Abstract
The first step in differentiation of pluripotent stem cell toward endoderm-derived cell/organ is differentiation to definitive endoderm (DE) which is the central issue in developmental biology. Based on several evidences, we hypothesized that activin-A optimization as well as replacement of fetal bovine serum (FBS) with knockout serum replacement (KSR) is important for differentiation of induced pluripotent stem cell (iPSC) line into DE. Therefore, a stepwise differentiation protocol was applied on R1-hiPSC1 cell line. At first, activin-A concentration (30, 50, 70 and 100 ng/ml) was optimized. Then, substitution of FBS with KSR was evaluated across four treatment groups. The amount of differentiation of iPSC toward DE was determined by quantitative gene expression analyses of pluripotency (NANOG and OCT4), definitive endoderm (SOX17 and FOXA2) and endoderm-derived organs (PDX1, NEUROG3, and PAX6). Based on gene expression analyses, the more decrease in concentrations of activin-A can increase the differentiation of iPSC into DE, therefore, 30 ng/ml activin-A was chosen as the best concentration for the differentiation of R1-hiPSC1 line toward endoderm-derived organ. Moreover, complete replacement of FBS with gradually increased KSR improved the differentiation of iPSC toward DE. For this reason, the addition of 0% KSR at day 1, 0.2% at day 2 and 2% for the next 3 days was the best optimal protocol of the differentiation of iPSC toward DE. Overall, our results demonstrate that optimization of activin-A is important for differentiation of iPSC line. Furthermore, the replacement of FBS with KSR can improve the efficiency of iPSC differentiation toward DE.
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Acknowledgements
The authors are grateful to the Organ Transplant Research Center, Shiraz University of medical sciences and University of Sistan and Baluchestan, for their executive and financial support of this project.
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Conceptualization was done by NA. Laboratory experiments was done by SG-D, SL and AS. Statistical analyses were done by SG-D and MS. Funding acquisition was done by NA and MHS. The first draft of the article was written by SG-D and NA. Methodology and investigation were done by HS-L, RY and IHA-A. All authors reviewed, edited and approved the manuscript.
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Ghorbani-Dalini, S., Azarpira, N., Sangtarash, M.H. et al. Optimization of activin-A: a breakthrough in differentiation of human induced pluripotent stem cell into definitive endoderm. 3 Biotech 10, 215 (2020). https://doi.org/10.1007/s13205-020-02215-3
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DOI: https://doi.org/10.1007/s13205-020-02215-3