Transdifferentiated Mesenchymal Stem Cells as Alternative Therapy in Supporting Nerve Regeneration and Myelination

Abstract

1. Aims: Demyelination plays a crucial role in neurodegenerative processes and traumatic disorders. One possibility to achieve remyelination and subsequent restoration of neuronal function is to provide an exogenous source of myelinating cells via transplantation. In this context, mesenchymal stem cells (MSCs) have attracted interest. They are multipotent stem cells that differentiate into cells of the mesodermal lineage like bone, cartilage, fat, and muscle. Although adult, their differentiation potential is remarkable, and they are able to transdifferentiate.

2. Methods: We transformed cultivated rat MSCs into myelinating cells by using a cytokine cocktail. Transdifferentiated MSCs were characterized by an enhanced expression of LNGF-receptor, Krox20, and CD104, and a decreased expression of BMP receptor-1A as compared to untreated MSCs. The myelinating capacity was evaluated in vitro and in vivo. Therefore, PC12 cells, normally unmyelinated, were cocultivated with MSCs, transdifferentiated MSCs, and Schwann cells, or the respective cells were grafted into an autologous muscle conduit bridging a 2-cm gap in the rat sciatic nerve. Myelination of PC12 cells was demonstrated by electron microscopy. In vivo, after 3 and 6 weeks regeneration including myelination was monitored histologically and morphometrically. Autologous nerves and cell-free muscle grafts were used as control.

3. Results: Schwann cells and transdifferentiated MSCs were able to myelinate PC12 cells after 14 days in vitro. In vivo, autologous nerve grafts demonstrated the best results in all regenerative parameters. An appropriate myelination was noted in the Schwann cell groups and, albeit with restrictions, in the transdifferentiated MSC groups, while regeneration in the MSC groups and in the cell-free groups was impaired.

4. Conclusion: Our findings demonstrate that it may be possible to differentiate MSCs into therapeutically useful cells for clinical applications in myelin defects.

INTRODUCTION

Myelinating cells, oligodendrocytes in the central nervous system (CNS), and Schwann cells in the peripheral nervous system (PNS), play a crucial role in neurodegenerative and regenerative processes. Multiple sclerosis (MS) and spinal cord injury both present variable amounts of axonal demyelination and transection. Furthermore, peripheral nerve lesions are connected with myelin defects. Currently, therapeutic approaches for such neurological disorders are limited. Generally, remyelination and subsequent restoration of neuronal function can be achieved by either promoting endogenous repair mechanisms or providing an exogenous source of myelinating cells via transplantation. A number of cell types like oligodendrocytes and Schwann cells, neural stem cells or stem cell derived oligodendrocytes, transplanted into the injured areas, can remyelinate demyelinated axons and can encourage axonal regeneration. Substantial axonal regeneration, however, can occur only in the PNS. Part of this regenerative capacity has been linked to several abilities of Schwann cells, including the production of extracellular matrix and trophic factors for the sprouting nerve and the lack of growth inhibitors that are expressed in oligodendrocytes (Ide, 1996). But Schwann cells are of special interest not only as central player in peripheral nerve regeneration; also in MS therapy they seem to be a therapeutic option. Although Schwann cells contain myelin basic protein, one of the main targets of the immune cells in MS (Deber and Reynolds, 1991), they are not affected by this disease. Furthermore, Schwann cells are able to break down devastated myelin and to clear debris by phagocytosis (Liu et al., 1995), an important prerequisite for successful remyelination (Stoll and Müller, 1999), and they can remyelinate demyelinated axons in the CNS (Blakemore, 1977; Honmou et al., 1996). Despite their high efficiency in inducing nerve regeneration, the clinical use of Schwann cells is limited. One practical difficulty with the potential use of Schwann cells for autologoues transplantation 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; Calderon-Martinez et al., 2002). And, one or more functioning nerves must be sacrificed with the consequence of loss of sensation, scarring, and possibly, neuroma formation. Although special techniques to cultivate adult Schwann cells have been established (Keilhoff et al., 1999, 2000; Bachelin et al., 2005), alternative cell systems are desirable.

Mesenchymal stem cells (MSCs) may be an alternative source for Schwann cells. They reside in the bone marrow and differentiate mainly in cell lineages of mesodermal origin to form, for example, muscle, bone, cartilage, fat, and tendon (Prockop, 1997; Pittenger et al., 1999). MSCs are easily accessible through aspiration of bone marrow. They readily adhere in plastic culture dishes (Phinney et al., 1999). With appropriate stimuli and environmental conditions, MSC can differentiate into nonmesenchymal lineages, including astrocytes (Kopen et al., 2001), myocardium (Pittenger and Martin, 2004), endothelial cells (Oswald et al., 2004), neurons (Woodbury et al., 2000; Deng et al., 2001), and myelinating cells of the PNS (Dezawa et al., 2001; Mimura et al., 2004; Kamada et al., 2005). Recently, however, transdifferentiation has been debated and several other biological explanations, e.g. cell fusion, have been put forward (Rutenberg et al., 2004; Kashofer and Bonnet, in press).

