Induction of Midbrain Dopaminergic Neurons from Primate Embryonic Stem Cells by - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:04 |只看该作者 |倒序浏览 |打印

作者：Fengming Yue, Li Cui, Kohei Johkura, Naoko Ogiwara, Katsunori Sasaki作者单位：Department of Anatomy and Organ Technology, Institute of Organ Transplants, Reconstructive Medicine and Tissue Engineering, Shinshu University Graduate School of Medicine, Asahi, Matsumoto, Nagano, Japan

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      【摘要】) R: U5 s6 T1 E! T- y8 z6 T0 j
   The aim of this study was to produce dopaminergic neurons from primate embryonic stem (ES) cells following coculture with mouse Sertoli cells. After 3 weeks of induction, immunostaining revealed that 90% ¡À 9% of the colonies contained tyrosine hydroxylase-positive (TH ) neurons, and 60% ¡À 7% of the tubulin &szlig; III-positive (Tuj III ) neurons were TH . Reverse transcription-polymerase chain reaction analyses showed that Sertoli-induced neurons expressed midbrain dopaminergic neuron markers, including TH, dopamine transporter, aromatic amino acid decarboxylase (AADC), receptors such as TrkB and TrkC, and transcription factors NurrI and Lmx1b. Neurons that had been differentiated on Sertoli cells were positive for Pax2, En1, and AADC, midbrain-related markers, and negative for dopamine-&szlig;-hydroxylase, a marker of noradrenergic neurons. These Sertoli cell-induced dopaminergic cells can release dopamine when depolarized by high K . Sertoli cell-conditioned medium contained glial cell line-derived neurotrophic factor (GDNF) and supported neuronal differentiation. After pretreatment with anti-GDNF antibody, the percentage of Tuj III  colonies was reduced to 14%. Thus, GDNF contributed significantly to inducing primate ES cells into dopaminergic neurons. When transplanted into a 6-hydroxydopamine-treated Parkinson¡¯s disease model, primate-derived dopaminergic neurons integrated into the mouse striatum. Two weeks after transplantation, surviving TH  cells were present. These TH  cells survived for 2 months. Therefore, the induction method of coculture ES cells with Sertoli cells provides an unlimited source of primate cells for the study of pathogenesis and transplantation in Parkinson¡¯s disease.
      【关键词】 Embryonic stem cells Primate Dopaminergic neuron Differentiation Sertoli cells Coculture
   INTRODUCTION

Neurodegenerative diseases present severe problems due to the limited repair capability of the nervous system .6 u6 }: D9 X* V3 P0 F. Y

Embryonic stem (ES) cells, derived from the inner cell mass of preimplantation embryos, can proliferate indefinitely in culture and are able to differentiate into cell types of all three germ layers in vivo and in vitro .

Dopaminergic neurons have been efficiently generated from ES cells by several different methods. One is a multiple-step method involving in embryoid body (EB) formation followed by sonic hedgehog (Shh) and fibroblast growth factor 8 (FGF8) treatment and selection for nestin  cells  demonstrated that coculture of human ES cell line with the stromal cell lines MS5 or S2 generated large "rosettes" of cells of neuroectodermal lineage. Extended propagation of dissociated cells in FGF8, Shh, and related differentiation factors resulted in significant numbers of dopaminergic neurons. Simple and straightforward means of obtaining mature dopaminergic neurons from ES cells would be extremely valuable for both clinical application and for in vitro studies.( H  _4 Z& {2 h* M9 l! ?
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GDNF promotes the survival of the embryonic dopaminergic neurons of the midbrain, that is, exactly those neurons that degenerate in Parkinson¡¯s disease  also demonstrated that the presence of additional factors, such as GDNF, is required for induction of dopaminergic neurons derived from human ES cells.

Testicular Sertoli cells secreted GDNF normally . The transplantation effectiveness of the Sertoli cells as cografts may be because they confer local immunoprotection for cotransplanted cells, as well as secreting GDNF and possibly other neurotrophic factors. These characteristics of Sertoli cells could provide a means of overcoming the obstacles associated with cell transplantation.

