# Construction and Influence of Induced Pluripotent Stem Cells on Early Embryo Development in Black Bone Sheep

## TL;DR

This study successfully reprogrammed black bone sheep cells into stem cells and showed they can develop into early embryos, offering insights into pluripotency in sheep.

## Contribution

The study demonstrates the use of the piggyBac+TET-on system for efficient reprogramming of black bone sheep fibroblasts into iPSCs with tri-lineage differentiation potential.

## Key findings

- iPSCs showed higher cleavage and blastocyst rates in somatic cell nuclear transfer compared to fibroblasts.
- iPSCs exhibited enriched oxidative phosphorylation and cell proliferation pathways with distinct gene expression.
- iPSCs expressed higher levels of trilineage marker genes, indicating pluripotency and tri-lineage differentiation potential.

## Abstract

The piggyBac+TET-on transposon system was used to reprogram black bone sheep fibroblasts into iPSCs and explore the mechanism of their influence on early embryonic development. The iPSC clones have a good morphology, positive alkaline phosphatase staining, and normal karyotype. The transcriptome analysis showed that it was significantly enriched in oxidative phosphorylation and cell proliferation pathways, and the expression of related genes was significantly different from that of fibroblasts. In the somatic cell nuclear transfer experiment, the cleavage rate and blastocyst rate of the iPSC were higher than those of fibroblasts, and the difference was obvious. The expression of three-layer marker genes in iPSCs was much higher than that in fibroblasts. These results indicated that the obtained iPSCs had pluripotent and triploblastic development potential and revealed the mechanism of action of the reprogrammed iPSCs on early embryonic development, which laid a solid foundation for the study of pluripotent stem cells in sheep.

The piggyBac+TET-on transposon induction system has a high efficiency in integrating exogenous genes in multiple cell types, can precisely integrate to reduce genomic damage, has a flexible gene expression regulation, and a strong genetic stability. When used in conjunction with somatic cell nuclear transfer experiments, it can precisely and effectively reveal the intrinsic mechanisms of early biological development. This study successfully reprogrammed black-boned sheep fibroblasts (SFs) into induced pluripotent stem cells (iPSCs) using the piggyBac+TET-on transposon system and investigated their impact on early embryonic development. Seven exogenous reprogramming factors (bovine OCT4, SOX2, KLF4, cMyc, porcine NANOG, Lin-28, and SV40 Large T) were delivered into SFs, successfully inducing iPSCs. A growth performance analysis revealed that iPSC clones exhibited a raised or flat morphology with clear edges, positive alkaline phosphatase staining, and normal karyotypes. The transcriptome analysis indicated a significant enrichment of iPSCs in oxidative phosphorylation and cell proliferation pathways, with an up-regulated expression of the ATP5B, SDHB, Bcl-2, CDK1, and Cyclin D1 genes and a down-regulated expression of BAX (p < 0.05). Somatic cell nuclear transfer experiments demonstrated that the cleavage rate (85% ± 2.12) and blastocyst rate (52% ± 2.11) of the iPSCs were significantly higher than those of the SFs (p < 0.05). The detection of trilineage marker genes confirmed that the expression levels of endoderm (DCN, NANOS3, FOXA2, FOXD3, SOX17), mesoderm (KDR, CD34, NFH), and ectoderm (NEUROD) markers in iPSCs were significantly higher than in SFs (p < 0.01). The findings demonstrate that black-boned sheep iPSCs possess pluripotency and the potential to differentiate into all three germ layers, revealing the mechanisms by which reprogrammed iPSCs influence early embryonic development and providing a critical foundation for research on sheep pluripotent stem cells.