Epigenetic mechanisms preside over our genetic information to enable development from the fertilized, totipotent oocyte to the adult body. The astonishing reprogramming experiment published by Shinya Yamanaka in 2006 demonstrates the profound flexibility of the mammalian epigenome: in less than one month's time, a handful of transcription factors can reprogram differentiated mouse cells back to a pluripotent state, referred to as induced pluripotent stem (iPS) cell state 1
. Only one year, after the publication of this seminal study using mouse cells, human iPS cells were generated with very similar combinations of transcription factors 2, 3, 4, 5
. Because human and mouse iPS cells represent an inexhaustible source of cells, highly similar to embryonic stem (ES) cells, the Yamanaka era of stem cell biology is driven by tremendous medical interest. Patient-specific pluripotent cells have already been created and will hopefully be used as substrates for modeling disease pathogenesis and provide immune-matched sources for cell or tissue grafts 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12
The Yamanaka screening strategy to find factors that can induce pluripotency is surprisingly simple and affordable 1
. The first reprogramming experiment involved retroviral-mediated overexpression of two dozen well-defined pluripotency regulators in mouse embryonic fibroblasts, and led to emergence of cells that morphologically resemble ES cells upon selection for expression of a resistance gene inserted into the Fbx15
locus, which encodes an ES cell-specific gene. Subsequent experiments, in which factors were dropped from the original mix, showed that induction of pluripotency is more efficient when only four factors, Oct4, Sox2, Klf4 and c-Myc, are co-expressed in fibroblasts 1
. A characterization of the resulting iPS cell clones demonstrated, however, that not all of the genes typically expressed in ES cells were strongly upregulated. In agreement with this notion, these original iPS cells self-renewed and differentiated into diverse cell types of all three germ layers, but did not support adult chimerism upon blastocyst injection. Subsequent improvements of methods for the selection of faithfully reprogrammed cells allowed the derivation of iPS cells that are able to contribute to all three germ layers and the germline in mice 13, 14, 15
, bringing them closer to the developmental potential of mouse ES cells. Some newer mouse iPS cell lines can even generate purely iPS cell-derived animals by tetraploid complementation, which is the most stringent pluripotency test available 16, 17, 18, 19, 20
. Many mouse and human iPS cell lines induced by overexpression of Oct4, Sox2, Klf4 and c-Myc were extensively characterized at the molecular level, and are similar to ES cells in their expression and chromatin signatures 15, 21, 22, 23, 24
. Thus, reprogramming leads to the silencing of somatically expressed genes and upregulation of ES cell genes, concomitant with the resetting of chromatin structure.
To understand the reprogramming process, one could look at the role that Oct4, Sox2, Klf4 and c-Myc play in ES cells. These transcription factors are all important for the establishment and/or maintenance of pluripotent state during early embryonic development (see recent review 25
for further reading about their function). Importantly, Oct4, Sox2 and Klf4 are thought to maintain the pluripotent, self-renewing state of ES cells by co-occupying the promoter and enhancer regions of a large set of highly expressed ES cell-specific genes, often referred to as pluripotency genes 26, 27, 28, 29, 30
. Co-occupancy of Oct4, Sox2 and Klf4, is often predictive for co-occupancy by Nanog, another ES cell-specific transcription factor 21, 27, 29, 30, 31
. Thus, it has been suggested that Oct4, Sox2 and Klf4 cooperate over the course of reprogramming to establish functional enhancosomes required for upregulation of the ES cell-specific transcriptome. In contrast, solitary binding of these factors in ES cells is generally associated with transcriptional repression and this may explain how Oct4, Sox2 and Klf4 are able to silence somatic gene expression early in the course of reprogramming. In contrast, c-Myc, a well-known oncogene and cell cycle regulator, has a largely distinct set of target genes from Oct4, Sox2 and Klf4 in ES cells, including numerous cell cycle, metabolism genes etc., thus, forming a separate transcriptional network 28, 29, 32
. Though c-Myc can co-occupy some target genes with Oct4, Sox2 and Klf4, it is believed that these transcription factors constitute two largely separate transcriptional networks in ES cells 32
. Interestingly, ectopic c-Myc is dispensable for the creation of iPS cells, but acts as an enhancer of kinetics and efficiency of reprogramming 33, 34
, supporting the idea that pluripotency gene activation does not directly depend on c-Myc.
In this review, we will discuss the current knowledge of how the reprogramming factors accomplish the mammoth change in gene expression leading to iPS cell induction. Recent reprogramming reviews cover the historic events that led to the iPS cell reprogramming strategy, improved reprogramming methods and disease modeling with iPS cells in depth 35, 36
. Notably, new reprogramming methods that convert one differentiated cell into another, without establishing an intermediate pluripotent state (lineage conversion) 37, 38, 39
, point us to alternative approaches of induced cell fate change that we will discuss at the end.