Rab32 Enhances Mouse IPSCs Induction By Promoting Lipid Synthesis

The direct reprogramming of differentiated somatic cells to induced pluripotent stem cells (iPSCs) can be realized through the overexpression of a set of defined transcription factors such as Oct4 (O), Sox2 (S), Klf4 (K), c-Myc (M) and so on. iPSCs technology is a breakthrough in the stem cells fields, which makes obtaining pluripotent stem cells without using the eggs and embryos, so iPSCs technology avoids the ethical problems. Therefore, iPSCs have greater application potential in human medicine in the future. However, as the research continues, scientists have found that there are quality differences among these iPSCs, and the molecular mechanism is not clear. To solve this problem, we chose different quality of iPSCs and embryonic stem cells (ESCs) (High-quality iPSCs/ESCs can produce chimeras with germ line transmission, while low-quality iPSCs/ESCs cannot form a chimera) to carry out the transcriptome analysis by microarray assay, then selected the genes may affect the iPSCs quality or reprogramming efficiency as candidate genes. We then detected the generation efficiency of iPSCs induction by OSK plus one of the candidate genes.The screening results showed that Rab32 improved the OG2 MEFs (Oct4 promoter driven GFP expression) reprogramming efficiency. The same results can be observed in TF4 MEFs (cells express OSKM when they are cultured in medium with Dox). When the expression level of Rab32 was knockdown in TF4 MEFs, the efficiency of iPSCs induction significantly decreased. The OSKR iPSCs (iPSCs generated with O, S, K and Rab32) are pluripotent and can differentiate into three germ line layers in vitro and in vivo. Furthermore, the chimera mice were obtained. Autophagosomes and lipid droplets number were increased when Rab32 was overexpressed in MEFs. So we tested the changes of autophagy formation and lipid droplets during reprogramming. First, we established a detection system, the system can detect the cell organelle morphological changes in transforming cells. Second, we detected the morphology of autophagy and lipid droplets during reprogramming with OSK, OSKR, OSKM (O, S, K and c-Myc). Finally, the expression levels of genes which regulate autophagy and lipid metabolism were detected. Results show that it needs to consume large amounts of lipid droplets for iPSCs induction in early and middle stages of reprogramming, and Rab32 can improve lipid synthesis at early and middle stages. In addition, Rab32 can promote autophagosomes formation at middle stage of reprogramming. The addition of autophagy and lipid synthesis inhibitors (SP600125 and TOFA) in the process of reprogramming could inhibit the formation of iPSCs, but Rab32 only rescued the inhibition caused by TOFA. Thus, Rab32 can provide more lipid substances for cells, thereby improving the efficiency of cell reprogramming. During reprogramming, the pluripotent genes are gradually expressed and the regulatory network is established, then the stable iPSCs cell lines are formed. The results of pluripotent genes expression showed that Rab32 upregulated expression of c-Myc, Tbx3, Klf2 and Nr5a2 during reprogramming, and the same results can be observed in ESCs overexpressed Rab32.In summary, both autophagy and lipid metabolism play very important roles during reprogramming, and Rab32 can improve reprogramming efficiency through regulating of lipid metabolism and pluripotent genes expression.