Towards a defined ECM and small molecule based monolayer culture system for the expansion of mouse and human intestinal stem cells

Abstract

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).

Milk osteopontin promotes brain development by up‐regulating osteopontin in the brain in early life

“…HIECs were isolated from normal human small intestine (jejunum and ileum) ranging from 17 to 19 wk gestation (65). Because of rapid growth and high demand for nutrients and functional proteins in early human infancy (66), neonatal absorption of OPN is likely mediated by integrins, known OPN receptors (2), which are expressed in human intestinal epithelial cells (HIEC and Caco-2) (67, 68) and mouse intestine (69,70).…”

A fully automated high-throughput workflow for 3D-based chemical screening in human midbrain organoids

“…The established protocols tend not to focus on scalable, homogeneous organoids with quantitatively predictable morphology, cellular composition, and local cell organization. Obligatory extensive manual handling often including cumbersome matrix embedding steps render them challenging for scale-up ( Tong et al, 2018 ). Furthermore, common analysis methods (e.g.…”

Human Microphysiological Models of Intestinal Tissue and Gut Microbiome

“…To solve these issues, several authors have enzymatically lysed organoids into single cells and seeded them in monolayers onto 2D substrates made of collagen or Matrigel (Wang et al, 2017a;Thorne et al, 2018). Some went further with the use of Transwell plates coated with collagen or Matrigel matrix where dissociated cells from organoids are seeded (Tong et al, 2018). These monolayers demonstrated apical-basal organization, gave access for chemical or viral treatment of either cell side differentially (Foulke-Abel et al, 2014;Ettayebi et al, 2016).…”