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Tolloid-Like 1 Negatively Regulates Hepatic Differentiation of Human Induced Pluripotent Stem Cells Through Transforming Growth Factor Beta Signaling.

ABSTRACT: Single nucleotide polymorphisms in Tolloid-like 1 (TLL1) and the expression of TLL1 are known to be closely related to hepatocarcinogenesis after hepatitis C virus elimination or liver fibrosis in patients with nonalcoholic fatty liver disease. TLL1 is a type of matrix metalloprotease and has two isoforms in humans, with the short isoform showing higher activity. However, the functional role of TLL1 in human liver development is unknown. Here, we attempted to elucidate the function of human TLL1 using hepatocyte-like cells generated from human pluripotent stem cells. First, we generated TLL1-knockout human induced pluripotent stem (iPS) cells and found that hepatic differentiation was promoted by TLL1 knockout. Next, we explored TLL1-secreting cells using a model of liver development and identified that kinase insert domain receptor (FLK1)-positive cells (mesodermal cells) highly express TLL1. Finally, to elucidate the mechanism by which TLL1 knockout promotes hepatic differentiation, the expression profiles of transforming growth factor beta (TGF?), a main target gene of TLL1, and its related genes were analyzed in hepatic differentiation. Both the amount of active TGF? and the expression of TGF? target genes were decreased by TLL1 knockout. It is known that TGF? negatively regulates hepatic differentiation. Conclusion: TLL1 appears to negatively regulate hepatic differentiation of human iPS cells by up-regulating TGF? signaling. Our findings will provide new insight into the function of TLL1 in human liver development.

SUBMITTER: Kiso A

PROVIDER: S-EPMC6996343 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Tolloid-Like 1 Negatively Regulates Hepatic Differentiation of Human Induced Pluripotent Stem Cells Through Transforming Growth Factor Beta Signaling.

Kiso Ayumi A Toba Yukiko Y Tsutsumi Susumu S Deguchi Sayaka S Igai Keisuke K Koshino Saki S Tanaka Yasuhito Y Takayama Kazuo K Mizuguchi Hiroyuki H

Hepatology communications 20200102 2

Single nucleotide polymorphisms in Tolloid-like 1 (TLL1) and the expression of <i>TLL1</i> are known to be closely related to hepatocarcinogenesis after hepatitis C virus elimination or liver fibrosis in patients with nonalcoholic fatty liver disease. TLL1 is a type of matrix metalloprotease and has two isoforms in humans, with the short isoform showing higher activity. However, the functional role of TLL1 in human liver development is unknown. Here, we attempted to elucidate the function of hum ...[more]

PMID: 32025609

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Project description:BackgroundThe 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.MethodsiPSCs 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.ResultsWhen 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.ConclusionWe 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.

Dataset Information

Tolloid-Like 1 Negatively Regulates Hepatic Differentiation of Human Induced Pluripotent Stem Cells Through Transforming Growth Factor Beta Signaling.

Publications

Tolloid-Like 1 Negatively Regulates Hepatic Differentiation of Human Induced Pluripotent Stem Cells Through Transforming Growth Factor Beta Signaling.

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