Issue 91, 2015

Optimizing Wnt-3a and R-spondin1 concentrations for stem cell renewal and differentiation in intestinal organoids using a gradient-forming microdevice

Abstract

Epithelial renewal in the colonic crypt is a tightly controlled process and essential for normal digestive function. Crypts contain both proliferative and differentiated cells with the balance orchestrated by the interplay of proliferation and differentiation signals. While critically important to homeostatic renewal, the threshold concentrations of factors such as Wnt-3a and R-spondin1 that promote stem cell renewal are unknown. A simple, linear gradient-generating device was used to screen a wide range of Wnt-3a and R-spondin1 concentrations for their impact on a large number of colon organoids (colonoids) to achieve statistically significant results while minimizing the amounts of costly reagents and primary tissue samples. Matrigel-embedded colonoids were tracked for the presence of stem/transit-amplifying cells, differentiated cells and overall colonoid size in response to varying concentrations of Wnt-3a and R-spondin1, presented singly and in combination. A Wnt-3a concentration of 60 ng mL−1 and R-spondin1 concentration of 88 ng mL−1 were identified as the critical concentrations required for stem-cell renewal and colonoid expansion. At these concentrations, the colonoids possessed similar viability, size and passage efficiency compared to colonoids cultured under standard growth concentrations. The gradient culture system enabled efficient determination of the minimal growth factor concentrations needed to produce a physiologically-relevant colonoid phenotype. Relative to traditional culture conditions, lower factor concentrations yielded the added benefit of a more morphologically appropriate colonoid possessing columnar cells surrounding a central lumen with active crypt-like bud formation.

Graphical abstract: Optimizing Wnt-3a and R-spondin1 concentrations for stem cell renewal and differentiation in intestinal organoids using a gradient-forming microdevice

Supplementary files

Article information

Article type
Paper
Submitted
27 Jul 2015
Accepted
26 Aug 2015
First published
27 Aug 2015

RSC Adv., 2015,5, 74881-74891

Optimizing Wnt-3a and R-spondin1 concentrations for stem cell renewal and differentiation in intestinal organoids using a gradient-forming microdevice

A. A. Ahmad, Y. Wang, C. E. Sims, S. T. Magness and N. L. Allbritton, RSC Adv., 2015, 5, 74881 DOI: 10.1039/C5RA14923A

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