In the present study, we investigated whether dopaminergic neurons could be induced from primate ES cells by coculture with Sertoli cells. In addition, the effect of GDNF secreted by the Sertoli cells on the differentiation potential of dopaminergic neurons was assessed. We also discussed the possibilities for therapeutic application of this method, by which dopaminergic neurons are efficiently produced.
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MATERIALS AND METHODS. k' ]. |9 x5 A5 w1 Q

Maintenance of Primate ES Cells and Formation of Primate Embryoid Bodies: @$ G2 S" V$ i' a

Undifferentiated cynomolgus primate ES cells (Stemtech; Asahi Technoglass, Chiba, Japan) were maintained on a feeder layer of mitomycin C (Mutamycin; Bristol-Myers Squibb; Princeton, NJ)-treated mouse embryonic fibroblasts in knockout Dulbecco¡¯s modified Eagle¡¯s medium (DMEM) supplemented with 20% knockout serum replacement, 2 mM L-glutamine, 0.1 mM nonessential amino acids (Gibco, Grand Island, NY, http://www.invitrogen.com), and 0.1 mM 2-mercaptoethanol (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com). For subculturing, cells were released by 0.25% trypsin in Dulbecco¡¯s phosphate-buffered saline (DPBS; Gibco) with 20% knockout serum replacement and 1 mM CaCl2 as previously described.

Sertoli Cell Culture and Conditioned Medium
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The Sertoli cells were isolated from 16¨C19-day-old Sprague-Dawley male rats as previously described . The cells were gently trypsinized and centrifuged twice at 800 rpm for 2¨C3 minutes in DMEM:Ham¡¯s F-12 medium, and viability was assessed using trypan blue dye exclusion. The cell concentration was adjusted to approximately 80% confluence and cocultured with primate ES cells.

To prepare Sertoli cell-conditioned medium, Sertoli cells were cultured alone until they reached confluence. After another 3 days without changing the medium, the conditioned medium was collected and retained in the incubator for 3 more days. The supernatant was stored at ¨C20¡ãC after centrifugation at 5,000g for 15 minutes.

Induction of Neural Differentiation of Primate ES Cells and EBs
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Sertoli cells were plated on gelatin-coated dishes and used as feeder cells. As a control, PA6 cells were also plated as feeder cells. To strictly avoid contamination by incidentally differentiating cells, we manually selected undifferentiated ES cell colonies with stem cell-like morphology, characterized by tightly packed cells with a high nucleus/cytoplasm ratio (Fig. 1A). Undifferentiated ES cell colonies were washed twice with 0.1 M phosphate-buffered saline (PBS). Unlike mouse ES cells, primate ES cells do not form colonies from single cells on feeder layers. It is essential to remove serum from the media since the addition of fetal calf serum strongly inhibits neural differentiation . Therefore, after trypsinization for 5 minutes at 37¡ãC, partially dissociated ES cell clumps of 10¨C50 cells per clump were plated on Sertoli cells at a density of 15 clumps per well in 12-well plate. Other clumps were similarly plated on PA6 cells. Cultures were maintained in the ES medium for 3 weeks or other indicated periods. The culture medium was changed every second day.

Figure 1. Sertoli-induced neural differentiation of monkey embryonic stem (ES) cells. (A): Undifferentiated monkey ES cells grown in mouse embryonic fibroblasts were tightly packed in the colony and had a high nucleus-to-cytoplasm ratio. Undifferentiated cells typically expressed alkaline phosphatase activity (B) and SSEA-4 (C). (D¨CF): Characterization of monkey ES colony induced by Sertoli cells. Expression of NCAM (D), Tuj III (E), and NeuN (F) confirmed the neural identity of cells in ES colony. (G): The presynapse-specific marker Syn (red) was present on the induced neurons (Tuj III, green). (H): Few colonies expressed glial marker GFAP. (I): After 3 weeks, approximately 97% of the ES cell colonies cultured with Sertoli cells were Tuj III , whereas 95% of those cultured on PA6 stromal cells and only 6% of those cultured on gelatin were Tuj III . (J): Within the colonies, 41% ¡À 8% of the Sertoli-treated ES cells (n = 6,600) and 34% ¡À 5% of the PA6-treated ES cells (n = 5,900) expressed Tuj III. Scale bars (bars represent different scales) = 50 µm. Abbreviations: ALP, alkaline phosphatase; GFAP, glial fibrillary acidic protein; NCAM, neural cell adhesion molecule; NeuN, neuron-specific nuclear protein; PA6, stromal cell derived from skull bone marrow; SSEA-4, stage-specific embryonic antigen-4; Syn, synaptophysin; Tuj III, tubulin ß III.

