Enhancement of regeneration with glia cell line–derived neurotrophic factor–transduced human amniotic fluid mesenchymal stem cells after sciatic nerve crush injury [RETRACTED]

Laboratory investigation

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Object

Human amniotic fluid–derived mesenchymal stem cells (AFMSCs) have been shown to promote peripheral nerve regeneration, and the local delivery of neurotrophic factors may additionally enhance nerve regeneration capacity. The present study evaluates whether the transplantation of glia cell line–derived neurotrophic factor (GDNF)–modified human AFMSCs may enhance regeneration of sciatic nerve after a crush injury.

Methods

Peripheral nerve injury was produced in Sprague-Dawley rats by crushing the left sciatic nerve using a vessel clamp. Either GDNF-modified human AFMSCs or human AFMSCs were embedded in Matrigel and delivered to the injured nerve. Motor function and electrophysiological studies were conducted after 1 and 4 weeks. Early or later nerve regeneration markers were used to evaluate nerve regeneration. The expression of GDNF in the transplanted human AFMSCs and GDNF-modified human AFMSCs was monitored at 7-day intervals.

Results

Human AFMSCs were successfully transfected with adenovirus, and a significant amount of GDNF was detected in human AFMSCs or the culture medium supernatant. Increases in the sciatic nerve function index, the compound muscle action potential ratio, conduction latency, and muscle weight were found in the groups treated with human AFMSCs or GDNF-modified human AFMSCs. Importantly, the GDNF-modified human AFMSCs induced the greatest improvement. Expression of markers of early nerve regeneration, such as increased expression of neurofilament and BrdU and reduced Schwann cell apoptosis, as well as late regeneration markers, consisting of reduced vacuole counts, increased expression of Luxol fast blue and S100 protein, paralleled the results of motor function. The expression of GDNF in GDNF-modified human AFMSCs was demonstrated up to 4 weeks; however, the expression decreased over time.

Conclusions

The GDNF-modified human AFMSCs appeared to promote nerve regeneration. The consecutive expression of GDNF was demonstrated in GDNF-modified human AFMSCs up to 4 weeks. These findings support a nerve regeneration scenario involving cell transplantation with additional neurotrophic factor secretion.

Abbreviations used in this paper: AFMSC = amniotic fluid–derived mesenchymal stem cell; BDNF = brain-derived neurotrophic factor; CMAP = compound muscle action potential; ELISA = enzyme linked immunosorbent assay; GDNF = glia cell line–derived neurotrophic factor; GFP = green fluorescent protein; MOI = multiplicity of infection; NGF = nerve growth factor; NT-3 = neurotrophin-3; SFI = sciatic nerve function index.

Article Information

Address correspondence to: Hung-Chuan Pan, M.D., Ph.D., Department of Neurosurgery, Taichung Veterans General Hospital, No. 160, Section 3, Taichung-Kang Road, Taichung 407, Taiwan, Republic of China. email: hcpan2003@yahoo.com.tw.

Please include this information when citing this paper: published online October 9, 2009; DOI: 10.3171/2009.8.JNS09850.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    In vitro assay of GDNF in GDNF-modified AFMSCs. The AFMSCs were transfected either with rAd-GFP or rAd-GDNF for 48 hours. A: Photomicrographs in light phase (upper row) and corresponding images (bottom row) showing expression of GFP in AFMSCs after rAd-GFP infection. B: Western blot analyses of GDNF in Ad-GFP AFMSCs and Ad-GDNF AFMSCs. C: Graph showing results of ELISA analysis of supernatant with respect to Ad-GFP AFMSCs and Ad-GDNF AFMSCs.

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    Evaluation of SFI, electrophysiological function, and muscle weight. The left sciatic nerve was crushed; SFI, CMAP, conduction latency, and muscle weight were investigated in 6 animals per group. Upper: Graph showing representative scores of SFI. Lower: Ratio of CMAP, conduction latency, and muscle weight. AFS = AFMSC Group; AFS-G = GDNF-AFMSC Group (rats treated with GDNF-modified AFMSCs); Crush = Crush Group; Crush + M = Crush + Matrigel Group; LT = left; Rt = right. *** p < 0.001; ### p < 0.001 relative to AFMSCs.

