Abstract
SummaryInduced pluripotency requires the expression of defined factors and culture conditions that support the self-renewal of embryonic stem (ES) cells [1]. Small molecule inhibition of MAP kinase (MEK) and glycogen synthase kinase 3 (GSK3) with LIF (2i/LIF) provides an optimal culture environment for mouse ES cells [2] and promotes transition to naive pluripotency in partially reprogrammed (pre-iPS) cells [3]. Here we show that 2i/LIF treatment in clonal lines of pre-iPS cells results in the activation of endogenous Nanog and rapid downregulation of retroviral Oct4 expression. Nanog enables somatic cell reprogramming in serum-free medium supplemented with LIF, a culture condition which does not support induced pluripotency or the self-renewal of ES cells, and is sufficient to reprogram epiblast-derived stem cells to naive pluripotency in serum-free medium alone. Nanog also enhances reprogramming in cooperation with kinase inhibition or 5-aza-cytidine, a small molecule inhibitor of DNA methylation. These results highlight the capacity of Nanog to overcome multiple barriers to reprogramming and reveal a synergy between Nanog and chemical inhibitors that promote reprogramming. We conclude that Nanog induces pluripotency in minimal conditions. This provides a strategy for imposing naive pluripotency in mammalian cells independently of species-specific culture requirements.
Highlights
Transfer and passaging in serum-free 2i/LIF medium generated a culture of iPS cells with uniform Oct4-GFP reporter activity (Figure 1A) and the capacity to contribute to adult mice
Individual GFP events in pre-iPS cells, were significantly less intense than in iPS cells obtained from the same clones in 2i/LIF
GFP-positive pre-iPS cells expressed Fgf4 and Nr0b1, which are recurrently detected in partially reprogrammed cells [3, 4]
Summary
Transfer and passaging in serum-free 2i/LIF medium generated a culture of iPS cells with uniform Oct4-GFP reporter activity (Figure 1A) and the capacity to contribute to adult mice (see Figure S1A available online). These results demonstrate that weak Oct4-GFP activity in clonal lines of pre-iPS cells in serum/LIF is not a sign of complete reprogramming. Switching to serum-free medium with LIF alone did not give rise to stable Oct4-GFP reporter activity (Figure 1I).
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