the compound in a polymer matrix or gel.

[0172] Injectable formulations of pharmaceutical compositions, such as sterile injectable aqueous or oily suspensions, are formulated according to known techniques using appropriate dispersants, wetting agents, and suspending agents. Sterile injectable preparations may also be sterile injectable solutions, suspensions, or emulsions in non-toxic, parenterally acceptable diluents or solvents, for example, as a solution in 1,3-butanediol. Acceptable vehicles and solvents that may be employed include water, Ringer's solution, USP, and isotonic sodium chloride solution. Furthermore, sterile non-volatile oils are conventionally employed as solvents or suspension media. For this purpose, non-irritating non-volatile oils containing synthetic monoglycerides or diglycerides are used. Additionally, fatty acids, such as oleic acid, are used in injectable preparations. Injectable formulations are sterilized before use, for example, by filtration with a bacterial-retaining filter, irradiation, or by incorporating a sterilizing agent in the form of a sterile solid composition (dissolved or dispersed in sterile water or other sterile injectable media). Delayed absorption of the drug via subcutaneous or intratumoral injection has been observed to prolong the effect of the activator. Delayed absorption of parenterally administered activators is achieved by dissolving or suspending the drug in an oily vehicle. Injectable depot formulations are prepared by forming a microcapsule matrix of the drug in a biodegradable polymer, such as polylactide-polyglycolide. The rate of activator release is controlled depending on the activator-to-polymer ratio and the properties of the specific polymer used. Other examples of biodegradable polymers include poly(orthoester) and poly(anhydrous). Depot injection formulations are also prepared by incorporating the drug into liposomes or microemulsions that are compatible with body tissues.

[0173] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In solid dosage forms, the activator is mixed with at least one inert, pharmaceutically acceptable excipient or carrier, e.g., sodium citrate, dicalcium phosphate; fillers and / or bulkers, e.g., starch, sucrose, glucose, mannitol, and silicic acid; binders, e.g., carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidinone, sucrose, and acacia; humectants, e.g., glycerol; disintegrants, e.g., agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; dissolution retarders, e.g., paraffin; absorption enhancers, e.g., quaternary ammonium compounds; wetting agents, e.g., cetyl alcohol and glycerol monostearate; absorbents, e.g., kaolin and bentonite clay; and lubricants, e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof.

[0174] Similar types of solid compositions may also employ excipients, such as lactose and high molecular weight PEG, as fillers in the soft and hard gelatin capsules used. Solid dosage forms of tablets, sugar-coated tablets, capsules, pills, and granules are prepared using coatings and shells, such as enteric coatings, controlled-release coatings, and other coatings known in the field of pharmaceutical formulation. In these solid dosage forms, the activator(s) is mixed with at least one inert diluent, such as sucrose or starch. Such dosage forms also include additional substances other than the inert diluent, such as tableting lubricants and other tableting aids, such as magnesium stearate and microcrystalline cellulose, as in standard practices. In the case of capsules, tablets, and pills, the dosage form may also include a buffer. The composition may optionally contain an opacifying agent that releases only the activator(s), preferably to certain parts of the intestinal tract, and optionally in a delayed manner. Examples of embedding compositions include polymeric substances and waxes.

[0175] Designed heart tissue The cells according to the present invention as described herein can be used, for example, in the context of designed cardiac tissue to generate a cardiac patch. The present invention encompasses designed cardiac tissue comprising the cells according to the present invention as described herein.

[0176] Generally, tissue engineering is a branch of engineering science that focuses on the development of living tissue matrices under laboratory conditions. These tissue matrices can consist of cell-containing scaffolds that can be used as model systems for drug testing or applied as in-vivo grafts or patches to repair damaged tissue or organs. Cardiac tissue engineering aims to facilitate cell assembly and construct functional tissue by manipulating the microenvironment in which cells interact. Its primary objective is to provide functional human cardiomyocytes for drug discovery, research into cardiac pathophysiology, and ultimately for cell therapy by repairing diseased or damaged cardiomyocytes.

[0177] In vitro approaches utilize different scaffolds, as well as different cell types or combinations of cell types. Human pluripotent stem cell-derived cardiomyocytes are used in this context and are expected to represent the future of cardiac regenerative medicine. The objective is to cultivate human pluripotent stem cell-derived cardiomyocytes to a size sufficient for their intended use and to achieve a contractile force (≧2~4 mN / mm²). 2 The goal is to organize cells into functional tissue capable of generating and propagating electrical signals (conduction velocity ≥ 25 cm / s). Cells can be cultured on scaffolds (structural and logistical templates for tissue formation) in tissue culture wells or bioreactors (bulk culture systems that provide conditions designed to achieve a desired degree of functionality).

[0178] Engineered cardiac tissues can be induced by experimental manipulation of pluripotent stem cells, such as induced pluripotent stem cells (hiPSCs) and embryonic stem cells (hESCs) to differentiate into human cardiomyocytes. Interest in these bioengineered cardiac tissues is growing due to their potential use in cardiovascular research and clinical therapy. These tissues provide unique in vitro models for studying cardiac physiology that are species-specifically advantageous over cultured animal cells in experimental studies. Engineered cardiac tissues also have therapeutic potential for in vivo regeneration of myocardium. Engineered cardiac tissues can replicate the normal development of human cardiac tissue, provide a valuable resource for understanding the pathogenesis of human cardiovascular disease (CVD), and lead to engineered tissue-based therapies for CVD patients.

[0179] The precise size of the designed cardiac tissue is selected by the individual physician, taking into account the subject being treated. The number of cells required to design such a patch is adjusted to provide sufficient contractility, proper conduction and / or coverage, or to maintain the desired effect in the subject. Additional factors that may be taken into consideration include the severity of the disease state, e.g., liver function, cancer progression, and / or the mid- or advanced stage of macular degeneration; age; weight; sex; diet and timing; frequency of administration; route of administration; drug combinations; response sensitivity; level of immunosuppression; and tolerance / response to treatment.

[0180] The administration of the designed cardiac tissue may be consistent with the administration described herein.

[0181] The activator can be mixed with a pharmaceutically acceptable carrier under sterile conditions. A preservative or buffer may be required. Dosage may be in therapeutic or prophylactic forms. Certain embodiments of the present invention can be combined with implantable devices (e.g., pacemakers) and methods for manufacturing or using such devices or products.

[0182] The engineered heart tissue can be administered, for example, in the form of a patch, depending on the survival of the patch and the progression of the disease, for example, monthly, twice a year, once a year, once every two years, or even as a single administration. For example, repeated patch administrations may be required because multiple administrations are needed for the desired effect in the patient, regardless of the survival of the patch and the stabilization of the disease.

