Early reprogramming regulators identified by prospective isolation and mass cytometry

Abstract

In the context of most induced pluripotent stem (iPS) cell reprogramming methods, heterogeneous populations of nonproductive and staggered productive intermediates arise at different reprogramming time points1–11. Despite recent reports claiming substantially increased reprogramming efficiencies using genetically modified donor cells12,13 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 properties1,2,7,8,10. 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 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 phase2. Expression profiling revealed early upregulation of the transcriptional regulators Nr0b1 and Etv5 in this reprogramming state, preceding activation of key pluripotency regulators such as Rex1, Dppa2, Nanog and Sox2. Both factors are required for the generation of the early intermediate state and fully reprogrammed iPS cells, and thus mark some of the earliest known regulators of iPS cell induction. Our study deconvolutes the first steps in a hierarchical series of events that lead to pluripotency acquisition.

SALL4, the missing link between stem cells, development and cancer

“…Furthermore, SALL4 can interact with the Nucleosome Remodeling and Histone Deacetylation (NuRD) complex 3949 . MBD3, an essential structural component of the NuRD complex, has been shown to be a key regulator in the reprogramming process 50515253 . Though it has been suggested that the SALL4/NuRD interaction may not be required in iPSC reprogramming in a cell fusion model 45 , it remains to be seen whether the same conclusion can be drawn from other approaches in generating iPSCs.…”

From cloned frogs to patient matched stem cells: induced pluripotency or somatic cell nuclear transfer?

“…In contrast, intermediates of iPS reprogramming have a unique molecular signature [21], without an in vivo equivalent. These differences in the path to pluripotency could in principle affect the outcome.…”

Lessons learned from functional assessment of pluripotency-associated transcription factors during early embryogenesis and embryonic stem cells

“…Alternatively, DAX1 contributes to cellular reprogramming by facilitating the reactivation of other pluripotency genes (Zhang et al, 2014;Lujan et al, 2015), although the requirement of its activity to maintain pluripotency remains controversial (Khalfallah et al, 2009;Zhang et al, 2014). The understanding of DAX1 downstream targets in both pluripotent cells and during reprogramming may shed light on how this PATF play such contrasting roles.…”