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Towards a defined ECM and small molecule based monolayer culture system for the expansion of mouse and human intestinal stem cells
Name
nihms918854.pdf
Description
Accepted version
Size
2.24 MB
Format
Adobe PDF
Checksum (MD5)
50454ac26cf13d7d6162d858ba1f843b
Author(s)
Tong, Zhixiang
Martyn, Keir
Yang, Andy
Yin, Xiaolei
Mead, Benjamin E
Joshi, Nitin
Sherman, Nicholas E
Langer, Robert S
Karp, Jeffrey M
Date Issued
2018
Journal
Biomaterials
Publisher
Elsevier BV
Version
Author's final manuscript
Abstract
© 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).
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Creative Commons Attribution-NonCommercial-NoDerivs License
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DOI of Published Version
10.1016/J.BIOMATERIALS.2017.10.038
