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Differentiation of rat multipotent adult progenitor cells to functional hepatocyte-like cells by mimicking embryonic liver development

Abstract

Differentiation of stem cells to hepatocytes has industrial applications, as well as the potential to develop new therapeutic strategies for liver disease. The protocol described here, sequentially using cytokines that are known to have a role in liver embryonic development, efficiently differentiates rat multipotent adult progenitor cells (rMAPCs) to hepatocyte-like cells by directing them through defined embryonic intermediates, namely, primitive streak/mesendoderm/definitive endoderm, hepatoblast and hepatocyte-like phenotype. After 20 days, the final differentiated multipotent adult progenitor cell progeny is a mixture of cells, comprising cells with the characteristics of hepatoblasts and a smaller cell fraction with the morphological and phenotypical features of mature hepatocytes, as well as other mesodermal cells and some persistent undifferentiated rMAPCs. A detailed functional characterization of the stem cell progeny is also described; this should be used to confirm that differentiated cells display the functional characteristics of mature hepatocytes, including albumin secretion, glycogen storage and several detoxifying functions such as urea production, bilirubin conjugation, glutathione S-transferase activity and cytochrome activity.

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Figure 1
Figure 2: Bright field microscopy of rMAPC progeny during liver differentiation.
Figure 3: RT-qPCR analysis of rMAPC progeny during liver differentiation, depicted as deltaCT compared with Gapdh (DeltaCT = CTgene − CTGAPDH).
Figure 4: Immunocytochemistry analysis.
Figure 5: Functional assays on rMAPC progeny (one representative experiment) during liver differentiation.

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Acknowledgements

We thank F. Nevens, J. van Pelt, P. Windmolders, I. Vander Elst and M. Zeegers (Hepatology Lab, K.U. Leuven) for their help with functional assays and T. Notelaers (SCIL) for help with differentiation studies. This work was supported by grants to C.V. from the NIH (RO1-HL67932, RO1 DK58295, RO1-HL073221 and U19-DK61244), the Alpha 1 Foundation, FWO G.0667.07 Odysseus award and a K.U. Leuven SCIL Center of Excellence Award. C.V. also received research funding from Athersys Inc. (Cleveland, OH, USA). P.R. was funded by IWT and VVGE. P.S.-B. was funded by FWO (KAN 1.5163.09) and Agència de Gestió d'Ajuts Universitaris i de Recerca, Beatriu de Pinós (2006 BP-A 10104). K.P. was funded by FWO, BAEF and VVGE.

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P.R. and P.S.-B. prepared the manuscript. K.P. and C.V. designed the protocol. P.R., P.S.-B. and K.P. conducted the experiments, RT-qPCR, immunocytochemistry and functional assays. C.V. supervised the project.

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Correspondence to Catherine Verfaillie.

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The authors declare no competing financial interests.

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Roelandt, P., Sancho-Bru, P., Pauwelyn, K. et al. Differentiation of rat multipotent adult progenitor cells to functional hepatocyte-like cells by mimicking embryonic liver development. Nat Protoc 5, 1324–1336 (2010). https://doi.org/10.1038/nprot.2010.80

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