Lipid-mediated Wnt protein stabilization enables serum-free culture of human organ stem cells

Nesrin Tüysüz,Eric Braakman,Lijian Hui,Robert Vries,Luis J Cruz,Stieneke Van Den Brink,Monique M A Verstegen,Harry Begthel,Luc J W Van Der Laan,Hans Clevers,Enrico Mastrobattista,Jan J Cornelissen,Louis Van Bloois,Jeroen De Jonge,Derk Ten Berge

doi:10.1038/ncomms14578

Abstract

Wnt signalling proteins are essential for culture of human organ stem cells in organoids, but most Wnt protein formulations are poorly active in serum-free media. Here we show that purified Wnt3a protein is ineffective because it rapidly loses activity in culture media due to its hydrophobic nature, and its solubilization requires a detergent, CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate), that interferes with stem cell self-renewal. By stabilizing the Wnt3a protein using phospholipids and cholesterol as carriers, we address both problems: Wnt activity remains stable in serum-free media, while non-toxic carriers allow the use of high Wnt concentrations. Stabilized Wnt3a supports strongly increased self-renewal of organ and embryonic stem cells and the serum-free establishment of human organoids from healthy and diseased intestine and liver. Moreover, the lipophilicity of Wnt3a protein greatly facilitates its purification. Our findings remove a major obstacle impeding clinical applications of adult stem cells and offer advantages for all cell culture uses of Wnt3a protein.

Highlights

Wnt signalling proteins are essential for culture of human organ stem cells in organoids, but most Wnt protein formulations are poorly active in serum-free media
We found that purified Wnt3a protein performed poorly in the establishment and propagation of human organ stem cell cultures in serum-free conditions
On dilution in cell culture medium, the CHAPS concentration drops below the level required to maintain Wnt activity, and the protein rapidly loses activity[11]

Summary

Introduction

Wnt signalling proteins are essential for culture of human organ stem cells in organoids, but most Wnt protein formulations are poorly active in serum-free media. We show that purified Wnt3a protein is ineffective because it rapidly loses activity in culture media due to its hydrophobic nature, and its solubilization requires a detergent, CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate), that interferes with stem cell self-renewal. Stabilized Wnt3a supports strongly increased self-renewal of organ and embryonic stem cells and the serum-free establishment of human organoids from healthy and diseased intestine and liver. On dilution in cell culture media, the detergent concentration drops below the level required to maintain Wnt solubility This leads to rapid aggregation and loss of activity of the protein, in particular in the absence of serum[11]. We found that purified Wnt3a protein performed poorly in the establishment and propagation of human organ stem cell cultures in serum-free conditions. Our findings constitute an important step towards the use of human organ stem cells in clinical scenarios

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Conclusion