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Sanchez del Valle, Rodrigo (2013): In vitro direct conversion of somatic cells from the adult human brain into functional neurons by defined factors. Dissertation, LMU München: Fakultät für Biologie



Reprogramming of somatic cells into neurons provides a new approach toward cell-based therapy of neurodegenerative diseases. Conversion of postnatal astroglia from the cerebral cortex of mice into functional neurons in vitro can be achieved by forced expression of a single transcription factor. Also skin fibroblasts have been successfully reprogrammed into functional neurons yet through the synergistic action of several transcription factors. A major challenge for the translation of neuronal reprogramming into therapy concerns the feasibility of this approach in adult human tissues. This work demonstrates the potential of perivascular cells isolated from the adult human brain to serve as a substrate prompted to neuronal reprogramming by forced co-expression of neurogenic transcription factors, namely the SRY-related HMG box protein Sox2 and the basic helix loop helix (bHLH) mammalian homologue of achaete-schute-1 Mash1 (also known as Ascl1). The cells used in this study display characteristics of pericytes assessed by immunocytochemistry, fluorescence-activated cell sorting (FACS) and real time RT-PCR. The presence of neural progenitor cells was excluded by real time RT-PCR analysis of mRNAs typically expressed by these cell lineages. Upon expression of Sox2 and Mash1, these cells adopt a neuronal phenotype characterized by the expression of neuronal markers such us ßIII-Tubulin, MAP2, NeuN, GABA and calretinin. Electrophysiological recordings reveal the ability of these cells to fire repetitive action potentials and to integrate into neuronal networks when co-cultured with mouse embryonic neurons. The pericytic nature of the reprogrammed cells was further demonstrated by isolation of PDGFRß-positive cells from adult human brain cultures by FACS and monitoring the Mash1/Sox2-induced neuronal conversion by time-lapse video microscopy. Genetic fate-mapping in mice expressing an inducible Cre recombinase under the tissue non-specific alkaline phosphatase promoter corroborated that pericytes from the adult cerebral cortex can be expanded and reprogrammed in vitro into neurons by co-expression of Sox2 and Mash1. These results demonstrate the feasibility of an in vitro neuronal reprogramming approach on somatic cells isolated from the adult human cerebral cortex which could have important implications in the development of in vivo direct repair strategies in neurodegenerative diseases and brain injury.