Establishment of Bovine-Induced Pluripotent Stem Cells.

Yue Su,Irina Polejaeva,Zhiqiang Fan,Jiaqi Zhu,Ling Wang,Young Tang,Deborah Kaback,Ying Liu,Tayler Patrick,Xiuchun (Cindy) Tian,Siu Pok Yee,Julia Oudiz

doi:10.3390/ijms221910489

Abstract

Pluripotent stem cells (PSCs) have been successfully developed in many species. However, the establishment of bovine-induced pluripotent stem cells (biPSCs) has been challenging. Here we report the generation of biPSCs from bovine mesenchymal stem cells (bMSCs) by overexpression of lysine-specific demethylase 4A (KDM4A) and the other reprogramming 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 media 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 integration into extra-embryonic tissues. Finally, at least 24.5% cloning efficiency could be obtained in nuclear transfer (NT) experiment using late passage biPSCs as nuclear donors. Our report represents a significant advance in the establishment of bovine PSCs.

Highlights

Since 2006, induced pluripotent stem cells rapidly emerged as another type of pluripotent stem cells (PSCs), which were reprogrammed from somatic cells by exogenous expression of OCT4, SOX2, KLF4, and c-MYC (OSKM), or OCT4, SOX2 (OS) plus LIN28 and NANOG (LN) [1,2,3]
We report the induction of bovine-induced pluripotent stem cells (biPSCs) using a similar approach, with OSKMLN plus the additional expression of lysine-specific demethylase 4A (KDM4A) for reprogramming (Kd OSKMLN), and their pluripotency characterization
In this study, using a combination of seven factors (Kd OSKMLN), and the reprogramming medium containing inhibitors to WNT (IWR1) and H3K79 methyltransferase Dot1L, we report the induction of primed-like biPSCs, which do not rely on the continuous exogenous transgene activity for self-renewal

Summary

Introduction

Since 2006, induced pluripotent stem cells (iPSCs) rapidly emerged as another type of pluripotent stem cells (PSCs), which were reprogrammed from somatic cells by exogenous expression of OCT4 ( known as POU5F1), SOX2, KLF4, and c-MYC (OSKM), or OCT4, SOX2 (OS) plus LIN28 and NANOG (LN) [1,2,3]. There have been numerous efforts in the derivation of bovine pluripotent stem cells (PSCs) including bovine embryonic stem cells (bESCs) and induced pluripotent stem cells (biPSCs). Bovine ESC-like cells capable of long-term culture were reported [8]. These bESCs showed the expression of pluripotent genes and an epigenetic landscape similar to the human primed-pluripotent state ESCs (basic fibroblast growth factor (bFGF) dependent)

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Discussion

Conclusion