A “Hit and Run” Approach to Inducible Direct Reprogramming of Astrocytes to Neural Stem Cells

Abstract

Temporal and spatial control of gene expression can be achieved using an inducible system as a fundamental tool for regulated transcription in basic, applied and eventually in clinical research. We describe a novel “hit and run” inducible direct reprogramming approach. In a single step, 2 days post-transfection, transiently transfected Sox2FLAG under the Leu3p-αIPM inducible control (iSox2) triggers the activation of endogenous Sox2, redirecting primary astrocytes into abundant distinct nestin-positive radial glia cells. This technique introduces a unique novel tool for safe, rapid and efficient reprogramming amendable to regenerative medicine.

Direct cell-fate conversion of somatic cells: Toward regenerative medicine and industries

“…In addition to these methods, plasmid transfection has also been widely tested. 27),29),32),78),116),117) Although there is a risk of genome integration and a problem of low transduction efficiency, novel tools, such as a polysaccharide-based gene co-delivery system, 119),120) a Leu3p-,IPM system, 121) or episomal vectors, 122) might improve the plasmid-based transduction technologies.…”

A Role for Sox2 in the Adult Cerebellum

“…Mice were described elsewhere [7,14,16]. All animals were handled in strict accordance with good animal practice as defined by the Animals Act 160/03.05.1991 applicable in Greece, revised according to the 86/609/EEC/24.11.1986 EU directive regarding the proper care and use of laboratory animals and in accordance to the Hellenic License for Animal Experimentation at the BSRC “Alexander Fleming” (Prot.…”

“…Tail, yolk sack or embryonic tissues were isolated and processed according to previously described methodology. PCR amplification conditions and primers used are described elsewhere [7,14,16].…”

“…In an effort to understand the role of Sox2 in neural stem and progenitor cells, we have conditionally ablated the function of Sox2 in radial glial cells via Sox 2COIN/COIN [7], to transgenic Tg(hGFAP-CRE) mouse intercrosses [7,14,16]. Tg(hGFAP-CRE) mice express CRE recombinase at embryonic day E13.5 in radial glial cells at dorsal and middle regions of the telencephalon, while at an early birth stage, hGFAP is ubiquously expressed throughout the CNS, in all neural cell types derived from radial glial cells.…”