WO2025191098A1 - Method for the production of cardiac organoids, the cardiac organoids thus obtained and uses thereof - Google Patents

Abstract

The present invention relates to a system for the culture of cardiac organoids, the relative method for the production of cardiac organoids, the cardiac organoids thus obtained and their uses as in vitro cardiac models.

Description

TITLE

METHOD FOR THE PRODUCTION OF CARDIAC ORGANOIDS, THE CARDIAC ORGANOIDS THUS OBTAINED AND USES THEREOF

ABSTRACT

The present invention relates to a system for the culture of cardiac organoids, the relative method for the production of cardiac organoids, the cardiac organoids thus obtained and their uses as in vitro cardiac models.

BACKGROUND

Despite the considerable steps forward in the field, cardiovascular diseases are still one of the major causes of death and disability worldwide, with a strong impact on health care costs (Vaduganathan M.G. et al., 2022). For these reasons, both the decryption of the mechanisms at the basis of cardiac pathologies and the identification of novel treatments are still hot research topics.

Research sustainability is tightly linked to the availability of models successfully recapitulating the pathological and the physiological scenarios. Indeed, research and development costs are still highly affected by costs of compound discovery, of pre -clinical and clinical trials. In particular, pre-clinical trials often rely on the use of animal models, which are linked to high costs (e.g. housing and handling costs), complex management, ethical concerns (3Rs principles of replacement, reduction and refinement call for their limitation), and, more importantly, due to interspecies variability, are often unable to successfully recapitulate some relevant aspects of human cardiac pathophysiology (Cesarovic N. et al., 2020).

Advanced in vitro models could be an alternative to overcome some of these limitations. In particular, the implementation of induced pluripotent stem cell (iPSC), which carry patient genetic background and which can be obtained from somatic, fully differentiated cells, collected from peripheral districts, avoiding invasive biopsies, opened the path to personalize medicine. Moreover, the joint use of iPSC and CRISPR/Cas9 technology, simplifying the cell lines genetic corrections, enables the design of complex experimental set up, allowing the investigation of the effects induced by specific genetic defects.

The full exploitation of the great potential of these technologies is secondary to the ability to design in vitro models, allowing to recapitulate relevant organ features, such as complex geometry, multi cel lularity, intercellular interaction, organ functionality, thus making the in vitro model representative of the physio-pathological human cardiac scenario. Indeed, numerically cardiomyocytes are only the 20-30% of cells in the myocardium, where endothelial cells (64%), cardiac fibroblasts (27%) and leukocytes (9%) play a relevant role for cardiac structure and homeostasis (Guo Y. et al., 2020). The interaction between the different resident cardiac cells is guided by cell -cell junctions, cell-ECM interactions, released soluble factors, geometrical and mechanical cues (Pesce M. et al., 2017, Park H. J. et al., 2021, Paraskevaidis I. et al., 2023, Zeitz M.J. et al., 2023).

The use of traditional 2D static cultures of iPSC-derived single cell types (e.g. cardiomyocytes), despite demonstrating their relevance for modelling activation of biological mechanisms in response to stimuli, have been limited by the low level of maturity of iPSC-derived cardiomyocytes, mainly representative of a fetal developmental stage (Karakikes I. et al., 2015). iPSC-derived cell maturity can be enhanced by geometrical cues, interaction with other cardiac cell types and physical stimuli (Scuderi G.J. et al., 2017, Karbassi E. et al., 2020).

At this aim, several 3D culture methods have been designed (Cho S. et al., 2022, Min S. et al., 2023), based on the assembly of either primary cells or iPSC-derived cardiac subsets obtained by previous differentiation in 2D, with the final goal to better mimic cardiac tissue morphological, biochemical and mechanical features to improve maturity.

Cells are assembled either in spheroids or microtissues (Filippo Buono M. et al. 2020, Richards D. J. et al. 2020), with or without the addition of biomaterials (e.g. Matrigel, collagen), deposited through bioprinting methods or assembled in microwells, cultured statically, by means of microfluidic chambers or exposed to mechanical training (e.g. engineered heart tissues) (Voges H.K. et al., 2017, Mills R.J. et al. 2019, Mills R.J. et al. 2021).

Each of these approaches has its own specific strength and pitfail, for example the spheroids are high-throughput models, while the use of microfluidic systems allows more efficient environmental condition control. The common limitation of these methods is due to the need to previously obtain different cardiac cell subsets, thus making the procedures long and expensive.

Based on the experience in other fields, including neurology, intestinal and cancer (Monteduro A. G. et al. 2023, Saorin G. et al. 2023), during the last years, an increasing interest has risen on the development of cardiac organoids.

Organoids are 3D structures generated taking advantage of the natural iPSC capability to self-assembly rebuilding organ parts, by following a special restricted lineage commitment. Similar to spheroids, organoids are composed of different tissue specific cell types, but, in addition, they have also a tissue specific geometrical organization. This approach bypasses the need to pre -differentiate and pre-culture cell subsets, and to artificially force their integration and organization in the tissue.

While in literature the use of organoids for the generation of relevant models of heart organogenesis has been reported (Rossi G. et al., 2018; Silva A.C. et al., 2020; Olmsted Z.T. et al., 2022), the development of adult cardiac models is still a challenge.

Indeed, several cardiac features are potentially relevant for in vitro recapitulation of physio- pathological scenarios, including the presence of the plurality of cardiac cell subsets, geometrically organized, forming cardiac chambers with an endocardium layer, showing segregation of atrial and ventricular cardiomyocytes, implementing vascularization and innervation.

The generation of organoids showing cardiac chambers also including the presence of CMs, endocardial cells, epicardial cells and fibroblasts, preferentially localized in distinct layers has been reported by Lewis-Israeli Y. R. et al., 2021; Hofbauer P. et al., 2021; Drakhlis L. et al., 2021 and Hoang P. et al., 2021.

In these organoids, despite the presence of chambers, their identity has not been fully characterized.

Attempts in this direction have been reported by Feng W. et al., 2022 and Meier et al., 2023, respectively focusing on the generation of 3D organoids recapitulating atrial and ventricular myocardium. These studies provided relevant data on the factors guiding each differentiation, but did not integrate the two myocardium specifications in a unique organoid. A protocol for the generation of an epicardium -myocardium organoid, recapitulating a functional pro-epicardium supporting CM maturation has been proposed by Branco M.A. et al., 2022; the protocol includes the differentiation of epicardium and CM components in different spheroids, successively assembled in a new spheroid.

The implementation of innervation has been proposed by Olmsted Z.T. et al., 2022 who guided, in a gastruloid model, the differentiation towards a neurocardiac lineage, but in a model missing the other relevant features previously discussed.

The potential of strictly controlled geometrical confinement for the generation of cardiac organoids has been highlighted by the work of Hoang P. et al., 2018, who, using a micropatterned stencil, identified in 600 pm diameter holes the optimal shape/size for the formation of early-developing cardiac organoids with contracting CM in the center, surrounded by a layer of stromal cells.

Indeed, it has been widely reported how, through the regulation of mechano -transduction pathways, cell differentiation can be guided in vitro by geometrical and mechanical stimuli, including confinement, substrate rigidity and mechanical load, in combination, but also independently, from administration of soluble factors (Engler A. J. et al. 2006, Santoro et al. 2019, Wong et al. 2019).

US2022397564 Al discloses cardiac organoids formed by differentiating a quantity of human induced pluripotent stem cells (hiPSC) on a micropatterned stencil with a plurality of circles with a diameter of 200-1000 pm on top of a (PEG)-based substrate coated with hESC-qualified matrix. These cardiac organoids have neither two separate asymmetric cardiac chambers nor atrial and ventricular tissue-specific cardiomyocytes. Moreover, the organoids disclosed in the patent application are used to carry out embryotoxicity studies. No relevance has been attributed to the relevant role of the properties of the substrate and the focus was on the geometric confinement wherein the pattern geometry of the microwells dictates structural morphology and contractile physiology of cardiac organoids.

