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TRANSLATIONAL AND CLINICAL RESEARCH

Olfactory Mucosa Is a Potential Source for Autologous Stem Cell Therapy for Parkinson's Disease

^aNational Centre for Adult Stem Cell Research, Eskitis Institute for Cell and Molecular Therapies, Griffith University, Brisbane, Australia;
^bNICN, CNRS UMR6184, IFR Jean Roche & Centre d'Investigations Cliniques en Biothérapie CIC-B 150, AP-HM-Institut Paoli Calmettes-Inserm, Université de la Méditerranée, Marseille, France;
^cSchool of Biomedical Sciences, The University of Queensland, Brisbane, Queensland, Australia;
^dDepartment of Otolaryngology, Princess Alexandra Hospital, Brisbane, Australia;
^eSchool of Medicine, The University of Queensland, Brisbane, Queensland, Australia

Key Words. Parkinson's disease • Olfactory stem cell • Autologous cell therapy • Dopaminergic

Correspondence: Wayne Murrell, B.Sc. (Hons), Ph.D., Vilhelm Magnus Center, Institute for Surgical Research, Rikshospital, University of Oslo, Oslo, Norway 0027. Telephone: 47-23071405; Fax: 47-23071397; e-mail: Wayne.Murrell{at}rr-research.no

Received January 24, 2008; accepted for publication May 23, 2008.
First published online in STEM CELLS EXPRESS   June 5, 2008.

	ABSTRACT

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
Parkinson's disease is a complex disorder characterized by degeneration of dopaminergic neurons in the substantia nigra in the brain. Stem cell transplantation is aimed at replacing dopaminergic neurons because the most successful drug therapies affect these neurons and their synaptic targets. We show here that neural progenitors can be grown from the olfactory organ of humans, including those with Parkinson's disease. These neural progenitors proliferated and generated dopaminergic cells in vitro. They also generated dopaminergic cells when transplanted into the brain and reduced the behavioral asymmetry induced by ablation of the dopaminergic neurons in the rat model of Parkinson's disease. Our results indicate that Parkinson's patients could provide their own source of neuronal progenitors for cell transplantation therapies and for direct investigation of the biology and treatments of Parkinson's disease.

Disclosure of potential conflicts of interest is found at the end of this article.

	INTRODUCTION

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
Parkinson's disease is a movement disorder, the debilitating symptoms of which are due to degeneration of dopaminergic neurons in the substantia nigra [1]. There are other neuropathological changes in the disease, but replacement of these neurons remains the focus of attempts at cellular therapies because the most successful drug therapies target these neurons and their synaptic targets. The rat model for Parkinson's disease is one in which dopaminergic input to the caudate nucleus is destroyed unilaterally by injection of the selective neurotoxin, 6-hydroxydopamine (6OHDA). This results in circling locomotion, the amelioration of which is the focus of most tests of proposed therapies.

The primary, and early, signs and symptoms of Parkinson's are slow movement, rigidity, and tremor due to loss of dopaminergic neurons from the substantia nigra that project to the striatum. There are other neuropathological changes in the disease, but replacement of dopamine in the striatum remains the focus of attempts at cellular therapies because the most successful drug therapies target these neurons and their synaptic targets. The relatively localized nature of this lesion has led to it being considered a prime target for cell transplantation therapy. This can be accomplished by placing a source of dopaminergic cells into the striatum. The most well-studied tissue for clinical trials of transplantation in Parkinson's disease has been fetal substantia nigra [2]. At present there is debate regarding the efficacy of this procedure after double-blind studies revealed only moderate reduction in Parkinson's symptoms [2]. The utility of fetal substantia nigra as a source of cells is severely limited by the logistics of supply. Two to five aborted fetuses are required per side per patient; the fetuses must be 6 to 8 weeks after conception [3]; and the cells must be both harvested by dissection from fetuses fragmented by the abortion procedure and transplanted within hours of harvest [4]. Apart from community ethical concerns, these supply requirements are unlikely to be routinely achieved and some researchers are investigating xenografts of porcine fetal nigral tissue as an alternative [2, 5]. It is commonly believed that stem cell biology may provide the source for many cell transplantation therapies, including Parkinson's disease [2, 6]. The present study explores the hypothesis that multipotent stem cells obtained autologously from the olfactory mucosa can differentiate into dopaminergic neurons.

Neurogenesis continues throughout adult life in the human olfactory mucosa [7], providing an accessible source of neuroblasts and stem cells [7–10]. Human olfactory neural stem cells can be grown in neurospheres [11], are multipotent, and differentiate into neurons, astrocytes, and oligodendrocytes in vitro [9, 12] as well as many non-neural cell types in vitro and in vivo [12]. We hypothesized that adult olfactory stem cells, or neural progenitors derived from them, would differentiate into dopaminergic neurons and may be useful for autologous transplantation in Parkinson's disease. Differentiation potential of olfactory stem cells was assessed in vitro and after transplantation into the hemiparkinsonian rat [13], the most frequently used model for Parkinson's disease.

