WO2023104813A1 - Methods of cardiomyocyte production - Google Patents

Abstract

The present invention relates to in vitro methods for producing cardiomyocytes, cardiomyocytes produced by such methods and methods for the use of such cardiomyocytes. The invention provides methods for producing a population of cardiomyocytes, as well as populations of cardiomyocytes so produced. Also provided are methods of identifying or classifying a genetic variants associated with a cardiac condition, of screening compounds, and of predicting a subject's response to one or more treatments for a cardiac condition using the populations of cardiomyocytes produced by the methods of the invention. Other aspects of the invention relate to therapeutic compositions comprising the population of cardiomyocytes produced by the methods of the invention, for use in the treatment of a cardiac condition.

Description

Methods of Cardiomvocvte Production

Field

The present invention relates to in vitro methods for producing cardiomyocytes, cardiomyocytes produced by such methods and methods for the use of such cardiomyocytes.

Background

Cardiac diseases are the most prevalent cause of death worldwide (World Health Organisation 2018) and understanding the molecular mechanisms underlying cardiac afflictions and identifying drug sensitivities is an active field of research. Hypertrophic Cardiomyopathy (HCM) is the most common inherited heart disease, with an estimated prevalence of 1 :200 to 1 :500 individuals worldwide (Maron et al. 2018).

The recent emergence of human induced pluripotent stem cells (hiPSCs) provided an important human cell source to develop alternatives to animal models that do not fully recapitulate the features of human disease, based on their in vitro expansion potential and capability to differentiate into any somatic cell-type, including cardiomyocytes (hiPSC-CMs) (Takahashi et al. 2007; Yoshida and Yamanaka, 2017).

Protocols for cardiac differentiation were improved by the modulation of key signalling pathways associated with embryonic heart development that enabled, to some extent, recapitulation of the critical stages of cardiac specification, leading to a higher differentiation efficiency and yield. One of the most widely applied differentiation protocols is based on the temporal modulation of the Wnt signalling pathway in 2D hiPSC culture models (commonly termed GIWI protocols - for GSK3 inhibition, Wnt Inhibition) (Karakikes et al. 2015; Lian et al. 2013; Burridge et al. 2014). However, hiPSC-CMs produced by current 2D GIWI protocols tend to display high levels of heterogen icity and have immature CM characteristics that resemble fetal rather than adult CMs.

Since differentiation protocols have been mostly applied in monolayer cultures, they do not consider the 3D configuration of the embryo, which provides appropriate spatial, temporal and mechanical cues that are crucial for the human heart development. Recently, cardiac differentiation and maturation strategies that use a 3D culture to mimic the embryonic development of the heart in vitro have emerged. These approaches demonstrated a faster and more reproducible way to generate hiPSC-CMs with higher levels of maturity than the ones obtained from similar 2D strategies (Branco et al. 2019; Correia et al. 2018). Maintenance of hiPSC-CMs in culture for long periods of time has been associated with increasing maturity (Kamakura et al. 2013). Nevertheless, aggregates produced using these 3D protocols cannot be maintained in culture for long periods of time due to loss of cell viability, which prevents the production of hiPSC-CMs with more mature phenotypes. Moreover, standard 2D and 3D protocols also give rise to heterogenous cell populations.

Transplantation of cardiac progenitors (CPCs) derived from ESC or IPSC has been explored in a series of preclinical models (Chong et al. 2014; Shiba et al. 2016; JJ et al.2014; Liu et al. 2018). A small clinical trial (ESCORT) transplanted a fibrin patch embedded with hESC-derived cardiac progenitors in patients with severe left ventricular ischemia. An hESCs line was committed to a cardiac fate for 4 days in culture and CD 15+ lsl-1 + progenitors were subsequently selected for transplant. No tumor was detected in the 18-month follow-up and patients did not present arrythmias, one of the main concerns raised by previous animal studies. This trial demonstrated that hESC-CPCs transplantation was well-tolerated, although three patients developed silent alloimmunization and larger studies are required to further confirm safety and efficacy of this strategy (Menasche et al. 2018; Bellamy et al. 2015). Currently, there are five ongoing clinical trials using hiPSC-derived cardiomyocyte transplantation. The first was approved in Japan (NCT04696328) using transplant of an allogenic iPSC-CMs sheet, following positive results in mice and pig models, in which heart function was improved (Menasche et al. 2018; Bellamy et al. 2015). In the phase l/ll HEAL-CHF trial, allogenic hiPSC-CMs will be injected directly in the myocardium of patients with HF and the same strategy will be used in another trial for congestive HF and DCM (NCT04982081) (Guan et al. 2020). The BioVAT-HF trial will explore implantation of engineered heart muscle produced from iPSC-CMs and stromal cells in a hydrogel, after proof-of-concept preclinical studies having demonstrated its potential in remuscularization and cardiac repair (Tiburcy et al. 2017; Yildirim et al. 2007). The LAPis study will transplant iPSC-CM spheroids (HS-10001) in patients with ICM following a report in small and large animal models supporting feasibility and efficacy (Kawaguhi et al. 2021). One of the major issues still limiting clinical application of hESC or hiPSC-derived CPCs is that they present an immature phenotype when compared to adult cardiomyocytes. This is hypothesized to be the reason underlying the occurrence of arrhythmias in animal models following transplantation, as the hiPSC-CMs may not efficiently integrate and synchronize with the host CMs (Chong et al. 2014; Shiba et al. 2016).

Despite the advances in methods for generating hiPSC-CM, the immaturity of the hiPSC-CMs produced by current 2D GiWi protocols and 3D culture strategies limits the generation of reliable cellular disease models to study genotype-phenotype relationships, the impact of so-called variants of uncertain significance (VUS) or new therapeutic approaches (Hoes, Borner, and van der Meer 2019; Purevjav 2019; Musunuru et al. 2018). This immaturity is of particular importance as teratoma formation due to the presence of highly proliferating immature cells is a main concern in transplantation, raising the need to further develop differentiation and enrichment strategies of cardiac progenitor cells for transplantation applications (Liu et al. 2018; Nussbaum et al. 2007).

Summary

The present inventors have developed improved methods to produce populations of cardiomyocytes from pluripotent stem cells in vitro. In particular, the methods may generate, in a short period of time compared to previous methods, homogeneous populations of cardiomyocytes that are more mature than cardiomyocytes produced by previous 2D and 3D methods and which are capable of recapitulating the phenotype of genetic cardiac conditions, such as-hypertrophic cardiomyopathy (HCM). The cardiac differentiation protocol herein described may be useful, for example in disease modelling.

A first aspect of the invention provides a method for producing a population of cardiomyocytes comprising;

(i) culturing a 3-dimensional aggregate of pluripotent stem cells in a cardiomyocyte induction medium in the presence of a GSK30 inhibitor;

(ii) culturing the 3-dimensional aggregate from step (i) in the cardiomyocyte induction medium; (iii) culturing the 3-dimensional aggregate from step (ii) in the cardiomyocyte induction medium in the presence of a Wnt antagonist;

(iv) culturing the 3-dimensional aggregate from step (iii) in the cardiomyocyte induction medium;

(v) culturing the 3-dimensional aggregate from step (iv) in a cardiomyocyte maturation medium to produce a 3-dimensional aggregate of immature cardiomyocytes,

(vi) isolating VCAM1 -positive cells from the 3-dimensional aggregate from step (v); and

(vii) culturing the VCAM1 -positive cells from step (vi) in a monolayer in the cardiomyocyte maturation medium to produce a population of cardiomyocyte cells.

In some embodiments, the method further comprises step (viii) comprising replating the cardiomyocytes from step (vii) in micropatterned coverslips in cardiomyocyte maturation medium.

A second aspect of the invention provides a-population of cardiomyocytes produced by a method of the first aspect.

A third aspect of the invention provides a therapeutic composition comprising a population of cardiomyocytes of the second aspect of the invention for use in a method of treating a cardiac condition.

A fourth aspect of the invention provides a method of identifying or classifying a genetic variant associated with a cardiac condition, comprising; providing a first population of cardiomyocytes produced by the method of the first aspect, wherein the cardiomyocytes in the population comprises a genetic variant; and comparing one or more of the morphology, function, gene expression, transcriptome and proteome profile of the population of cardiomyocytes with a second population of cardiomyocytes, wherein the-second population does not comprise the genetic variant; wherein a difference in one or more of the morphology, function, gene expression, transcriptome and proteome profile between the first population and the second population is indicative that the genetic variant is associated with a cardiac condition. For example, the population may display morphology characterised by one or more of an increased size, rounder morphology, disorganized sarcomeres and higher percentage of multinucleated cells.

A fifth aspect of the invention provides a method of identifying a gene associated with a cardiac condition comprising; providing a population of cardiomyocytes produced by the method of the first aspect from induced pluripotent stem cells (IPSCs) derived from an individual with a cardiac condition, and comparing the gene expression profile of one or more genes in the population of cardiomyocytes with the gene expression profile of one or more genes of a control population of cardiomyocytes, wherein a difference in the expression of a gene in the population relative to the control population is indicative that the gene is associated with a cardiac condition.

A sixth aspect of the invention provides a method of screening a compound comprising; contacting an isolated population of cardiomyocytes of the second aspect with a test compound; and determining the effect the test compound on said cardiomyocytes. A seventh aspect of the invention provides a method of assessing a cardiac condition in an individual comprising; providing a population of cardiomyocytes produced by the method of the first aspect from induced pluripotent stem cells (IPSCs) derived from the individual, comparing the morphology of the population of cardiomyocytes with a control population of cardiomyocytes; wherein a difference in morphology between the population and the control population is indicative that the individual has or is at risk of a cardiac condition.

An eight aspect of the invention provides an in vitro method of predicting a subject’s response to one or more treatment regimens for treating cardiac condition comprising; providing a population of cardiomyocytes according to claim 19, wherein the population is derived from the subject; and comparing one or more of the morphology, function, gene expression, transcriptome and proteome profile of the population of cardiomyocytes with a control population of cardiomyocytes to predict the response of the subject to one or more treatment regimens.

Other aspects and embodiments of the invention are described in more detail below.

Brief Description of the Figures

Figure 1 shows schematics illustrating the culture conditions used to induce differentiation of hiPSCs to cardiomyocytes with the 3D2D differentiation protocol. DF6, TCLab and Gibco cells were forced to aggregate in AggreWell™800 plates. CM differentiation was performed following temporal modulation of Wnt signalling pathway. On day 7 of differentiation, aggregates were transferred to ultra-low attachment plates; then, on day 13, aggregates were dissociated and VCAM-1 positive cells, sorted by FACS, were replated on wells coated with Matrigel, and kept in 2D monolayer culture until day 31 of cardiac differentiation. Alternatively, on day 27, cardiomyocytes were replated into micropatterned 4DCell coverslips and maintained in culture until day 31 of cardiac differentiation.

Figure 2 shows changes in gene expression in cells at different stages of cardiomyocyte differentiation using the 3D2D protocol. Figure 2 (A) shows the culture conditions and representative IF images of cells at Day 0 (top panel), day 7 (upper middle panel) and day 13 (bottom middle panel) and day 31 (bottom panel). Day 0 hiPSCs aggregates were stained for pluripotent stem cell markers OCT4 (green) and Nanog (red). Day 7 hiPSC-CMs were stained for Troponin T(green), VCAM-1 , and nuclei (blue). Day 13 hiPSC-CMs were sorted for VCAM-1 and stained for Troponin T (green), VCAM-1 (red) and nuclei (blue). Day 31 hiPSC-CMs plated in micropatterned coverslips were stained for myosin binding protein C (cMyBP-C, green), F-actin (red) and nuclei (blue). Figure 2 (B) shows the change in gene expression of pluripotency associated markers Nanog and OCT4, the mesoderm associated marker Brachyury, cardiac precursor markers Isl1 and Nkx2.5, and the cardiac associated sarcomere gene TNNT2 over the first 13 days of differentiation. Figure 3 shows representative IF image of hiPSC-CMs generated using the 2D or the 3D2D protocol. Cells were stained for: MYBPC3 Myosin binding protein C, also known as cMyBP-C, (a and b), Troponin T (c and d) or a-Actinin (e and f) (green) and F-actin (red). Nuclei are stained with DAPI (in blue). On the right side, schematic illustrations depict the location of the analysed sarcomeric proteins, with a magnification of the sarcomeric pattern formed among them.

Figure 4 shows sarcomere length measurement using IF against a-Actinin. (A) Example of a sarcomere measurement. (B) Quantitative plot of the data collected from 200 and 330 sarcomere measurements in 2D or 3D2D TCLab hiPSC-CMs, respectively. Data represented as mean ± SD. ***p<0.001,

Figure 5 shows qRT-PCR analysis of sarcomeric genes expression in WT hiPSC-CMs (average of 3 independent cell lines), under the different experimental conditions: 2D protocol (2D_WT); 3D2D protocol without sorting (3D2D_Unsorted_WT) and 3D2D protocol VCAM-1 -positive cells (3D2D_VCAM+_WT), compared to human heart as a positive reference. (A) Data normalized against housekeeping U6 gene. (B) Data normalized against TNNT2 (n= 3 independent experiments, data represented as mean + SD).