[0183] A therapeutically effective dose refers to the size of the engineered heart tissue and its internal cell content that is sufficient to improve a symptom or condition or to prevent the progression of a disease or condition. The therapeutic efficacy and toxicity of an active agent are determined by standard pharmaceutical procedures in cell culture or experimental animals, e.g., ED 50 (the dose that has a therapeutic effect on 50% of the population) and LD 50 (the lethal dose for 50% of the population). The therapeutic effect is measured, for example, by evaluating cardiac function and the physical strength of the patient. The dose ratio of toxicity to therapeutic effect is the therapeutic index expressed as the LD 50 / ED 50 ratio. Pharmaceutical compositions having a large therapeutic index are preferred. Data obtained from cell culture assays and animal tests are used in formulating dosage ranges for human use.

[0184] The present invention encompasses engineered heart tissue containing cells according to the present invention described herein.

[0185] The engineered heart tissue can be generated using methods described in the art (e.g., Zimmermann et al., in 2004; Hansen et al., in 2010), for example, using modifications or new methods of these methods.

[0186] The designed cardiac tissue can be constructed by (1) growing one or more cell sheets in a monolayer and releasing them intact from the culture surface, (2) seeding cells into the extracellular matrix of decellularized tissue, e.g., decellularized cardiomyocyte tissue, or (3) suspending cells in a scaffold. The scaffold itself can be produced by 3D printing and is particularly useful for pre-patterning vascular channels within the scaffold. The designed cardiac tissue may also require the addition of non-cardiac cells, including but not limited to endothelial cells, endocardial cells, and fibroblasts.

[0187] As discussed herein, the designed cardiac tissue may include cells (one or more subtypes) that have been grown on or seeded on a scaffold.

[0188] In one aspect of the present invention, the cells according to the present invention can be combined with a scaffold. The present invention provides a designed cardiac tissue comprising the cells and scaffold according to the present invention as described herein. The present invention also provides a scaffold on which the cells according to the present invention are seeded.

[0189] Suitable scaffolds are commercially available and known to those skilled in the art. Scaffolds may be natural or synthetic. Suitable scaffolds may include materials such as collagen, fibrin, cellulose, polyglycolic acid, silk fibroin, hyaluronic acid, alginate, chitosan, heparin, gelatin methacryloyl, and / or polymer electrolyte complexes. It may also include piezoelectric polymers or piezoelectric ceramics, which are electrically active scaffolds used for tissue repair and regeneration. These types of scaffolds can deliver variable electrical stimuli without an external power source and are therefore advantageous for electrical signal propagation between cardiac cells in designed cardiac tissue constructs. In the case of nanofiber scaffolds, the fiber diameter can be controlled via electrospinning, a technique that enables the production of scaffolds with mechanical properties that very well mimic the natural extracellular matrix. Electrospinned scaffolds have a porous structure with a high surface area-to-volume ratio to facilitate cell adhesion and migration.

[0190] In one embodiment, the scaffold and / or cells according to the present invention may be supplemented with growth / differentiation factors, small molecules, microRNAs, or vesicles, such as exosomes.

[0191] In one embodiment, the designed cardiac tissue produced using the cells according to the present invention may include non-cardiac cell cells, such as endothelial cells, endocardial cells, and / or fibroblasts. In one embodiment, the present invention preferably provides a designed cardiac tissue comprising a scaffold and cells according to the present invention, as described herein, and optionally including additional supporting cells, such as endothelial cells, endocardial cells, and / or fibroblasts. The cells may be added at specific concentrations that can be determined by those skilled in the art.

[0192] In one embodiment, a suitable scaffold may be selected from the group consisting of those made using polymers; extracellular matrices; those manufactured, synthesized, or recovered from animal donors; extracellular matrix / polymer hybrids; natural extracellular matrices; or natural tissue constructs. The scaffold may be designed to attract cells, such as endothelial cells, for regrowth or seeding. Thus, the scaffold may contain living cells, i.e., the scaffold is regrowthed with or without additional supporting cells, including (plus or minus) cells suitable for the tissue, such as cells according to the present invention as described herein.

[0193] The designed cardiac tissue may take the form of a cardiac patch. Appropriate methods for producing cardiac patches are known to those skilled in the art.

[0194] In one embodiment, the designed cardiac tissue or patch may contain fibrin. The present invention provides a patch comprising a population of cells according to the present invention as described herein, for example, a fibrin patch.

[0195] Fibrin (also known as factor Ia) is a fibrous, non-globular protein involved in blood coagulation. It is formed by the action of thrombin, a protease, on fibrinogen, which then polymerizes. Polymerized fibrin, together with platelets, forms a hemostatic plug or blood clot throughout the wound site. Fibrin may be used as the basis for the patches described herein.

[0196] In one embodiment, the scaffold or patch may contain collagen / Matrigel. The present invention provides a patch containing a population of cells according to the present invention as described herein, for example, a collagen / Matrigel patch.

[0197] Collagen is a major structural protein in the extracellular matrix of various connective tissues in the body. More than 90% of the collagen in the human body is type I collagen. For example, type I collagen for use according to the present invention can be prepared from rat tails.

[0198] Matrigel is the trade name for a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. Suitable sources of Matrigel for use according to the present invention are commercially available and include, for example, BD Matrigel® basement membrane matrix (Becton Dickinson, catalog no. 356234) and solubilized basement membrane preparations extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcomas (tumors rich in extracellular matrix proteins including laminin (major component), collagen IV, heparan sulfate proteoglycan, and entactin / nidogen). The BD Matrigel matrix also contains TGF-beta, epidermal growth factor, insulin-like growth factor, fibroblast growth factor, tissue plasminogen activator, and other growth factors naturally present in EHS tumors.

[0199] Collagen / Matrigel can be used as a patch base as described herein.

[0200] In one embodiment, the patch may contain fibrinogen / Matrigel + thrombin. Fibrinogen (factor I) is a glycoprotein complex produced in the liver that circulates in the blood of all vertebrates. In the event of tissue and blood vessel damage, it is enzymatically converted to fibrin by thrombin. Thrombin is a serine protease that plays a physiological role in regulating hemostasis and maintaining blood coagulation. When converted from prothrombin, thrombin converts fibrinogen to fibrin.

[0201] In one embodiment, the scaffold or patch may contain gelatin methacryloyl (GelMA). The present invention provides a patch containing a population of cells according to the present invention as described herein, for example, a gelatin methacryloyl patch.

[0202] GelMA hydrogels are widely used in various biomedical applications due to their appropriate biological properties and tunable physical characteristics. Three-dimensional GelMA hydrogels closely resemble several essential properties of the natural extracellular matrix due to the presence of cell-adherent and matrix metalloproteinase-responsive peptide motifs, allowing cells to proliferate and spread within the GelMA-based scaffold. GelMA can be used as the basis for patches described herein.