	MATERIALS AND METHODS

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
Experimental Plan
Olfactory stem cells were grown from rat and human olfactory mucosa. These cells are propagated as neurosphere cultures [14], similar to other neural stem cells [11]. Olfactory neurospheres were grown in vitro and differentiated along the dopaminergic lineage, assessed by expression of dopamine neuron proteins and by release of dopamine and metabolites into the culture medium. Olfactory neurosphere cells were transplanted into the hemiparkinsonian rat, lesioned unilaterally in the striatum by stereotactic injection of 6OHDA [15]. Functional improvement was assessed by changes in rotational behavior after transplantation of human olfactory neurosphere-derived cells into the lesioned striatum. Engraftment was assessed histologically by identifying grafted cells in the transplanted striatum.

Animals
Female adult, Sprague-Dawley rats were obtained from the Royal Brisbane Hospital Animal House (Queensland, Australia). Animals were euthanized by lethal injection (pentobarbitone sodium, > 0.1 mg/g bodyweight). All aspects of experimentation were performed in accordance with the guidelines of Griffith University Animal Ethics Committee and the National Health and Medical Research Council of Australia.

Human Nasal Biopsies, Cell Preparation, and Neurosphere Culture
Human nasal mucosa was obtained by biopsy from Parkinson's disease patients and healthy controls with informed consent as described [8] and approved by the ethics committees of the hospital and university according to guidelines of the National Health and Medical Research Council of Australia. The participant with Parkinson's disease whose cells were used in the work reported here was diagnosed by a movement disorder neurologist (P.S.) according to standard criteria [16]. Olfactory mucosa cultures were established as described [8, 12], expanded in Dulbecco's modified Eagle's medium (DMEM; JRH Biosciences, Lenexa, KS, http://www.jrhbio.com) supplemented with 10% fetal bovine serum (JRH Biosciences) and passaged twice using 0.25% trypsin/0.02% EDTA. To generate neurospheres, cells were plated at 1,000 cells/cm² into 6-well plates (Nunc, Rochester, NY, http://www.nuncbrand.com) coated with poly-L-lysine (0.85 µg/cm²; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) in a serum-free medium of DMEM supplemented with epidermal growth factor (50 ng/ml; Sigma-Aldrich) and basic fibroblast growth factor-2 (bFGF; 25 ng/ml; Sigma-Aldrich) and insulin/transferrin/selenium (ITS; 1 g/l insulin, 0.55g/l transferrin, 0.67 mg/l sodium selenite; Gibco, Grand Island, NY, http://www.invitrogen.com). Cells under these latter conditions grow as multipotent neurospheres, the same as those described in our paper [14]. For in vitro differentiation and for transplantation into the striatum, neurospheres were dissociated by washing in Hanks' balanced saline solution (HBSS), prior to being resuspended in Dispase II (2.4 U/ml; Roche, Basel, Switzerland, http://www.roche-applied-science.com) and DNase I/II (10 µg/ml; Roche) and incubated on an orbital shaker at 37°C, 100 rpm for 15 minutes. Spheres were gently triturated 15 times. Cells were washed in HBSS.

For transplantation the cells were suspended in the serum-free culture medium (DMEM/ITS). For the analysis of survival and phenotype of transplanted cells some olfactory neurosphere-derived cells were labeled with green fluorescent protein (GFP) as described [12]. Briefly transduction experiments used a pFB-hrGFP, Viraport (Stratagene, La Jolla, CA, http://www.stratagene.com) retroviral control supernatant that contains an Moloney murine leukemia virus replication-defective retrovirus. As a precaution, cells targeted for transduction were tested using a reverse transcriptase assay in case of endogenous replication of competent retrovirus. When confirmed to be clear of endogenous retrovirus, they were transduced according to the manufacturer's instructions. Polymerase chain reaction was then used to confirm genomic integration of the viral insert. Transduction efficiency varied with these retroviral labeling reactions, but cells were subsequently fluorescence-activated cell sorted to be near 100% positive prior to assessment for multipotency and then transplantation. Neurospheres obtained from GFP-transduced cultures were tested and found capable of producing both neurons and glia prior to use in transplantation experiments.

Differentiation

Optimization of Differentiation Conditions in Rat Olfactory Mucosa Cultures.   Dissociated third-generation neurosphere cells were resuspended in DMEM-ITS and DMEM-ITS plus growth factors, including neurotrophin-3 (25 ng/ml), brain-derived neurotrophic factor (25 ng/ml), bFGF (25 ng/ml), interleukin-1β (IL-1β; 100 pg/ml), sonic hedgehog (200 ng/ml), and glial cell-derived neurotrophic factor (GDNF; 25 ng/ml) [17]. Cells were plated onto chamber slides coated with collagen IV at a cell density of 5,000 cells/well (7,140 cells/cm²). The media were changed on day 2, the cells were fixed with 4% paraformaldehyde on day 4, and immunocytochemistry was carried out for tyrosine hydroxylase (TH) and dopamine transporter.

