We have generated a doxycycline-inducible lentiviral system to transiently express the four reprogramming factors c-myc, Klf4, Oct4 and Sox2 in fibroblasts. iPS cells produced with this system are pluripotent and give rise to chimeras when injected into blastocysts, similar to retrovirally-produced iPS cells. We and others have recently shown that drug selection is not required to obtain iPS cells (Blelloch et al., 2007
; Maherali et al., 2007
; Meissner et al., 2007
). However, omission of drug selection increases the number of false positive colonies, such as transformed cells or cells that failed to receive all four viruses, and necessitates a trained eye to identify ES cell-like colonies. A major advantage of the inducible system over constitutive expression systems is that it allows for the “self-selection” of reprogrammed cells in the absence of drug selection and obviates the need for ES cell expertise. After stably reprogrammed cells have been generated and doxycycline has been withdrawn, cells that survive go on to reactivate the endogenous pluripotency program, while unstable reprogramming intermediates and transformed colonies disappear, likely through differentiation or apoptosis.
We have used the doxycycline-dependent system to assess the temporal requirement for exogenous factor expression to produce iPS cells. Our results show that the four factors are required for only about ten days, at which point the somatic genome becomes poised for conversion into a pluripotent state. This has been consistently seen in independent experiments using fibroblasts from different reporter strains, suggesting that reprogramming follows a defined rather than a random sequence of molecular events that transform a differentiated cell into a pluripotent cell. In agreement with this notion, we have identified and characterized cell populations that appear prior to and during this transition point by surface marker expression, reporter gene activity and transcriptional analyses (). Based on these results, we conclude that downregulation of fibroblast markers such as Thy1 is the earliest detectable change in cells undergoing reprogramming, followed by modest reactivation of the ES cell marker SSEA-1, which is further upregulated at the time when cells become doxycycline-independent. At this transition point, other major modifications take place including the reactivation of Oct4 and Sox2, telomerase, and the silenced X chromosome. Importantly, reactivation of these markers is not yet indicative of a fully reprogrammed phenotype since continuous expression of exogenous factors can still increase the number of iPS colonies. Interestingly, the ES cell specific Fbx15
gene is reactivated at day 3, before other pluripotency genes including Nanog
become detectable. This may explain why the initially derived iPS cells made with an Fbx15 selectable allele (Takahashi and Yamanaka, 2006
) appeared incompletely reprogrammed compared with the subsequently reported Nanog selection approach (Maherali et al., 2007
; Okita et al., 2007
; Wernig et al., 2007
). Support for the functional importance of the markers described here comes from the observation that FACS sorting of subpopulations defined by these markers allows for a significant enrichment for cells with the potential to become iPS cells.
Kinetics of marker expression during reprogramming.
Retroviral silencing during reprogramming appears to be a gradual process that occurs as early as four days after the induction of the transcription factors but is completed only in cells that have reached the transition point of doxycycline independence. In accordance with the observed early retroviral silencing is the transient upregulation of the de novo
while silencing at later stages coincides with the upregulation of Dnmt3b
(Okano et al., 1999
) and the retroviral silencing factor TRIM28 (Wolf and Goff, 2007
) . This finding implies that the frequency of obtaining iPS cells with retroviruses should be reduced due to premature silencing in some cells. Indeed, we have observed that infection of fibroblasts with lentiviruses, which are not subject to silencing, gives rise to ES-like colonies roughly 5 to 10 times more efficiently compared with retroviruses (unpublished observations).
The molecular mechanism underlying nuclear reprogramming remains enigmatic. Due to its low efficiency, it has thus far been impossible to capture intermediate stages of the reprogramming process. Our identification of intermediate cell population that are enriched for iPS-producing cells will help to solve some of the open questions surrounding nuclear reprogramming. For example, a genome-wide analysis of individual subpopulations at different stages of the reprogramming process will be instrumental for understanding how the three or four transcription factors induce pluripotency and epigenetic reprogramming. Lastly, the system described here provides the framework for the identification of genes and chemicals that replace or enhance the production of iPS cells.