[0203] The designed 3D environment of cardiac tissue enables in vivo-like cardiac organization of cellular types and better induces effective intercellular communication. The designed cardiac tissue allows for precise mechanical loading of sarcomeres. Mechanical tension cells, when incorporated into the designed cardiac tissue, may be at a critical step for maturation, and these 3D tissues have been shown to be suitable for physical conditioning, aiding further maturation of cardiomyocytes (Ronaldson-Bouchard et al., 2018; doi.org / 10.1038 / s41586-018-0016-3).

[0204] In one embodiment, the designed cardiac tissue produced using the cells according to the present invention can be subjected to physical conditioning with increasing intensity over time, that is, to induce contraction, it can be subjected to intensity pacing training for two weeks at a frequency increasing by 0.33 Hz / day from 2 Hz to 6 Hz, followed by one week at 2 Hz.

[0205] The cardiac tissue, scaffold, and / or patch designed according to the present invention can be used in any of the methods / uses described herein.

[0206] In one embodiment, the present invention provides a method for treating or preventing left ventricular dysfunction in a subject, comprising administering to the subject a designed cardiac tissue, scaffold, or patch as described herein.

[0207] In one embodiment, the present invention provides a designed cardiac tissue, scaffold, or patch described herein for use in the treatment or prevention of left ventricular disorders.

[0208] kit In one embodiment, the present invention provides a kit comprising a population of cells described herein, wherein at least about 60% of the cells are double-positive for the markers HAND1 and MLC2v.

[0209] In one embodiment, the present invention provides a kit comprising a population of cells described herein and components necessary for generating the designed cardiac tissue described herein, such as scaffold components and / or supporting cells.

[0210] Drug screening In one embodiment, the cell population described herein may be used to screen for drugs that may be useful as a treatment for treating or preventing left ventricular disorders.

[0211] In this regard, cell populations can be used for drug screening, for example, to identify safe drugs in preclinical trials, such as 1) drugs that can improve LV function in patients with congenital heart disease affecting the LV; and 2) drugs that are known to affect the proper function of the heart, i.e., the LV.

[0212] Accordingly, in one embodiment, the present invention provides a method for screening for drugs suitable for treating or preventing left ventricular disorders, comprising contacting a population of cells according to the present invention with a candidate drug. The method may further include analyzing the effect of the candidate drug on the population of cells.

[0213] The present invention also encompasses a method for screening for cardiotoxicity with respect to a drug, the method comprising contacting a population of cells described herein with the drug.

[0214] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this disclosure belongs. Singleton et al., *Dictionary of Microbiology and Molecular Biology*, 20th edition, John Wiley and Sons, New York (1994), and Hale & Marham, *The Harper Collins Dictionary of Biology*, Harper Perennial, NY (1991) provide general dictionaries for many of the terms used in this disclosure to those skilled in the art.

[0215] This disclosure is not limited to the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of embodiments of this disclosure. Numerical ranges include the numerical value defining the range. Unless otherwise indicated, any nucleic acid sequence is written from left to right in the 5' to 3' direction; and amino acid sequences are written from left to right in the amino to carboxy direction.

[0216] The headings provided herein are not limitations on the various or more aspects of this disclosure that can be obtained by referring to this entire specification. Therefore, the terms defined immediately below are more fully defined by referring to the entire specification.

[0217] Amino acids are referred to herein by their full name, a three-letter abbreviation, or a one-letter abbreviation.

[0218] As used herein, the term "protein" includes proteins, polypeptides, and peptides.

[0219] Definitions of other terms can be found throughout this specification. Before describing exemplary embodiments in more detail, it should be understood that this disclosure is not limited to the specific embodiments described and is subject to change. It should also be understood that the terms used herein are used only to describe specific embodiments and are not intended to limit the scope of this disclosure, as it is limited only by the appended claims.

[0220] Where a range of values ​​is provided, unless the context explicitly indicates otherwise, each intermediate value between the upper and lower limits of that range, up to one-tenth of the lower limit, is also understood to be specifically disclosed. Each smaller range between any given value or intermediate value within a given range and any other given value or intermediate value within that given range is included within the scope of this disclosure. The upper and lower limits of these smaller ranges may be independently included in or excluded from that range, and if there are any specifically excluded limits within the described range, each range in which any, neither, or both limits are included within the smaller range is included within this disclosure. If the described range includes one or both limits, the range excluding either or both of those included limits is also included within this disclosure.

[0221] As used herein and in the appended claims, unless the context explicitly indicates otherwise, the singular forms “a,” “an,” and “the” refer to multiple objects.

[0222] As used herein, the terms “comprising,” “comprises,” and “comprised of” are synonymous with “including,” “includes,” or “containing,” and are inclusive or open-ended, and do not exclude additional members, elements, or process steps not enumerated. The terms “comprising,” “comprises,” and “comprised of” also include the term “consisting of.”

[0223] The publications discussed herein are provided solely for disclosure prior to the filing date of this application. Nothing in this specification should be construed as constituting prior art of the claims attached herein. The present invention includes, for example, the following embodiments: [1] A population of cells comprising at least about 60% of cells that are double-positive for the markers HAND1 and MLC2v, wherein the cells are left ventricular cardiomyocytes. [2] A population of cells as described in [1], comprising at least about 65, 70, 75, 80, 85, 90, 95, or 100% of cells that are double-positive for the markers HAND1 and MLC2v. [3] A population of cells according to [1] or claim 2, comprising at least about 65, 70, 75, 80, 85, 90, 95, or 100% of cells that are positive for the marker TBX5, as well as for HAND1 and MLC2V. [4] A population of cells described in any of [1] to [3], comprising at least approximately 85% of cells that are double-positive for markers HAND1 and MLC2v. [5] A population of cells described in any of [1] to [4], comprising at least approximately 85% of cells that are positive for the markers TBX5, HAND1, and MLC2v. [6] A population of cells described in any of [1] to [5], comprising at least about 85% of cells that are positive for the markers IRX4, TBX5, HAND1, and MLC2v. [7] A population of cells described in any of [1] to [6], including mature cardiomyocytes. [8] The mature cells are preferably, (i) Ventricular action potential shape; (ii) Low heart rate and / or periodicity; (iii) Conduction velocity characteristic of neonatal ventricular cells; (iv) The average value of the pulse amplitude higher than (iv); (v) Field potential intervals that match the field potential intervals of ventricular cardiomyocytes; (vi) Fast action potential rise time (Trise); (vii) CaT similar to the calcium transient (CaT) found in adult human ventricular cardiomyocytes; and (viii) Improved CaT rise time (time to peak, Tpeak) and CaT A population of cells described in [7] that exhibits characteristics of maturity selected from the above. [9] The mature cells are preferably, (i) Organization, length, and function of sarcomeres; (ii) Indication of the presence of connexin-43, a mature gap junction marker; sarcomere alpha-actinin; telesonin, a mature Z-disk marker; M-protein, a mature M-band marker; and desmin, a cardiomyocyte-associated intermediate filament in the Z-disk; (iii) Display of an extensive, interconnected mitochondrial network typical of neonatal cardiomyocytes; and (iv) Increased activation of mitochondrial DNA A population of cells according to [7] or claim 9, which exhibits an improved marker of maturity, selected from among the following.

