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

Laboratory investigation

Restricted access


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.


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.


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.


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:

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.



  • View in gallery

    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.


  • 1

    Asahara TMurohara TSullivan ASilver Mvan der Zee RLi T: Isolation of putative progenitor endothelial cells for angiogenesis. Science 275:9649671997

  • 2

    Bryan DJWang KKChakalis-Haley DP: Effect of Schwann cells in the enhancement of peripheral-nerve regeneration. J Reconstr Microsurg 12:43961996

  • 3

    Deshpande DMKim YSMartinez TCarmen JDike SShats I: Recovery from paralysis in adult rats using embryonic stem cells. Ann Neurol 60:32442006

  • 4

    Goussetis ETheodosaki MPaterakis GPeristeri JPetropoulos DKitra V: A functional hierarchy among the CD34+ hematopoietic cells based on in vitro proliferative and differentiative potential of AC133+CD34(bright) and AC133(dim/)-CD34+ human cord blood cells. J Hematother Stem Cell Res 9:8278402000

  • 5

    Hadlock TASundback CAHunter DAVacanti JPCheney ML: A new artificial nerve graft containing rolled Schwann cell monolayers. Microsurgery 21:961012001

  • 6

    Heiss CKeymel SNiesler UZiemann JKelm MKalka C: Impaired progenitor cell activity in age-related endothelial dysfunction. J Am Coll Cardiol 45:144114482005

  • 7

    Iwasaki HKawamoto AIshikawa MOyamada ANakamori SNishimura H: Dose-dependent contribution of CD34-positive cell transplantation to concurrent vasculogenesis and cardiomyogenesis for functional regenerative recovery after myocardial infarction. Circulation 113:131113252006

  • 8

    Kawamoto AKatayama MHanda NKinoshita MTakano HHorii M: Intramuscular transplantation of G-CSFmobilized CD34+ cells in patients with critical limb ischemia: a phase I/IIa, multicenter, single-blinded, dose-escalation clinical trial. Stem Cells 27:285728642009

  • 9

    Kijima YIshikawa MSunagawa TNakanishi KKamei NYamada K: Regeneration of peripheral nerve after transplantation of CD133+ cells derived from human peripheral blood. Laboratory investigation. J Neurosurg 110:7587672009

  • 10

    Loges SFehse BBrockmann MALamszus KButzal MGuckenbiehl M: Identification of the adult human hemangioblast. Stem Cells Dev 13:2292422004

  • 11

    Mackinnon SEDellon AL: Clinical nerve reconstruction with a bioabsorbable polyglycolic acid tube. Plast Reconstr Surg 85:4194241990

  • 12

    Majka MJanowska-Wieczorek ARatajczak JEhrenman KPietrzkowski ZKowalska MA: Numerous growth factors, cytokines, and chemokines are secreted by human CD34+ cells, myeloblasts, erythroblasts, and megakaryoblasts and regulate normal hematopoiesis in an autocrine/paracrine manner. Blood 97:307530852001

  • 13

    Masuda HIwasaki HKawamoto AAkimaru HIshikawa MAsahara T: Development of serum-free quality and quantity control culture of colony forming endothelial progenitor cell expansion for vasculogenesis. Stem Cells Trans Med [epub ahead of print]2012

  • 14

    Matsumoto TKawamoto AKuroda RIshikawa MMifune YIwasaki H: Therapeutic potential of vasculogenesis and osteogenesis promoted by peripheral blood CD34-positive cells for functional bone healing. Am J Pathol 169:144014572006

  • 15

    Murakami TFujimoto YYasunaga YIshida OTanaka NIkuta Y: Transplanted neuronal progenitor cells in a peripheral nerve gap promote nerve repair. Brain Res 974:17242003

  • 16

    Peichev MNaiyer AJPereira DZhu ZLane WJRafii S: Expression of VEGFR-2 and AC133 by circulating human CD34+ cells identifies a population of functional endothelial precursors. Blood 95:9529582000

  • 17

    Rafii SLyden D: Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration. Nat Med 9:7027122003

  • 18

    Tohill MMantovani CWiberg MTerenghi G: Rat bone marrow mesenchymal stem cells express glial markers and stimulate nerve regeneration. Neurosci Lett 362:2002032004

  • 19

    Urbich CDimmeler S: Endothelial progenitor cells functional characterization. Trends Cardiovasc Med 14:3183222004

  • 20

    Vasa MFichtlscherer SAicher AAdler KUrbich CMartin H: Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease. Circ Res 89:E1E72001

  • 21

    Xu YTamamaki NNoda TKimura KItokazu YMatsumoto N: Neurogenesis in the ependymal layer of the adult rat 3rd ventricle. Exp Neurol 192:2512642005

  • 22

    Yang LYZheng JKWang CYLi WY: Differentiation of adult human bone marrow mesenchymal stem cells into Schwannlike cells in vitro. Chin J Traumatol 8:77802005

  • 23

    Yin AHMiraglia SZanjani EDAlmeida-Porada GOgawa MLeary AG: AC133, a novel marker for human hematopoietic stem and progenitor cells. Blood 90:500250121997




All Time Past Year Past 30 Days
Abstract Views 214 214 9
Full Text Views 125 125 1
PDF Downloads 200 200 8
EPUB Downloads 0 0 0


Google Scholar