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Main Text
Induced pluripotent stem cell (iPSC) technology, i.e. reprogramming somatic cells into pluripotent cells that closely resemble embryonic stem cells (ESCs) by introduction of defined transcription factors (TFs), holds great potential in biomedical research and regenerative medicine (Takahashi and Yamanaka, 2006, Takahashi et al., 2007 and Yu et al., 2007). Various strategies have been developed to generate iPSCs with fewer or no exogenous genetic manipulations, which represent a major hurdle for iPSC applications (Yamanaka, 2009). With the ultimate goal of generating iPSCs with a defined small molecule cocktail alone, substantial effort and progress have been made in identifying chemical compounds that can functionally replace exogenous reprogramming TFs and/or enhance the efficiency and kinetics of reprogramming (Shi et al., 2008, Huangfu et al., 2008, Lyssiotis et al., 2009, Ichida et al., 2009, Maherali and Hochedlinger, 2009, Lin et al., 2009, Li et al., 2009 and Esteban et al., 2010). To date, only neural stem cells (NSCs), which endogenously express SOX2 and cMYC at a high level, have been reprogrammed to iPSCs by exogenous expression of just OCT4 (Kim et al., 2009). However, human fetal NSCs are rare and difficult to obtain. It is therefore important to develop reprogramming conditions for other more accessible somatic cells. Here we report a small molecule cocktail that enables reprogramming of human primary somatic cells to iPSCs with exogenous expression of only OCT4. In addition, mechanistic studies revealed that modulation of cell metabolism from mitochondrial oxidation to glycolysis plays an important role in reprogramming.
Among several readily available primary human somatic cell types, keratinocytes can be isolated easily from human skin or hair follicle, and therefore represent an attractive cell source for reprogramming. Keratinocytes also endogenously express KLF4 and cMYC, and can be reprogrammed efficiently using the conventional four TFs or three TFs (without cMYC) ( Aasen et al., 2008 and Maherali et al., 2008). More recently, we reported that dual inhibition of TGFβ and MAPK/ERK pathways using small molecules (SB431542 and PD0325901, respectively) provided significantly enhanced conditions for reprogramming of human fibroblasts with the four TFs (i.e., OSKM) ( Lin et al., 2009). We have also shown that this dual pathway inhibition can also enhance reprogramming of human keratinocytes by two exogenous TFs (i.e., OK) with two small molecules, Parnate (an inhibitor of lysine-specific demethylase 1) and CHIR99021 (a GSK3 inhibitor) ( Li et al., 2009). With a goal of OCT4-only reprogramming, we developed a stepwise strategy for refining reprogramming conditions and identifying additional small molecules that enhance reprogramming.
We first attempted to further optimize the reprogramming process using four or three TFs (i.e., OSKM or OSK) in neonatal human epidermal keratinocytes (NHEKs) by testing various inhibitors of TGFβ and MAPK pathways at different concentrations using previously reported human iPSC characterization methods (Lin et al., 2009). Encouragingly, we found that the combination of 0.5 μM A-83-01 (a more potent and selective TGFβ receptor inhibitor) and 0.5 μM PD0325901 was more effective than previous small molecule combinations at enhancing reprogramming of human keratinocytes transduced with OSKM or OSK (Figure 1A). Remarkably, when we reduced the viral transduction to only two factors (OK), we could still generate iPSCs from NHEKs when they were treated with 0.5 μM A-83-01 and 0.5 μM PD0325901, although with low efficiency. We then began screening additional small molecules from a collection of known bioactive compounds at various concentrations as previously reported (Shi et al., 2008). Among more than 50 compounds tested, we found that a small molecule activator of 3′-phosphoinositide-dependent kinase-1 (PDK1), PS48 (5 μM), which has not previously been reported to have reprogramming activity, can enhance reprogramming efficiency by about 15-fold. Interestingly, we also found that 0.25 mM sodium butyrate (NaB, a histone deacetylase inhibitor) is much more reliable and efficient than the previously reported 0.5 mM VPA for the generation of iPSCs under OK conditions (Figure 1B). Subsequent follow-up studies demonstrated that a combination of 5 μM PS48 and 0.25 mM NaB could further enhance the reprogramming efficiency over 25-fold (Figure 1B).