Figure 6 (A) shows qRT-PCR analysis of sarcomeric splicing isoforms (TNNT2, TNN and TNNI) in wild type hiPSC-CMs (average of 3 independent cell lines), under different experimental conditions: 2D protocol (2D_WT); 3D2D protocol without sorting (3D2D_Unsorted_WT) and 3D2D protocol VCAM-1 -positive cells (3D2D_VCAM+_WT), compared to human heart as a positive reference. (B) Shows the ratio between fetal and adult and fetal and total sarcomeric splicing isoforms, n = 3 independent experiments, data represented as mean + SD.

Figure 7 shows RNA sequencing analysis of TTN and TNNT2 adult and fetal splicing isoforms expressed in wild type hiPSC-CMs differentiated using the 3D2D protocol with VCAM-1 sorting. hiPSC-CMs were differentiated form DF6, Gibco and TCLab cell lines.

Figure 8 shows classification of cell type based on single-cell RNA sequencing of differentiated DF6 wild type cells following the 3D2D differentiation protocol with VCAM-1 purification.

Figure 9 shows qRT-PCR analysis of sarcomeric gene expression in WT hiPSC-CMs (average of 3 independent cell lines) and hypertrophic cardiomyopathy hiPSC-CMs. n = 3 independent experiments, data represented as mean + SD.

Figure 10 shows qRT-PCR analysis of sarcomeric isoforms (TNNT2, TTN and TNNI) in wild type hiPSC-CMs (average of 3 independent cell lines), and hypertrophic cardiomyopathy hiPSC-CMs. n= 3 independent experiments, data represented as mean + SD.

Figure 11 shows representative immunofluorescence images of wild type (WT) and hypertrophic cardiomyopathy (HCM) hiPSC-CMs at day 31 of cardiac differentiation, platted into conventional coverslips coated with Matrigel. The cells are stained for a-actinin, cMyBP-C or Troponin T (green) and F-actin (red). Nuclei are stained with DAPI (blue). Figure 12 shows quantification of morphological parameters and percentage of binucleated cells in wild type (DF6, Gibco, TCLab) and hypertrophic cardiomyopathy (Ruah, Xutl, Jewl, Newl, Miov and looi) hiPSC-CMs using the 3D2D differentiation protocol. (A) to (D) shows the aspect ratio, circularity, cell size (as area) and percentage of binucleated cells, respectively, n = 2 independent experiments (105-234 cells/cell line). Data represented as mean + SD.

Figure 13 shows representative immunofluorescence images of wild type (WT) and hypertrophic cardiomyopathy (HCM) hiPSC-CMs after being replated into micropatterned 4DCell coverslips on day 27 of cardiac differentiation and kept in culture until day 31. The cells are stained for cMyBP-C (green) and F-actin (red). Nuclei are stained with DAPI (blue).

Figure 14 shows quantification of morphological parameters in wild type (WT, blue) and gene-edited hypertrophic cardiomyopathy (HCM, orange) hiPSC-CMs at day 31 of cardiac differentiation, derived from DF6 and TCLab cell lines and platted into conventional versus micropatterned 4DCell coverslips. (A) and (B) show the cells size (as area) and circularity (as aspect ratio), respectively.

Detailed Description

This invention relates to the production of cardiomyocytes through the in vitro differentiation of pluripotent stem cells. Pluripotent stem cells are cultured in 3-dimensional cell culture in a cardiomyocyte induction medium successively in the presence of a GSK3 inhibitor (step I); in cardiomyocyte induction medium alone (step II); in the presence of a Wnt antagonist (step ill); and finally in cardiomyocyte induction medium alone (step iv). The resulting 3-dimensional aggregate is then cultured in a cardiomyocyte maturation medium to produce a 3-dimensional aggregate of immature cardiomyocytes (step v). VCAM1 -positive cells are then isolated from the immature cardiomyocytes within the 3-dimensional aggregate (step vi) and cultured in 2- dimensional cell culture in the cardiomyocyte maturation medium to produce cardiomyocyte cells (step vii). These cardiomyocytes can be replated in micropatterned coverslips (step viii) for morphological and functional analysis. The use of micropatterned coverslips uniformizes the measurements of the morphological parameters, whilst still maintaining the difference in morphology between control and diseased cells.

The methods described herein may allow the rapid generation of cardiomyocytes. For example, a homogeneous population of cardiomyocytes may be produced in 31 days or less.

A population of cardiomyocytes produced by the methods described herein may be homogeneous. For example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% of the cells in a population produced as described herein may be cardiomyocytes. Homogeneity of a population of cardiomyocytes may be determined by any convenient means. For example, the percentage of cardiomyocytes in a cell population derived from hiPSCs following a cardiac differentiation protocol may be determined by quantifying the percentage of cells in the population that express cardiomyocyte-specific markers, such as TNNT2, for example, by performing immunofluorescence imaging, flow-cytometry, or single-cell RNA sequencing. A population of cardiomyocytes produced by the methods described herein may display a morphology, gene expression profile, functional proprieties, transcriptome profile and/or proteome profile that are characteristic of more mature cardiomyocytes when compared to those generated by previous in vitro differentiation methods. In some embodiments, a population of cardiomyocytes produced by the methods described herein may be more mature than fetal cardiomyocytes or cardiomyocytes produced by previous in vitro differentiation methods. For example, the population of cardiomyocytes may display a more mature morphology, gene expression profile, functional properties, transcriptome profile and/or proteome profile than fetal cardiomyocytes or cardiomyocytes produced by previous in vitro differentiation methods.

Previous in vitro differentiation methods may include GSK3 and Wnt Inhibition (GiWi) techniques in monolayer culture (2D), for example as described in Karakikes et al. 2015; Lian et al. 2013; Burridge et al. 2014; and aggregate or organoid-based techniques (3D), as described in Branco et al. 2019; Correia et al. 2018.

The increased expression of cardiomyocyte-specific genes and/or adult isoforms of cardiomyocyte specific genes, such as one or more of MYBPC3, MYH7, MYH6, ACTN2, TTN and TNNT2, in a population of cardiomyocytes produced by the methods described herein may be characteristic of mature cardiomyocytes or more characteristic of mature cardiomyocytes than fetal cardiomyocytes. In some embodiments, the cardiomyocytes in the population may display increased expression of cardiomyocyte-specific genes and/or increased expression of adult splicing isoforms, of cardiomyocyte specific genes, such as one or more of MYBPC3, MYH7, MYH6, ACTN2, TTN and TNNT2, relative to fetal cardiomyocytes or cardiomyocytes produced by previous in vitro differentiation methods.

A population of cardiomyocytes produced by the methods described herein may display a transcriptome profile characteristic of mature cardiomyocytes when compared to cardiomyocytes generated by previous in vitro differentiation methods. Transcriptome profiling can be performed by any well-known method in the art, for example microarray analysis (Barbulovic-Nad, I et al. 2006) or RNA-Seq (Wang, Z., 2009).

Transcriptome profiling may be performed on total RNA transcripts (e.g. including mRNA, tRNA, rRNA, miRNA, IncRNA etc.) or mRNA alone. The transcriptome profile of a population of cardiomyocytes produced by the methods described herein may be more similar to the transcriptome profile of mature cardiomyocytes, for example adult cardiomyocytes, compared to that of cardiomyocytes produced by previous methods.

The cardiomyocytes in the population may also display increased levels of cardiomyocyte-specific proteins and/or increased levels of adult isoforms of cardiomyocyte-specific proteins, such as one or more of Myosin Binding Protein C, Myosin Heavy Chain 7, Myosin Heavy Chain 6, Actinin Alpha 2, Titin, Titin N2B (adult splicing isoform) and/or Troponin 11, relative to cardiomyocytes produced by previous in vitro differentiation methods. Conversely, the cardiomyocytes in the population may display decreased expression of fetal isoforms of cardiac-specific proteins, such as Troponin T2 (fetal troponin T) and/or Titin N2BA (fetal splicing isoform), relative to fetal cardiomyocytes or cardiomyocytes produced by previous in vitro differentiation methods.

A population of cardiomyocytes produced by the methods described herein may display a proteome profile characteristic of mature cardiomyocytes when compared to cardiomyocytes generated by previous in vitro differentiation methods. Proteome profiling can be performed to analyse the entire set of proteins, or a subset of proteins, produced by a cell, cell population, or tissue. Proteome profiling can be performed by any well-known method in the art, for example mass spectrometry (Posch, A., 2021. Proteomic profiling. New York: Humana Press.) The proteome profile of a population of cardiomyocytes produced by the methods described herein may be more similar to the proteome profile of mature cardiomyocytes, for example adult cardiomyocytes, compared to that of cardiomyocytes produced by previous methods.

The cardiomyocytes produced by the methods described herein may display a morphology that is characteristic of mature cardiomyocytes; or more characteristic of mature cardiomyocytes than fetal cardiomyocytes. For example, cardiomyocytes produced by the methods described herein may be more elongated, display a more organized sarcomere and/or display an increased sarcomere length than fetal cardiomyocytes or cardiomyocytes produced by previous in vitro differentiation methods.

The cardiomyocytes produced by the methods described herein may display a function or activity that is characteristic of mature cardiomyocytes; or more characteristic of mature cardiomyocytes than fetal cardiomyocytes. For example, the cardiomyocytes may display contractility characteristic of mature cardiomyocytes; or more characteristic of mature cardiomyocytes than fetal cardiomyocytes. Cardiomyocytes produced by the methods described herein may have greater contractility than fetal cardiomyocytes or cardiomyocytes produced by previous in vitro differentiation methods.

A population of cardiomyocytes produced by the methods described herein may be capable of displaying a cardiomyocyte disease phenotype, such as hypertrophy (e.g. the cardiomyocytes may be hypertrophy- competent). For example, the cardiomyocytes may be sufficiently mature to display a disease phenotype, such as hypertrophy, when subjected to conditions in which mature cardiomyocytes display the disease phenotype, such as the presence of a causative genetic mutation.

Cardiomyocytes are produced as described herein from human pluripotent stem cells.

Pluripotent stem cells are capable of self-renewal in vitro, exhibit an undifferentiated phenotype; and are potentially capable of differentiating into any fetal or adult cell type of any of the three germ layers (endoderm, mesoderm and endoderm). A pluripotent stem cell is distinct from a totipotent cell and cannot give rise to extraembryonic cell lineages. The population of pluripotent stem cells may be clonal i.e. genetically identical cells descended from a single common ancestor cell.

Pluripotent stem cells may express one or more of the following pluripotency associated markers: Oct4, Sox2, ALPL, POU5f1, SSEA-3, Nanog, SSEA-4, Tra-1-60, KLF-4 and c-myc, preferably one or more of POU5f1, NANOG and SOX2. A pluripotent cell may lack markers associated with specific differentiative fates, such as Bra, Sox17, FoxA2, aFP, Sox1, NCAM, GATA6, GATA4, Handl and CDX2. In particular, a pluripotent cell may lack markers associated with endodermal, mesodermal or ectodermal and fates.

Suitable pluripotent stem cells include human pluripotent stem cells. In some embodiments, the pluripotent stem cells may comprise a genetic mutation associated with a cardiac condition, such as cardiomyopathy. The mutation may cause or contribute to the cardiac condition or may be suspected of causing or contributing to the cardiac condition. Cardiomyocytes produced as described herein from pluripotent stem cells with a genetic mutation associated with a cardiac condition, such as cardiomyopathy, may be useful for example in drug screening and disease modelling.

The pluripotent cells may be induced pluripotent stem cells (IPSCs). IPSCs are pluripotent cells, which are derived from non-pluripotent, fully differentiated donor or antecedent cells.

Preferably, the IPSCs are human IPSCs (hiPSCs). hiPSCs are pluripotent stem cells that are derived or reprogramed from donor cells, which may be somatic cells or other antecedent cells obtained from a source. Suitable donor cells for reprogramming into hiPSCs as described herein may be obtained from a donor individual. In some embodiments, the donor individual may have a cardiac condition, such as a cardiomyopathy, for example hypertrophic cardiomyopathy, dilated cardiomyopathy, restrictive cardiomyopathy, Arrhythmogenic right ventricular dysplasia, left ventricular hypertrophy (LVH), ischemic heart disease, hypertension, heart failure, valve disease and unclassified cardiomyopathy.

The donor cells may be mammalian, preferably human cells. Suitable donor cells include mesoderm cells, such as adult fibroblasts and blood cells, for example peripheral blood cells, such as peripheral blood mononuclear cells. A population of donor cells for reprogramming may be isolated, for example from a blood sample, such as an umbilical cord sample, or a skin biopsy, for example following dispersal using collagenase or trypsin and out-growth in appropriate cell culture conditions. Suitable methods for the isolation of donor cells are well-known in the art and include, for example magnetic activated cell sorting (see, for example, Gaudernack et al 1986 J Immunol Methods 90 179), fluorescent activated cell sorting (FACS: see for example, Rheinherz et al (1979) PNAS 764061), and cell panning (see for example, Lum et al (1982) Cell Immunol 72 122).

