DOI 10.1007/s11062-015-9501-6
Neurophysiology, Vol. 47, No. 1, February, 2015
Effects of Neural Crest-Derived Multipotent Stem Cells on Regeneration of an Injured Peripheral Nerve in Mice R. G. Vasyliev,1 A. E. Rodnichenko,1 S. N. Shamalo,2 A. S. Demidchouk,1,2 I. F. Labunets,1 Yu. B. Chaikovskii,2 and G. M. Butenko1 Received September 30, 2014. We studied the effect of transplantation of neural crest-derived multipotent stem cells (NC-MSCs) obtained from vibrissa germs on regeneration of the injured (transected) sciatic nerve in adult mice (strain FVB). After transplantation of the cells into the injured region, regeneration of the severed nerve was intensified, as compared with that in mice with no implantation. The intensity of vascularization and renewal of the endoneurium also increased. The density of nerve fibers in the distal part of the injured nerve in mice after transplantation of NC-MSCs (10522.8 ± 1044.0 mm –2) was significantly higher than that in mice with nerve injury but without transplantation (8409.5 ± 739.5 mm–2). Possible mechanisms of acceleration of regeneration of the injured peripheral nerve under conditions of transplantation of stem cells are discussed.
Keywords: stem cells, peripheral nerve, transplantation, regeneration.
INTRODUCTION The development of approaches promoting regeneration of the injured peripheral nerve trunks attracts significant attention of many researchers [1]. The extensive use of novel surgical technologies and pharmacological preparations for treatment of such damages open prospects for significant improvement of clinical outcomes; however, many aspects of the problem of complete regeneration of the structure and function of the nerves after the above traumas remain unresolved. To stimulate regenerative processes in the injured peripheral nerves, experimental approaches using cellular therapy are now developed [2, 3]. The ability of stem cells of different types to intensify regeneration of the traumatized sciatic nerve has been demonstrated [2, 4]. Stem cells implanted into the site of nerve injury differentiate turning into Schwann cells, and this process effectively accelerates the growth and myelination of regenerating axons. Neural crest-derived multipotent stem cells (NC-MSCs) are a promising type of the stem cells State Institute of Genetic and Regenerative Medicine, National Academy of Medical Sciences of Ukraine, Kyiv, Ukraine. 2 Bogomolets National Medical University, Ministry of Public Health of Ukraine, Kyiv, Ukraine. Correspondence should be addressed to Yu. B. Chaikovskii (e-mail:
[email protected]). 1
that can improve peripheral nerve regeneration using the techniques of cellular therapy and cell engineering. Such cell can be obtained from the bulbar region (BR) of the hair (vibrissa) follicles (HFs). NC-MSCs possess the ability to self-renewal and directed multilinear differentiation, including their transformation into Schwann cells [4-6]. In our study, we investigated the possibilities for intensification of the regenerative processes in the injured sciatic nerve of adult mice after transplantation of NC-MSCs obtained from HF BRs.
METHODS Experiments were carried out on wild-type and GFP transgenic mice belonging to the FVB strain. The animals were obtained from the vivarium of the Institute of Genetic and Regenerative Medicine (National Academy of Medical Sciences of Ukraine, Kyiv, Ukraine). The mice of the above FVB strain were divided into the following groups. In animals of group 1 (n = 5), a segment of the right sciatic nerve was only surgically isolated but not transected (sham-operated control). In mice of group 2 (n = 5), the right sciatic nerve was transected at the level of the middle third of its length. Mice of group 3 (n = 5) were initially subjected to transection of the sciatic nerve and then to transplantation of NC-MSCs in a carrier (fibrin gel; 10 6 cells in 50 µl
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Effects of Multipotent Stem Cells on Nerve Regeneration of the gel). For this purpose, we mixed 10 µl of a 2% solution of fibrinogen and 10 µl of cell-containing DMEM:F12 medium with 0.5 IU of thrombin added. Polymerization of the solution and formation of the fibrin gel occured immediately after introduction into the lesion site. All surgical interventions were performed under avertin anesthesia (0.4 ml of 2.5% solution, i.p.). NC-MSCs of GFP transgenic mice were obtained from BRs of the HFs of the vibrissae and cultured in DMEM:F12 medium supplemented with 10% fetal bovine serum, 5 ng/ml bFGF, and 2 mM glutamine (all agents from Sigma, USA) in a gas incubator with an artificial atmosphere containing 5% СО 2, 5% О 2, and 90% N 2. For transplantation, we used cells of the third passage. Biological properties of cultured in vitro NC-MSCs were described in detail in our earlier report [6]. Four weeks after the beginning of the experiment, mice of all studied groups were euthanized under ether anesthesia; samples of biological materials (parts of the intact sciatic nerve and fragments of the regenerative neuromas with the adjacent parts of the injured nerve) were examined using standard morphological techniques. For light microscopy, histological preparations were fixed for 24 h in a 10% solution of neutral formalin; after rinsing, frozen sections were made and impregnated with silver nitrate [7]. For morphometric analysis of the results of light
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microscopy of histological preparations, we used UTHSCSA Image Tool for Windows (Version 2.00) software and an Olympus microscope equipped with a standard ocular mount. The density of distribution of nerve fibers was estimated in the distal part of the injured nerve. Histological slices were photographed using a digital photocamera.