Wang Bin et al., 2018 disclose a system comprising embryoid bodies like structures cultured in confinement into a stencil of sterile patterned microwells (polydimethylsiloxane stencil, PDMS stencil) located on top of a micropillar substrate of moldable low stiffness material with a stiffness of 1-9 kPa. Such embryoid bodies like structures have only one inner chamber, which is obtained in all the culture condition on their substrate (see page 7 of the paper). On page 5 the paper refers to a comparative flat substrate named “flat PDMS” without pillars as a not-working embodiment because of the migration of cells. Therefore, the paper contains a teaching away from using a flat substrate and the importance of cell culture on pillar substrates.

The author of the present invention has now realized that substrate rigidity even onto a flat non-pillar based substrate guides differentiation and geometrical organization of complex organoids, in particular of cardiac organoids. It has been observed that the modulation of mechanical and geometrical features of the substrate leads towards the highly reproducible generation of a complex cardiac model showing cardiac chambers and an epicardial layer, to be implemented in a high-throughput platform, where other factors (e.g. metabolic environment role, compounds efficacy, cardiotoxicity) could be easily evaluated.

SUMMARY OF THE INVENTION

The present invention relates to a system comprising two chambers in vitro cardiac organoids cultured in confinement into a stencil of sterile patterned microwells located on top of a flat substrate of moldable low stiffness material with a stiffness of 1-9 kPa and attached on a glass coverslip, wherein said patterned microwells are made of a non cell-adhesive material, have a diameter of 350 pm-650 pm and a depth of 300 pm-450 pm.

The term “flat” referred to substrate employed in the system of the present invention means that the substrate does not contain microstructures, such as pillars.

In a preferred embodiment the moldable low stiffness material of said flat substrate has a stiffness of 4 kPa. According to another preferred embodiment said moldable low stiffness material is polyacrylamide gel, polydimethylsiloxane or PEG. In the most preferred embodiment of the invention said moldable low stiffness material is polyacrylamide gel.

According to another preferred embodiment the substrate of moldable low stiffness is coated to support cell attachment by mean of at least one cardiac matrix component.

It is underlined that moldable stiffness of the flat substrate brings three main advantages in the final cardiac organoid thus obtained:

1. enhanced functional differentiation of cardiomyocytes

2. presence of two specific and asymmetric cardiac chambers

3. presence of specific epicardial layer.

According to another preferred embodiment of the invention the non cell-adhesive material of said patterned microwells is polydimethylsiloxane or polystyrene. By using said non celladhesive material the cells will non adhere to the stencil but to the substrate.

In a further preferred embodiment said sterile patterned microwells are sterile polystyrene 6-well plates composed of approximately 150 microwells.

In another preferred embodiment said sterile patterned microwells have a diameter of 500 pm. According to another preferred embodiment said sterile patterned microwells have a depth of 350 pm.

According to a preferred embodiment the bottom surface of the sterile patterned microwells contains the substrate of moldable low stiffness material with a stiffness of 1-9 kPa coated with vitronectin, fibronectin or a mixture of ECM proteins. More preferably, vitronectin is human recombinant vitronectin.

According to a further embodiment of the invention the two chambers in vitro cardiac organoids comprise a plurality of cardiac cells including cardiomyocytes and cardiac fibroblasts.

In fact, the further characterization of the cardiac organoids obtained according to the present invention revealed the following characteristics:

1. enhanced functional differentiation of cardiomyocytes in the cardiac organoids (significant upregulation of gene ontology gene sets related to ECM organization, tissue morphogenesis, muscle structure development, heart development)

2. presence of different cardiac cell subsets, including contractile cells and fibroblasts

2. presence of two specific and asymmetric cardiac chambers 3. presence of specific epicardial layer.

The present invention further relates to the use of the system comprising two chambers in- vitro cardiac organoids onto the sterile patterned microwells above described as in vitro cardiac model for drug screening.

It is another object of the present invention the use of the system comprising two chambers in-vitro cardiac organoids onto the sterile patterned microwells above described as in vitro model of cardiomyopathy.

This in vitro model allows either to carry out studies on the mechanisms involved in the cardiomyopathies or to study the effect of drugs on the cardiomyopathy, also allowing development of individual plan of treatment for each patient.

The model of cardiac organoids of the invention is versatile and easy to handle and may be inserted in multiorgan in vitro systems, wherein is possible to simultaneously evaluate the activity/effect of a drug (for example, cardiotoxicity of antitumoral drugs). This may be achieved by inserting the cardiac organoids of the invention and a tumoral organoid on the same chip/plate.

It is a further object of the present invention a micropatterned plate comprising a stencil of sterile patterned microwells, wherein said stencil is a thin layer of non cell-adhesive material with a thickness of 300 pm - 400 pm being located on top of a flat substrate of moldable low stiffness material with a stiffness of 1-9 kPa attached on a glass coverslip, wherein said patterned microwells are made of a non cell-adhesive material, have a diameter in a range of 350 pm-650 pm and a depth of 300 pm-450 pm; wherein said sterile patterned microwells are seeded with mammal pluripotent stem cells (iPSC).

The micropatterned plate of the invention forces geometrical confinement from pluripotent stem cells seeding throughout the duration of the differentiation and maturation procedures.

In a preferred embodiment of the invention said mammal pluripotent stem cells are human induced pluripotent stem cells (hiPSC).

According to a preferred embodiment of the system of the invention said non cell- adhesive material is polydimethylsiloxane or polystyrene.

In another preferred form said moldable stiffness material has a stiffness of 4 kPa.

More preferably said moldable low stiffness material is polyacrylamide gel, polydimethylsiloxane or PEG. Even more preferably said moldable low stiffness material is polyacrylamide gel. In a particular preferred embodiment of the invention the substrate is a thin layer of polyacrylamide gel, with a thickness of 100 pm-200 pm. According to a preferred embodiment of the micropattemed plate of the invention said sterile patterned microwells diameter is 500 pm. In another preferred embodiment said sterile patterned microwells have a depth of 350 pm.

According to another embodiment of the invention the surface of the flat substrate is pre-treated, activated or coated with matrix components or proteins, to allow the attachment of seeded cells. According to a preferred embodiment of the invention the flat substrate is coated with at least one cardiac matrix component, such as vitronectin, fibronectin or a mixture of ECM proteins or a combination thereof. More preferably vitronectin is human recombinant vitronectin.

A further object of the present invention relates to the use of the micropatterned plate above disclosed for the production of organoids. In a preferred embodiment of the invention when the flat substrate of the micropatterned plate above disclosed is coated with a cardiac matrix component said organoid are cardiac organoids.

The present invention further relates to a two-chambers in vitro cardiac organoid with an epicardium layer formed by differentiating a quantity of human induced pluripotent stem cells, under geometrical confinement, on a flat substrate of moldable low stiffness material with a stiffness of 1-9 kPa coated with at least one cardiac matrix component.

The invention further relates to a process for the in-vitro production of cardiac organoids comprising the following steps: a) seeding mammal pluripotent stem cells into the sterile patterned microwells of the system of micropatterned plate above described, wherein said sterile patterned microwells bottom surface is constituted by a known-stiffness flat substrate coated with at least one cardiac matrix component; b) adding culture medium to the microwells until reaching 90% confluence in each micro well; c) inducing cardiac differentiation by sequential modulation of Wnt signaling by adding small molecules involved in the Wnt regulation; d) cultivating up to day 21. Typically beating cardiac organoids are obtained starting from day 7 in culture.

In a preferred embodiment of the invention said cardiac matrix component is vitronectin, fibronectin or a mixture of ECM proteins or a combination thereof. More preferably vitronectin in is human recombinant vitronectin.

In a preferred embodiment of the invention the mammal pluripotent stem cells are either mammal induced pluripotent stem cells or human induced pluripotent stem cells (hiPSC).

According to another preferred embodiment the culture medium is RPMI. In a preferred embodiment the sequential modulation of Wnt pathway signaling of step c) comprises the following steps:

- treating the induced pluripotent stem cells with 10 pmol/1 of CHIR99021 (a GSK3 inhibitor) in RPMI medium supplemented with insulin-free B27 added to each well; replacement of the medium with RPMI medium supplemented with insulin-free B27 after 24 h; on day 3 adding to each well combined medium containing 50% of conditioned media and 50% of 10 pmol/1 of IWP2 (a Wnt/beta-catenin inhibitor) in RPMI supplemented with insulin-free B27; replacement of the combined medium with RPMI supplemented with insulin-free B27 after 36 h; on day 7 replacing the medium with RPMI supplemented with B27 containing insulin.

