Purification of Schwann cells by selection of p75 low affinity nerve growth factor receptor expressing cells from adult peripheral nerve

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Abstract

The intrinsic capacity of Schwann cells to promote regeneration after limited peripheral nerve lesions has been successfully transferred to extensive peripheral nerve injuries and central nervous system lesions by autologous transplantation strategies. However, both the intrinsic ability of axotomized neurons to regenerate and the permissiveness of the parenchyma surrounding the acute injury site diminish over time. Therefore, the autologous transplantation mode requires a fast and effective method to isolate Schwann cells from peripheral nerve biopsies. Here, we report a method to purify p75 low affinity nerve growth factor receptor (p75LNGFr) expressing Schwann cells from peripheral nerve biopsies in adult rats using magnetic-activated cell separation (MACS). After 1 week of nerve degeneration in culture, nerve fragments were dissociated resulting in mixed cultures containing Schwann cells and fibroblasts. After incubation with specific anti-p75LNGFr antibodies and secondary magnetic bead conjugated antibodies followed by one cycle of MACS, 95% pure Schwann cell cultures were generated as confirmed by flow-cytometry and immunocytochemistry. In contrast to established methods, MACS separation of p75LNGFr expressing cells allows the reliable purification of Schwann cells within 9 days after biopsy employing direct selection of Schwann cells rather than fibroblast depletion assays. Therefore, this method represents an effective and fast means to generate autologous Schwann cells for clinical transplantation strategies aiming for axon repair and remyelination.

Introduction

After limited injury to the peripheral nervous system (PNS), endogenous Schwann cells are recruited to form a scaffold for regenerating axons to grow along, produce growth conducive extracellular matrix components, secrete neurotrophic factors and remyelinate regenerating axons in a phenotypical appropriate manner, which ultimately leads to reinnervation of the target and functional recovery (Griffin and Hoffman, 1993).

By transplanting Schwann cells, these favorable properties have been successfully transferred to more severe peripheral nerve injuries and even central nervous system (CNS) lesions. After severe peripheral nerve injuries endogenous recruitment of Schwann cells is not sufficient to promote morphological and functional restoration. The availability of peripheral nerve autograft material, which represents the standard therapy in these cases, is limited. Allografts on the other hand, are subject to graft rejection (Berger and Lassner, 1994). Artificially produced guidance channels seeded with autologous Schwann cells could overcome these limitations. Syngenic Schwann cells seeded into guidance channels have been successfully employed to support regeneration in animal models of peripheral nerve injury (Guenard et al., 1992, Rodriguez et al., 2000).

In the CNS, transplanted Schwann cells can regenerate and remyelinate axons, which have been completely interrupted (Kromer and Cornbrooks, 1985, Li and Raisman, 1994, Paino et al., 1994, Xu et al., 1995), or have been demyelinated by toxins or irradiation (Blakemore and Crang, 1985, Baron-Van Evercooren et al., 1992, Honmou et al., 1996, Kohama et al., 2001). Furthermore, the regenerative capacity of Schwann cells can be enhanced by ex vivo genetic modification to overexpress neurotrophic factors (Menei et al., 1998, Tuszynski et al., 1998, Weidner et al., 1999).

Ideally, Schwann cells are transplanted in an autologous fashion to avoid graft immune rejection. The disadvantage is that autologous Schwann cells will not be available immediately after a nerve lesion. The generation of sufficient quantities of Schwann cells for transplantation from the patient's own peripheral nerve biopsy requires at least 3–6 weeks according to established protocols (Morrissey et al., 1991, Verdu et al., 2000, Calderon-Martinez et al., 2002). However, the regeneration supportive capacity in the PNS and CNS decreases over time due to events such as cellular degeneration, scar formation and down-regulation of growth promoting molecules (Tuszynski and Kordower, 1999, Zochodne, 2000).

