Impact of the Epigenetically Regulated Hoxa-5 Gene in Neural Differentiation from Human Adipose-Derived Stem Cells

SIMPLE SUMMARY: A deep knowledge of the regulation of genes involved in human adipose-derived mesenchymal stem cells (hASCs) neuronal differentiation is essential to their application in neurological disorder treatment. hASCs were induced to neuronal differentiation using three differentiation protocols and modulation of specific neuronal biomarkers and epigenetic genes changes were determined. An excellent neuronal differentiation of hASCs was obtained after Neu1 media exposure accompanied by relevant epigenetic changes in six genes including Hoxa-5. Moreover, functional analysis overexpressing the Hoxa-5 gene by CRISPR/dCas9 and lentiviral systems induced neuronal differentiation in hASCs which was improved and accelerated with the use of Neu1 media. These results suggest that Hoxa-5 plays a crucial role in the differentiation process, highlighting it as a potential candidate for the development of therapeutic strategies aimed at cell therapy in diseases related to the nervous system. ABSTRACT: Human adipose-derived mesenchymal stem cells (hASCs) may be used in some nervous system pathologies, although obtaining an adequate degree of neuronal differentiation is an important barrier to their applicability. This requires a deep understanding of the expression and epigenetic changes of the most important genes involved in their differentiation. We used hASCs from human lipoaspirates to induce neuronal-like cells through three protocols (Neu1, 2, and 3), determined the degree of neuronal differentiation using specific biomarkers in culture cells and neurospheres, and analyzed epigenetic changes of genes involved in this differentiation. Furthermore, we selected the Hoxa-5 gene to determine its potential to improve neuronal differentiation. Our results showed that an excellent hASC neuronal differentiation process using Neu1 which efficiently modulated NES, CHAT, SNAP25, or SCN9A neuronal marker expression. In addition, epigenetic studies showed relevant changes in Hoxa-5, GRM4, FGFR1, RTEL1, METRN, and PAX9 genes. Functional studies of the Hoxa-5 gene using CRISPR/dCas9 and lentiviral systems showed that its overexpression induced hASCs neuronal differentiation that was accelerated with the exposure to Neu1. These results suggest that Hoxa-5 is an essential gene in hASCs neuronal differentiation and therefore, a potential candidate for the development of cell therapy strategies in neurological disorders.