Finally, the invention relates to two chambers in vitro cardiac organoids obtained by the process above described.

The present invention will now be described for illustrative but non-limiting purposes, according to a preferred embodiment with particular reference to the attached figures, in which:

- Figure 1, panel a) shows an exemplifying organoid culture platform according to the invention composed by an acrylamide gel, firmly attached on a glass coverslip and covered by a silicone stencil, with through holes of 500 pm diameter. iPSC seeding and culture into this platform leads to the generation of cardiac organoids following the protocol illustrated in panel b). Panel c) shows constructs cultured under geometrical confinement are spheroids characterized by reproducible dimension and shape, independently from substrate stiffness (G= H= 30 KPa, L= 4 KPa).

- Figure 2A-C shows Bulk RNA-seq analysis. Panel A) shows scatterplot of the first two principal components (PCI and PC2) obtained from the principal component analysis (PC A) performed on all expressed genes in the bulk RNA-seq dataset. PCI and PC2 together explained 38% of the variance and allowed the four spheroid types to be clearly distinguished. Panel B) shows cultures grown under geometric confinement with a large number of significantly differentially expressed (DE) genes compared to samples grown under non-confined conditions. The scatterplots show the log2 fold-change (FC) and the significance (x and y axis, respectively) for the confined (G, H and L, respectively) vs. non-confined (EB) condition comparisons. Significance: adjusted P-Value <0.05, |log2FC |> 1. Panel C) illustrates functional enrichment analysis shows a positive association of confinement conditions with the regulation of several functions related to muscle and heart development and morphogenesis; statistically enriched overview Gene Ontology terms with their identifiers are hierarchically clustered into a tree based on Kappa-statistical similarities among their gene memberships (kappa score threshold = 0.3). The significance of the enrichment is defined by the -loglO (adjusted P-Value) from high (dark) to low (light). Not significant = grey.

- Figure 3 shows hierarchical clustering based on cardiomyocyte marker genes. The clustering and heatmap shows that unsupervised clustering based on genes related to cardiomyocyte structure and function is strongly associated with the four spheroid types (first row), allowing 4 out of 5 EB, 5 out of 6 H and all G and L samples to be correctly grouped. Hierarchical clustering was performed using 1 -Pearson's correlation and the average linkage method.

- Figure 4 shows hierarchical clustering and heatmap based on a selection of specific cardiac- related pathways and biological processes suggesting an effect of substrate stiffness. Hierarchical clustering was performed using 1-Pearson's correlation and the average linkage method on loglO (P -value) of significant gene sets as obtained in the previous enrichment analysis depicted in Figure 2, panel C.

- Figure 5 A-B shows enrichment networks of bulk RNA-seq data. For the comparison of EB vs. L (Figure 5 A) and H vs. L (Figure 5 B) samples, enrichment networks were drawn to show the Gene Ontology (GO) biological processes (BP) gene sets (nodes) that are significantly associated (false discovery rate <0.01) with L, H and EB conditions. Node gradient color is proportional to the gene-set normalized enrichment score (NES), from lower (light) to higher (dark); node size is proportional to the gene-set size. Edges connect related GO-BPs. Edge thickness is proportional to the similarity between two GO-BPs, for a cutoff=0.25 of the combined Jaccard plus Overlap coefficient. Culture under confinement on soft gels as per L vs. EB comparison (Figure 5 A) positively regulates processes related to cardiac maturity, function and 3D organization (e.g. chamber development); H vs. L comparison (Figure 5 B) suggests that processes related to cardiac maturity and cardiomyocyte ultrastructure are significantly regulated by fine tuning of substrate stiffness.

- Figure 6 shows the immunofluorescence staining supports results of transcriptome analysis. Optical sections, of both top and equator level, of organoid stained for cardiac marker (NKX2.5) show the presence of internal cavities, non located in the spheroid center and divided by a septum (20X); staining for cell-cell junctions indicates presence of Zol and Cx45 (present in cardiomyocytes since the initial stages of differentiation) in both EB and ORG, while Cx43 (specific of mature ventricular cardiomyocytes) is expressed only in ORG samples (40X); staining for cTnT confirms the presence of cardiomyocytes in both EB and ORG (40X), but magnification boxes highlight enhanced cellular ultrastructure in ORG samples; presence of different cardiac fibroblasts subsets is both EB and ORG is indicated by staining for Colli Al and aSMA (40X); staining for TBX18 and WT1 indicates presence of a single cell layer of epicardia cells in ORG samples.

- Figure 7 A-B shows single cell RNA-seq analysis. Panel A. Data integration of conditions H and L identifies 9 clusters, matched in both culture conditions, reproducibly between the analyzed batches. Cell type annotation suggests identification of different cell clusters, including contractile cells and fibroblasts. Panel B. Functional annotation by cluster top markers, based on GO for biological processes, on scRNA-seq data integration of conditions H and L confirms the presence of clusters with a transcriptome signature linked either to contractile functions, or ECM deposition and remodeling.

- Figure 8 shows consensus analysis between proteomic and transcriptomic analysis, EB versus L. Gene Ontology biological process enrichment network were obtained by combining enrichment results from RNA-seq and proteomic data. Enrichment is calculated separately for each omics layer and then aggregated p-values are calculated to derive a composite multi-omics enrichment. The combined results confirm the concordance of the results obtained by both proteomic and RNA-seq analysis on independent samples. Node color refers to the association with the phenotype (L; EB); node gradient color is proportional to the gene-set normalized enrichment score (NES), from lower (light) to higher (dark); node size is proportional to the gene-set size. Edges connect related GO-BPs. Edge thickness is proportional to the similarity between two GO- BPs, for a cutoff=0.25 of the combined Jaccard plus Overlap coefficient.

- Figure 9 shows single cell RNA-seq data dimensional reduction using the Uniform Manifold Approximation and Projection (UMAP) method and cluster visualization before data integration of the 4 H and L conditions. Scatterplots of the two UMAP components show a similar representation of the 4 sample batches.

- Figure 10 show organoids cultured onto low substrates show functional response to cardioactive drugs in a dose dependent manner. A) Representative contraction amplitude plots and B) average beating rates of organoids untreated versus in response to increasing doses of Isoproterenol and Verapamil (n>5, Mean, SE, T-Student test, One-tailed, p<0.05).

- Figure 11 shows the results of an electrophysiology study. Panel a) Hierarchical clustering and heatmap based on a selected GOBP gene set, indicative of cardiac muscle cell potential suggesting differential regulation in ORG-L vs. EB samples. Panel b) Representative voltage traces of spontaneous action potential recorded in single cardiomyocytes at 21 day of differentiation 24 h post dissociation from Organoids (ORG-L red trace) and Embryoids (EB, blue trace). Panel c) Scatter plot of the firing rate (Hz) in ORG-L (red dots; N =8)) and EB-derived cardiomyocytes (blue dots; N =13). Panel d) Scatter plots of maximum diastolic potential (MDP), action potential amplitude (APA) color as in C. Panel e) scatter plots of action potential duration at 30 %, 50 % and 90 % of repolarization (APD30, APD50 and APD90, respectively) in ORG-L and EBs (color as in Panel c) Mean ±SEM values are reported as black squares and whiskers. Panel f) Representative traces of TTX-sensitive currents elicited by depolarizing voltages from ORG-L (red traces, top panel) and EB-derived cardiomyocytes (blue traces, bottom panel). Panel g) Mean sodium current density -voltage relations in ORG-L (red dots; N =11) vs. EB-derived cardiomyocytes (blue dots; N =10). Panel h) Mean activation and inactivation curves fitted by the Boltzmann equation (symbols and colors as in Panel g).

The following non-limiting examples are now provided for a better illustration of the invention, in which cardiac organoid with two chambers according to the invention are obtained.