Dissociated third-generation neurosphere cells were resuspended in DMEM-ITS supplemented with GDNF plus the neural growth supplement B27 (GDNF + B27) and DMEM-ITS + B27. Cells were again plated onto chamber slides coated with collagen IV, at the same cell density as previously used, and were cultured for 4 days, after which the cells were fixed and immunocytochemistry was carried out.

Human Differentiation Conditions.   The initial experiment tested the efficacy of a defined culture medium and culture surface coating. Cells from dissociated neurospheres were resuspended in DMEM/ITS with or without GDNF (25 ng/ml; Chemicon, Temecula, CA, http://www.chemicon.com) and plated into glass chamber slides (Labtek 2; Nunc). The wells of the slides were uncoated or coated with either collagen IV alone or with poly-L-ornithine plus laminin. Collagen IV (5 µg/cm²; Sigma-Aldrich) was applied in sterile H₂O and allowed to dry overnight at room temperature. Poly-L-ornithine (28 µg/cm², 0.1 mg/ml; Sigma-Aldrich) was incubated overnight in sterile H₂O and then washed three times in HBSS prior to laminin coating (2.9 µg/cm², 10 µg/ml; Gibco) in sterile H₂O. For dopamine assays, cells were plated at a density of 7,000 cells/cm².

Attempts to Improve Dopamine Production
Further defined culture media were assessed for their differentiation induction ability [18–21] when dopamine levels were sought: DMEM/ITS + GDNF (25 ng/ml) + insulin-like growth factor 1 (IGF1, 10 ng/ml); DMEM/ITS + GDNF (2 ng/ml) + IL-1β (200 ng/ml); ITS + trichostatin A (TSA, 10 ng/ml); ITS + TSA (10 ng/ml) + GDNF (25 ng/ml) + FGF8 (100 ng/ml) + forskolin (5 µM); ITS + neuropeptide Y (10 µM); and ITS + LiCl (10 nM). Controls included DMEM/ITS medium and media with 10% fetal bovine serum (FBS).

Immunocytochemistry and Quantification of In Vitro Differentiation
Cells were placed in differentiation medium on day 0. Fresh medium was applied on day 2, and the cells were fixed with 4% paraformaldehyde on day 4, after which immunocytochemistry was carried out to identify the cells expressing tyrosine hydroxylase or dopamine transporter. Cells were fixed in 4% paraformaldehyde for 7 minutes, washed, and stored in phosphate-buffered saline (PBS) + 0.1% sodium azide. Wells were blocked in 10% goat serum, 2% bovine serum albumin, 0.1% Triton X-100 (The Dow Chemical Company, Midland, MI, http://www.dow.com) in PBS for 1 hour at room temperature. Primary antibodies were rabbit anti-dopamine transporter (Sigma-Aldrich) (whole antiserum) and mouse anti-tyrosine hydroxylase (40.5 µg/ml) (Sigma-Aldrich). Primary antibodies were applied in blocking solution for 1 hour at room temperature, after which cells were washed in PBS. Secondary antibodies (goat anti-mouse IgG conjugated with AlexaFluor 488 and goat anti-rabbit IgG conjugated with AlexaFluor 594) were applied at 5 µg/ml for 1 hour at room temperature. The cells were washed in PBS, then the nuclei were stained with 1 mM Hoechst 33342 (Sigma-Aldrich) for 10 minutes, and the cells were washed again. Cell nuclei were counterstained with the nuclear stain Hoechst 33342. In each well, the total number of cell nuclei and the number of cells positive for tyrosine hydroxylase were counted. Three wells were counted for each growth condition. Control immunocytochemistry, without primary antibody, was carried out for each secondary antibody used in every experiment. The percentage of TH-positive cells was represented as a mean percentage ± SEM. The number of tyrosine hydroxylase-positive cells was expressed as a proportion of the total number of cells per well, and the data were expressed as mean and standard error of this proportion per well. The groups were compared by two-way analysis of variance to determine whether the groups differed in inducing a tyrosine hydroxylase phenotype. The alpha level was .05.

