Publications
Browse peer-reviewed literature, posters, webinars, blog articles, and more showing how we and others are using RepliGut Systems to support discovery.
2022
Breau, Keith A.; Ok, Meryem T.; Gomez-Martinez, Ismael; Burclaff, Joseph; Kohn, Nathan P.; Magness, Scott T.
Efficient transgenesis and homology-directed gene targeting in monolayers of primary human small intestinal and colonic epithelial stem cells Journal Article
In: vol. 17, no. 6, pp. 1493–1506, 2022, ISSN: 2213-6711.
Abstract | Links | BibTeX | Tags: 2D monolayer cultures, electroporation, Gene Editing, Gene Targeting, human {ISC} marker, Humans, Intestine, microphysiological device, Organoids, planar crypt-microarray, Small, stem cells, transfection, transgenic
@article{breau_efficient_2022,
title = {Efficient transgenesis and homology-directed gene targeting in monolayers of primary human small intestinal and colonic epithelial stem cells},
author = {Keith A. Breau and Meryem T. Ok and Ismael Gomez-Martinez and Joseph Burclaff and Nathan P. Kohn and Scott T. Magness},
doi = {10.1016/j.stemcr.2022.04.005},
issn = {2213-6711},
year = {2022},
date = {2022-06-14},
urldate = {2022-06-14},
volume = {17},
number = {6},
pages = {1493–1506},
abstract = {Two-dimensional (2D) cultures of intestinal and colonic epithelium can be generated using human intestinal stem cells (hISCs) derived from primary tissue sources. These 2D cultures are emerging as attractive and versatile alternatives to three-dimensional organoid cultures; however, transgenesis and gene-editing approaches have not been developed for hISCs grown as 2D monolayers. Using 2D cultured hISCs we show that electroporation achieves up to 80% transfection in hISCs from six anatomical regions with around 64% survival and produces 0.15% transgenesis by PiggyBac transposase and 35% gene edited indels by electroporation of Cas9-ribonucleoprotein complexes at the OLFM4 locus. We create OLFM4-emGFP knock-in hISCs, validate the reporter on engineered 2D crypt devices, and develop complete workflows for high-throughput cloning and expansion of transgenic lines in 3-4 weeks. New findings demonstrate small hISCs expressing the highest OLFM4 levels exhibit the most organoid forming potential and show utility of the 2D crypt device to evaluate hISC function.},
keywords = {2D monolayer cultures, electroporation, Gene Editing, Gene Targeting, human {ISC} marker, Humans, Intestine, microphysiological device, Organoids, planar crypt-microarray, Small, stem cells, transfection, transgenic},
pubstate = {published},
tppubtype = {article}
}
Two-dimensional (2D) cultures of intestinal and colonic epithelium can be generated using human intestinal stem cells (hISCs) derived from primary tissue sources. These 2D cultures are emerging as attractive and versatile alternatives to three-dimensional organoid cultures; however, transgenesis and gene-editing approaches have not been developed for hISCs grown as 2D monolayers. Using 2D cultured hISCs we show that electroporation achieves up to 80% transfection in hISCs from six anatomical regions with around 64% survival and produces 0.15% transgenesis by PiggyBac transposase and 35% gene edited indels by electroporation of Cas9-ribonucleoprotein complexes at the OLFM4 locus. We create OLFM4-emGFP knock-in hISCs, validate the reporter on engineered 2D crypt devices, and develop complete workflows for high-throughput cloning and expansion of transgenic lines in 3-4 weeks. New findings demonstrate small hISCs expressing the highest OLFM4 levels exhibit the most organoid forming potential and show utility of the 2D crypt device to evaluate hISC function.
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.
2020
Wang, Yuli; Sims, Christopher E.; Allbritton, Nancy L.
Enterochromaffin Cell-Enriched Monolayer Platform for Assaying Serotonin Release from Human Primary Intestinal Cells Journal Article
In: vol. 92, no. 18, pp. 12330–12337, 2020, ISSN: 1520-6882.
