This paper demonstrates the principle that fibroblasts can be directly converted into induced OPCs by forced expression of just three defined transcription factors. Following the systematic evaluation of 10 candidate transcription factors we identified a minimally required combination of Sox10, Olig2, and Zfp536 that is sufficient to robustly induce OPC-like cells, termed iOPCs. These cells have the typical morphology of OPCs, express the relevant OPC markers O4, NG2 and A2B5, respond to PDGF signaling like OPCs and show a global transcriptional reprogramming towards an OPC-like profile. Notably, they give rise to mature oligodendrocytes that can myelinate host axons in vivo. This demonstrates that iOPCs are functional precursor cells and can execute the highly coordinated and complex cell biological processes involved in myelination, such as axon recognition and attachment, membrane wrapping and compaction. We observed small graft sizes, which was due to the extremely low short-term survival rate of freshly immunopanned cells. The detection of MBP+ cells in all grafted sites argues therefore for a remarkably robust in vivo differentiation capacity of iOPCs.
Several lines of evidence suggest that iOPCs are similar but not identical to primary OPCs. Compared to neonatal OPCs, the fibroblast-derived iOPCs have a lower efficiency of differentiation into mature oligodendrocytes and their gene expression profile is similar to, yet still distinct from that of primary OPCs, although the fibroblast-specific transcriptional network programs appear to be effectively downregulated. These transcriptional differences could be explained by several not mutually exclusive reasons. First, the iOPC cultures (as defined by O4 immunoreactivity) could represent a fairly heterogeneous population of fully and partially reprogrammed cells, similar to what was found in the first reports of induced pluripotent stem (iPS) cells32-34
. However, while the transcriptional profile seems to be more similar to OPCs undergoing differentiation, the elevated mRNA levels of differentiation genes do not result in detectable protein or mature oligodendrocyte morphology, suggesting that they might not be functionally important. In addition, we observed a small group of aberrantly expressed genes unique to iOPCs. These might be direct or indirect targets of the reprogramming factors, which have documented roles in regulating other cell lineages (e.g. Sox10 in neural crest lineages, Olig2 in motor and ventral forebrain neurons). Future studies could address whether these fairly subtle differences may lead to detectable functional consequences.
Notably, we believe it to be highly unlikely that the described iOPCs are derived from contaminating neural crest stem cells or Schwann cell precursor cells. First, we have carefully dissected the tissue before fibroblast generation, minimizing contamination by neural crest progenitor cells. Second, we previously performed an extensive molecular characterization of these primary fibroblast cultures and found no evidence of neural crest or Schwann cell marker expression14
. Third, we have demonstrated that iOPCs differentiate into myelinating cells, that are PLP+
, a marker that is specific to central oligodendrocytes, and P0−
, a specific marker of peripheral Schwann cells. Finally, we found that multiple host axons can be ensheathed by single engrafted cells, which is a typical feature of oligodendrocytes, whereas Schwann cells can only myelinate one axon per cell.
Given the strong clinical interest in OPCs for regenerative therapies, one of the most important next steps is to translate our findings to human fibroblasts. Based on our experience with iPS and iN cells, we predict that generation of human iOPCs is possible but may require additional reprogramming factors, such as other transcriptional regulators or microRNAs15, 35-37
. Substantial optimization will likely be required to reliably generate large numbers of iOPCs form human cells that are functionally indistinguishable from OPCs of the human brain.