Pluripotent stem cells (PSCs) have been successfully developed in many species. However, generating bovine induced pluripotent stem cells (biPSCs) has been challenging. Here we report the generation of biPSCs by overexpression of lysine-specific demethylase 4A (KDM4A) and repro-gramming factors OCT4, SOX2, KLF4, cMYC, LIN28, and NANOG (KdOSKMLN). These biPSCs exhibited silenced transgene expression at passage 10, and had prolonged self-renewal capacity for over 70 passages. The biPSCs have flat, primed-like PSC colony morphology in combined me-dia of knockout serum replacement (KSR) and mTeSR, but switched to dome-shaped, naïve-like PSC colony morphology in mTeSR medium and 2i/LIF with single cell colonization capacity. These cells have comparable proliferation rate to the reported primed- or naïve-state human PSCs, with three-germ layer differentiation capacity and normal karyotype. Transcriptome analysis revealed a high similarity of biPSCs to reported bovine embryonic stem cells (ESCs) and embryos. The naïve-like biPSCs can be incorporated into mouse embryos, with the extended capacity of in-tegration into extra-embryonic tissues. Finally, 24.6% cloning efficiency could be achieved in nu-clear transfer (NT) experiment using late passage biPSCs as nuclear donors. Our report represents a significant advance in the establishment of bovine PSCs.
Overall design: Comparing mRNA profiles of biPSCs and bMSCs with the previously published data of 4 bovine ESCs from GSE110036 (GSM2976768, GSM2976772, GSM2976773, GSM2976777), 3 bovine 16-cell embayos from GSE52415 (GSM1265770, GSM1265771, GSM1265772), and 3 bovine blastocysts (GSM1265773 from GSE52415, GSM1265774 from GSE52415, GSM2976764 from GSE110036). For these previously published raw data, sequencing adapters and reads with low quality were trimmed using Cutadapt before mapping. The quality of reads after filtering was examined using fastQC. For mapping, bovine genomic sequence and RefSeq gene coordinate (ARS-UCD1.2/bosTau9) were downloaded from the UCSC genome browser. All filtered reads were aligned to bovine reference genome by RNA STAR (Galaxy Version 2.7.8a) with default parameters. The number of reads per gene was counted by featureCounts (Galaxy Version 2.0.1). Differentially expressed genes between different samples were identified using default parameters in DESeq2 (Galaxy Version 2.11.40.6+galaxy1), which generated a principal component analysis plot and the heatmap of the sample-to-sample distance matrix. The most differentially expressed genes (adjusted p-value < 0.05) were extracted from DESeq2 results with an absolute fold change > 5. The normalized counts for those differentially expressed genes and the Z-score of the counts were calculated on the galaxy platform and exhibited as heatmaps by Heatmap2 (Galaxy Version 3.0.1).
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