Regeneration of peripheral nerve after transplantation of CD133+ cells derived from human peripheral blood

Laboratory investigation

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Object

Despite intensive efforts in the field of peripheral nerve injury and regeneration, it remains difficult to achieve full functional recovery in humans following extended peripheral nerve lesions. In this study, the authors examined the use of blood-derived CD133+ cells in promoting the repair of peripheral nerve defects.

Methods

The authors transplanted phosphate-buffered saline (control), mononuclear cells, or CD133+ cells embedded in atelocollagen gel into a silicone tube that was used to bridge a 15-mm defect in the sciatic nerve of athymic rats (12 animals in each group). At 8 weeks postsurgery, molecular, histological, and functional evaluations were performed in regenerated tissues.

Results

The authors found that sciatic nerves in which a defect had been made were structurally and functionally regenerated within 8 weeks after CD133+ cell transplantation. From macroscopic evaluation, massive nervelike tissues were confirmed only in rats with CD133+ cell transplantation compared with the other groups. Morphological regeneration in the samples after CD133+ cell transplantation, as assessed using toluidine blue staining, was enhanced significantly in terms of the number of myelinated fibers, axon diameter, myelin thickness, and percentage of neural tissue. Compound muscle action potentials were observed only in CD133+ cell–treated rats. Furthermore, it was demonstrated that the transplanted CD133+ cells differentiated into Schwann cells by 8 weeks after transplantation.

Conclusions

The results show that CD133+ cells have potential for enhancement of histological and functional recovery from peripheral nerve injury. This attractive cell source could be purified easily from peripheral blood and could be a feasible autologous candidate for peripheral nerve injuries in the clinical setting.

Abbreviations used in this paper: CMAP = compound muscle action potential; DAPI = 4′6-diamidino-2-phenylindole; FACS = fluorescence-activated cell sorter; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; HNA = human nuclear antigen; IgG = immunoglobulin G; MNC = mononuclear cell; PBS = phosphatebuffered saline; RT-PCR = reverse transcriptase polymerase chain reaction; vWF = von Willebrand factor.

Article Information

Address correspondence to: Masakazu Ishikawa, M.D., Ph.D., 1-2-3 Kasumi, Hiroshima, 734-8551, Japan. email: goemon1217@hotmail.co.jp.

Please include this information when citing this paper: published online November 14, 2008; DOI: 10.3171/2008.3.17571.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    Representative FACS and RT-PCR analysis of human peripheral blood–derived MNCs and CD133+ cells. Upper: Charts showing the positivity for CD133 in MNCs (left) and CD133+ cells (right). Numbers are percentages of the cells positive for CD133. Lower: Blots showing results of RT-PCR analysis for human-specific genes of neural stem cell, Schwann cell, neuronal cell, and endothelial lineage in peripheral blood–derived MNCs and CD133+ cells.

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    Intraoperative photographs showing the macroscopic appearance of the regenerated tissue inside the silicone tube at 8 weeks after transplantation. A: Scarlike tissue is shown in the PBS group. B: Regenerated thin nervelike structures bridging both stumps are shown in the MNC group. C: Massive regenerated nervelike structures are shown in the CD133+ group. Vessels are shown on the surface of the regenerated tissue. Bar = 5 mm.

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    Confirmation of engraftment of transplanted human CD133+ cells in regenerated tissues. Left: Photomicrograph of tissue section with representative immunofluorescence staining using the antibody for HNA and DAPI at 1 week after transplantation. Human cells were identified as double-positive cells for HNA (red) and DAPI (blue). Bar = 50 μm. Right: Blots showing that at the transcriptional level, human GAPDH was detected only in the CD133 group from regenerated tissue at 8 weeks after graft implantation by RT-PCR analysis in which human-specific primers were used.

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    Representative light micrographs of cross-sectional view in midportion of harvested tissues stained with toluidine blue. A and B: Photomicrographs of tissue sections obtained in the PBS group. Myelinated fibers are shown; however, they are few and small in diameter. C and D: Photomicrographs of tissue sections obtained in the MNC group. Myelinated fibers with a small diameter are shown. E and F: Photomicrographs of tissue sections obtained in the CD133+ group. Myelinated fibers with a large diameter are shown. Fibers are surrounded with thick myelin. Bar = 50 μm.

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    Bar graphs showing results of histomorphometric evaluations of nerve regeneration among the groups. All data were analyzed using an image analyzer. *p < 0.05, **p < 0.01.

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    Confirmation of engraftment and differentiation of CD133+ cells into Schwann cells. A: Representative immunological staining of regenerated tissues of CD133 group performed using anti-S100 protein antibody at Week 8; S100-positive cells (green), as Schwann cells, are shown around the atelocollagen gel in low magnification. Bar = 200 μm. B–E: Differentiated human Schwann cells (arrows) were identified in Zone I, the area of atelocollagen gel, as S100 positive (green) and as HNA positive (red) in animals receiving CD133+ cells. Bar = 50 μm. F–I: In Zone II, around the atelocollagen gel, no HNA-positive cell was identified. Bar = 50 μm. J: According to RT-PCR analysis, rat S100 was expressed abundantly in the CD133 group from regenerated tissue obtained 8 weeks after graft implantation.

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    Staining with vWF for detection of endothelial cells. A: Representative immunological staining in the CD133+ group performed using anti-vWF antibody at Week 8. Endothelial cells are shown as vWF positive (green). Bar = 200 μm. B–E: Photomicrographs in higher magnification of vessel structures stained with DAPI (B), anti-vWF antibody (C), antibody to HNA (D), and merged (E). Although several vessel structures are shown, differentiation of human cells into endothelial cells could not be confirmed. Bar = 50 μm.

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