Generation of Functional Dopamine Neurons from Neural Precursor Cells Isolated f - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:54 |只看该作者 |倒序浏览 |打印

作者：Jae-Won Shima,i, Chang-Hwan Parkb,f, Yong-Chul Baeg, Jin-Young Baeg, Seungsoo Chungh, Mi-Yoon Changa,e,f, Hyun-Chul Kohc,f, Hyun-Seob Leea,e,f, Se-Jin Hwangd, Ki-Hwan Leea,e,f, Yong-Sung Leea,e,f, Cha-Yong Choih, Sang-Hun Leea,e,f作者单位：Departments of aBiochemistry and Molecular Biology,


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      【摘要】" P1 O0 n7 f7 {0 A
   Neural precursor (NP) cells from adult mammalian brains can be isolated, expanded in vitro, and potentially used as cell replacement source material for treatment of intractable brain disorders. Reduced ethical concerns, lack of teratoma formation, and possible ex vivo autologous transplantation are critical advantages to using adult NP donor cells over cells from fetal brain tissue or embryonic stem cells. However, the usage of adult NP cells is limited by the ability to induce specific neurochemical phenotypes in these cells. Here, we demonstrate induction of a dopaminergic phenotype in NP cells isolated from the subventricular zone (SVZ) and white matter of rodent adult brains using overexpression of the nuclear receptor Nurr1 in vitro. Forced expression of Nurr1, a transcriptional factor specific to midbrain dopamine (DA) neuron development, caused in the adult cells an acquisition of the DA neurotransmitter phenotype and sufficient differentiation toward morphologically, phenotypically, and ultrastructurally mature DA neurons. Co-expression of neurogenic factor Mash1 and treatment with neurogenic cytokines brain-derived neurotrophic factor and neurotrophin-3 greatly enhanced Nurr1-induced DA neuron yield. The Nurr1-induced DA neurons demonstrated in vitro presynaptic DA neuronal functionality, releasing DA neurotransmitter in response to depolarization stimuli and specific DA reuptake. Furthermore, Nurr1-engineered adult SVZ NP cells survived, integrated, and differentiated into DA neurons in vivo that can reverse the behavioral deficit in the host striatum of parkinsonian rats. These findings open the possibility for the use of precursor cells from adult brains as a cell source for neuronal replacement treatment of Parkinson disease.* @/ Z5 S3 l" S% {. ^
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Disclosure of potential conflicts of interest is found at the end of this article. * }9 K" o; X0 c5 g
      【关键词】 Adult neural stem cell Nurr Dopamine neurons Parkinson disease7 [+ p6 }* S. D# E. d0 K! v
   INTRODUCTION

Multipotent undifferentiated stem or precursor cells with the capacity for self-renewal play major roles in mammalian development. Most adult tissues harbor tissue-specific precursor cells even after the termination of developmental processes. The brain was long thought to be an exception, until neural precursor (NP) cells were discovered in the subventricular zone (SVZ) of rodent adult forebrain , which is mostly occupied by neuronal fibers and a minor population of oligodendrocyte progenitors.& ?! z! [, |5 N6 r
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Parkinson disease (PD) is a neurodegenerative disorder characterized by progressive and specific loss of dopamine (DA)-secreting neurons in the substantia nigra of the midbrain. Given the distinct loss of this single neuronal subtype in a confined brain region, cell-based replacement approaches for PD have garnered much attention over the past decade. Precursor cells from fetal and adult brains can be isolated, maintained in culture in an undifferentiated state, and guided to differentiate into a variety of neurons and glia . The usage of NP cells isolated from the adult brain would circumvent these ethical issues and complications. Furthermore, adult NP cell-based autologous transplantation could involve isolating precursors from intact brain regions of the patient and ex vivo expansion of these precursor cells.* O: A& |# Z' k- m: u

In this study, we isolated NP cells from the SVZ of the lateral ventricle and subcortical WM of rodent adult brain and examined whether such precursor cells could generate functional DA neurons relevant for cell therapeutic treatment of PD. Historically, NP cells derived from adult brain have primarily differentiated into astroglial cells, and the induction of DA neurons from naïve adult precursor cells has seldom been achieved (reviewed in , to promote DA neuronal differentiation in adult NP cells. Nurr1-expressing adult precursors efficiently differentiated toward DA cells, demonstrating the functionalities of presynaptic DA neurons in vitro and in vivo by engrafting into the striatum of PD-model rats. These findings suggest that NPs derived from adult brain, in conjunction with Nurr1 gene manipulation, could be a useful strategy for PD therapy.
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MATERIALS AND METHODS

Culture of Neural Precursor Cells Isolated from the SVZ and WM of Adult Rat Brains# \/ w( c, e$ V3 O

