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  3. Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 05/06 Podcast
  4. In vitro direct conversion of somatic cells from the adult human brain into functional neurons by defined factors
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.

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