Early reprogramming regulators identified by prospective isolation and mass cytometry

Identification of transient early induced pluripotency reprogramming intermediates allows for mechanistic insight into the reprogramming process. Cell markers for iPS cell potential Previous attempts to isolate cells en route to successful induced pluripotent stem (iPS) cell reprogramming have been based on the assumption that cells progressively lose donor cell identity and gradually acquire iPS cell properties. Here Marius Wernig and colleagues identify surface markers appearing early in the process that are not expressed in the original fibroblasts nor in the resulting pluripotent cells. By isolating and profiling the expression of the cells bearing these markers (including CD73, CD49d and CD200), the authors have identified novel transcriptional regulators required for reprogramming, gaining mechanistic insights into the process. In the context of most induced pluripotent stem (iPS) cell reprogramming methods, heterogeneous populations of non-productive and staggered productive intermediates arise at different reprogramming time points 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 . Despite recent reports claiming substantially increased reprogramming efficiencies using genetically modified donor cells 12 , 13 , prospectively isolating distinct reprogramming intermediates remains an important goal to decipher reprogramming mechanisms. Previous attempts to identify surface markers of intermediate cell populations were based on the assumption that, during reprogramming, cells progressively lose donor cell identity and gradually acquire iPS cell properties 1 , 2 , 7 , 8 , 10 . Here we report that iPS cell and epithelial markers, such as SSEA1 and EpCAM, respectively, are not predictive of reprogramming during early phases. Instead, in a systematic functional surface marker screen, we find that early reprogramming-prone cells express a unique set of surface markers, including CD73, CD49d and CD200, that are absent in both fibroblasts and iPS cells. Single-cell mass cytometry and prospective isolation show that these distinct intermediates are transient and bridge the gap between donor cell silencing and pluripotency marker acquisition during the early, presumably stochastic, reprogramming phase 2 . Expression profiling reveals early upregulation of the transcriptional regulators Nr0b1 and Etv5 in this reprogramming state, preceding activation of key pluripotency regulators such as Rex1 (also known as Zfp42 ), Dppa2 , Nanog and Sox2. Both factors are required