Here we aimed to evaluate the transdifferentiation potential of MSCs into myelinating “Schwann cell-like” cells to offer new therapeutic strategies for myelin defects. A few reports describe the possible impact of bone marrow cells on PNS, all using different approaches to lesion and regeneration characteristics. Tohill and Terenghi, for example, have studied the effect of grafting glial-differentiated bone marrow stromal cells into a rat model of a peripheral nerve injury. They report a beneficial effect on Schwann cell growth within regenerating nerves (Wiberg and Terenghi, 2003; Tohill and Terenghi, 2004; Tohill et al., 2004). Cuevas et al. (2002) were able to induce peripheral nerve regeneration using undifferentiated bone marrow stromal cells, and Kocsis et al. (2002) examined the remyelinating potential of “peripheral-myelin-forming” cells derived from bone marrow to repair the injured spinal cord. The group of Dezawa, one of the pioneers in the field of stem cell derived therapies for peripheral nerve injuries, presented an artificial nerve graft made with bone marrow stromal cell derived Schwann cells as an alternative for the difficult reconstruction of peripheral nerves (Dezawa et al., 2001; Dezawa, 2002; Mimura et al., 2004; Kamada et al., 2005).

We used our well established rat model (Fansa et al., 1999, 2001; Fansa and Keilhoff, 2004): undifferentiated or transdifferentiated (Schwann cell-like) MSCs, or alternatively, genuine Schwann cells were implanted into devitalized muscle graft to bridge a 2-cm gap in the rat sciatic nerve. This model has two advantages. Muscle conduits are free of endogenous Schwann cells, thus excluding the possibility that regeneration is assisted by endogenous Schwann cells or that implanted MSCs fuse with the remaining host Schwann cells imitating a transdifferentiation effect. Moreover, the gap length of 2 cm minimizes the effect of spontaneous regeneration in rats, that is known to be highly effective and reaches 2–3 mm per day (Near et al., 1992). Moreover, the myelinating capacity of the transdifferentiated Schwann cell-like cells was studied in PC12 cell cultures, normally not myelinated.

METHODS

Chemicals

α-MEM (Biochrom, Berlin, Germany), 3,3′-diaminobenzidine (Sigma, Saint Louis, Missouri), 3-isobutyl-1-methyl-xanthine (IBMX; Merck, Darmstadt, Germany), albumin (PAA Laboratories GmbH, Cölbe, Germany), all-trans-retinoic acid (Sigma), cacodylate buffer (Sigma), collagenase (Sigma), dexamethasone (Sigma), dispase (Boehringer-Mannheim), Dulbecco's modified Eagle's medium (DMEM; Biochrom), Durcupan (Sigma), Evans blue (Sigma), fetal calf serum (FCS; Biochrom), Fluorescein diacetate (Serva, Heidelberg, Germany), forskolin (Merck), glutaraldehyde (Sigma), human insulin-like growth factor 1 (IGF-1; Biosource, Camarillo, CA), human recombinant heregulin-β EGF domain (Her-β; Upstate Biotechnology, Lake Placid, NY), hyaluronidase (Sigma), insulin (Sigma), Isoflurane (Abbott, Wiesbaden, Germany), laminin (Sigma), l-ascorbic acid (Merck), l-glutamine (Sigma), osmium (Merck), paraformaldehyde (Merck), penicillin/streptomycin (Invitrogen, Carlsbad, CA), pentobarbital (Abbott), platelet derived growth factor (PDGF; Sigma), poly-d-lysine (Sigma), recombinant human basic fibroblast growth factor (bFGF; Chemicon, Temecula, CA), β-glycerol phosphate (Sigma), β-mercaptoethanol (Merck), RevertAidTM-H Minus First Strand cDNA Synthesis Kit (Fermentas, Burlington, Canada), Sudan red (Merck), Taq-DNA-polymerase (Peqlab, Erlangen, Germany), Toluidine blue (Chroma, Munster, Germany), Triton X-100 (Ferak Berlin, Berlin, Germany), TRIzol® reagent (Invitrogen), TURBO DNA-free™-Kit (Ambion, Austin, Texas), trypsin/EDTA (Invitrogen), uranyl acetate (EMS, Fort Washington, PA).