Detection of Dopamine Release from Differentiated Cells by High-Performance Liquid Chromatography
Dopamine release by differentiated neurons was achieved by potassium chloride (KCl) depolarization [22]. Briefly, differentiated cells were washed with HBSS twice prior to depolarization with 200 µL of 56 mM KCl in HBSS for 15 minutes. The supernatant was then collected and transferred to a clean tube. Fifty microliters of 0.1 M of perchloric acid was added and the supernatant was stored at –80°C. Intracellular dopamine was also examined by cell lysis. Depolarized cells were scraped off the culture flask using a sterile cell scraper (Greiner Bio-One, Frickenhausen, Germany, http://www.gbo.com/en), and the detached cells were prepared for SDS-polyacrylamide gel electrophoresis (PAGE) and Western blot analysis (see below). Two hundred microliters of 0.1 M perchloric acid was added to the cells assigned for dopamine analysis. The cells were sonicated in ice for two cycles (20 seconds on and 45 seconds off) in an MSE sonicator (Fisher Scientific, Loughborough, U.K., http://www.fisher.co.uk) using the 10-mm sonication probe at maximum power (30 µm amplitude). Cell debris was then removed by centrifuging at 17,500g for 20 minutes at 4°C. Supernatant was collected and stored at –80°C. Dopamine release and intracellular dopamine levels were analyzed by high-performance liquid chromatography (HPLC). This system consisted of an Autosampler and pump (Model 1100; Agilant Technologies, Inc., Palo Alto CA, http://www.agilent.com), a Sunfire C18 column (4.6 x 150 mm, 5 µm packing; Waters Corp., Milford, MA, http://www.waters.com), and an electrochemical detector (Coulochem III; ESA Laboratories, Inc., Chelmsford, MA, http://www.esainc.com). The mobile phase consisted of a 10% acetonitrile/75 mM potassium dihydrogen phosphate buffer containing 25 µM EDTA and 1.7 mM octane sulfonic acid adjusted to pH 3 with phosphoric acid. Flow rate was 1 ml/minute. Detector settings were as follows: conditioning cell (Model 5020; ESA Laboratories, Inc.) at +350 mV; analytical cell (Model 5014B; ESA Laboratories, Inc.) with the first and second electrodes maintained at –150 and +250 mV, respectively. Data were stored and processed with Chemstation software (Rev B.01.03; Agilent Technologies, Inc.). The pheochromocytoma 12 (PC12) cell line was used as a positive control for the dopamine assay as described previously [23]. In brief, 1 x 10⁶ of PC12 cells were seeded on poly-L-ornithine-precoated 6-well plates for 1 day. After the cells attached, cells were washed twice with HBSS prior the addition of 200 µL of depolarization media containing 51.5 mM KCl and 2 mM barium chloride. Cells were incubated for 15 minutes at room temperature. After depolarization, the supernatant was collected and stored at –80°C. PC12 cells were analyzed alongside differentiated human neurosphere cells via HPLC.

Detection of Tyrosine Hydroxylase in Differentiated Cells by Western Analysis
Protein samples (containing equivalent total protein) were prepared using TRIzol Reagent (Invitrogen, Carlsbad, CA, http://www.invitrogen.com), as per the manufacturer's instructions, from 10⁵ olfactory neurosphere-derived cells and from 10⁶ PC12-positive control cell line, respectively, and separated by SDS-PAGE. Proteins were transferred to a nitrocellulose membrane that had been soaked in chilled transfer buffer (10 mM NaHCO₃/3 mM Na₂CO₃, pH 9.9, 20% [vol/vol] methanol) for 5 minutes. The Bio-Rad transfer apparatus for mini-gel system II (Hercules, CA, http://www.bio-rad.com) was used in accordance with the manufacturer's instructions, with protein transfer performed at 100 V for 1 hour. Protein transfer was assessed by the complete transfer of prestained low-molecular-weight markers (Bio-Rad) to the membrane. The membrane was then blocked for at least 2 hours at room temperature with gentle agitation in 10% BLOTTO (10% nonfat milk powder in TBST [50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Triton X-100]). The blocking solution was then decanted and replaced with the primary antibody diluted appropriately in 10% BLOTTO. The primary antibody (anti-TH, 10.1 µg/ml; Sigma-Aldrich) was incubated overnight at 4°C with gentle agitation. The membrane was then washed five times for 5 minutes in TBST, after which a horseradish peroxidase-conjugated secondary antibody was added 1:5000 in 10% BLOTTO. The secondary antibody was incubated at room temperature for 1 hour with gentle agitation and the membrane then washed in TBST for 5 x 5 minutes. Bound antibodies on the membrane were detected using enhanced chemiluminescence reagent (Amersham Biosciences, Piscataway, NJ, http://www.amersham.com) in accordance with the manufacturer's instructions. The blot was read using ImageReader LAS-3000 software (Bucher Biotech, Basel, Switzerland, http://www.bucher.ch/en/home) and Fujifilm machine LAS-3000 (Fuji Film Australia, Sydney, Australia, http://www.fujifilm.com.au).

Hemiparkinsonian Rat Model
Eighteen rats (6 months of age) were injected i.p. 30 minutes prior to lesion surgery with two drugs: pargyline (50 mg/kg; Sigma-Aldrich), to inhibit endogenous monoamine oxidase, and desipramine hydrochloride (25 mg/kg; Sigma-Aldrich), to protect noradrenergic neurons from 6OHDA toxicity [24]. Animals were anesthetized with ketamine/xylazine (60 mg/kg and 3 mg/kg, respectively; ilium, Troy Laboratories Pty Ltd., Sydney, Australia, http://www.troylab.com.au), placed in a stereotaxic headholder, and injected unilaterally into four sites in the right striatum with 6OHDA (3.0 µg/µl, 2 µl/site, total dose 24 mg; Sigma-Aldrich) using a 30-gauge, 10-µl Hamilton syringe (Hamilton Co., Reno, NV, http://www.hamiltoncompany.com). The injection sites were determined from pilot studies to reliably target the lateral and dorsal aspects of the striatum [25]. After injection the small hole in the skull was covered in a collagen matrix (SpongeStan; Johnson & Johnson, Langhorne, PA, http://www.jnj.com) and the scalp sutured.