EXAMPLES

EXAMPLE 1 : Generation of cardiac organoids

MATERIALS AND METHODS iPSC Generation and Characterization

All investigations were conducted on an iPSC line generated from healthy adult dermal fibroblasts obtained from tebu-bio (Le-Perray-en-Yvelines, France). Generation and characterization of the used iPSC line was previously reported in Farini et al., 2019. Briefly, expanded fibroblasts were transfected with four episomal vectors (pCXLE-hUL, pCXLE- hSK, pCXLE-hOCT3/4-shp53-F, and a positive control pCXLE-EGFP) by electroporation (1650 V, 10 ms, 3 pulses) with the Neon transfection system (Invitrogen, Carlsbad, CA). iPSC colonies were manually isolated between post -transfection days 21 to 30; from postoperative day 4 onward cells were passaged every 3 to 4 days with ReLeSR (Stemcell Technologies) a non-enzymatic solution, and they were plated as cell aggregates onto human recombinant vitronectin-coated multiwell plates in Stem-Flex (Thermo Fisher Scientific) containing RevitaCell (Thermo Fisher Scientific) substituted 24 hour later with plane Stem- Flex.

For the analysis of pluripotency protein expression, immunofluorescence staining for stagespecific embryonic antigen-4 (SSEA4) was performed and images were acquired with 20X objective (LSM710; Zeiss), while Tra-1-60 staining was detected by FACS analyses using a FACSCalibur (BD Biosciences, Franklin Lakes, NJ) or gallios (Beckman Coulter Life Sciences, Indianapolis, IN) flow cytometers. Finally, the capacity of this iPSC line to differentiate in the cardiac lineage was assessed by inducing differentiation in cardiomyocytes, following the Lian X. et al., 2013 differentiation protocol and characterizing cells by immunofluorescence, FACS and multielectrode array analysis. Cardiac Organoid Platform

Cardiac organoids were produced by mean of on-purpose designed micropatterned plates (Figure 1; panel a), forcing geometrical confinement from iPSC seeding throughout the duration of the cardiac differentiation and maturation procedures.

The micropatterned plate is constituted by a stencil (a) located on top of a substrate (b).

In particular:

(a) The stencil is a thin layer (between 300-400 pm thickness) of material non celladhesive (e.g. silicone), patterned with through holes of the diameter of 500 pm (±150 pm), that will serve for cell confinement when located onto the substrate (b).

(b) The substrate is a moldable stiffness material, with a stiffness of 5±4 kPa. As example it could be a thin layer (approximately 100 pm) of polyacrylamide gel. It must be feasible to treat the substrate in order to allow the attachment of the seeded cells (e.g. the surface is activated and coated it by cardiac matrix component, such as vitronectin).

Following manufacturer instructions, silicone (10: 1 mix ratio of base and cure reagents of Sylgard 184 silicone elastomer, Dow Corning) disks of 26 mm diameter and 350 pm in thickness were polymerized, to produce stencil for organoid geometrical confinement. Using a 500 pm diameter puncher, through-holes with a center-to-center distance of 1.2 mm were realized, in order to obtain around 150 microchambers per each stencil. Stencil sterility was obtained by autoclave treatment (120°C, 20 minutes).

Each silicon stencil was assembled on top of a 100 pm thick polyacrylamide gel (PAAg) substrate. The PAAg substrate was produced as previously described in Santoro R. et al., 2018. Briefly, a thin layer of polyacrylamide was deposited onto a 30 mm diameter glass coverslip.

The stiffness of the final gel was tuned by varying the percentage of acrylamide and the ratio acrylamide/bisacrylammide. Aiming to allow cell attachment and proliferation, the polyacrylamide surface was activated by treatment with sulfosuccinimidyl-6 (49-azido-29- nitrophenyl-amino hexanoate (Sulfo-SANPAH; Pierce) and coated by overnight incubation at 4°C with 5 pg/ml human recombinant vitronectin (as used for iPSCs culture and maintenance). Before use, PAAg were sterilized by UV treatment (15 min, 245 nm) and stored for two days in 20% BASE128 solution in PBS (Al. Chi. Mi. A. Sri), an antibiotic/mitotic solution. In conclusion, the system, accommodated in sterile polystyrene 6-well plates, is composed of 150 microwells, of 500 pm diameter and 350 pm depth, with vitronectin-coated low-stiffness bottom surface.

Cardiac Organoid Generation PSC are seeded into the microwell generated by the micropatterned plate and kept in maintenances medium until reaching approximately 90% confluence in the each microwell. When this confluence was reached, a cardiac differentiation was induced through a dynamic modulation of Wnt pathway, using small molecules (e.g. Lian X. et al. 2013).

Beating organoids are obtained in 7 days of culture and organoids can be kept in culture for long term (full organoid characterization performed at day 21, but organoids kept in culture up to 90 days). iPSC expanded in vitronectin-coated plastic culture wells were passaged with ReLeSR and plated as cell aggregates either on agarose coated polystyrene multiwells (preventing cell to adhesion on the plate for the production of embroid bodies) or onto the organoid platform, using Stem -Flex containing RevitaCell.

The day after, medium was substituted to Stem-Flex; the differentiation protocol was initiated 3 days after, when approximately 90% confluence in the each microwell was reached.

On day 0 of differentiation, iPSCs were treated with 10 mmol/L CHIR99021 (Selleck Chemicals LLC, Houston, TX) in RPMI 1640 medium supplemented with insulin-free B27 (Invitrogen). The media was replaced with RPMI supplemented with insulin-free B27 after 24 hours.

On day 3, a combined medium was prepared that contained 50% of conditioned media of each well and 50% of 10 mmol/L IWP2 (a Wnt signaling inhibitor, final concentration of 5 mmol/L) in RPMI supplemented with insulin-free B27. 36 hours later, the combined medium was replaced with RPMI supplemented with insulin-free B27.

On day 7, the medium was changed to RPMI supplemented with B27 containing insulin. From this point, the medium was changed every 3 days.

On day 21 both embryoid bodies and organoids were processed for further analysis.

RESULTS

Confined culture regulates 3D construct geometrical features

Current literature identifies in the sequential modulation of the Wnt pathway an effective method for cardiac differentiation of iPSC both in 2D and 3D applications (Lian X. et al., 2013).

At first, the effect of geometrical confinement on the efficacy of this differentiation protocol in the formation of 3D cardiac microstructures was verified.

As described in Figure 1, iPSCs were seeded either onto standard multi-well plates treated for low cell attachment, or in home-made microfabricated plates, describable as 500 pm diameter microwell array, with a vitronectin coated substrate of known stiffness.

All samples were cultured with addiction of the same small molecules (Lian et al. , 2013) and were kept in culture for 21 days before their collection for the performed analysis.

Macroscopically, samples cultured in all conditions showed spontaneous contraction, but, due to geometrical confinement, ORG samples were characterized by highly reproducible geometrical features, stable over time (Figure 1 b), summarized by Area, describing their size, and Circularity factor, describing their shape (Figure 1 c).

There is a significant difference in these parameters between ORG and EB (Figure 1 c): samples cultured under confinement are significantly smaller (EB: 570900±39440 n=l l; G: 212800±27190 n=5; H: 21400±31450 n=9; L: 271600±29180 n=9; avg±SEM, T-test: unpaired, p<0.05) and rounder (EB: 0,70±0,04 n=l l; G: 0.86±0,03 n=5; H: 0.83±0.02 n=9; L: 0,84±0,02 n=9; avg±SEM, T-test: unpaired, p<0.05) then EB, while the geometrical parameters are not affected by substrate rigidity. Indirectly, confinement affects also collateral parameters.

Finally, the protocol here proposed allows high throughput production of cardiac organoids: in a 30 mm diameter dish, in parallel, up to 100 organoids can be produced and cultured, with 4 ml medium.

EXAMPLE 2: Cardiac Organoid Characterization

Cardiac organoids thus obtained were characterized by:

Immunofluorescence: detection of markers of cardiomyocytes (cTNT, Cx43, Cx45, ZO1), epicardium (WT1, TBX18) and fibroblasts (ColllAl, aSMA).

Transcriptomic bulk: Total RNA was extracted from single organoid and RNAseq was performed to profile the transcriptome.