Histochemistry and Immunostaining' q7 ?- z- e2 N$ P# j. X

For detecting alkaline phosphatase activity, primate ES cells were cultured on gelatin-coated glass coverslips without Sertoli cells. They were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 1 hour at room temperature and then rinsed three times with 20 mM PBS, pH 7.4. The cells were incubated in a nitroblue tetrazolium/bromochloroindolyl phosphate solution (Bio-Rad, Hercules, CA, http://www.bio-rad.com) for 15 minutes in a dark box.  L3 o& [; T! o! F( A0 J
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The following antibodies were used for immunohistochemistry: monoclonal neural cell adhesion molecule (NCAM; 1:300; Chemicon, Temecula, CA, http://www.chemicon.com); polyclonal TH (1:300; Chemicon); monoclonal neuron-specific nuclear protein (NeuN; 1:100; Chemicon); monoclonal stage-specific embryonic antigen-4 (SSEA-4; 1:200; Chemicon); monoclonal tubulin ß III (Tuj III; 1:300; Covance, Princeton, NJ, http://www.covance.com); polyclonal Synaptophysin (prediluted; Biomeda, Foster City, CA, http://www.biomeda.com); monoclonal glial fibrillary acidic protein (GFAP; 1:800; Sigma); polyclonal vesicular transporters of acetylcholine (VAChT; 1:200; Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com); polyclonal glutamate decarboxylase65/67 (GAD65/67; 1:200; Chemicon); monoclonal serotonin (1:100; Chemicon); polyclonal dopamine ß-hydroxylase (DBH; 1:250; Chemicon); polyclonal Pax2 (1:200; Zymed Laboratories, San Francisco, http://www.invitrogen.com); polyclonal En1 (1:200; Chemicon); polyclonal aromatic amino acid decarboxylase (AADC) (1:1,000; Protos Biotech, New York, http://www.protosantibody.com); monoclonal human nucleolar antigen (HNA; 1:50; Acris, Hiddenhausen, Germany, http://www.acris-antibodies.com) and monoclonal GDNF (1:500; R&D Systems Inc., Minneapolis, http://www.rndsystems.com). The specificity of these antibodies was tested by using appropriate tissues or cells as positive controls. Cells were fixed in 4% paraformaldehyde for 20 minutes at 4¡ãC, washed with 0.01 M PBS, and incubated with primary antibodies at 4¡ãC overnight. Localization of antigens was visualized with anti-rabbit or anti-mouse IgG secondary antibodies conjugated with fluorescein (Alexa 568 and 488; Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com).

Positive colonies were defined as colonies containing stained cells regardless of the number. To estimate the efficiency of differentiation at the cell level, colonies were randomly selected, and the numbers of positive cells were counted at different stages of differentiation. The total cell number was determined by 4,6-diamidino-2-phenylindole (DAPI) (Molecular Probes) nuclear staining. When appropriate, data are given as mean ¡À standard deviation obtained from at least three independent experiments.

RNA Analysis5 w6 \" ~$ A. w4 H, t
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Total cellular RNA was prepared using the TRIzol reagent (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). For reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, 5 µg of RNA was reverse-transcribed into cDNA with the SuperScript preamplification kit (Invitrogen Life Technologies). The cDNA was then diluted 1:100 and amplified by polymerase chain reaction (PCR) using the primers listed in Table 1. The cycle parameters were as follows: 94¡ãC for 1 minute, 55¡ãC for 1 minute, 72¡ãC for 1 minute; 30 cycles. The PCR cycle was preceded by an initial denaturation of 3 minutes at 94¡ãC and followed by a final extension of 10 minutes at 72¡ãC.