Five months after unilateral lesion the animals were assessed for amphetamine-induced rotation (see below). The nine animals with the fastest rates of ipsilateral turning were selected for transplantation (see below). There were three groups of animals (n = 3 per group): (a) those that received a transplant of human olfactory neurosphere-derived cells from a healthy human volunteer, (b) those that received a transplant of human olfactory neurosphere-derived cells from a human volunteer with Parkinson's disease, and (c) a sham surgery control group receiving injections of culture medium only. A further four animals received a transplant of human olfactory neurosphere-derived cells to investigate the cell survival 2 days to 9 months following transplantation.

Cell Transplantation Procedure
The animals were lesioned at 6 months of age and grafted 5 months later, making them 11 months of age at the time of transplantation. Preoperatively animals were given acepromazine (3.3 mg/ kg, i.m.; PromAce, Fort Dodge Animal Health Fort Dodge, IA, http://www.wyeth.com/divisions/Fort_dodge.asp). Twenty minutes later they were anesthetized with ketamine/xylazine (60 mg/kg and 3 mg/kg, respectively, i.p.; Ilium, Troy Laboratories). Animals were placed in a stereotaxic headholder and cells were transplanted into the striatum in two sites using a 22-gauge, 10-µl Hamilton syringe (2.0 µl per injection, 0.25 µl/minute). The injection sites were determined from pilot studies to target the centers of the previous 6OHDA lesions. The cells were suspended in DMEM/ITS. Animals that underwent transplantation received 37,000–210,000 cells in total. To prevent rejection of grafted cells, all animals (including controls) were injected with cyclosporine A (15 mg/kg, s.c.; Sandimmune; Sandoz Pharmaceutical, Princeton, NJ, http://www.sandoz.com) from the day prior to transplantation and for the following 12 weeks, until they were euthanized.

Amphetamine-Induced Rotation
Rotational behavior was measured at 5 months after the 6OHDA lesion, prior to grafting, and at 3, 7, and 12 weeks following cell transplantation. Rotation was measured in a Rotometer (San Diego Instruments, San Diego, http://www.sandiegoinstruments.com) for 90 minutes following injection of amphetamine (5 mg/kg, i.p) [26]. The numbers of ipsiversive and contraversive rotations were summed for each 10-minute time bin and expressed as mean and standard error. The rotational behavior of the three groups of animals was compared by repeated measures analysis of variance.

Tissue Processing, Histology, and Immunohistochemistry
Animals were sacrificed and perfused transcardially with 4% paraformaldehyde in PBS. The brain was removed and soaked in 4% paraformaldehyde/PBS and allowed to equilibrate in 30% sucrose/PBS/1% sodium azide at 4°C overnight. Brains were cryoprotected with optimal cutting temperature (Tissue Tek, Sakura Finetek, Torrance, CA, http://www.sakuraus.com) and cryosectioned at 8 µm. Sections were permeabilized in 0.3% Triton X-100 in PBS for 1 hour at room temperature. Sections were blocked in 10% goat serum in PBS for 30 minutes before incubation with primary antibodies for 1 hour at room temperature. Sections were subsequently washed three times before incubation of the secondary fluorescent antibodies in 10% goat serum in PBS for 30 minutes.

For immunohistochemistry of the brain sections, primary antisera used were rabbit anti-dopamine transporter (1/100; Sigma-Aldrich), mouse monoclonal anti-tyrosine hydroxylase (1/2000; ImmunoStar Inc., Hudson, WI, http://www.immunostar.com), mouse monoclonal anti-human nucleus (1/20; Chemicon), mouse monoclonal anti-neurofilament-200 (1/200; Sigma-Aldrich), and mouse monoclonal anti-human Tom22 (1/100; Sigma-Aldrich). Secondary antibodies used were AlexaFluor 594 or 488 highly cross-absorbed anti-rabbit or anti-mouse IgG (H+L) (1/400; Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com). Cell nuclei were counterstained with Hoechst 33342 (10 µg/ml) or 4',6-diamidino-2-phenylindole (in Vectashield mounting medium; Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com). The specificity of all primary antibodies used was confirmed using controls incubated with non-immune sera of the species used to raise those antibodies. Positive and negative controls were performed for each antibody. Sections incubated with secondary antisera enabled assessment of only any nonspecific binding, and sections without fluorophore eliminated any effects of autofluorescence. Slides were examined using an Olympus BX50 fluorescence microscope (Olympus, Tokyo, http://www.olympus-global.com) fitted with an Apogee KX85 digital camera and digital software (PMIS; Apogee Electronics Corp., Santa Monica, CA, http://www.apogeedigital.com) or a Zeiss AxioImager Z1 and Zeiss (Monochrome) Camera model number - Axiocam MRm (Carl Zeiss, Jena, Germany, http://www.zeiss.com). Images were adjusted for contrast and labeled using Adobe Photoshop (Adobe Systems, San Jose, CA, http://www.adobe.com).

