Abstract
The availability of disease-specific induced pluripotent stem cells (iPSCs) offers a unique opportunity for studying and modeling the effects of specific gene defects on human liver development in vitro and for testing small molecules or other potential therapies for relevant liver disorders. Here we report, for the first time, the derivation of iPSCs by the retroviral transduction of Yamanaka’s factors in serum and feeder-free culture conditions from liver-specific patients with tyrosinemia, glycogen storage disease, progressive familial hereditary cholestasis, and two siblings with Crigler-Najjar syndrome. Furthermore, they were differentiated into functional hepatocyte-like cells efficiently. These iPSCs possessed properties of human embryonic stem cells (hESCs) and were successfully differentiated into three lineages that resembled hESC morphology, passaging, surface and pluripotency markers, normal karyotype, DNA methylation, and differentiation. The hepatic lineage-directed differentiation showed that the iPSC-derived hepatic cells expressed hepatocyte-specific markers. Their functionality was confirmed by glycogen and lipid storage activity, secretion of albumin, alpha-fetoprotein, and urea, CYP450 metabolic activity, as well as LDL and indocyanin green uptake. Our results provide proof of principal that human liver-disease specific iPSCs present an exciting potential venue toward cell-based therapeutics, drug metabolism, human liver development and disease models for liver failure disorders.
Change history
06 April 2021
A Correction to this paper has been published: https://doi.org/10.1007/s12015-021-10158-2
Abbreviations
- hiPSCs:
-
Human induced pluripotent stem cells
- hESCs:
-
Human embryonic stem cells
- TYR:
-
Tyrosinemia
- GSD:
-
Glycogen storage disease
- HER:
-
Hereditary cholestasis
- CNS:
-
Crigler-Najjar syndrome
- EB:
-
Embryoid body
- AFP:
-
Alpha-fetoprotein
- ALB:
-
Albumin
- LDL:
-
Low-density lipoprotein
- PAS:
-
Periodic Acid-Schiff
- ICG:
-
Indocyanin green
- HDFs:
-
Human dermal fibroblasts
- HLCs:
-
Hepatocyte like cells
- ALP:
-
Alkaline phosphatase
References
Seifinejad, A., Tabebordbar, M., Baharvand, H., Boyer, L. A., & Salekdeh, G. H. (2010). Progress and promise towards safe induced pluripotent stem cells for therapy. Stem Cell Reviews, 6, 297–306.
Chen, M., Tomkins, D. J., Auerbach, W., et al. (1996). Inactivation of Fac in mice produces inducible chromosomal instability and reduced fertility reminiscent of Fanconi anaemia. Nature Genetics, 12, 448–451.
Asgari, S., Pournasr, B., Salekdeh, G. H., Ghodsizadeh, A., Ott, M., & Baharvand, H. (2010). Induced pluripotent stem cells: A new era for hepatology. Journal of Hepatology, doi:10.1016/j.jhep.2010.05.009.
Tuschl, G., Lauer, B., & Mueller, S. O. (2008). Primary hepatocytes as a model to analyze species-specific toxicity and drug metabolism. Expert Opinion on Drug Metabolism & Toxicology, 4, 855–870.
Totonchi, M., Taei, A., Seifinejad, A., Tabebordbar, M., Rassouli, H., Farrokhi, A., et al. (2010). Feeder- and serum-free establishment and expansion of human induced pluripotent stem cells. International Journal of Developmental Biology, 54, 877–886.
Mollamohammadi, S., Taei, A., Pakzad, M., et al. (2009). A simple and efficient cryopreservation method for feeder-free dissociated human induced pluripotent stem cells and human embryonic stem cells. Human Reproduction, 24, 2468–2476.
Baharvand, H., Salekdeh, G. H., Taei, A., & Mollamohammadi, S. (2010). An efficient and easy-to-use cryopreservation protocol for human ES and iPS cells. Nature Protocols, 5, 588–594.
Baharvand, H., Ashtiani, S. K., Taee, A., et al. (2006). Generation of new human embryonic stem cell lines with diploid and triploid karyotypes. Development, Growth & Differentiation, 48, 117–128.
