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.
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, ...
2017
Wang, Yuli; DiSalvo, Matthew; Gunasekara, Dulan B.; Dutton, Johanna; Proctor, Angela; Lebhar, Michael S.; Williamson, Ian A.; Speer, Jennifer; Howard, Riley L.; Smiddy, Nicole M.; Bultman, Scott J.; Sims, Christopher E.; Magness, Scott T.; Allbritton, Nancy L.
Self-renewing Monolayer of Primary Colonic or Rectal Epithelial Cells Journal Article
In: vol. 4, no. 1, pp. 165–182.e7, 2017, ISSN: 2352-345X.
Abstract | Links | BibTeX | Tags: Colonic Epithelial Cells, Compound Screening, Monolayer, Organoids
@article{wang_self-renewing_2017,
title = {Self-renewing Monolayer of Primary Colonic or Rectal Epithelial Cells},
author = {Yuli Wang and Matthew DiSalvo and Dulan B. Gunasekara and Johanna Dutton and Angela Proctor and Michael S. Lebhar and Ian A. Williamson and Jennifer Speer and Riley L. Howard and Nicole M. Smiddy and Scott J. Bultman and Christopher E. Sims and Scott T. Magness and Nancy L. Allbritton},
url = {https://www.sciencedirect.com/science/article/pii/S2352345X17300462},
doi = {10.1016/j.jcmgh.2017.02.011},
issn = {2352-345X},
year = {2017},
date = {2017-07-01},
urldate = {2023-02-20},
volume = {4},
number = {1},
pages = {165–182.e7},
abstract = {Background & Aims
Three-dimensional organoid culture has fundamentally changed the in vitro study of intestinal biology enabling novel assays; however, its use is limited because of an inaccessible luminal compartment and challenges to data gathering in a three-dimensional hydrogel matrix. Long-lived, self-renewing 2-dimensional (2-D) tissue cultured from primary colon cells has not been accomplished.
Methods
The surface matrix and chemical factors that sustain 2-D mouse colonic and human rectal epithelial cell monolayers with cell repertoires comparable to that in vivo were identified.
Results
The monolayers formed organoids or colonoids when placed in standard Matrigel culture. As with the colonoids, the monolayers exhibited compartmentalization of proliferative and differentiated cells, with proliferative cells located near the peripheral edges of growing monolayers and differentiated cells predominated in the central regions. Screening of 77 dietary compounds and metabolites revealed altered proliferation or differentiation of the murine colonic epithelium. When exposed to a subset of the compound library, murine organoids exhibited similar responses to that of the monolayer but with differences that were likely attributable to the inaccessible organoid lumen. The response of the human primary epithelium to a compound subset was distinct from that of both the murine primary epithelium and human tumor cells.
Conclusions
This study demonstrates that a self-renewing 2-D murine and human monolayer derived from primary cells can serve as a physiologically relevant assay system for study of stem cell renewal and differentiation and for compound screening. The platform holds transformative potential for personalized and precision medicine and can be applied to emerging areas of disease modeling and microbiome studies.},
keywords = {Colonic Epithelial Cells, Compound Screening, Monolayer, Organoids},
pubstate = {published},
tppubtype = {article}
}
Background & Aims
Three-dimensional organoid culture has fundamentally changed the in vitro study of intestinal biology enabling novel assays; however, its use is limited because of an inaccessible luminal compartment and challenges to data gathering in a three-dimensional hydrogel matrix. Long-lived, self-renewing 2-dimensional (2-D) tissue cultured from primary colon cells has not been accomplished.
Methods
The surface matrix and chemical factors that sustain 2-D mouse colonic and human rectal epithelial cell monolayers with cell repertoires comparable to that in vivo were identified.
Results
The monolayers formed organoids or colonoids when placed in standard Matrigel culture. As with the colonoids, the monolayers exhibited compartmentalization of proliferative and differentiated cells, with proliferative cells located near the peripheral edges of growing monolayers and differentiated cells predominated in the central regions. Screening of 77 dietary compounds and metabolites revealed altered proliferation or differentiation of the murine colonic epithelium. When exposed to a subset of the compound library, murine organoids exhibited similar responses to that of the monolayer but with differences that were likely attributable to the inaccessible organoid lumen. The response of the human primary epithelium to a compound subset was distinct from that of both the murine primary epithelium and human tumor cells.
Conclusions
This study demonstrates that a self-renewing 2-D murine and human monolayer derived from primary cells can serve as a physiologically relevant assay system for study of stem cell renewal and differentiation and for compound screening. The platform holds transformative potential for personalized and precision medicine and can be applied to emerging areas of disease modeling and microbiome studies.
Three-dimensional organoid culture has fundamentally changed the in vitro study of intestinal biology enabling novel assays; however, its use is limited because of an inaccessible luminal compartment and challenges to data gathering in a three-dimensional hydrogel matrix. Long-lived, self-renewing 2-dimensional (2-D) tissue cultured from primary colon cells has not been accomplished.
Methods
The surface matrix and chemical factors that sustain 2-D mouse colonic and human rectal epithelial cell monolayers with cell repertoires comparable to that in vivo were identified.
Results
The monolayers formed organoids or colonoids when placed in standard Matrigel culture. As with the colonoids, the monolayers exhibited compartmentalization of proliferative and differentiated cells, with proliferative cells located near the peripheral edges of growing monolayers and differentiated cells predominated in the central regions. Screening of 77 dietary compounds and metabolites revealed altered proliferation or differentiation of the murine colonic epithelium. When exposed to a subset of the compound library, murine organoids exhibited similar responses to that of the monolayer but with differences that were likely attributable to the inaccessible organoid lumen. The response of the human primary epithelium to a compound subset was distinct from that of both the murine primary epithelium and human tumor cells.
Conclusions
This study demonstrates that a self-renewing 2-D murine and human monolayer derived from primary cells can serve as a physiologically relevant assay system for study of stem cell renewal and differentiation and for compound screening. The platform holds transformative potential for personalized and precision medicine and can be applied to emerging areas of disease modeling and microbiome studies.