Abstract | Links | BibTeX | Tags: Enterochromaffin Cells, Humans, Intestinal Mucosa, Serotonin
@article{wang_enterochromaffin_2020,
title = {Enterochromaffin Cell-Enriched Monolayer Platform for Assaying Serotonin Release from Human Primary Intestinal Cells},
author = {Yuli Wang and Christopher E. Sims and Nancy L. Allbritton},
doi = {10.1021/acs.analchem.0c02016},
issn = {1520-6882},
year = {2020},
date = {2020-09-15},
volume = {92},
number = {18},
pages = {12330–12337},
abstract = {Enteroendocrine (EE) cells within the intestinal epithelium produce a range of hormones that have key roles in modulating satiety and feeding behavior in humans. The regulation of hormone release from EE cells as a potential therapeutic strategy to treat metabolic disorders is highly sought after by the pharmaceutical industry. However, functional studies are limited by the scarcity of EE cells (or surrogates) in both in vivo and in vitro systems. Enterochromaffin (EC) cells are a subtype of EE cells that produce serotonin (5HT). Here, we explored simple strategies to enrich EC cells in in vitro monolayer systems derived from human primary intestinal stem cells. During differentiation of the monolayers, the EC cell lineage was significantly altered by both the culture method [air-liquid interface (ALI) vs submerged] and the presence of vasoactive intestinal peptide (VIP). Compared with traditional submerged cultures without VIP, VIP-assisted ALI culture significantly boosted the number of EC cells and their 5HT secretion by up to 430 and 390%, respectively. The method also increased the numbers of other subtypes of EE cells such as L cells. Additionally, this method generated monolayers with enhanced barrier integrity, so that directional (basal or apical) 5HT secretion was measurable. For all donor tissues, the enriched EC cells improved the signal-to-background ratio and reliability of 5HT release assays. The enhancement in the 5HT secretion behavior was consistent over time from a single donor, but significant variation in the amount of secreted 5HT was present among tissues derived from five different donors. To demonstrate the utility of the EC-enriched monolayer system, 13 types of pungent food ingredients were screened for their ability to stimulate 5HT secretion. Curcumin found in the spice turmeric derived from the Curcuma longa plant was found to be the most potent secretagogue. This EC-enriched cell monolayer platform can provide a valuable analytical tool for the high-throughput screening of nutrients and gut microbial components that alter the secretion of 5HT.},
keywords = {Enterochromaffin Cells, Humans, Intestinal Mucosa, Serotonin},
pubstate = {published},
tppubtype = {article}
}
Enteroendocrine (EE) cells within the intestinal epithelium produce a range of hormones that have key roles in modulating satiety and feeding behavior in humans. The regulation of hormone release from EE cells as a potential therapeutic strategy to treat metabolic disorders is highly sought after by the pharmaceutical industry. However, functional studies are limited by the scarcity of EE cells (or surrogates) in both in vivo and in vitro systems. Enterochromaffin (EC) cells are a subtype of EE cells that produce serotonin (5HT). Here, we explored simple strategies to enrich EC cells in in vitro monolayer systems derived from human primary intestinal stem cells. During differentiation of the monolayers, the EC cell lineage was significantly altered by both the culture method [air-liquid interface (ALI) vs submerged] and the presence of vasoactive intestinal peptide (VIP). Compared with traditional submerged cultures without VIP, VIP-assisted ALI culture significantly boosted the number of EC cells and their 5HT secretion by up to 430 and 390%, respectively. The method also increased the numbers of other subtypes of EE cells such as L cells. Additionally, this method generated monolayers with enhanced barrier integrity, so that directional (basal or apical) 5HT secretion was measurable. For all donor tissues, the enriched EC cells improved the signal-to-background ratio and reliability of 5HT release assays. The enhancement in the 5HT secretion behavior was consistent over time from a single donor, but significant variation in the amount of secreted 5HT was present among tissues derived from five different donors. To demonstrate the utility of the EC-enriched monolayer system, 13 types of pungent food ingredients were screened for their ability to stimulate 5HT secretion. Curcumin found in the spice turmeric derived from the Curcuma longa plant was found to be the most potent secretagogue. This EC-enriched cell monolayer platform can provide a valuable analytical tool for the high-throughput screening of nutrients and gut microbial components that alter the secretion of 5HT.
2019
Dutton, Johanna S.; Hinman, Samuel S.; Kim, Raehyun; Wang, Yuli; Allbritton, Nancy L.
Primary Cell-Derived Intestinal Models: Recapitulating Physiology Journal Article
In: Trends in Biotechnology, vol. 37, no. 7, pp. 744, 2019, (Publisher: NIH Public Access).