Bock, C., Reither, S., Mikeska, T., Paulsen, M., Walter, J., & Lengauer, T. (2005). BiQ Analyzer: visualization and quality control for DNA methylation data from bisulfite sequencing. Bioinformatics, 21, 4067–4068.
Basma, H., Soto-Gutierrez, A., Yannam, G. R., et al. (2009). Differentiation and transplantation of human embryonic stem cell-derived hepatocytes. Gastroenterology, 136, 990–999.
Tzanakakis, E. S., Hsiao, C. C., Matsushita, T., Remmel, R. P., & Hu, W. S. (2001). Probing enhanced cytochrome P450 2B1/2 activity in rat hepatocyte spheroids through confocal laser scanning microscopy. Cell Transplantation, 10, 329–342.
Adewumi, O., Aflatoonian, B., Ahrlund-Richter, L., et al. (2007). Characterization of human embryonic stem cell lines by the international stem cell initiative. Nature Biotechnology, 25, 803–816.
Hotta, A., & Ellis, J. (2008). Retroviral vector silencing during iPS cell induction: an epigenetic beacon that signals distinct pluripotent states. Journal of Cellular Biochemistry, 105, 940–948.
Park, I. H., Zhao, R., West, J. A., et al. (2008). Reprogramming of human somatic cells to pluripotency with defined factors. Nature, 451, 141–146.
Takahashi, K., Tanabe, K., Ohnuki, M., et al. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 131, 861–872.
Cowan, C. A., Atienza, J., Melton, D. A., & Eggan, K. (2005). Nuclear reprogramming of somatic cells after fusion with human embryonic stem cells. Science, 309, 1369–1373.
Song, Z., Cai, J., Liu, Y., Zhao, D., Yong, J., Duo, S., et al. (2009). Efficient generation of hepatocyte-like cells from human induced pluripotent stem cells. Cell Research, 19, 1233–1242.
Sullivan, G. J., Hay, D. C., Park, I. H., Fletcher, J., Hannoun, Z., Payne, C. M., et al. (2010). Generation of functional human hepatic endoderm from human induced pluripotent stem cells. Hepatology, 51, 329–335.
Si-Tayeb, K., Noto, F. K., Nagaoka, M., et al. (2010). Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells. Hepatology, 51, 297–305.
Liu, H., Ye, Z., Kim, Y., Sharkis, S., & Jang, Y. Y. (2010). Generation of endoderm-derived human induced pluripotent stem cells from primary hepatocytes. Hepatology, 51, 1810–1819.
Saha, K., & Jaenisch, R. (2009). Technical challenges in using human induced pluripotent stem cells to model disease. Cell Stem Cell, 5, 584–595.
Acknowledgements
This study was funded by a grant provided from Royan Institute and Iranian Stem Cell Council.
Financial support
This study was funded by a grant provided from Royan Institute and Iranian Stem Cell Council.
Disclosures
None of the authors have any conflicts of interest to disclose and all authors support submission to this journal.
Author information
Authors and Affiliations
Corresponding author
Additional information
Arefeh Ghodsizadeh and Adeleh Taei contributed equally in this work.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Supplementary Table 1
Relative mRNA levels of lineage-specific hepatic markers [albumin (ALB), hepatic nuclear factor 4α (HNF4α) and CYP3a4], endoderm markers (SOX 17 and AFP), and markers for undifferentiated cells (Oct4) in differentiated and undifferentiated states. Data are normalized to β-actin and depicted as mean ± standard error of mean. (DOC 31 kb)
Supplementary Table 2
Primer sequences and conditions of real time RT-PCR. (DOC 37 kb)
Supplementary Fig. 1
A schematic view of protocol for the directed differentiation of hiPSCs toward HLCs. (DOC 107 kb)
Supplementary Fig. 2
PROD assay on hiPSC-HLCs of three lines at day 18, before and after CYP450 induction with phenobarbital. (DOC 501 kb)
(MP4 1746 kb)
Rights and permissions
About this article
Cite this article
Ghodsizadeh, A., Taei, A., Totonchi, M. et al. Generation of Liver Disease-Specific Induced Pluripotent Stem Cells Along with Efficient Differentiation to Functional Hepatocyte-Like Cells. Stem Cell Rev and Rep 6, 622–632 (2010). https://doi.org/10.1007/s12015-010-9189-3
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12015-010-9189-3