[10] A population of cells according to any one of [1] to [9], wherein the cells can be paced.

[11] A method for treating or preventing left ventricular dysfunction in a subject, comprising administering to the subject a population of cells containing at least about 60% of cells that are double-positive for the markers HAND1 and MLC2v described in any of [1] to

[10] .

[12] The method according to

[11] , wherein the left ventricular disorder is selected from myocardial infarction, heart failure, left ventricular hypertrophy, hypoplastic left heart syndrome, and left ventricular noncompaction cardiomyopathy (LVNC).

[13] The method according to

[12] , wherein the left ventricular disorder is myocardial infarction.

[14] The method according to

[12] , wherein the left ventricular disorder is heart failure.

[15] A population of cells according to any of [1] to

[10] for use in the treatment or prevention of left ventricular disorders.

[16] A population of cells for use as described in

[15] , wherein the left ventricular disorder is selected from myocardial infarction, heart failure, left ventricular hypertrophy, hypoplastic left heart syndrome, and left ventricular noncompaction cardiomyopathy (LVNC).

[17] A population of cells for use according to

[16] , wherein the left ventricular disorder is myocardial infarction.

[18] A population of cells for use as described in

[16] , wherein the left ventricular disorder is heart failure.

[19] A method for preparing a cell population containing at least about 60% of cells that are double-positive for the markers HAND1 and MLC2v, comprising the step of culturing pluripotent stem cells in a medium containing a retinoic acid receptor antagonist or an inverse agonist.

[20] The method according to

[19] , having a duration of 15 to 25 days, preferably 20 days, wherein the population of cells preferably exhibits the maturity characteristics / markers described in [8] and / or [9].

[21] The method according to claim 18, wherein the pluripotent stem cells are embryonic stem cells or induced pluripotent stem cells.

[19]

[22] The method according to any one of

[19] to

[21] , comprising culturing the cell population with a glycogen synthase kinase-3 (Gsk3) inhibitor, preferably Chiron, BMP (preferably BMP4), activin (preferably activin A), and FGF (preferably FGF2).

[23] The cell population is divided into the following quantities: (i) A glycogen synthase kinase-3 (Gsk3) inhibitor in a concentration of approximately 1-6 μM / ml, preferably Chiron, preferably approximately 2-4 or 2-3 μM / ml of Chiron; (ii) BMP of about 1 to 10 ng / ml, preferably BMP4, preferably about 1 to about 6 ng / ml, or about 3 to about 5 ng / ml; (iii) Activin in a concentration of approximately 1 to 10 ng / ml, preferably activin A, preferably approximately 3 to approximately 10 ng / ml of activin, preferably approximately 5 ng / ml of activin; (iv) FGF in a concentration of approximately 1 to 10 ng / ml, preferably FGF2, preferably approximately 3 to approximately 10 ng / ml, preferably approximately 5 ng / ml of FGF The method according to

[22] , which includes culturing together with

[24] The method according to any one of

[19] to

[23] , comprising culturing the cell population together with a Wnt inhibitor.

[25] The method according to any one of

[19] to

[24] , which then comprises culturing the cells in a medium that does not contain vitamin A.

[26] A method according to any of

[19] to

[25] , including the protocols shown in Table 1. A population of cells obtained or obtainable by any of the methods described in

[27]

[19] ~

[26] .

[28] A population of cells according to any one of [1] to

[10] and claim 25 for use in the treatment or prevention of left ventricular disorders.

[29] A method for treating or preventing left ventricular dysfunction in a subject, comprising administering to the subject a population of cells according to any one of [1] to

[10] and claim 25.

[30] A method for screening drugs suitable for treating or preventing left ventricular disorders, comprising contacting a population of cells described in any of [1] to

[10] and

[27] with a candidate drug.

[31] A method for screening for cardiotoxicity of a drug, comprising contacting a population of cells described in any of [1] to

[10] and

[27] with the drug. A scaffold on which a population of cells described in any of

[32] [1]~

[10] and

[27] has been seeded. Designed cardiac tissue comprising a population of cells described in any of

[33] [1] to

[10] and

[27] or a scaffold described in

[32] . A cardiac patch comprising a population of cells described in any of

[34] [1] to

[10] and

[27] or a scaffold described in

[32] .

[35] A designed cardiac tissue, patch, or scaffold according to any one of

[32] to

[34] , comprising one or more of fibrinogen, fibrin, Matrigel, thrombin, collagen, and gelatin methacryloyl.

[36] Preferably further comprising non-cardiac cells selected from endothelium, endocardium, and fibroblasts, the designed cardiac tissue, patch, or scaffold according to any one of

[32] to

[35] .

[0224] The present invention will be described only by reference to the following examples, but it is intended to be useful in assisting those skilled in the art in carrying out the invention and is not intended to limit the scope of the invention in any sense.

[0225] [Examples] [Example 1] Studying the first stage of heart development The inventors characterized the gene expression phenotypes of different chambers of the mouse heart. Samples were analyzed from microscopically dissected hearts, as illustrated in the right-hand diagram of Figure 1A. RV corresponds to the presumptive right ventricle, LV to the presumptive left ventricle, and A to the presumptive atrial chamber. The analyzed stages (ST0-ST3) correspond to a series of cardiac developmental stages from the early heart bub (E8.25) to the end of looping (E8.5), as shown in the schematic diagram on the right of Figure 1A. Genes that are consistently differentially expressed in a given region across all analyzed stages are highlighted in Figure 1A, as indicated by color coding.

[0226] Genes specifically upregulated in atrial samples were analyzed using the Reactome analysis tool. These results demonstrate a significant enrichment of genes involved in retinoid metabolism and transport pathways, constituting 16 of the top 25 most relevant pathways (Figures 1B and C). Genes specifically upregulated in atrial samples were subjected to Gorilla GO-term analysis. Figure 1D shows the results for GO-terms related to the retinoic acid (RA) pathway that were found to be enriched within the gene set.

[0227] We evaluated differential expression of retinoic acid directed to target genes and genes involved in the retinoic acid pathway across three lumens (highlighted by Reactome analysis) (Figure 1E). Results represent the mean of three replicates at three different stages (i.e., nine data points). Expression of retinoic acid-related genes was consistently higher in the putative atrium, in contrast to the left or right ventricle.

