Protracted Fate Acquisition and Epigenetic De‐Aging During Induced Neural Stem Cell Conversion of Human Blood Cells

ABSTRACT Transcription factor‐based direct conversion of somatic cells represents an interesting avenue for generating induced neural stem cells (iNSCs) from peripheral blood without transit through a pluripotent stage. While this paradigm has been shown to be associated with epigenetic de‐aging, the dynamics of this process have remained unclear. Here, we used overexpression of the two reprogramming factors SOX2 and cMYC to generate iNSCs from erythroid progenitors of donors ranging from neonatal to 101 years of age. Using an epigenetic aging clock algorithm, we corroborated our previous finding that iNSCs generated from aged donors show pronounced epigenetic de‐aging, preserving around 13% and 5% of the original donor age at low and high passages, respectively. Studying the dynamics of epigenetic de‐aging during iNSC conversion across time, we found that this process is largely protracted, continuing for several weeks and even under proliferation‐inhibiting conditions. Transcriptomic differences between young and old donor‐derived iNSCs dissipate with extended time in conversion, too. Concordant with this observation, established iNSC lines lack age‐associated cellular hallmarks, similar to induced pluripotent stem cells and their derivatives. Interestingly, time course analysis of DNA methylation and RNA sequencing data revealed that acquisition of a bona fide NSC signature extends greatly beyond the time point when proliferative PAX6‐positive iNSCs emerge. The unexpected slow dynamics of these processes make iNSC conversion an attractive model for dissecting the mechanisms underlying somatic transdifferentiation and epigenetic de‐aging.