Animals

Ninety inbred Wistar rats were used (Shoe: Wist/Shoe, DIMED Schönwalde GmbH, 20 for cell cultivation, 70 for the 10 experimental groups). The rats were kept in accordance with the guidelines of the German Animal Welfare Act. The experimental protocol was approved by a review committee of the state of Sachsen-Anhalt, Germany. The animals were housed under temperature-controlled conditions at 21±1°C, relative air humidity 55–60%, with a 12-h light/dark cycle, with free access to standard rat chow (Altromin 1324™, Altromin GmbH, Lage, Germany) and tap water. All surgical procedures were performed under general anaesthesia with intraperitoneal injection of pentobarbital (60 mg/kg).

Cultivation, Transdifferentiation, and Characterization of Mesenchymal Stem Cells

Isogenic MSCs were obtained from the femur of rats aged 25–30 days as described in Keilhoff et al. (in press). The primary cultures were termed passage 0 (P0). On every 10th day, cells were detached with 0.25% trypsin and 1 mM EDTA for 5 min at 37°C and plated in new flasks (P1 and P2, respectively).

For transdifferentiation, MSCs of P2 were plated at 500 cells/cm² and expanded in growth medium (α-MEM supplemented with 20% FCS, 2 mM l-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin) for 3 days and further for 1 day with additional 1 mM β-mercaptoethanol. Then medium was replaced with transdifferentiation medium (α-MEM, 10% FCS, 2 mM l-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin, 35 ng/mL all-trans-retinoic acid). For the final transdifferentiation step, cultures were incubated in transdifferentiation medium supplemented with forskolin 5 μM, bFGF 10 ng/mL, PDGF-AA 5 ng/mL, Her-β 200 ng/mL, and IGF-1 10 ng/mL for 8 days with a media change every other day.

MSCs as such were characterized by immuno-labeling for the stem cell markers BMPR-1A and Stro-1 and by their ability to differentiate into mesodermal cell lineages. For immuno-labeling, cultures were fixed in 4% paraformaldehyde in PBS for 30 min. Nonspecific antigens were blocked with 3% fetal calf serum in PBS for 45 min and cultures were incubated overnight with the primary antibodies (polyclonal anti-BMPR-1A, rabbit, 1:100, Santa Cruz Biotechnology, Santa Cruz, CA; monoclonal anti-Stro-1, mouse, 1:100, R&D Systems, Minneapolis, Minnesota). After washing (thrice for 5 min with PBS) cells were incubated with the secondary antibodies (Alexa Fluor® anti-rabbit IgG; Alexa Fluor® anti-mouse IgG, goat, 1:500, Molecular Probes, Eugene, Oregon) at room temperature for 3 h. Cultures were examined using a fluorescence microscope (Axiophot; Zeiss, Jena, Germany) equipped with phase contrast, fluoresceine, rhodamine and DAPI optics and documented with a color camera AxioCam MRc (Zeiss, Jena). To demonstrate the differentiation potential, cells of P2 were disseminated at a density of 500 cells/cm² and maintained in growth medium for 3 days. Then medium was replaced with differentiation medium (growth medium enriched with either 10 nM dexamethasone, 50 μg/mL l-ascorbic acid, and 10 mM β-glycerol phosphate for osteogenic differentiation, or 10 nM dexamethasone, 5 μg/mL insulin, and 0.5 μM IBMX for adipogenic differentiation). After 21 days, cultures were stained with Sudan red (adipogenic differentiation) or Toluidin-Alizarin (osteogenic differentiation). Moreover, an RT-PCR for osteopontin and the fatty acid binding protein 4 (FABP4) was carried out (primers and technique see the following text).

A comparative characterization of undifferentiated and transdifferentiated MSCs was done by PT-PCR. Total RNA was isolated using TRIzol reagent according to the manufacturer's instructions followed by DNAse treatment. For RT-step 5 μg RNA was used for first strand cDNA synthesis. PCR was performed under the respective conditions with 0.5 μg cDNA, a Taq-DNA-polymerase, and the respective primers:

• BMPR-1A, forward 5′-CAGCCCTACATCATGGCTGAC-3′, reverse 5′-gcttcaaaacggctcgaagac-3′ (229 bp, 40 cycles, gene bank No. NM_030849);

• IGF-1R, forward 5′-tcccaagctgtgtgtctctgaa-3′, reverse 5′-gtgccacgttatg atgatgcg-3′ (178 bp, 36 cycles, gene bank No. NM_052807);

• ErbB2, forward 5′-aatgccagcctctcattcctg-3′, reverse 5′-gacttcgaagctgcagctcc-3′ (235 bp, 40 cycles, gene bank No. NM_017003);

