Naïve-like conversion of bovine induced pluripotent stem cells from Sertoli cells

Pluripotent stem cells (PSCs) such as embryonic stem cells (ESCs) and induced PSCs (iPSCs) hold great promise for cell-based therapies and drug discovery [1]. From a biomedical perspective, iPSCs offer several advantages over ESCs because they can be generated without using embryos from virtually any individual, i.e., in a patient- and disease-specific manner, for subsequent therapeutic or research applications. In the last decade, iPSCs were generated from various somatic cells using exogenous transcriptional factors combining with small chemical inhibitors. Studies in primates, porcine and human have indicated that iPSCs can be classified into two distinct and stable pluripotent states, the naïve and the primed states [[2], [3], [4]]. Considering their attractive applications in animal farming, animal conservation and environmental protection, generation of livestock iPSCs has been continuously tried. More importantly, naïve live stock iPSCs may have similar totipotency with mouse iPSCs. Thus, researchers have been devoting themselves to derive the naïve state iPSCs of domestic animals, which can form the dome-shaped colonies and express the naïve markers. Bovine iPSCs had been generated from varied somatic cells with several pluripotency transcript factors and small molecules [[5], [6], [7], [8]], but they shared multiple defining features with post-implantation mouse epiblast stem cells rather than mouse ESCs in terms of gene expression profiles, signaling pathways required for proliferation, flat morphology and intolerance to single-cell passaging. Therefore, whether and how the naïve pluripotent state in bovine iPSCs can be established is an important issue. Recent studies have reported that ESC/iPSC pluripotency and reprogramming efficiency are thought to be regulated in part by histone H3 lysing (K4/27) methylation (H3K4/27) [9,10], but its role in the regulation of transformation from bovine primed iPSCs to naïve iPSCs still remains unclear.

The pluripotent status of iPSCs/ESCs is also related to the conditions of the feeder cells. Thus it's necessary to prepare suitable feeder layers for iPSC/ESC cultivation [11]. Feeder cells can not only supply the growth environment but also afford the nutrients and essential factors especially leukemia inhibitory factor (LIF) for the stem cells cultured on themselves. It's demonstrated that feeder is crucial for human ESC conversion from the primed to the naïve state, otherwise the colonies will detach from the plate easily [12]. Mouse embryonic fibroblasts (MEF) are the acknowledged feeder cells for iPSC/ESC generation and cultivation in many species. However, MEF feeder may cause the species interference for the iPSCs/ESCs from other animals and the basic substances secreted by MEF feeder may not be the most suitable materials for domestic animal iPSCs in long-term culture. Till now, several other cell types were also used as feeder cells for stem cell cultivation, e.g., NIH3T3 [13] and granulosa cells [14] for ESCs/iPSCs, bovine embryonic fibroblasts (BEF) for undifferentiated spermatogonial stem cells [15], and human amniotic epithelial cells for human iPSCs [16]. However, whether BEF feeder layers could be more beneficial for the primed bovine iPSC cultivation and even converse them to the naïve-like state than MEF feeder are still unknown.

Previously, studies reported that culture of primed ESCs/iPSCs in a naïve medium (NM) generated naïve-like monkey and human ESCs/iPSCs [2,17]. However, it's unclear that whether bovine primed iPSCs can be converse into naïve-like state and generate dome-shaped colonies in NM on MEF and BEF feeders (termed as MNM and BNM conditions thereafter). Based on our previous work [18], In this study, we reported that bovine iPSCs grew better in NM-based medium and formed naïve-like colonies both on MEF and BEF feeders, and BEF feeder improved the conversion efficiency.

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