Animals were housed and cared for according to NIH guidelines. Brains were freshly obtained in the course of surgical resection from adult male Sprague-Dawley rats (7¨C8 weeks old; Koatech, Seoul, Korea, http://www.ekoatech.com), washed several times in Ca2 -, Mg2 -free Hanks' balanced salt solution (CMF-HBSS), and sliced anteroposteriorly in a brain matrix (coronal-type; Stoelting, Wooddale, IL, http://www.stoeltingco.com). The SVZ and WM tissues, as indicated in supplemental online Figure 1a, were dissected from the rest of the tissue, free of adjacent cortex and ventricular epithelium. The tissues were mechanically chopped and digested in a solution containing papain (2.5 U/ml papain; Worthington, Freehold, NJ, http://www.worthington-biochem.com), dispase II (1 U/ml; Roche, Indianapolis, http://www.roche.com), and DNase I (2.5 U/ml; Worthington) by incubating at 37¡ãC for 45 minutes on a rocking shaker (model 1012; SHEL LAB, Cornelius, OR, http://www.shellab.com). The pellets were collected by centrifugation at 200g, rinsed once with CMF-HBSS, and resuspended in 1 ml of serum-free N2 medium . Immunocytochemical analyses were performed on the cryosectioned spheres or coverslip preparations as described below. In most of the other experiments, neurospheres were dissociated into single cells as described above, and plated at 500 cells per mm2 onto PLO/FN-coated coverslips in 24-well plates or 6-cm tissue culture dishes (for cell transplantation) in mitogen-supplemented N2. Retroviral transductions were carried out at 50%¨C60% cell confluence (typically 1 day after plating) as described below. Cell differentiation was induced for 7¨C14 days by withdrawal of the mitogens in N2 medium supplemented with B27 supplement (1x; Invitrogen, Grand Island, NY, http://www.invitrogen.com) and ascorbic acid (200 µM; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com). In certain experiments, brain-derived neurotrophic factor (BDNF) (20 ng/ml), glial cell-derived neurotrophic factor (GDNF) (20 ng/ml), or neurotrophin-3 (NT3) (20 ng/ml; all from R&D Systems) was added to the medium.% V$ }' X: E4 t; V$ B* ]. D

Cells were isolated from rat cortices at embryonic day 14 (E14) and cultured in suspension in N2 supplemented with the mitogens. Neurospheres formed after 3¨C6 days of cell expansion were dissociated, plated, and differentiated in conditions identical to those of the adult cultures as described above.  k$ {: Z, M7 x% w

Retroviral Construction and Transduction
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The retroviral vectors expressing Nurr1 (pNurr1-IRES-LacZ, pNurr1-IRES-green fluorescent protein . The retroviral vectors were transfected into 293gpg packaging cells (Lipofectamine; Invitrogen), and supernatant containing viral particles (VSV-G pseudotyped recombinant retrovirus) was harvested 72 hours after incubation. Viral titer was adjusted to 5 x 106 particles per ml. For retroviral transduction, cells on coverslips or 6-cm dishes were incubated with the viral supernatant containing polybrene (1 µg/ml) for 3 hours. After 1¨C2 days of further cell expansion in the presence of the mitogens described above, cells were induced to differentiate or prepared for cell transplantation. Retroviral infection per se did not affect cell survival, proliferation, or differentiation.* I  y& r2 |' `& L4 C8 `9 A5 `
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Immunostaining of Cultured Cells and Brain Slices

Cultured cells, cryosectioned brain slices, or neurospheres were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) and incubated with primary antibodies overnight at 4¡ãC. The following primary antibodies were used: tyrosine hydroxylase (TH) anti-rabbit (1:250; Pel-Freez, Rogers, AK, http://www.pel-freez.com) or anti-mouse (1:1000; Sigma-Aldrich), neuron-specific class III ¦Â-tubulin (TuJ1) anti-mouse (1:500) or anti-rabbit (1:2000; both from Covance, Princeton, NJ, http://www.crpinc.com), anti-rabbit Nurr1 (1:200; Chemicon, Temecula, CA, http://www.chemicon.com), nestin (1:50, provided by Martha Marvin and Ron Mckay, NIH, Bethesda, MD), vesicular monoamine transporter 2 (VMAT2) (1:500; Pel-Freez), anti-mouse GFP (1:400; Roche), Ki67 (1:100; Novocastra Ltd., Newcastle upon Tyne, U.K., http://www.novocastra.co.uk), glial fibrillary acidic protein (GFAP) (1:200; MP Biomedicals, Solon, OH, http://www.mpbio.com), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) (1:500; Sigma-Aldrich), microtubule-associated protein (MAP2) (1:1,000; Sigma-Aldrich), neuron-specific RNA-binding protein (HuC/D, 1:100; Invitrogen), and anti-rat dopamine transporter (DAT) (1:5,000; Chemicon). For detection of primary antibodies, fluorescently labeled (fluorescein isothiocyanate or Cy3) secondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com) were used according to the specifications of the manufacturer. Cells or brain slices were mounted in Vectashield containing 4',6 diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) and analyzed under an epifluorescent (Nikon, Tokyo, http://www.nikon.com) or confocal (LMS 510; Carl Zeiss, Feldbach, Switzerland, http://www.zeiss.com) microscope.
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Immunoelectron Microscopy Analysis" c  p6 X1 M$ n4 w

Cells grown on Aclar embedding film (Electro Microscopy Science, Hatfield, PA, http://www.emsdiasum.com) were fixed in 4% paraformaldehyde/0.01% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4 (PB), cryoprotected in 30% sucrose in PB, and then incubated with anti-TH antibody (Pel-Freez) 4¡ãC overnight. TH  cells were visualized with peroxidase reaction with diaminobenzidine (DAB) substrate. TH-stained cells were postfixed with 1% osmium tetroxide in 0.1 M PB for 30 minutes, dehydrated in a graded series of alcohols, embedded in Durcupan ACM (Fluka, Buchs SG, Switzerland, http://www.sigmaaldrich.com) between strips of Aclar film and polymerized at 60¡ãC for 48 hours. Small pieces containing TH  cells cut and glued onto the plastic block. Ultrathin sections were cut, mounted on formvar-coated single-slot nickel grids and examined on a Hitachi H 7500 electron microscope (Hitachi, Tokyo, http://www.hitachi.com). For specificity of immunoreactivity, a negative control test was carried out. Omission or replacement of the primary antiserum with normal rabbit serum showed an absence of immunoreactivity.- g+ [, S4 @5 F
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Reverse Transcription-Polymerase Chain Reaction- l6 r' e/ f4 j3 A' E
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Total cellular RNA was isolated using Tri Reagent (Molecular Research Center, Inc. Cincinnati, OH, http://www.mrcgene.com) and cDNA was synthesized from 5 µg of total RNA in a 20-µl reaction using the Superscript kit (Invitrogen). Optimal polymerase chain reaction (PCR) conditions for each primer set were determined by various MgCl2 concentrations, annealing temperatures, and cycle numbers to determine a linear amplification range. Information about primer sequences and optimum conditions provided upon request.
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Assays for DA Release and Reuptake6 H, V% U, @" j+ d, H) h

