The therapeutic potential of ex vivo expanded CD133+ cells derived from human peripheral blood for peripheral nerve injuries

Laboratory investigation

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Object

CD133+ cells have the potential to enhance histological and functional recovery from peripheral nerve injury. However, the number of CD133+ cells safely obtained from human peripheral blood is extremely limited. To address this issue, the authors expanded CD133+ cells derived from human peripheral blood using the serum-free expansion culture method and transplanted these ex vivo expanded cells into a model of sciatic nerve defect in rats. The purpose of this study was to determine the potential of ex vivo expanded CD133+ cells to induce or enhance the repair of injured peripheral nerves.

Methods

Phosphate-buffered saline (PBS group [Group 1]), 105 fresh CD133+ cells (fresh group [Group 2]), 105 ex vivo expanded CD133+ cells (expansion group [Group 3]), or 104 fresh CD133+ cells (low-dose group [Group 4]) embedded in atelocollagen gel were transplanted into a silicone tube that was then used to bridge a 15-mm defect in the sciatic nerve of athymic rats (10 animals per group). At 8 weeks postsurgery, histological and functional evaluations of the regenerated tissues were performed.

Results

After 1 week of expansion culture, the number of cells increased 9.6 ± 3.3–fold. Based on the fluorescence-activated cell sorting analysis, it was demonstrated that the initial freshly isolated CD133+ cell population contained 93.22% ± 0.30% CD133+ cells and further confirmed that the expanded cells had a purity of 59.02% ± 1.58% CD133+ cells. However, the histologically and functionally regenerated nerves bridging the defects were recognized in all rats in Groups 2 and 3 and in 6 of 10 rats in Group 4. The nerves did not regenerate to bridge the defect in any of the rats in Group 1.

Conclusions

The authors' results show that ex vivo expanded CD133+ cells derived from human peripheral blood have a therapeutic potential similar to fresh CD133+ cells for peripheral nerve injuries. The ex vivo procedure that can be used to expand CD133+ cells without reducing their function represents a novel method for developing cell therapy for nerve defects in a clinical setting.

Abbreviations used in this paper:CMAP = compound muscle action potential; FACS = fluorescence-activated cell sorting; PBS = phosphate-buffered saline.

Article Information

Address correspondence to: Shin Ohtsubo, M.D., Department of Orthopaedic Surgery, Graduate School of Biomedical Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan. email: ohtsubo0529@yahoo.co.jp.

Please include this information when citing this paper: published online August 10, 2012; DOI: 10.3171/2012.7.JNS111503.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    A: The increase in cell numbers after expansion culture. The cell numbers increased 9.6-fold on average. B: The flow cytometric analysis of fresh CD133+ cells and expanded cells. The purity of the CD133+ cells was reduced from 93.22% ± 0.30% to 59.02% ± 1.58% by the expansion (upper). Additionally, the purity of the CD133+/CD34+ cells was reduced from 91.16% ± 0.37% to 32.22% ± 0.74% by the expansion (lower).

  • View in gallery

    Intraoperative photographs showing the macroscopic appearance of the regenerated tissue inside the silicone tubes at 8 weeks after transplantation. A: Scarlike tissue is observed in Group 1. B: Regenerated nervelike structures are observed in Group 2. C: Nervelike tissues are also recognized in all cases in Group 3. D and E: The regenerated structures are shown for Group 4. There are moderate nervelike tissues in 6 rats, (D) and slight continuities are observed in 4 rats (E). Bar = 5 mm.

  • View in gallery

    Compound muscle action potentials recorded in the gastrocnemius muscle as the functional axon regeneration of the excised sciatic nerve. A: Representative CMAP waves. “Control” refers to the contralateral normal side. B: The peak-to-peak amplitudes of CMAPs. There is no significant difference in the amplitude values between the fresh group (Group 2) and the expansion group (EXPAN, Group 3). However, the amplitudes in the low-dose group (Group 4) are significantly smaller than those in Groups 2 and 3. *p < 0.05; **p < 0.01.

  • View in gallery

    Representative light micrographs of cross-sectional views in the midportion of harvested tissues stained with toluidine blue. A and B: Photomicrographs of tissue sections obtained from Group 1. Myelinated fibers are shown; however, they are few and small in diameter. C and D: Photomicrographs of tissue sections obtained from Group 2. Myelinated fibers with a large diameter are shown. The fibers are surrounded with myelin. E and F: Photomicrographs of tissue sections obtained from Group 3. They are approximately equal to those in Group 2. G and H: Photomicrographs of tissue sections obtained from Group 4. Myelinated fibers with a moderate diameter are shown; however, they are sparse. Bar = 50 μm.

  • View in gallery

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

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