Abstract
Pluripotent stem cells in the naïve state are highly useful in regenerative medicine and tissue engineering. A robust reprogramming of the primed murine Epiblast Stem Cells (EpiSCs) to naïve pluripotency is feasible via chemical-only approach. This protocol described a method to reprogram murine EpiSCs by MM-401 treatment, which blocks histone H3K4 methylation by MLL1/KMT2A.
Keywords: Naïve pluripotency, EpiSCs, MLL1, H3K4 methylation, Chemical inhibitor
Background
Previous protocols on EpiSC reprogramming depended on the genetic manipulations of transcriptional factors or chemical inhibition of the signaling pathways, albeit with varying efficiency and duration. Based on a recent mechanistic study that links the MLL1 complex to the naïve state, this protocol provides a straightforward and robust method to restore naïve pluripotency from EpiSCs via targeting MLL1 mediated H3K4 methylation and subsequent transcription regulation. The reprogramming efficiency is significantly higher than previous published method, achieving 50% conversion rate in two weeks.
Materials and Reagents
Equipment
Procedure
A schematic summary of the reprogramming procedure described in this protocol can be found in Figure 1. Figure 1. The schematic view of general procedures for the reversion of EpiSCs by MM-401 treatment
Notes
Recipes
Acknowledgments
This protocol was originally published as part of Zhang et al. (2016). The authors wish to thank all present and past members developing/functional testing MM-401 in the Dr. Yali Dou, Dr. Sundeep Kalantry and Dr. Shaomeng Wang laboratories. Special thanks are obligated to Dr. Saunders and Ms. Hughes at the University of Michigan for technical support, and Dr. Brady and Dr. Tesar for sharing materials. This work is supported by National Institute of General Medical Sciences (NIGMS) (GM082856), Leukemia and Lymphoma Society and the National Cancer Institute (CA117307-04) to Dr. Yali Dou.
References
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