Smad6-Mediated Suppression of TGF-β/BMP Signaling Facilitates Oocyte Extract-Induced Reprogramming

Stem cells serve as foundational seed cells in regenerative medicine, yet their limited availability remains a significant constraint. The emergence of somatic cell reprogramming has provided a promising alternative for generating pluripotent stem cells. However, challenges such as low efficiency, unpredictable outcomes, and safety concerns persist, largely due to an incomplete understanding of the underlying molecular mechanisms, particularly the dynamic regulation of gene networks during reprogramming. In our previous work, we developed a novel reprogramming strategy using extracts from fish oocytes, which successfully induced induced multipotent stem cells (iMS) with robust differentiation potential by activating endogenous pluripotency circuits and stabilizing epigenetic states. This approach offers a safer and more efficient platform for regenerative applications. In this study, we investigated the temporal gene expression changes and signaling dynamics during fish oocyte extract-induced reprogramming of human skin fibroblasts (HSFs). Through transcriptomic analysis, we identified key genes and pathways involved in this process, with a focus on Smad6, a critical inhibitor of the TGF-β/BMP pathway. Our results demonstrate a biphasic "inhibition-then-activation" pattern of TGF-β/BMP signaling during reprogramming. Overexpression of Smad6 suppressed TGF-β/BMP signaling and enhanced the expression of core pluripotency markers, including Oct4 and Nanog. In contrast, knockdown of Smad6 activated the pathway and inhibited pluripotency marker expression. Mechanistic studies revealed that Smad6 blocks the phosphorylation and nuclear translocation of Smad1/5/8 and Smad2/3, thereby alleviating their transcriptional repression on the core pluripotency network. These findings uncover a key molecular mechanism by which Smad6-mediated suppression of TGF-β/BMP signaling licenses somatic cell reprogramming, providing new insights and potential targets for improving reprogramming strategies.