DA release was quantified using high-performance liquid chromatography (HPLC) analyses as described previously . Briefly, cells in 24-well plates were incubated in 400 µl/well of freshly prepared N2 (basal release) or N2 supplemented with 56 mM KCl (evoked release) for 30 minutes. The media were collected, stabilized with 0.1 mM EDTA, and analyzed. Samples (100 µl) were injected into a reverse phase µ-Bondapak C18 column (150 x 3.0 mm; Eicom, Tokyo, http://www.eicom-usa.com), and analyzed with an amperometric electrochemical detector (model ECD-300; Eicom). DA levels were calculated using external DA standard (50 nM methyl DOPA) injected immediately before and after each experiment.  P0 @) t* S) P+ N( C

Assays for DAT-mediated specific DA uptake were conducted as described previously DA (51 Ci/mmol; Amersham Biosciences, Buckinghamshire, U.K., http://www.amersham.com) with or without 10 µM nomifensine (Sigma-RBI, Natick, MA), a DAT blocker, to determine nonspecific uptake. After incubation for 10 minutes at 37¡ãC, cells were washed with cold PBS and lysed in 0.5 M NaOH. Radioactivity was measured by liquid scintillation counting (MicroBeta TriLux version 4.4; PerkinElmer, Waltham, MA, http://www.perkinelmer.com). Specific DA uptake was calculated by subtracting nonspecific uptake (with nomifensine) from uptake without nomifensine. The level of DA release and uptake per TH  cell was calculated by dividing each value by the TH  cell number obtained from cultures conducted in parallel to the DA release and uptake assays.

Electrophysiology8 o4 [+ }3 p6 P6 o

Nurr1-expressing cells were identified by GFP expression under a GFP-filtered microscope in the differentiated cultures for SVZ NP cells transduced with Nurr1-IRES-GFP viruses and subjected to electrophysiological analysis using the whole-cell recording configuration of the conventional "dialyzed" whole-cell patch-clamp technique . Patch electrodes were fabricated from a borosilicate glass capillary (Sutter Instrument Co., San Rafael, CA, http://www.sutter.com) by using a vertical micropipette puller (Narishige, Tokyo, http://www.narishige.co.jp). Resistances of patch electrodes were set to 2¨C4 M. Cell membrane capacitance and series resistance were compensated for (typically >80%) electronically using a patch-clamp amplifier (Axopatch-200A; Axon Instruments/Molecular Devices Corp., Foster City, CA, http://www.moleculardevices.com). Current protocol generation and data acquisition were performed using pClamp 8.2 software on an IBM computer equipped with an analog-to-digital converter (Digidata 1322A; Axon Instruments). Voltage traces were filtered at 5 KHz by using the four-pole Bessel filter in the clamp amplifier and stored on the computer hard drive for later analysis. All experiments were performed at room temperature (21¨C24¡ãC). For recording in voltage clamp mode, the patch pipette solution contained (in mM): KCl, 134; MgCl2, 1.2; MgATP, 1; Na2GTP, 0.1; EGTA, 10; glucose, 14; and HEPES, 10.5 (pH adjusted to 7.2 with KOH). The bath solution contained (in mM): NaCl, 134; KCl, 5; CaCl2, 2.5; MgCl2, 1.2; glucose, 14; and HEPES 10.5 (pH adjusted to 7.4 with NaOH).
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In Vivo Transplantation( l! J* P7 z$ J1 e" R

Generation of 6-hydroxydopamine-lesioned PD model rats and an amphetamine-induced rotation test were performed as described previously .2 x' d& \( T6 |1 W2 C1 [" ~
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Cell Enumeration and Statistical Analysis
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Cells were counted in microscopic fields randomly chosen across the culture area, using an eyepiece grid at a final magnification of x200 or x400. Three to six culture wells were analyzed in each experiment. Data are expressed as mean ¡À SEM. Statistical comparisons were made by one-way analysis of variance with Tukey post hoc analysis (SPSS 11.0; SPSS, Inc., Chicago, http://www.spss.com) when more than two groups were involved.
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RESULTS( V# S/ L$ N- `+ |
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Isolation and Characterization of NP Cells Derived from the SVZ and WM of Adult Rat Brain/ V1 F8 v- X  s# Y$ o( \% E

We have attempted an in vitro expansion of NP cells from the SVZ of the lateral ventricles and subcortical WM of rat adult brains to test whether adult NP cells could provide a renewable source of DA neurons for cell replacement strategy in PD. The culture of SVZ precursor cells was carried out in serum-free N2 medium supplemented with the mitogen bFGF as described previously . These protocols are highly complex, however, and difficult to replicate. We tested whether WM NPs could be simply isolated, without sorting, through proliferation in vitro with appropriate mitogens. SVZ cells were induced to proliferate by the mitogenic action of bFGF, and neurospheres appeared within 7 DIV. In contrast, no neurospheres were formed in the bFGF-supplemented WM cultures. Neither supplementation with EGF nor the combination of bFGF EGF caused efficient proliferation of WM cells. However, bFGF PDGF did promote the expansion of WM cells, resulting in conspicuous neurospheres within 7¨C14 DIV. The minimum periods required for visible sphere formation were 3¨C4 days in SVZ and 6¨C8 days in WM cultures. Usually, at 7 (SVZ) and 14 (WM) DIV, the spheres were transferred into fresh dishes using a pipette under the microscope. This procedure was a critical modification that allowed further culturing while keeping cultures free from tissue debris and potential contaminants. No spheres were observed from attempts to culture cells from the cortex or striatum under any of the culture conditions tested (data not shown), confirming that identification and isolation of NP from the SVZ and WM was tissue specific.

