Reprogramming of Equine Adipose-Derived Stem Cells into Induced Neuronal and Schwann Cells by Lentiviral Vector Expression of Specific Transcription Factors

Neurological disorders are encountered frequently in equine medicine, yet despite this, little is known about them. This often leads to euthanisation of affected animals, thus emphasising the need to develop suitable models for understanding equine neuropathogenesis. At their root, neurological disorders result from the loss of critical populations of cells that the body is unable to replace due to the limited regenerative capacity of the mammalian central nervous system. As a result, there has been much interest in identifying methods of generating clinically relevant numbers of functional cells to replace those that have been damaged or lost. Adipose-derived stem cells have been proposed as an ideal candidate, due to their relative abundance and ability to be
harvested from non-critical locations using minimally invasive methods, as well as their self-renewing, highly proliferative, and multipotent nature, with evidence of their transdifferentiation into induced neuronal and Schwann cells under specific experimental conditions. Studies have also revealed the induction of neuronal transdifferentiation in somatic cells following lentiviral vector expression of neuronal transcription factors, providing further opportunities for the generation of neuronal cell types.

As such, we aimed to investigate the transdifferentiation of equine adipose-derived stem cells (EADSC) into neuronal cell types. Subcutaneous adipose tissue was first harvested from the fat pad adjacent to the base of the tail of donor horses, and cells were isolated. Characterisation assays were performed to identify the isolated cells as EADSC; plastic adherent mononuclear spindle-shaped cells expressing the cell surface markers CD29, CD44, and CD90, and exhibiting trilineage mesenchymal differentiation potential. Gene delivery to EADSC was found to be optimal using lentiviral vectors pseudotyped with
VSV-G, resulting in efficient and sustained transgene expression, with minimal effects on cell viability and doubling time, and an unaltered chondrogenic differentiation potential. Additionally, 3D culture was found to enhance the osteogenic differentiation of EADSC, demonstrating faster and more extensive mineralisation, with cellular morphology consistent with that in vivo.

Using VSV-G pseudotyped lentiviral vectors to deliver the neuronal transcription
factors Brn2, Ascl1, Myt1l, and NeuroD1, EADSC were transdifferentiated into beta III tubulin positive neuron-like cells, expressing the neuronal markers Nestin and MAP2. Furthermore, this process was enhanced in 3D culture, with an increased conversion efficiency and enhanced neuronal maturation, providing the first examples of transcription factor mediated neuronal transdifferentiation of EADSC, and transcription factor mediated neuronal transdifferentiation in 3D culture. Similarly, the Schwann cell transcription factors Krox20, Oct6, Sox10, and Brn2 were used to transdifferentiate EADSC into Schwann cell-like cells, with characteristic aligned cells of bipolar spindleshaped morphology, and a gene expression phenotype of MPZ + , GFAP ¯, L1CAM ¯, NGFR ¯ consistent with myelinating Schwann cells, providing the first examples of transcription factor mediated Schwann cell transdifferentiation, and the ability of EADSC to transdifferentiate into cells of a myelinating Schwann cell-like phenotype.
The results of this study demonstrate the ability of EADSC to be transdifferentiated into induced neuronal and Schwann cells, with prospective roles in the investigation of equine neurological disorders, including neurodegenerative disease modelling, drug screening, and cellular replacement for regenerative medicine applications.