Abstract
Induced pluripotent stem cells (iPSCs) have variable expression levels of a series of genes that affect their pluripotent potential, but the regulatory mechanisms controlling reprogramming remain unclear. By testing the efficiency of iPSC generation using Oct4, Sox2, Klf4 (termed OSK) plus one additional gene, we found that Rab32 improved reprogramming efficiency. We established a system for detecting the number and the size of lipid droplets and autophagosomes per cell for tracking their morphological changes during reprogramming. Our results showed that Rab32 increased lipid storage during the early and middle stages, and also increased autophagy during the middle stage of reprogramming. These findings were further confirmed by the up-regulation of lipid biosynthesis and autophagosome formation related genes, of which their expression could improve iPSC induction. The inhibition of lipid biosynthesis and autophagosome formation significantly reduced reprogramming efficiency, and the inhibition of lipid synthesis phenotype could be rescued by the overexpression of Rab32. In addition, the expression of pluripotency genes such as Klf2, Nr5a2 and Tbx3, was up-regulated by Rab32. These results demonstrated that Rab32 could improve the induction of iPSCs through the enhancement of lipid biosynthesis, highlighting the importance of lipid metabolism during reprogramming.
Highlights
Induced pluripotent stem cells can be obtained by the forced expression of four transcription factors, Oct[4], Sox[2], Klf[4], and c-Myc in somatic cells[1,2]
We found that Rab[32] was able to efficiently facilitate Induced pluripotent stem cells (iPSCs) reprogramming, and additional studies were performed to explore the mechanism of this action
To better identify and characterize candidate genes that could facilitate the generation of iPSCs, we cloned a panel of full-length mouse genes into the pMXs retroviral vector[2] and established a 6-well plate-based gene screening system (Fig. 1a) for reprogramming using various factor combinations
Summary
Induced pluripotent stem cells (iPSCs) can be obtained by the forced expression of four transcription factors, Oct[4] ( known as Pou5f1), Sox[2], Klf[4], and c-Myc (termed OSKM) in somatic cells[1,2]. High-quality iPSCs can produce iPSC-derived mice by the tetraploid complementation and undergo germ line transmission, while low-quality iPSCs can only produce chimeras with low coat-color contribution. We selected high-quality iPSC lines such as the OSKT cell line (which was generated by the factors Oct[4], Sox[2], Klf[4] and Tbx3) and the SKR cell line (Sox[2], Klf[4] and Nr5a2) and low-quality iPSC lines such as OSK (Oct[4], Sox[2] and Klf4) and SKRM (Sox[2], Klf[4], Nr5a2 and c-Myc), which have been reported in our previous studies[8,9]. To uncover factors that improve the reprogramming efficiency, candidate genes were selected based on their higher expression levels in high-quality iPSCs. We assessed the efficiency of iPSC generation using OSK plus one additional candidate gene. The inhibition of lipid synthesis and autophagy formation by specific inhibitors could significantly reduce iPSC colony numbers, underscoring the importance of lipid metabolism during reprogramming
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