Generation of Schwann cell-derived multipotent neurospheres isolated from intact sciatic nerve.
Schwann cells (SCs) are the supporting cells of the peripheral nervous system and originate from the neural crest. They play a unique role in the regeneration of injured peripheral nerves and have themselves a highly unstable phenotype as demonstrated by their unexpectedly broad differentiation potential. Thus, SCs can be considered as dormant, multipotent neural crest-derived progenitors or stem cells. Upon injury they de-differentiate via cellular reprogramming, re-enter the cell cycle and participate in the regeneration of the nerve. Here we describe a protocol for efficient generation of neurospheres from intact adult rat and murine sciatic nerve without the need of experimental in vivo pre-degeneration of the nerve prior to Schwann cell isolation. After isolation and removal of the connective tissue, the nerves are initially plated on poly-D-lysine coated cell culture plates followed by migration of the cells up to 80% confluence and a subsequent switch to serum-free medium leading to formation of multipotent neurospheres. In this context, migration of SCs from the isolated nerve, followed by serum-free cultivation of isolated SCs as neurospheres mimics the injury and reprograms fully differentiated SCs into a multipotent, neural crest-derived stem cell phenotype. This protocol allows reproducible generation of multipotent Schwann cell-derived neurospheres from sciatic nerve through cellular reprogramming by culture, potentially marking a starting point for future detailed investigations of the de-differentiation process.
Molecular Neurobiology, Department of Cell Biology Faculty of Biology, University of Bielefeld, Bielefeld, Germany.
Ina
I
Martin
The Duy
TD
Nguyen
Vivien
V
Krell
Johannes F W
JF
Greiner
Janine
J
Müller
Stefan
S
Hauser
Peter
P
Heimann
Darius
D
Widera
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D054337:Cell Dedifferentiation; D002465:Cell Movement; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D006801:Humans; D051379:Mice; D039902:Multipotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D009432:Neural Crest / Q000166:cytology / Q000378:metabolism; D058953:Neural Stem Cells / Q000166:cytology* / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012583:Schwann Cells / Q000166:cytology* / Q000378:metabolism; D012584:Sciatic Nerve / Q000166:cytology* / Q000378:metabolism; D018874:Spheroids, Cellular / Q000166:cytology* / Q000378:metabolism