[0228] [Example 2] We will use the teachings from embryos to generate left ventricular cardiomyocytes in human embryonic stem cells. Considering the low expression of retinoic acid-related genes in the ventricular region of the heart (Figure 1), the inventors hypothesized that the retinoic acid pathway needs to be turned off in order to generate ventricular cardiomyocytes.

[0229] In light of prior art (Bardot et al., 2017; DOI: 10.1038 / ncomms14428) showing that ventricular cardiomyocytes (primarily left ventricular cardiomyocytes) originate from a specific mid-to-anterior region of the primitive streak, we further hypothesized that it is necessary to mimic such streak locations in vitro in order to generate the mesoderm that gives rise to ventricular cardiomyocytes, particularly left ventricular cardiomyocytes.

[0230] From the above, a protocol for generating left ventricular-specific cardiomyocytes can be devised. The inventors began by adjusting the levels of activin, BMP, and Wnt in the first step of a differentiation protocol aimed at generating mesoderm with left ventricular cardiomyocyte potentials (Figure 2A). The inventors also adjusted the retinoic acid signaling pathway from day 0 of differentiation to achieve a homogeneous population of left ventricular cardiomyocytes (Figure 2A); this was important during the first 8 days of differentiation. Subsequently, the cells had to be maintained in a medium that did not contain vitamin A or retinoic acid (Figure 2A). A schematic diagram of the hPSC cardiomyocyte differentiation protocol described herein can be found in Figure 2A, which indicates the different steps and components used in the protocol.

[0231] According to this protocol, cells undergo specific developmental stages, namely, through appropriate mesoderm patterning, induction of the first heart field cardiac progenitor cells, followed by patterning and maturation of left ventricular cardiomyocytes. Cells can be further matured over time in culture. Beating is first observed on day 6 of differentiation. By day 10, the cells beat rapidly. By day 20, the inventors obtain a homogeneous population of left ventricular cardiomyocytes identified by the dual expression of slowly beating MLC2V and HAND1 (Figures 3A, 3C, 3D). As expected, the cells also express the pan-cardiomyocyte marker TNNT2 (Figures 3A and 3B). The cells show relatively organized myofibrils (Figure 3C), and further express the pan-ventricular marker IRX4 as well as the left ventricular and atrial marker TBX5 (Figure 3E). Furthermore, the cells express very high levels of MLC2v (Figure 3E). Representative images of various steps along the left ventricular cardiomyocyte differentiation protocol can be found in Figure 2B.

[0232] The protocol for manufacturing cells is as described below.

[0233] Differentiation of pluripotent stem cells Maintenance cells were grown for 4 - 5 days (WA09 is usually 5 days) until the colonies reached confluence. Cells were passaged around lunchtime each time at an average split ratio of 1:10 - 1:20.

[0234] Experimental setup: 1. Split the cells with TrypLE at 37°C for 5 minutes. 2. Quench the cells with 4 volumes of medium, take a small aliquot to count the cells, and spin down the remaining cells at 900 - 1200 rpm for 5 minutes. 3. Resuspend the cells in medium containing 10 μM Rock inhibitor (RI). 4. 1 cm 2 per 0.3 - 0.8×10 5 Coat the cells with ES cell grade Matrigel (or Matrigel with reduced growth factors) for 1 hour and plate them in wells containing medium with 10 μM Rock inhibitor. Let the cells settle overnight. 5. The following day, supply the cells with hPSC medium lacking radioisotopes. 6. Feed the cells daily until you are ready to begin the experiment (this can be up to 4 days). Note that the cell density should be approximately 80% confluent at the start. 7. Cells should be pluripotent on day 0, with co-expression of OCT4, NANOG, and SOX2 in more than 90% of cells and absence of SSEA1.

[0235] Cardiomyocyte differentiation protocol: 1. On day 0, remove the hPSC medium and initiate differentiation by adding 1 ml of cardiac medium 1 containing activin A (0-25 ng / ml), FGF2 (5-25 ng / ml), BMP4 (0-25 ng / ml), and Chiron (1-12 μM). The concentration depends on the cell density and cell line. 2. On day 1, add 2 ml of cardiac medium 1, which is devoid of extraneous factors. 3. On the second day, add 2 ml of cardiac medium 1 containing L-AA and IWR (1-5 μM). 4. On day 4, add 2 ml of cardiac medium 1 containing L-AA. 5. On day 6, add 2 ml of cardiac medium 1 containing L-AA. At this stage, the cells should begin to pulsate. 6. On day 8, add 2 ml of cardiac medium 2 containing L-AA. At this stage, the entire dish should pulsate rapidly, and the pulsating cell bundles should connect to each other. 7. On day 10, add 2 ml of cardiac medium 3 containing L-AA. Normally, the culture is relatively homogeneous with respect to cardiomyocytes, but depending on the cell line, some additional cells that are distinguishable on the dish may be present (usually appearing as a monolayer beneath the cardiomyocytes). Cardiac medium 3 is designed to metabolically select cardiomyocytes. Because this medium can be toxic to cells, the amount of time cells are exposed to it may vary from cell line to cell line, or even between experiments. 8. Feed the cells 2 ml of Cardiac Medium 3 containing L-AA every other day until mostly cardiomyocytes remain in the cultures. If cells show signs of struggle in the metabolic selection medium (Cardiac Medium 3), remove them immediately and feed the cells Cardiac Medium 2 instead. Note: The best results, i.e., a more homogeneous population, can be achieved if the cells are exposed to metabolic selection for at least 2 days. Typically, cells are maintained in Cardiac Medium 3 for only 2 days and never longer than 4-6 days. 9. On day 15, cells were isolated in fresh dishes coated with Matrigel with reduced growth factors. Using the STEMdiff cardiomyocyte dissociation kit (Stem cell technologies, #05025), the cells were isolated and replated onto the provided support medium supplemented with 10 μM Rock inhibitor. Note: Some cell lines may require more or less replated cells, and the optimal outcome is to have enough cells on the dish to generate a network of cardiomyocytes beating as a monolayer. 10. Starting on day 16, supply the cells with cardiac culture medium 2 containing L-AA every other day. 11. The cells will continue to mature on the dish and will be ready for analysis around day 20.

[0236] Cardiac culture medium 1: 1. RPMI 1640 medium (11875, Life Technologies) containing glucose and L-glutamine. 2. B27 supplement - (negative) insulin (1 ml per 50 ml of RPMI). 3. AGN193109 (final concentration 20-200nM).

[0237] Cardiac culture medium 2: 1. RPMI 1640 medium (11875, Life Technologies) containing glucose and L-glutamine. 2. Vitamin B27 supplement + insulin - Vitamin A (1 ml per 50 ml of RPMI). 3. Optional: AGN (final concentration 20-200 nM).

