The analysis of X chromosome activity of porcine embryonic stem Cells: Study based on parthenogenetic embryonic stem cells with LCDM medium

Abstract

The derivation of porcine embryonic stem cell (pESC) lines remains a major challenge in this field. To date, the porcine naïve ESCs have yet to be successfully established, and standardized criteria for their characterization and evaluation are still lacking. The regulation of X-chromosome activity integrates information from embryonic development and the dosage of sex chromosomes, which is closely associated with the pluripotent state of embryonic stem cells. In this study, we aimed to establish pESC lines in LCDM medium from porcine blastocyst-stage embryos, and analyzed the features of ESCs from the sight of X chromosome activity. We assessed molecular markers and epigenetic characteristics to confirm pluripotency and X chromosome activity in porcine parthenogenetic ESCs (named as ppLCDM) using XIST RNA-FISH, immunofluorescence staining, single-cell RNA sequencing (scRNA-seq), and other techniques. Results showed that ppLCDM cells expressed most pluripotent markers. The percentage of ppLCDM cells exhibiting H3K27me3 and XIST aggregation signals increased with passage, indicating the progressive establishment of X-chromosome inactivation (XCI). Meanwhile, the pluripotency of most ppLCDM cells gradually declined during extended passaging. However, two distinct patterns of ppLCDM cells were observed from passage 35 (type I cells, P35-I) displayed normal XCI states, while type II cells (P35-II) exhibited X-chromosome erosion-like state, characterized by the loss of aggregation signals, abnormal X-linked gene ratios. Particularly, the pluripotency of ppLCDM cells with an X-chromosome erosion-like state undergoes unusual changes compared to normal cells. These findings indicate that X chromosome activity is closely associated with the pluripotent state of porcine ESCs and that heterogeneity in X chromosome activity arises during passaging. Our research provides crucial insights into X chromosome dynamics in large-animal ESC models and contribute to ongoing efforts to establish stable naïve pESC lines.