At 14 DIV, there was a great difference in the number of spheres per gram of tissue formed in SVZ (14,007.0 ¡À 1,471.3) and WM cultures (3,202.3 ¡À 1,985.5; n = 3) (Fig. 1A). Furthermore, the average neurosphere size (cells assembled in a sphere) in the SVZ cultures (3,130 cells) was greater than those in the WM cultures (1,220 cells) (Fig. 1B). These findings suggest both the presence of more self-renewable cells and a greater in vitro proliferation capacity in SVZ cells compared with WM cells. When the spheres were dissociated into single cells and seeded at a clonal density (25 cells per milliliter), subsequent (secondary) spheres were generated from 91.2% ¡À 8.9% and 75.0% ¡À 16.7% of the dissociated cells in SVZ and WM cultures, respectively (Fig. 1C). Similarly, 63.2% ¡À 9.5% (SVZ) and 49.6% ¡À 17.9% (WM) of cells dissociated from the respective secondary spheres gave rise to tertiary spheres. Further reductions were shown in the sphere forming units of fourth to ninth SVZ and WM sphere-forming cultures.
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Figure 1. In vitro self-renewal and multipotent properties of neural precursor cells derived from the SVZ and WM of adult rat brain. (A, B): Frequency and proliferation capacity of proliferating neural precursor cells were determined by the number of neurospheres per gram of tissue at 14 days in vitro (DIV) (A) and neurosphere size (average number of cells in a sphere) over the culture period (B). (C): Sphere forming rate. Neurospheres at DIV 12 were dissociated into single cells and seeded at a clonal density (25 cells per milliliter) in 96-well plates. Only the wells containing a single cell were marked for the analysis. The sphere forming rate represents percentage of wells containing spheres compared with total wells marked, at 3¨C4 days after cell seeding. Similarly, the sphere forming rates for the third through the ninth spheres were evaluated. *, Significantly different from WM at p ) b$ g# ~. N+ s8 E7 l  O0 K! O

Disregarding the differences in the sphere formation rate and proliferation capacity, the phenotypical properties and in vitro differentiation potentials of the SVZ- and WM-derived spheres were indistinguishable. Immunostaining performed after 14 DIV showed that 99.2% ¡À 1.0% (SVZ) and 98.5% ¡À 1.3% (WM) of assembled cells in spheres were positive for nestin, an intermediate filament specific to NP cells. Proliferation cell marker Ki67 was colocalized in 95.0% ¡À 2.2% and 94.3% ¡À 1.0% of nestin  cells in the SVZ and WM cultures, respectively (Fig. 1D, 1F). Interestingly, GFAP, a marker for astrocytic progeny and NP cells in the adult brain, was colocalized in 48.9% ¡À 3.3% and 51.9% ¡À 2.6% of nestin  cells in the SVZ and WM cultures, respectively (Fig. 1E, 1G). Virtually all GFAP  cells expressed nestin, and the GFAP /nestin  cells were always assembled in the center portions of the spheres.

Cell differentiation was induced by dissociating the spheres and plating the cells onto FN-coated plates in the absence of mitogens. After 7 days of differentiation, SVZ cultures consisted of 18.0% ¡À 1.9% TuJ1  neurons, 44.2% ¡À 2.3% GFAP  astrocytes, and 15.6 ¡À 1.6% CNPase  oligodendrocytes. WM cultures consisted of 16.0% ¡À 2.1% TuJ1  neurons, 48.1% ¡À 2.3% GFAP  astrocytes, and 16.9% ¡À 1.8% CNPase  oligodendrocytes (Fig. 1H, 1I). These findings, taken together, suggest that NP cells with self-renewal and multipotent properties from adult brain WM and SVZ can be derived simply but efficiently.
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To examine whether proliferative and developmental properties of the adult NP cells can be maintained after long-term cell expansion in vitro, SVZ and WM spheres were further cultured in the presence of the same mitogens for 70 days with 10 passages (as described in Materials and Methods). Total cell numbers increased during the two initial passages of SVZ cultures but leveled off and rather decreased after fourth cell passages (supplemental online Fig. 1d). However, significant increase of total cell numbers was not achieved by the extended period of WM cultures. When cells on each passage were plated and differentiated, more proportions of cells underwent cell apoptosis in the cultures expanded longer in vitro (percentage of cells with apoptotic nuclei in SVZ cultures: 38.4% ¡À 9.2% in passage 3  vs. 63.1% ¡À 6.8% in P6 at differentiation day 7.) Furthermore TuJ1  neuron yields from SVZ NP cells were gradually decreased over passages (supplemental online Fig. 1f). These findings suggest that further in vitro cell expansion of adult NP cells by a longer period of culture does not offer substantial benefit in the generation of NP cells with self-renewal and multipotent capacities. The following experiments, therefore, were done in unpassaged cultures (neurospheres cultured for 7 days after transferring into freshly prepared dishes), unless otherwise noted.
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Dopaminergic Differentiation of SVZ- and WM-Derived NP Cells by Nurr1 Transgene Expression: l$ v  u: {& O3 J+ p7 m+ L; W

