Towards a defined ECM and small molecule based monolayer culture system for the expansion of mouse and human intestinal stem cells
© 2017 Elsevier Ltd Current ISC culture systems face significant challenges such as animal-derived or undefined matrix compositions, batch-to-batch variability (e.g. Matrigel-based organoid culture), and complexity of assaying cell aggregates such as organoids which renders the research and clinical...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier BV
2021
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| Online Access: | https://hdl.handle.net/1721.1/134688 |
| _version_ | 1826205258288726016 |
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| author | Tong, Zhixiang Martyn, Keir Yang, Andy Yin, Xiaolei Mead, Benjamin E Joshi, Nitin Sherman, Nicholas E Langer, Robert S Karp, Jeffrey M |
| author_facet | Tong, Zhixiang Martyn, Keir Yang, Andy Yin, Xiaolei Mead, Benjamin E Joshi, Nitin Sherman, Nicholas E Langer, Robert S Karp, Jeffrey M |
| author_sort | Tong, Zhixiang |
| collection | MIT |
| description | © 2017 Elsevier Ltd Current ISC culture systems face significant challenges such as animal-derived or undefined matrix compositions, batch-to-batch variability (e.g. Matrigel-based organoid culture), and complexity of assaying cell aggregates such as organoids which renders the research and clinical translation of ISCs challenging. Here, through screening for suitable ECM components, we report a defined, collagen based monolayer culture system that supports the growth of mouse and human intestinal epithelial cells (IECs) enriched for an Lgr5+ population comparable or higher to the levels found in a standard Matrigel-based organoid culture. The system, referred to as the Bolstering Lgr5 Transformational (BLT) Sandwich culture, comprises a collagen IV-coated porous substrate and a collagen I gel overlay which sandwich an IEC monolayer in between. The distinct collagen cues synergistically regulate IEC attachment, proliferation, and Lgr5 expression through maximizing the engagement of distinct cell surface adhesion receptors (i.e. integrin α2β1, integrin β4) and cell polarity. Further, we apply our BLT Sandwich system to identify that the addition of a bone morphogenetic protein (BMP) receptor inhibitor (LDN-193189) improves the expansion of Lgr5-GFP+ cells from mouse small intestinal crypts by nearly 2.5-fold. Notably, the BLT Sandwich culture is capable of expanding human-derived IECs with higher LGR5 mRNA levels than conventional Matrigel culture, providing superior expansion of human LGR5+ ISCs. Considering the key roles Lgr5+ ISCs play in intestinal epithelial homeostasis and regeneration, we envision that our BLT Sandwich culture system holds great potential for understanding and manipulating ISC biology in vitro (e.g. for modeling ISC-mediated gut diseases) or for expanding a large number of ISCs for clinical utility (e.g. for stem cell therapy). |
| first_indexed | 2024-09-23T13:09:51Z |
| format | Article |
| id | mit-1721.1/134688 |
| institution | Massachusetts Institute of Technology |
| language | English |
| last_indexed | 2024-09-23T13:09:51Z |
| publishDate | 2021 |
| publisher | Elsevier BV |
| record_format | dspace |
| spelling | mit-1721.1/1346882021-10-28T03:55:53Z Towards a defined ECM and small molecule based monolayer culture system for the expansion of mouse and human intestinal stem cells Tong, Zhixiang Martyn, Keir Yang, Andy Yin, Xiaolei Mead, Benjamin E Joshi, Nitin Sherman, Nicholas E Langer, Robert S Karp, Jeffrey M © 2017 Elsevier Ltd Current ISC culture systems face significant challenges such as animal-derived or undefined matrix compositions, batch-to-batch variability (e.g. Matrigel-based organoid culture), and complexity of assaying cell aggregates such as organoids which renders the research and clinical translation of ISCs challenging. Here, through screening for suitable ECM components, we report a defined, collagen based monolayer culture system that supports the growth of mouse and human intestinal epithelial cells (IECs) enriched for an Lgr5+ population comparable or higher to the levels found in a standard Matrigel-based organoid culture. The system, referred to as the Bolstering Lgr5 Transformational (BLT) Sandwich culture, comprises a collagen IV-coated porous substrate and a collagen I gel overlay which sandwich an IEC monolayer in between. The distinct collagen cues synergistically regulate IEC attachment, proliferation, and Lgr5 expression through maximizing the engagement of distinct cell surface adhesion receptors (i.e. integrin α2β1, integrin β4) and cell polarity. Further, we apply our BLT Sandwich system to identify that the addition of a bone morphogenetic protein (BMP) receptor inhibitor (LDN-193189) improves the expansion of Lgr5-GFP+ cells from mouse small intestinal crypts by nearly 2.5-fold. Notably, the BLT Sandwich culture is capable of expanding human-derived IECs with higher LGR5 mRNA levels than conventional Matrigel culture, providing superior expansion of human LGR5+ ISCs. Considering the key roles Lgr5+ ISCs play in intestinal epithelial homeostasis and regeneration, we envision that our BLT Sandwich culture system holds great potential for understanding and manipulating ISC biology in vitro (e.g. for modeling ISC-mediated gut diseases) or for expanding a large number of ISCs for clinical utility (e.g. for stem cell therapy). 2021-10-27T20:08:40Z 2021-10-27T20:08:40Z 2018 2019-09-09T13:11:21Z Article http://purl.org/eprint/type/JournalArticle https://hdl.handle.net/1721.1/134688 en 10.1016/J.BIOMATERIALS.2017.10.038 Biomaterials Creative Commons Attribution-NonCommercial-NoDerivs License http://creativecommons.org/licenses/by-nc-nd/4.0/ application/pdf Elsevier BV PMC |
| spellingShingle | Tong, Zhixiang Martyn, Keir Yang, Andy Yin, Xiaolei Mead, Benjamin E Joshi, Nitin Sherman, Nicholas E Langer, Robert S Karp, Jeffrey M Towards a defined ECM and small molecule based monolayer culture system for the expansion of mouse and human intestinal stem cells |
| title | Towards a defined ECM and small molecule based monolayer culture system for the expansion of mouse and human intestinal stem cells |
| title_full | Towards a defined ECM and small molecule based monolayer culture system for the expansion of mouse and human intestinal stem cells |
| title_fullStr | Towards a defined ECM and small molecule based monolayer culture system for the expansion of mouse and human intestinal stem cells |
| title_full_unstemmed | Towards a defined ECM and small molecule based monolayer culture system for the expansion of mouse and human intestinal stem cells |
| title_short | Towards a defined ECM and small molecule based monolayer culture system for the expansion of mouse and human intestinal stem cells |
| title_sort | towards a defined ecm and small molecule based monolayer culture system for the expansion of mouse and human intestinal stem cells |
| url | https://hdl.handle.net/1721.1/134688 |
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