cARLA: a small molecule cocktail for robust induction of blood-brain barrier properties
Gergo Porkolab 1,2 , Maria Meszaros 1 , Aniko Szecsko 1,2 , Judit P. Vigh 1,2 , Fruzsina R. Walter 1 , Ricardo Figueiredo 3 , Ildiko Kalomista 4 , Gaszton Vizsnyiczai 1 , Jeng-Shiung Jan 5 , Fabien Gosselet 6 , Monika Vastag 4 , Szilvia Veszelka 1 & Maria A. Deli 1.
1 Institute of Biophysics, Biological Research Centre, Eötvös Loránd Research Network, Szeged, Hungary
2 Doctoral School of Biology, University of Szeged, Szeged, Hungary
3 GenXPro GmbH, Frankfurt am Main, Germany
4 In vitro Metabolism Laboratory, Gedeon Richter Plc., Budapest, Hungary
5 Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan
6 Laboratoire de la Barriére Hémato-Encéphalique (LBHE), Université d’Artois, Lens, France
Correspondence: Gergo Porkolab – <
Blood-brain barrier (BBB) models derived from human stem cells are powerful tools to improve our understanding of human cerebrovascular diseases and facilitate drug development for the brain. Yet providing endothelial cells with the appropriate molecular cues to both retain a vascular identity and acquire BBB characteristics remains challenging. Here we present cARLA, an easy-to-use and affordable small molecule cocktail that robustly induces BBB properties in vitro. By activating cyclic AMP and Wnt/β-catenin signaling while inhibiting the transforming growth factor beta (TGF-β) pathway, cARLA synergistically enhances barrier tightness in a range of BBB models. We demonstrate that, upon cARLA treatment, human stem cell-derived endothelial cells have lower rates of transcytosis, higher glycocalyx density and increased efflux pump activity with a shift in gene expressional profile towards the in vivo brain endothelial signature. Our work provides mechanistic insight into how endothelial signaling is orchestrated during BBB maturation and leverages this to advance the prediction of drug delivery to the human brain.