Donor cells are typically reprogrammed into iPSCs by the introduction of reprogramming factors, such as Oct4, Sox2 and Klf4 into the cell. The reprogramming factors may be proteins or encoding nucleic acids and may be introduced into the differentiated cells by any suitable technique, including plasmid, transposon or more preferably, viral transfection or direct protein delivery. Other reprogramming factors, for example Klf genes, such as Klf-1, -2, -4 and -5; Myc genes such as C-myc, L-myc and N-myc, Nanog; SV40 Large T antigen; Lin28; and short hairpins (shRNA) targeting genes such as p53, may also be introduced into the cell to increase induction efficiency. Following introduction of the reprogramming factors, the donor cells may be cultured. Cells expressing pluripotency markers may be isolated and/or purified to produce a population of IPSCs. Techniques for the production of hiPSCs are well-known in the art (Yamanaka et al Nature 2007; 448:313-7; Yamanaka 62007 Jun 7; 1(1):39-49; Kim et al Nature. 2008 Jul 31 ; 454(7204):646-50;

Takahashi Cell. 2007 Nov 30; 131(5):861-72. Park et al Nature. 2008 Jan 10; 451(7175):141-6; Kimet et al Cell Stem Cell. 2009 Jun 5;4(6):472-6; Vallier, L., et al. Stem Cells, 2009. 9999(999A): p. N/A; Baghbaderani et al 2016; Stem Cell Rev. 2016 Aug; 12(4):394-420; Baghbaderani et al. (2015) Stem Cell Reports, 5(4), 647-659). Conventional techniques may be employed for the culture and maintenance of IPSCs (Vallier, L. et al Dev. Biol. 275, 403-421 (2004), Cowan, C.A. et al. N. Engl. J. Med. 350, 1353-1356 (2004), Joannides, A. et al. Stem Cells 24, 230-235 (2006) Klimanskaya, I. et al. Lancet 365, 1636-1641 (2005), Ludwig, T.E. et al. Nat. Biotechnol. 24, 185-187 (2006)). iPSCs for use in the present methods may be grown in defined conditions or on feeder cells. For example, iPSCs may be conventionally cultured in a culture dish on a layer of feeder cells, such as irradiated mouse embryonic fibroblasts (MEF), at an appropriate density (e.g. 10^s to 10⁶ cells/60mm dish), or on an appropriate substrate, in a feeder conditioned or defined IPSC maintenance medium. IPSCs for use in the present methods may be passaged by enzymatic, chemical or mechanical means. In some embodiments, IPSCs may be passaged using an EDTA dissociation buffer, for example using EDTA dissociation buffer (Life Technologies) at a concentration of about 0.5 mM. In some embodiments, IPSCs may be passaged on matrigel™ or an ECM protein, such as vibronectin, and in an IPSC maintenance medium, such as mTeSR Plus ™, mTeSR™1 orTeSR™2 (StemCell Technologies) or E8 flex (Life Thermo) culture medium.

A population of pluripotent stem cells suitable for use in the present methods may be heterogeneous or may be substantially free from one or more other cell types (i.e. homogenous). Pluripotent cells may, for example, be separated from other cell types, using any technique known to those skilled in the art, including those based on the recognition of extracellular epitopes by antibodies and magnetic beads or fluorescence activated cell sorting (MACS or FACS) including the use of antibodies against extracellular regions of molecules found on stem cells, such as SSEA4.

Differentiation and maturation of the cell populations in the steps of the methods described herein is induced by culturing the cells in culture medium in a series of steps. In some steps, the cells may be cultured in the presence of one or more differentiation factors. The differentiation factor(s) that is listed for each culture medium is preferably exhaustive and medium may be devoid of other differentiation factors. In preferred embodiments, the culture media are chemically defined media. For example, a culture medium may consist of a chemically defined nutrient medium that is supplemented with an effective amount of one or more differentiation factors, as described below. A chemically defined nutrient medium may comprise a basal medium that is supplemented with one or more serum-free culture medium supplements.

Differentiation factors are factors which modulate, for example promote or inhibit, a signalling pathway which mediates differentiation in a mammalian cell, for example factors which inhibit or increase the Wnt signalling pathway. Differentiation factors may include growth factors, cytokines and small molecules. Differentiation factors which are used in one or more of the media described herein include Wnt antagonists, such as IWP-4 and GSK-3 inhibitors (such as CHIR-99021). A differentiation factor may be present in a medium described herein in an amount that is effective to modulate a signalling pathway in cells cultured in the medium.

The extent of differentiation of the cell population during each step may be determined by monitoring and/or detecting the expression of one or more cell markers in the population of differentiating cells. For example, an increase in the expression of markers characteristic of the more differentiated cell type or a decrease in the expression of markers characteristic of the less differentiated cell type may be determined. The expression of cell markers may be determined by any suitable technique, including immunocytochemistry, immunofluorescence, RT-PCR, immunoblotting, flow cytometry, fluorescence activated cell sorting (FACS), and enzymatic analysis. In preferred embodiments, a cell may be said to express a marker if the marker is detectable on the cell surface. For example, a cell which is stated herein not to express a marker may display active transcription and intracellular expression of the marker gene, but detectable levels of the marker may not be present on the surface of the cell.

Cells may be cultured in steps (i) to (v) in three-dimensional (3D) cell culture. 3D cell culture is performed in an artificial environment that allows the cultured cells to grow and interact with their surroundings in all three dimensions. Cells cultured in 3D cell culture may self-assemble into 3D aggregates or clusters. 3D aggregates may include embryoid bodies and spheroids.

In some embodiments, a method described herein may comprise providing a population of pluripotent stem cells in the form of 3D cell aggregates.

In other embodiments, a method described herein may comprise providing a population of pluripotent stem cells and aggregating the population to form 3D aggregates. Suitable techniques and reagents are readily available in the art (e.g. Aggrewell™ microwell plates, Stemcell Technologies™, CA).

In some embodiments, pluripotent stem cells may be incubated in maintenance medium supplemented with 0.1 to 100 pM Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor, preferably about 10 pM. The pluripotent stem cells may then be dissociated, for example using Acutase and seeded at an appropriate density in low adhesion plates, micropatterned surfaces or microwell plates, or hanging drops to facilitate the formation of 3D cell aggregates (e.g. 10⁵to 10⁶cells/microwell in AggreWell™800 plates). For example, the dissociated IPSCs may be seeded into a microwell plate at a density of between 10^s and 10⁶ cells/cm², preferably between 0.4x10⁶ and 0.9x10⁶cells/cm².

Preferably, the maintenance medium is a chemically defined medium, such as the commercially available mTeSR Plus™ maintenance medium (StemCell Technologies). Suitable ROCK inhibitors are readily available in the art and include Y-27632 ((R)-(+)-trans-4-(1-Aminoethyl)-N-(4- Pyridyl)cyclohexanecarboxamide).

The seeded pluripotent stem cells may then be cultured in maintenance medium supplemented with ROCK inhibitor for between 0.5 and 4 days, preferably 1 day, and then transferred to maintenance medium without ROCK inhibitor for between 1 and 5 days, preferably 2 days, to promote IPSC aggregate formation. For example, the seeded IPSCs may be incubated for 1 days in maintenance medium supplemented with 0.1 to 100 pM ROCK inhibitor, preferably about 10 pM, and then transferred to maintenance medium without ROCK inhibitor for 2 days to produce 3D aggregates of pluripotent stem cells for use in the methods described herein.

In the initial steps of the method described herein, aggregates of pluripotent stem cells are cultured in a cardiomyocyte induction medium.

Cardiomyocyte induction medium is an insulin-free nutritive cell culture medium. Preferably the cardiomyocyte induction medium is a chemically defined serum free medium. A chemically defined medium (CDM) is a nutritive solution for culturing cells which contains only specified components, preferably components of known chemical structure. A CDM is devoid of undefined components or constituents, which include undefined components, such as feeder cells, stromal cells, serum, serum albumin and complex extracellular matrices, such as Matrigel™. For example, a CDM does not contain stromal cells, such as OP9 cells, expressing Notch ligands, such as DLL1 or DLL4.

The cardiomyocyte induction medium may comprise a chemically defined basal medium. Suitable basal media include RPMI-1640 (Moore, G.E. and Woods L.K., (1976) Tissue Culture Association Manual. 3, 503- 508), E8 basal medium (Lin, Y. and Zou, J., (2020) STAR Protoc. 1(1)) and DMEM-F12 (Sequiera, G.L. et al., (2013) A Simple Protocol for the Generation of Cardiomyocytes from Human Pluripotent Stem Cells. In: Turksen K. (eds) Human Embryonic Stem Cell Protocols. Methods in Molecular Biology, vol 1307. Humana Press, New York, NY.).

The basal medium may be supplemented by a serum-free culture medium supplement in the cardiomyocyte induction medium. Both the basal medium and the supplement of the cardiomyocyte induction medium are insulin free. Suitable supplements are described above and may include biotin, vitamin A, bovine serum albumin, catalase, human transferrin, superoxide dismutase, corticosterone, galactose, glutathione, T3 hormone, linoleic acid, L-glutamine or substitutes, such as GlutaMAX-1™, ascorbic acid, monothiolglycerol (MTG), antibiotics such as penicillin and streptomycin, human serum albumin, for example recombinant human serum albumin, such as Cellastim™ (Merck/Sigma) and Recombumin™ (albumedix.com), transferrin and 2-mercaptoethanol. A basal medium may be supplemented with a serum substitute, such as Knockout Serum Replacement (KOSR; Invitrogen).

Suitable serum-free media supplements include B27 (Brewer et al Brain Res (1989) 49465-74; Brewer et al J. Neurosci Res 35567-576 (1993); Brewer et al Focus 16 1 6-9; Brewer et al (1995) J. Neurosci. Res. 42:674-683; Roth et al J Trace Elem Med Biol (2010) 24 130-137) and NS21 (Chen et al J. Neurosci Meths (2008) 171 239-247). Serum-free media supplements, such as B27 and N21, are well known in the art and widely available commercially (e.g. Invitrogen; Sigma Aldrich Inc). Mixtures of agents for production of cardiomyocytes may be used in place of commercially available serum-free supplements. Suitable mixtures are well known in the art. In some embodiments, if the induction medium comprises E8 basal medium, serum-free supplements or mixtures need not be added.

Preferably the serum-free media supplement is B27 without insulin. For example, the cardiomyocyte induction medium may comprise between 1 % and 5% (v/v) B-27 minus insulin, preferably 2% (v/v) B-27 minus insulin.

Suitable techniques for 2D and 3D cell culture are well-known in the art (see, for example, Basic Cell Culture Protocols, C. Helgason, Humana Press Inc. U.S. (15 Oct 2004) ISBN: 1588295451; Human Cell Culture Protocols (Methods in Molecular Medicine S.) Humana Press Inc., U.S. (9 Dec 2004) ISBN: 1588292223; Culture of Animal Cells: A Manual of Basic Technique, R. Freshney, John Wiley & Sons Inc (2 Aug 2005) ISBN: 0471453293, Ho WY et al J Immunol Methods. (2006) 310:40-52, Handbook of Stem Cells (ed. R.

Lanza) ISBN: 0124366430) Basic Cell Culture Protocols’ by J. Pollard and J. M. Walker (1997), ‘Mammalian Cell Culture: Essential Techniques’ by A. Doyle and J. B. Griffiths (1997), ‘Human Embryonic Stem Cells’ by A. Chiu and M. Rao (2003), Stem Cells: From Bench to Bedside’ by A. Bongso (2005), Peterson & Loring (2012)Human Stem Cell Manual: A Laboratory Guide Academic Press and ‘Human Embryonic Stem Cell Protocols’ by K. Turksen (2006). Media and ingredients thereof may be obtained from commercial sources (e.g. Gibco, Roche, Sigma, Europa bioproducts, R&D Systems). Standard mammalian cell culture conditions may be employed for the above culture steps, for example 37°C, 21 % Oxygen, 5% Carbon Dioxide. Media is preferably changed every two days and cells allowed to settle by gravity.

In the methods described herein, aggregates of pluripotent stem cells are cultured in 3D cell culture in the cardiomyocyte induction medium. The aggregates may be cultured under conditions that promote and maintain iPSC aggregation and expansion. For example, the aggregates may be cultured on aggregation promoting culture plates, such as AggreWell™ plates, or in a bioreactor, such as PBS MINI 0.1™. Aggregates of pluripotent cells described herein may express pluripotency markers (e.g. Nanog and OCT4) and lack expression of mesoderm markers (e.g. Brachyury).

The 3D aggregate of pluripotent stem cells is cultured in the cardiomyocyte induction medium in the presence of a GSK3 inhibitor in the first step of the methods described herein.