[0238] Cardiac culture medium 3: 1. RPMI 1640 medium (11879, Life Technologies) containing L-glutamine but without glucose. 2. Vitamin B27 supplement + insulin - Vitamin A (1 ml per 50 ml of RPMI). 3. Optional: AGN (final concentration 20-200 nM). 4. 4 mM L-lactic acid

[0239] [Example 3] Functional characterization of left ventricular cardiomyocytes The inventors characterized the functionality of cardiomyocytes produced using a left ventricular cardiomyocyte protocol by examining the electrophysiological properties of the cells and calcium transients.

[0240] Local extracellular action potential (LEAP) signals of cardiomyocytes were acquired using the Axion Biosystems MEA system. Cardiomyocytes on day 20 exhibited a ventricular action potential shape, namely a plateau followed by a sharp repolarization phase (Figure 4A). This result was confirmed using the Maxwell Biosystems MEA system (data not shown) and optical mapping (CellOptic, data not shown). Furthermore, principal component analysis of field potentials determined using the Maxwell Biosystems MEA system revealed that it was possible to identify only one cell population (Figures 4Bi and 4Bii). The mean beat rate (bpm) of cardiomyocytes on day 20 was determined using di-4-ANEPPS and optical mapping (CellOptic). Even at the early stage of differentiation (day 20), these cardiomyocytes exhibited a low beat rate, indicating that the cardiomyocytes were maturing in early culture (Figure 4C). In addition, the cardiomyocytes showed a regular and uniform beating periodicity (Figure 4D).

[0241] Conduction velocity analysis showed that LV-like cardiomyocytes have a conduction velocity close to that of neonatal cardiomyocytes (0.3 m / s; Figure 4E). This represents an improvement over prior art (Zhu et al., 2017; Scientific Reports, Vol. 7: pp. 43210) that achieved conduction velocities between 0.035 m / s at day 18 and 0.12 m / s at day 28.

[0242] Pulse amplitude averaging analysis shows that LV-like cardiomyocytes have stronger contractility than commercially available cardiomyocytes (Figure 4F). We further demonstrated through excitation-contraction delay analysis that LV-like cardiomyocytes take longer to contract after the onset of the action potential than commercially available cardiomyocytes (Figure 4G). This is likely because LV-like cardiomyocytes exhibit a long plateau associated with slow calcium channel opening and closing, which is typical of ventricular cardiomyocytes (Figure 4A).

[0243] Field potential interval analysis revealed that LV-like cardiomyocytes exhibited an FPD range of 300 ms to 500 ms (Figure 4H), which was, on average, longer than that described in the literature for hPSC-derived cardiomyocytes generated using protocols known in the art, and was approximately the length of a typical ventricular action potential (Coppini et al., 2014; doi:10.3791 / 51116). Furthermore, these 20-day-old cardiomyocytes possessed a rapid firing capacity, as determined by action potential rise time (Trise) analysis (Figure 4I).

[0244] The action potential triangulations, determined by the action potential durations at 30% (ADP30), 50% (ADP50), and 90% (ADP90) depolarization, as well as the ratio between ADP50 and ADP90, are consistent with those described for ventricular cells (Figure 4J). Specifically, they highlight that ADP50 and ADP90 are not far off, consistent with the presence of a plateau and subsequent rapid repolarization, which are key features of the ventricular action potential shape. This is in contrast to commercially available cardiomyocytes, which have a triangular action potential shape, i.e., no plateau (Figure A), and therefore exhibit a smaller triangulation ratio.

[0245] The average calcium transient (CaT) was determined using Fura-4F (Figs. 4K and 4L), and the results showed a CaT peak towards the end of the action potential plateau (Figs. 4A and 4K), followed by cell contraction (Fig. 4G), demonstrating that the cells respond appropriately to calcium, i.e., they exhibit excitation-contraction coupling. The CaT is similar to that seen in human adult ventricular cardiomyocytes.

[0246] [Example 4] Intracellular characterization of left ventricular cardiomyocytes The inventors further characterized the intracellular structure of cardiomyocytes produced using the left ventricular cardiomyocyte protocol.

[0247] Transmission electron microscopy (TEM) was used to analyze the ultrastructure of cardiomyocytes on day 20 of differentiation, particularly the nucleus, mitochondria, and sarcomeres (Fig. 5A). Notably, there is a remarkable organization of sarcomeres on day 20 of differentiation, which can be seen in Fig. 5A(iii), and this has not been reported in monolayer cultures before day 60 of differentiation. The average length of typical human adult ventricular cardiomyocytes is 1.7 μm (Nguyen et al., 2017; doi: 10.3389 / fphys.2017.01073). The average sarcomere length of cardiomyocytes produced using the left ventricular cardiomyocyte protocol approached 1.7 μm on day 20 and was shown to be close to the length of fully mature ventricular sarcomeres by day 60 (Fig. 5B; gray dotted line).

[0248] The average mitochondrial DNA copy numbers at days 0, 10, 20, 40, and 60 of differentiation were also characterized. There was a sharp increase between days 10 and 20, suggesting an increase in subsequent mitochondrial activity, which is consistent with cells showing activated metabolism (Fig. 5C).

[0249] Cardiomyocytes were stained with the mitochondrial marker MitoTracker, and the mitochondrial network of these cells was determined. To identify thick sarcomere filaments in living cells, the inventors performed these experiments using cells endogenously GFP-tagged at the MLC2v locus, and identified the living nuclear stain Hoechst to identify the nucleus. Confocal microscopy analysis of these cells showed that the cardiomyocytes presented an extensive interconnected mitochondrial network typical of neonatal cardiomyocytes (Figure 5D) (Eisner et al., 2017; doi.org / 10.1073 / pnas.1617288114).

[0250] Next, the inventors focused on characterizing the cellular structure of left ventricular cardiomyocytes at day 20. For this purpose, cardiomyocytes were stained with a series of antibodies and analyzed by confocal microscopy. Consistent with TEM results, the cells showed a well-developed myofibrils arrangement already present at day 20 of differentiation, as indicated by staining for sarcomere alpha-actinin, a Z-disk protein (Figure 6A), with relatively little pre-myofibrils, the earliest stage of myofibril assembly in cultured cardiomyocytes (Rhee et al., 1994; doi: 10.1002 / cm.970280102). Myofibrils arrangement was also evaluated by staining for desmin, an intermediate filament protein, and mature cardiomyocytes showed Z-disk level striations compared to filamentous arrays extending throughout the cytoplasm of immature cells (Ehler et al., 1999; PMID:10212147; Kim 1996; PMID: 8888968). Although not as complete as in mature hearts, desmin striae are already evident in 20-day cardiomyocytes, in addition to desmin filamentous signaling and accumulation in cell-cell contact (Figure 6D).