Statistical Methods

Differentiation Experiments.   Statistical analysis compared the effect of serum-free medium with or without GDNF on either the number of tyrosine hydroxylase-positive cells or the total cells per well as indicated by two-tailed, Mann-Whitney U test. Further similar analysis was made for the effect of substrate.

Rotameter Data.   A one-way analysis of variance tested for differences among the groups in rotational behavior before transplantation. After transplantation, group differences were measured for 12 weeks. A repeated measures analysis of variance was used with main effect of group and repeated measure of time. Significant differences between groups were analyzed with a posthoc Bonferroni test.

	RESULTS

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
Rat and Human Olfactory Neurosphere-Derived Cells Expressed Dopaminergic Neuron Proteins In Vitro
A variety of procedures (see Materials and Methods), some based in part on reports in the literature for differentiation of neural and embryonic stem cells into dopaminergic cells, were tested on rat olfactory neurosphere-derived cells to derive an optimal protocol. Different culture conditions using a collagen IV coating were manipulated to optimize the percentage of cells expressing tyrosine hydroxylase. No conditions, including those with serum, expressed TH without the presence of the coating. Expanded olfactory epithelial cultures grown in 10% FBS were included for comparison and did not express markers of dopaminergic phenotype. These results are summarized in Table 1.

View larger version (17K): [in this window] [in a new window]   Figure 1. Control and Parkinson's olfactory neurospheres can be isolated, expanded, and differentiated similarly. Cells were cultured on poly-L-ornithine/laminin in glial cell-derived neurotrophic factor (25 ng/ml) for 4 days. (A): Expression of tyrosine hydroxylase (red). (B): Merged image of same cells double labeled for neurofilament 200 (green). The neurofilament expression pattern reveals the neuronal morphology and phenotype. (C): Tyrosine hydroxylase and (D) dopamine transporter expression in the same cells. Nuclei were stained blue with Hoechst blue. Scale bar (A, B) = 200 µm. (C, D) = 50 µm. This figure depicts differentiation of human Parkinson's disease patient's olfactory neurosphere cells into dopaminergic neurons. Cells from a non-Parkinson's disease control were differentiated the same way.

View larger version (18K): [in this window] [in a new window]   Figure 2. Differentiation of tyrosine hydroxylase-positive cells from neurosphere-derived cells from a patient with Parkinson's disease. Dissociated neurosphere cells were plated into control uncoated wells or wells coated with collagen IV, or poly-L-ornithine plus laminin. Cells were grown in serum-free medium (Dulbecco's modified Eagle's medium/insulin/transferrin/selenium) with or without glial cell-derived neurotrophic factor (GDNF). Data are expressed as the percentage of all cells that were immunopositive for tyrosine hydroxylase. Each bar shows the mean ± SEM for three wells. Note that no dopaminergic phenotype was expressed in the absence of the coatings. Abbreviations: Lam, laminin; PLO, poly-L-ornithine; TH, tyrosine hydroxylase; Unc, uncoated.

Expression of tyrosine hydroxylase protein in positive cell cultures was confirmed by SDS-PAGE and Western analysis. Cell lysates of cells grown in optimized conditions contained a protein identified on the Western blot as tyrosine hydroxylase (not shown). A similar band was identified in the positive control, PC12 cell line.

Cultures Released Dopamine Under Some Experimental Conditions
The presence of dopamine and its metabolites was measured after depolarization with high extracellular potassium concentration, using HPLC analysis of cell supernatants. Molecules present in the supernatant were compared with "spiked" media and with a positive control cell line (PC12) [27].

Based on studies reported in the literature [18–21] a number of different culture media modifications were tested as detailed in Materials and Methods. After cell depolarization, dopamine and related metabolites were produced in various culture scenarios at picomolar levels. Addition of the various culture components was made to basal media (DMEM/ITS) with and without GDNF (see Materials and Methods). Results compared with these media alone were not significantly different. Dopamine or its metabolites (5-hydroxytryptamine, homovanillic acid, 5-hydroxyindoleacetic acid) were released in 9/9 culture scenarios including controls containing FBS. Cultures on plastic alone released no dopamine or its metabolites.

Olfactory Neurosphere-Derived Cells Ameliorated Ipsiversive Rotation in Hemiparkinsonian Rat
As expected, unilateral 6OHDA lesion of the striatum induced ipsiversive turning in all animals after amphetamine (Fig. 3, before graft). Nine lesioned animals, fast turners chosen for the study, were divided into three groups: receiving a sham graft with culture medium injection only (n = 3); receiving human olfactory neurosphere-derived cell transplants (n = 3); and receiving similar transplants derived from a patient with Parkinson's disease (n = 3). Net ipsilateral turns were compared for the 90-minute test period before and after transplantation. These data were normally distributed (Komolgorov-Smirnov test, z = 1.1, p = .19) with equal sample sizes in all groups (n = 3). A one-way analysis of variance indicated that there were no differences among the groups in rotational behavior before transplantation (F_2,6 = 1.7, p = .25). After transplantation, the sham-operated animals maintained a stable rotational bias for 12 weeks following sham surgery (Fig. 3). In contrast, all animals that received a transplant of human neurosphere-derived cells had a dramatic reduction in the rate of ipsiversive rotation within 3 weeks that was maintained for 12 weeks (Fig. 3). Repeated measures analysis of variance indicated a significant difference between the groups after transplantation (F_2,6 = 11.8, p < .01) but there was no significant effect of period since transplantation (F_2,12 = 1.6, p = .25), indicating that the grafts were effective by 3 weeks. There were significant differences between human to sham (p = .015) and Parkinson's to sham (p = .020) using a posthoc Bonferroni test for multiple comparisons. Cells derived from a Parkinson's disease patient were similarly effective in reducing amphetamine-induced rotation compared with cells from a healthy control (Fig. 3).

