Singh et al. reply

Abstract

Replying to: H. F. Jørgensen & A. G. Fisher Nature 467, doi:10.1038/nature09305 (2010)

Jørgensen and Fisher¹ suggest that the discrepancy with our results² could be attributable to our use of E14Tg2a (or its derivatives, such as OS25) rather than E14Tg2a.4 as the parental control line for the REST^+/− cells (YHC and RRC). We have now reconfirmed our use of E14Tg2a.4 clonal cells as the control cells. Also, we have found that the YHC and RRC cells used in our experiments², originally purchased from Bay Genomics, differ from the YHC and RRC cells used by Jørgensen and Fisher¹ with respect to pluripotency-based on alkaline phosphatase/self-renewal assays (N. Song and S.K.S., unpublished results). We are currently using other assays to confirm these observations.

Main

We have discovered that the original YHC and RRC cells from Bay Genomics were expanded without drug selection at MMRRC before being made available for distribution; these were the cells used by Jørgensen and Fisher¹. Such propagation of mutant cells under self-renewal culture conditions could create an adaptive response, in which mutant cells produce changes to dampen the REST-mediated pathway and activate counteracting mechanisms to increase the self-renewing cell population.

To eliminate the effect of adaptive response of mutant cells to growth conditions, we grew wild-type E14Tg2a.4 mouse ES cells² and knocked-down Rest with small interfering RNA or short hairpin RNA. The results indicated a clear dependence of pluripotency of these cells on REST protein and that this pathway involved microRNA21, supporting our original observation².

We therefore believe that growth conditions such as media, duration of growth and presence of fibroblasts can influence REST’s role in pluripotency. We would welcome exchange of cells and information on culture conditions to help clarify the situation.