Efficient dopaminergic differentiation from adult brain-derived NP cells has been difficult to achieve. Consistently, we found that no differentiated SVZ and WM cells were positive for TH, a key enzyme for DA biosynthesis. Neither treatment of cytokines, such as SHH, FGF8 (early inducers for midbrain DA neuronal development), GDNF, BDNF, and NT3 (differentiation and survival factors for DA neurons), nor alteration of culture conditions, such as cell density or culture period and type (suspension or adhesive culture), was effective in generating TH  cells (data not shown), suggesting no DA neurogenic potential of naïve adult NP cells.
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Genetic manipulation of key transcription factors also has the potential to induce acquisition of DA neurogenic potential in adult NP cells. Nurr1 is an orphan nuclear receptor-type transcription factor with a critical role in midbrain DA neuron development that has been demonstrated in vivo . We retrovirally induced Nurr1 transgene expression in SVZ- and WM-derived NP cells using Nurr1-IRES-GFP or Nurr1-IRES-LacZ vector. Efficiency of the retroviral transduction was 46.0% ¡À 4.9% and 37.9% ¡À 2.7% of total cells based on the estimation of Nurr1  and GFP  cells, respectively, at day 2 postinfection. Of the GFP  cells, 22.6% ¡À 2.2% and 24.0% ¡À 2.3% of cells were positive for TH in the cultures for SVZ- and WM-derived NP cells (Fig. 2A, 2B, arrows), respectively, and TH  cell numbers were maintained without significant loss up to at least 14 days of in vitro differentiation (data not shown). Subpopulations of TH  cells expressed DAT, a protein specific to DA homeostasis in presynaptic DA neurons (Fig. 2E, 2F, arrows); after 7 days of differentiation, 30.4% ¡À 14.6% (SVZ) and 25.3% ¡À 12.6% (WM) of TH  cells were TH /DAT . In addition, in semiquantitative reverse transcription-PCR analyses, expression of DA neuron-specific markers TH, DAT, and aromatic acid L-amino acid decarboxylase mRNAs were strikingly enhanced in Nurr1-transduced cultures, compared with the untransduced control (Fig. 2G). These findings suggest a role for Nurr1 in inducing dopaminergic differentiation of adult NP cells.

Figure 2. Dopaminergic differentiation from adult SVZ and WM-neural precursor cells by Nurr1 overexpression. Neurospheres derived from adult rat SVZ and WM were dissociated into single cells and cultured on fibronectin-coated coverslips. Cells were transduced with Nurr1-expression viruses (Nurr1-IRES-GFP  or Nurr1-IRES-LacZ), and cell differentiation was induced the following day by withdrawing the mitogens' basic fibroblast growth factor (bFGF) (SVZ) and bFGF platelet-derived growth factor-AA (WM). Dopaminergic differentiation of the Nurr1-transduced precursor cells was determined at day 7 of in vitro differentiation by reverse transcription-polymerase chain reaction (RT-PCR) (G) and immunocytochemical analyses for TH/GFP (A, B), TH/Nurr1 (C, D), and TH/DAT (E, F). Arrows indicate representative TH  cells expressing GFP (A, B), Nurr1 (C, D), and DAT (E, F), whereas arrowheads indicate GFP /TH¨C (A, B), Nurr1 /TH¨C (C, D), and TH /DAT¨C (E, F) cells. Scale bar = 20 µm. (G): Semiquantitative RT-RCR analyses to examine Nurr1-induced expression of DA neuronal markers TH, DAT, and AADC. Abbreviations: AADC, aromatic acid L-amino acid decarboxylase; DAT, dopamine transporter; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; SVZ, subventricular zone; TH, tyrosine hydroxylase; WM, white matter.. e/ g/ A% ~; w( d3 }9 g2 P6 v+ H0 ?

Morphologic, Phenotypic, and Functional Properties of DA Neurons Derived from Nurr1-Expressing Adult NP Cells

