A non-canonical aryl hydrocarbon receptor pathway authorizes and safeguards clinical-scale expansion of functional human endothelial cells.

Tissue-specific endothelial cells (ECs) regulate metabolism, inflammation, coagulation, organ development and regeneration. However, therapeutic application of EC transplantation requires scalable expansion of engraftable ECs that sustain their angiogenic and angiocrine functions. Here we identify a non-canonical aryl hydrocarbon receptor (AHR) pathway switched on by canonical AHR inhibitors that reactivates quiescent EC proliferation. Incubation of tissue-specific human ECs with AHR inhibitors, such as StemRegenin1 (SR1), increased EC proliferation by three-fold within an 8-day period. AHR inhibitors induced 100-fold greater expansion of 200,000 primary human adipose ECs to 2.4 × 10(12) ECs, retaining in vivo vessel-forming and homeostatic functions in the recipient mice. AHR inhibitors induce a non-canonical AHR pathway by ornithine decarboxylase 1 (ODC1)-dependent synthesis of polyamines that drives EC cell cycle progression, detoxification of reactive oxygen species and oxidative phosphorylation metabolism, thereby recruiting hibernating ECs to accompany expanding EC populations without imposing replicative senescence. Therefore, AHR inhibitors, through transcriptional-independent protein-protein interactions, shepherd unrestricted human-scalable functional EC expansion, enabling cell therapies.