[0251] The inventors also investigated connexin-43, a major gap junction protein in adult cardiomyocytes, and analyzed the maturity of intercellular communication using confocal microscopy (Hirschy et al., 2006; doi: 10.1016 / j.ydbio.2005.10.046). In cardiomyocytes on day 20, punctate signals could occasionally be detected, and the location and signal intensity of these points in cell-to-cell contact between cardiomyocytes confirmed that they were indeed gap junctions (Figure 6A).

[0252] Finally, the inventors examined both Z-disk and M-band maturation markers and analyzed sarcomere maturity using a confocal microscope.

[0253] Telesonin (also known as the T-cap, named after the titin cap) provides extremely close connections between the N-terminuses of titin in the Z-disk in a sandwich-like structure (Zou et al., 2006; doi: 10.1038 / nature04343). In adult cardiomyocytes, telesonin is absent in transitional junctions, which are sites where new sarcomeres can be inserted during stress to cope with increasing demands. Telesonin expression is also upregulated only during late embryonic development, and in primary neonatal cultures of rat cardiomyocytes, 1 / 3 to 2 / 3 of cardiomyocytes remain telesonin-negative, depending on the rat strain and the exact age of the offspring. In 20-day cardiomyocyte cultures, a subset of cells express telesonin in the Z-disk (yellow signal when overlaid with sarcomere alpha-actin, a Z-disk marker) (Figure 6B). Telesonin has not been previously observed in monolayer cultures of cardiomyocytes produced using the methods described herein, and has been reported to be present in only 9% of commercially available cardiomyocytes via Axol (Zuppinger et al., 2017; doi:10.4081 / ejh.2017.2763). Therefore, the methods described herein confirm that they produce more mature cardiomyocytes.

[0254] The structure of the M-band, which connects to the elastic filament system composed of thick structures (myosin and related proteins) and titin in the center of the sarcomere, changes depending on the developmental stage (Lange et al., 2020; doi: 10.1016 / j.bbamcr.2019.02.003). Myomesin is constitutively expressed as the main crosslinking agent between myosin and titin, but the maturation state of the M-band in the ventricle is indicated by the upregulation of M-protein expression before and after birth. A subset of cardiomyocytes at day 20 is positive for M-protein, similar to what is observed in neonatal rat cardiomyocytes (Figure 6C). M-protein has been observed in monolayer cultures of cardiomyocytes produced using methods described in the art, but not in the early stages of differentiation up to day 20 (Kamakura et al., 2013; 10.1253 / circj.cj-12-0987; Fleischer et al., 2019; doi.org / 10.1016 / j.bios.2018.10.061).

[0255] The results above demonstrate that cardiomyocytes produced using the left ventricular cardiomyocyte protocol reach full functional and phenotypic maturity.

[0256] [Example 5] Generation and characterization of designed cardiac tissue using cardiomyocytes on day 40. Designed cardiac tissue (EHT) was generated according to a protocol developed by the Eschenhagen lab (Hansen et al., 2010; 10.1161 / CIRCRESAHA.109.211458).

[0257] The inventors characterized EHTs generated using 40-day cardiomyocytes produced using the left ventricular cardiomyocyte protocol (LV-EHT). Notably, LV-EHTs began beating on day 9, but the spontaneous beat rate was low and remained low or absent thereafter (Figure 7B). In other experiments, no beating was observed (data not shown). Importantly, LV-EHTs were able to be paced (Figure 7B). This indicates a relatively mature ventricular phenotype of left ventricular cardiomyocytes obtained in vitro. This is because adult ventricular cardiomyocytes only beat when stimulated, while LV-EHTs can be paced even if they cannot beat spontaneously. The characteristics of LV-EHTs during spontaneous beating were compared to those of EHTs generated from other hPSC-derived cardiomyocytes (Figures 7C-F). The results confirmed the intrinsic, more mature, and lower beat rate of LV-EHTs. The LV-EHT produced average force compared to other EHTs tested, showing an average time to reach 20% contraction and 20% relaxation.

[0258] In conclusion, the results described herein demonstrate that, using left ventricular cardiomyocytes obtained in vitro, it is possible to generate engineered cardiac tissue with a mild spontaneous pacemaker capability, consistent with what would be expected from more mature ventricular cardiomyocytes. These tissues will pulsate when induced and produce some degree of contractility.

[0259] Consideration Heart disease is a leading cause of death in developed industrial nations, and myocardial infarction is the most common cause of heart injury. Myocardial infarction involves a dramatic loss of contractile myocardium (cardiomyocardium cells), generally affecting the left ventricle in particular. In post-infarction heart failure, the only treatment is a heart transplant, but this is a fatal condition and a significant financial burden for hospitals. The key to improving the long-term outcomes of these patients is to regenerate the left ventricle with functional cardiomyocytes that exhibit physiological characteristics as close as possible to those of adult left ventricular cardiomyocytes.

[0260] Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine, and indeed, generating cardiomyocytes in vitro from hPSCs is now commonplace in many laboratories. However, current protocols produce heterogeneous populations of atrial and ventricular cardiomyocytes, making them unsuitable for replacement therapy. Atrial and ventricular cardiomyocytes are significantly different. Furthermore, while right and left ventricular cardiomyocytes are similar, they arise from different progenitor cells and exhibit several structural, electrophysiological, metabolic, and calcium handling differences.

[0261] To overcome the problem of heterogeneity, the inventors utilized their knowledge of mammalian embryonic development to generate a nearly homogeneous population of left ventricular-like cardiomyocytes from hPSCs. The inventors followed a two-step approach, first generating mesodermal progenitor cells capable of generating left ventricular cardiomyocytes. In the second step, the inventors promoted left ventricular identity by switching off the retinoic acid pathway, matching the inactivation state of the retinoic acid pathway in mouse ventricular cardiomyocytes. The inventors' approach generated over 90% left ventricular-like cardiomyocytes (determined by co-expression of MLC2V and HAND1) at day 20 of differentiation, which was validated in four different cell lines. These cells exhibited ventricular-like action potential shapes with long plateaus followed by rapid repolarization phases (mean triangulation = 0.87), possessed conduction velocities close to those of neonatal cardiomyocytes, were pacable, and showed calcium transients similar to those found in adult human ventricular cardiomyocytes (mean Tpeak = 190 ms).

[0262] Cells at day 20 exhibit a remarkable level of maturity despite such a short differentiation time. They show a well-developed myofibrils arrangement, including a distinct Z-disk, sarcomere length close to that of fully mature ventricular cardiomyocytes, and an extensive interconnected mitochondrial network similar to that found in neonatal ventricular cardiomyocytes. They express high levels of mitochondrial DNA. Furthermore, various cells express telesonin, a maturation Z-disk marker, and M-protein, a maturation M-band marker, while some cells also express connexin 43, a maturation gap junction marker. Importantly, these cells exhibit a low heart rate during culture time, and when used to generate designed cardiac tissue at day 40, they barely beat but can still pace. This loss of spontaneous pacemaker is consistent with what is expected in more mature ventricular cardiomyocytes.

