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Neural Stem Cells from iPSCs – Not so Safe After All?

Review of “Neural stem cells differentiated from iPS cells spontaneously regain pluripotency” from Stem Cells by Stuart P. Atkinson.

Virally transfer of reprogramming factors is a widely used strategy to generate induced pluripotent stem cells (iPSCs) and relies on the integration and expression of transgenes which are then silenced after differentiation. Even though many “safer” non-viral methods of gene transduction exist, viruses are still used due to their high efficiency of reprogramming. A recent report in Stem Cells, from the laboratory of Jeong Tae Do (Konkuk University, Seoul, Republic of Korea), has studied transgene expression in neural stem cells (NSCs) differentiated from iPSCs generated via retroviral gene transduction. This study has found that initially silenced transgenes can reactivate spontaneously and mediate a reversion to pluripotency, representing a possible tumorigenic threat [1].

Retroviral transduction of Oct4, Sox2, Klf4, and c‐Myc (OSKM) into mouse embryonic fibroblasts (MEFs) generated iPSCs which the authors then differentiated into NSCs [2], and further differentiated into neurons (Tuj1), astrocytes (GFAP), and oligodendrocytes (O4). The group examined three separate cultures of NSCs, and while two remained NSC-like during long-term culture, one culture changed presenting with aggregated cells. These cells expressed Sox2, but not Nestin, and therefore did not represent neurospheres or NSCs, and after changing culture conditions to that suited for ESCs, an Oct4-GFP transgene reporter became activated (iPSC-NSC-GFP+) followed by endogenous Oct4 and Nanog expression, the detection of two active x chromosomes, and the formation of germline chimeras. Interestingly, this did not occur in the terminally differentiated neuronal cell types, suggesting that this reversion may only occur in stem/progenitor cell types.

The generation of iPSCs is normally associated with the upregulation of three DNA methyltransferases (Dnmt1, 3a and 3b) and the silencing of transgene expression [3], followed by their downregulation as iPSCs differentiate to NSCs. Here, the researchers link this downregulation of Dnmts, and therefore the downregulation of DNA methylation, to the reactivation of the silenced transgenes, after which Dnmts become activated again during the transformation of iPSC-NSCs to iPSC-NSC-GFP+ pluripotent cells (See Figure).

Overall, this suggests that iPSCs generated via virally-mediated transgene transfer are not suitable for therapeutic uses, and also may not be useful in disease modelling or drug testing. The researchers ruled out the existence of a contaminating undifferentiated cell type overall suggesting that the iPSC-derived NSCs could regain pluripotent characteristics and therefore may represent a tumorigenic threat under some circumstances. The researchers do note that terminally differentiated cell types from NSCs did not revert, and so may be a safer cell type for therapeutic uses, but this would however mitigate the immunomodulatory/growth factor support role which stem cells appear to exert after transplantation.

Points to Discuss:

  • Does this represent an immediate threat to stem cell based therapeutics?
  • Do all iPSC-derived stem/progenitor cells suffer in the same way?
  • Is this effect specific to the retroviral system?
  • Would removal of Myc inhibit this effect?
  • Would a different reprogramming cocktail (e.g. Sall4, Nanog, Esrrb, and Lin28 (SNEL)) mitigate the reversion to pluripotency?
  • Does this occur in human iPSCs?

References

  1. Choi HW, Kim JS, Choi S, et al. Neural Stem Cells Differentiated From iPS Cells Spontaneously Regain Pluripotency. Stem Cells 2014;32:2596-2604.
  2. Conti L, Pollard SM, Gorba T, et al. Niche-independent symmetrical self-renewal of a mammalian tissue stem cell. PLoS biology 2005;3:e283.
  3. Wernig M, Meissner A, Foreman R, et al. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 2007;448:318-324.