Transcriptomic single cell: organoids were dissociated, through tissue mechanical and enzymatic digestion in order to extract and sequence the transcriptome profile of single cells.

Proteomic: the proteome of single organoids was evaluated through mass spectrometry.

Drug response: organoids were incubated with drug (Isoproterenol and Verapamil, range between 0.001-1 pM) supplemented medium for 15 minutes (37°C, 5% CO2, humidified incubator) before live imaging to monitor drug dose response (beating rate frequency was evaluated).

MATERIALS AND METHODS

Immunofluorescence Samples (both organoids and embryoid bodies) were fixed in 4% PFA at room temperature for 20 minutes, then washed in PBS.

After blocking and permeabilizing for 1 hour at room temperature with PBS containing 3% bovine serum albumin and 0.1% Triton-X, cells were incubated for 1 hour at room temperature with the primary antibodies listed in the following Table:

Table 1. List of antibodies used for immunofluorescence

Samples were then incubated for 1 hour at room temperature with either Alexa Fluor 488 - or Alexa Fluor 633 -conjugated secondary antibodies (Invitrogen). Nuclear staining was performed by incubating samples with Hoechst 33342 (Invitrogen). Images were acquired using LSM710 confocal microscope (Carl Zeiss, Jena, Germany).

Transcriptome

Total RNA was extracted from single organoids or single embryoid bodies. Each organoid, after washing in PBS, was digested in 0.1 ml of TRI Reagent and RNA was extracted by Direct-Zol RNA Microprep kit (Zymo Research, CA, USA), according to producer instructions, including DNase treatment for genomic DNA removal and, finally, resuspended in 15 pl of water. Total RNA concentration, quality and integrity were evaluated, respectively, using microvolume spectrophotometry and microfluidics electrophoresis with an RNA 6000 Nano Assay Kit on a 2100 Bioanalyzer system (Agilent, Santa Clara, CA, USA), a- and P-globin mRNA depletion was performed using the GLOBINclear Human kit (Applied Biosystems). RNA libraries were prepared and sequenced by Illumina NovaSeq 6000. Reads, aligned to hgl9 genome were counted for each gene. Category enrichment and network analyses were used to sort differentially expressed genes.

Single cell RNA-seq

After 21 days of culture, 3D microtissues (either cardiac organoids produced on different substrates or embryoid bodies cultured in suspension) were carefully washed in PBS and collected in tubes (5 samples per tube) for tissue mechanical and enzymatic digestion (incubation for 15 min at 37°C in Collagenase II 0.2 mg/ml solution in PBS without Ca²⁺ and Mg²⁺, followed by Vortex). The obtained single cell suspension, after cell counting, was resuspended in PBS without Ca²⁺ and Mg²⁺ with 0.04% BSA. Approximately 2000 cells/pl from each sample were used for the analysis. Briefly, every sample was loaded into one channel of Single Cell Chip A using a Chromium Single Cell 3' v2 Reagent Kit (lOx Genomics). After capture and lysis, complementary DNA was synthesized and amplified over 14 cycles according to the manufacturer’s protocol (lOx Genomics). Libraries were prepared from 50 ng amplified cDNA. Sequencing was performed using a NovaSeq 6000 System (Illumina). An average sequencing depth of at least 50,000 reads per cell was obtained for each sample.

Label-free mass spectrometry (LC-MSE) analysis

Organoids were dissolved in 25 mmol/L NH4HCO3 containing 0.1% RapiGest (Waters Corporation), sonicated, and centrifuged at 13,000 g for 10 min. Protein concentration was evaluated using the DC protein assay (BioRad laboratories). After 15 min of incubation at 80°C, 5 pg of proteins were reduced with 5 mmol/L DTT at 60°C for 15 min, and carbamidom ethylated with 10 mmol/L iodoacetamide for 30 min at room temperature in the darkness. Overnight digestion has been performed with sequencing grade trypsin (Promega) (1 pg every 20 pg of proteins) at 37°C. After digestion, 2% TFA was added to hydrolyze RapiGest and inactivate trypsin. Tryptic peptides were analyzed by a label-free mass spectrometry-based approach, LC-MSE, performed on a hybrid quadrupole -time of flight mass spectrometer (SYNAPT-XS) coupled with an UPLC Mclass system and equipped with a nanosource (Waters Corporation). Samples were first trapped into a Symmetry C18 nanoACQUITY trap column, 100 A, 5 pm, 180 pm><2 cm (Waters Corporation, Milford, MA, USA) and subsequently separated on the analytical column HSS T3 C18, 100 A, 1.7 pm, 75 pm* 150 mm (Waters Corporation, Milford, MA, USA), at a flow rate of 300 nL/min by increasing the organic solvent B concentration from 3 to 40 % over 90 min, using 0. l%v/v formic acid in water as reversed phase solvent A, and 0.1% v/v formic acid in acetonitrile as reversed phase solvent B (Brioschi M. et al., 2014). All of the analyses were made in triplicate. LC-MSE has been performed by ion mobility-enhanced data-independent acquisition (IMS-DIA). In particular, in the low-energy MS mode, the data were collected at constant collision energy of 6 eV, while in high energy mode, fragmentation was obtained by applying drift time-specific collision energies (Distler U. et al. 2016).

Drug response

Isoproterenol hydrochloride (0.2 mg/ml, injectable solution, S.A.L.F.) and Verapamil hydrochloride (Isoptin 5 mg/ml injectable solution, Mylan) injectable solutions were diluted in RPMI/B27 +Insulin media to make final concentrations between 0.001 -1 pM.

Organoids were incubated with drug supplemented medium for 15 minutes (37°C, 5% CO2, humidified incubator) before live imaging. Imaging set up was constituted by a Zeiss Axiovert 200M microscope, equipped with 10X long distance objective and Axiocam 503 camera; contraction frequency was calculated by automated open -source software-based method (Imaged, Musclemotion plugin (Sala L. et al., 2018).

Bioinformatic analysis

Data processing

Sequential aligning of raw reads of bulk RNA-sequencing as produced by the Ion GeneStudio S5 Prime System was performed against the GROG 8 Human Genome reference using the “Spliced Transcripts Alignment to a Reference (STAR)” (Dobin A. et al, 2013) and the “Bowtie2” software (Langmead B. et al. 2012;) to align locally any reads not mapped by STAR. Gene expression quantification and annotation were computed by “featureCounts” (Liao Y. et al. 2014;). Data exploration was performed through the EDASeq (Risso D. et al. 2011;) and DaMiRseq (Chiesa M. et al. 2018) Bioconductor packages.

Bulk transcriptome analysis

Raw counts were imported into the R software v4.1.0. and filtered to retain genes with a minimum of 5 counts in at least 20% samples. Differential expression analysis (DEA) was performed by a negative binomial GLM approach (edgeR/Bioconductor package) (Robinson M.D. et al. 2010; McCarthy D.J. et al. 2012) along with the estimation of latent (confounding) variables, including technical batch effects or biological ones, for adjusting the statistical model (RUVSeq R/B ioconductor package) (Risso D. et al. 2014). The RUVr method was applied to estimate the latent variables (W) by comparing unadjusted vs. adjusted expression data by the use of diagnostic plots, ie., relative log expression (RLE) plot, scatter plot of the first two principal components derived from principal components analysis (PCA) performed on total data, and histogram of the P -value distribution for testing the differential gene expression between the different 3D model types. The design matrix was set to handle the different type of Organoids and EB, as the primary variable of interest.

A W=5 latent variables showed the best trade-off between data adjustment and the risk of data overcorrection and were, thus, used as covariates for model adjustment for DEA. A contrast matrix was set to identify gene expression differences between each organoid type (G, H, and L) v.s. EB; and H vs. L. Genes were deemed as significantly different for FDR-adjusted P-value < 0.05 and |log2 Fold Change (FC)|>0.58. The reliability of the DEA results was further assessed by exploring the histograms of the P-value distribution, which showed a uniformly flat distribution across the unit interval (null P-values) with a peak near zero (P -values for alternative hypotheses) (Leek J.T. et al. 2008;). DEA results for each comparison were graphically summarized by the use of Volcano plots, i.e. scatterplots of the magnitude of the difference between conditions (expressed as log2 FC) and the significance (expressed as -logic of nominal P-value). Volcano plots were generated with the Enhanced Volcano R/B ioconductor package vl.10.0.

