Plasmid-Based Generation of Induced Neural Stem Cells from Adult Human Fibroblasts

Abstract

Direct reprogramming from somatic to neural cell types has become an alternative to induced pluripotent stem cells. Most protocols employ viral expression systems, posing the risk of random genomic integration. Recent developments led to plasmid-based protocols, lowering this risk. However, these protocols either relied on continuous presence of a variety of small molecules or were only able to reprogram murine cells. We therefore established a reprogramming protocol based on vectors containing the Epstein-Barr virus (EBV)-derived oriP/EBNA1 as well as the defined expression factors Oct3/4, Sox2, Klf4, L-myc, Lin28, and a small hairpin directed against p53. We employed a defined neural medium in combination with the neurotrophins bFGF, EGF and FGF4 for cultivation without the addition of small molecules. After reprogramming, cells demonstrated a temporary increase in the expression of endogenous Oct3/4. We obtained induced neural stem cells (iNSC) 30 days after transfection. In contrast to previous results, plasmid vectors as well as a residual expression of reprogramming factors remained detectable in all cell lines. Cells showed a robust differentiation into neuronal (72%) and glial cells (9% astrocytes, 6% oligodendrocytes). Despite the temporary increase of pluripotency-associated Oct3/4 expression during reprogramming, we did not detect pluripotent stem cells or non-neural cells in culture (except occasional residual fibroblasts). Neurons showed electrical activity and functional glutamatergic synapses. Our results demonstrate that reprogramming adult human fibroblasts to iNSC by plasmid vectors and basic neural medium without small molecules is possible and feasible. However, a full set of pluripotency-associated transcription factors may indeed result in the acquisition of a transient (at least partial) pluripotent intermediate during reprogramming. In contrast to previous reports, the EBV-based plasmid system remained present and active inside the cells at all time points.

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“…Using this excisable reprogramming vector system, the authors could derive transgene-free iNSCs by direct conversion of human adult peripheral blood monocytes, as well as dermal and fetal pancreas fibroblasts. Similar oriP/EBNA of EBV-based episomal vectors were used to express OKSM and LIN28 in combination with a small hairpin against p53 in adult human fibroblasts [42] or Brn4, Klf4, Sox2, and c-Myc in mouse embryonic fibroblasts [58]. Mauksch et al reported the generation of iNSCs by nonviral means employing plasmid transfection and recombinant protein transduction using SOX2 and PAX6 [54].…”

“…Self-renewal capacity was also assessed through immunocytochemical staining for proliferation markers, such as Ki-67, in combination with key markers for NSCs [25,26,30,34,57,59,74]. Two studies also included FGF4 in their neural expansion media [11,42]. However, iNSC populations also showed slight differences in their marker expression ( Table 2).…”

“…However, maturity of these neurons varies considerably in respect of resting membrane potential and capability of firing repetitive action potentials [11,13,14,25,28,30,[32][33][34]36,38,48,[57][58][59][60]62,73] [81]. Moreover, several studies strongly suggest the presence of functional synapses, as indicated by their response to excitatory or inhibitory neurotransmitters, as well as of spontaneous postsynaptic currents, and the ability to (partially) abolish synaptic activity with specific blockers [11,25,28,38,42,48,57,59,75,80]. In addition to electrophysiological experiments, the ability to form synapses was also investigated through immunocytochemical approaches showing colocalization of pre-and postsynaptic markers like synapsin 1, vGLUT1, or PSD-95 ( Table 2 [13,14,25,28,30,32,36,38,48,57,73,75]).…”

“…Induced neural stem cells are generated through direct conversion, a process in which cells acquire the expression of NSC markers in a stepwise fashion, theoretically without passing through a pluripotent state [40]. However, most protocols for iNSC generation involve the Yamanaka factors OSKM [11,13,25,26,41,42], suggesting that cells undergo a transient pluripotent state during the reprogramming process. Various efforts have been made to dissect the trajectory of somatic cells undergoing conversion into iNSCs.…”

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“…Studies have described the use of pluripotency factors to directly reprogram somatic cells into alternative lineages, such as neural stem cells, apparently without the generation of a pluripotent intermediate. This involves curtailed expression of some or all of the Yamanaka factors in conjunction with neural inducing cytokines such as epidermal growth factor (EGF) and FGF2, and the absence of pluripotency‐inducing conditions . Initially, it was suggested that the mechanism for PF‐mediated direct‐to‐iNSC reprogramming was the induction of a plastic or unstable intermediate cell type that could be directed to different cell fates, including NSCs, depending on other cues .…”