Year 4 (NCE)
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 targeting gene regulatory elements known as promoters using small activating RNA (saRNA) can induce gene activity. Our strategy of iPS cell derivation relies on the use of saRNAs to stimulate the endogenous expression of several stem cell factors including OCT4, NANOG, SOX2, KLF4 and C-MYC which are known to be able to induce iPS cells if they are forced to express using viral vectors. We took a three-step approach involving first screening saRNAs that could activate individual stem cell factors, then using the identified saRNAs to replace one viral factor at a time, and eventfully replace all viral factors to reprogram iPS cells from somatic cells. We have identified saRNAs for OCT4, KLF4, NANOG and C-MYC. 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). We also found that a NANOG saRNA which induces NANOG expression can antagonize retinoic acid-induced differentiation of stem cells. These results provided proof of principle that saRNAs can be used for cell fate manipulation. Despite promising results have been achieved, significant obstacles need to be circumvented before we reach our final goal of deriving virus-free iPS cells. Ongoing research is optimizing saRNA-induced reprogramming by tweaking saRNA design and iPS reprogramming protocols and use of small molecule compounds. Alternative strategy of direct conversion of one cell type to another by RNAa is also being tested.