SUMMARY Somites form during embryonic development and give rise to unique cell and tissue types, such as skeletal muscles and bones and cartilage of the vertebrae. Using somitogenesis stage human embryos, we performed transcriptomic profiling of human presomitic mesoderm as well as nascent and developed somites. In addition to conserved pathways such as WNT/β-catenin, we also identified BMP and TGFβ signaling as major regulators unique to human somitogenesis. This information enabled us to develop an efficient protocol to derive somite cells in vitro from human pluripotent stem cells (hPSCs). Importantly, the in vitro differentiating cells progressively expressed markers of distinct developmental stages known during in vivo somitogenesis. Furthermore, when subjected to lineage-specific differentiation conditions, the hPSC-derived somite cells were multipotent in generating somite derivatives, including skeletal myocytes, osteocytes and chondrocytes. This work improves our understanding of human somitogenesis and may enhance our ability to treat diseases affecting somite derivatives.

Highlights d Human atlas of limb skeletal muscle in embryonic, fetal, and adult tissues d Human limb skeletal muscle populations and supportive cells vary across development d PAX7 muscle progenitor and stem cells are not identical across developmental states d hPSC-PAX7 cells align to the embryonic-to-fetal transition in human development

Rationale: Coronary artery disease (CAD) is the leading cause of death worldwide, but there are currently no methods to stimulate artery growth or regeneration in diseased hearts. Studying how arteries are built during development could illuminate strategies for re-building these vessels during ischemic heart disease. We previously found that Dach1 deletion in mouse embryos resulted in small coronary arteries. However, it was not known whether Dach1 gain-of-function would be sufficient to increase arterial vessels and whether this could benefit injury responses. Objective: We investigated how Dach1 overexpression in endothelial cells affected transcription and artery differentiation, and how it influenced recovery from myocardial infarction (MI). Methods and Results: Dach1 was genetically overexpressed in coronary endothelial cells (ECs) in either developing or adult hearts using ApjCreER. This increased the length and number of arterial end branches expanded arteries during development, in both the heart and retina, by inducing capillary ECs to differentiate and contribute to growing arteries. Single-cell RNA sequencing (scRNAseq) of ECs undergoing Dach1-induced arterial specification indicated that it potentiated normal artery differentiation, rather than functioning as a master regulator of artery cell fate. ScRNAseq also showed that normal arterial differentiation is accompanied by repression of lipid metabolism genes, which were also repressed by Dach1. In adults, Dach1 overexpression did not cause a statistically significant change artery structure prior to injury, but increased the number of perfused arteries in the injury zone post-MI. Conclusions: Our data demonstrate that increasing Dach1 is a novel method for driving artery specification and extending arterial branches, which could be explored as a means of mitigating the effects of CAD.

Collateral arteries act as natural bypasses, which re-route blood flow to ischemic regions and facilitate tissue regeneration. In the heart, injured neonatal artery endothelial cells orchestrate a systematic series of cellular events, which includes their outward migration, proliferation, and coalescence into fully functional collateral arteries. This process, called Artery Reassembly, aids complete cardiac regeneration in neonatal hearts, but is absent in adults. The reason for this age-dependent disparity in artery cell response is completely unknown. In this study, we investigated if regenerative potential of coronary arteries, like in cardiomyocytes, is dictated by their ability to de-differentiate. We performed single cell RNA sequencing of coronary endothelial cells and identified differences in molecular profiles of neonatal and adult endothelial cells. Neonates show significant increase in actively cycling artery cells that expressed prominent de-differentiation markers. Using both, in silico pathway analyses and in vivo experiments, we show that cell cycle re-entry of pre-existing artery cells and subsequent collateral artery formation, is dependent on arterial VegfR2. This sub-population of de differentiated and proliferating artery cells is absent in non-regenerative P7 or adult hearts. Together, our data indicate that adult artery endothelial cells fail to drive collateral artery development due to their limited ability to de-differentiate and proliferate.