GSK30 inhibitors inhibit the activity of glycogen synthase kinase 30 (Gene ID 2932: EC2.7.11.26). GSK30 phosphorylates 0-catenin and targets it for degradation. Inhibition of GSK30 activates canonical Wnt/0- catenin signalling. Preferred inhibitors specifically inhibit the activity of glycogen synthase kinase 30. Suitable inhibitors include CHIR99021 (6-((2-((4-(2,4-Dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2- yl)amino)ethyl)amino)nicotinonitrile; Ring D. B. et al., Diabetes, 52:588-595 (2003)) alsterpaullone, kenpaullone, BIO(6-bromoindirubin-3'-oxime (Sato et al Nat Med. 2004 Jan;10(1):55-63), SB216763 (3-(2,4- dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2, 5-dione), Lithium and SB415286 {3-[(3-chloro-4- hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrole-2, 5-dione; Coghlan et al Chem Biol. 2000 Oct;7(10):793- 803). Preferably, the GSK30 inhibitor is CHIR99021.Suitable glycogen synthase kinase 30 inhibitors may be obtained from commercial suppliers (e.g. Stemgent Inc. MA USA; Cayman Chemical Co. Ml USA;

Selleckchem, MA USA). For example, the aggregate may be cultured in the cardiomyocyte induction medium in the presence of 0.1 to 1 OOpM of a GSK30 inhibitor, such as CHIR99021 , preferably from about 9 pM to 11 pM, e.g. about 10 pM.

The 3D aggregate of pluripotent cells may be cultured in the cardiomyocyte induction medium in the presence of GSK30 inhibitor for 12 hours to 2 days, preferably about 24 hours.

The pluripotent cells in the 3D aggregate may differentiate into mesoderm cells in the cardiomyocyte induction medium with the GSK30 inhibitor. For example, the pluripotent cells may become committed to mesodermal lineages and may express one or more mesoderm markers. Mesoderm markers may include Brachyury, Mix/1, MyoD, MyF5 and Pecaml. For example, following culture in the cardiomyocyte induction medium with the GSK30 inhibitor, the cells in the 3D aggregate may display increased Brachyury expression. Following culture in cardiomyocyte induction in the presence of a GSK30 inhibitor as described above, the 3D aggregate is then cultured in the cardiomyocyte induction medium in the absence of differentiation factors, such a GSK30 inhibitor or Wnt antagonist. The 3D aggregate may be cultured in the cardiomyocyte induction medium for between 24 hours and 3 days, preferably for about 2 days.

Following culture in cardiomyocyte induction medium as described above, the 3D aggregate of pluripotent stem cells is cultured in cardiomyocyte induction medium in the presence of a Wnt antagonist.

Wnt antagonists inhibit canonical Wnt/0-catenin signalling in cells. Suitable Wnt antagonists are well-known in the art and include Inhibitor of WNT Production (IWP)-2 (N-(6-Methyl-2-benzothiazolyl)-2-[(3, 4,6,7- tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]-acetamide), IWP-3 (2-[[3-(4-Fluorophenyl)-3, 4,6,7- tetrahydro-4-oxothieno[3,2-d]pyrimidin-2-yl]thio]-N-(6-methyl-2-benzothiazolyl)-acetamide), and IWP-4 (N-(6- Methyl-2-benzothiazolyl)-2-[[3,4,6,7-tetrahydro-3-(2-methoxyphenyl)-4-oxothieno[3,2-d]pyrimidin-2-yl]thio]- acetamide). In some preferred embodiments, the Wnt antagonist is IWP-4. Suitable Wnt antagonists may be obtained from commercial suppliers (e.g. Stemgent Inc. MA USA). For example, the aggregate may be cultured in the cardiomyocyte induction medium in the presence of 0.5 pM to 50 pM of Wnt antagonist, such as IWP-4, preferably about 5 pM.

The aggregate may be cultured in the cardiomyocyte induction medium for 24 hours to 4 days, preferably about 2 days.

The mesoderm cells in the 3D aggregate may differentiate into cardiac progenitor cells in the cardiomyocyte induction medium with the Wnt antagonist. For example, the mesoderm cells may become committed to cardiac lineages and may express one or more cardiac markers. Cardiac markers may include Isl1 and Nkx2.5. For example, following culture in the cardiomyocyte induction medium with the Wnt antagonist, the cells in the 3D aggregate may display increased Isl1 and Nkx2.5 expression. The cardiac progenitor cells in the 3D aggregate may display no expression or substantially no expression of pluripotency markers, such as Nanog and OCT4, and mesoderm markers, such as Brachyury.

Following culture in cardiomyocyte induction medium in the presence of a Wnt antagonist as described above, the 3D aggregate of cardiac progenitor cells is then cultured in the cardiomyocyte induction medium in the absence of differentiation factors, such a GSK30 inhibitor or Wnt antagonist. The 3D aggregate may be cultured in the cardiomyocyte induction medium for between 24 hours and 3 days, preferably for about 2 days.

Following culture in cardiomyocyte induction medium as described above, the 3D aggregate is cultured in cardiomyocyte maturation medium.

The 3D aggregate may be cultured in the cardiomyocyte induction medium in the absence of differentiation factors, such GSK30 inhibitors or Wnt antagonists.

Cardiomyocyte maturation medium is an insulin containing nutritive cell culture medium. Preferably, the cardiomyocyte maturation medium is a chemically defined serum free medium. The cardiomyocyte maturation medium may comprise a chemically defined basal medium. Suitable basal media include RPMI-1640 (Moore, G.E. and Woods L.K., (1976) Tissue Culture Association Manual. 3, 503- 508), E8 basal medium and DMEM-F12. The basal medium may be supplemented by a serum-free culture medium supplement in the cardiomyocyte induction medium. The basal medium may be insulin free.

Suitable supplements are described above and may include insulin, biotin, vitamin A, BSA, catalase, human transferrin, superoxide dismutase, corticosterone, galactose, glutathione, T3 hormone, and/or linoleic acid, L- glutamine or substitutes, such as GlutaMAX-1™, ascorbic acid, monothiolglycerol (MTG), antibiotics such as penicillin and streptomycin, human serum albumin, for example recombinant human serum albumin, such as Cellastim™ (Merck/Sigma) and Recombumin™ (albumedix.com), insulin, transferrin and 2-mercaptoethanol. A basal medium may be supplemented with a serum substitute, such as Knockout Serum Replacement (KOSR; Invitrogen). Suitable serum-free media supplements include B27 (Brewer et al Brain Res (1989) 494 65-74; Brewer et al J. Neurosci Res 35567-576 (1993); Brewer et al Focus 16 1 6-9; Brewer et al (1995) J. Neurosci. Res. 42:674-683; Roth et al J Trace Elem Med Biol (2010) 24 130-137) and NS21 (Chen et al J. Neurosci Meths (2008) 171 239-247). Serum-free media supplements, such as B27 and N21, are well known in the art and widely available commercially (e.g. Invitrogen; Sigma Aldrich Inc). Preferably, the serum-free media supplement is B27 with insulin. For example, the cardiomyocyte maturation medium may comprise between 1% and 4% (v/v) B-27 with insulin, preferably 2% (v/v) B-27 with insulin.

The aggregates may be cultured in the cardiomyocyte maturation medium for between 3 days and 7 days, preferably for about 5 days.

The aggregates may be cultured using any conventional suspension cell culture method in the presence of cardiomyocyte maturation medium. For example, the aggregates may be cultured in a culture plate or dish that is not amenable to cell adhesion, for example in low attachment cell culture plates. Suitable culture vessels are readily available, for example from commercial suppliers.

The cardiac progenitor cells in the 3D aggregate may differentiate into immature cardiomyocytes in the cardiomyocyte maturation medium. For example, the cardiac progenitor cells may become committed to cardiomyocyte lineages and may express one or more cardiomyocyte markers. Cardiomyocyte markers may include MYBPC3, MYH7, MYH6, ACTN2, TTN and TNNT2. For example, following culture in the cardiomyocyte induction medium with the GSK30 inhibitor, the cells in the 3D aggregate may display increased expression of one or more cardiomyocyte markers.

Following culture in the cardiomyocyte maturation medium, the 3D aggregates may be dissociated or disrupted to produce a dissociated population of cells.

Suitable techniques for dissociating or disrupting cell aggregates are well known in the art and include mechanical or enzymatic disruption. For example, the 3-dimensional aggregates may be dissociated by exposing the aggregates to a solution comprising a dissociation reagent, such as Trypsin-EDTA (Gibco), for example at a final concentration of 0.25% Trypsin-EDTA. Other suitable dissociation reagents are well known in the art and include TrypLE, Accutase and Accumax. Following dissociation of the aggregates, cells that express vascular cell adhesion molecule 1 (VCAM-1 -positive cells) may be isolated from the dissociated population of immature cardiomyocytes.

VCAM-1 (also known as CD106; Gene ID 7412) is a cell-surface sialoglycoprotein expressed by cytokine- activated endothelium that mediates leukocyte-endothelial cell adhesion and signal transduction. VCAM-1 may have the reference amino acid sequence of NP_001069.1 or an isoform thereof and may be encoded by the reference nucleic acid sequence of NM_001078.4 or an isoform thereof. Suitable methods for isolating VCAM-1 -positive cells from the dissociated population of immature cardiomyocytes are well known in the art and include, for example, flow cytometry techniques, such as fluorescence-activated cell sorting (FACS) or magnetic activated cell sorting (MACS). Preferably, VCAM-1 -positive cells are isolated by FACS.

The isolated VCAM-1 -positive cardiomyocytes may be further cultured in the cardiomyocyte maturation medium to mature and/or expand the population.

The VCAM-1 -positive cardiomyocytes may be cultured in adherent cell culture in the cardiomyocyte maturation medium. For example, the aggregates may be cultured on surface amenable to cell adhesion, for example a hydrogel-coated cell culture plate. Suitable culture vessels are readily available, for example from commercial suppliers and include Matrigel™ coated culture vessels.

The isolated VCAM-1 -positive cardiomyocytes may be seeded in the cardiomyocyte maturation medium at a density of between 10,000 to 50,000 cells/cm², preferably between 20,000 to 40,000 cells/cm², preferably 20,000 to 26,000 cells/cm².

The VCAM-1 -positive cardiomyocytes may be cultured for between 12 days and 24 days, preferably for about 19 days.

The VCAM-1 -positive cardiomyocytes cultured in adherent cell culture may show increased expression of cardiomyocyte-markers and/or adult isoforms of cardiac genes over time. For example, the VCAM-1 -positive cardiomyocytes may display increased expression of one or more of MYBPC3, MYH7, MYH6, ACTN2, TTN and TNNT2. The VCAM-1 -positive cardiomyocytes may display increased expression of cardiac-associated ion channels and/or Ca²⁺ handling genes. The VCAM-1 -positive cardiomyocytes cultured in adherent cell culture may also show an increase in size, sarcomere organisation and/or sarcomere length over time.

Following production as described herein the cardiomyocytes may be used as appropriate. For example, the cardiomyocytes may be further cultured, manipulated, stored, or used in suitable applications.

Also provided is a population of isolated cardiomyocytes produced by a method described above. The cardiomyocytes in the population may display increased expression of one or more, preferably all of MYBPC3, MYH7, MYH6, ACTN2, TTN and TNNT2, relative to the expression in a population of cardiomyocytes produced by standard 2D and 3D differentiation protocols. The cardiomyocytes may also display increased expression of cardiac-associated ion channels, such as Nav1.5, Nav1.8, L-Type calcium channels (e.g. Cav1.2, Cav1.3), N-type calcium channels (e.g. Cav2.2), T-type calcium channels (e.g.

Cav3.1 and Cav3.2), and/or voltage-gated potassium channels (e.g. Kv1.5, Kv1.5, Kv4.3, Kv7.1 , Kv11.1), relative to the expression in cardiomyocytes produced by standard 2D and 3D differentiation protocols. The cardiomyocytes may also display increased expression of Ca²⁺ related genes, such as Calmodulin (CaM), Ca²⁺/calmodulin-dependent kinase II (CAMKII), and/or sarcoplasmic reticulum Ca²⁺-ATPases (SERCA), relative to expression in cardiomyocytes produced by standard 2D and 3D differentiation protocols. The cardiomyocytes may also display a cardiomyocyte morphology more similar to mature cardiomyocytes compared to cardiomyocytes produced by standard 2D and 3D differentiation protocols. For example, the cardiomyocytes produced by a method described herein may be more elongated, have an increased aspect ratio, decreased circularity, increased sarcomere length, and/or increased sarcomere organisation compared to cardiomyocytes produced by standard 2D and 3D differentiation protocols.

Cardiomyocytes produced by the methods described herein may include atrial and ventricular cardiomyocytes, as showcased by single-cell RNA seq analysis.

A population of cardiomyocytes produced by a method described herein may find use in a method of treatment of the human or animal body, for example, in the treatment of a cardiac condition. Also provided is a method of treating a cardiac condition comprising administering a population of cardiomyocytes produced by a method described herein to an individual in need thereof and the use of a population of cardiomyocytes produced by a method described herein in the manufacture of a medicament for use in the treatment of a cardiac condition. Cardiac conditions include, for example, cardiomyopathy, hypertrophic cardiomyopathy, dilated cardiomyopathy, restrictive cardiomyopathy, Arrhythmogenic right ventricular dysplasia, left ventricular hypertrophy (LVH), ischemic heart disease, hypertension, heart failure, valve disease and unclassified cardiomyopathy.