• LNGF-R, forward 5′-cgacaacctcattcctgtctattgc-3′, reverse 5′-gtgccacgttat gatgatgcg-3′ (227 bp, 40 cycles, gene bank No. NM_012610);

• S100b, forward 5′-gagagagggtgacaagcacaa-3′, reverse 5′-ggccataaactcctgg aagtc-3′ (169 bp, 28 cycles, gene bank No. NM_013191);

• CD104, forward 5′-gctctgctggaaatactgtgc-3′, reverse 5′-caggcttcatgaggttctcag-3′ (beta4-integrin, 317 bp, 40 cycles, gene bank No. NM_013180);

• Krox20, forward 5′-agataccatcccaggctcagt-3′, reverse 5′-ctctccggtcatgtcaatgtt-3′ (300 bp, 40 cycles, gene bank No. NM_053633);

• L1, forward 5′-tggaagtggaggaaggagaat-3′, reverse 5′-aagtgggcattgcagatgtag-3′ (202 bp, 40 cycles, gene bank No. NM_017345);

• FABP4, forward 5′-aaagaagtgggagttggcttc-3′, reverse 5′-accatccagggttat gatgct-3′ (fatty acid binding protein 4, 204 bp, 33 cycles, gene bank No. NM_053365);

• Osteopontin, forward 5′-tccgatgaatctgatgagtcc-3′, reverse 5′-gcaactgggat gaccttgata-3′ (236 bp, 22 cycles, gene bank No. NM_012881);

• GAPDH, forward 5′-ttagcacccctggccaagg-3′, reverse 5′-cttactccttggaggccatg-3′ (glyceraldehyde-3-phosphate dehydrogenase, house-keeping gene, 531 bp, 24 cycles, gene bank No. NM_017008).

Cultivation of PC12 and Schwann Cells

PC12 cells (rat pheochromocytoma cell line) were cultured at 37°C and 5% CO₂ in MEM containing 10% FCS, 2 mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin at a density of 500 cells/cm². The cells were allowed to differentiate for 4 days. During the experiments the medium was changed every 2 days and the additives were added each time.

Isogenic Schwann cells were obtained after predegeneration of rat sciatic nerves as described in Keilhoff et al. (1999). Before implantation random samples of the cultures were characterized by double labeling with antibodies directed against S100 protein, specific for Schwann cells (polyclonal, rabbit, 1:500, Dako, Hamburg, Germany), and against fibronectin (monoclonal, mouse, 1:10, Boehringer Mannheim), specific for fibrocytes (description see earlier text).

Preparation and Characterization of the Muscle-Cell Grafts

There were 10 groups, 5 with a survival time of 3 weeks and 5 with a survival time of 6 weeks. Each group consisted of seven Wistar rats, 8 weeks old at the beginning of the experiments. An identical operating protocol was performed for each group. The right sciatic nerve was exposed and a 2 cm nerve segment was completely removed. In control groups I and II, the nerve segment was orthotopically reimplanted with 10/0 monofilament nylon epineural sutures. In groups III–VIII, the gracilis muscle was harvested. This muscle offers a longitudinal fiber orientation, which is similar to the endoneural tube structure. The muscles were acellularized by freezing in liquid nitrogen (−96°C) until thermal equilibrium was achieved and thawing in PBS (pH 7.2, 22°C). This was repeated for three times. A suspension of MSCs, transdifferentiated MSCs, and Schwann cells (2×10⁶ cells/mL DMEM) was transferred longitudinally into the acellular muscle with a microsyringe (29G×1/2″ needle) immediately before implantation. For control groups IX and X only DMEM was used to fill the muscles.

Evaluation of the Myelinating Capacity

In vitro myelination assay: Co-cultures were established by removing the media from the PC12 cells and seeding 500 dissociated Schwann cells, 500 MSCs, or 500 transdifferentiated MSCs, respectively, into each dish. PC12/Schwann cells and PC12/MSCs were cultured in the “PC12-medium.” PC12/transdifferentiated cells were cultured in “PC12-medium” or in transdifferentiated medium. During the experiments the medium was changed every 2–3 days. After 14 days, cocultures were fixed for 30 min in 0.2 M cacodylate buffer containing 2.5% glutaraldehyde, then osmicated (2% OsO₄), dehydrated, en-bloc stained with 7% uranyl acetate and finally, embedded in Durcupan. All steps were carried out in the culture dishes. Ultra-thin sections (50–70 nm) were cut and mounted on Formvar-coated slot grids. For examination an E 900 transmission electron microscope (Zeiss, Germany) was used.