Abstract | Links | BibTeX | Tags: Biological, Cell Culture Techniques, Cells, Cultured, Humans, in vitro models, Intestine, Intestines, Models, monolayers, organ-on-chips, Organoids, stem cells, Tissue Engineering
@article{dutton_primary_2019,
title = {Primary Cell-Derived Intestinal Models: Recapitulating Physiology},
author = {Johanna S. Dutton and Samuel S. Hinman and Raehyun Kim and Yuli Wang and Nancy L. Allbritton},
url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6571163/},
doi = {10.1016/j.tibtech.2018.12.001},
year = {2019},
date = {2019-07-01},
journal = {Trends in Biotechnology},
volume = {37},
number = {7},
pages = {744},
abstract = {The development of physiologically relevant intestinal models fueled by breakthroughs in primary cell-culture methods, has enabled successful recapitulation of key features of intestinal physiology. These advances when paired with engineering methods, ...},
note = {Publisher: NIH Public Access},
keywords = {Biological, Cell Culture Techniques, Cells, Cultured, Humans, in vitro models, Intestine, Intestines, Models, monolayers, organ-on-chips, Organoids, stem cells, Tissue Engineering},
pubstate = {published},
tppubtype = {article}
}
The development of physiologically relevant intestinal models fueled by breakthroughs in primary cell-culture methods, has enabled successful recapitulation of key features of intestinal physiology. These advances when paired with engineering methods, ...
Wang, Y.; Kim, R.; Sims, C. E.; Allbritton, N. L.
Building a Thick Mucus Hydrogel Layer to Improve the Physiological Relevance of In Vitro Primary Colonic Epithelial Models Journal Article
In: Cellular and Molecular Gastroenterology and Hepatology, vol. 8, no. 4, pp. 653–655.e5, 2019, ISSN: 2352-345X.
Links | BibTeX | Tags: Cell Culture Techniques, Colon, Humans, Hydrogels, Intestinal Mucosa, Mucus
@article{wang_building_2019,
title = {Building a Thick Mucus Hydrogel Layer to Improve the Physiological Relevance of In Vitro Primary Colonic Epithelial Models},
author = {Y. Wang and R. Kim and C. E. Sims and N. L. Allbritton},
doi = {10.1016/j.jcmgh.2019.07.009},
issn = {2352-345X},
year = {2019},
date = {2019-01-01},
journal = {Cellular and Molecular Gastroenterology and Hepatology},
volume = {8},
number = {4},
pages = {653–655.e5},
keywords = {Cell Culture Techniques, Colon, Humans, Hydrogels, Intestinal Mucosa, Mucus},
pubstate = {published},
tppubtype = {article}
}
2018
Gunasekara, Dulan B.; Speer, Jennifer; Wang, Yuli; Nguyen, Daniel L.; Reed, Mark I.; Smiddy, Nicole M.; Parker, Joel S.; Fallon, John K.; Smith, Philip C.; Sims, Christopher E.; Magness, Scott T.; Allbritton, Nancy L.
A monolayer of primary colonic epithelium generated on a scaffold with a gradient of stiffness for drug transport studies Journal Article
In: vol. 90, no. 22, pp. 13331, 2018, (Publisher: NIH Public Access).
Abstract | Links | BibTeX | Tags: Atenolol, Biological Transport, Caco-2 Cells, Colon, Epithelium, Humans, Propranolol, Riboflavin, Tissue Engineering
@article{gunasekara_monolayer_2018,
title = {A monolayer of primary colonic epithelium generated on a scaffold with a gradient of stiffness for drug transport studies},
author = {Dulan B. Gunasekara and Jennifer Speer and Yuli Wang and Daniel L. Nguyen and Mark I. Reed and Nicole M. Smiddy and Joel S. Parker and John K. Fallon and Philip C. Smith and Christopher E. Sims and Scott T. Magness and Nancy L. Allbritton},
url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6339567/},
doi = {10.1021/acs.analchem.8b02845},
year = {2018},
date = {2018-11-11},
urldate = {2018-11-11},
volume = {90},
number = {22},
pages = {13331},
abstract = {Animal models are frequently used for in vitro physiologic and drug transport studies of the colon, but there exists significant pressure to improve assay throughput as well as achieve tighter control of experimental variables than can be achieved with ...},
note = {Publisher: NIH Public Access},
keywords = {Atenolol, Biological Transport, Caco-2 Cells, Colon, Epithelium, Humans, Propranolol, Riboflavin, Tissue Engineering},
pubstate = {published},
tppubtype = {article}
}
Animal models are frequently used for in vitro physiologic and drug transport studies of the colon, but there exists significant pressure to improve assay throughput as well as achieve tighter control of experimental variables than can be achieved with ...