Hormonally and chemically defined expansion conditions for organoids of biliary tree Stem Cells

Wencheng Zhang; Yangyang Cui; Mengqi Lu; Mingyang Xu; Yuting Li; Haimeng Song; Yi Luo; Jinjia Song; Yong Yang; Xicheng Wang; Lijun Liao; Yunfang Wang; Lola Reid; Zhiying He

doi:10.1016/j.bioactmat.2024.08.010

Hormonally and chemically defined expansion conditions for organoids of biliary tree Stem Cells

Bioact Mater. 2024 Sep 10:41:672-695. doi: 10.1016/j.bioactmat.2024.08.010. eCollection 2024 Nov.

Authors

Wencheng Zhang^{1

2

3}, Yangyang Cui^{1

4}, Mengqi Lu^{1

4}, Mingyang Xu^{1

2

3}, Yuting Li^{1

2

3}, Haimeng Song^{1

2

3}, Yi Luo^{1

2

3}, Jinjia Song^{1

2

3}, Yong Yang⁵, Xicheng Wang^{1

2

3}, Lijun Liao⁶, Yunfang Wang⁷, Lola Reid⁸, Zhiying He^{1

2

3}

Affiliations

¹ Institute for Regenerative Medicine, Medical Innovation Center and State Key Laboratory of Cardiology, Shanghai East Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, 200123, China.
² Shanghai Engineering Research Center of Stem Cells Translational Medicine, Shanghai, 200335, China.
³ Shanghai Institute of Stem Cell Research and Clinical Translation, Shanghai, 200120, China.
⁴ Postgraduate Training Base of Shanghai East Hospital, Jinzhou Medical University, Jinzhou, Liaoning, 121001, China.
⁵ Department of General Surgery, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, 330006, China.
⁶ Department of Anesthesiology and Pain Management, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, 200123, China.
⁷ Hepatobiliary and Pancreatic Center, Medical Research Center, Beijing Tsinghua Changgung Hospital, Beijing, 102218, China.
⁸ Departments of Cell Biology and Physiology, Program in Molecular Biology and Biotechnology, UNC School of Medicine, Chapel Hill, NC, 27599, United States.

Abstract

Wholly defined ex vivo expansion conditions for biliary tree stem cell (BTSC) organoids were established, consisting of a defined proliferative medium (DPM) used in combination with soft hyaluronan hydrogels. The DPM consisted of commercially available Kubota's Medium (KM), to which a set of small molecules, particular paracrine signals, and heparan sulfate (HS) were added. The small molecules used were DNA methyltransferase inhibitor (RG108), TGF- β Type I receptor inhibitor (A83-01), adenylate cyclase activator (Forskolin), and L-type Ca²⁺ channel agonist (Bay K8644). A key paracrine signal proved to be R-spondin 1 (RSPO1), a secreted protein that activates Wnts. Soluble hyaluronans, 0.05 % sodium hyaluronate, were used with DPM to expand monolayer cultures. Expansion of organoids was achieved by using DPM in combination with embedding organoids in Matrigel that was replaced with a defined thiol-hyaluronan triggered with PEGDA to form a hydrogel with a rheology [G*] of less than 100 Pa. The combination is called the BTSC-Expansion-Glycogel-System (BEX-gel system) for expanding BTSCs as a monolayer or as organoids. The BTSC organoids were expanded more than 3000-fold ex vivo in the BEX-gel system within 70 days while maintaining phenotypic traits indicative of stem/progenitors. Stem-cell-patch grafting of expanded BTSC organoids was performed on the livers of Fah^-/- mice with tyrosinemia and resulted in the rescue of the mice and restoration of their normal liver functions. The BEX-gel system for BTSC organoid expansion provides a strategy to generate sufficient numbers of organoids for the therapeutic treatments of liver diseases.

Keywords: BTSC-Expansion-Glycogel (BEX-gel); Biliary tree stem cells (BTSCs); Defined Proliferative Medium (DPM); Organoids; Paracrine signals.