Table 1. Genes and primers, b# Q8 F6 L, ?7 k- {6 ^6 h. V! ]

Immunogold Labeling and Electron Microscopy8 v0 Z# [- x% o* }2 `# h6 u; G
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Sertoli-induced ES cells were fixed in 0.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 1 hour and then washed with 0.1 M lysine buffer. After complete dehydration with increasing concentrations of ethanol, the samples were taken through a graded series of Lowicryl K4M (Polysciences Inc., Warrington, PA, http://www.polysciences.com):100% ethanol mixtures (1:1 and 2:1 for 1 hour, pure overnight, pure for 2 hours). At last, the samples were infiltrated with pure Lowicryl K4M overnight. Polymerization was performed by UV irradiation for 48 hours. Ultrathin sections were cut and then mounted on nickel grids coated with 2% Neoprene (Nisshin EM, Tokyo). Immunostaining was performed as follows. The grids were immersed in a mixture of 0.5% bovine serum albumin and 0.05% Triton X-100 in 50 mM Tris-buffered saline buffer (pH 7.4) for 30 minutes at room temperature, incubated overnight with the primary antibody (Ab) in the above buffer (rabbit anti-TH, 1:50; Chemicon). Ab was visualized with gold-labeled (15-nm gold particles) goat-anti-rabbit IgG (British BioCell, Cardiff, U.K.) for 2 hours, and electron microscopy was performed after contrasting the sections with uranyl acetate (5 minutes) and lead citrate (2 minutes). The result was examined with a JEOL JEM-1200 transmission electron microscope at an accelerating voltage of 80 kV.- m0 C9 s  @. U4 d
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Dopamine Assay
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Primate ES cells cultured on Sertoli cells for 3 weeks were incubated for 15 minutes in HBSS (3 ml per 10-cm dish) containing 56 mM KCl to induce depolarization of the neurons. The medium was then stabilized with 0.4 M perchloric acid and 5 mM EDTA and kept at ¨C80¡ãC until analyzed for dopamine by high-performance liquid chromatography (HPLC) with fluorescence detection as described previously . Results were validated by elution with authentic substances.

Transfer Filter Assay
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To determine whether ES neuronal differentiation was the result of one or more products secreted by the Sertoli cells, ES cells were seeded onto gelatin-coated six-well plates. Filter membrane inserts (0.22-µm filter; Millipore, Billerica, MA, http://www.millipore.com) were laid over the ES cells, and Sertoli cells were then cultured on the filter membrane.

Determination of GDNF Expression in Sertoli Cells and Conditioned Medium by Western Blot Analysis

The presence of GDNF in Sertoli cells and the conditioned medium was analyzed using mouse anti-human GDNF antibody. Sertoli cells were collected and disrupted with lysis buffer composed of 1 ml of PBS, 0.5 µl of 0.5 M EDTA, 10 µl of 25% Triton X-100, and 2.5 µl of 10% sodium dodecyl sulfate (SDS). The cell lysate was centrifuged at 12,000g for 10 minutes at 4¡ãC, and the supernatant was boiled in SDS sample buffer. The conditioned medium derived from Sertoli cells and conditioned medium blocked by anti-GDNF antibody (see below) were concentrated by lyophylization. The supernatant and the conditioned medium were subjected to electrophoresis in a 12% SDS polyacrylamide gel and electrotransferred to a nitrocellulose membrane (Bio-Rad) for Western blotting. To prevent nonspecific binding, the membrane was treated with a blocking solution containing 10% nonfat dry milk powder dissolved in PBS and 0.2% Tween 20 (PBS-t). After three washes with PBS-t, the membrane was probed with mouse monoclonal GDNF antibody (1:500; R&D Systems) in PBS-t overnight. Unbound antibody was removed by washing with PBS-t, and the membrane was subsequently incubated with peroxidase-conjugated rabbit anti-mouse IgG secondary antibody (1:1,000; Bio-Rad). The result was observed by enhanced chemiluminescence Western blotting analysis system (Amersham Biosciences, Piscataway, NJ, http://www.amersham.com).- q4 e" e$ u6 m5 H% R5 l& T

GDNF Antibody Blocking Experiments and Extraneous GDNF Treatment, @3 Z: J* E+ n2 l

