Comparison of the therapeutic potential of adult and embryonic neural precursor cells in a rat model of Parkinson disease

Laboratory investigation

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Object

The therapeutic effects of adult and embryonic neural precursor cells (NPCs) were evaluated and their therapeutic potential compared in a rat model of Parkinson disease.

Methods

Adult NPCs were obtained from the subventricular zone and embryonic NPCs were taken from the ganglionic eminence of 14-day-old embryos. Each NPC type was cultured with epidermal growth factor. The in vitro neuronal differentiation rate of adult NPCs was approximately equivalent to that of embryonic NPCs after two passages. Next, the NPCs were transfected with either green fluorescent protein or glial cell line–derived neurotrophic factor (GDNF) by adenoviral infection and transplanted into the striata in a rat model of Parkinson disease (PD) induced by unilateral intrastriatal injection of 6-hydroxydopamine. An amphetamine-induced rotation test was used to evaluate rat behavioral improvement, and immunohistochemical analysis was performed to compare grafted cell survival, differentiation, and host tissue changes.

Results

The rats with GDNF-transfected NPCs had significantly fewer amphetamine-induced rotations and less histological damage. Except for the proportion of surviving grafted cells, there were no significant differences between adult and embryonic NPCs.

Conclusions

Adult and embryonic NPCs have a comparable therapeutic potential in a rat model of PD.

Abbreviations used in this paper:ANOVA = analysis of variance; CNS = central nervous system; E14 = 14-day-old embryos; EGF = epidermal growth factor; ELISA = enzyme-linked immunosorbent assay; GDNF = glial cell line–derived neurotrophic factor; GFAP = glial fibrillary acidic protein; GFP = green fluorescent protein; IgG = immunoglobulin G; MHM = modified Hanks medium; NeuN = neuron-specific nuclear protein; NPC = neural precursor cell; PBS = phosphate-buffered saline; PD = Parkinson disease; SEM = standard error of the mean; SNC = substantia nigra pars compacta; TH = tyro-sine hydroxylase; 6-OHDA = 6-hydroxydopamine.

Article Information

Address correspondence to: Kenichiro Muraoka, M.D., Department of Neurological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikatacho, Okayama 700-8558, Japan. email: ken-ichi@zj8.so-net.ne.jp.

Isao Date received grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology in Japan, and a grant from the Project for Realization of Regenerative Medicine from the Ministry of Education, Culture, Sports, Science and Technology in Japan.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    Schematic of the experimental protocol for the in vitro assessment of neuronal differentiation in this study. A neural stem cell is expanded by the formation of a clonally derived cell cluster, called a sphere, in EGF-containing growth medium. The primary spheres generated are dissociated and plated in populations (5 × 104 cells/ml) for neuronal differentiation assessment or reseeded for secondary sphere generation in EGF medium. The neuronal differentiation rates of both secondary and tertiary spheres were assessed in the same manner.

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    Bar graph showing the differentiation rates after passaging (P) of adult (A-) and embryonic (E-) primary (0), secondary (1), and tertiary spheres (2). Repeated passages remarkably reduced the neuronal differentiation rates of embryonic NPCs (E-P0 compared with E-P1, p < 0.0001, 3 rats). Error bar: + SEM. ***p < 0.0001.

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    Left: Amount of GDNF produced in vitro. The adult and embryonic GDNF-transfected cells produced significantly higher amounts of GDNF than both GFP-transfected groups. There was no statistical difference between the GDNF-or GFP-transfected groups. **p < 0.0001; error bar + SEMs. Right: Amount of GDNF produced in vivo. In vivo evaluation also revealed a significantly higher amount of GDNF in the groups that received adult and embryonic GDNF-transfected cells than in both groups receiving GFP-transfected cells. There was no statistical difference, however, between the GDNF- or GFP-transfected groups. **p < 0.0001; error bar + SEMs.

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    Graph of the time course of the amphetamine-induced rotational behavior in rats with 6-OHDA–induced loss of dopaminergic neurons. There was a statistically significant reduction in amphetamine-induced rotations in animals implanted with GDNF-transfected cells (adult and embryonic) compared with animals that received GFP-transfected cells. Data are shown as means ± SEMs expressed as rotation numbers; 6 animals/group. ** p < 0.01.

