Intestinal organoids morphologically resemble intestinal tissues and are expected to be used in both regenerative medicine and drug development studies, including pharmacokinetic studies. However, the pharmacokinetic properties of these organoids remain poorly characterized. In this study, we aimed to generate pharmacokinetically functional intestinal organoids from human induced pluripotent stem (iPS) cells. Human iPS cells were induced to differentiate into the midgut and then seeded on EZSPHERE plates (AGC Techno Glass Inc., Shizuoka, Japan) to generate uniform spheroids, and the floating spheroids were subsequently differentiated into intestinal organoids using small-molecule compounds. Exposure to the small-molecule compounds potently increased the expression of intestinal markers and pharmacokinetic-related genes in the organoids, and the organoids also included various intestinal cells such as enterocytes, intestinal stem cells, goblet cells, enteroendocrine cells, Paneth cells, smooth muscle cells, and fibroblasts. Moreover, microvilli and tight junctions were observed in the organoids. Furthermore, we detected not only the expression of drug transporters but also efflux transport activity through ABCB1/MDR1 and the induction of the drug-metabolizing enzyme CYP3A4 by ligands of nuclear receptors. Our results demonstrated the successful generation of pharmacokinetically functional intestinal organoids from human iPS cells. Thus, these intestinal organoids could be used as a pharmacokinetic evaluation system in drug development studies.
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.
Background: In vitro blood-brain barrier (BBB) models using human induced pluripotent stem (iPS) cell-derived brain microvascular endothelial-like cells (iBMELCs) have been developed to predict the BBB permeability of drug candidates. For the differentiation of iBMELCs, Matrigel, which is a gelatinous protein mixture, is often used as a coating substrate. However, the components of Matrigel can vary among lots, as it is obtained from mouse sarcoma cells with the use of special technics and also contains various basement membranes. Therefore, fully defined substrates as substitutes for Matrigel are needed for a stable supply of iBMELCs with less variation among lots. Methods: iBMELCs were differentiated from human iPS cells on several matrices. The barrier integrity of iBMELCs was evaluated based on transendothelial electrical resistance (TEER) values and permeability of fluorescein isothiocyanate-dextran 4 kDa (FD4) and Lucifer yellow (LY). Characterization of iBMELCs was conducted by RT-qPCR and immunofluorescence analysis. Functions of efflux transporters were defined by intracellular accumulation of the substrates in the wells of multiwell plates. Results: iBMELCs differentiated on laminin 221 fragment (LN221F-iBMELCs) had higher TEER values and lower permeability of LY and FD4 as compared with iBMELCs differentiated on Matrigel (Matrigel-iBMELCs). Besides, the gene and protein expression levels of brain microvascular endothelial cells (BMEC)-related markers were similar between LN221F-iBMELCs and Matrigel-iBMELCs. Moreover, both Matrigel-and LN221F-iBMELCs had functions of P-glycoprotein and breast cancer resistance protein, which are essential efflux transporters for barrier functions of the BBB. Conclusion: The fully defined substrate LN221F presents as an optimal coating matrix for differentiation of iBMELCs. The LN221F-iBMELCs had more robust barrier function for a longer period than Matrigel-iBMELCs with characteristics of BMECs. This finding will contribute the establishment of an iBMELC supply system for pharmacokinetic and pathological models of the BBB.
Endothelial cells (ECs) and endothelial progenitor cells (EPCs) play crucial roles in maintaining vascular health and homeostasis. Both cell types have been used in regenerative therapy as well as in various in vitro models; however, the properties of primary human ECs and EPCs are dissimilar owing to differences in genetic backgrounds and sampling techniques. Human induced pluripotent stem cells (hiPSCs) are an alternative cell source of ECs and EPCs. However, owing to the low purity of differentiated cells from hiPSCs, purification via an antigen-antibody reaction, which damages the cells, is indispensable. Besides, owing to limited expandability, it is difficult to produce these cells in large numbers. Here we report the development of relatively simple differentiation and purification methods for hiPSC-derived EPCs (iEPCs). Furthermore, we discovered that a combination of three small molecules, that is, Y-27632 (a selective inhibitor of Rho-associated, coiled-coil containing protein kinase [ROCK]), A 83-01 (a receptor-like kinase inhibitor of transforming growth factor beta [TGF-β]), and CHIR-99021 (a selective inhibitor of glycogen synthase kinase-3β [GSK3β] that also activates Wnt), dramatically stimulated protein synthesis-related pathways and enhanced the proliferative capacity of iEPCs. These findings will help to establish a supply system of EPCs at an industrial scale.
These results demonstrated that the cephalic vein at the cubital fossa is a relatively safe venipuncture site because of its distance from the median nerve and brachial artery. When puncturing the cephalic vein is difficult because it is not visible, the median cubital vein at the cubital fossa may be selected for venipuncture due to its cross-sectional area and visibility; however, care is needed to avoid penetrating the vein because the median nerve and brachial artery are located underneath.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.
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