We have previously demonstrated the effect of forced Nurr1 expression in NP cells derived from rat embryonic brains. Nurr1 efficiently induced DA neurotransmitter identity in rat embryonic NP cells, but the Nurr1-induced DA cells were morphologically and functionally immature as presynaptic DA neurons . Consistently, short or absent neurite extensions were observed from the TH  cells differentiated from Nurr1-transduced precursor cells isolated from rat cortical tissues at E14 (Fig. 3G¨C3I). By contrast, Nurr1-induced TH  cells in the differentiated cultures for Nurr1-transduced adult SVZ- and WM-derived NP cells showed mature neuronal morphology with extensive and multiple neurite outgrowths (Fig. 3A¨C3F). Neurites per TH  cell at day 7 of differentiation had an average length of 512.7 ¡À 133.6 µm (SVZ), 531.7 ¡À 136.2 µm (WM), and 36.1 ¡À 13.2 µm (E14 cortex) (Fig. 3J; p " v" C3 g4 L9 U4 l* s
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Figure 3. Morphological and phenotypic differentiation of dopamine cells derived from Nurr1-transduced adult neural precursor cells. SVZ- and WM-derived neural precursor cells were cultured, transduced with Nurr1 (Nurr1-IRES-LacZ), and differentiated, as described in the legend of Figure 2. The same procedures were performed with precursor cells isolated from E14 rat cortices. At day 7 of in vitro differentiation, morphologic maturation of TH  cells and colocalization of the neuronal markers TuJ1, HuC/D, and MAP2 were assessed. Shown in (A¨CI) are the representative images for TH/TuJ1 (A, D, G), TH/HuC/D (B, E, H), and TH/MAP2 (C, F, I). Arrows indicate TH  cells expressing TuJ1 (A, D, E), HuC/D (B, E, H), and MAP2 (C, F, I). Scale bar = 20 µm. Note that TH  cells in adult SVZ (A¨CC) and WM (D¨CF) cultures exhibit mature neuronal morphology with multiple TH fibers and that the neuronal markers are colocalized in a major population of TH  cells derived from the adult cells (A¨CF). This is in a clear contrast to short or absent processes in TH  cells and separated localization of TH and neuronal markers in different cells in E14 cortical cultures (G¨CI). Graphs (J, K) depict total length of neurites per TH  cell and percentage of double-positive cells out of total TH  cells, respectively. *, Significantly different from E14 cortical cultures at p
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We next tested whether enhanced morphometric maturation was correlated with the expression of mature neuronal or dopaminergic markers. Quantification of the percentage of Nurr1-induced DA cells expressing neuronal markers TuJ1, HuC/D, and MAP2 confirmed our morphometric data on cell differentiation shown in Figure 3. Only a minor proportion (10.5% ¡À 2.1%) of Nurr1-TH  cells expressed TuJ1 in the cultures for E14 cortical precursors (Fig. 3G, 3K). In contrast, TH /TuJ1  cells were 91.9% ¡À 4.4% and 87.0% ¡À 6.5% of total TH  cells in the cultures for SVZ- and WM-derived precursor cells, respectively (Fig. 3A, 3D, 3K). Similar results were obtained in 87.1% ¡À 5.2% (SVZ), 84.2% ¡À 5.7% (WM) of TH  cells coexpressing HuC/D and 86.2% ¡À 6.6% (SVZ), 85.3% ¡À 7.4% (WM) of TH  cells coexpressing MAP2 specific to mature neuronal cells (Fig. 3B, 3C, 3E, 3F, 3K). In addition, there was a clear contrast in the expression of DAT, which is expressed at later stages of DA neuron development . As shown in Figure 2, DAT protein expression was colocalized in the subpopulations of TH  cells derived from SVZ and WM NP. In contrast, none of TH  cells in E14 cultures expressed DAT (data not shown). The generation of synapses is a definite marker for maturity of neuronal cells. Synapsin and synaptophysin, specific for synaptic vesicles, were colocalized in TH  cells differentiated from Nurr1-transduced SVZ or WM NP cells (Fig. 4A, 4B; data not shown). Immunoelectron microscopic analyses showed that TH-stained axon terminals, which contain neurotransmitter-containing vesicles, have synaptic contacts with distinct postsynaptic densities (Fig. 4C, 4D). Furthermore, ultrastructures were shown for the synapses generated between TH  dendrites and neighboring axons (Fig. 4E, 4F).& O/ M. O' e- y8 ^# d

Figure 4. Ultrastructures of synapses generated by Nurr1-SVZ-derived dopamine neurons. (A, B): Double immunofluorescent images for TH with the markers for At, synaptophysin (A), and synapsin (B) in the differentiated cultures for Nurr1-SVZ cells. Boxed areas are shown magnified in the insets at top left. Note that synaptophysin and synapsin were localized in the boutons on the TH-positive axons. (C¨CF): Light microscopy (C, E) and electron microscopy (EM) (D, F) for the immunoperoxidase staining of TH-stained SVZ cells. Boxed areas in (C) and (E) represent the areas for the EM observation in (D) and (F), respectively. The image shown in (D) demonstrates that a TH-positive At (asterisk) containing round vesicles forms synaptic contact with a TH-negative dt. (F): Ultrastructures of synapses are also observed between TH-positive dt (asterisk) with two TH-negative At. Arrows in (D) and (F) indicate the sites of synaptic contacts. Scale bar = 20 µm (A¨CC, E) and 0.5 µm (D, F). Abbreviations: At, axon terminal; dt, dendrite; TH, tyrosine hydroxylase.
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Presynaptic DA neuron function was assessed by measuring DA release in vitro in Nurr1-transduced cultures 10 days after differentiation. HPLC analysis revealed that DA was readily detected in the medium conditioned for 30 minutes in the differentiated SVZ and WM cultures (basal release, SVZ: 10.0 ¡À1.7 pg per TH  cell; WM: 8.0 ¡À 0.4 pg per TH  cell; n = 6 for each value; Fig. 5A, 5B). Elevated potassium was used to determine whether DA release could be evoked by depolarization. DA releases of Nurr1-tranduced SVZ and WM cells were strikingly increased by high potassium treatment. DA levels were 48.1 ¡À 7.8 pg per TH  cell (SVZ) and 26.7 ¡À 0.8 pg per TH  cell (WM) in the medium supplemented with 56 mM KCl (n = 6). In contrast, basal release of DA was undetectable, and DA level in the KCl-supplemented medium was only 0.6 ¡À 0.1 pg per TH  cell in Nurr1-transduced E14 cortical cultures. DA was not detected in any untransduced naïve cultures (Fig. 5A). DAT-mediated high affinity reuptake of DA is a critical process in DA metabolism and could be an indicator for functional presynaptic DA neurons. Although reuptake in Nurr1-transduced E14 cells was at the limit of detection (E14: 0.07 ¡À 0.01 fmol per minute per 1,000 TH  cells), SVZ and WM cells transduced with Nurr1 exhibited robust DA uptake of 22.75 ¡À 1.54 and 19.75 ¡À 0.68 fmol per minute per 1,000 TH  cells, respectively (Fig. 5C). In whole-cell recording on cells differentiated from Nurr1-SVZ NP cells (transduced with Nurr1-IRES-GFP), depolarizing voltage steps elicited both large outward potassium currents and fast inward Na  currents. (Fig. 5D), which are hallmark features of differentiated neurons.! o6 @! I/ N  w" T3 B6 a" s1 T) W