Unsupervised hierarchical clustering analysis was performed on normalized expression values of differentially expressed genes or genes of interest, based on the Euclidean and 1 -pearson correlation metric, for samples and genes, respectively, and the average linkage method, as implemented in the GENE-E software v3.0.213.

For functional inference analysis, we took advantage of prior knowledge of genes grouped by biological processes (BP) as per the Gene Ontology (GO) gene-set collection (Ashbumer M. et a. 2000;) in the Metascape software (Zhou Y. et al. 2019). Enrichment analysis was performed based on the up- and down-regulated genes as found by the comparison between organoids vs. EB. The genes expressed in the present RNA-seq dataset was used as the background. Parameters for analysis were set with Min Overlap=3, P-Value Cutqff=0.01 and Min Enrichment=2. Significant terms were hierarchically clustered in a tree based on Kappa-statistical similarities (threshold = 0.3) among gene memberships. The most statistically significant terms within each cluster is chosen as overview terms and were then visualized as heatmaps.

To unveil even more subtle associations between ORG-L and EB, an enrichment analysis was also performed using the GO-BP and the Gene Set Enrichment Analysis (GSEA, v4.2.3) method (Subramanian A. et al. 2005).

For GSEA, the “Human_GO_bp_no_GO_iea_symbol.gmt” gene set collection (release February 2021) from the repository of the Bader Lab (http://download.baderlab.org/EM_Genesets) was used.

Specific markers of adult cardiac cell types as retrieved by the work by Tucker N.R. et al., 2020, were instead used to (i) construct a reference gene set collection of cardiac cell types for GSEA analysis to identify possible associations of more differentiated and mature cardiac cell sub-types within each organoid and EB, and (ii) to perform an unsupervised hierarchical clustering of organoids and EB based on the expression levels of well-defined specific cardiac cell types.

Proteomics

The proteomic data obtained from LC-MSE included 1197 proteins and were log2 transformed. The proteomic log2 expression matrix was used for differential analysis between organoids vs. EB using the limma R/Bioconductor package (Ritchie M.E. et al., 2015). Proteins were considered differentially expressed if the adjusted P-value was <0.05. Enrichment analysis was performed using the GSEA approach as described above for bulk RNA sequencing. To obtain a functional consensus from the transcriptomic and proteomic data, the GSEA results of both bulk RNA- sequencing and LC-MSE were first filtered to retain commonly detected GO-BPs (regardless of the significance level), and then the P-values were combined using the Stouffer method (combinePvalues function of the multiGSEA R/Bioconductor package). Combined P-values were further adjusted for multiple testing using the Benjamini -Hochberg method.

Single cell RNA-sequencing

For scRNA-sequencing, alignment, filtering, barcode counting, and Unique Molecular Identifier counting were performed using the Cell Ranger count module. Filtered feature (gene)-barcode matrices of two H and L organoids were imported into the R software v4.1.0 as a SingleCellExperiment object (DropletUtils R/Bioconductor package) to perform the following analysis. To discard any cell with very few expressed genes, the data were first filtered to retain genes with at least of 5 counts in more than 5 cells. Quality control (QC) metrics were assessed using the perCellQCMetrics and isOutlier functions (scater and scuttle R/Bioconductor packages) (McCarthy J. et al., 2017) to identify cells expressing too many mitochondrial genes, indicating low-quality cells. Data were then normalized for library size, log2-transformed and modelled for the mean-variance relationship by sizeFactors, logNormCounts, and modelGeneVar functions, respectively (scater R/Bioconductor package). Data from the 4 organoids were integrated by: first, intersecting the common genes and renormalizing to adjust for differences in depth; second, correcting for batch effects in single-cell expression data using a fast version of the mutual nearest neighbors (MNN) method (the two steps were performed by the multiBatchNorm and fastMNN functions of the batchelor R/Bioconductor package, respectively) (Haghverdi L. et al., 2018). Uniform Manifold Approximation and Projection (UMAP) was performed as the dimensional reduction method to visualize individual cells in a two-dimensional space, while multi-sample kmeans clustering was performed using the KmeansParam function (bluster R/Bioconductor package) with number of centres=9 as a trade-off between the ability to identify putative different cell types or those with nuances in functional properties. To interpret the clustering results and assign biological significance to each cluster, marker detection and scoring was performed by applying the fmdMarkers and scoreMarkers functions (scran R/Bioconductor package) to the integrated data, using the four batches of H and L scRNA-seq experiments as a blocking factor. The ScType R package (A. lanevski A. et al., 2022) was used to annotate single-cells of organoids based on their similarity to a priori known markers of specific cell types.

RESULTS

Geometrical confinement is not sufficient for the regulation of cardiac features

Despite the above described absence of differences in the macroscopic geometrical features, we further investigated the effects induced by substrate rigidity on the generation of cardiac organoids. To this aim, we compared by a genome-wide approach the gene expression differences of organoids obtained through the same confinement system, but onto substrates of different stiffness (ORG-G, E~25GPa; ORG-H, E=30kPa; ORG-L, E=4kPa) (Figure 2). As shown by the scatterplot of the first two principal components of the PCA (principal component analysis) on all expressed genes, there is substantial difference between the four spheroid types (Figure 2a). The effect of geometrical confinement can be observed in the gene expression differences between the non-confined and each confined condition, all characterized by huge number of differentially expressed genes (DEG) (Figure 2b). A more detailed functional investigation based on the enrichment analysis for gene ontology biological processes allows to qualify these differences and understand their relevance for the generation of cardiac structures. Geometrical confinement positively regulates cellular activities related to the structural organization and remodeling of the extracellular surrounding, i.e. extracellular matrix organization, tissue morphogenesis, blood vessel development and tissue morphogenesis (Figure 2c). Nevertheless, gene sets related to cardiac maturity, such as heart development, muscle structure development and muscle system process, are only slightly or non-significantly upregulated when confinement is associated with high substrate stiffness (G condition). Unsupervised hierarchical clustering based on gene sets related to adult cardiac cells structure and function (Figure 2c) showed a clear separation of the four spheroid types with the wider clustering distance between ORG- H and ORG-L conditions with respect to the EB and ORG-G. This result is even more appreciable if the unsupervised hierarchical cluster is performed to gene sets related to the subset of cardiomyocytes (Figure 3). Since the reference cardiac gene set was built up from single nuclei analysis of an adult heart, this result suggests a relationship between an increased maturity of cardiac cell subsets and the ORG-H and -L conditions, as a probable combined effect of confinement and substrate mechanical features.

Finally, hierarchical clustering based on a selection of specific cardiac-related signaling pathways and biological processes, confirms, under confinement conditions, a positive effect of low (L) to moderate (H) substrate rigidity in the regulation of complex cardiac structures features (e.g. cardiac septum morphogenesis, cardiac chamber morphogenesis, cardiac atrium/ventricle morphogenesis) and maturity (e.g. response to mechanical stimulus, cardiac muscle tissue morphogenesis), possibly guided by YAP-TAZ dependent cascade (Figure 4). Low substrate stiffness significantly affects organoid cardiac features

Assessed the relevance of controlling substrate rigidity in our in vitro model, the effect of its fine tuning was evaluated.

Polyacrylamide gel substrates H and L were chosen as representative respectively of myocardium material constant in adult heart (10-30 kPa) and during early cardiogenesis (1 - 3 kPa) (Engler A. J. et al. 2008, Filas B. A. et al. 2011, Sharifi-Sanjani M. et al. 2017, Querceto S. et al. 2022). We chose to test these two stiffnesses in order to verify two alternative hypotheses: i) feeling the rigidity of the mature cardiac tissue would guide towards cardiomyocytes maturation or ii) exposed to a substrate rigidity similar to the one acting during development would guide stem cell self-organization, self-differentiation and subsequent maturation.

