Publications
Browse peer-reviewed literature, posters, webinars, blog articles, and more showing how we and others are using RepliGut Systems to support discovery.
2022
Gomez-Martinez, Ismael; Bliton, R. Jarrett; Breau, Keith A.; Czerwinski, Michael J.; Williamson, Ian A.; Wen, Jia; Rawls, John F.; Magness, Scott T.
A Planar Culture Model of Human Absorptive Enterocytes Reveals Metformin Increases Fatty Acid Oxidation and Export Journal Article
In: Cellular and Molecular Gastroenterology and Hepatology, vol. 14, no. 2, pp. 409–434, 2022, ISSN: 2352-345X.
Abstract | Links | BibTeX | Tags: Absorptive Enterocyte Monolayers, Caco-2 Cells, Diabetes Mellitus, Drug Screening, Enterocytes, Fatty Acid Oxidation, Fatty Acids, Humans, Hypoglycemic Agents, intestinal stem cells, Metformin, Type 2
@article{gomez-martinez_planar_2022,
title = {A Planar Culture Model of Human Absorptive Enterocytes Reveals Metformin Increases Fatty Acid Oxidation and Export},
author = {Ismael Gomez-Martinez and R. Jarrett Bliton and Keith A. Breau and Michael J. Czerwinski and Ian A. Williamson and Jia Wen and John F. Rawls and Scott T. Magness},
doi = {10.1016/j.jcmgh.2022.04.009},
issn = {2352-345X},
year = {2022},
date = {2022-01-01},
urldate = {2022-01-01},
journal = {Cellular and Molecular Gastroenterology and Hepatology},
volume = {14},
number = {2},
pages = {409–434},
abstract = {BACKGROUND & AIMS: Fatty acid oxidation by absorptive enterocytes has been linked to the pathophysiology of type 2 diabetes, obesity, and dyslipidemia. Caco-2 and organoids have been used to study dietary lipid-handling processes including fatty acid oxidation, but are limited in physiological relevance or preclude simultaneous apical and basal access. Here, we developed a high-throughput planar human absorptive enterocyte monolayer system for investigating lipid handling, and then evaluated the role of fatty acid oxidation in fatty acid export, using etomoxir, C75, and the antidiabetic drug metformin.
METHODS: Single-cell RNA-sequencing, transcriptomics, and lineage trajectory was performed on primary human jejunum. In vivo absorptive enterocyte maturational states informed conditions used to differentiate human intestinal stem cells (ISCs) that mimic in vivo absorptive enterocyte maturation. The system was scaled for high-throughput drug screening. Fatty acid oxidation was modulated pharmacologically and BODIPY (Thermo Fisher Scientific, Waltham, MA) (B)-labeled fatty acids were used to evaluate fatty acid handling via fluorescence and thin-layer chromatography.
RESULTS: Single-cell RNA-sequencing shows increasing expression of lipid-handling genes as absorptive enterocytes mature. Culture conditions promote ISC differentiation into confluent absorptive enterocyte monolayers. Fatty acid-handling gene expression mimics in vivo maturational states. The fatty acid oxidation inhibitor etomoxir decreased apical-to-basolateral export of medium-chain B-C12 and long-chain B-C16 fatty acids, whereas the CPT1 agonist C75 and the antidiabetic drug metformin increased apical-to-basolateral export. Short-chain B-C5 was unaffected by fatty acid oxidation inhibition and diffused through absorptive enterocytes.
CONCLUSIONS: Primary human ISCs in culture undergo programmed maturation. Absorptive enterocyte monolayers show in vivo maturational states and lipid-handling gene expression profiles. Absorptive enterocytes create strong epithelial barriers in 96-Transwell format. Fatty acid export is proportional to fatty acid oxidation. Metformin enhances fatty acid oxidation and increases basolateral fatty acid export, supporting an intestine-specific role.},
keywords = {Absorptive Enterocyte Monolayers, Caco-2 Cells, Diabetes Mellitus, Drug Screening, Enterocytes, Fatty Acid Oxidation, Fatty Acids, Humans, Hypoglycemic Agents, intestinal stem cells, Metformin, Type 2},
pubstate = {published},
tppubtype = {article}
}
BACKGROUND & AIMS: Fatty acid oxidation by absorptive enterocytes has been linked to the pathophysiology of type 2 diabetes, obesity, and dyslipidemia. Caco-2 and organoids have been used to study dietary lipid-handling processes including fatty acid oxidation, but are limited in physiological relevance or preclude simultaneous apical and basal access. Here, we developed a high-throughput planar human absorptive enterocyte monolayer system for investigating lipid handling, and then evaluated the role of fatty acid oxidation in fatty acid export, using etomoxir, C75, and the antidiabetic drug metformin.