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    A: Bar graph showing the preservation of TH-positive fibers in the host striatum in the 4 groups of rats. No statistical difference was detected between the adult and embryonic GDNF groups or between the adult and embryonic GFP groups. Data are shown as means ± SEMs expressed as percentages of the contralateral side. ** p < 0.0001. B–F: Photomicrographs. Staining for TH in the striatum on tissue obtained from the nonlesioned side (B), adult GDNF (C), adult GFP (D), embryonic GDNF (E), and embryonic GFP (F) are shown. In the GDNF groups there were more TH fibers than in the GFP groups, but there were no morphological differences between adult and embryonic NPCs in any of the groups.

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    A: Bar graph showing the preservation of TH-positive neurons in the host SNC. Adult and embryonic GDNF transplantation induced a greater preservation of these neurons and had more neuroprotective effects in vivo than adult and embryonic GFP cell transplantation. On the other hand, there was no statistical difference between adult and embryonic GDNF groups or between adult and embryonic GFP groups. Data are shown as means ± SEMs expressed as percentages of the contralateral side. **p < 0.0001. B–F: Photomicrographs. Staining for TH in the SNC on the nonlesioned side (B), adult GDNF (C), adult GFP (D), embryonic GDNF (E), and embryonic GFP (F) are shown.

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    Photomicrographs of the appearance of intrastriatal grafts 6 weeks after implantation. Adult GDNF (A, E), adult GFP (B, F), embryonic GDNF (C, G), and embryonic GFP (D, H). Upper images at low magnification show that grafted cells migrated in a dorsoventral direction along the needle track and also dispersed laterally, settling within the host striatum. Lower images at higher magnification show that in both GDNF-transfected grafts, the grafted cells extended longer and smoother processes than the GFP-transfected grafts. A–D: bar = 1.0 mm; E–H: bar = 100 μm. LV = lateral ventricle.

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    Photomicrographs showing differentiation of adult GFP grafts. The GFP-positive cell clusters disperse and arrange within the host striatum (A and G). No positivity for NeuN was observed within the cluster (B). The GFP/NeuN double-positive cells are located mainly in the periphery and outside of the cluster (C is a merged view of A and B). D–F: High magnification images showing a GFP/NeuN double-positive cell extending a long, thick process (arrowhead). D: GFP. E: NeuN. F: Merged view of D and E, indicating grafted cells that differentiated into mature neurons and integrated into the host tissue. G–I: Although astrocytic differentiation of grafted cells is observed inside the cluster, they are located mainly in the cluster periphery (G: GFP; H: GFAP; I is a merged view of G and H). J–L: High magnification images showing GFP/GFAP double-positive cells extending multiple short processes that are typical of astrocytes (arrowheads, J: GFP; K: GFAP; L is a merged view of J and K). A–C and G–I: bar = 200 mm; D–F, J–L: bar = 30 mm.

References

  • 1

    Akerud PCanals JMSnyder EYArenas E: Neuroprotection through delivery of glial cell line-derived neurotrophic factor by neural stem cells in a mouse model of Parkinson's disease. J Neurosci 21:810881182001

    • Search Google Scholar
    • Export Citation
  • 2

    Asahara TKalka CIsner JM: Stem cell therapy and gene transfer for regeneration. Gene Ther 7:4514572000

  • 3

    Bakshi AShimizu SKeck CACho SLeBold DGMorales D: Neural progenitor cells engineered to secrete GDNF show enhanced survival, neuronal differentiation and improve cognitive function following traumatic brain injury. Eur J Neurosci 23:211921342006

    • Search Google Scholar
    • Export Citation
  • 4

    Barker RAWidner H: Immune problems in central nervous system cell therapy. NeuroRx 1:4724812004

  • 5

    Bjorklund ALindvall O: Cell replacement therapies for central nervous system disorders. Nat Neurosci 3:5375442000

  • 6

    Brederlau ACorreia ASAnisimov SVElmi MPaul GRoybon L: Transplantation of human embryonic stem cell-derived cells to a rat model of Parkinson's disease: effect of in vitro differentiation on graft survival and teratoma formation. Stem Cells 24:143314402006

    • Search Google Scholar
    • Export Citation
  • 7

    Date IFelten SYFelten DL: The nigrostriatal dopaminergic system in MPTP-treated mice shows more prominent recovery by syngeneic adrenal medullary graft than by allogeneic or xeno-geneic graft. Brain Res 545:1911981991

    • Search Google Scholar
    • Export Citation
  • 8

    Doetsch FPetreanu LCaille IGarcia-Verdugo JMAlvarez-Buylla A: EGF converts transit-amplifying neurogenic precursors in the adult brain into multipotent stem cells. Neuron 36:102110342002