Figure 5. In vitro presynaptic neuronal functions of DA cells differentiated from Nurr1-transduced adult neural precursor (NP) cells. (A¨CC): At day 10 of in vitro differentiation, DA releases and uptakes specific for Nurr1-induced DA cells were obtained by subtracting the respective untransduced control values from those of Nurr1-transduced cultures. Graphs (B, C) represent DA release and uptake per unit of Nurr1-induced TH  cells, calculated by dividing the specific values by TH  cell numbers in the respective dishes cultured in parallel. Shown in (A) is a representative result of high-performance liquid chromatography for DA level in the medium of untransduced control SVZ (blue) and Nurr1-transduced SVZ (red) cultures. Graph (B) shows DA levels in the medium (basal release; sky blue) and medium containing 56 mM KCl (evoked release; dark blue). *, Significantly different from the respective values of E14 cortical cultures at p

Neurogenic Factors Enhance DA Neuron Yields from Nurr1-Expressed Adult NPs9 n3 \& W, ^+ J4 y" [! f

Although most Nurr1-TH  cells expressed neuronal markers in adult NP cultures, as described above, none of the TH  cells were positive for glial markers GFAP and CNPase (data not shown). These findings imply that Nurr1-induced TH expression occurs only in neuronal progeny and that DA neuronal yield in the Nurr1-transduced cultures could be further enhanced by neurogenic factor treatments. Treatment with neurogenic cytokines BDNF and NT-3 resulted in two- to threefold increases in TuJ1  neurons (Fig. 6A, 6D, 6J). Yields of TH  cells were also strikingly enhanced in the cultures treated with these cytokines (Fig. 6B, 6E, 6J); 5.9% ¡À 2.0% (untreated), 19.8% ¡À 4.7% (BDNF), and 15.7% ¡À 5.4% (NT3) of total cells were TH . Mash1 is a proneural basic helix-loop-helix transcriptional factor that plays an essential role during neuronal specification of precursor cells (reviewed in ). Forced expression of Mash1 robustly enhanced TuJ1  neuronal yields from SVZ- and WM-NP cells (Fig. 6G, 6J). Co-expression of Mash1 in Nurr1-transduced cultures by co-infection using the mixture of the viruses expressing Nurr1 (Nurr1-IRES-LacZ) and Mash1 (Mash1-IRES-LacZ) individually resulted in an increased yield of TH  cells (5.8% ¡À 2.2% in the SVZ¨C cultures transduced with Nurr1 alone vs. 21.3% ¡À 7.2% in the cultures transduced with Nurr1 Mash1). The percentage of TH  cells out of total cells was further enhanced by co-expression of Nurr1 and Mash1 in the same cells using retroviral transduction with bicistronic Nurr1-IRES-Mash1 vector (37.4% ¡À 12%; Fig. 6B, 6H, 6J). These findings, taken together, suggest that Nurr1-induced DA differentiation is correlated with neurogenesis of adult NP cells and specifically occurs in the cells of neuronal lineages. These results also indicate that neurogenic factor treatment and expression might be used for efficient derivation of DA neurons from adult NP cells.
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Figure 6. Neurogenic factors enhance TH  neuronal yields from Nurr1-subventricular zone (SVZ)- and white matter (WM)-derived neural precursor cells. Cultured SVZ and WM precursor cells were transduced with Nurr1 or Nurr1 Mash1 (Nurr1-IRES-Mash1) and differentiated in the absence or presence of neurogenic cytokines BDNF and NT3. The number of TuJ1  and TH  cells were determined 7 days after differentiation. Shown in (A¨CI) are representative images for TuJ1  (left column), TH  (middle), and merged TuJ1 /TH  (right) immunocytochemistry on the cells transduced with Nurr1 (upper row), Nurr1 Mash1 (bottom row), and Nurr1 BDNF treatment (middle row). Scale bar = 40 µm. The graph in (J) represents the percentage of TuJ1  and TH  cells out of total (4',6 diamidino-2-phenylindole  ) cells. Note that co-expression of proneural basic helix-loop-helix Mash1 and BDNF/NT3 treatments greatly enhanced Nurr1-induced DA neuronal yields in the cultures of adult neural precursor cells. *, Significantly different from the respective Nurr1-transduced control values at p
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In Vivo Survival, Differentiation, and Function of Nurr1-Transduced SVZ NPs
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In vivo analyses were carried out in the hemi-parkinsonian rats grafted with SVZ-NPs transduced with Nurr1 and Nurr1 Mash1. In histological assessment at 6 weeks after transplantation, the numbers of TH  cells were 412 ¡À 122 (n = 4) and 2,704 ¡À 368 (n = 8) in the animals grafted with Nurr1 and Nurr1 Mash1, respectively. Subpopulations of TH  cells within the grafts exhibited morphologically mature neuronal shape with extensive neurite arborization, and neuronal markers MAP2 and VMAT2 were localized in the TH  cell populations (Fig. 7C, 7D). Behavioral assessment in the PD rats grafted was performed using an amphetamine-induced rotation test for 6 weeks after transplantation (Fig. 6G). None of the sham-operated rats (PBS injected; n = 8) exhibited a reduction in amphetamine-induced rotation 6 weeks post-transplantation. By contrast, two out of six Nurr1-grafted rats and 9 out of 14 Nurr1 Mash1-grafted rats exhibited >40% reduction in rotation scores. The average rotation scores 6 weeks after transplantation, compared with the pretransplantation values, were 914 ¡À 129 in the sham controls (n = 8) versus 734 ¡À 259 in the Nurr1-grafted animals (n = 6; p 9 }/ N( u* H  \  L4 k
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Figure 7. In vivo transplantation study. (A, B): Representative confocal images of TH  cells in the striatal grafts of PD rats transplanted with SVZ-neural precursors (NPs) transduced with Nurr1 (A) and Nurr1 Mash1 (B) at 6 weeks after transplantation. (C, D): Co-expression of TH with mature neuronal (MAP2 ). (E): Quantification of viable TH  cell numbers in the grafts generated by Nurr1- and Nurr1 Mash1-transduced SVZ cells at 6 weeks after transplantation. *, Significantly different from Nurr1-grafted at p