METHODS: Single-cell RNA-sequencing, transcriptomics, and lineage trajectory was performed on primary human jejunum. In vivo absorptive enterocyte maturational states informed conditions used to differentiate human intestinal stem cells (ISCs) that mimic in vivo absorptive enterocyte maturation. The system was scaled for high-throughput drug screening. Fatty acid oxidation was modulated pharmacologically and BODIPY (Thermo Fisher Scientific, Waltham, MA) (B)-labeled fatty acids were used to evaluate fatty acid handling via fluorescence and thin-layer chromatography.
RESULTS: Single-cell RNA-sequencing shows increasing expression of lipid-handling genes as absorptive enterocytes mature. Culture conditions promote ISC differentiation into confluent absorptive enterocyte monolayers. Fatty acid-handling gene expression mimics in vivo maturational states. The fatty acid oxidation inhibitor etomoxir decreased apical-to-basolateral export of medium-chain B-C12 and long-chain B-C16 fatty acids, whereas the CPT1 agonist C75 and the antidiabetic drug metformin increased apical-to-basolateral export. Short-chain B-C5 was unaffected by fatty acid oxidation inhibition and diffused through absorptive enterocytes.
CONCLUSIONS: Primary human ISCs in culture undergo programmed maturation. Absorptive enterocyte monolayers show in vivo maturational states and lipid-handling gene expression profiles. Absorptive enterocytes create strong epithelial barriers in 96-Transwell format. Fatty acid export is proportional to fatty acid oxidation. Metformin enhances fatty acid oxidation and increases basolateral fatty acid export, supporting an intestine-specific role.
METHODS: Single-cell RNA-sequencing, transcriptomics, and lineage trajectory was performed on primary human jejunum. In vivo absorptive enterocyte maturational states informed conditions used to differentiate human intestinal stem cells (ISCs) that mimic in vivo absorptive enterocyte maturation. The system was scaled for high-throughput drug screening. Fatty acid oxidation was modulated pharmacologically and BODIPY (Thermo Fisher Scientific, Waltham, MA) (B)-labeled fatty acids were used to evaluate fatty acid handling via fluorescence and thin-layer chromatography.
RESULTS: Single-cell RNA-sequencing shows increasing expression of lipid-handling genes as absorptive enterocytes mature. Culture conditions promote ISC differentiation into confluent absorptive enterocyte monolayers. Fatty acid-handling gene expression mimics in vivo maturational states. The fatty acid oxidation inhibitor etomoxir decreased apical-to-basolateral export of medium-chain B-C12 and long-chain B-C16 fatty acids, whereas the CPT1 agonist C75 and the antidiabetic drug metformin increased apical-to-basolateral export. Short-chain B-C5 was unaffected by fatty acid oxidation inhibition and diffused through absorptive enterocytes.
CONCLUSIONS: Primary human ISCs in culture undergo programmed maturation. Absorptive enterocyte monolayers show in vivo maturational states and lipid-handling gene expression profiles. Absorptive enterocytes create strong epithelial barriers in 96-Transwell format. Fatty acid export is proportional to fatty acid oxidation. Metformin enhances fatty acid oxidation and increases basolateral fatty acid export, supporting an intestine-specific role.
2019
Speer, Jennifer E.; Wang, Yuli; Fallon, John K.; Smith, Philip C.; Allbritton, Nancy L.
Evaluation of human primary intestinal monolayers for drug metabolizing capabilities Journal Article
In: J. Biol. Eng, vol. 13, pp. 82, 2019, ISSN: 1754-1611.