Blocking antibodies to GDNF (monoclonal mouse anti-human GDNF-neutralizing antibody; R&D Systems) were reconstituted according to the supplier¡¯s instructions. The appropriate quantities of Sertoli cell-conditioned medium and 3 µg/ml blocking antibodies (final) were incubated together. After 1 hour in the CO2 incubator at 37¡ãC, a suspension of primate ES cell clumps composed of 10 to 50 cells per clump were plated at a density of 150 clumps per well in a six-well plate. The contents of each well were aspirated once and then gently expelled to ensure uniform distribution of ES cell clumps. The conditioned medium neutralized by the monoclonal anti-GDNF antibody was changed every 2 days. The number of induced TH  cells was counted after immunohistochemistry and DAPI staining. On the other hand, to further investigate whether treatment of ES cells with GDNF could influence the differentiation of dopaminergic neurons, primate ES cells were cultured on a gelatin-coated 12-well plate alone or in coculture with Sertoli cells or PA6 cells with or without the addition of GDNF (10 µg/ml, Sigma) in differentiation media for 3 weeks.
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Transplantation Experiments  s- [# w, l; |& Z$ k! Y; w8 N4 B
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All animal experiments were performed in accordance with institutional guidelines. For 6-hydroxydopamine (6-OHDA) treatment, nonfasted 6-week-old male BALB/c nude mice (SLC, Shizuoka, Japan) were anesthetized with 0.04 mg/kg pentobarbital sodium solution and fixed on a stereotactic device (Narishige, Tokyo). The 6-OHDA was dissolved in PBS (8 µg/µl), and 0.5 µl was injected unilaterally with a glass needle into three sites in the striatum. Using the bregma as the reference , the injection coordinates were as follows: (A:  0.5, L:  2.0, V:  3.0), (A:  1.2, L:  2.0, V:  3.0), and (A:  0.9, L:  1.4, V:  3.0). Seven days after 6-OH dopamine injection, cultured cells were implanted into the striatum as described below.

ES cells were cultured on Sertoli cells for 3 weeks and then incubated with mitomycin C for 2 hours to eliminate mitotic cells but not postmitotic neurons. The differentiated colonies were collected by exposure to 0.25% trypsin, 1 mM CaCl2, and 20% knockout serum replacement in DPBS and then suspended in knockout DMEM. Using a blunt-ended 26-gauge Hamilton syringe, 4 x 105 cells were slowly injected into the striatum (A:  0.9, L:  2.0, V:  3.0) over a 3-minute period. As a control, the suspension medium alone was injected. Transplanted recipients were killed either 2 weeks or 2 months after the transplantation, and the midbrains were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 24 hours at 4¡ãC. The tissue was embedded in paraffin, and 5-µm serial sections were cut. After deparaffinization and rehydration, the sections were stained with specific antibodies against TH, and implantation primate ES cells were labeled with HNA. Localization of antigens was visualized with anti-rabbit IgG secondary antibodies conjugated with fluorescein (Alexa 488; Molecular Probes).
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RESULTS3 i  W# W: O, A: m
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Induction of Neural Differentiation in Primate ES Cells on Sertoli Cells0 \$ C6 D6 p. o1 G) X: g
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The primate ES cell line plated on mouse embryonic fibroblasts in serum replacement medium displayed immature cell morphologies (Fig. 1A). The colonies expressed high levels of undifferentiated cell markers such as alkaline phosphatase and SSEA-4 (Fig. 1B, 1C).9 @5 C- \+ r8 n' V

We screened several primary culture cells for inducing neural differentiation of primate ES cells under serum-free conditions. The cell types used in these experiments included mouse embryonic fibroblasts, Cos7 cells, and HEK293, none of which significantly induced neural markers, such as NCAM (pan-neural). In contrast, PA6 cells and bone marrow stromal cells derived from rats .
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After 3 weeks of coculture with Sertoli cells, extensive neurites formed in the majority of primate ES cell colonies that contained a large number of neural precursors and neurons positive for NCAM (Fig. 1D) and postmitotic neurons positive for Tuj III (Tuj III ) (Fig. 1E). The Tuj III  cells also expressed another neuronal marker, NeuN (Fig. 1F), and the presynaptic marker synaptophysin (Fig. 1G). Very few colonies contained GFAP  cells (3%, n = 200) (Fig. 1H). Most colonies expressed Tuj III (97% ¡À 3%, n = 60; Fig. 1I). Within the colonies, 41% ¡À 8% of the Sertoli-treated ES cells (n = 6,600) expressed Tuj III (Fig. 1J). For cells cocultured with PA6 cells, the number of Tuj III  colonies and cells was 95% ¡À 4% (n = 60) (Fig. 1I) and 34% ¡À 5% (n = 5,900), respectively (Fig. 1J)., }+ Z# U% {0 L, ]( H# X