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    Expression of neurofilament and results of TUNEL and BrdU assay in crushed nerve 7 days after injury. A: Representative example of results of staining for neurofilament, TUNEL, and BrdU assay in crushed nerve. Bar = 50 μm. Insets: Merging of TUNEL and BrdU staining with S100 staining (red). B and C: Quantitative analyses of expression of neurofilament levels (B), TUNEL results (C), and BrdU staining in 6 animals. *** p < 0.001; ** p < 0.01; ### p < 0.001 relative to AFMSCs.

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    Vacuole counts and Luxol fast blue and S100 staining in 6 animals 4 weeks postinjury. A: Representative photomicrographs illustrating vacuoles, Luxol fast blue staining, and S100 staining in each group. Bar = 50 μm. B: Quantitative analysis of vacuole counts. C: Quantitative analyses of Luxol fast blue and S100 staining. *** p < 0.001; ** p < 0.01; ### p < 0.001; ## p < 0.01 relative to AFMSCs.

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    Expression of GDNF in GDNF-modified AFMSCs. Left sciatic nerve specimens obtained at 7-day intervals were subjected to immunohistochemical analysis with antibodies to GDNF and the GDNF receptor. A: Photomicrographs showing decreased expression of GDNF (green) colocalized with GDNF-modified AFMSCs (red) at different time points, with AFMSCs (AFS) as a control. Bar = 50 μm. B: Representative photomicrographs showing expression of GDNF receptor in the distal end of crushed nerve from animals in the 4 different groups 7 days after injury. Bar = 50 μm. C: Quantitative analyses of GDNF and CM Dil counts. D: Quantitative analysis of GDNF receptor expression in the distal end of the crushed nerve. *** p < 0.001; ** p < 0.01; ### p < 0.001 relative to the AFMSC Group.

References

  • 1

    Barras FMPasche PBouche NAebischer PZurn AD: Glial cell line-derived neurotrophic factor released by synthetic guidance channels promotes facial nerve regeneration in the rat. J Neurosci Res 70:7467552002

  • 2

    Berns KIGiraud C: Adenovirus and adeno-associated virus as vectors for gene therapy. Ann N Y Acad Sci 772:951041995

  • 3

    Blesch AConner JMTuszynski MH: Modulation of neuronal survival and axonal growth in vivo by tetracycline-regulated neurotrophin expression. Gene Ther 8:9549602001

  • 4

    Blesch ATuszynski MH: Cellular GDNF delivery promotes growth of motor and dorsal column sensory axons after partial and complete spinal cord transections and induces remyelination. J Comp Neurol 467:4034172003

  • 5

    Boyd JGGordon T: Neurotrophic factors and their receptors in axonal regeneration and functional recovery after peripheral nerve injury. Mol Neurobiol 27:2773242003

  • 6

    Buj-Bello ABuchman VLHorton ARosenthal ADavies AM: GDNF is an age-specific survival factor for sensory and autonomic neurons. Neuron 15:8218281995

  • 7

    Chen ZYChai YFCao LLu CLHe C: Glial cell line-derived neurotrophic factor enhances axonal regeneration following sciatic nerve transection in adult rats. Brain Res 902:2722762001

  • 8

    de Medinaceli LFreed WJWyatt RJ: An index of the functional condition of rat sciatic nerve based on measurement made from walking tracks. Exp Neurol 77:6346431982

  • 9

    Dezawa MTakahashi IEsaki MTakano MSawada H: Sciatic nerve regeneration in rats induced by transplantation of in vitro differentiated bone-marrow stromal cells. Eur J Neurosci 14:177117762001

  • 10

    Fine EGDecosterd IPapaloizos MZurn ADAebischer P: GDNF and NGF released by synthetic guidance channels support sciatic nerve regeneration across a long gap. Eur J Neurosci 15:5896012002

  • 11

    Hammarberg HPiehl FCullheim SFjell JHokfelt TFried K: GDNF mRNA in Schwann cells and DRG satellite cells after chronic sciatic nerve injury. Neuroreport 7:8578601996

  • 12

    Henderson CEPhillips HSPollock RADavies AMLemeulle CArmanini M: GDNF: a potent survival factor for motoneurons present in peripheral nerve and muscle. Science 266:106210641994

  • 13

    Hendriks WTRuitenberg MJBlits BBoer GJVerhaagen J: Viral vector-mediated gene transfer of neurotrophins to promote regeneration of the injured spinal cord. Prog Brain Res 146:4514762004