DISCUSSION
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In this study, we have demonstrated that NP cells derived from adult rat SVZ and WM can be expanded in vitro and efficiently differentiated toward functional DA neurons, which could potentially be used in basic and translational studies for PD. An adult precursor-based approach has the critical merit of circumventing ethical concerns over using embryos or fetuses as a source of precursor cells. Furthermore, an autologous transplantation strategy using adult brain-derived NP cells might work by isolating and expanding adult NP cells in vitro and then reimplanting them into the damaged site of the patient brain. In an autologous grafting approach, the SVZ and WM would be the most likely regions from which NP could be isolated without significant damage to brain function, as these regions are less specific in their physiological functions relative to other NP-containing regions, such as the hippocampus and midbrain substantia nigra, which play critical roles in memory and motor control, respectively., S9 J: a& ^; ]- M

Nunes et al. .' G0 X3 F7 A& J- T! y
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Despite differences in sphere formation rate and proliferation capacity of SVZ and WM NP cells in vitro, the phenotypic properties and general differentiation potentials of these cells were indistinguishable. Over 90% of cells in the neurospheres derived from both brain regions expressed the NP-specific marker nestin. Interestingly, the astroglial marker GFAP was colocalized in approximately 48%¨C52% of nestin  cells, and these cells were localized in the center portion of the spheres. Considering the nature of astroglial neural stem cells in the adult SVZ and hippocampal dentate gyrus  or C-cell-corresponding cells in the WM. However, it is also possible that the nestin /GFAP  cells are differentiated progenitor cells with a differentiation phenotype of astrocytic progeny. This concept assumes that cell differentiation could occur preferentially in the center of spheres, where mitogenic signals from the media might not extend through the multilayer cell barrier of the spheres.

Consistent with preferred in vitro astroglial differentiation potentials of adult brain-derived neurospheres  that most WM NP cells derived from purified oligodendrocyte progenitors retained differentiation toward oligodendrocytic progeny. Further research is needed to clarify which experimental parameters are responsible for the difference in differentiation potentials between the two approaches.
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Efficient DA differentiation of cultured NP cells has been demonstrated to be confined to cells derived from ventral mesencephalon early in embryonic development ). Furthermore DA cells differentiated from the Nurr1-transduced SVZ and WM NPs were not only morphologically, ultrastructurally, and phenotypically mature but also demonstrated robust presynaptic functions of DA release and DAT-mediated specific DA uptake in vitro. These findings collectively suggest the possible large-scale generation of functional DA neurons from Nurr1-engineered adult brain-derived precursor cells.9 B' O3 S+ b+ {

The Nurr1 effect in adult NP cell cultures is in contrast to that of embryonic brain-derived NP cells demonstrating DA phenotype expression in Nurr1-transduced embryonic NP cells without neuronal differentiation and acquisition of presynaptic DA neuron functionality (Figs. 3, 5) , was rather decreased in the SVZ and WM cultures compared with that of E14 NP cells. Additional studies are required for an understanding of the molecular basis for the difference of the Nurr1 effects between adult and embryonic NP cells.4 D4 K) x! X, l. l  `: O7 q, t, v
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We further demonstrated that SVZ NPs transduced with Nurr1 or Nurr1 Mash1 survived, differentiated into TH  DA neurons in the striatum of PD animals for 6 weeks post-transplantation, and functioned to restore the behavioral deficit of PD rats. However, the numbers of viable TH  cells were only 412 and 2,704 cells out of 1 x 106 cells injected in the animals grafted with Nurr1 and Nurr1 Mash1, respectively: survival rates, calculated by dividing the TH  cell numbers in vivo by the numbers of TH  cells counted after 7 days of in vitro differentiation in parallel cultures, were 0.8% ¡À 0.2% in Nurr1 and 1.1% ¡À 0.4% in Nurr1 Mash1 grafts, indicating insufficient in vivo survival or differentiation of these cells. In addition, TH  cells in the grafts demonstrated a diversity of morphologic differentiation: subpopulations of TH  cells in the grafts exhibited immature neuronal morphologies with short or no processes, whereas certain populations of TH  cells had fully differentiated neuronal shapes and phenotypes with extensive and elongated fibers and co-expression of mature neuronal or DA neuronal markers (Fig. 7A¨C7D). This is in contrast to the in vitro cultures, where the majority of TH  cells derived from Nurr1-transuced adult NPs were morphologically, phenotypically, and functionally mature DA neurons (Figs. 3¨C5). Consistent with our observations in vivo, poor survival and limited neuronal differentiation of adult SVZ cells have previously been reported after ectopic grafting into the adult brain . The poor survival and neuronal differentiation might be attributable to limited senescence of adult NP cells per se, incompatible donor-host interactions, or inadequate cell manipulation during cell dissociation, retroviral transduction, and cell transplantation. Further investigation into ensuring better in vivo survival and differentiation is needed.

In conclusion, our study demonstrates the first example of an efficient derivation of functional DA neurons from adult brain-derived precursor cells using genetic manipulation of dopaminergic and neurogenic genes. The use of genetically manipulated adult NP cells could provide an interesting alternative over the use of fetal midbrains as a DA neuron source and opens the possibility of autologous cell transplantation for PD patients. However, it remains to be determined whether our approach is applicable to human cells and whether the risk of introducing transgenes is manageable in a therapeutic setting.0 B* H0 o5 z2 z4 z# G* i

DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST
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The authors indicate no potential conflicts of interest.7 H3 ^; ~1 c9 V/ Y- {# g
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ACKNOWLEDGMENTS

This work was supported by SC2150 (Stem Cell Research Center of the 21st Century Frontier Research Program) funded by the Ministry of Science and Technology, Korea.
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