At the same time, we determined whether neural differentiation induced by Sertoli cells was accompanied by endodermal and mesodermal induction. Primate ES cells plated on Sertoli cells formed neuroepithelial structures. At 1 week of differentiation, Oct-4  cells still surrounded around neuroepithelium. After an additional 2 weeks of differentiation, 97% of the ES cells on Sertoli cells expressed neural markers. However, rare clusters of persisting Oct-4  cells could still be detected. In contrast to the high rate of neural differentiation, very few colonies expressed endodermal markers, such as GATA-4 (all , }: p+ T; r/ G

Induction of Dopaminergic Neurons from Primate ES Cells
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After 3 weeks of induction by Sertoli cells, immunocytochemical analyses of the Sertoli-induced neurons revealed that 90% ¡À 9% of the colonies contained TH  neurons (n = 50; Fig. 2A). At the cellular level, 60% ¡À 7% of the Tuj III  neurons were TH  (n = 6,000; Fig. 2B). A time course study showed that TH  neurons appeared between day 7 and day 9 of the induction period, following the appearance of Tuj marker. After that, TH  colonies and cells progressively increased (Fig. 2C). These TH  neurons proved to be dopaminergic, as they were negative for DBH (marker for norepinephrine and epinephrine neurons). In addition, the neurons expressed midbrain-related markers, such as Pax2, En1, and AADC in a developmentally characteristic sequence. Pax2, without En1, first appeared on day 3 and developed progressively thereafter (Fig. 2D). From day 7, cells co-expressed Pax2 and En1 (Fig. 2E). Pax2-expressing cells were negative for TH (Fig. 2F), whereas cells positive for En1/TH were readily detected (Fig. 2G). The data suggest that primate dopaminergic neurons in vitro are derived from proliferating progenitors that sequentially express Pax2, and upon exiting the cell cycle, they become positive for En1 and eventually TH. AADC, an additional midbrain-dopaminergic marker, was also expressed in TH  cells (Fig. 2H). As a control, PA6 induced neurons showed 74% ¡À 10% (n = 60) (Fig. 2A) TH  colonies and 31% ¡À 7% (n = 6,100) TH  cells among Tuj III  neurons (Fig. 2B).

Figure 2. Derivation of TH  Neurons from Sertoli-induced monkey ES cells. (A): After 3 weeks of culture, 90% of the Sertoli cell cocultured colonies were TH , whereas only 2% were positive when cultured on gelatin. Shown on the right is double staining of Tuj III and TH. (B): Similarly, 60% of the Tuj III  cells were TH  when cocultured with Sertoli cells, whereas only 6% were positive when cultured on gelatin. Shown on the right is double staining of Tuj III and TH. (C): Immunostaining of Sertoli-induced neurons with anti-TH antibody at 1, 2, and 3 weeks. (D): Pax2-positve cells (red) at day 3 did not co-express En1 (green). (E): From day 7, cells coexpressed Pax2 (red) and En1 (green). (F): Pax2  (red) immature precursors next to TH  (green) neurons. (G): Coexpression of En1 (red) and TH (green). (H): TH  (green) cells coexpressing AADC (red). (I): Reverse transcriptase-polymerase chain reaction analysis of markers of dopaminergic neurons, receptors, other neurons, and neural precursors using RNA from Sertoli cells (lane 1), undifferentiated primate ES cells (lane 2), and Sertoli-induced cells (lane 3) at 3 weeks. (J): Electron microscopy showed a large number of synaptic vesicles clustered in the proximity of the cell membrane. Numerous boutons filled with vesicles (arrowheads and magnification inset). (K): These structures often made axodentric and axosomatic contacts and displayed pre- and postsynaptic densities. TH ImmunoGold particles (15 nm) were associated with small vesicles presumably containing neurotransmitters located at the presynaptic terminal (arrowheads). (L): A representative high-performance liquid chromatogram showed high levels of DOPAC and DA after 15 minutes of KCL-evoked depolarization. The DA and DOPAC peaks were not detected in control medium conditioned with Sertoli cells only. Immunostaining, scale bars (bars represent different scales) = 50 µm; immnoelectron microscopy, scale bars = 100 nm. Abbreviations: AADC, aromatic amino acid decarboxylase; CHAT, choline acetyltransferase; DA, dopamine; DAT, dopamine transporter; DBH, dopamine ß-hydroxylase; DOPAC, 3,4-dihydroxyphenylacetic acid; En1, engrailed 1; ES, embryonic stem; GAD67, glutamate decarboxylase67; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Nrp-1, neuropilin 1; Nurr1, orphan nuclear receptor; PA6, stromal cell derived from skull bone marrow; Pax2a, paired box gene 2a; TH, tyrosine hydroxylase; Tuj, tubulin ß III; VAChT, vesicular transporters of acetylcholine; w, week.3 @9 z) W- P  j% w" L