Also provided is a pharmaceutical composition, medicament, drug or other composition comprising the cardiomyocytes produced by the methods of the invention. The pharmaceutical composition may find use in the treatment of a cardiac condition. Methods of treatment include the preventative treatment of a cardiac condition. Also provided is a method of making a pharmaceutical composition comprising admixing said cardiomyocytes with a pharmaceutically acceptable excipient, vehicle or carrier, and optionally one or more other ingredients. For therapeutic applications, the cardiomyocytes are preferably clinical grade cells. In some embodiments, disease associated mutations or genetic defects in the IPSCs may be corrected before differentiation into cardiomyocytes, as described above. Alternatively, disease associated mutations or genetic defects may be corrected after differentiation into cardiomyocytes.

A pharmaceutical composition comprising the cardiomyocytes produced in accordance with the invention may comprise one or more additional components. For example, the composition may comprise a pharmaceutically acceptable excipient, carrier, buffer, preservative, stabiliser, anti-oxidant, biodegradable scaffold, or other material well known to those skilled in the art. Such materials are preferably non-toxic and do not interfere with the activity of the cardiomyocytes. The precise nature of the carrier or other material will depend on the route of administration.

Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, tissue or cell culture media, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

The composition may be in the form of a parenterally acceptable aqueous solution, which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride, Ringer's Injection, or Lactated Ringer's Injection.

The composition may be transplanted, implanted, injected, infused or otherwise administered into the individual. For example, a subject may receive an autologous or allogeneic transplant of a population of cardiomyocytes produced by the first aspect. In some embodiments, the subject may receive an allogeneic transplant of a population of cardiomyocytes derived from a healthy donor. In some embodiments, the subject and the donor are matched for blood type and/or matched for human leukocyte antigen.

Cardiomyocytes may be transplanted, implanted or infused into a patient by any technique known in the art (e.g. Yildirim et al. 2007; Bellamy et al. 2015; Tiburcy et al. 2017; Menasche et al. 2018; Guan et al. 2020; Kawaguhi et al. 2021 ).

Administration of a composition in accordance with the present invention is preferably in a "prophylactically effective amount" or a "therapeutically effective amount" (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors.

A composition comprising cardiomyocytes produced by the methods of the invention may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

In some embodiments, the cardiomyocytes in the population produced as described herein may display a normal phenotype. For example, cells may be obtained from an individual with a cardiac disorder and used to produce IPS cells. In some embodiments, the IPS cells may contain a mutation or genetic defect and this mutation or defect may be corrected using conventional recombinant techniques to produce IPS cells with a normal phenotype. Alternatively, IPS cells with a normal genotype may be obtained from the individual with the cardiac disorder. Cardiomyocytes with a normal phenotype may be produced from these IPS cells as described herein and implanted into the patient to repair or ameliorate the disorder.

In other embodiments, the cardiomyocytes in the population produced as described herein may display a disease phenotype. For example, cells may be obtained from an individual with a cardiac disorder and used to produce disease-specific IPS (ds-IPS) cells. Cardiac progenitors with a disease phenotype may be produced from these IPS cells as described herein. These cells may then be treated to restore a normal phenotype. For example, the genetic mutation or defect which is responsible for the disease phenotype may be corrected in vitro. Various techniques are available to correct genetic mutations or defects in isolated mammalian cells, for example, CRISPR-Cas gene editing systems. Once the defect or mutation is corrected and the normal phenotype restored, the cardiomyocytes may be implanted into the patient to repair or ameliorate the disorder.

Also provided are in vitro methods of identifying a genetic cause of a cardiac condition. A method of identifying a genetic mutation or variant associated with a cardiac condition may comprise providing a first population of cardiomyocytes produced by a method described herein that comprise a genetic mutation or variant. Considering that a large percentage of patients suffering from heart conditions do not have a genetic diagnosis, characterization of variants of unknown significance (VUS) may be crucial to provide a genetic diagnosis to the patients, allowing for further screening of related family members.

The first population of cardiomyocytes may be compared with a second population of cardiomyocytes that does not comprise the genetic mutation or variant. A difference between the first and second populations may be indicative that the genetic mutation or variant is associated with a cardiac condition.

For example, the morphology, gene expression, function, transcriptome or proteome of the first population of cardiomyocytes may be compared with the second population.

Cardiomyocyte morphology may include the shape, structure, form, aspect ratio, length-to-width ratio, circularity, area, sarcomere length, sarcomere organisation, sarcomere density, percentage multinucleation or other morphological parameters of the cardiomyocytes. For example, cardiomyocytes produced by the methods described herein that have a genetic mutation associated with a cardiac condition, such as HCM, may have a larger cell area, decreased aspect ratio, increased circularity, decreased sarcomere length, decreased sarcomere organisation and/or a higher percentage of multinucleated cells compared to wild-type cardiomyocytes produced by the same methods that do not comprise the genetic mutation. Cardiomyocyte morphology may be determined using any convenient technique.

Cardiomyocyte gene expression may include expression of sarcomere genes, for example, Myosin Binding Protein C (MYBPC3), Myosin Heavy Chain 7 (MYH7), Myosin Heavy Chain 6 (MYH6), Actinin Alpha 2 (ACTN2), Titin (TTN), Troponin T2 (TNNT2; fetal troponin T), Titin N2BA (TTN N2BA; fetal splicing isoform), Titin N2BA N2B (TTN N2B adult splicing isoform) and/or Troponin 11 (TNNIT). For example, the sample population of cardiomyocytes produced by the methods described herein may display lower expression levels of one or more of the sarcomere genes compared to a control population. Gene expression may be determined using any convenient transcriptomic or proteomic technique.

Cardiomyocyte function may include contractility. For example, a sample population of cardiomyocytes may have a hypercontractility phenotype compared to a control population of cardiomyocytes.

Suitable control (or second) populations of cardiomyocytes may be produced by a method described herein.

In some embodiments, both the first and second populations of mature cardiomyocytes may be derived from a patient suffering from a cardiac condition, such as a cardiomyopathy. The patient may be suspected of having a genetic mutation or variant suspected of causing or contributing to the cardiac condition. The genetic mutation or variant may be corrected in vitro in the second population of mature cardiomyocytes. The genetic mutation or variant may be corrected in vitro in precursor cells of the second population of mature cardiomyocytes. For example, the genetic mutation or variant may be corrected in IPSCs of the second population before differentiation in to mature cardiomyocytes.

In other embodiments, the first and second population of mature cardiomyocytes may be derived from a subject not suffering from a cardiac condition, such as a cardiomyopathy. A genetic mutation or variant suspected of causing or contributing to a cardiac condition may be introduced in vitro in the first population of mature cardiomyocytes. The genetic mutation or variant may be introduced in vitro in precursor cells of the first population of mature cardiomyocytes. For example, the genetic mutation or variant may be introduced into IPSCs of the first population before differentiation in to mature cardiomyocytes.

Any suitable method may be used to introduce or correct a genetic mutation or variant in the mature cardiomyocytes, or precursor cells of the mature cardiomyocytes, such as IPSCs. Methods and technologies for genetically modifying cells are well known in the art and include, for example, base editing CRISPR- Cas9, TALENS, and Zinc finger nucleases. For example, genetic mutations or variants of interest may be introduced or corrected in IPSC populations by a CRISPR-Cas approach. In one example, single stranded DNAs (ssDNAs) harbouring mutations of interest can be used as homologous recombination templates to repair a cut in the DNA of an IPSC generated by a Cas nuclease. In another example, ssDNAs may be used as a homologous recombination template to correct a genetic mutation, or replace a variant of interest, in a target gene. Gene-edited IPSCs can be further isolated and differentiated into mature cardiomyocytes as described herein.

In other embodiments, genetic mutations may be introduced or corrected in mature cardiomyocytes by a CRISPR base-editing approach. This approach can allow for a single nucleotide substitution in a target gene of interest.

Cardiac conditions may include diseases associated with hypertrophy or a hypertrophic phenotype. For example, diseases associated with hypertrophy include cardiomyopathy, left ventricular hypertrophy (LVH), ischemic heart disease, hypertension, heart failure, and valve disease.

Also provided are in vitro methods of identifying a gene associated with a cardiac condition. A method of identifying a gene associated with a cardiac condition may comprise providing a sample population of cardiomyocytes produced by the method of the first aspect from induced pluripotent stem cells (IPSCs) derived from an individual with a cardiac condition. In a preferred embodiment, the IPSCs are human IPSCs. The expression of one or more genes in the sample population of cardiomyocytes may be compared to the gene expression of one or more genes in a control population of cardiomyocytes. A difference in the expression of a gene in the sample population relative to the control population may be indicative that the gene is associated with a cardiac condition. The control population may be produced by a method of the first aspect of the invention. A difference in gene expression may be an upregulation or downregulation of the one or more genes relative to the control population. The difference in gene expression may be associated with a change in the morphology and/or function of the sample population. For example, the sample population may have an increased cell area, decreased aspect ratio, increased circularity, decreased sarcomere length, decreased sarcomere organisation, a higher percentage of multinucleated cells, increased contractility, and/or decreased contractility compared to control cardiomyocytes.

Other aspects of the invention relate to methods of screening to identify compounds that are potentially useful in treating a cardiac condition, as described above. For example, a method of screening a compound may comprise contacting an isolated population of cardiomyocytes produced as described herein with a test compound; and determining the effect of the test compound on said cardiomyocytes.

The isolated population may comprise a genetic mutation suspected of causing or contributing to a cardiac condition, such as cardiomyopathy.

The isolated population of cardiomyocytes may display morphology, gene expression, and/or function associated with a cardiac condition. For example, the isolated population may display an HCM phenotype. A test compound that affects the morphology, gene expression, and/or function associated with the cardiac condition may be useful in treating or developing therapeutics to treat the cardiac condition. For example, a test compound may reduce or abrogate a morphology, gene expression, and/or function that is associated with a cardiac condition in the isolated population.

In some embodiments, the isolated population of cardiomyocytes may display morphology associated with a cardiac condition, such as increased area, decreased aspect ratio, increased circularity, decreased sarcomere length, decreased sarcomere organisation and/or a higher percentage of multinucleated cells. If the morphology associated with the cardiac condition is reduced following contact with the test compound, for example, such that the isolated population displays wild-type cardiomyocyte morphology, this may be indicative the test compound is useful in treating a cardiac condition. For example, the cardiomyocytes may display decreased area, increased aspect ratio, decreased circularity, increased sarcomere length, increased sarcomere organisation and/or a lower percentage of multinucleated cells following contact with the test compound.

The isolated population of cardiomyocytes may display gene expression associated with a cardiac condition, such as decreased expression of one or more sarcomere genes, for example, MYBPC3, MYH7, MYH6, ACTN2, TTN, TNNT2 (fetal troponin T), TTN N2BA (fetal splicing isoform), TTN N2B (adult splicing isoform) and/or TNNI1. If the gene expression associated with the cardiac condition is reduced following contact with the test compound, for example, such that the sample population displays wild-type cardiomyocyte gene expression this may be indicative the test compound is useful in treating a cardiac condition. For example, the cardiomyocytes may display increased expression of one or more sarcomere genes following contact with the test compound.

The isolated population of cardiomyocytes may display function associated with a cardiac condition, such as hypercontractility. If hypercontractility or other function associated with the cardiac condition is reduced following contact with the test compound, for example, such that the isolated population displays wild-type contractility, then this may be indicative the test compound is useful in treating a cardiac condition. The precise format of any of the screening or assay methods of the present invention may be varied by those of skill in the art using routine skill and knowledge. The skilled person is well aware of the need to employ appropriate control experiments.

A test compound may be an isolated molecule or may be comprised in a sample, mixture, or extract, for example, a biological sample. Compounds which may be screened using the methods described herein may be natural or synthetic chemical compounds used in drug screening programmes. Extracts of plants, microbes, or other organisms, which contain several characterised or uncharacterised components may also be used.

Combinatorial library technology provides an efficient way of testing a potentially vast number of different compounds for ability to affect cardiomyocytes. Such libraries and their use are known in the art, for all manner of natural products, small molecules, and peptides, among others. The use of peptide libraries may be preferred in certain circumstances.

The amount of test compound which may be added to an assay of the invention will normally be determined by trial and error depending upon the type of compound used. Typically, from about 0.001 nM to 1 mM or more concentrations of putative inhibitor compound may be used, for example from 0.01 nM to 100pM, e.g. 0.1 to 50 pM, such as about 10 pM. Even a compound which has a weak effect may be a useful lead compound for further investigation and development.

A test compound identified as affecting cardiomyocytes may be investigated further. For example, the selectivity of a compound for cardiomyocytes may be determined by screening against other cell types. Suitable methods for determining the effect of a compound on cells are well known in the art.

A test compound identified as affecting cardiomyocytes may be isolated and/or purified or alternatively, it may be synthesised using conventional techniques of recombinant expression or chemical synthesis. Furthermore, it may be manufactured and/or used in preparation, i.e. manufacture or formulation, of a therapeutic composition such as a medicament, pharmaceutical composition or drug. Methods described herein may thus comprise formulating the test compound in a pharmaceutical composition with a pharmaceutically acceptable excipient, vehicle, or carrier for therapeutic application.

