Year 2

We proposed to generate human induced pluripotent stem (iPS) cells using a virus-free technique called RNA activation (RNAa). RNAa is a newly identified gene regulation mechanism by which promoter-targeting double-stranded small RNA also known as small activating RNA (saRNA) can induce gene expression. Our approach to iPS cell derivation is through simultaneous activation of stem cell factors including OCT4, NANOG, SOX2 and KLF4 using RNAa. Currently we are focusing on Aim 1 and 2 of this project to screen saRNAs that can activate individual stem cell factors and to use the identified saRNAs to replace one viral factor at a time to reprogram iPS cells from somatic cells. We have identified saRNAs for OCT4, KLF4 and NANOG. Replacing OCT4 virus in the OSKM four factor reprogramming recipe (OCT4, SOX2, KLF4 and MYC) with an OCT4 saRNA led to the derivation of iPS-like colonies from adipose tissue derived stem cells (ADSCs). These results provided proof of principle that saRNAs can be used for iPS reprogramming. Ongoing research is identifying saRNAs for additional stem cell factors and maximizing gene activation by optimizing iPS reprogramming protocol. Eventually all viruses will be replaced with their corresponding saRNAs to generate virus-free iPS cells.