    • Search Google Scholar
    • Export Citation
  • 9

    Eriksson CBjorklund AWictorin K: Neuronal differentiation following transplantation of expanded mouse neurosphere cultures derived from different embryonic forebrain regions. Exp Neurol 184:6156352003

    • Search Google Scholar
    • Export Citation
  • 10

    Falk AHolmstrom NCarlen MCassidy RLundberg CFrisen J: Gene delivery to adult neural stem cells. Exp Cell Res 279:34392002

    • Search Google Scholar
    • Export Citation
  • 11

    Fricker RACarpenter MKWinkler CGreco CGates MABjörklund A: Site-specific migration and neuronal differentiation of human neural progenitor cells after transplantation in the adult rat brain. J Neurosci 19:599060051999

    • Search Google Scholar
    • Export Citation
  • 12

    Korochkin LIRevishchin AVOkhotin VE: Neural stem cells and their role in recovery processes in the nervous system. Neurosci Behav Physiol 36:4995122006

    • Search Google Scholar
    • Export Citation
  • 13

    Kuhn HGWinkler JKempermann GThal LJGage FH: Epidermal growth factor and fibroblast growth factor-2 have different effects on neural progenitors in the adult rat brain. J Neurosci 17:582058291997

    • Search Google Scholar
    • Export Citation
  • 14

    Kukekov VGLaywell EDSuslov ODavies KScheffler BThomas LB: Multipotent stem/progenitor cells with similar properties arise from two neurogenic regions of adult human brain. Exp Neurol 156:3333441999

    • Search Google Scholar
    • Export Citation
  • 15

    Lindvall O: Parkinson disease. Stem cell transplantation. Lancet 358:SupplS482001

  • 16

    Lindvall OKokaia ZMartinez-Serrano A: Stem cell therapy for human neurodegenerative disorders—how to make it work. Nat Med 10:SupplS42S502004

    • Search Google Scholar
    • Export Citation
  • 17

    Lu PJones LLSnyder EYTuszynski MH: Neural stem cells constitutively secrete neurotrophic factors and promote extensive host axonal growth after spinal cord injury. Exp Neurol 181:1151292003

    • Search Google Scholar
    • Export Citation
  • 18

    Moe MCVarghese MDanilov AIWesterlund URamm-Pettersen JBrundin L: Multipotent progenitor cells from the adult human brain: neurophysiological differentiation to mature neurons. Brain 128:218921992005

    • Search Google Scholar
    • Export Citation
  • 19

    Muraoka KShingo TYasuhara TKameda MYuan WHayase H: The high integration and differentiation potential of autologous neural stem cell transplantation compared with allogeneic transplantation in adult rat hippocampus. Exp Neurol 199:3113272006

    • Search Google Scholar
    • Export Citation
  • 20

    Nishino HHida HTakei NKumazaki MNakajima KBaba H: Mesencephalic neural stem (progenitor) cells develop to dopaminergic neurons more strongly in dopamine-depleted striatum than in intact striatum. Exp Neurol 164:2092142000

    • Search Google Scholar
    • Export Citation
  • 21

    Nunes MCRoy NSKeyoung HMGoodman RRMcKhann G IIJiang L: Identification and isolation of multipotential neural progenitor cells from the subcortical white matter of the adult human brain. Nat Med 9:4394472003

    • Search Google Scholar
    • Export Citation
  • 22

    Ostenfeld TTai YTMartin PDeglon NAebischer PSvendsen CN: Neurospheres modified to produce glial cell line-derived neurotrophic factor increase the survival of transplanted dopamine neurons. J Neurosci Res 69:9559652002

    • Search Google Scholar
    • Export Citation
  • 23

    Palmer TDTakahashi JGage FH: The adult rat hippocampus contains primordial neural stem cells. Mol Cell Neurosci 8:3894041997

  • 24

    Park KIHimes BTStieg PETessler AFischer ISnyder EY: Neural stem cells may be uniquely suited for combined gene therapy and cell replacement: evidence from engraftment of neurotrophin-3-expressing stem cells in hypoxic-ischemic brain injury. Exp Neurol 199:1791902006

    • Search Google Scholar
    • Export Citation
  • 25

    Parmar MSjoberg ABjorklund AKokaia Z: Phenotypic and molecular identity of cells in the adult subventricular zone. in vivo and after expansion in vitro. Mol Cell Neurosci 24:7417522003