Following identification of a compound that is potentially useful in modulating cardiomyocyte morphology, gene expression and/or function as described herein, a method may further comprise modifying the compound to optimise its pharmaceutical properties. Suitable methods of optimisation, for example by structural modelling, are well known in the art. Further optimisation or modification can then be carried out to arrive at one or more final compounds for in vivo or clinical testing.

Also provided is an in vitro method of assessing a cardiac condition in an individual. A method of assessing a cardiac condition in an individual may comprise providing a population of cardiomyocytes produced by the first aspect of the invention from IPSCs derived from the individual. The method may comprise comparing the morphology of the sample population with a control population of cardiomyocytes. A difference in morphology between the sample population and the control population may be indicative that the individual is at risk of a cardiac condition. For example, a sample population may have a larger cell area, decreased aspect ratio, increased circularity, decreased sarcomere length, decreased sarcomere organisation and/or a higher percentage of multinucleated cells compared to control population. The method may further comprise identifying the suitability of an individual to receive treatment for a cardiac condition, i.e. the method may be useful as a companion diagnostic method.

For example, in one aspect, the invention also provides an in vitro method of predicting a subject’s response to one or more treatments for a cardiac condition. The method comprises providing a population of cardiomyocytes from the subject, produced as described herein, and comparing one or more characteristics of the population of cardiomyocytes with a control population of cardiomyocytes to predict the response of the subject to a particular treatment plan. In some embodiments, the method may be used to determine the suitability of the subject to receive a treatment with cardiomyocytes produced by the methods as described herein.

Other aspects and embodiments of the invention provide the aspects and embodiments described above with the term “comprising” replaced by the term “consisting of’ and the aspects and embodiments described above with the term “comprising” replaced by the term “consisting essentially of’.

It is to be understood that the application discloses all combinations of any of the above aspects and embodiments described above with each other unless the context demands otherwise. Similarly, the application discloses all combinations of the preferred and/or optional features either singly or together with any of the other aspects, unless the context demands otherwise.

Modifications of the above embodiments, further embodiments and modifications thereof will be apparent to the skilled person on reading this disclosure, and as such, these are within the scope of the present invention.

All documents and sequence database entries mentioned in this specification are incorporated herein by reference in their entirety for all purposes.

“and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, “A and/or B” is to be taken as specific disclosure of each of (I) A, (ii) B and (ill) A and B, just as if each is set out individually herein.

Experimental data

Materials and Methods

Maintenance of human iPSCs

Three commercially available human IPSC lines were used as wild-type (WT) control cell lines, namely DF6.9.9 T.B cell line (DF6, WiCell®), reprogrammed from foreskin fibroblasts (Junying et al. 2009), F002.1A.13 (TCLab, Portugal), derived from female donor fibroblasts (Takahashi et al. 2007) and IPSC6.2 (Gibco®), derived from CD34+ cord blood (Burridge et al. 2011 ). Six hypertrophic cardiomyopathy (HCM) patient-derived hiPSC lines, reprogrammed from fibroblasts using Sendai virus, were also analysed (Table D- All human IPSCs were cultured on Matrigel™(Corning)-coated plates with mTeSR™1 Medium (StemCell Technologies). Medium was changed daily, and cells were passaged every three to four days (at a confluence of approximately 85% of the surface area of the culture dish) using 0.5mM EDTA dissociation buffer (Life Technologies). Two to three passages were performed before starting the differentiation process.

Cardiac differentiation: 2D culture conditions

For 2D culture, hiPSCs were seeded onto Matrigel-coated 12-well tissue culture plates and cultured in mTeSR™1. Medium was changed daily until a confluence of around 90% was attained.

From day 0 to day 6, cells were cultured in RPM1 1640 (Thermo Fisher Scientific) supplemented with 2%(v/v) B-27 minus insulin (Thermo Fisher Scientific). Then, from day 7 until the end of differentiation, cells were cultured in RPMI supplemented with 2%(v/v) B-27 (Thermo Fisher Scientific).

At day 0 of differentiation, the Wnt signaling pathway was activated using the GSK3 inhibitor CHIR99021 (Stemgent) at a final concentration of 6 pM. After 24 hours (day 1 ), full-volume medium replacement with RPMI + B27 minus insulin was performed. At day 3, half of the medium in each well was replaced and cells were supplemented with Wnt inhibitor IWP-4 (Stemgent) at a final concentration of 5 pM, for two days. At day 5, a total volume of medium change was performed. On day 7, medium was changed for RPMI + B27 and total volume of RPMI + B27 medium was replaced every 2-3 days thereafter until cell harvest at day 31 of differentiation.

Cardiac differentiation-. 3D/2D culture conditions

For 3D aggregate formation, before seeding hiPSCs were incubated with ROCKi (Y-27632, 10 pM, StemCell Technologies) for 1 h at 37°C and then treated with Accutase (Sigma) for 7 min at 37°C. After dissociation, cells were forced to aggregate using microwell plates (AggreWell™800, StemCell Technologies) according to the manufacturer’s instructions. To control the size of 3D aggregates, cells were plated at a density of 0.9 x 10⁶ cells/well (3,000 cells/microwell) and cultured in 1.5 mL/well of mTeSR™1 supplemented with 10 pM ROCKi. The day on which the cell seeding was performed was defined as day -3. After 24 hours, full volume of the medium was replaced, and aggregates were maintained in mTeSR™1 without ROCKi for an additional 48h.

From day 0 to day 6, cells were cultured in RPM1 1640 (Thermo Fisher Scientific) supplemented with 2%(v/v) B-27 minus insulin (Thermo Fisher Scientific). Then, from day 7 until the end of differentiation, cells were cultured in RPMI supplemented with 2%(v/v) B-27 (Thermo Fisher Scientific).

At day 0 of differentiation, the Wnt signalling pathway was activated using the GSK3 inhibitor CHIR99021 (Stemgent) at a final concentration of 6 pM in 2D conditions, and 11 pM in the 3D aggregates. After 24 hours (day 1 ), full-volume medium replacement with RPMI + B27 minus insulin was performed. At day 3, half of the medium in each well was replaced and cells were supplemented with Wnt inhibitor IWP-4 (Stemgent) at a final concentration of 5 pM, for two days. At day 5, a total volume of medium change was performed. On day 7, medium was changed for RPMI + B27 and aggregates were flushed from AggreWell™800 plates and transferred to ultra-low attachment 6-well plates (Costar, Corning). At day 10, full-volume medium replacement was performed.

At day 13 aggregates were dissociated using 0.25% Trypsin-EDTA (Gibco) for 7 min at 37°C. After dissociation, cells were washed with 2% fetal bovine serum in phosphate buffered saline (1xPBS, 0.1 M), resuspended in anti-VCAM-1 antibody (BioLegend, 1:50) diluted in 1xPBS/2% FBS and incubated for 30 min at 37°C. Afterwards, cells were washed and resuspended in with 1xPBS/2% FBS prior to FACS sorting. VCAM-1 -positive cells were plated on wells coated with Matrigel, at a seeding density between 20,000 - 40,000 cells/cm2. For the remaining time in culture, two-thirds of the total volume of RPMI + B27 medium was replaced at every 2-3 days, until cell harvest at day 31 of differentiation.

For some experiments, at day 13 aggregates were dissociated and cultured as above until day 27. At day 27 the monolayer was harvested and replated on 4Dcell micropatterned coverslips and cultured in RPMI + B27 medium until day 31 of differentiation.

Quantitative real time (qRT)-PCR

Expression profiles of sarcomeric genes were assessed by qRT-PCR analysis in hiPSC-CMs at day 31 of differentiation. As a positive cardiac control, a commercially available human heart RNA was used (cat # AM7966, Ambicon, Invitrogen). Briefly, total RNA was extracted using NZYol (NZYTech®) with a standard protocol, and contaminating DNA was removed by DNase I (Roche®) treatment. Complementary cDNA synthesis was achieved with the Transcriptor High Fidelity cDNA Synthesis Kit (Roche®). qRT-PCR was performed using the Universal SYBR Green Supermix (Bio-Rad) and specific primers for each gene and/or developmental isoform. All PCR reactions were run in triplicate, using the ViiA™7 RT-PCR systems (Applied 176 BioSystems). Data was normalized against the U6 housekeeping gene (or cardiac specific TNNT2) and depicted in histograms with mean and standard deviation from at least three independent experiments.

Immunofluorescence (IF) assays and morphologic characterization of hiPSC-CMs

IF assays were performed using different antibodies against several sarcomeric proteins. At day 31 of differentiation, hiPSC-CMs were fixed with 3.7% PFA/1xPBS and permeabilized with 0.5% Tx100/1XPBS. Next, a double stain with (1) phalloidin conjugated with TxRed (Thermo Fisher, T7471) (for detection of actin) and (2) a specific antibody against a given sarcomeric protein (Table 2), detected by an anti-mouse Alexa Fluor 488-conjugated (A-11018 Thermo Fisher Scientific) was performed overnight at 37°C, in a moist chamber. Nuclei counterstaining was performed using 4’,6-diamidino-2-phenylindole (DAPI, 1pg/mL; Enzo Life Sciences). After brief drying, coverslips were mounted in VECTASHIELD® Mounting Medium. Fluorescence images were acquired with Zeiss LSM 710 Confocal Laser Point-Scanning Microscope.

RNA sequencing

Total RNA was extracted from hiPSC-CMs at day 31 of differentiation using NZYol (NZYTech®), described above under “Quantitative real time (qRT)-PCR”. Strand-specific RNA libraries were prepared for Illumina sequencing using standard protocols. Raw data was pre-processed with TrimGalore vO.4.4. Splicing isoform quantification was performed with Salmon vO.13.1. Results

Cardiac differentiation of hi PSCs using a combined 3D/2D approach

Human IPSCs were differentiated into cardiomyocytes using a 3D/2D combined approach of differentiation, purification and maturation steps as described in Example 1. The initial 3D differentiation steps forced aggregation of hiPSCs in AggreWell™800 plates and at this stage the cells of the 3D aggregate displayed high levels of expression of pluripotent stem cell markers Nanog and OCT4 (Figure 2A, top panel). The aggregated hiPSCs were induced to differentiate into cardiomyocytes (CMs), as size-controlled aggregates via temporal modulation of the Wnt signalling pathway, by sequential addition of a GSK3 inhibitor (CHIR99021) and a Wnt inhibitor (IWP-4). By day 7, the cardiomyocytes began to express the cardiac- associated sarcomere protein Troponin T (Figure 2A, upper middle panel). After this step, on day 13 of differentiation, aggregates were dissociated and cells expressing VCAM-1 were isolated by FACS (Uosaki et al. 2011). Immunofluorescence imaging of these cells revealed high levels of Troponin T and VCAM-1 expression (Figure 2A, bottom middle panel). These purified CMs were replated on wells coated with Matrigel and further maintained in 2D culture until cell harvest at day 31 of cardiac differentiation (Figure 1). Alternatively, on day 27, cardiomyocytes were replated into micropatterned 4DCell coverslips and maintained in culture until day 31 of cardiac differentiation, where a high level of sarcomere organization is observed with commitment expression of cMyBP-C protein (Figure 2A, bottom panel).

RNA expression levels of Nanog, OCT4, Brachyury (mesoderm marker), Isl1 and Nkx2.5 (cardiac precursor markers) and TNNT2 were analysed over the course of the first 13 days of the differentiation protocol (Figure 2B). Consistent with a progressive maturation of hiPSCs to a mesoderm fate, from day 0 to day 5 Nanog and OCT4 expression disappears, with a peak in Brachyury expression at day 1 , which is indicative of a commitment to a mesoderm fate. By day 5, Brachyury expression disappears and between day 5 and day 7 Isl1 and Nkx2.5 expression is switched on indicating the cells have adopted a cardiac fate. By day 7, the cells were also seen to express TNNT2, with the level of expression increasing up until day 13 indicating successful generation of immature cardiomyocytes.

3D2D cardiac differentiation of hiPSCs gives rise to a population of pure CMs with a high degree of maturation

The three WT hiPSC lines were differentiated using the 2D and 3D2D protocols described in Example 1. hiPSC-CMs at day 31 of differentiation were collected and analysed. To assess the impact of the FACs purification step in the combined 3D2D approach, a fraction of dissociated 3D aggregates not subjected to FACS purification (unsorted), as well as cells that were negative for VCAM-1 (VCAM-1 -) were also collected and further analysed.

Comparative IF analysis of cells generated through each of the protocols and stained for sarcomeric proteins (cMyBP, Troponin T and a-actinin) revealed a marked improvement in cell elongation and sarcomere organization, which was evidenced by higher density and more visible striation alignment across the cells, in the 3D2D hiPSC-CMs. In turn, cells differentiated through the 2D protocol are rounder, smaller and present less organized and aligned sarcomeres (Figure 3).