Interestingly, despite the low number of genes claimed to be differentially expressed in the whole transcriptome between ORG-H vs. ORG-L, a finer enrichment analysis allowed to unveil substantial functional differences between organoids cultured onto the two substrates (Figure 5B). At first, the enrichment network of bulk RNA-seq data comparing spheroids obtained in absence of confinement with organoids generated combining confinement and low substrate stiffness, underlined several Gene Ontology biological processes significantly associated with the second condition. In particular, ORG-L condition upregulates processes linked to complex geometrical organization (e.g. heart morphogenesis, cardiac chamber development and morphogenesis), extracellular matrix remodeling (e.g. collagen metabolic process, positive regulation of smooth muscle cell proliferation, positive regulation of supramolecular fiber organization, mesenchyme development), cardiac maturity and functionality (e.g. positive regulation of muscle cell differentiation, regulation of heart rate, heart contraction, cardiac conduction, sarcomere organization, cell junction assembly and organization) (Figure 5A).

Immunofluorescence staining confirms these data (Figure 6). Indeed, optical section of organoids show presence of two internal chambers, not localized in the middle of the spheroid, but asymmetrically and divided by a septum, thus suggesting an origin different from necrotic core due to nutrient diffusion limitations and possibly ascribable to “cardiac chamber development and morphogenesis” functions highlighted by transcriptome study. Staining for cell junctions confirms inferential analysis of functional enrichment: in ORG-L samples can be observed areas co-staining for Cx45, a connexin expressed in early stages of development, and Cx43, specific of ventricular cardiomyocytes. This last protein could not be found in spheroids generated in absence of confinement. The staining for ZO1, regularly present in ORG-L, is another sign of presence of more developed and functional cardiac junction, such as intercalated disks (Dai W. et al. 2020).

Staining for cardiac troponin shows presence of cardiomyocytes in both spheroids, but, as appreciable in the magnification box, the ultrastructural organization is enhanced in ORG- L; staining for collagen and smooth muscle actin indicates presence of cardiac fibroblasts in both spheroid models.

Even more interestingly, enrichment network of bulk RNA-seq data comparing ORG-L and ORG-H (Figure 5B) indicates that the low stiffness condition further enriches the gene sets related to cardiac maturity (e.g. cardiac muscle cell action potential, cardiac muscle contraction, heart contraction, cardiac muscle tissue development, ATP metabolic process) and cardiomyocyte ultrastructure (e.g. muscle filament sliding, myofibril assembly, sarcomere organization, cell communication involved in cardiac conduction).

In order to further characterize the differences between ORG-L and ORG-H, providing a quantitative description of the plurality and the maturity of the cell subsets constituting them an RNA-seq analysis at the single cell level was performed (Figure 9). At a first sight, single cell RNA seq data dimensional reduction using the UMAP suggests absence of major differences between the analyzed sample batches (2 ORG-L vs 2 ORG-H) (Figure 7A): data integration of conditions ORG-L and ORG-H identifies 9 clusters, matched in both culture conditions and reproducible between the analyzed batches. As expected, cell type annotation of identified clusters by GO marker gene database suggests the identification mainly of different contractile cell subsets and fibroblasts, besides neurons and adipocytes (Figure 7 A).

Cluster identity has been further described by the functional annotation by cluster top markers based on GO for biological processes (Figure 7B), confirming the presence of cell subsets deputed to contractile functions (Cl, C4, C6, C8) or to matrix deposition and remodeling (C9, C3, C2), and a general upregulation of processes related to tissue geometrical organization.

Cardiac organoids generated by geometrical confinement in low stiffness substrates enhance cardiac maturity and functionality

Assessed the relevance in the protocol of both geometrical confinement and substrate stiffness in defining the final properties of cardiac organoids, a deeper characterization of organoids produced using the most promising protocol has been carried out.

To this aim, the organoids generated on a low stiffness substrate with EB, growth in suspension and in absence of confinement were compared.

In conjunction with the gene expression signature previously described, the proteomic signature was performed by a bulk proteome analysis. The consensus analysis reported in Figure 8 shows the high level of agreement between the two ‘omic approaches. Indeed, both signatures describe superiority of organoid model when enrichment analysis is performed both for adult heart resident cardiac subsets and cardiac functions. Beside these indications, the consensus analysis demonstrates the high level of reproducibility of the novel protocol assessed for organoid generation: organoids obtained in different experiments, analyzed independently with different approaches, generated highly reproducibility results, thus, finally, demonstrating the high robustness of the proposed in vitro method.

The comparison between the transcriptome signature of ORG-L to EB reveals that low stiffness substrates favorably regulates functions related to electrophysiological activity, including “cardiac muscle cell action potential” and “action potential involved in contraction”.

Finally, the functionality of the organoids as a whole was assessed, in order to evaluate their possible future application as in vitro model of cardiomyopathy. By means of nondestructive on-line video recording of organoids and their computer-based analysis, we analyzed organoid beating rate in response to medium supplement with different drug doses. In particular, we evaluated positive and negative chronotropic effects of isoproterenol and verapamil, two widely used cardioactive drugs (Guo Y. et al. 2011, Mannhardt et al. 2016). Organoids in culture showed spontaneous contraction, measurable with our video set-up, and a dose dependent response to the drug administration (Figure 10): treatment with isoproterenol significantly increased beating rate already at O.OluM, reaching the maximum at luM, while treatment with verapamil showed inhibition of contraction at O.OluM. Data resulted in accordance with previous reports in other 3D microtissues (Ergir E. et al., 2022). EXAMPLE 3 : Electrophysiological analysis MATERIALS AND METHODS

EBs and ORG-L were collected at 21 days of culture, both samples were washed twice with Low calcium solution (pH 6.9) containing (mM): 120 NaCl, 5.4 KC1, 5 MgSO4, 5 Na- Pyruvate, 20 Glucose, 20 Taurine, 10 HEPES-NaOH. Samples were then enzymatically digested in the low calcium solution adding 400U/ml of Collagenase B (Sigma Aldrich) and

3 pM CaCL. Following three rounds of enzymatic digestion, each lasting 9 min at 37°C, the enzymatic solution was replaced with potassium solution (pH 7.2) that contained (mM): 85 KC1, 30 KH2PO4, 5 MgSO₄, 1 EGTA-KOH, 2 ATP-2Na, 5 Na-Pyruvate, 20 Glucose, 5 Creatine, 20 taurine; in which mechanical disruption was carried out pipetting up and down until the cellular clumps completely disappear. The potassium solution with single cells was adjusted to a high concentration of calcium by adding 100 pl of complete medium (RPMI/B27) every 5 min for five times. The fibroblasts-like cells were allowed to spread for half an hour, plating the cell suspensions in a 60 mm dish (Corning) with 4 ml of RPMI/B27. After collecting the remaining floating cells, the enriched cardiac cells were centrifuged for

4 min at 200 g. The pellets were suspended in a complete medium containing 1 pM Rock Inhibitor (Y27632- Tocris) and cells were spread on dishes coated with fibronectin (5 pg/cm²-Sigma Aldrich).

After 24-30 h dissociated CMs were recorded by the patch-clamp technique in current clamp or voltage clamp mode using the whole- cell configuration at physiological temperature (36 ±1°C). Data acquisition was performed using the amplifier Axopatch 200 B, the Digidata 1550 and the pClamp 10.0 software (Molecular Devices, LLC). Data were filtered at 1 -5 kHz and sampled at 10 kHz.

The analysis of the data was carried out by Clampfit 10.0 (Molecular Devices, LLC) in combination with Origin Pro 9 (OriginLab). Cells were perfused with the following extracellular-like solutions:

- Tyrode solution (pH 7.4) containing (mM): 137 NaCl, 5 KC1, 2 CaCh, 1 MgCh, 10 D- glucose, 10 Hepes-NaOH to record spontaneous action potentials.

- Tyrode solution was supplemented with 0.01 mM Nifedipine and with or without 0.03 mM tetrodotoxin (TTX) to dissect sodium current (INa).

- Potassium extracellular solution (pH 7.4) containing: 110 mM NaCl, 0.5 mM MgCh, 1.8 mM CaCL, 5 mM Hepes-NaOH, 30 mM KC1, supplemented with 1 mM BaCh and 2 mM MnCh in order to dissect the funny current (If).

