Species origin of exogenous transcription factors affects the activation of endogenous pluripotency markers and signaling pathways of porcine induced pluripotent stem cells

Meng Zhou; Manling Zhang; Tianxu Guo; Lihua Zhao; Xiyun Guo; Zhibao Yin; Linxin Cheng; Han Liu; Lixia Zhao; Xihe Li; Rongfeng Li

doi:10.3389/fcell.2023.1196273

Species origin of exogenous transcription factors affects the activation of endogenous pluripotency markers and signaling pathways of porcine induced pluripotent stem cells

Front Cell Dev Biol. 2023 Apr 21:11:1196273. doi: 10.3389/fcell.2023.1196273. eCollection 2023.

Authors

Meng Zhou¹, Manling Zhang¹, Tianxu Guo¹, Lihua Zhao^{1

2}, Xiyun Guo¹, Zhibao Yin¹, Linxin Cheng¹, Han Liu¹, Lixia Zhao³, Xihe Li³, Rongfeng Li^{1

2}

Affiliations

¹ Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing, China.
² Key Laboratory of Targeted Intervention of Cardiovascular Disease, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, Nanjing, China.
³ The State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, College of Life Science, Inner Mongolia University, Hohhot, Inner Mongolia, China.

Abstract

The incomplete silencing of exogenous transcription factors (TFs) and the lack of endogenous counterpart activation hampers the application of porcine induced pluripotent stem cells (piPSCs). We used porcine, bovine and murine TFs to reprogram porcine fetal fibroblasts. Porcine TFs-derived piPSCs (ppiPSCs) showed the highest levels of endogenous pluripotency markers activation, were able to differentiate into three germ layers and primordial germ cell-like cells (PGCLCs) and integrated into neural ectoderm of E7.5 mouse embryos in vitro. The bovine TFs derived piPSCs (bpiPSCs) expressed endogenous pluripotency markers higher than murine TFs derived piPSCs (mpiPSCs), but both had limited differentiation ability in vitro and depended on continuous expression of exogenous TFs for the maintenance. RNA sequencing confirmed ppiPSCs had distinct global transcriptional profiling, upregulated Hippo, PI3K-Akt, MAPK and relevant pluripotency signaling pathways as porcine blastocyst inner cell mass and expressed PGC early related genes. In addition, a positive and a negative correlation between exogenous and endogenous TFs' expression level were observed in ppiPSCs and bpiPSCs lines, respectively. The TFs' protein structures in pig were more similar to cattle than to mouse. In conclusion, the species affinity of the exogenous TFs is a key element, and the own species origin of TFs is optimal for iPSCs generation and application.

Keywords: activation; endogenous pluripotency marker; exogenous transcription factors; porcine induced pluripotent stem cells; species.

Grants and funding

This study was supported by the National Natural Scientific Foundation of China (31971379) and Science and Technology Major Project of Inner Mongolia Autonomous Region of China (2021ZD0048).