Abstract
During the process of differentiation from induced pluripotent stem cells (iPSCs) to hepatocytes it is crucial to monitor the levels of cellular maturation. We present a new method to evaluate the stage of differentiation based on the monitoring of the ratio between two plasma proteins typically secreted by hepatocytes, that is, albumin and alpha-fetoprotein. This ratio is particularly useful for the direct comparison of cells grown in different conditions, avoiding typical processes of standardization for the cell number (i.e., variation of cell quantity due to the use of different seeding densities and different growth vessels/supports or difficulties in establishing the effective cell viability due to the use of bioreactors or other 3D devices). Our analysis is performed via liquid chromatography–tandem mass spectrometry which allows a precise, selective, and reproducible quantitation of low-abundance proteins.
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References
Sullivan GJ, Hay DC, Park IH et al (2010) Generation of functional human hepatic endoderm from human induced pluripotent stem cells. Hepatology 51:329–335
Guigoz Y, Werffeli P, Favre D et al (1987) Aggregate cultures of foetal rat liver cells: development and maintenance of liver gene expression. Biol Cell 60:163–171
Kurosawa H, Yuminamochi E, Yasuda R, Amano Y (2003) Morphology and albumin secretion of adult rat hepatocytes cultured on a hydrophobic porous expanded polytetrafluoroethylene membrane. J Biosci Bioeng 95:59–64
Buyl K, De Kock J, Bolleyn J et al (2015) Measurement of albumin secretion as functionality test in primary hepatocyte cultures. In: Vinken M, Rogiers V (eds) Protocols in in vitro hepatocyte research. Methods in molecular biology (methods and protocols), vol 1250. Humana Press, New York
Yu LR, Zeng R, Shao XX et al (2000) Identification of differentially expressed proteins between human hepatoma and normal liver cell lines by two-dimensional electrophoresis and liquid chromatography-ion trap mass spectrometry. Electrophoresis 21:3058–3068
Pressey JG, Pressey CS, Robinson G et al (2011) 2D-difference gel electrophoretic proteomic analysis of a cell culture model of alveolar rhabdomyosarcoma. J Proteome Res 10:624–636
Shi T, Su D, Liu T et al (2012) Advancing the sensitivity of selected reaction monitoring-based targeted quantitative proteomics. Proteomics 12:1074–1092
Liebler DC, Zimmerman LJ (2013) Targeted quantitation of proteins by mass spectrometry. Biochemistry 52:3797–3806
Acknowledgments
This work was supported by StemBANCC (support from the Innovative Medicines Initiative joint undertaking under grant 115439-2, whose resources are composed of financial contribution from the European Union [FP7/2007-2013] and EFPIA companies’ in-kind contribution).
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Mazzacuva, F. et al. (2019). Mass Spectrometry Measurement of Albumin–Alpha Fetoprotein Ratio as an Indicator of iPSC-Derived Hepatocyte 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_13
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DOI: https://doi.org/10.1007/978-1-4939-9477-9_13
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