Claims

1. A population of cells comprising at least about 60% of cells that are double-positive for the markers HAND1 and MLC2v, wherein the cells are left ventricular cardiomyocytes.

2. The cell population according to claim 1, comprising at least about 65, 70, 75, 80, 85, 90, 95, or 100% of cells that are double-positive for the markers HAND1 and MLC2v.

3. A population of cells according to claim 1 or 2, comprising at least about 65, 70, 75, 80, 85, 90, 95, or 100% of cells that are positive for the marker TBX5, as well as for HAND1 and MLC2V.

4. A population of cells according to any one of claims 1 to 3, comprising at least about 85% of cells that are double-positive for the markers HAND1 and MLC2v.

5. A population of cells according to any one of claims 1 to 4, comprising at least about 85% of cells that are positive for the markers TBX5, HAND1, and MLC2v.

6. A population of cells according to any one of claims 1 to 5, comprising at least about 85% of cells that are positive for the markers IRX4, TBX5, HAND1, and MLC2v.

7. A population of cells according to any one of claims 1 to 6, comprising mature cardiomyocytes.

8. The aforementioned mature cells exhibit characteristics of maturity, The aforementioned characteristics of maturity are, (i) Ventricular action potential shape; (ii) Low heart rate and / or periodicity; (iii) Conduction velocity characteristic of neonatal ventricular cells; (iv) The average value of the pulse amplitude higher than (iv); (v) Field potential intervals that match the field potential intervals of ventricular cardiomyocytes; (vi) Fast action potential rise time (Trise); (vii) CaT similar to the calcium transient (CaT) found in adult human ventricular cardiomyocytes; and (viii) Improved CaT rise time (time to peak, Tpeak) and CaT Selected from, A population of cells according to claim 7.

9. The aforementioned mature cells show improved maturity markers. The aforementioned maturity marker is (i) Organization, length, and function of sarcomeres; (ii) Indication of the presence of connexin-43, a mature gap junction marker; sarcomere alpha-actinin; telesonin, a mature Z-disk marker; M-protein, a mature M-band marker; and desmin, a cardiomyocyte-associated intermediate filament in the Z-disk; (iii) Display of an extensive, interconnected mitochondrial network typical of neonatal cardiomyocytes; and (iv) Increased activation of mitochondrial DNA Selected from, A population of cells according to claim 7 or 8.

10. A population of cells according to any one of claims 1 to 8, wherein the cells can be paced.

11. A population of cells according to any one of claims 1 to 10, for use in the treatment or prevention of left ventricular disorders.

12. A population of cells for use according to claim 11, wherein the left ventricular disorder is selected from myocardial infarction, heart failure, left ventricular hypertrophy, hypoplastic left heart syndrome, and left ventricular noncompaction cardiomyopathy (LVNC).

13. A population of cells for use according to claim 12, wherein the left ventricular disorder is myocardial infarction.

14. A population of cells for use according to claim 12, wherein the left ventricular disorder is heart failure.

15. A method for preparing a cell population containing at least about 60% left ventricular cardiomyocytes that are double-positive for markers HAND1 and MLC2v, comprising the step of culturing pluripotent stem cells selected from embryonic stem cells or induced pluripotent stem cells in a medium containing a retinoic acid receptor antagonist or inverse agonist, Chiron, BMP4, activin A, and FGF2, The culture medium is as follows: (i) Chiron in concentrations of approximately 2–6 μM, 2–4 μM, or 2–3 μM; (ii) BMP4 in a concentration of approximately 1 to 6 ng / ml, or approximately 3 to 5 ng / ml; (iii) Activin A at approximately 3 to 10 ng / ml; (iv) FGF2 at approximately 3 to 10 ng / ml Includes, A method further comprising the step of culturing the cells in a medium containing a retinoic acid receptor antagonist or inverse agonist, a Wnt inhibitor, and L-ascorbic acid.

16. It has a duration of 15 to 25 days, and the cell population exhibits characteristics of maturity. The aforementioned characteristics of maturity are, (i) Ventricular action potential shape; (ii) Low heart rate and / or periodicity; (iii) Conduction velocity characteristic of neonatal ventricular cells; (iv) The average value of the pulse amplitude higher than (iv); (v) Field potential intervals that match the field potential intervals of ventricular cardiomyocytes; (vi) Fast action potential rise time (Trise); (vii) CaT similar to the calcium transient (CaT) found in adult human ventricular cardiomyocytes; and (viii) Improved CaT rise time (time to peak, Tpeak) and CaT Selected from, The method according to claim 15.

17. Having a duration of 15 to 25 days, the cell population exhibits a marker of maturity, The aforementioned maturity marker is (i) Organization, length, and function of sarcomeres; (ii) Indication of the presence of connexin-43, a mature gap junction marker; sarcomere alpha-actinin; telesonin, a mature Z-disk marker; M-protein, a mature M-band marker; and desmin, a cardiomyocyte-associated intermediate filament in the Z-disk; (iii) Display of an extensive, interconnected mitochondrial network typical of neonatal cardiomyocytes; and (iv) Increased activation of mitochondrial DNA Selected from, The method according to claim 15 or 16.

18. The method according to any one of claims 15 to 17, further comprising the step of culturing the cells in a medium containing a retinoic acid receptor antagonist or inverse agonist, a Wnt inhibitor and L-ascorbic acid, and then culturing the cells in a medium that does not contain vitamin A but contains L-ascorbic acid.

19. The method according to any one of claims 15 to 18, comprising the protocol shown in Table 1.

20. A population of cells obtained or obtainable by the method described in any one of claims 15 to 19.

21. A population of cells according to claim 20 for use in the treatment or prevention of left ventricular disorders.

22. A method for screening drugs suitable for treating or preventing left ventricular disorders, comprising contacting a population of cells according to any one of claims 1 to 10 and 20 with a candidate drug.

23. A method for screening for cardiotoxicity with respect to a drug, comprising contacting a population of cells according to any one of claims 1 to 10 and 20 with the drug.

24. A scaffold on which a population of cells according to any one of claims 1 to 10 and 20 is seeded.

25. A designed cardiac tissue comprising a population of cells according to any one of claims 1 to 10 and 20 or a scaffold according to claim 24.

26. A cardiac patch comprising a population of cells according to any one of claims 1 to 10 and 20, or a scaffold according to claim 24.

27. A designed cardiac tissue, patch, or scaffold according to any one of claims 24 to 26, comprising one or more of fibrinogen, fibrin, Matrigel, thrombin, collagen, and gelatin methacryloyl.

28. A designed cardiac tissue, patch, or scaffold according to any one of claims 24 to 27, further comprising non-cardiac cells.

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