Characterisation of human mesenchymal stem cells following differentiation into Schwann cell-like cells

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Abstract

Cell-based therapies provide a clinically applicable and available alternative to nerve autografts. Our previous studies have characterised rat-derived mesenchymal stem cells (MSC) and here we have investigated the phenotypic, molecular and functional characteristics of human-derived MSC (hMSC) differentiated along a Schwann cell lineage. The hMSC were isolated from healthy human donors and the identity of the undifferentiated hMSC was confirmed by the detection of MSC specific cells surface markers. The hMSC were differentiated along a glial cell lineage using an established cocktail of growth factors including glial growth factor-2. Following differentiation, the hMSC expressed the key Schwann cell (SC) markers at both the transcriptional and translational level. More importantly, we show the functional effect of hMSC on neurite outgrowth using an in vitro co-culture model system with rat-derived primary sensory neurons. The number of DRG sprouting neurites was significantly enhanced in the presence of differentiated hMSC; neurite length and density (branching) were also increased. These results provide evidence that hMSC can undergo molecular, morphological and functional changes to adopt a SC-like behaviour and, therefore, could be suitable as SC substitutes for nerve repair in clinical applications.

Introduction

Peripheral nerve injuries are an economic burden for society in general and despite advanced microsurgical reconstruction of the damaged nerves the functional result is unsatisfactory with poor sensory recovery and reduced motor functions (Wiberg and Terenghi, 2003). In the treatment of nerve injuries transplantation of a nerve graft is often necessary, especially in nerve gap injuries. More recently, promising functional results have been achieved using different types of conduits containing cultured Schwann cells (SCs) and in vitro differentiated bone marrow stromal cells with Schwann cell properties (Dezawa et al., 2001, Mosahebi et al., 2002, Keilhoff et al., 2006, Pfister et al., 2007, Shimizu et al., 2007). Schwann cells are the key facilitators of peripheral nerve regeneration and are responsible for the formation and maintenance of the myelin sheath around axons in peripheral nerve fibres. They are essential for nerve regeneration after nerve injuries as they produce extracellular matrix molecules, integrins and trophic factors providing guidance and trophic support for regenerating axons (Bunge, 1994, Ide, 1996, Mahanthappa et al., 1996, Terenghi, 1999, Wiberg and Terenghi, 2003). However, the use of ex vivo cultured SC within conduits is limited in its clinical application because of the concomitant donor site morbidity and the slow growth of these cells in vitro (Tohill et al., 2004).

Mesenchymal stem cells (MSC or bone marrow stromal cells) are easily accessible non-haematopoietic stem cells that have proved essential for research purposes due to their plasticity and ability to differentiate into several functional cell types. In vitro, they display a fibroblastic morphology and readily adhere to plastic surfaces (Pittenger et al., 1999, Krampera et al., 2007). By nature, these cells are a heterogeneous population, thus there is problem finding a specific marker that defines their origin. MSCs are CD14, CD34, CD45 negative and CD44, CD54, CD90 and Stro-1 positive (Barry, 2003, Bobis et al., 2006, Phinney, 2007).

Studies by our group and others have shown that following differentiation with a cocktail of growth factors, MSCs express glial cell markers, such as glial fibrillary protein (GFAP), low-affinity neutrophin factor p75 and calcium binding protein S100 (Dezawa et al., 2001, Tohill et al., 2004, Caddick et al., 2006, Zurita et al., 2007, Lin et al., 2008). Clearly, it is of considerable clinical importance to establish the differentiation of human-derived MSC (hMSC) into SC-like cells (dhMSC). This alternative source of cells, which is relatively simple to isolate and expand in culture, should provide nerve fibre support and guidance during nerve regeneration. Shimizu et al. (2007) have examined the clinical potential of SC-like cells in a rat sciatic nerve injury model. They have demonstrated that MSCs express SC markers in vivo and also come in close physical contact with the regenerating axons.

The purpose of this study is to identify the phenotypic, molecular and functional characteristics of hMSC differentiated into cells with a SC-like phenotype. We also assessed the function of the dhMSC as SC substitutes in a functional co-culture assay with dissociated rat primary sensory neurons. From a clinical standpoint, it is also important to assess the effects of patient age and gender on efficacy of hMSC as SC substitutes.

Section snippets

Culture of bone marrow stromal cells

Samples of human bone marrow were obtained from the iliac crests of three healthy donors during reconstructive surgery with informed consent. The patients were designated as follows: P1 female aged 59 years, P2 male aged 58 years and P3 male aged 32 years. Procedures were approved by the Local Ethical Committee for Clinical Research in Umeå University (no. 03-425).

A modification of previously described protocol (Azizi et al., 1998) was used to isolate and to prepare primary cultures of hMSC.

Identification of MSC markers

Immunocytochemical labelling of the uhMSC showed positive staining for Stro-1 in 80% uhMSC of the three patients (Fig. 1). Staining for haemopoietic stem cell surface markers CD14 (Fig. 2A, E and I) and CD45 (Fig. 2B, F and J) was negative in the uhMSC of all three patients (P1–P3). More than 80% of uhMSC were positive for MSC surface markers CD54 (Fig. 2C, G and K) and CD90 (Fig. 2D, H and L). Flow-cytometry showed that approximately 40% of the uhMSC from each of the three patients expressed

Discussion

In this study we have confirmed that the MSC from three healthy human donors express characteristic MSC cell surface markers and also demonstrated the multilineage potential of MSC (Pittenger et al., 1999, Krampera et al., 2007). Clear evidence is provided to show that human-derived MSC have the ability to differentiate along a glial lineage and express cell markers which are typical for glial cells including Schwann cells. Similar results have previously been reported for rat MSC (Tohill et

Acknowledgments

This study was supported by the Rosetrees Trust, Medical Research Council (UK), Swedish Medical Research Council, Umeå University, County of Västerbotten, Åke Wibergs Stiftelse, Magn. Bergvalls Stiftelse, Clas Groschinskys Minnesfond, Anna-Stina och John Mattsons Minnesstiftelse för sonen Johan and the Gunvor and Josef Anér Foundation. The authors also wish to thank Acorda Therapeutics for their generous gift of GGF-2.

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