After MET, a subset of E-cadherin+ cells proceed to the next reprogramming phase that larger colonies are formed, many embryonic genes regulating housekeeping functions are upregulated (Samavarchi-Tehrani et al., 2010; Sridharan et al., 2009), and ESC makers (e.g. SSEA-1) are induced (Li et al., 2010; Stadtfeld et al., 2008b). Studies tracing the SSEA-1-positive and SSEA-1-negative populations during reprogramming process demonstrate that only SSEA-1-positive cells give rise to faithfully iPS cells and only a few of them can make the final transition.
Lujan et al. (Lujan et al., 2015) preformed a high-dimensional mass cytometry to identify the surface markers of cells at different phases of reprogramming and demonstrated that cells reprogrammed to faithful iPS cells acquire surface marker expression in a stepwise manner. In this study, they performed a systematic functional surface maker screening on the cell lines representing the early, intermediate and late pluripotency stages and identified 21 candidate surface markers, of which the expression was then characterized in primary cells by mass cytometry over the reprogramming process. Consistent with previous reports, they also demonstrate that the transient, intermediate stage is a general property of iPS reprogramming that presents across multiple reprogramming systems. They showed that the intermediate cells can be identified by a unique set of surface markers, including CD73, CD49d and CD200, which are absent in both parental fibroblasts and the generated iPS cells.
Partially reprogrammed (pre-iPS) cells, which have already efficiently silenced somatic genes but have not induced the endogenous pluripotency program, arise from this intermediate stage. These pre-iPS cells have ESC-like colonies morphology, but not yet gained Nanog expression or induced pluripotency-related genes. However, pre-iPS cells are capable of transition to iPS cells with additional treatments, such as inhibition of MAPK and GSK3; TGFb inhibition and Nanog overexpression (Silva et al., 2009; Theunissen et al., 2011).
Studies mapping the binding sites of 4 reprogramming factors in pre-iPS cells revealed that c-Myc is largely engaged at this intermediate phase and many of its target genes have already been bound, while the pluripotency network hasn’t yet been established (Sridharan et al., 2009).