Patch-clamp pipettes had resistances of 3-6 MQ when filled with the intracellular-like solution containing (mM): 120 KC1, 20 Na-HEPES, 10 MgATP, 0.1 EGTA-KOH, and 2 MgCh; pH 7.1 with KOH.

The following AP parameters were analyzed: rate (Hz), action potential amplitude (APA, mV), maximum diastolic potential (MDP, mV), and action potential duration at 30%-50 % and 90 % of repolarization (APD30, APD50, and APD90). Sodium current (INa) was recorded as the TTX- sensitive current, applying depolarizing voltage steps, 10 mV each, to the range of -80/+60 mV, followed by a step at -20 mV (hp of -90 mV).

Funny current (If) was activated from a holding potential (hp) of -30 mV applying 10 mV hyperpolarizing voltage steps to the range of -35/-125 mV long enough to reach steady-state of activation, followed by a fully activating step at -125 mV.

Current densities were calculated for each step of voltage protocols as the ratio of current intensity versus the cell capacitance. The activation and inactivation curves were obtained from normalized tail currents or from normalized conductance at each voltage step and fitted to the Boltzmann equation.

Electrophysiological analysis

The comparison between the transcriptome and proteome signature of ORG-L vs. EB reveals that low stiffness substrates favorably regulate genes and transcripts related to electrophysiological activity, including “cardiac muscle cell action potential” and “regulation of cardiac muscle cell action potential” (Figure 11, Panel a). As validation of these data, we performed patch clamp recording-based study on single CMs isolated either from ORG-L or EBs.

Spontaneously beating cells have been observed after 24 h post-dissociation of both sample groups. Representative spontaneous action potentials (AP) recorded from these isolated cells are shown in Figure 11, Panel b. ORG-derived CMs exhibit a significantly higher AP rate (Figure 11 Panel c), while there is no change between ORG and EB-derived AP in the amplitude (APA) and in the maximal diastolic potential (MDP) that is still depolarized (Figure 11, Panel d). Based on these data, we proceeded by analyzing the pacemaker current (If) linked to HCN4 gene expression, that did not differ between EB and ORG-derived (data not shown). Despite a higher AP rate, ORG-L-derived CMs present longer action potential duration (APD) at 30-50 and 90 % of repolarization (Figure 11, Panels b-e), thus supporting a different regulation of calcium handling (RYR2 and ATP2A2 mediated) and of the membrane expression of sodium and potassium channels. In particular, since it is known that a prolongation of the AP duration may depend on an increase in the inward sodium current (Grant A.O. et al., 2009), and the transcriptomic results indicate a positive regulation of sodium ionic channels with an increased expression of the beta-subunits SCN4B, we evaluated the fast sodium current (INa). Notably, patch clamp analysis demonstrated a significant increase in sodium current density in ORG-derived CM compared to EBs, as shown by representative traces and the mean current density -voltage relations in Figure 11, Panels f-g. No changes in steady state activation and inactivation curves were observed (Figure 11, Panel h). The differences between sodium current and a prolongation of the action potential duration support the omics data of a different commitment of the CMs populating the ORG-L at day 21 in culture.

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Claims

1. A system comprising two chambers in vitro cardiac organoids cultured in confinement into a stencil of sterile patterned microwells located on top of a flat substrate of moldable low stiffness material with a stiffness of 1 -9 kPa, attached on a glass coverslip, wherein said patterned microwells are made of a non cell-adhesive material, have a diameter of 350 pm- 650 pm and a depth of 300 pm-450 pm.

2. A system according to claim 1, wherein said moldable low stiffness material has a stiffness of 4 kPa.

3. A system according to anyone of claim 1 -2, wherein said moldable low stiffness material is polyacrylamide gel, polydimethylsiloxane or PEG.

4. A system according to anyone of claim 1-3, wherein said flat substrate of moldable low stiffness material is coated with at least one cardiac matrix component.

5. A system according to anyone of claim 1-4, wherein said non cell-adhesive material is polydimethylsiloxane or polystyrene.

6. A system according to anyone of claim 1-5, wherein said sterile patterned microwells are polystyrene 6-well plates composed of 150 microwells.

7. A system according to anyone of claim 1-6, wherein said sterile patterned microwells have a diameter of 500 pm.

8. A system according to anyone of claim 1 -7, wherein the bottom surface of the sterile patterned microwells contains the flat substrate coated with vitronectin, fibronectin or a mixture of ECM proteins.

9. Use of the system according to anyone of claims 1-8 as in vitro model for drug screening or cardiomyopathy.

10. A micropatterned plate comprising a stencil of sterile patterned microwells, wherein said stencil is a thin layer of non cell-adhesive material with a thickness of 300 pm - 400 pm being located on top of a flat substrate of moldable low stiffness material with a stiffness of 1-9 kPa and attached on a glass coverslip, wherein said patterned microwells are made of a non cell-adhesive material, have a diameter in a range of 350 pm-650 pm and a depth of 300 pm-450 pm; wherein said sterile patterned microwells are seeded with mammal pluripotent stem cells (iPSC).

11. A micropatterned plate wherein said mammal pluripotent stem cells are human induced pluripotent stem cells (hiPSC).

12. A micropatterned plate according to anyone of claim 10-11, wherein said non celladhesive material is polydimethylsiloxane or polystyrene.

13. A micropatterned plate according to anyone of claim 10-12, wherein said moldable stiffness material has a stiffness of 4 kPa.

14. A micropatterned plate according to anyone of claim 10-13, wherein said moldable low stiffness material is polyacrylamide gel, polydimethylsiloxane or PEG.

15. A micropatterned plate according to anyone of claim 10-14, wherein said sterile patterned microwells diameter is 500 pm.

16. A micropatterned plate according to anyone of claim 10-15, wherein the surface of the flat substrate is pre-treated, activated or coated with at least one matrix components or proteins to allow the attachment of seeded cells.

17. A micropatterned plate according to claim 16, wherein the flat substrate is coated with at least one cardiac matrix component, such as vitronectin, fibronectin or a mixture of ECM proteins.

18. Use of the micropatterned plate according to anyone of claims 10-17, for the production of organoids.

19. Use of the micropatterned plate according to claim 17, for the production of two- chambers in vitro cardiac organoids.

20. A two-chambers in vitro cardiac organoid with an epicardium layer formed by differentiating a quantity of human induced pluripotent stem cells, under geometrical confinement, on a flat substrate of moldable low stiffness material with a stiffness of 1 -9 kPa coated with at least one cardiac matrix component.

21. A process for the production of two-chambers in vitro cardiac organoids comprising the following steps: a) seeding induced pluripotent stem cells into the sterile patterned microwells of the system of micropatterned plate according to anyone of claims 10-17, wherein said sterile patterned microwells is located on top of the flat substrate coated with at least one cardiac matrix component; b) adding culture medium to the microwells until reaching 90% confluence in each microwell; c) inducing cardiac differentiation by sequential modulation of Wnt signaling by adding small molecules involved in the Wnt regulation; d) cultivating until beating organoids are obtained.

22. A process for the in vitro production of cardiac organoids according to claim 21, wherein said cardiac matrix component is vitronectin, fibronectin or a mixture of ECM proteins.

23. A process for the production of two-chambers in vitro cardiac organoids according to anyone of claims 21-22, wherein the sequential modulation of Wnt pathway signaling of step c) comprises the following step: i) treating the induced pluripotent stem cells with 10 pmol/1 of CHIR99021 in RPMI medium supplemented with insulin-free B27 added to each well; ii) replacement of the medium with RPMI medium supplemented with insulin -free B27 after 24 h; iii) on day 3 adding to each well combined medium containing 50% of conditioned media and 50% of 10 pmol/1 of IWP2 in RPMI supplemented with insulin-free B27; iv) replacement of the combined medium with RPMI supplemented with insulin-free B27 after 36 h; v) on day 7 replacing the medium with RPMI supplemented with B27 containing insulin.

24. A two chambers in vitro cardiac organoid obtained by the process according to anyone of claims 20-23.