Induced pluripotent stem cell–derived hepatocytes have the functional and proliferative capabilities needed for liver regeneration in mice
Silvia Espejel, … , Shinya Yamanaka, Holger Willenbring
Silvia Espejel, … , Shinya Yamanaka, Holger Willenbring
Published August 25, 2010
Citation Information: J Clin Invest. 2010;120(9):3120-3126. https://doi.org/10.1172/JCI43267.
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Research Article

Induced pluripotent stem cell–derived hepatocytes have the functional and proliferative capabilities needed for liver regeneration in mice

Abstract

The ability to generate induced pluripotent stem (iPS) cells from a patient’s somatic cells has provided a foundation for organ regeneration without the need for immune suppression. However, it has not been established that the differentiated progeny of iPS cells can effectively reverse failure of a vital organ. Here, we examined whether iPS cell–derived hepatocytes have both the functional and proliferative capabilities needed for liver regeneration in mice with fumarylacetoacetate hydrolase deficiency. To avoid biases resulting from random genomic integration, we used iPS cells generated without viruses. To exclude compensation by hepatocytes not derived from iPS cells, we generated chimeric mice in which all hepatocytes were iPS cell derived. In vivo analyses showed that iPS cells were intrinsically able to differentiate into fully mature hepatocytes that provided full liver function. The iPS cell–derived hepatocytes also replicated the unique proliferative capabilities of normal hepatocytes and were able to regenerate the liver after transplantation and two-thirds partial hepatectomy. Thus, our results establish the feasibility of using iPS cells generated in a clinically acceptable fashion for rapid and stable liver regeneration.

Authors

Silvia Espejel, Garrett R. Roll, K. John McLaughlin, Andrew Y. Lee, Jenny Y. Zhang, Diana J. Laird, Keisuke Okita, Shinya Yamanaka, Holger Willenbring

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Figure 3

iPS cell–derived hepatocytes have normal proliferative capabilities.

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iPS cell–derived hepatocytes have normal proliferative capabilities. (A)...
(A) FAH immunostaining (red, both images) shows approximately 90% liver repopulation with FAH-expressing hepatocytes in a chimeric mouse at P28. Additional Ki67 immunostaining (green, right image) shows proliferating FAH-positive hepatocytes bordering growth-arrested FAH-negative hepatocytes. (B and C) Analysis of liver regeneration after two-thirds partial hepatectomy in 3 wild-type and 3 FAH-deficient mice repopulated to approximately 100% with transplanted iPS cell–derived hepatocytes (FAH immunostaining, green or red). (B) BrdU labeling (red) 40 hours after two-thirds partial hepatectomy shows that timing and magnitude of DNA synthesis are indistinguishable between wild-type hepatocytes (left) and iPS cell–derived hepatocytes (middle). Quantification of BrdU-labeled hepatocytes in all mice (right). (C) Immunostaining for phosphorylated histone H3 (p-hH3, green) 48 hours after two-thirds partial hepatectomy shows that wild-type hepatocytes (left) and iPS cell–derived hepatocytes (middle) progress into mitosis with similar efficiency. Quantification of p-hH3–labeled hepatocytes in all mice (right). (D) Combined FAH immunostaining (red) and X-gal staining (blue) shows 2 nodules of similar size derived from a single iPS cell–derived hepatocyte or a Rosa26 hepatocyte transplanted together into an immune-deficient, FAH-deficient mouse. The blue X-gal staining masks the red FAH signal. Both FAH immunostaining and X-gal staining are highly sensitive and specific (Figure 1, E and F, and Supplemental Figure 6). Classifying repopulating nodules based on the number of hepatocytes visible in 2-dimensional liver sections (cells per nodule) indicates that cotransplanted Rosa26 hepatocytes and iPS cell–derived hepatocytes are equally effective in clonal expansion. The number of cell divisions required for formation of repopulating nodules composed of 11–25, 26–50, 51–75, 76–100, or 101–125 hepatocytes is 7, 8, 9, 10, or 11, respectively. This calculation is based on the assumption that each hepatocyte in the nodule proliferates to the same extent and that nodules are 3-dimensional spheres (35). Results of 3 transplantation experiments are shown. Nuclei are stained blue. Scale bars: 100 μm (A, left image, and D); 50 μm (B and C); 25 μm (A, right image). Data represent mean ± SEM. #P > 0.05.