In vivo myelination assay: Nerve material was taken from the middle of the graft 3 and 6 weeks after reconstruction. Specimens were fixed as described earlier. Ultra-thin sections (50–70 nm) were cut for electron microscopy and semi-thin (1000 nm) sections for morphometric analysis. Ten arbitrarily selected grids from each nerve segment were evaluated. Semi-thin sections were stained with toluidine blue and examined with an Axiophot microscope.

For statistical analysis the groups of the nerve sections were blinded to the examiner. Five details of five cross-sections per nerve or graft segment were scanned using a CCD camera and all morphologically vital appearing axons were counted manually. Morphometric evaluation was carried out with a computer-assisted system (Image C, Imtronic, Münster, Germany). To assess the maturity of the fibers the g-ratio was calculated, expressing the relation of axonal diameter to the entire fiber diameter. Statistical analysis was performed with the nonparametric Kruskal–Wallis test. The Mann–Whitney U-test was used as a post-hoc test.

To identify the donor derived cells, a prelabeling with 5-bromo-2′-deoxy-uridine (BrdU) was performed. Therefore, BrdU (final concentration of 10 μM) was added to the culture medium of MSCs and transdifferentiated MSCs for the final 24 h of cultivation. The prepared cells were implanted into muscle grafts and these were transplanted as described (three animals per group). After a survival time of 3 or 6 weeks, cryosections (20 μm) of the transplant (5 mm distal to the proximal suture) were incubated in 2 N HCl for 1 h at 37°C for DNA denaturation, neutralized by 0.1 M borate buffer (pH 8.5), incubated with a rat monoclonal antibody to BrdU (Oxford Biotechnology Ltd., 1:100, in PBS containing 0.3% Triton X-100) for 1 h at 37°C and double immuno-stained with an antibody against myelin basic protein (MBP, monoclonal, mouse, 1:100, Dako). Following incubation with primary antibodies, slices were washed in PBS (thrice for 5 min), incubated with a combination of secondary antibodies (goat anti-rat-IgG Alexa Fluor 546/goat anti-mouse-IgG Alexa Fluor 350, 1:500, Molecular Probes), mounted, and examined on an Axiophot microscope.

RESULTS

Cell Characterization

Prepared bone marrow contained a heterogeneous population of cells. The proportion of MSCs increased from less than 1% at the time of dissemination to 97.4±4.5% BMPR-1A receptor immuno-positive cells in P2, from which 88.3±11.7% were immuno-positive for the stem cell marker Stro-1. When MSCs were grown at a low density, cells showed a triangular shape (Fig. 1A). But morphology changed to a fibroblast-like morphology, when cultures reached confluence (Fig. 1B). More than 30% of the MSCs incubated with adipogenic induction medium differentiated to adipocytes (Fig. 1C). Calcification started 1 week after application of the osteogenic differentiation medium and showed an extensive deposition of bone material after 3 weeks. Among the studied cells, only adipocytes derived from MSCs expressed the FABP4 gene. mRNA expression levels of osteopontin increased after the MSCs differentiated into osteoblasts. The mRNA cannot be found in Schwann cells.

Fig. 1.

(A) In the phase-contrast microscope MSCs were heterogeneous in their morphology. (B) At a low density cells showed a triangular shape, which changed to a fibroblast-like morphology when cultures reached confluence. (C) MSCs incubated with adipogenic induction medium were able to differentiate into lipid-containing adipocytes (arrows, Sudan staining). (D) At the beginning of cultivation, PC12 cells were round or polygonal with only short, if at all, processes. (E) After 4 days in culture, cells were multiform with noticeable processes (arrowheads). (F) Phase-contrast micrograph demonstrating a spindle-shaped bi- or tri-polar morphology of Schwann cells (magnification for all 200×).

All transdifferentiation experiments were carried out in cultures of P2. During transdifferentiation, approximately 50% of the transforming cells, obviously dead, detached from the surface. From the remaining adherent cells about 5% showed morphological similarities with oligodendrocytes and about 60% of the total MSCs adopted Schwann cell-like morphology. Transdifferentiation was reversible. Approximately 5% of the transdifferentiated MSCs redifferentiated to MSCs on the first day after return to growth media and 3 days after the medium was changed; only separate cells remained in a transdifferentiated shape. After cells were redifferentiated, we were able to induce transdifferentiation again by adding the respective cytokines.