View larger version (22K): [in this window] [in a new window]   Figure 3. Amphetamine-induced rotational bias was eliminated in the 6-hydroxydopamine (6OHDA) lesioned rat after transplantation with human olfactory stem cells derived from a patient with Parkinson's disease or a non-Parkinson's control (n = 3 for each group). (A): Sham graft. (B): Non-Parkinson's disease control graft. (C): Parkinson's disease graft. Each graph shows the number of ipsiversive (Ipsi) turns (black dots) and contraversive (Con) turns (white dots) in each 10-minute time period during the 90-minute test period following amphetamine injection at time 0. For each group three graphs are shown, the pregraft rotation, 5 months after 6OHDA lesion, and at 3, 7, and 12 weeks following transplantation.

View larger version (96K): [in this window] [in a new window]   Figure 4. Human cells (from a non-Parkinson's disease control) survive and differentiate into dopaminergic phenotype after being transplanted into the 6-hydroxydopamine lesioned rat brain. (A): Green fluorescent protein (GFP)-labeled human neurosphere-derived cells (green) 48-hour after transplantation clearly visible near the graft site in fresh frozen sections. (B): The same image taken simultaneously with a differential interference contrast image to show the rat tissue (gray). (C): The same field after immunochemistry using the anti-human Tom22 antibody (red). Bar = 10 µm. (D): GFP-labeled human neurosphere-derived cells (green) 3 weeks after transplantation. Nuclei are labeled with Hoechst blue. (E): The same field showing Tom22 immunoreactivity in the same cells, confirmed by merging the images (F). Bar = 50 µm. (G, H): Fluorescence and fluorescence/differential interference contrast overlay low power images showing end of needle track (white arrow) and transplanted GFP-labeled cells just within the striatum 3 weeks after transplantation. Bar = 10 µm. One animal that received a transplant of human olfactory neurosphere-derived cells was euthanized 9 months after transplantation. Human-derived cells were detected using the anti-human nuclear antibody (red, I) that expressed tyrosine hydroxylase (green, J). (K): Differential interference contrast image of the same region. (L): Overlay of (J) with nuclei stained with Hoechst blue. Bar = 10 µm.

View larger version (105K): [in this window] [in a new window]   Figure 5. Human Parkinson's disease patient's neurosphere-derived cells differentiated into dopaminergic phenotype after being transplanted into the 6-hydroxydopamine lesioned rat brain. (A, B): Striata of a lesioned rat brain showing tyrosine hydroxylase immunoreactivity (red) in the control, left side (A) and lesioned, right side (B). Bar = 1 mm. (C): Human neurosphere-derived cells labeled with an anti-human nucleus antibody (green) overlayed with differential interference contrast image showing the brain tissue (gray) in the right striatum. (D): The same field overlayed with anti-tyrosine hydroxylase staining in the red channel detecting a cluster of immunopositive fibers in the vicinity. Bar = 50 µm. (E–G): Human neurosphere-derived cells (in the vicinity of [C] and [D] above) in the right striatum expressing tyrosine hydroxylase (red, E) and the human nuclear antigen (green, F). Images are overlaid in (G). Nuclei are stained with 4',6-diamidino-2-phenylindole (DAPI; blue). Bar = 10 µm. (H–J): Human neurosphere-derived cell in the right striatum expressing dopamine transporter (red, H) and the human nuclear antigen (green, I). Images are overlaid in (J). Nuclei are stained with DAPI (blue). Bar = 10 µm.

	DISCUSSION

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

Interestingly, there have been reports in the popular press of private clinics transplanting fresh olfactory epithelium-derived cell preparations in human patients. It is noteworthy therefore that we were unable to provoke any significant dopaminergic differentiation from olfactory epithelial cell preparations or cultures without first generating neurospheres.