    • Search Google Scholar
    • Export Citation
  • 26

    Paxinos GWatson C: The Rat Brain in Stereotaxic Coordinates New YorkAcademic Press1998

  • 27

    Reynolds BATetzlaff WWeiss S: A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes. J Neurosci 12:456545741992

    • Search Google Scholar
    • Export Citation
  • 28

    Richardson RMBroaddus WCHolloway KLFillmore HL: Grafts of adult subependymal zone neuronal progenitor cells rescue hemiparkinsonian behavioral decline. Brain Res 1032:11222005

    • Search Google Scholar
    • Export Citation
  • 29

    Rossi FCattaneo E: Opinion: neural stem cell therapy for neurological diseases: dreams and reality. Nat Rev Neurosci 3:4014092002

    • Search Google Scholar
    • Export Citation
  • 30

    Roy NSCleren CSingh SKYang LBeal MFGoldman SA: Functional engraftment of human ES cell-derived dopaminergic neurons enriched by coculture with telomerase-immortalized midbrain astrocytes. Nat Med 12:125912682006

    • Search Google Scholar
    • Export Citation
  • 31

    Shingo TDate IYoshida HOhmoto T: Neuroprotective and restorative effects of intrastriatal grafting of encapsulated GDNF-producing cells in a rat model of Parkinson's disease. J Neurosci Res 69:9469542002

    • Search Google Scholar
    • Export Citation
  • 32

    Shingo TSorokan STShimazaki TWeiss S: Erythropoietin regulates the in vitro and in vivo production of neuronal progenitors by mammalian forebrain neural stem cells. J Neurosci 21:973397432001

    • Search Google Scholar
    • Export Citation
  • 33

    Stenman JToresson HCampbell K: Identification of two distinct progenitor populations in the lateral ganglionic eminence: implications for striatal and olfactory bulb neurogenesis. J Neurosci 23:1671742003

    • Search Google Scholar
    • Export Citation
  • 34

    Studer LTabar VMcKay RD: Transplantation of expanded mesencephalic precursors leads to recovery in parkinsonian rats. Nat Neurosci 1:2902951998

    • Search Google Scholar
    • Export Citation
  • 35

    Toda HTsuji MNakano IKobuke KHayashi TKasahara H: Stem cell-derived neural stem/progenitor cell supporting factor is an autocrine/paracrine survival factor for adult neural stem/progenitor cells. J Biol Chem 278:35491355002003

    • Search Google Scholar
    • Export Citation
  • 36

    Uchida NBuck DWHe DReitsma MJMasek MPhan TV: Direct isolation of human central nervous system stem cells. Proc Natl Acad Sci U S A 97:14720147252000

    • Search Google Scholar
    • Export Citation
  • 37

    Verkhratsky AToescu EC: Neuronal-glial networks as substrate for CNS integration. J Cell Mol Med 10:8268362006

  • 38

    Weiss SDunne CHewson JWohl CWheatley MPeterson AC: Multipotent CNS stem cells are present in the adult mammalian spinal cord and ventricular neuroaxis. J Neurosci 16:759976091996

    • Search Google Scholar
    • Export Citation
  • 39

    Westerlund USvensson MMoe MCVarghese MGustavsson BWallstedt L: Endoscopically harvested stem cells: a putative method in future autotransplantation. Neurosurgery 57:7797842005

    • Search Google Scholar
    • Export Citation
  • 40

    Winkler CFricker RAGates MAOlsson MHammang JPCarpenter MK: Incorporation and glial differentiation of mouse EGF-responsive neural progenitor cells after transplantation into the embryonic rat brain. Mol Cell Neurosci 11:991161998

    • Search Google Scholar
    • Export Citation
  • 41

    Wu PTarasenko YIGu YHuang LYCoggeshall REYu Y: Region-specific generation of cholinergic neurons from fetal human neural stem cells grafted in adult rat. Nat Neurosci 5:127112782002

    • Search Google Scholar
    • Export Citation
  • 42

    Yasuhara TShingo TMuraoka KKobayashi KTakeuchi AYano A: Early transplantation of an encapsulated glial cell line-derived neurotrophic factor-producing cell demonstrating strong neuroprotective effects in a rat model of Parkinson disease. J Neurosurg 102:80892005

    • Search Google Scholar
    • Export Citation
  • 43

    Zhang SCWernig MDuncan IDBrustle OThomson JA: In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat Biotechnol 19:112911332001

    • Search Google Scholar
    • Export Citation

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