Generation of Schwann Cell-Derived Multipotent Neurospheres Isolated from Intact Sciatic Nerve

Abstract

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.

Translational Research and Therapeutic Applications of Neural Crest-Derived Stem Cells in Regenerative Periodontology

“…During the last 15 years, however, an emerging line of evidence supported the hypothesis that at least a limited number of adult NCSCs may exist in the human body even in the adulthood. NCSCs can be found in different tissues and organs, especially within the craniofacial region [1][2][3][4][5][6][7]. Such adult human NCSCs are able to undergo self-renewal and possess a surprisingly high differentiation potential in vitro and in vivo (reviewed in [8]).…”

An efficient system for selection and culture of Schwann cells from adult rat peripheral nerves

“…Given the identity of SCs, the neural crest stem cells medium was considered and adapted to SCs culture through addition of chemically defined ingredients. (Achilleos and Trainor 2012;Li et al 2007;Martin et al 2012). Ingredients such as bFGF and EGF are common mitogens for almost all kinds of cells including SCs (Monje et al 2009).…”

Concise Review: Cellular and Molecular Mechanisms of Postnatal Injury-Induced Enteric Neurogenesis

“…Sustained Sox2 expression in vivo blocks peripheral nerve myelination and maintains Schwann cells in a proliferating, nondifferentiated state . Schwann cell de‐differentiation is recapitulated in vitro, in that isolated Schwann cells revert to a stem‐like state in culture, and form neurospheres that express Nestin, p75, and Sox10 .…”