Several different media and culture conditions [28–34] were examined to optimize the proportion of tyrosine hydroxylase-positive cells in the cultures. For dopaminergic differentiation in vitro, the results demonstrate that coated tissue culture plastic is necessary and that serum-free medium alone is effective to induce differentiation, with a further marginal increase with the addition of GDNF in the medium. Dopamine release by these cells would seem to suggest that serum-free medium alone may be the optimal medium in which to induce a dopaminergic phenotype in these cells, as additional components tested (such as GDNF, IGF1, IL-1β, trichostatin A, FGF8, forskolin, neuropeptide Y, and LiCl [18–21]) or serum did not significantly increase dopamine release. However, results described in our paper demonstrate a capability to produce dopamine, albeit in low amounts. It was not the aim here to arrive at an efficient differentiation regime but merely to demonstrate capability for dopamine production in tandem with a demonstration of in vivo induction because autologous cell transplantation will require competent cells from the patients. Pursuit of the mechanisms regulating dopamine production is a worthy experimental goal but a major study in itself requiring comprehensive in vitro culture to isolate the effective induction protocol. These studies are under way worldwide using a variety of progenitor cells and have yet to provide a definitive solution.

Further evidence for functional differentiation of olfactory neurosphere-derived cells was demonstrated by their ability to ameliorate the locomotor effects of unilateral 6OHDA lesion of dopaminergic neurons projecting to the striatum. The mechanism for this behavioral effect has not been determined. It may be due to dopamine release by the transplanted cells, given the presence of tyrosine hydroxylase-positive, human-derived cells within the transplants and given our evidence that these cells can release dopamine in vitro. A quantitative analysis of the grafted cells was not undertaken but is required for a clearer understanding of the mechanism of the success of the grafts. Another plausible hypothesis is that the grafted cells produced GDNF, which then induced surviving striatal axons and terminals to grow into the lesioned region, thereby increasing the dopamine available after amphetamine [35–38]. Significantly, none of the transplants led to formation of tumors or teratomas in the host rats, as has occurred after embryonic stem cell transplantation in a similar model [39]. Longer-term studies with a larger number of animals are required to fully assess the risk of tumorigenesis after olfactory cell transplantation, but the present result is encouraging, particularly because the rat hosts were immune suppressed.

The 6-hydroxydopamine lesioned, hemiparkinsonian rat is one of the most frequently used models for Parkinson's disease partly because assessment of amphetamine-induced, ipsiversive rotations provides an automated readout of the efficacy of the experimental therapy. Therapeutic strategies have included direct injection of growth factors to stimulate endogenous stem cells [12], injection of genetically modified cells as delivery vehicles [40], as well as many attempts at cellular transplant therapy [22, 39, 41–51], including embryonic stem cells [39]. In other studies embryonic stem cells have been differentiated into a neural progenitor state [46, 51–53] or a dopaminergic state prior to transplantation [22, 41, 42]. Neural progenitors from human [43] or porcine [44] fetuses have also been transplanted into this model, including a demonstration that transplantation of predifferentiated neural progenitors toward the dopaminergic lineage was more effective than nondifferentiated progenitors [45]. Our results indicate that predifferentiation was not necessary for a therapeutic effect of olfactory neurosphere-derived progenitors, although it is possible that the effect can be improved. Nevertheless, successful olfactory cell transplantation was observed in these relatively old animals, which were 6 months of age at the time of lesion and 9 months at the time of cell transplantation.

Adult, tissue-derived stem cell transplantations into the hemiparkinsonian rat include bone marrow stromal cells predifferentiated into dopaminergic neurons [47] prior to transplantation and similar cells transplanted without predifferentiation [48]. Transplanted cells were effective in both studies. In the latter study the bone marrow stromal cells were as effective as fetal mesencephalic dopaminergic neuron transplants [47]. In the former study dopaminergic neuron differentiation was induced by gene transfection and growth in a medium containing GDNF, with this combination of treatments most effective in the hemiparkinsonian rat [47]. Other, potentially autologous sources of cells have been transplanted into this model. Autologous transplantation of adrenal medulla led to transient improvements in motor behavior [54, 55], although they disappeared within 18 months [56], and led to a high rate of mortality and morbidity [57]. Autologous transplantation of adrenal chromaffin cells had limited efficacy [58, 59]. Autologous transplantations of cell aggregates of carotid bodies were moderately successful in human clinical trials, with some improvement in symptoms in younger patients [60]. The mechanism for the improvement may be via trophic support of remaining dopaminergic neurons rather than supply of dopamine [61].

	CONCLUSION

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
The results of the present study suggest that olfactory neurosphere-derived cells should be explored further as a potential autologous source of cells for cell transplantation therapy in Parkinson's disease. These cells are readily accessible via biopsy of the human olfactory mucosa [8], they can differentiate along the dopaminergic lineage in vitro and in vivo, and they are therapeutic after transplantation into the lesioned striatum of the hemiparkinsonian rat.

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
The authors thank C. Cahill, B. Baldwin, K. Splatt, R. Sutharson, Will Young, and J. Kan for technical assistance. This work was supported by The National Health and Medical Research Council of Australia and the Australian Department of Health and Ageing.

	FOOTNOTES

 
Author contributions: W.M.: conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; A.W.: provision of study material, collection and assembly of data, data analysis and interpretation, manuscript writing; M.D. and J.B.: provision of study material, collection and assembly of data; F.F. and C.P.: provision of study material; T.B. and J.K.: data analysis and interpretation; A.M.: collection of data; P.S.: provision of patients, financial support; A.M.-S.: conception and design, financial support, administrative support, manuscript writing.

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