Human induced pluripotent stem cells (hiPSCs) represent an almost limitless source of cells for disease modelling and drug screening applications. Here we established an efficient and robust 3D platform for cardiomyocyte (CMs) production from hiPSCs, solely through small-molecule-based temporal modulation of the Wnt signalling, which generates more than 90% cTNT⁺ cells. The impact of performing the differentiation process in 3D conditions as compared to a 2D culture system, was characterized by transcriptomic analysis by using data collected from sequential stages of 2D and 3D culture. We highlight that performing an initial period of hiPSC aggregation before cardiac differentiation primed hiPSCs towards an earlier mesendoderm lineage differentiation, via TGF-β/Nodal signaling stabilization. Importantly, it was also found that CMs in the 3D microenvironment mature earlier and show an improved communication system, which we suggested to be responsible for a higher structural and functional maturation of 3D cardiac aggregates.

The epicardium, the outer epithelial layer that covers the myocardium, derives from a transient organ known as pro-epicardium, crucial during heart organogenesis. The pro-epicardium develops from lateral plate mesoderm progenitors, next to septum transversum mesenchyme, a structure deeply involved in liver embryogenesis. Here we describe a self-organized human multilineage organoid that recreates the co-emergence of pro-epicardium, septum transversum mesenchyme and liver bud. Additionally, we study the impact of WNT, BMP and retinoic acid signaling modulation on multilineage organoid specification. By co-culturing these organoids with cardiomyocyte aggregates, we generated a self-organized heart organoid comprising an epicardium-like layer that fully surrounds a myocardium-like tissue. These heart organoids recapitulate the impact of epicardial cells on promoting cardiomyocyte proliferation and structural and functional maturation. Therefore, the human heart organoids described herein, open the path to advancing knowledge on how myocardium-epicardium interaction progresses during heart organogenesis in healthy or diseased settings.

The knowledge acquired throughout the years concerning the in vivo regulation of cardiac development has promoted the establishment of directed differentiation protocols to obtain cardiomyocytes (CMs) and other cardiac cells from human pluripotent stem cells (hPSCs), which play a crucial role in the function and homeostasis of the heart. Among other developments in the field, the transition from homogeneous cultures of CMs to more complex multicellular cardiac microtissues (MTs) has increased the potential of these models for studying cardiac disorders in vitro and for clinically relevant applications such as drug screening and cardiotoxicity tests. This review addresses the state of the art of the generation of different cardiac cells from hPSCs and the impact of transitioning CM differentiation from 2D culture to a 3D environment. Additionally, current methods that may be employed to generate 3D cardiac MTs are reviewed and, finally, the adoption of these models for in vitro applications and their adaptation to medium- to high-throughput screening settings are also highlighted.

Coronary vascularization and sympathetic innervation are tightly coordinated during heart development and are essential for normal cardiac function. Here, we present a self-organized human iPSC-derived epicardium-myocardium organoid (EMO) that mimics heart vascular plexus development and integration with myocardium sympathetic innervation. Through the modulation of VEGF and PDGFβ signaling, EMOs develop a functional, self-generated coronary-like vascular plexus (V-EMOs). and display active epicardial-to-mesenchymal transition trajectories into fibroblast, mural, and vascular cell lineages, mirroring in vivo processes. Assembly of these vascularized EMOs with human iPSC-derived sympathetic neuron spheroids yields innervated EMOs exhibiting integrated neurovascular organization and functional responses to nicotine stimulation (iV-EMOs). This modular, developmentally guided organoid system features coordinated human cardiac, vascular, and neural development, providing a physiologically relevant platform for studying heart morphogenesis, neurovascular interactions, and regenerative therapeutic strategies.

Today, it is recognized that medicines will eventually be needed during pregnancy to help prevent to, ameliorate or treat an illness, either due to gestation-related medical conditions or pre-existing diseases. Adding to that, the rate of drug prescription to pregnant women has increased over the past few years, in accordance with the increasing trend to postpone childbirth to a later age. However, in spite of these trends, information regarding teratogenic risk in humans is often missing for most of the purchased drugs. So far, animal models have been the gold standard to obtain teratogenic data, but inter-species differences have limited the suitability of those models to predict human-specific outcomes, contributing to misidentified human teratogenicity. Therefore, the development of physiologically relevant in vitro humanized models can be the key to surpassing this limitation. In this context, this review describes the pathway towards the introduction of human pluripotent stem cell-derived models in developmental toxicity studies. Moreover, as an illustration of their relevance, a particular emphasis will be placed on those models that recapitulate two very important early developmental stages, namely gastrulation and cardiac specification.

SUMMARY: Coronary vascularization and sympathetic innervation of the myocardium is a concomitant event during embryonic heart development and both systems are crucial to ensure normal adult heart function. Here we describe a self-organized hiPSC-derived heart organoid that recreates both the coronary vascular plexus and the sympathetic neuronal network of the ventricle myocardium, with a physiologically relevant in-vivo-like structural organization and function. Through modulation of PDGF-β and VEGF signalling pathways, we attained a heart organoid that incorporates 1) an external epicardial layer (mesothelium) of DACH1, NR2F2 and WT1 positive cells, 2) a sub-epicardial space from where a functional primary coronary vascular plexus of CD31 ⁺ /DACH1 ⁺ cells emerge, 3) a compact myocardial region adjacent to the epicardium, enriched in proliferative cardiomyocytes and ECM deposition, and 4) a sympathetic neuronal network that controls heart organoid contraction. Therefore, the human heart organoid described herein, is a unique model to study new regenerative medicine-based approaches to restore innervation and promote re-vascularization in adult heart after ischemic events and to perform adult and developmental cardiotoxicity studies.