At mRNA-level (a survey is given in Fig. 2), BMPR-1A, the marker for bone precursor cells, including MSC, was intensely expressed in MSCs, whereas in transdifferentiated MSCs BMPR-1A expression was lower and failed in Schwann cells. IGF-1R and erbB2, the respective receptors for the applied growth factors IGF-1 and Her-β, were expressed at high levels in all samples. In MSCs, mRNA of the LNGF-R was not detectable, whereas in transdifferentiated MSCs and in Schwann cells it was markedly expressed. S100b-mRNA, intensely expressed in Schwann cells, was detectable in both types of MSCs at lower levels. CD104 was not expressed in MSCs. In transdifferentiated MSCs, the expression of CD104 was increased, but did not reach the level of Schwann cells. Krox20, a transcription factor for myelin genes, was expressed in MSCs at detectable levels; its expression was induced in transdifferentiated MSCs, and reached the highest expression in Schwann cells. The L1 expression pattern was the same as for S100b.

Fig. 2.

Expression pattern of several genes in MSCs, transdifferentiated MSCs, and Schwann cells at mRNA level. For product sizes see “Methods” section.

At the beginning of cultivation, PC12 cells were round or polygonal. The great majority of the cells had none or short processes (Fig. 1D). Only a few cells had processes greater than or equal to the long diameter of the cell body. After 4 days in culture, about 20% of the cells became spindle, triangle, or irregular shaped with noticeable processes stretching out (Fig. 1E).

Schwann cells in culture developed a typical bi- or tri-polar spindle-shaped morphology, easily distinguishable from the flattened fibrocytes (Fig. 1F). Coimmunostaining for S-100 and fibronectin demonstrated that the cultures mainly consisted of Schwann cells with tolerable contamination of fibrocytes (∼10%).

Myelinating Capacity and Regenerative Outcome

In vitro myelination assay: Addition of Schwann cells and, with curtailments, transdifferentiated MSCs induced a rapid neuron-like differentiation of the PC12 cells, as revealed by an increase in cell size and by the extension of neurites. Electron microscopy confirmed myelin structures in PC12/Schwann-cultures at 14 days in vitro (Fig. 3A). This myelin was compacted and the presence of intraperiodic lines was seen. In the PC12/transdifferentiated MSCs cultures, the grade of myelination depended on the culture medium. A remarkable myelination, like in PC12/Schwann cell cultures with a mean of 7±2 layers, was noted when the cells were cultivated in transdifferentiation medium (Fig. 3B). Poor myelination was found when the “PC12-medium” was used (Fig. 3C). These cultures produced myelin membranes with less layers which were not compacted. MSCs were not able to myelinate PC12 cell neurites.

Fig. 3.

Electron microscopy of 14-day-old cocultures from (A) PC12 and Schwann cells, (B) PC12 and transdifferentiated MSCs in transdifferentiation medium, and (C) PC12 and transdifferentiated MSCs in “PC12-medium.” In (A) and (B), compact myelin membranes composed of a mean 7±2 layers could be seen, whereas in (C) only less compacted myelin membranes were detectable. Electron micrographs of the respective graft segments (∼10 mm distal to the proximal suture) after a regeneration time of 3 (D, F, H, J, N) and 6 (E, G, I, K, O) weeks: (D, E) The control group (nerve graft) showed the best regeneration indicated by a high number of axons with a proper myelin sheath, a fascicular structure and no signs of cellular infiltration in the graft. (F, G) The cell-free muscle grafts showed an impaired regeneration with only a few thin myelinated fibers and a massive connective tissue fibrosis. (H, I) The muscle-MSC grafts showed a poor regeneration with similar to the cell-free muscle characteristics. (J, K) The additional implantation of transdifferentiated MSCs into muscle grafts significantly improved the regeneration outcome, although the electron microscopical evaluation indicated fibers with a more irregular shape and thinner myelin sheaths in comparison to the control and the muscle-Schwann cell groups. (L) Here and in the muscle-Schwann cell group, a mini-fascicle formation was evident. (M) In contrast to Schwann cells, which always wrap only one axon transdifferentiated MSCs are able to myelinate two and even more axons. (N, O) In the muscle-Schwann cell group, a quite good regeneration was evident. Axons were regularly shaped and well myelinated (A, B, C, M 20.000×; all others 7000×).

The myelinating capacity in vivo was evaluated by the regenerative outcome after bridging a 2-cm gap of rat sciatic nerve with a graft tissue engineered from devitalized muscle enriched with the respective stem cells in comparison to muscle-Schwann cell grafts after survival times of 3 and 6 weeks. Tolerance to the operations was good. No clinical signs of pain or discomfort were observed. No trophic ulcerations on the operated leg were visible.

