Journal Article

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

Yamashita, Misaki, Iwao, Takahiro, Matsunaga, Tamihide, Aoki, Hiromasa, Hashita, Tadahiro

2020

Overview

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.

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: Analysis / Antibodies / Biomedical and Life Sciences / Biomedicine / Blood-brain barrier / Blood-Brain Barrier - drug effects / Blood-Brain Barrier - physiology / Bone morphogenetic proteins / Brain / Brain research / Brain stem / Cell culture / Cell differentiation / Cell Differentiation - drug effects / Cell Differentiation - physiology / Cryopreservation / Cytological Techniques / Drug development / Electric properties / Electrical resistivity / Endothelial cells / Endothelial Cells - drug effects / Endothelial Cells - physiology / Endothelium / Endothelium, Vascular - cytology / Endothelium, Vascular - drug effects / Endothelium, Vascular - physiology / Freezing / Growth factors / Hematology / Humans / Immunofluorescence / Induced pluripotent stem cells / Induced Pluripotent Stem Cells - drug effects / Induced Pluripotent Stem Cells - physiology / Inhibitory postsynaptic potentials / Kinases / Low density lipoprotein / Membrane permeability / Microvasculature / Microvessels - cytology / Microvessels - drug effects / Microvessels - physiology / Models, Neurological / Neovascularization / Neurobiology / Neurosciences / Parenchyma / Permeability / Pharmacokinetics / Physiology / Pluripotency / Signal transduction / Signal Transduction - drug effects / Signal Transduction - physiology / Stem cell research / Stem cells / Thawing / Transforming growth factor beta / Transforming Growth Factor beta - antagonists & inhibitors / Transforming Growth Factor beta - physiology / Transforming growth factor-b / Transforming growth factors