Abstract | Links | BibTeX | Tags: Absorptive Enterocyte Monolayers, Bioavailability, drug metabolising enzymes ({DME}), epithelial barrier, Fg, Intestinal Epithelial Cells, permeability
@article{speer_evaluation_2019,
title = {Evaluation of human primary intestinal monolayers for drug metabolizing capabilities},
author = {Jennifer E. Speer and Yuli Wang and John K. Fallon and Philip C. Smith and Nancy L. Allbritton},
url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6829970/},
doi = {10.1186/s13036-019-0212-1},
issn = {1754-1611},
year = {2019},
date = {2019-11-04},
urldate = {2019-11-04},
journal = {J. Biol. Eng},
volume = {13},
pages = {82},
abstract = {Background
The intestinal epithelium is a major site of drug metabolism in the human body, possessing enterocytes that house brush border enzymes and phase I and II drug metabolizing enzymes (DMEs). The enterocytes are supported by a porous extracellular matrix (ECM) that enables proper cell adhesion and function of brush border enzymes, such as alkaline phosphatase (ALP) and alanyl aminopeptidase (AAP), phase I DMEs that convert a parent drug to a more polar metabolite by introducing or unmasking a functional group, and phase II DMEs that form a covalent conjugate between a functional group on the parent compound or sequential metabolism of phase I metabolite. In our effort to develop an in vitro intestinal epithelium model, we investigate the impact of two previously described simple and customizable scaffolding systems, a gradient cross-linked scaffold and a conventional scaffold, on the ability of intestinal epithelial cells to produce drug metabolizing proteins as well as to metabolize exogenously added compounds. While the scaffolding systems possess a range of differences, they are most distinguished by their stiffness with the gradient cross-linked scaffold possessing a stiffness similar to that found in the in vivo intestine, while the conventional scaffold possesses a stiffness several orders of magnitude greater than that found in vivo.
Results
The monolayers on the gradient cross-linked scaffold expressed CYP3A4, UGTs 2B17, 1A1 and 1A10, and CES2 proteins at a level similar to that in fresh crypts/villi. The monolayers on the conventional scaffold expressed similar levels of CYP3A4 and UGTs 1A1 and 1A10 DMEs to that found in fresh crypts/villi but significantly decreased expression of UGT2B17 and CES2 proteins. The activity of CYP3A4 and UGTs 1A1 and 1A10 was inducible in cells on the gradient cross-linked scaffold when the cells were treated with known inducers, whereas the CYP3A4 and UGT activities were not inducible in cells grown on the conventional scaffold. Both monolayers demonstrate esterase activity but the activity measured in cells on the conventional scaffold could not be inhibited with a known CES2 inhibitor. Both monolayer culture systems displayed similar ALP and AAP brush border enzyme activity. When cells on the conventional scaffold were incubated with a yes-associated protein (YAP) inhibitor, CYP3A4 activity was greatly enhanced suggesting that mechano-transduction signaling can modulate drug metabolizing enzymes.
Conclusions
The use of a cross-linked hydrogel scaffold for expansion and differentiation of primary human intestinal stem cells dramatically impacts the induction of CYP3A4 and maintenance of UGT and CES drug metabolizing enzymes in vitro making this a superior substrate for enterocyte culture in DME studies. This work highlights the influence of mechanical properties of the culture substrate on protein expression and the activity of drug metabolizing enzymes as a critical factor in developing accurate assay protocols for pharmacokinetic studies using primary intestinal cells.
Graphical abstract},
keywords = {Absorptive Enterocyte Monolayers, Bioavailability, drug metabolising enzymes ({DME}), epithelial barrier, Fg, Intestinal Epithelial Cells, permeability},
pubstate = {published},
tppubtype = {article}
}
Background
The intestinal epithelium is a major site of drug metabolism in the human body, possessing enterocytes that house brush border enzymes and phase I and II drug metabolizing enzymes (DMEs). The enterocytes are supported by a porous extracellular matrix (ECM) that enables proper cell adhesion and function of brush border enzymes, such as alkaline phosphatase (ALP) and alanyl aminopeptidase (AAP), phase I DMEs that convert a parent drug to a more polar metabolite by introducing or unmasking a functional group, and phase II DMEs that form a covalent conjugate between a functional group on the parent compound or sequential metabolism of phase I metabolite. In our effort to develop an in vitro intestinal epithelium model, we investigate the impact of two previously described simple and customizable scaffolding systems, a gradient cross-linked scaffold and a conventional scaffold, on the ability of intestinal epithelial cells to produce drug metabolizing proteins as well as to metabolize exogenously added compounds. While the scaffolding systems possess a range of differences, they are most distinguished by their stiffness with the gradient cross-linked scaffold possessing a stiffness similar to that found in the in vivo intestine, while the conventional scaffold possesses a stiffness several orders of magnitude greater than that found in vivo.
