Human heart-forming organoids recapitulate early heart and foregut development.

Organoid models of early tissue development have been produced for the intestine, brain, kidney and other organs, but similar approaches for the heart have been lacking. Here we generate complex, highly structured, three-dimensional heart-forming organoids (HFOs) by embedding human pluripotent stem cell aggregates in Matrigel followed by directed cardiac differentiation via biphasic WNT pathway modulation with small molecules. HFOs are composed of a myocardial layer lined by endocardial-like cells and surrounded by septum-transversum-like anlagen; they further contain spatially and molecularly distinct anterior versus posterior foregut endoderm tissues and a vascular network. The architecture of HFOs closely resembles aspects of early native heart anlagen before heart tube formation, which is known to require an interplay with foregut endoderm development. We apply HFOs to study genetic defects in vitro by demonstrating that NKX2.5-knockout HFOs show a phenotype reminiscent of cardiac malformations previously observed in transgenic mice.

Femtosecond laser-based nanosurgery reveals the endogenous regeneration of single Z-discs including physiological consequences for cardiomyocytes.

A highly organized cytoskeleton architecture is the basis for continuous and controlled contraction in cardiomyocytes (CMs). Abnormalities in cytoskeletal elements, like the Z-disc, are linked to several diseases. It is challenging to reveal the mechanisms of CM failure, endogenous repair, or mechanical homeostasis on the scale of single cytoskeletal elements. Here, we used a femtosecond (fs) laser to ablate single Z-discs in human pluripotent stem cells (hPSC) -derived CMs (hPSC-CM) and neonatal rat CMs. We show, that CM viability was unaffected by the loss of a single Z-disc. Furthermore, more than 40% of neonatal rat and 68% of hPSC-CMs recovered the Z-disc loss within 24 h. Significant differences to control cells, after the Z-disc loss, in terms of cell perimeter, x- and y-expansion and calcium homeostasis were not found. Only 14 days in vitro old hPSC-CMs reacted with a significant decrease in cell area, x- and y-expansion 24 h past nanosurgery. This demonstrates that CMs can compensate the loss of a single Z-disc and recover a regular sarcomeric pattern during spontaneous contraction. It also highlights the significant potential of fs laser-based nanosurgery to physically micro manipulate CMs to investigate cytoskeletal functions and organization of single elements.

Continuous WNT Control Enables Advanced hPSC Cardiac Processing and Prognostic Surface Marker Identification in Chemically Defined Suspension Culture.

Aiming at clinical translation, robust directed differentiation of human pluripotent stem cells (hPSCs), preferentially in chemically defined conditions, is a key requirement. Here, feasibility of suspension culture based hPSC-cardiomyocyte (hPSC-CM) production in low-cost, xeno-free media compatible with good manufacturing practice standards is shown. Applying stirred tank bioreactor systems at increasing dimensions, our advanced protocol enables routine production of about 1 million hPSC-CMs/mL, yielding ∼1.3 × 108 CM in 150 mL and ∼4.0 × 108 CMs in 350-500 mL process scale at >90% lineage purity. Process robustness and efficiency is ensured by uninterrupted chemical WNT pathway control at early stages of differentiation and results in the formation of almost exclusively ventricular-like CMs. Modulated WNT pathway regulation also revealed the previously unappreciated role of ROR1/CD13 as superior surrogate markers for predicting cardiac differentiation efficiency as soon as 72 h of differentiation. This monitoring strategy facilitates process upscaling and controlled mass production of hPSC derivatives.