Evaluation of different media compositions promoting hepatocyte differentiation in the canine liver organoid model
Vojtech Gabriel,
Addison Lincoln,
Christopher Zdyrski,
Abigail Ralston,
Hannah Wickham,
Sydney Honold,
Basant H. Ahmed,
Karel Paukner,
Ryan Feauto,
Maria M. Merodio,
Pablo Piñeyro,
David Meyerholz,
Karin Allenspach,
Jonathan P. Mochel
Affiliations
Vojtech Gabriel
SMART Lab, Department of Biomedical Sciences, Iowa State University, Ames, IA, USA; Corresponding author.
Addison Lincoln
SMART Lab, Department of Biomedical Sciences, Iowa State University, Ames, IA, USA
Christopher Zdyrski
SMART Lab, Department of Biomedical Sciences, Iowa State University, Ames, IA, USA; 3D Health Solutions Inc., Ames, IA, USA; Precision One Health Initiative, Department of Pathology, University of Georgia College of Veterinary Medicine, 30602, Athens, GA, USA
Abigail Ralston
3D Health Solutions Inc., Ames, IA, USA
Hannah Wickham
SMART Lab, Department of Biomedical Sciences, Iowa State University, Ames, IA, USA
Sydney Honold
SMART Lab, Department of Biomedical Sciences, Iowa State University, Ames, IA, USA
Basant H. Ahmed
SMART Lab, Department of Biomedical Sciences, Iowa State University, Ames, IA, USA
Karel Paukner
Laboratory for Atherosclerosis Research, Institute for Clinical and Experimental Medicine, Prague, CZ, Czech Republic
Ryan Feauto
SMART Lab, Department of Biomedical Sciences, Iowa State University, Ames, IA, USA
Maria M. Merodio
SMART Lab, Department of Biomedical Sciences, Iowa State University, Ames, IA, USA
Pablo Piñeyro
Veterinary Diagnostic Laboratory, Iowa State University, Ames, IA, USA
David Meyerholz
Department of Pathology, University of Iowa, Iowa City, IA, USA
Karin Allenspach
SMART Lab, Department of Biomedical Sciences, Iowa State University, Ames, IA, USA; 3D Health Solutions Inc., Ames, IA, USA; Precision One Health Initiative, Department of Pathology, University of Georgia College of Veterinary Medicine, 30602, Athens, GA, USA; Corresponding author. Precision One Health Initiative, Department of Pathology, University of Georgia College of Veterinary Medicine, 30602, Athens, GA, USA.
Jonathan P. Mochel
3D Health Solutions Inc., Ames, IA, USA; Precision One Health Initiative, Department of Pathology, University of Georgia College of Veterinary Medicine, 30602, Athens, GA, USA; Corresponding author. Precision One Health Initiative, Department of Pathology, University of Georgia College of Veterinary Medicine, 30602, Athens, GA, USA.
Organoids are 3-dimensional (3D) self-assembled structures capable of replicating the microanatomy and physiology of the epithelial components of their organ of origin. Adult stem cell (ASC) derived organoids from the liver have previously been shown to differentiate into primarily mature cholangiocytes, and their partial differentiation into functional hepatocytes can be promoted using specific media compositions. While full morphological differentiation of mature hepatocytes from ASCs has not yet been reported for any species, the functional differentiation can be approximated using various media compositions.Six differentiation media formulations from published studies on hepatic organoids were used for the differentiation protocol. Target species for these protocols were humans, mice, cats, and dogs, and encompassed various combinations and concentrations of four major hepatocyte media components: Bone morphogenetic protein 7 (BMP7), Fibroblast Growth Factor 19 (FGF19), Dexamethasone (Dex), and Gamma-Secretase Inhibitor IX (DAPT). Additionally, removing R-spondin from basic organoid media has previously been shown to drive the differentiation of ASC into mature hepatocytes. Differentiation media (N = 20) were designed to encompass combinations of the four major hepatocyte media components. The preferred differentiation of ASC-derived organoids from liver tissue into mature hepatocytes over cholangiocytes was confirmed by albumin production in the culture supernatant.Out of the twenty media compositions tested, six media resulted in the production of the highest amounts of albumin in the supernatant of the organoids. The cell lines cultured using these six media were further characterized via histological staining, transmission electron microscopy, RNA in situ hybridization, analysis of gene expression patterns, immunofluorescence, and label-free proteomics. The results indicate that preferential hepatocyte maturation from canine ADC-derived organoids from liver tissue is mainly driven by Dexamethasone and DAPT components. FGF19 did not enhance organoid differentiation but improved cell culture survival. Furthermore, we confirm that removing R-spondin from the media is crucial for establishing mature hepatic organoid cultures.