Results
The monolayers on the gradient cross-linked scaffold expressed CYP3A4, UGTs 2B17, 1A1 and 1A10, and CES2 proteins at a level similar to that in fresh crypts/villi. The monolayers on the conventional scaffold expressed similar levels of CYP3A4 and UGTs 1A1 and 1A10 DMEs to that found in fresh crypts/villi but significantly decreased expression of UGT2B17 and CES2 proteins. The activity of CYP3A4 and UGTs 1A1 and 1A10 was inducible in cells on the gradient cross-linked scaffold when the cells were treated with known inducers, whereas the CYP3A4 and UGT activities were not inducible in cells grown on the conventional scaffold. Both monolayers demonstrate esterase activity but the activity measured in cells on the conventional scaffold could not be inhibited with a known CES2 inhibitor. Both monolayer culture systems displayed similar ALP and AAP brush border enzyme activity. When cells on the conventional scaffold were incubated with a yes-associated protein (YAP) inhibitor, CYP3A4 activity was greatly enhanced suggesting that mechano-transduction signaling can modulate drug metabolizing enzymes.
Conclusions
The use of a cross-linked hydrogel scaffold for expansion and differentiation of primary human intestinal stem cells dramatically impacts the induction of CYP3A4 and maintenance of UGT and CES drug metabolizing enzymes in vitro making this a superior substrate for enterocyte culture in DME studies. This work highlights the influence of mechanical properties of the culture substrate on protein expression and the activity of drug metabolizing enzymes as a critical factor in developing accurate assay protocols for pharmacokinetic studies using primary intestinal cells.
Graphical abstract
The intestinal epithelium is a major site of drug metabolism in the human body, possessing enterocytes that house brush border enzymes and phase I and II drug metabolizing enzymes (DMEs). The enterocytes are supported by a porous extracellular matrix (ECM) that enables proper cell adhesion and function of brush border enzymes, such as alkaline phosphatase (ALP) and alanyl aminopeptidase (AAP), phase I DMEs that convert a parent drug to a more polar metabolite by introducing or unmasking a functional group, and phase II DMEs that form a covalent conjugate between a functional group on the parent compound or sequential metabolism of phase I metabolite. In our effort to develop an in vitro intestinal epithelium model, we investigate the impact of two previously described simple and customizable scaffolding systems, a gradient cross-linked scaffold and a conventional scaffold, on the ability of intestinal epithelial cells to produce drug metabolizing proteins as well as to metabolize exogenously added compounds. While the scaffolding systems possess a range of differences, they are most distinguished by their stiffness with the gradient cross-linked scaffold possessing a stiffness similar to that found in the in vivo intestine, while the conventional scaffold possesses a stiffness several orders of magnitude greater than that found in vivo.
Results
The monolayers on the gradient cross-linked scaffold expressed CYP3A4, UGTs 2B17, 1A1 and 1A10, and CES2 proteins at a level similar to that in fresh crypts/villi. The monolayers on the conventional scaffold expressed similar levels of CYP3A4 and UGTs 1A1 and 1A10 DMEs to that found in fresh crypts/villi but significantly decreased expression of UGT2B17 and CES2 proteins. The activity of CYP3A4 and UGTs 1A1 and 1A10 was inducible in cells on the gradient cross-linked scaffold when the cells were treated with known inducers, whereas the CYP3A4 and UGT activities were not inducible in cells grown on the conventional scaffold. Both monolayers demonstrate esterase activity but the activity measured in cells on the conventional scaffold could not be inhibited with a known CES2 inhibitor. Both monolayer culture systems displayed similar ALP and AAP brush border enzyme activity. When cells on the conventional scaffold were incubated with a yes-associated protein (YAP) inhibitor, CYP3A4 activity was greatly enhanced suggesting that mechano-transduction signaling can modulate drug metabolizing enzymes.
Conclusions
The use of a cross-linked hydrogel scaffold for expansion and differentiation of primary human intestinal stem cells dramatically impacts the induction of CYP3A4 and maintenance of UGT and CES drug metabolizing enzymes in vitro making this a superior substrate for enterocyte culture in DME studies. This work highlights the influence of mechanical properties of the culture substrate on protein expression and the activity of drug metabolizing enzymes as a critical factor in developing accurate assay protocols for pharmacokinetic studies using primary intestinal cells.
Graphical abstract