Comparison of Umbilical Cord Mesenchymal Stem Cells and Fibroblasts as Donor Nuclei for Handmade Cloning in Sheep Using a Single-Cell Transcriptome

Abstract

Oocytes are efficient at reprogramming terminally differentiated cells to a totipotent state. Nuclear transfer techniques can exploit this property to produce cloned animals. However, the overall efficiency is low. The use of umbilical cord mesenchymal stem cells (UC-MSCs) as donor nuclei may increase blastocyst rates, but the exact reasons for this remain unexplored. A single-cell transcriptomic approach was used to map the transcriptome profiles of eight-cell embryos that were in vitro-fertilized and handmade-cloned using umbilical cord mesenchymal stem cells and fibroblasts as nuclear donors. Differences were examined at the chromatin level, the level of differentially expressed genes, the level of histone modifications and the level of DNA methylation. This research provides critical information regarding the use of UC-MSCs as a preferred donor nucleus for nuclear transfer techniques. It also offers unique insights into the mechanism of cellular reprogramming.

Keywords: handmade cloning; nuclear transplantation; sheep; single-cell transcriptome; umbilical cord mesenchymal stem cells.

Figure 1

RNA-seq base quality and comparison statistics: (A) Q30 rate of base measured for each sample. (B) Proportion of reads mapped to exon, intron, and intergenic regions. (C) Distribution of TPM for each sample. (D) Filtering statistics for reads.

Figure 2

RNA-seq inter- and intra-group correlation analysis: (A) Intra-group correlation violin plot. (B) PCA analysis plot. (C) Hierarchical clustering plot.

Figure 3

Statistical analysis of differentially expressed genes: (A–C) Volcano plot of differentially expressed genes. (D) Statistical histogram of differentially expressed genes between samples. (E) Upset plot of differentially expressed genes. (F) GO enrichment analysis of 133 specific differentially expressed genes in DFUC vs. IVF.

Figure 4

GO enrichment analyses: (A–C) indicate the bubble plots of GO enrichment analyses for DFF vs. DFUC, DFUC vs. IVF, and DFF vs. IVF, respectively.

Figure 5

DFF vs. DFUC, DFUC vs. IVF, DFF vs. IVF KEGG enrichment analysis bubble plot, respectively.

Figure 6

GSEA enrichment analysis ridge diagrams: (A–C) indicate the GSEA enrichment analysis ridge diagrams for DFF vs. DFUC, DFUC vs. IVF, and DFF vs. IVF, respectively.

Figure 7

GSEA enrichment analysis results: (A–C) indicate GSEA enrichment analysis results of the ribosome pathway for DFF vs. DFUC, DFUC vs. IVF, and DFF vs. IVF. (D–F) indicate GSEA enrichment analysis results of the oxidative phosphorylation pathway for DFF vs. DFUC, DFUC vs. IVF, and DFF vs. IVF.

Figure 8

Heatmap and PPI network analysis: (A) indicates the heatmap of differentially expressed genes of IVF, DFUC, and DFF. (B) indicates the results of the respective GO enrichment analysis of each group in the heatmap. (C) Analysis of Group 2–3 PPI network in the heatmap.

Figure 9

Chromosome-level differential expression analysis: (A) Graph indicating the expression level points of each chromosome. (B) Graph of the expression level lines of chromosome X. (C) Graph of the expression level lines of chromosome 4. (D) Graph of the major gene box patterns of the four differentially expressed regions of chromosome 4. The symbol * indicates p < 0.05, and ns indicates p > 0.05.

Figure 10

Analysis of histone methylation and acetylation with DNA methylation and demethylation gene expression patterns: (A–C) Each indicates a graph of changes in the expression of lysine methylase writer, lysine methylase eraser, and DNA methylation-related enzymes. (D) indicates box plots of the expression of several major histone acetylases and deacetylases. The symbol * indicates p < 0.05, ** indicates p < 0.01, and ns indicates p > 0.05.

Figure 11

Validation of RNA-seq using RT-qPCR.