Reprogramming of the somatic epigenome to a pluripotent, embryonic state through ectopic expression of the four transcription factors Klf4, Sox2, c-Myc and Oct4 is a slow and inefficient process. The current method for induction of reprogramming is through retroviral gene delivery, which results in heterogeneous cell populations with proviral integrations that vary in both number and genomic location, offering an explanation for the variability and inefficiency of direct reprogramming.
Here we describe a system for reprogramming genetically homogeneous cell populations. Reprogramming with dox-inducible lentiviral vectors and subsequent chimera formation yields tissues comprised of genetically homogeneous cells that harbor identical proviral integrations and re-express the reprogramming factors upon exposure to dox. This strategy selects for cells that carry the correct number of proviruses inserted at genomic loci that are favorable to drug-induced activation and eliminates the heterogeneity inherent in de novo
viral infection of target cells. Surprisingly, the timing of reprogramming in this system was similar to that of directly infected primary fibroblasts. The minimum length of time that dox was required to initiate reprogramming ranged from 9 to 13 d. This timescale is consistent with the 10- to 14-d time frame observed in cells that have been directly infected with vectors13,14
. We also observed that when dox was withdrawn from the cultures as early as day 9, GFP+
secondary iPS cell colonies continually appeared for the next several weeks in the absence of dox. These results support the notion that reprogramming is driven by a stochastic sequence of epigenetic modifications requiring a minimum period of transgene expression.
The observed reprogramming efficiency of secondary MEFs was as high as 4%, which is similar to the reprogramming efficiency of mature B cells22
, vastly higher than the estimated 0.1% efficiency using de novo
infection and drug selection, and about eightfold higher than what has been reported using morphological selection criteria1,11,12
. It has been well documented that iPS cells derived from infected MEFs carry on average 15 different proviral copies, suggesting strong selection for the small fraction of the infected cells that carry the ‘correct’ number of proviruses or that express the four factors with the appropriate stoichiometry for successful reprogramming. Thus, the reprogramming frequency of secondary MEFs would be expected to be higher because these cells have been clonally derived from infected cells that carried the ‘correct’ combination of proviruses. If so, why would 4% but not most, or all, dox-treated secondary cells give rise to secondary iPS cells? We consider two non–mutually exclusive explanations. (i) It has been established that genetically identical subclones of directly infected MEFs become reprogrammed at significantly different times or not at all11,20
. As discussed previously, this suggests that reprogramming involves a sequence of stochastic events such that cells carrying an identical number of proviral copies will activate the endogenous pluripotency genes at different times. (ii) Our data also show that dox treatment does not activate the proviruses uniformly in all cells but rather that differences in induction levels exist between individual cells. Because of these variegated expression levels, only a fraction of secondary MEFs may achieve high enough expression levels of the factors or the correct relative expression levels between the factors and therefore be capable of generating secondary iPS cells.
Whereas reprogramming is induced by viral transduction of the four factors, the maintenance of the pluripotent state depends on reestablishment of the autoregulatory loop involving the activation of the four endogenous pluripotency factors Oct4, Nanog, Sox2 and Tcf3 (refs. 20,23
) and silencing of exogenous factors. Similarly, secondary MEFs were capable of being fully reprogrammed to a pluripotent state that was maintained in the absence of transgene expression.
We also used the secondary system to examine the reprogramming potential of several additional adult somatic cell types. iPS cells could be derived from many other tissues, including brain, epidermis, intestinal epithelium, mesenchymal stem cells, tail tip fibroblasts, kidney, muscle and adrenal gland through dox treatment, indicating that the proviruses were appropriately activated in cell types other than MEFs. This demonstrates that the four reprogramming factors can mediate epigenetic reprogramming in cells with different developmental origins and epigenetic states and highlights the usefulness of the secondary system for the study of reprogramming in a broad range of cell types. Although special care was taken to avoid other contaminating cell types, we cannot unequivocally demonstrate the cells of origin of iPS cells from these various tissue types. Genetic lineage tracing experiments have in fact demonstrated that iPS cells can be derived from liver and pancreas cells after transduction with Oct4
). However, not all cell types are permissive to reprogramming by these four factors. We have shown that reprogramming of mature but not of immature B cells requires the transduction of an additional factor (c/EBP-alpha) or the inhibition of the B cell–specific transcription factor Pax5 (ref. 22
). It is possible that additional and as yet unknown factors are required to reprogram certain cell types. One practical advantage of the system described here is that cell types including those that might be refractory to ex vivo
culture and retroviral infection, such as intestinal epithelial cells, can be studied.
Our drug-inducible reprogramming system demonstrates predictable and highly reproducible kinetics and efficiencies () that should facilitate the study of early molecular events leading to epigenetic reprogramming. In addition, the genetic homogeneity of secondary cell types increases the feasibility of chemical and genetic screening to enhance reprogramming efficiency. For example, we demonstrate that the DNA demethylating agent 5-Aza-deoxycytidine substantially enhances reprogramming efficiency. Such screens could also be applied to identify compounds that replace the original reprogramming factors. Because the reprogrammed state is not dependent on the exogenous factors, the transgenes could be genetically excised, and secondary cells that lack a particular reprogramming factor could be generated by chimera formation15