The length of sarcomeres within the contractile apparatus was measured, for one of the differentiated cell lines, using cells stained with a-actinin (present in the Z-lines of sarcomeres) (Figure 4A.). The statistically significant elongation of sarcomere length, from 1.5910.008 pm in 2D hiPSC-CMs to 1.765 ± 0.008 pm in 3D2D hiPSC-CMs, further confirms a more mature phenotype of the generated 3D2D hiPSC-CMs (Figure 4B).

To evaluate the performance of the two cardiac differentiation protocols used, the expression of sarcomeric genes was also assessed by qRT-PCR analysis. Results show that all analysed sarcomeric genes MYBPC3, MYH7, MYH6, ACTN2, TTN, TNNT2 are expressed in the different experimental conditions (2D protocol; 3D2D protocol without purification; and 3D2D protocol VCAM-1 positive cells). However, in 3D2D VCAM-1 + conditions expression levels are significantly higher (Figure 5A).

Interestingly, when normalized to TNNT2 expression the differences amongst the expression of the remaining sarcomeric genes is attenuated, suggesting the presence of a more homogeneous hiPSC-CM population in 3D2D VCAM-1 + cells (Figure 5B). In contrast, VCAM-1 -negative cells show very low to nonexistent expression levels of cardiac sarcomeric genes (qRT-PCR analysis), and absence of sarcomere formation (IF assays) (data not shown), confirming that hiPSC-CMs were positively selected by FACS.

To characterise the degree of maturation of the hiPSC-CMs produced under the test conditions above, the expression levels of different developmental cardiac isoforms of selected sarcomeric genes were analysed. Specifically, the levels of fetal troponin T (TNNT2), fetal and adult titin (TTN) and fetal and adult troponin I (TNNI) were analysed by qRT-PCR. Results show that there is an increased expression of the adult cardiac isoforms in 3D2D VCAM-1 + hiPSC-CMs populations compared to either 2D differentiated hiPSC-CMs or unsorted 3D2D hiPSC-CMs (Figure 6A). This is further supported by comparing the ratios between fetal and adult (or total) isoforms in each hiPSC-CM population, which are higher in the 2D hiPSC-CMs and unsorted 3D2D hiPSC-CMs compared to 3D2D VCAM-1 + hiPSC-CMs. This further demonstrates that 3D2D VCAM- 1+ hiPSC-CMs are more mature than both 2D hiPSC-CMs and unsorted 3D2D hiPSC-CMs (Figure 6B).

RNA-sequencing also demonstrates that 3D2D VCAM-1 + hiPSC-CMs preferentially express adult heart splicing isoforms of both TNNT2 and TTN as opposed to fetal heart isoforms further demonstrating the relative maturity of the CM produced using the 3D2D VCAM-1 + culture conditions (Figure 7).

Single-cell RNA sequencing of a population of DF6 wild type cardiomyocytes produced under 3D2D conditions with purification of VCAM-1 cells generates a largely homogenous population of cardiomyocytes with 88% of cells in the population being of a cardiomyocyte lineage (Figure 8).

3D2D patient derived hiPSC-CMs recapitulate HCM phenotypes at the cellular level

The expression of cardiac sarcomeric genes in hiPSC-CMs, generated through the previously described 3D2D protocol, either from WT or HCM patient derived hiPSC lines, was further evaluated by qRT-PCR. Both HCM and WT hiPSC-CMs show similar levels of expression of the analysed sarcomeric genes, confirming successful generation of hiPSC-CMs from all tested cell lines (Figure 9).

When evaluating the maturity of WT and HCM hiPSC-CMs through the expression of developmental cardiac splicing isoforms, it can be observed that the fetal splicing TNNT2 isoform appears enriched in some HCM hiPSC-CMs relative to WT hiPSC-CMs (Figure 10). Moreover, the majority of HCM hiPSC-CMs show reduced levels of the adult splicing N2B TTN isoform relative to control WT hiPSC-CMs (Figure 10).

Patient-derived hiPSC-CMs can be used to study HCM genotype-phenotype relationships Comparative immunofluorescence of WT and HCM hiPSC-CMs generated using the 3D2D differentiation protocol with selection of VCAM-1 -positive cells shows that HCM hiPSC-CMs are larger, rounder and have a less organized sarcomeric structure compared to WT hiPSC-CMs (Figure 11 ).

To further characterize hiPSC-CMs morphology in a quantitative manner, several cell parameters were analysed, namely shape descriptors (circularity and aspect ratio) and area. The percentage of binucleated cells was also analysed (Figure 12A, B and C). Results show that HCM hiPSC-CMs are larger (area) and rounder than the wild type hiPSC-CMs (circularity and aspect ratio) (Figure 12A, B and C). Moreover, the percentage of binucleated was found to be elevated in the HCM hiPSC-CMs population when compared with the wild type hiPSC-CMs (Figure12D).

To reduce the variability in morphology that was observed on normal coverslips, WT and HCM hiPSC-CMs were plated on 4Dcell micropatterned coverslips at day 27 of differentiation until analysis at day 31. HCM hiPSC-CMs are larger, rounder and have a less organized sarcomeric structure compared to WT hiPSC- CMs (Figure 13).This approach considerably reduces the heterogeneity found in hiPSC-CMs platted on conventional coverslips, allowing a more reliable and consistent analysis of morphological cellular parameters, namely cell area and aspect ratio (Figure 14).

The difference in cell area and aspect ratio between wild type and HCM hiPSC-CMs is maintained and in some cases highlighted when replated on micropatterned coverslips (Figure 14A and B). For example, WT and HCM DF6 cultured on non-patterned coverslips do not display any significant difference in aspect ratio, whereas a comparison of the same cell types replated on micropatterned coverslips do show a significant difference in aspect ratio (Figure 14B). It is also apparent that cells replated on micropatterned coverslips generally display less variability in their morphology.

Together, these results confirm that patient-derived hiPSC-CMs obtained using the 3D2D differentiation and VCAM-1 selection recapitulate HCM-specific features at the single-cell level and as such can be used to study HCM genotype-phenotype relationships, the impact of variants of uncertain significance (VUS) and/or for testing new therapeutic approaches. Table I

Table II

References

A number of publications are cited above in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. The entirety of each of these references is incorporated herein.

Baghbaderani et al. 2015. Manufactured Human Induced Pluripotent Stem Cells Are Available for Pre-clinical and Clinical Applications. Stem Cell Reports. 2015 Oct 13;5(4):647-59. doi: 10.1016/j.stemcr.2O15.08.015. Epub 2015 Sep 24. PMID: 26411904; PMCID: PMC4624993.

Baghbaderani et al 2016. Detailed Characterization of Human Induced Pluripotent Stem Cells Manufactured for Therapeutic Applications. Stem Cell Rev Rep. 2016 Aug; 12(4):394-420. doi: 10.1007/s12015-016-9662- 8. PMID: 27283945; PMCID: PMC4919381

Barbulovic-Nad, I et al 2006 “Bio-microarray fabrication techniques — a review". Critical Reviews in Biotechnology. 26 (4): 237-59. CiteSeerX 10.1.1.661.6833. doi: 10.1080/07388550600978358.

Bellamy, V., et al 2015 “Long-term functional benefits of human embryonic stem cell-derived cardiac progenitors embedded into a fibrin scaffold,” J. Hear. Lung Transplant., vol. 34, no. 9, pp. 1198-1207, Sep. 2015, doi: 10.1016/J.HEALUN.2014.10.008.

Bongso, A 2005. Stem Cells: From Bench to Bedside. 1st Ed, World Scientific Publishing Co Pte Ltd.

Branco et al. 2019. “Transcriptomic Analysis of 3DCardiac Differentiation of Human Induced Pluripotent Stem Cells Reveals Faster Cardiomyocyte Maturation Compared to 2D Culture.” Scientific Reports 9 (1): 1- 13. https://doi.Org/10.1038/S41598-019-45047-9.

Brewer GJ, Cotman CW. Survival and growth of hippocampal neurons in defined medium at low density: advantages of a sandwich culture technique or low oxygen. Brain Res. 1989 Aug 7;494(1):65-74. doi: 10.1016/0006-8993(89)90144-3. PMID: 2765923.

Brewer GJ, Torricelli JR, Evege EK, Price PJ. Optimized survival of hippocampal neurons in B27- supplemented Neurobasal, a new serum-free medium combination. J Neurosci Res. 1993 Aug 1;35(5):567- 76. doi: 10.1002/jnr.490350513. PMID: 8377226.

Brewer GJ. Serum-free B27/neurobasal medium supports differentiated growth of neurons from the striatum, substantia nigra, septum, cerebral cortex, cerebellum, and dentate gyrus. J Neurosci Res. 1995 Dec;42(5):674-83. doi: 10.1002/jnr.490420510. PMID: 8600300.

Burridge et al. 2014. “Chemically Defined Generation of Human Cardiomyocytes.” Nature Methods 11 (8): 855-60. https://doi.org/10.1038/nmeth.2999.

Chen Y, Stevens B, Chang J, Milbrandt J, Barres BA, Hell JW. NS21: re-defined and modified supplement B27 for neuronal cultures. J Neurosci Methods. 2008 Jun 30;171(2):239-47. doi: 10.1016/j.jneumeth.2008.03.013. Epub 2008 Apr 1. PMID: 18471889; PMCID: PMC2678682.

Chong et al 2014. “Human embryonic-stem-cell-derived cardiomyocytes regenerate non-human primate hearts,” Nat. 20145107504, vol. 510, no. 7504, pp. 273-277, Apr. 2014, doi: 10.1038/nature13233.

Coghlan MP, Culbert AA, Cross DA, Corcoran SL, Yates JW, Pearce NJ, Rausch OL, Murphy GJ, Carter PS, Roxbee Cox L, Mills D, Brown MJ, Haigh D, Ward RW, Smith DG, Murray KJ, Reith AD, Holder JC. Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription. Chem Biol. 2000 Oct;7(10):793-803. doi: 10.1016/s1074-5521 (00)00025-9. PMID: 11033082. Correia et al. 2018. “3D Aggregate Culture Improves Metabolic Maturation of Human Pluripotent Stem Cell Derived Cardiomyocytes.” Biotechnology and Bioengineering 115 (3): 630-44. https://doi.Org/10.1002/bit.26504.

Cowan, C.A. et al. 2004. Derivation of embryonic stem-cell lines from human blastocysts. N Engl J Med. 350(13): 1353-6. doi: 10.1056/NEJMsr040330. Epub 2004 Mar 3. PMID: 14999088.

Chiu, A and Rao, M. 2003. Human Embryonic Stem Cells. 1st Ed. Great Britain: Humana Doyle, A and Griffiths, J. B. 1997. Mammalian Cell Culture: Essential Techniques. Wiley.

Freshney, R 2005. Culture of Animal Cells: A Manual of Basic Technique, John Wiley & Sons Inc ISBN: 0471453293

Gaudernack et al 1986. Isolation of pure functionally active CD8+ T cells. Positive selection with monoclonal antibodies directly conjugated to monosized magnetic microspheres. J Immunol Methods. 90(2): 179-87. doi: 10.1016/0022-1759(86)90074-8. PMID: 3088118.

Guan, X., et al. 2020 “Transplantation of human induced pluripotent stem cell-derived cardiomyocytes improves myocardial function and reverses ventricular remodelling in infarcted rat hearts,” doi:

10.1186/s13287-020-01602-0.

Helgason, C. (2004) Basic Cell Culture Protocols, Humana Press Inc. U.S. ISBN: 1588295451

Hoes, Borner, and van der Meer 2019. “The Current State of Human In Vitro Cardiac Disease Modeling: A Focus on Gene Editing and Tissue Engineering.” STEM CELLS Translational Medicine 8 (1): 66-74. https://d0i.0rg/l 0.1002/sctm.18-0052.

Ho WY, Nguyen HN, Wolfl M, Kuball J, Greenberg PD. In vitro methods for generating CD8+ T-cell clones for immunotherapy from the naive repertoire. J Immunol Methods. 2006 Mar 20;310(1-2):40-52. doi: 10.1016/j.jim.2005.11.023. Epub 2006 Jan 26. PMID: 16469329.

Human Cell Culture Protocols (Methods in Molecular Medicine S.) Humana Press Inc., U.S. (9 Dec 2004) ISBN: 1588292223

Joannides, A. et al. 2006. Automated mechanical passaging: a novel and efficient method for human embryonic stem cell expansion. Stem Cells. 2006 Feb;24(2):230-5. doi: 10.1634/stemcells.2005-0243. PMID: 16510428.

Junying et al. 2009. “Human Induced Pluripotent Stem Cells Free of Vector and Transgene Sequences.” Science 324 (5928): 797-801. https://doi.org/10.1126/science.1172482.

Karakikes et al. 2015. “Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Insights Into Molecular, Cellular, and Functional Phenotypes.” Circulation Research 117 (1): 80-88.192 https://doi.Org/10.1161 /Cl RCRESAHA.117.305365.

Kawaguchi, S., et al. 2021 “Intramyocardial Transplantation of Human iPS Cell-Derived Cardiac Spheroids Improves Cardiac Function in Heart Failure Animals,” JACC Basic to Transl. Sci., vol. 6, no. 3, pp. 239-254, doi: 10.1016/J. JACBTS.2020.11.017.