  • 14

    Höke AHo TCrawford TOLeBel CHilt DGriffin JW: Glial cell line-derived neurotrophic factor alters axon schwann cell units and promotes myelination in unmyelinated nerve fibers. J Neurosci 23:5615672003

  • 15

    In‘t Anker PSScherjon SAKleijburg-van der Keur CNoort WAClaas FHWillemze R: Amniotic fluid as a novel source of mesenchymal stem cells for therapeutic transplantation. Blood 102:154815492003

  • 16

    Jubran MWidenfalk J: Repair of peripheral nerve transections with fibrin sealant containing neurotrophic factors. Exp Neurol 181:2042122003

  • 17

    Krampera MPasini APizzolo GCosmi LRomagnani SAnnunziato F: Regenerative and immunomodulatory potential of mesenchymal stem cells. Curr Opin Pharmacol 6:4354412006

  • 18

    Li HTerenghi GHall SM: Effects of delayed re-innervation on the expression of c-erbB receptors by chronically denervated rat Schwann cells in vivo. Glia 20:3333471997

  • 19

    Mata MAlessi DFink DJ: S100 is preferentially distributed in myelin-forming Schwann cells. J Neurocytol 19:4324421990

  • 20

    May FMatiasek KVroemen MCaspers CMrva TArndt C: GDNF-transduced Schwann cell grafts enhance regeneration of erectile nerves. Eur Urol 54:117911872008

  • 21

    Menei PMontero-Menei CWhittemore SRBunge RPBunge MB: Schwann cells genetically modified to secrete human BDNF promote enhanced axonal regrowth across transected adult rat spinal cord. Eur J Neurosci 10:6076211998

  • 22

    Milunsky A: Amniotic fluid cell culture. Genetic Disorders and the Fetus: Diagnosis Prevention and Treatment New YorkPlenum Press1979. 7584

  • 23

    Moertel CAStupca PJDewald GW: Pseudomosacism, true mosaicism, and maternal cell contamination in amniotic fluid processed with in situ culture and robotic harvesting. Prenat Diagn 12:6716831992

  • 24

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

  • 25

    Naveilhan PElShamy WMErnfors P: Differential regulation of mRNAs for GDNF and its receptors Ret and GDNFR alpha after sciatic nerve lesion in the mouse. Eur J Neurosci 9:145014601997

  • 26

    Omura KOhbayashi MSano MOmura THasegawa TNagano A: The recovery of blood-nerve barrier in crush nerve injury—a quantitative analysis utilizing immunohistochemistry. Brain Res 1001:13212004

  • 27

    Oppenheim RWHouenou LJJohnson JELin LFLi LLo AC: Developing motor neurons rescued from programmed and axotomy-induced cell death by GDNF. Nature 373:3443461995

  • 28

    Pan HCChen CJCheng FCHo SPLiu MJHwang SM: Combination of G-CSF administration and human amniotic fluid mesenchymal stem cell transplantation promotes peripheral nerve regeneration. Neurochem Res 34:5185272009

  • 29

    Pan HCCheng FCChen CJLai SZLee CWYang DY: Post-injury regeneration in rat sciatic nerve facilitated by neurotrophic factors secreted by amniotic fluid mesenchymal stem cells. J Clin Neurosci 14:108910982007

  • 30

    Pan HCChin CSYang DYHo SPChen CJHwang SM: Human amniotic fluid mesenchymal stem cells in combination with hyperbaric oxygen augment peripheral nerve regeneration. Neurochem Res 34:130413162009

  • 31

    Pan HCYang DYChiu YTLai SZWang YCChang MH: Enhanced regeneration in injured sciatic nerve by human amniotic mesenchymal stem cell. J Clin Neurosci 13:5705752006

  • 32

    Piquilloud GChristen TPfister LAGander BPapaloizos MY: Variations in glial cell line-derived neurotrophic factor release from biodegradable nerve conduits modify the rate of functional motor recovery after rat primary nerve repairs. Eur J Neurosci 26:110911172007

  • 33

    Pollock M: Nerve regeneration. Curr Opin Neurol 8:3543581995

  • 34

    Prusa ARMarton ERosner MBernaschek GHengstschlager M: Oct-4-expressing cells in human amniotic fluid: a new source for stem cell research?. Hum Reprod 18:148914932003

