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Hepatic differentiation of mouse iPS cells and analysis of liver engraftment potential of multistage iPS progeny

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Abstract

Hepatocyte transplantation is considered a promising therapy for patients with liver diseases. Induced pluripotent stem cells (iPSCs) are an unlimited source for the generation of functional hepatocytes. While several protocols that direct the differentiation of iPSCs into hepatocyte-like cells have already been reported, the liver engraftment potential of iPSC progeny obtained at each step of hepatic differentiation has not yet been thoroughly investigated. In this study, we present an efficient strategy to differentiate mouse iPSCs into hepatocyte-like cells and evaluate their liver engraftment potential at different time points of the protocol (5, 10, 15, and 20 days of differentiation). iPSCs were differentiated in the presence of cytokines, growth factors, and small molecules to finally generate hepatocyte-like cells. These iPSC-derived hepatocyte-like cells exhibited hepatocyte-associated functions, such as albumin secretion and urea synthesis. When we transplanted iPSC progeny into the spleen, we found that 15- and 20-day iPSC progeny engrafted into the livers and further acquired hepatocyte morphology. In contrast, 5- and 10-day iPSC progeny were also able to engraft but did not generate hepatocyte-like cells in vivo. Our data may aid in improving current protocols geared towards the use of iPSCs as a new source of liver-targeted cell therapies.

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Acknowledgments

We thank Dr. Miguel Barajas for providing us retroviral plasmids for reprogramming. We thank Dr. Angelo Porciuncula for not only correcting the English but also for some valuable comments. This work is supported by Foundation for Applied Medical Research (FIMA), University of Navarra, Spain.

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Authors and Affiliations

  1. Gene Therapy and Hepatology Area, FIMA University of Navarra, Pamplona, Spain

    Anangi Balasiddaiah, Daniel Moreno, Jesús Prieto & Rafael Aldabe

  2. Morphology Lab, Center for Applied Medical Research (CIMA), FIMA University of Navarra, Pamplona, Spain

    Laura Guembe

  3. CIBER-EHD, University of Navarra, Pamplona, Spain

    Jesús Prieto

  4. Liver Unit, University Clinic, University of Navarra, Pamplona, Spain

    Jesús Prieto

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  1. Anangi Balasiddaiah
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  2. Daniel Moreno
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  5. Rafael Aldabe
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Corresponding author

Correspondence to Rafael Aldabe.

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Supplementary Fig. 1

a Characterisation of iPSCs by immunofluorescence. Images show the immunodetection of Oct4 (×20), Nanog (×20), and SSEA-1 (×20) in iPSC clones (clones 1.38 and 1.39). The red signal indicates the stem cell markers Oct4, Nanog, and SSEA-1 in iPSCs. The blue signal indicates the nuclei of iPSCs and feeder cells. Immunofluorescence without primary antibody was used as the negative control (named as control). No stem cell markers were detected in feeder cells. b Detection of alkaline phosphatase in iPSC clones 1.38 and 1.39. Images of alkaline phosphatase-stained iPSC clones grown on feeder cells and feeder cells alone are shown (magnification, ×10). The violet stain indicates the presence of alkaline phosphatase in the iPSC clones. Feeder cells were used as the negative control. c qPCR analysis of c-Myc levels in primary mouse tail tip fibroblasts and hepatocytes. The gene expression values of c-Myc in mouse tail tip fibroblasts are set to 1. (TIFF 10821 kb)

Supplementary Fig. 2

qPCR expression analysis of hepatic markers. iPSCs differentiated for 5, 10, 15, and 20 days were analysed for the expression of Arginase-1, Hnf1α, Hnf1β, C/EBPα, and C/EBPβ. The CT values of all genes were normalised to the CT values of β-actin. The y-axis represents the fold change of gene expression compared with undifferentiated iPSCs. One-way ANOVA test was used for statistical analysis (*p < 0.05; **p < 0.01; ***p < 0.001). Error bars represent the standard deviation. The data represent the average values of triplicates of the same experiment. (TIFF 10820 kb)

Supplementary Table 1

Primer sequences used in this study. (TIFF 10,801 kb)

Supplementary Table 2

Increased gene expression of hepatic markers in 20-day iPSC progeny and mouse hepatocytes compared with the observed in undifferentiated iPSCs. (TIFF 10,818 kb)

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Balasiddaiah, A., Moreno, D., Guembe, L. et al. Hepatic differentiation of mouse iPS cells and analysis of liver engraftment potential of multistage iPS progeny. J Physiol Biochem 69, 835–845 (2013). https://doi.org/10.1007/s13105-013-0260-9

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  • DOI: https://doi.org/10.1007/s13105-013-0260-9

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