Pluripotent stem cell-derived epithelium misidentified as brain microvascular endothelium requires ETS factors to acquire vascular fate

Abstract

Cells derived from pluripotent sources in vitro must resemble those found in vivo as closely as possible at both transcriptional and functional levels in order to be a useful tool for studying diseases and developing therapeutics. Recently, differentiation of human pluripotent stem cells (hPSCs) into brain microvascular endothelial cells (ECs) with blood–brain barrier (BBB)-like properties has been reported. These cells have since been used as a robust in vitro BBB model for drug delivery and mechanistic understanding of neurological diseases. However, the precise cellular identity of these induced brain microvascular endothelial cells (iBMECs) has not been well described. Employing a comprehensive transcriptomic metaanalysis of previously published hPSC-derived cells validated by physiological assays, we demonstrate that iBMECs lack functional attributes of ECs since they are deficient in vascular lineage genes while expressing clusters of genes related to the neuroectodermal epithelial lineage (Epi-iBMEC). Overexpression of key endothelial ETS transcription factors (ETV2, ERG, and FLI1) reprograms Epi-iBMECs into authentic endothelial cells that are congruent with bona fide endothelium at both transcriptomic as well as some functional levels. This approach could eventually be used to develop a robust human BBB model in vitro that resembles the human brain EC in vivo for functional studies and drug discovery.

Human Induced Pluripotent Stem Cell-Derived Brain Endothelial Cells: Current Controversies

“…Before a functional hPSC-derived vascular BBB model can be developed, stable hPSC-derived vascular ECs must be generated. In a recent study (Lu et al, 2021), our group demonstrated that a vascular fate can be induced in iBMECs through introduction of certain EC-specific ETS TFs (ETV2, ERG, and FLI1). These reprogrammed cells (rECs) harbor an EC transcriptomic profile, retaining a PECAM1 + CDH5 + KDR + EC immunophenotype during passaging and expansion.…”

Are In Vitro Human Blood–Brain–Tumor-Barriers Suitable Replacements for In Vivo Models of Brain Permeability for Novel Therapeutics?

“…iPSC-EC models have been noted to exhibit morphological and physiological characteristics closest to in vivo characteristics, with comparable barrier integrity, protein and transporter expression and barrier permeability. It should be highlighted that pluripotent-derived endothelial cells should be confirmed as bona fide endothelial cells before use in in vitro model systems [49]. The inclusion of multiple cell types, including ECs, astrocytes, pericytes and neurons, improved barrier integrity, and the conditioning of these cells in brain-specific media (i.e., astrocyte and neuron) further strengthened the barrier.…”

Wnt signaling mediates acquisition of blood-brain barrier properties in naïve endothelium derived from human pluripotent stem cells

“…hPSCs are an attractive model system to complement in vivo animal studies because they (i) are human, (ii) permit investigation of developmental processes in contrast to primary or immortalized cells, (iii) are highly scalable, (iv) can be derived from patients to facilitate disease modeling and autologous coculture systems, and (v) are genetically tractable. While widely used hPSC-based BBB models are useful for measuring molecular permeabilities and have been employed to understand genetic contributions to barrier dysfunction (Vatine et al, 2016(Vatine et al, , 2019Lim et al, 2017), they have not been shown to proceed through a definitive endothelial progenitor intermediate (Lippmann et al, 2012;Lu et al, 2021) and express epithelial-associated genes (Qian et al, 2017;Delsing et al, 2018;Vatine et al, 2019;Lu et al, 2021). Thus, new models with developmentally relevant differentiation trajectories and definitive endothelial phenotype are needed for improved understanding of developmental mechanisms.…”