  • 35

    Santos XRodrigo JHontanilla BBilbao G: Local administration of neurotrophic growth factor in subcutaneous silicon chambers enhances the regeneration of the sensory component of the rat sciatic nerve. Microsurgery 19:2752801999

  • 36

    Sayers STKhan NAhmed YShahid RKhan T: Preparation of brain-derived neurotrophic factor- and neurotrophin-3-secreting Schwann cells by infection with a retroviral vector. J Mol Neurosci 10:1431601998

  • 37

    Shen ZLLassner FBecker MWalter GFBader ABerger A: Viability of cultured nerve grafts: an assessment of proliferation of Schwann cells and fibroblasts. Microsurgery 19:3563631999

  • 38

    Syroid DEMaycox PJSoilu-Hanninen MPetratos SBucci TBurrola P: Induction of postnatal schwann cell death by the low-affinity neurotrophin receptor in vitro and after axotomy. J Neurosci 20:574157472000

  • 39

    Tai MHCheng HWu JPLiu YLLin PRKuo JS: Gene transfer of glial cell line-derived neurotrophic factor promotes functional recovery following spinal cord contusion. Exp Neurol 183:5085152003

  • 40

    Torigoe KTanaka HFTakahashi AAwaya AHashimoto K: Basic behavior of migratory Schwann cells in peripheral nerve regeneration. Exp Neurol 137:3013081996

  • 41

    Trupp MBelluardo NFunakoshi HIbanez CF: Complementary and overlapping expression of glial cell line-derived neurotrophic factor (GDNF), c-ret proto-oncogene, and GDNF receptor-alpha indicates multiple mechanisms of trophic actions in the adult rat CNS. J Neurosci 17:355435671997

  • 42

    Tsai MSHwang SMTsai YLCheng FCLee JLChang YJ: Clonal amniotic fluid-derived stem cells express characteristics of both mesenchymal and neural stem cells. Biol Reprod 74:5455512006

  • 43

    Tsai MSLee JLChang YJHwang SM: Isolation of human multipotent mesenchymal stem cells from second-trimester amniotic fluid using a novel two-stage culture protocol. Hum Reprod 19:145014562004

  • 44

    Tuszynski MHWeidner NMcCormack MMiller IPowell HConner J: Grafts of genetically modified Schwann cells to the spinal cord: survival, axon growth, and myelination. Cell Transplant 7:1871961998

  • 45

    Varejao ASCabrita AMMeek MFBulas-Cruz JMelo-Pinto PRaimondo S: Functional and morphological assessment of a standardized rat sciatic nerve crush injury with a non-serrated clamp. J Neurotrauma 21:165216702004

  • 46

    Varejao ASMelo-Pinto PMeek MFFilipe VMBulas-Cruz J: Methods for the experimental functional assessment of rat sciatic nerve regeneration. Neurol Res 26:1861942004

  • 47

    Vejsada RTseng JLLindsay RMAcheson AAebischer PKato AC: Synergistic but transient rescue effects of BDNF and GDNF on axotomized neonatal motoneurons. Neuroscience 84:1291391998

  • 48

    Weidner NBlesch AGrill RJTuszynski MH: Nerve growth factor-hypersecreting Schwann cell grafts augment and guide spinal cord axonal growth and remyelinate central nervous system axons in a phenotypically appropriate manner that correlates with expression of L1. J Comp Neurol 413:4955061999

  • 49

    Yan QMatheson CLopez OT: In vivo neurotrophic effects of GDNF on neonatal and adult facial motor neurons. Nature 373:3413441995

  • 50

    Yan QWang JMatheson CRUrich JL: Glial cell line-derived neurotrophic factor (GDNF) promotes the survival of axotomized retinal ganglion cells in adult rats: comparison to and combination with brain-derived neurotrophic factor (BDNF). J Neurobiol 38:3823901999

  • 51

    Young CMiller ENicklous DMHoffman JR: Nerve growth factor and neurotrophin-3 affect functional recovery following peripheral nerve injury differently. Restor Neurol Neurosci 18:1671752001

  • 52

    Yuan QWu WSo KFCheung ALPrevette DMOppenheim RW: Effects of neurotrophic factors on motoneuron survival following axonal injury in newborn rats. Neuroreport 11:223722412000

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