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Open AccessArticle

Optimized Approaches for the Induction of Putative Canine Induced Pluripotent Stem Cells from Old Fibroblasts Using Synthetic RNAs

1
Veterinary Medical Center and College of Veterinary Medicine, Laboratory of Veterinary Embryology and Biotechnology (VETEMBIO), Chungbuk National University, Cheongju 28644, Korea
2
Institute of Stem Cell & Regenerative Medicine (ISCRM), Chungbuk National University, Cheongju 28644, Korea
3
Abu Dhabi Biotech Research Foundation, 64 Kyungin-ro, Guro-gu, Seoul, Korea
*
Authors to whom correspondence should be addressed.
Animals 2020, 10(10), 1848; https://doi.org/10.3390/ani10101848
Received: 7 September 2020 / Revised: 2 October 2020 / Accepted: 7 October 2020 / Published: 11 October 2020
(This article belongs to the Special Issue Stem Cells in Domestic Animals: Applications in Health and Production)
A non-integrating and self-replicating Venezuelan equine encephalitis RNA replicon system can potentially make a great contribution to the generation of clinically applicable canine induced pluripotent stem cells. Our study shows a new method to utilize the synthetic RNA-based approach for canine somatic cell reprogramming regarding transfection and reprogramming efficiency.
Canine induced pluripotent stem cells (ciPSCs) can provide great potential for regenerative veterinary medicine. Several reports have described the generation of canine somatic cell-derived iPSCs; however, none have described the canine somatic cell reprogramming using a non-integrating and self-replicating RNA transfection method. The purpose of this study was to investigate the optimal strategy using this approach and characterize the transition stage of ciPSCs. In this study, fibroblasts obtained from a 13-year-old dog were reprogrammed using a non-integrating Venezuelan equine encephalitis (VEE) RNA virus replicon, which has four reprogramming factors (collectively referred to as T7-VEE-OKS-iG and comprised of hOct4, hKlf4, hSox2, and hGlis1) and co-transfected with the T7-VEE-OKS-iG RNA and B18R mRNA for 4 h. One day after the final transfection, the cells were selected with puromycin (0.5 µg/mL) until day 10. After about 25 days, putative ciPSC colonies were identified showing TRA-1-60 expression and alkaline phosphatase activity. To determine the optimal culture conditions, the basic fibroblast growth factor in the culture medium was replaced with a modified medium supplemented with murine leukemia inhibitory factor (mLIF) and two kinase inhibitors (2i), PD0325901(MEK1/2 inhibitor) and CHIR99021 (GSK3β inhibitor). The derived colonies showed resemblance to naïve iPSCs in their morphology (dome-shaped) and are dependent on mLIF and 2i condition to maintain an undifferentiated phenotype. The expression of endogenous pluripotency markers such as Oct4, Nanog, and Rex1 transcripts were confirmed, suggesting that induced ciPSCs were in the late intermediate stage of reprogramming. In conclusion, the non-integrating and self-replicating VEE RNA replicon system can potentially make a great contribution to the generation of clinically applicable ciPSCs, and the findings of this study suggest a new method to utilize the VEE RNA approach for canine somatic cell reprogramming. View Full-Text
Keywords: canine; reprogramming; iPSCs; VEE RNA; integration-free canine; reprogramming; iPSCs; VEE RNA; integration-free
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MDPI and ACS Style

Kim, M.; Hwang, S.-U.; Yoon, J.D.; Jeong, Y.W.; Kim, E.; Hyun, S.-H. Optimized Approaches for the Induction of Putative Canine Induced Pluripotent Stem Cells from Old Fibroblasts Using Synthetic RNAs. Animals 2020, 10, 1848. https://doi.org/10.3390/ani10101848

AMA Style

Kim M, Hwang S-U, Yoon JD, Jeong YW, Kim E, Hyun S-H. Optimized Approaches for the Induction of Putative Canine Induced Pluripotent Stem Cells from Old Fibroblasts Using Synthetic RNAs. Animals. 2020; 10(10):1848. https://doi.org/10.3390/ani10101848

Chicago/Turabian Style

Kim, Mirae; Hwang, Seon-Ung; Yoon, Junchul D.; Jeong, Yeon W.; Kim, Eunhye; Hyun, Sang-Hwan. 2020. "Optimized Approaches for the Induction of Putative Canine Induced Pluripotent Stem Cells from Old Fibroblasts Using Synthetic RNAs" Animals 10, no. 10: 1848. https://doi.org/10.3390/ani10101848

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