Abstract
We have developed a method to bulk culture definitive endoderm cells generated from human iPSCs which can be stored and differentiated to hepatocytes. Human iPSC-derived definitive endoderm cells were sorted based on the expression of CXCR4. The sorted cells were able to proliferate for extended periods and can be cryopreserved. The definitive endoderm cells were subsequently utilized to generate functional hepatocytes expressing albumin and α-fetoprotein in different multiwell formats. This provides a method to reliably produce more consistent hepatocytes in greater quantities and has enabled the development of high-throughput screening strategies.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Palakkan AA, Hay DC, Anil Kumar PR, Kumary TV, Ross JA (2013) Liver tissue engineering and cell sources: issues and challenges. Liver Int 33(5):666–676. https://doi.org/10.1111/liv.12134
Palakkan AA, Nanda J, Ross JA (2017) Pluripotent stem cells to hepatocytes, the journey so far. Biomed Rep 6(4):367–373. https://doi.org/10.3892/br.2017.867
Pettinato G, Ramanathan R, Fisher RA, Mangino MJ, Zhang N, Wen X (2016) Scalable differentiation of human iPSCs in a multicellular spheroid-based 3D culture into hepatocyte-like cells through direct Wnt/β-catenin pathway inhibition. Sci Rep 6:32888. https://doi.org/10.1038/srep32888
Hay DC, Fletcher J, Payne C, Terrace JD, Gallagher RC, Snoeys J, Black JR, Wojtacha D, Samuel K, Hannoun Z, Pryde A, Filippi C, Currie IS, Forbes SJ, Ross JA, Newsome PN, Iredale JP (2008) Highly efficient differentiation of hESCs to functional hepatic endoderm requires ActivinA and Wnt3a signaling. Proc Natl Acad Sci U S A 105(34):12301–12306. https://doi.org/10.1073/pnas.0806522105
Liu J, Brzeszczynska J, Samuel K, Black J, Palakkan A, Anderson RA, Gallagher R, Ross JA (2015) Efficient episomal reprogramming of blood mononuclear cells and differentiation to hepatocytes with functional drug metabolism. Exp Cell Res 338(2):203–213. https://doi.org/10.1016/j.yexcr.2015.08.004
Cameron K, Tan R, Schmidt-Heck W, Campos G, Lyall MJ, Wang Y, Lucendo-Villarin B, Szkolnicka D, Bates N, Kimber SJ, Hengstler JG, Godoy P, Forbes SJ, Hay DC (2015) Recombinant laminins drive the differentiation and self-organization of hESC-derived hepatocytes. Stem Cell Reports 5(6):1250–1262. https://doi.org/10.1016/j.stemcr.2015.10.016
Acknowledgments
The study leading to these results has received support from the Innovative Medicines Initiative Joint Undertaking under (grant no. 115439), resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2007-2013) and EFPIA companies. This publication reflects only the author’s views and neither the IMI JU nor EFPIA nor the European Commission are liable for any use that may be made of the information contained therein.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Palakkan, A.A., Nanda, J., Ross, J.A. (2019). Human Induced Pluripotent Stem Cell-Derived Definitive Endoderm Bulk Culture and Hepatic Differentiation. In: Mandenius, CF., Ross, J. (eds) Cell-Based Assays Using iPSCs for Drug Development and Testing. Methods in Molecular Biology, vol 1994. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9477-9_4
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9477-9_4
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9476-2
Online ISBN: 978-1-4939-9477-9
eBook Packages: Springer Protocols