Kim et al Nature. 2008. Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors. Nature. 454(7204):646-50. doi: 10.1038/nature07061. Epub 2008 Jun 29. PMID: 18594515.

Lian et al. 2013. “Directed Cardiomyocyte Differentiation from Human Pluripotent Stem Cells by Modulating Wnt/0-Catenin Signaling under Fully Defined Conditions.” Nature Protocols 8 (1): 162-75. https://d0i.0rg/l 0.1038/nprot.2012.150.

Liu, YW et al. 2018. “Human embryonic stem cell-derived cardiomyocytes restore function in infarcted hearts of non-human primates,” Nat. Biotechnol. 2018 367, vol. 36, no. 7, pp. 597-605, Jul. 2018, doi: 10.1038/nbt.4162. Kamakura et al. 2013. “Ultrastructural Maturation of Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes in a Long-Term Culture” Circulation Journal. 77(5): 1307-14. doi: 10.1253/circj.cj-12-0987. Epub 2013 Feb 9.

Kimet et al. 2009 Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell. 4(6):472-6. doi: 10.1016/j.stem.2009.05.005. Epub 2009 May 28. PMID: 19481515; PMCID: PMC2705327.

Klimanskaya, I. et al. 2005. Lancet. 13;365(9471): 1636-41. doi: 10.1016/S0140-6736(05)66473-2. PMID: 15885296.

Ludwig TE, Levenstein ME, Jones JM, Berggren WT, Mitchen ER, Frane JL, Crandall LJ, Daigh CA, Conard KR, Piekarczyk MS, Lianas RA, Thomson JA. Derivation of human embryonic stem cells in defined conditions. Nat Biotechnol. 2006 Feb;24(2): 185-7. doi: 10.1038/nbt1177. Epub 2006 Jan 1. PMID: 16388305.

Lum et al (1982). In vitro regulation of immunoglobulin synthesis by T-cell subpopulations defined by a new human T-cell antigen (9.3). Cell Immunol. 72(1):122-9. doi: 10.1016/0008-8749(82)90289-1. PMID: 6216957. Menasche, P., et al. 2018 “Transplantation of Human Embryonic Stem Cell-Derived Cardiovascular Progenitors for Severe Ischemic Left Ventricular Dysfunction,” J. Am. Coll. Cardiol., vol. 71 , no. 4, pp. 429- 438, doi: 10.1016/J.JACC.2017.11.047.

Moore, G.E. and Woods L.K., (1976) Tissue Culture Association Manual. 3, 503-508

Musunuru et al. 2018. “Induced Pluripotent Stem Cells for Cardiovascular Disease Modeling and Precision Medicine: A Scientific Statement From the American Heart Association.” Circulation. Genomic and Precision Medicine. NLM (Medline). https://doi.org/10.1161/HCG.0000000000000043.

Nussbaum, J. et al. 2007 ‘ ransplantation of undifferentiated murine embryonic stem cells in the heart: teratoma formation and immune response,” FASEB J., vol. 21, no. 7, pp. 1345-1357, doi: 10.1096/FJ.06- 6769COM.

Park et al Nature. 2008. Reprogramming of human somatic cells to pluripotency with defined factors. Nature. 451 (7175): 141 -6. doi: 10.1038/nature06534. Epub 2007 Dec 23. PMID: 18157115.

Peterson & Loring 2012. Human Stem Cell Manual: A Laboratory Guide. 1st Ed Academic Press.

Pollard, J and Walker JM. 1997. Basic Cell Culture Protocols. 2nd Ed Humana Press.

Posch, A., 2021. Proteomic profiling. New York: Humana Press.

Purevjav 2019. “Animal Models of Cardiomyopathies.” In Animal Models in Medicine and Biology [Working Title], 58:233-39. IntechOpen. https://doi.org/10.5772/intechopen.89033.Takahashi et al. 2007. “Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors.” Cell 131 (5): 861-72. https://doi.Org/10.1016/j.cell.2007.11.019.

Rheinherz et al., 1979. Separation of functional subsets of human T cells by a monoclonal antibody. Proc Natl Acad Sci U S A. 76(8):4061-5. doi: 10.1073/pnas.76.8.4061. PMID: 315070; PMCID: PMC383977.

Ring DB, Johnson KW, Henriksen EJ, Nuss JM, Goff D, Kinnick TR, Ma ST, Reeder JW, Samuels I, Slabiak T, Wagman AS, Hammond ME, Harrison SD. Selective glycogen synthase kinase 3 inhibitors potentiate insulin activation of glucose transport and utilization in vitro and in vivo. Diabetes. 2003 Mar;52(3):588-95. doi: 10.2337/diabetes.52.3.588. PMID: 12606497.

Roth S, Zhang S, Chiu J, Wirth EK, Schweizer U. Development of a serum-free supplement for primary neuron culture reveals the interplay of selenium and vitamin E in neuronal survival. J Trace Elem Med Biol. 2010 Apr;24(2): 130-7. doi: 10.1016/j.jtemb.2010.01.007. Epub 2010 Feb 2. PMID: 20413072. Sato N et al. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat Med. 2004 Jan;10(1):55-63. doi: 10.1038/nm979. Epub 2003 Dec 21. PMID: 14702635.

Y. Shiba et al., “Allogeneic transplantation of IPS cell-derived cardiomyocytes regenerates primate hearts,” Nat. 20165387625, vol. 538, no. 7625, pp. 388-391 , Oct. 2016, doi: 10.1038/nature19815.

Takahashi Cell. 2007. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell.131(5):861-72. doi: 10.1016/j.cell.2007.11.019. PMID: 18035408.

Tiburcy, M. et al. ,2017 “Defined Engineered Human Myocardium With Advanced Maturation for Applications in Heart Failure Modeling and Repair,” Circulation, vol. 135, no. 19, pp. 1832-1847, doi:

10.1161/CIRCULATIONAHA.116.024145.

Turksen, K. 2006. Human Embryonic Stem Cell Protocols. 1st Ed. Humana Press.

Uosaki et al. 2011. “Efficient and Scalable Purification of Cardiomyocytes from Human Embryonic and Induced Pluripotent Stem Cells by VCAM1 Surface Expression.” PLoS ONE 6 (8): e23657. https://doi.org/10.1371/journal.pone.0023657.

Vallier, L. et al. 2004. Nodal inhibits differentiation of human embryonic stem cells along the neuroectodermal default pathway. Dev Biol. 275(2):403-21. doi: 10.1016/j.ydbio.2004.08.031. PMID: 15501227.

Vallier, L., et al. 2009. Signaling pathways controlling pluripotency and early cell fate decisions of human induced pluripotent stem cells. Stem Cells. 27(11):2655-66. doi: 10.1002/stem.199. PMID: 19688839. Yamanaka et al., 2007. Generation of germline-competent induced pluripotent stem cells. Nature.

448(7151 ):313-7. doi: 10.1038/nature05934. Epub 2007 Jun 6. PMID: 17554338. Yamanaka 62007 Jun 7; 1(1):39-49

Wang Z, Gerstein M, Snyder M 2009. "RNA-Seq: a revolutionary tool for transcriptomics". Nature Reviews Genetics. 10 (1): 57-63. doi:10.1038/nrg2484. PMC 2949280.

Yildirim, Y., et al. 2007 “Development of a biological ventricular assist device: preliminary data from a small animal model,” Circulation, vol. 116, no. 11 Suppl, doi: 10.1161/CIRCULATIONAHA.106.679688.

Yoshida and Yamanaka, 2017. “Induced Pluripotent Stem Cells 10 Years Later.” Circulation Research 120 (12): 1958-68. https://doi.org/10.1161/CIRCRESAHA.117.311080.

Claims

Claims:

1. A method for producing a population of cardiomyocytes comprising;

(i) culturing a 3-dimensional aggregate of pluripotent stem cells in a cardiomyocyte induction medium in the presence of a GSK30 inhibitor;

(ii) culturing the 3-dimensional aggregate from step (I) in the cardiomyocyte induction medium;

(ill) culturing the 3-dimensional aggregate from step (ii) in the cardiomyocyte induction medium in the presence of a Wnt antagonist;

(iv) culturing the 3-dimensional aggregate from step (ill) in the cardiomyocyte induction medium;

(v) culturing the 3-dimensional aggregate from step (iv) in a cardiomyocyte maturation medium to produce a 3-dimensional aggregate of immature cardiomyocytes,

(vi) isolating VCAM-1 -positive cells from the 3-dimensional aggregate of from step (v); and

(vii) culturing the VCAM-1 -positive cells from step (vi) in a monolayer in the cardiomyocyte maturation medium; thereby producing a population of cardiomyocyte cells.

2. A method according to claim 1 wherein the cardiomyocyte induction medium is an insulin-free chemically defined medium.

3. A method according to any one of the preceding claims wherein the cardiomyocyte induction medium comprises a basal medium supplemented with a serum-free culture medium supplement.

4. A method according to claim 3 wherein the basal medium is RPMI-1640.

5. A method according to claim 3 or claim 4 wherein the serum-free culture medium supplement is insulin-free B27.

6. A method according to any one of the preceding claims wherein the GSK30 inhibitor is CHIR-99021.

7. A method according to any one of the preceding claims wherein the Wnt antagonist is IWP-4.

8. A method according to any one of the preceding claims wherein the 3-dimensional aggregate of pluripotent stem cells is cultured in step (I) for at least 24 hours.

9. A method according to any one of the preceding claims wherein the 3-dimensional aggregate is cultured in steps (ii), (ill) and (iv) for 2 days each.

10. A method according to any one of the preceding claims wherein the cardiomyocyte induction medium is a chemically defined medium.

11. A method according to claim 10 wherein the cardiomyocyte maturation medium comprises a basal medium supplemented with a serum-free culture medium supplement.

12. A method according to claim 11 wherein the basal medium is RPMI-1640.

13. A method according to claim 11 or claim 12 wherein the serum-free culture medium supplement is B27.

14. A method according to any one of the preceding claims wherein the 3-dimensional aggregate is cultured in step (v) for 5 days.

15. A method according to any one of the preceding claims wherein the VCAM-1 -positive cells are cultured in step (vii) for 19 days.

16. A method according to any one of claims 1 to 14 further comprising the steps of;

(viii) isolating cells of the monolayer of step (vii); and

(lx) culturing the cells of step (viii) on a micropatterned surface in the cardiomyocyte maturation medium.

17. A method according to claim 16 wherein the VCAM-1 -positive cells are cultured in step (vii) for about 15 days.

18. A method according to claim 16 or claim 17 wherein the cells are cultured in step (lx) for 4 days.

19. A population of isolated cardiomyocytes produced by a method of any one of claims 1 to 18.

20. A method of identifying or classifying a genetic variant associated with a cardiac condition comprising;

(I) providing a first population of cardiomyocytes produced by the method of any one of claims 1 to 18, wherein the population comprises a genetic variant; and

(ii) comparing one or more of the morphology, function, gene expression, transcriptome and proteome profile of the population of cardiomyocytes with a second population of cardiomyocytes, wherein the second population does not comprise the genetic variant; wherein a difference in one or more of the morphology, function, gene expression, transcriptome or proteome between the first population and the second population is indicative that the genetic variant is associated with a cardiac condition.

21. The method according to claim 20, wherein step (I) further comprises introducing the genetic variant into a first population of pluripotent stem cells before producing the first population of cardiomyocytes by the method of any one of claims 1 to 18 from said first population of pluripotent stem cells.

22. The method according to claim 20 further comprising step (la), wherein a first population of pluripotent stem cells comprising a genetic variant are genetically altered to correct the genetic variant before producing the second population of cardiomyocytes according to the method of any one of claims 1 to 18 from said first population of pluripotent stem cells.

23. A method of screening a compound comprising; contacting an isolated population of cardiomyocytes produced by the method of any one of claims 1 to 18 with a test compound; and determining the effect of the test compound on said cardiomyocytes.

24. A method of screening according to claim 23, wherein the effect of the test compound on the isolated cardiomyocytes is determined by single cell analysis, RNA sequencing, morphological and/or functional characterisation.

25. A therapeutic composition comprising a population of cardiomyocytes according to claim 19 for use in the treatment of a cardiac condition.

26. A therapeutic composition for use according to claim 25, wherein the cardiac condition is one or more of cardiomyopathy, hypertrophic cardiomyopathy, dilated cardiomyopathy, restrictive cardiomyopathy, Arrhythmogenic right ventricular dysplasia, left ventricular hypertrophy (LVH), ischemic heart disease, hypertension, heart failure, valve disease and unclassified cardiomyopathy.

27. An in vitro method of predicting a subject’s response to one or more treatments for a cardiac condition, wherein the method comprises;

(I) providing a population of cardiomyocytes according to claim 19, wherein the population is derived from the subject; and

(ii) comparing one or more of the morphology, function, gene expression, transcriptome and proteome profile of the population of cardiomyocytes with a control population of cardiomyocytes to predict the response of the subject to a treatment regimen.