RESULTS Properties of NC-MSCs. In in vitro culture, the obtained NC-MSCs possessed a typical stellate shape (Fig. 1A). They demonstrated properties typical of neural stem cells. In particular, a specific marker, protein of intermediate filaments (nestin), was expressed in these units (B), and neurospheres were formed under specific conditions of serumfree culturing (C) [6]. During culturing in the medium promoting induction of glial differentiation, NS-MSCs differentiated into Schwann cells; this was confirmed by typical changes in their morphological characteristics and expression of the specific protein S-100 (D) [6]. Peculiarities of Regeneration of the Injured Sciatic Nerve in Experimental Mice. After transection of the nerve, regeneration neuromas were formed in experimental animals at the nerve injury site. These structures included nerve fibers, blood vessels, cell elements (predominantly
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50 µm
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20 µm
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D
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F i g. 1. Biological properties of neural crest-derived multipotent stem cells (NCMSCs) from bulbar regions of the vibrissa follicles of adult mice. A) NC-MSCs cultured in vitro (phase-contrast microscopy); B) expression of the NC marker nestin (fluorescence microscopy); C) NC-MSCs formed under conditions of culturing with no serum (phasecontrast microscopy); D) differentiation of NC-MSCs into Schwann cells (expression of protein S-100 in the cells; fluorescence microscopy).
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82 fibroblasts), collagen fibers, and ground substance of the connective tissue (Fig. 2). In mice of group 3, regenerating nerve fibers were located appreciably more compactly than those in mice of group 2 (B and C, respectively). In animals of group 2 (without implantation of MSCs), many fibers were oriented at various angles with respect to the longitudinal axis of the nerve. In mice of group 3, the orientation of fibers was stricter, i.e., a great majority of them was located in the longitudinal direction. The distal part of the injured nerve contained regenerating myelinated and nonmyelinated nerve fibers. Results of the morphometric study of distal parts of the injured nerve showed that the density of nerve fibers in the injured zone in mice of group 2 was significantly smaller than that in animals of group 1 (8410.0 ± 740.0 and 11024.0 ± ± 628.0 mm –2, respectively; P < 0.05). In mice of group 3, the density (10523.0 ± 1044.0 mm –2) practically did not differ from that in the control (in sham-operated mice of group 1). Therefore, after transplantation of NC-MSCs in mice with the injured nerve, we observed a clearly pronounced intensification of the processes of regeneration and repair of structural elements of the injured nerve, as compared with that in animals with the injured nerve but with no transplantation of MSCs. The more qualitative regeneration of nerve fibers in mice after transplantation of NC-MSCs was reflected not only in more significant recovery of fibers of the affected sciatic nerve but also in more intense vascularization and renewal of endoneural envelopes around separate groups of the nerve fibers.
DISCUSSION It is known that regeneration of the injured
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B
peripheral nerves depends on a number of the influences of structural, cellular, vascular, and humoral factors on the distal part of the injured nerve where regeneration and elongation of the axons develop [1]. The mechanisms of action of stem cells on the reparation processes in the injured peripheral nerves remain understudied [2]. We believe that intensification of regeneration of the traumatized sciatic nerve in mice after transplantation of NC-MSCs is related both to a trophic effect of the grafted cell elements and to their immediate differentiation with subsequent transformation into Schwann cells. It has been demonstrated that Schwann cells are a source of trophic and growth factors promoting the nerve fiber growth [8]. The abilities of stem cells transplanted into the region of nerve injury to differentiate with their transformation into Schwann cells and to intensify myelination of regenerating axons [4] are also important. In future, we intend to carry out a study using detection of transplanted GFP-positive cells in the regenerating sciatic nerve in mice. In our study, we observed parallel intensification of angiogenesis in the injured segment of the nerve under conditions of transplantation of NC-MSCs. On the one hand, it was demonstrated that the blood-nerve barrier in mice is disturbed 30 days after transection of the sciatic nerve [9]. On the other hand, it was also found that vessels of the peripheral part of the nerve, despite their changes after injury, continue to function and participate in its regeneration [1]. The elements of the bloodnerve barrier in the injured nerve recover only in the case of sufficient expression of trophic factors in the course of regeneration. At the same time, it is important to emphasize that, for objectivization of the level of regeneration of injured peripheral nerves, the comprehensive approach should be used, including physiological
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F i g. 2. Distal part of the sciatic nerve four weeks after surgery. A) After sham surgery; B) after nerve transection, and C) after nerve injury and transplantation of neural crest-derived multipotent stem cells into the injury region. Silver nitrate impregnation; magnification 400.
Effects of Multipotent Stem Cells on Nerve Regeneration and electrophysiological techniques, analysis of the functional state of spinal cord neurons, etc. [2]. The phenomenon of proliferation of endogenous Schwann cells after nerve injury and transplantation of NC-MSCs should also be taken into account. The NC-MSCs possess a clearly pronounced ability to be directly transformed in vitro into Schwann cells. Transplantation of NC-MSCs into the site of injury (transection) of the sciatic nerve in mice provides a clear positive influence on its successful regeneration, which is manifested in an increase in the number of nerve fibers in the distal part of the nerve and optimization of their spatial orientation. Our observations indicate that further studies of the biological properties of these cells are promising, especially from the aspect of their possible use in regenerative medicine. All experiments on animals were carried out in accordance with the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes, as well as with the regulations of the Ethics Committees of the State Institute of Genetic and Regenerative Medicine, National Academy of Medical Sciences of Ukraine (Kyiv, Ukraine) and Bogomolets National Medical University, Ministry of Public Health of Ukraine (Kyiv, Ukraine). The authors, R. G. Vasyliev, A. E. Rodnichenko, S. N. Shamalo, A. S. Demidchouk, I. F. Labunets, Yu. B. Chaikovskii, and G. M. Butenko, confirm that they have no conflict of interest.
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