The best regenerative outcome was demonstrable in control groups I and II (Fig. 4A). Electron microscopy demonstrated a large number of remyelinated axons with a regular symmetry, wide cross-sections and in some cases thick myelin isolations. There was no connective tissue fibrosis (Fig. 3D, E). The regenerative capacity of the muscle conduits depended on the respective cell settlement. Poor regeneration was evident in the cell-free muscle conduits (Figs. 4A and 3F, G), and in the ones with MSCs (Figs. 4A and 3H, I). They had significantly fewer axons when compared with control groups, and the prominent feature was a massive connective tissue fibrosis. The ability of transdifferentiated MSCs to support regeneration decreased with increasing regeneration period. After a regeneration period of 3 weeks these conduits offered a quite good regenerative outcome (Fig. 3J) with significant numbers of newly myelinated fibers (Fig. 4A). With prolonged regeneration time, the axon counts reduced in comparison to the control group (Fig. 3K), and the organization in mini-fascicles was evident (Fig. 3L). Fibers developed a more irregular shape and thinner myelin sheaths. In contrast to Schwann cells, one transdifferentiated MSC was able to myelinate two or even more axons (Fig. 3M). In both types of muscle-Schwann cell grafts significantly more newly myelinated fibers, grouped in mini-fascicles, were seen when compared to all other muscle conduits (Figs. 4A and 3N, O).

Fig. 4.

(A) Morphometric analysis of the fiber counts showed a statistically significant reduction of fibers in all muscle grafts compared to the control nerve graft (not indicated in the graph, ^*** p < 0.0005 for cell-free and MSC muscle grafts, ^* p < 0.05 for the graft with Schwann cells or transdifferentiated MSCs), thereby the grafts with Schwann cells and thus with transdifferentiated MSCs developed a significant higher axon counts compared to the cell-free and MSC muscle grafts (^** p < 0.005, Mann–Whitney test). (B) The g-ratio (axon diameter/total fiber diameter, usually between 0.6 and 0.7) indicates a reduced myelin sheath thickness in the muscle grafts. Data are mean values±S.E.M.

The g-ratio, expressing the quality of the myelin sheaths, was between 0.6 and 0.7 in the control (proximal) nerve and tended to increase in the graft segments of all groups, indicating thinner myelin sheaths compared to the proximal nerve stump (Fig. 4B).

Before transplantation, MSCs and transdifferentiated MSCs were incubated with BrdU. After a survival time of 3 weeks as well as 6 weeks, we were able to demonstrate colabeling for BrdU and MBP in the muscle-transdifferentiated MSCs grafts (Fig. 5A, B), indicating that transdifferentiated MSCs were involved in the process of myelination. A double-labeling of undifferentiated MSCs was seen only in a very few cases (Fig. 5C, D).

Fig. 5.

Grafted cells (A, B: transdifferentiated MSC; C, D: undifferentiated MSC), detected by BrdU-labeling (red), 3 (A, C) and 6 (B, D) weeks after transplantation. In muscle/transdifferentiated MSC grafts immuno-staining for MBP (blue) was colocalized with the BrdU-fluorescence (arrows), indicating an active role of transdifferentiated MSCs in remyelination. Undifferentiated MSCs could be found double-labeled only in a very few cases indicating their defective myelinating capacity (magnification 100×).

DISCUSSION

Differentiation of MSCs into myelinating cells could be achieved by application of a growth factor cocktail, consisting of bFGF, PDGF-AA 5, heregulin-β, and IGF-1. These factors are known to induce ontogenetic development from Schwann cell precursors into early Schwann cells (Cheng et al., 1999; Cohen et al., 1999). Prerequisite for successful use of the growth factors was the preincubation with β-mercaptoethanol and all-trans-retinoic acid and coincubation with forskolin. β-Mercaptoethanol was used to promote formation of neurite-like outgrowth (Woodbury et al., 2000; Deng et al., 2001). Retinoic acid is known to induce differentiation of embryonic stem cells into neural lineages cells (Fraichard et al., 1995). An increase in cAMP, and thus, an elevated expression of mitogenic genes can be achieved when cells are treated with forskolin (Fortino et al., 2002).

The success of transdifferentiation was demonstrated by RT-PCR. The expression of the LNGF receptor, CD104, and Krox20, all accepted markers for Schwann cells (Mirsky and Jessen, 1999; Jessen and Mirsky, 2002; Chan et al., 2004), was induced by transdifferentiation. The expression of the MSC marker BMPR-1A (Otsuka et al., 1999; Hoffmann and Gross, 2001), however, was significantly reduced after transdifferentiation and reversed after recovery to the undifferentiated cell fate. Surprisingly, the “classic” Schwann cell genes S100b and L1 (Magnaghi et al., 2001; Jessen and Mirsky, 2002) were expressed in MSCs as well.

