Inhibition of transforming growth factor beta signaling pathway promotes differentiation of human induced pluripotent stem cell-derived brain microvascular endothelial-like cells

Abstract

Background: The blood-brain barrier (BBB) plays an important role as a biological barrier by regulating molecular transport between circulating blood and the brain parenchyma. In drug development, the accurate evaluation of BBB permeability is essential to predict not only the efficacy but also the safety of drugs. Recently, brain microvascular endothelial-like cells derived from human induced pluripotent stem cells (iPSCs) have attracted much attention. However, the differentiation protocol has not been optimized, and the enhancement of iPSC-derived brain microvascular endothelial-like cells (iBMELCs) function is required to develop highly functional BBB models for pharmaceutical research. Thus, we attempted to improve the functions of differentiated iBMELCs and develop a versatile BBB model by modulating TGF-β signaling pathway without implementing complex techniques such as co-culture systems. Methods: iPSCs were differentiated into iBMELCs, and TGF-β inhibitor was used in the late stage of differentiation. To investigate the effect of TGF-β on freezing-thawing, iBMELCs were frozen for 60-90 min or 1 month. The barrier integrity of iBMELCs was evaluated by transendothelial electrical resistance (TEER) values and permeability of Lucifer yellow. Characterization of iBMELCs was conducted by RT-qPCR, immunofluorescence analysis, vascular tube formation assay, and acetylated LDL uptake assay. Functions of efflux transporters were defined by intracellular accumulation of the substrates. Results: When we added a TGF-β inhibitor during iBMELCs differentiation, expression of the vascular endothelial cell marker was increased and blood vessel-like structure formation was enhanced. Furthermore, TEER values were remarkably increased in three iPSC lines. Additionally, it was revealed that TGF-β pathway inhibition suppressed the damage caused by the freezing-thawing of iBMELCs. Conclusion: We succeeded in significantly enhancing the function and endothelial characteristics of iBMELCs by adding a small molecular compound, a TGF-β inhibitor. Moreover, the iBMELCs could maintain high barrier function even after freezing-thawing. Taken together, these results suggest that TGF-β pathway inhibition may be useful for developing iPSC-derived in vitro BBB models for further pharmaceutical research.

Hypoxia‐induced blood‐brain barrier dysfunction is prevented by pericyte‐conditioned media via attenuated actomyosin contractility and claudin‐5 stabilization

“…Thus, TGF‐β signaling in iBMECs during hypoxia increases fibrillogenesis and decreases barrier function. These results are consistent with a recent study demonstrating improved TEER and TJ protein localization resulting from TGF‐β inhibition 50 …”

Next-generation in vitro blood–brain barrier models: benchmarking and improving model accuracy

“…iBMECs can be reprogrammed with ETS TFs (e.g., ETV2 , ERG , FLI1 ) to improve endothelial identity, however this process is associated with a loss of barrier function and leads to gene expression profiles more closely resembling non-brain specific iECs [ 21 , 27 ]. TGF-β inhibition can enhance endothelial and BBB-specificity of iECs and iBMECs [ 24 , 25 ], and activation of Wnt/β-catenin signaling (using agonist of Wnt/β-catenin signaling, Wnt ligands, or conditioned media from neural progenitor cells) in endothelial progenitor cells upregulates BBB-specific gene expression [ 26 ]. While these approaches hold promise and have shown improvements in accuracy of specific BBB or endothelial genes and/or functional properties, they have so far been unable to achieve the physiological barrier properties typical of iBMEC monolayers, and show TEER values comparable to primary and immortalized BMEC sources.…”

Multi-disciplinary Approach for Drug and Gene Delivery Systems to the Brain

“…To take advantage of this, grafting nanoparticles or drugs with ApoE could target LRP1 in the brain. These receptors could be triggered with antibodies to optimize CNS drug delivery through receptor-mediated transcytosis in an otherwise typical BBB (5,43,44). The ApoE receptors have been identified as acetylated low-density lipoproteins (LDL) which binds to antibodies FC5-Fc and IGF1R5-Fc allowing for endocytosis across the BBB (5).…”