Retinoic acid signaling modulation guides in vitro specification of human heart field-specific progenitor pools

Abstract

Cardiogenesis relies on the precise spatiotemporal coordination of multiple progenitor populations. Understanding the specification and differentiation of these distinct progenitor pools during human embryonic development is crucial for advancing our knowledge of congenital cardiac malformations and designing new regenerative therapies. By combining genetic labelling, single-cell transcriptomics, and ex vivo human-mouse embryonic chimeras we uncovered that modulation of retinoic acid signaling instructs human pluripotent stem cells to form heart field-specific progenitors with distinct fate potentials. In addition to the classical first and second heart fields, we observed the appearance of juxta-cardiac field progenitors giving rise to both myocardial and epicardial cells. Applying these findings to stem-cell based disease modelling we identified specific transcriptional dysregulation in first and second heart field progenitors derived from stem cells of patients with hypoplastic left heart syndrome. This highlights the suitability of our in vitro differentiation platform for studying human cardiac development and disease.

Cardiac differentiation roadmap for analysis of plasticity and balanced lineage commitment

“…Our findings corroborate these observa�ons and demonstrate that these popula�ons can emerge independently of RA treatment (Drakhlis et al, 2021;Silva et al, 2021). This was confirmed by the expression of markers of the juxta-cardiac field and pre-epicardial cells (Meier et al, 2023), including the expression of the newly iden�fied epicardial markers such as ITGA8 (Zawada et al, 2023). Paracrine signaling from endodermal and epicardial popula�ons has been reported to be beneficial for both the epicardial and CM cell popula�ons (Quijada et al, 2020).…”

“…Epicardial and anterior endoderm cell populations have been observed in previous studies (Drakhlis et al, 2021; Hofbauer et al, 2020; Meier et al, 2023; Silva et al, 2021; Zawada et al, 2023). Our findings corroborate these observations and demonstrate that these populations can emerge independently of RA treatment (Drakhlis et al, 2021; Silva et al, 2021).…”

“…In our experiments, the EBs showcased a plasticity of multipotent progenitor cells that was susceptible to paterning by RA. Because of this high level of plasticity in cardiac cultures and the ability to culture CMs of various specific subtypes (Devalla et al, 2015; Schmidt et al, 2023; Schwach et al, 2017; Zawada et al, 2023), detailed deconstruction of the developmental steps within the culture is necessary, to understand and optimize the cell type composition. The specification of posterior SHF progenitors directed CMs towards an atrial identity, as expected; however, other cell lineages (endothelial, epicardial, fibroblast and SMC-like cells) were minimally affected.…”

“…Early trajectories with SHF and early CM markers ISL1 and NXK2-5 and (pre-)epicardial marker expression. Per culture protocol UMAP representation with ventricular monolayer culture (top), ventricular embryoid body (EB) (middle) and atrial EB (botom) labeled for cell types (A) and expression levels of SHF marker ISL1 (B), epicardial marker WT1 (C) and genes expressed in pre-epicardial cells HAND1 (D), BNC2 (E), NKX2-5 (F), as well as the new epicardial surface marker ITGA8 (G) (Zawada et al, 2023). …”

“…Single-cell transcriptomic studies have elucidated the differentiation processes of hPSCs towards CMs (Ameen et al, 2022; Churko et al, 2018; Friedman et al, 2018; Grancharova et al, 2021; Hofbauer et al, 2021b; Lewis-Israeli et al, 2021; Meier et al, 2023; Mononen et al, 2020; Ruan et al, 2019; Sahara et al, 2019; Zawada et al, 2023). Several of these studies highlighted the presence of non-myocyte subpopulations within the culture (Churko et al, 2018; Friedman et al, 2018; Mononen et al, 2020), while others shed light on the specification of particular cardiomyocyte subtypes, such as atrial, right and left ventricular CMs, and node-like CMs (Funakoshi et al, 2021; Pezhouman et al, 2021; Wiesinger et al, 2022; Yang et al, 2022).…”