Poster Presentation 6th Annual Meeting for Australasian Society for Stem Cell Research 2013

Direct Generation of Neural Precursors Following Sox2/Pax6 Transduction of Adult Human Fibroblasts (#141)

Rui Liu 1 , Erin Firmin 1 , Christof Maucksch 1 2 , Kathryn Jones 1 , Bronwen Connor 1
  1. Department of Pharmacology and Clinical Pharmacology, University of Auckland, Auckland, New Zealand
  2. ethris GmbH, Martinsried, Germany

Somatic cell reprogramming is an innovative field with considerable potential to enhance our understanding of neural development. We have demonstrated that adult human dermal fibroblasts (aHDFs) can be directly converted to neural precursors by over-expression of the transcription factors SOX2 and PAX6, using non-viral plasmid transfection 1 . Induced neural precursors (iNPs) express a wide range of neural stem/precursor and pro-neural genes and upon differentiation give rise to GFAP+ astrocytes and functionally mature neurons expressing TUJ1, MAP2, NSE, and subtype specific markers TH, calbindin, DARRP32 and GAD65/67. The aim of the current study was to compare the use of lentiviral (LV) gene delivery to non-viral plasmid delivery for the generation of iNPs. We also investigated whether both SOX2 and PAX6 is required to induce neural precursors with the potential to generate mature neurons. Transduction of aHDFs with LV vectors expressing SOX2-zsGreen and PAX6-tdTomato increased the rate of iNP colony formation compared to non-viral transfection (~21 days vs ~45 days). In addition, qPCR analysis confirmed LV-induced iNPs expressed a comparable range of neural precursor and pro-neural genes as plasmid-induced iNPs. To compare the transcriptional requirement for neural precursor induction, FACS was used to sort LV-transduced cells expressing either: SOX2 only, PAX6 only or both SOX2 and PAX6. Following reprogramming and neuronal differentiation, we observed that cells expressing both SOX2 and PAX6, or SOX2 only exhibited a mature neuronal morphology with complex dendritic branches than cells expressing PAX6 only or negative controls which appeared rounded with minimal processes. The expression of mature neuronal markers for each of the sorted cell populations has been examined and will be discussed in detail. These results suggest that co-expression of both SOX2 and PAX6 is required to direct the generation of mature neurons from iNPs. Further, the results support the concept that SOX2 has a critical role in direct reprogramming of fibroblasts into a neural lineage, potentially acting as a “master regulator” 2 .

  1. Maucksch, C., E. Firmin, et al. (2012). "Non-Viral Generation of Neural Precursor-like Cells from Adult Human Fibroblasts." Journal of Stem Cells & Regenerative Medicine 8: 162-170.
  2. Maucksch, C., K. Jones, et al. (2013). "Concise Review: The Involvement of SOX2 in Direct Reprogramming of Induced Neural Stem/Precursor Cells." Stem cells translational medicine.