Bone marrow-derived Schwann cells achieve fate commitment – a prerequisite for remyelination therapy
Introduction
Protocols for in vitro derivation of Schwann cell-like cells from the bone marrow (BM) have been developed with the intention of therapeutic transplantation as in spinal cord injury (Caddick et al., 2006, Dezawa et al., 2001, Keilhoff et al., 2006). The Schwann cell-like phenotype as indicated by marker gene/protein expression depends on a cocktail of such extrinsic factors as forskolin, platelet derived growth factor (PDGF), basic fibroblast growth factor (bFGF) and β-heregulin for maintenance. Upon withdrawal of the factors, the cells revert to a stem-cell-like state. This instability is undesirable, as apart from reducing the yield of Schwann cells among the transplanted cell population, there is the risk of in vivo dedifferentiation and tumorigenesis (Cogle et al., 2007, Pawelek & Chakraborty, 2008, Takaishi et al., 2008). A means of stable, ex vivo differentiation to maturity must be achieved before the derived Schwann cells can be considered safe for therapeutic applications.
We turned to events that specify Schwann cell fate in vivo for insight. Schwann cells are developmentally derived from neural crest cells after delamination from the neural tube, migration along ventromedial routes, and condensation in target dorsal root ganglia (DRGs). It is yet unclear how regional and temporal changes govern cell competence and commitment to the glial fate. The molecular phenotype that defines developmental progression from Schwann cell precursors, through immature Schwann cells and finally, mature Schwann cells is however emerging (Mirsky et al., 2008). Conversion of Schwann cell precursors to immature Schwann cells in embryonic sciatic nerves occurs in proximity to DRG neurons as early as E14/15 in rats; beyond this stage, Schwann cells in the developing nerve acquire irreversible commitment to glial fate (Jessen & Mirsky, 2005, Morrison et al., 2000) and develop autocrine loops such that Schwann cells survive even in the absence of axons as in the distal stump of transected nerves (Meier et al., 1999). Of the three molecules which have been implicated in fate commitment, only bFGF is a paracrine cue (Dong et al., 1999) whereas Delta1 (Morrison et al., 2000) and NRG1 type III (Leimeroth et al., 2002) are present on neuronal membranes and therefore are juxtacrine cues requiring direct cell-cell contact to be instructive. We therefore hypothesize that Schwann cell-like cells derived by in vitro exposure to extrinsic factors require paracrine and contact-dependant cues, from neurons of E14/15 DRGs for commitment to the Schwann cell fate.
Despite their in vitro instability, Schwann cell-like cells that result from culture in medium containing forskolin, PDGF, bFGF and β-heregulin have been shown to promote axonal regeneration and remyelination in a rat spinal cord injury model if transplantation is timely (Dezawa et al., 2001, Keilhoff et al., 2006). This suggests that the injured environment nevertheless harbors trophic factors that support survival of the Schwann cell-like cells such that these cells provide chemoattraction to regrowing axons. In turn, contact of Schwann cell-like cells with regrowing axons is critical for the juxtacrine signaling that accomplishes differentiation and commitment to the Schwann cell fate.
Here, we tested our hypothesis by coculture with neurons purified from E14.5-15.5 DRGs. We exploited conditions used for maintenance of neural stem cells (Wislet-Gendebien et al., 2003, Xu et al., 2008) to select for a sphere-forming subpopulation of BM cells. Then culture on laminin in medium supplemented with forskolin, PDGF, bFGF and β-heregulin directed differentiation along the Schwann cell lineage. Subsequent co-culture that provided direct contact with neurons of E14.5-15.5 DRGs was necessary for fate commitment of the derived Schwann cells.
Section snippets
Culture of BM cells
Rat BM cells were isolated as described by Dezawa et al. (2001). Briefly, young adult Sprague Dawley (SD) rats (200-250 g) were sacrificed by pentobarbital sodium overdose (240 mg/kg, ip). Femurs were dissected, cleansed of connective tissue, and sterilized in ethanol. All procedures were in strict accordance with the NIH Guide for the care and use of laboratory animals and approved by the Committee on Use of Live Animals for Teaching and Research, LKS Faculty of Medicine, The University of Hong
Results
Stromal cells recovered from adult rat bone marrow formed densely packed fibroblast-like colonies adherent on tissue culture plastic (Fig. 1A). Immunocytochemistry demonstrated that 88.8% ± 2.5% (n = 6) of the cells expressed STRO-1 (Fig. 1, Fig. 2), a marker of BM stromal cells (Goncalves et al., 2006) while 16.7% ± 1.6% (n = 6) expressed nestin (Fig. 1, Fig. 2), a marker of neural stem/progenitor cells. The latter suggested that our BM cell preparation contained a minor but significant subpopulation
Discussion
The ultimate goal of in vitro derivation of Schwann cells from adult tissues is to use them autologously in nerve guidance channels for post-traumatic nerve regeneration in both the PNS and CNS. Protocols for deriving Schwann cell-like cells from BM cells fall short in stability of the acquired phenotype and functional capacity of the derived cells to myelinate axons. The present study addressed these issues with a new protocol that (1) selected and expanded neuro-ectodermal progenitor cells in
Acknowledgments
Supported in part by funding from Strategic Research Theme of The University of Hong Kong and Croucher Foundation MBBS/PhD scholarship to GKH Shea.
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