Alternative Routes to Induced Pluripotent Stem Cells Revealed by Reprogramming of the Neural Lineage

Abstract

SummaryDuring the reprogramming of mouse embryonic fibroblasts (MEFs) to induced pluripotent stem cells, the activation of pluripotency genes such as NANOG occurs after the mesenchymal to epithelial transition. Here we report that both adult stem cells (neural stem cells) and differentiated cells (astrocytes) of the neural lineage can activate NANOG in the absence of cadherin expression during reprogramming. Gene expression analysis revealed that only the NANOG+E-cadherin+ populations expressed stabilization markers, had upregulated several cell cycle genes; and were transgene independent. Inhibition of DOT1L activity enhanced both the numbers of NANOG+ and NANOG+E-cadherin+ colonies in neural stem cells. Expressing SOX2 in MEFs prior to reprogramming did not alter the ratio of NANOG colonies that express E-cadherin. Taken together these results provide a unique pathway for reprogramming taken by cells of the neural lineage.

Zfp281 orchestrates interconversion of pluripotent states by engaging Ehmt1 and Zic2

“…Transcriptional and epigenomic profiling of the reprogramming process has revealed an ordered series of events that include the transient and sequential activation of late and early developmental genes (Takahashi et al , ; Cacchiarelli et al , ; Amlani et al , ). Although the specific trajectory is dictated by the identity of the starting somatic cell type (Jackson et al , ; Nefzger et al , ) and the experimental regime (Chantzoura et al , 2015; Stuart et al , ), iPSC formation may involve the reversion of natural developmental mechanisms (Takahashi & Yamanaka, ). Consistent with this possibility, a mesenchymal‐to‐epithelial transition is necessary for iPSC formation (Li et al , ; Samavarchi‐Tehrani et al , ), while the converse epithelial‐to‐mesenchymal transition is crucial for embryogenesis, e.g., during gastrulation and neural crest formation (Acloque et al , ).…”

Dot1L methyltransferase activity is a barrier to acquisition of pluripotency but not transdifferentiation

“…Strikingly, H3K79me2 is the most differentially enriched modification more abundant in mouse embryonic fibroblasts (MEFs) compared to PSCs (Sridharan et al, 2013). Dot1L was shown to be a barrier for human fibroblast (Onder et al, 2012) and mouse neural stem cell (Jackson et al, 2016) reprogramming Collectively, these phenotypes provide evidence for the importance of Dot1L in cell fate determination; however, the function of H3K79me in mammals has remained elusive.…”

Defining reprogramming checkpoints from single-cell analysis of induced pluripotency

“…We have previously shown that the addition of ascorbic acid (AA) and 2i increases reprogramming efficiency of both embryonic and adult fibroblasts (Tran et al, 2015). A small molecule screen of chemicals (data not shown) revealed that the addition of an inhibitor to the H3K79 methyltransferase, Dot1L, called SGC0946 (Jackson et al, 2016) to the AA+2i combination boosted iPSC generation from reprogrammable MEFs. By day 6, ~1900 Nanog+ iPSC colonies were obtained at an efficiency of ~42% (Methods) ( Fig 1B).…”

“…The chemicals we used for high-efficiency reprogramming include signaling inhibitors and two epigenetic modulatorsascorbic acid, which is thought to regenerate 2-oxoglutarate-dependent epigenetic enzymes (Hore et al, 2016), and SGC, an inhibitor of Dot1L-mediated histone H3K79 methylation (Jackson et al, 2016). To understand the relative contribution of each component, we subjected MEFs to every dual combination of chemicals and assessed reprogramming efficiency on day 6.…”