Transcriptomic insights and feeder-free culturing of porcine expanded potential stem cells from cloned embryos

Abstract

Background Generating expanded potential stem cells from cloned porcine embryos (pEPSCsNT) represents a notable advancement in regenerative medicine and agricultural biotechnology. However, challenges, including low derivation efficiency, limited understanding of transcriptomic features, and unknown feasibility of culturing under feeder-free conditions, remain. This study aimed to generate pEPSCs using blastocysts derived from parthenogenetic activation, in vitro fertilization, and somatic cell nuclear transfer (SCNT) using a modified culture system. Methods We derived pEPSCNT lines using an optimized culture system. We characterized the pEPSC lines from all three origins by analyzing pluripotent marker expression, performing karyotyping, and assessing their differentiation potential into the three germ layers. Furthermore, we performed a comparative transcriptomic analysis using in vivo and cloned embryo data, with a major focus on cell lines derived from SCNT (pEPSCsNT). We optimized feeder-free culture conditions for the pEPSCNT line and derived the pEPSCNT lines using an optimized culture system with an efficiency of ~14%. Results The cells were closely correlated with 8-cell to morula-stage embryos and exhibited significant enrichment of EPSC signature genes, suggesting a unique pluripotent state relatively close to the naïve state, specifically within a formative state. The pEPSCsNT possessed broad differentiation capacity, indicative of Hippo signaling pathway enrichment, blastocyst-like structure formation ability, and potential differentiation into trophoblast lineage cells. Conclusions Our modified culture medium combined with the 2× Matrigel coating system facilitated the transition to feeder-independent culture conditions. These findings facilitate the establishment of a feeder-free culture system while preserving pluripotency and differentiation potential.

1. Introduction

Pluripotent stem cells (PSCs) are versatile and invaluable tools for various research applications because of their intrinsic ability to self-renew and differentiate into multiple cell types [1, 2, 3]. Among the various PSC types, those derived from domestic pigs (pPSCs) are the most promising. The physiological, genetic, and immunological similarities between pigs and humans render pigs an ideal large-animal model for comparative biology, disease modeling, and regenerative medicine [4, 5, 6, 7]. Despite this considerable potential, establishing bona fide pPSCs has been challenging over the past few decades, often resulting in the generation of cells referred to as embryonic stem (ES)-like cells instead [8, 9, 10, 11, 12].

Since 2019, important breakthroughs have shown promise in generating stable pPSCs [13, 14, 15, 16, 17]. Similar to those from other species, the characteristics and status of pPSCs in terms of transcriptomic expression vary depending on the established strategies employed [13, 14, 15, 16, 17]. For instance, Choi et al. [14] demonstrated that the activation of the fibroblast growth factor 2 (FGF2), ACTIVIN/NODAL, and WNT signaling pathways enabled the derivation of authentic pPSCs. These cells exhibited a transcriptome developmentally similar to that of late epiblasts rather than that of an inner cell mass (ICM), suggesting a primed pluripotent state achieved by seeding hatched blastocysts under feeder cells [14]. These primed pPSCs formed morphologically flat colonies, maintained stability for over 50 passages, exhibited teratoma formation, and retained two active X chromosomes [14]. In the same year, Gao et al. [18] demonstrated the establishment of more advanced cell lines, known as porcine-expanded potential stem cells (pEPSCs).

These cells exhibited a remarkable ability to differentiate into embryonic and extra-embryonic lineages. This was achieved by inhibiting GSK3, SRC, and Tankyrase, activating the Activin A and TGFβ pathways, and adding vitamin C [18]. These pEPSCs formed compact colonies with smooth edges, remained genetically stable over multiple passages, showed extensive DNA demethylation, and exhibited transcriptomic features similar to those of the early embryo.