The separation of Schwann cells from rapidly dividing fibroblasts, which build the protecting layers (epi-, perineurium) surrounding bundles of nerve fibers, represents the major time consuming factor (Bunge et al., 1989, Levi, 1996). Thus far, established methods for Schwann cell purification from peripheral nerve fragments are based on fibroblast depletion as opposed to direct selection of Schwann cells. As an initial step to separate Schwann cells from fibroblasts, nerve fragments are maintained under cell culture conditions on an adhesive substrate such as laminin for several weeks. This procedure allows fibroblasts to migrate out, while Schwann cells remain in the nerve fragment. Each consecutive transfer of these nerve fragments reduces the number of fibroblasts (Morrissey et al., 1991). The number of rapidly dividing fibroblasts can be reduced by adding antimitotic drugs such as cytosine arabinoside (Ara-C) to the cell culture medium or by maintaining serum-free primary cultures (Wood, 1976, Needham et al., 1987). The yield of enriched Schwann cells can be further enhanced by predegeneration of peripheral nerves in vivo before biopsy (Keilhoff et al., 1999, Verdu et al., 2000). Besides the time required to enrich Schwann cells, these purification methods are rather unspecific (Calderon-Martinez et al., 2002). More specific purification methods, such as fibroblast depletion using specific antibodies against the cell-surface antigen Thy-1 coupled to either complement activation or immunopanning, have only been reported for neonatal peripheral nerves (Brockes et al., 1979, Assouline et al., 1983).

Magnetic-activated cell separation (MACS) represents a highly effective and fast method to select individual cell populations from a mixed cell population. Cell type specific cell surface antigens are labeled with magnetic bead conjugated antibodies followed by separation on a high gradient magnetic column (Miltenyi et al., 1990). A suitable cell surface antigen to select Schwann cells is the p75 low affinity nerve growth factor receptor (p75LNGFr), which is widely expressed on Schwann cells in vitro, but not on fibroblasts (Yasuda et al., 1987). A similar approach using magnetic Dynabeads to enrich p75LNGFr expressing olfactory ensheathing cells has been described recently (Barnett et al., 2000). Vice versa, the cell surface antigen Thy-1 is expressed on fibroblasts, but not on Schwann cells, and thus can be used to enrich Schwann cells by depleting Thy-1 expressing fibroblasts (Fields and Raine, 1982).

In the present experiment, we investigated the efficacy of MACS to purify Schwann cells from adult sciatic nerve biopsies by using either specific antibodies (1) against p75LNGFr to select Schwann cells or (2) against Thy-1 to deplete fibroblasts. Results from this study indicate that only MACS selection of p75LNGFr expressing Schwann cells represents a highly effective procedure to establish primary Schwann cell cultures for autologous transplantation strategies in the PNS and CNS.

Section snippets

Schwann cell isolation

Sciatic nerve fragments with a length of approximately 35 mm were taken bilaterally from deeply anesthetized (0.5 ml of a combination of ketamine (50 mg/kg), xylazine (2.6 mg/kg) and acepromazine (0.5 mg/kg)) adult Fischer 344 rats (average weight 160–180 g). The sciatic nerves were washed with ice-cold Hank's balanced salt solution (HBSS, PAA Laboratories, Austria) and the epineurium was stripped off with a fine forceps. Each sciatic nerve fragment weighed on average 29.6 mg (±1.7). Nerves

Schwann cell isolation

After obtaining sciatic nerve biopsies from adult rats, nerve fragments were kept in culture for 7 days before dissociation (Fig. 1). After 7 days of in vitro degeneration, dissociation of peripheral nerve fragments and another 2 days in culture, 0.86×106 (±0.09×106, n=6) total viable cells per animal were counted, which corresponds to 1.5×104 cells/mg nerve tissue. Out of all dissociated cells less than 5% were non-viable cells. Dissociation before day 7 post biopsy resulted in a low yield of

Discussion

The results of the present study demonstrate that Schwann cells can be purified by MACS from adult peripheral nerve biopsies using the expression of the cell surface antigen p75LNGFr for selection. This method allows the fast and reliable purification of adult Schwann cells directly through positive selection rather than indirectly through depletion of fibroblasts. MACS depletion of Thy-1 expressing fibroblasts lacks the sensitivity and specificity to establish Schwann cell cultures from adult

Acknowledgements

This work was supported by Institute International de Recherche en Paraplegie, Geneva, on behalf of an anonymous donation, Deutsche Stiftung Querschnitt, Deutsche Forschungsgemeinschaft and by the ReForM-Program, University of Regensburg, School of Medicine. We thank L. Aigner, H.G. Kuhn, J. Winkler, E. Marani for helpful comments and K. Wewetzer for providing the 27C7 antibody.

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