Purchase this article with an account.
Ahmad Al Moujahed, Bo Tian, Eleni Konstantinou, Haijiang Lin, Demetrios G. Vavvas; The Role of Necroptosis Kinases in the Induction of iPSCs from Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4575.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
The molecular mechanisms involved in induced pluripotent stem cells (iPSCs) derivation are not completely understood. Caspases 3 and 8 , protease enzymes that play essential roles in apoptosis, have been shown to facilitate nuclear reprogramming in iPSCs induction. However, the role of receptor-interacting protein kinases 1 and 3 (RIPK1 and RIPK3), essential regulators of programmed necrosis (necroptosis), in the derivation of iPSCs has not been studied. We wanted to investigate the role of RIPK1 and RIPK3 in iPSCs induction.
Early passage Mouse Embryonic Fibroblasts (MEFs) isolated from wild type (WT), Ripk1, Ripk3, and Ripk3/ Caspase8 knockout mice and from Rip1D138N, in which the kinase domain RIPK1 is inactive, were reprogrammed to iPSCs using a single lentiviral vector (STEMCCA) expressing the four Yamanaka transcription factors Oct-4, Klf4, SOX-2, and c-Myc (OKSM) and the generated iPSCs colonies were quantified using alkaline phosphatase staining. Immunoblot analysis was used to check the expression levels of RIPK1 and RIPK3.
Immunoblot analysis showed that both RIPK1 and RIPK3 were activated during the iPSCs induction process. However, RIPK1 was found to be downregulated, while RIPK3 upregulated in established mature iPSCs compared to MEFs. Deletion of Ripk1 increased the induction of iPSCs, although this increase was not significant. Surprisingly, however, Rip1D138N MEFs or treating WT MEFs with Necrostatin-1, a potent RIPK1 inhibitor, led to about 40% suppression in the frequency of iPSC induction. Interestingly, deletion of RIPK3 dramatically suppressed the induction of iPSCs (about 82%) and this reduction was further enhanced to almost complete suppression (about 95%) in Ripk3/ Caspase8 knockout MEFs.The growth rate of Ripk3 KO MEFs was significantly lower than WT MEFs. Furthermore, RNA-seq analysis and qRT-PCR showed that RIPK3 KO MEFs expressed lower levels of genes that control cell cycle progression (e.g., CDK1, CDK6, CDC6, and CDC7) and higher levels of extracellular matrix genes (e.g., Col3a1, Col8a1, Col12a, and Hapln1) compared to WT MEFs.
RIPK1 and RIPK3 play an important role in nuclear reprogramming during iPSC induction. Rip3 KO was associated with slower growth rate, slow cell cycle progression, and upregulation of many extracellular matrix genes, which can, at least partially, explain the inhibitory effects of RIPK3 deletion on iPSCs induction.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
This PDF is available to Subscribers Only