Non-Viral Generation of Neural Precursor-like Cells from Adult Human Fibroblasts

Abstract

Recent studies have reported direct reprogramming of human fibroblasts to mature neurons by the introduction of defined neural genes. This technology has potential use in the areas of neurological disease modeling and drug development. However, use of induced neurons for large-scale drug screening and cell-based replacement strategies is limited due to their inability to expand once reprogrammed. We propose it would be more desirable to induce expandable neural precursor cells directly from human fibroblasts. To date several pluripotent and neural transcription factors have been shown to be capable of converting mouse fibroblasts to neural stem/precursor-like cells when delivered by viral vectors. Here we extend these findings and demonstrate that transient ectopic insertion of the transcription factors SOX2 and PAX6 to adult human fibroblasts through use of non-viral plasmid transfection or protein transduction allows the generation of induced neural precursor (iNP) colonies expressing a range of neural stem and pro-neural genes. Upon differentiation, iNP cells give rise to neurons exhibiting typical neuronal morphologies and expressing multiple neuronal markers including tyrosine hydroxylase and GAD 65/67 . Importantly, iNP-derived neurons demonstrate electrophysiological properties of functionally mature neurons with the capacity to generate action potentials. In addition, iNP cells are capable of differentiating into glial fibrillary acidic protein (GFAP)-expressing astrocytes. This study represents a novel virusfree approach for direct reprogramming of human fibroblasts to a neural precursor fate.

Transgene and Chemical Transdifferentiation of Somatic Cells for Rapid and Efficient Neurological Disease Cell Models

“…Transdifferentiation has previously been carried out using fibroblasts or peripheral blood mononuclear cells (PBMCs) to neural stem cells, neurons, astrocytes, oligodendrocytes, microglia, and skeletal muscle cells ( Table 1 ). Direct differentiation is not limited to a terminally differentiated endpoint; for neurogenesis, reprogramming of fibroblasts to multipotent neural precursor cells can be performed with the overexpression of transcription factors SOX2, OCT3, KLF4, and MYC ( Meyer et al, 2014 ), SOX2 and PAX6 ( Maucksch et al, 2012 ), or even a single transcription factor, such as PTF1A ( Xiao et al, 2018 ).…”

Cell Reprogramming to Model Huntington’s Disease: A Comprehensive Review

“…In 2012, we reported the generation of iNPs from adult human fibroblasts using SOX2 and PAX6 cDNA plasmids or SOX2 and PAX6 recombinant protein expression [ 131 ]. Plasmid-derived iNPs expressed SOX2, PAX6, ASCL1, and NGN2 mRNA and protein, as well as the neural markers SOX1 , HOXB9 , NKX6.1 , and SIX3 .…”

“…iNPs differentiated into both GABAergic and glutamatergic neurons and a small proportion of astrocytes within 30 days and exhibited neuronal functionality. However, the reprogramming efficiency was low (0.05%), and the duration was 85 days long [ 131 ]. In 2018, our research group reported the generation of iNPs from adult human fibroblasts with a higher reprogramming efficiency (0.3%) and shorter time frame (21–28 days) following the transient expression of chemically-modified mRNA (cmRNA) encoding SOX2 and PAX6 [ 132 ].…”

Reprogramming of adult human dermal fibroblasts to induced dorsal forebrain precursor cells maintains aging signatures

“…We have developed a novel protocol that utilizes stable and non-immunogenic, chemically modified mRNA (cmRNA) to convert adult HDF to induced dorsal forebrain precursor cells (hiDFPs; Edwards et al, 2022 ). By transiently over-expressing the neural genes SOX2 and PAX6 using cmRNA we can generate hiDFPs which can subsequently be differentiated into cortical neurons (Maucksch et al, 2012 ; Connor et al, 2018 ; Playne et al, 2018 ; Edwards et al, 2022 ). Following 21 days of reprogramming, we have demonstrated that hiDFPs express the dorsal forebrain markers FOXG1, NGN2, BRN2, TBR1, TBR2, DLX2, CTIP2 , and SATB2 (Edwards et al, 2022 ).…”