A further proof of successful transdifferentiation is to demonstrate the myelinating capacity. This we have done in vitro and in vivo. In vitro, PC12 cells were used as reliable assay for detecting ensheathment of neurites. When the cocultures were processed for electron microscopy, it could be demonstrated that Schwann cells and transdifferentiated MSCs were able to wrap P12 cell neurites normally unmyelinated. After 2 weeks in vitro, the myelin membranes were composed of several layers. Cocultures of PC12 and transdifferentiated MSCs cultured in “PC12-medium,” also produced myelin-like membranes, but these were composed of less layers that were loosely compacted. Obviously, the “PC12-medium,” lacking the transdifferentiation cytokine cocktail, was not able to stabilize transdifferentiation of MSCs to a needed for myelination extent.

Our biogenic grafts, prepared from devitalized muscle and transdifferentiated MSCs, supported regeneration of the sciatic nerve in a Schwann cell-like manner, thus demonstrating the ability of transdifferentiated cells to produce an environment needed for regeneration processes. Unlike others (Akiyama et al., 2002; Tohill et al., 2004) we did not find any nerve regeneration in the muscle grafts enriched with undifferentiated MSCs, but rather a massive connective tissue fibrosis that suggests an arbitrary differentiation of MSCs. Since the muscle grafts used were free of endogenous Schwann cells, it could be excluded that regeneration was assisted by endogenous Schwann cells or that implanted MSCs fused with remaining host Schwann cells pretending a transdifferentiation effect. In the grafts enriched with Schwann cells or transdifferentiated MSCs, the bundles of regenerated nerve fibers were arranged in mini-fascicles. These structures might be a result of newly formed perineurial cells isolating the regenerated clusters from the surrounding connective tissue. Such minifascicular reorganization is characteristic for nerve grafts in which the original perineurial barrier has been eliminated, as resembled by our muscle grafts (Schröder, 2001).

The demonstrated differences in myelination capacity of transdifferentiated MSCs compared with Schwann cells were reflected in the respective expression patterns of CD 104 (beta4 integrin) and Krox20. CD104 is a cell-surface extracellular matrix receptor, and several authors suggested a correlation between its expression and myelination, as Schwann cells from beta4 integrin knock-out mice form only rudimental myelin (Previtali et al., 2003). Krox20 (Egr2) is a transcription factor that controls Schwann cell myelination (Parkinson et al., 2004). Schwann cells in Krox20 knock-out mice failed to myelinate, and unlike myelinating Schwann cells, continued to proliferate and were susceptible to death.

Moreover, the reduced myelination capacity of transdifferentiated MSCs compared to Schwann cells may be attributed to the different expression patterns of the cell adhesion molecule L1. In our experiments, it is heavily expressed only in Schwann cells. This molecule is important for ensheathment and myelination in peripheral nerve development. In mice, lacking the sixth domain of the L1 protein, myelin was thinner and abnormal (Itoh et al., in press). Interestingly, these mice, like our rats, showed the phenomenon that multiple axons wrapped in a single myelin sheath. Furthermore, the differences in supporting extensive nerve regeneration including myelination may be attributed to the different levels of the calcium-binding protein S100b that released into the extracellular space, stimulates neuronal survival, proliferation, and differentiation. Blocking of S100 receptors suppresses peripheral nerve regeneration (Donato, 2001; Rong et al., 2004).

Another explanation might be the demonstrated instability of MSC transdifferentiation. In this study, a stable transdifferentiation was only achieved when the cytokine cocktail was applied continuously. In vivo, this cannot be assumed. Normally, in a distal segment of transected peripheral nerves a microenvironment allowing regeneration is formed by reprogrammed Schwann cells (Stoll and Müller, 1999). If transdifferentiated MSCs were transplanted into such an environment, their long-term fate will be stable defined as Schwann cell-like by cell–cell interaction as it was shown in the literature (Dezawa et al., 2001; Dezawa, 2002; Kocsis et al., 2002; Mimura et al., 2004; Tohill and Terenghi, 2004; Tohill et al., 2004; Kamada et al., 2005). In the presented experiments, however, the microenvironment for transplanted cells mainly consisted of devitalized muscle lacking most of the cues for peripheral nerve regeneration and subsequently for differentiation of MSCs into myelinating Schwann cell-like cells.

In conclusion, the experiments presented here demonstrate that it is possible to induce myelinating capacity of MSCs by chemically induced transdifferentiation. The transdifferentiated MSCs developed a Schwann cell-like morphology and expressed respective biochemical markers. Although the results must be interpreted with caution, we may speculate that this technique provides a tool to manipulate adult stem cells for cell-based therapy of myelin defects.