The expression of neuronal markers in Sertoli-induced cells was also analyzed by RT-PCR. Several dopaminergic markers, including TH, dopamine transporter (DAT), AADC, and the transcription factors that are known to control dopaminergic differentiation, Nurr1 and lumxb, were all detected. Receptors such as TrkA, TrkB, and TrkC were also expressed in the Sertoli-induced cultures but not in undifferentiated ES cells. Several markers of nondopaminergic neuronal subtypes were also present. VAChT, GAD67, and cholinergic marker choline acetyltransferase (ChAT) were detected in differentiated cells. DBH was not detected (Fig. 2I).
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A definitive marker of neuronal fate is the generation of synapses. ES cell-derived neurons frequently contained aggregates of synaptic vesicles (50¨C100 nm) in the vicinity of the cell membrane (Fig. 2J). Synaptic contacts were seen between dendritic and axonal process, or between an axonal process and a cell body. TH-ImmunoGold-labeled particles were associated with neurotransmitter-containing vesicles and clustered near the cell membrane (Fig. 2K). In addition to ultrastructural evidence of synapse formation, HPLC revealed that Sertoli-induced dopaminergic neurons released dopamine. In response to a depolarizing stimulus of 56 mM K , ES cell-derived neurons released a significant amount of dopamine into the medium (Fig. 2L). These data showed that functional dopaminergic neurons were generated with this method.' @; I# q5 X; D& O/ y5 {

We next examined characteristics of TH-negative neurons induced by Sertoli cells with various markers. In addition to TH  neurons, the Sertoli cell-treated primate ES cells gave rise to GAD-positive neurons (GABAergic; 10% ¡À 7%), ChAT-positive neurons (cholinergic; 3% ¡À 2%), and serotonin (serotonergic; 0.6% ¡À 0.4%) in Tuj  neurons at the cell level (n = 3,000). As a control, TH  cell value in ES/PA6 culture was also significantly higher than percentages of GABAergic, cholinergic, and serotonergic neurons in Tuj  neurons (19% ¡À 10%, 10% ¡À 6%, and 3% ¡À 1%, respectively) at the cell level. Primate ES cells plated on MEFs were devoid of neural markers.3 t/ f/ ?* t1 y
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The Role of Sertoli Cells in the Induction of Dopaminergic Neurons from Primate ES Cells

When cultured on the gelatin-coated dish in the same medium but without Sertoli cells, ES cells differentiated into neurons at a low frequency compared with the rate obtained with ES cells cultured on Sertoli cells (Fig. 3A, lanes 1 and 2). This suggested that Sertoli cells had an active role in the promotion of neural differentiation of ES cells. We tested whether direct physical contact between ES cells and Sertoli cells was essential for the induction. ES cells cultured on gelatin-coated dishes and separated from cocultured Sertoli cells by a 0.22-µm filter membrane were still able to induce significant neural differentiation of ES cells (Fig. 3A, lane 3; Fig. 3B).

Figure 3. The physical and chemical role of Sertoli cells in the induction of dopaminergic neurons. (A): Sertoli cells induced the expression of neuronal marker Tuj III in co-cultured monkey embryonic stem (ES) cells even when separated by a filter membrane. CM induced neural differentiation in ES cells cultured on gelatin-coated dish. However the differentiation rate was lower than with direct coculture with Sertoli cells. (B, C): Tuj III (green) and TH (red) double staining of ES cells when separated from Sertoli cells by a filter (B) and cultured in conditioned medium alone (C). Scale bar = 50 µm. Abbreviations: CM, medium conditioned by Sertoli cells for 3 days; TH, tyrosine hydroxylase; Tuj, tubulin ß III.
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When Sertoli-conditioned medium was used to culture ES cells without Sertoli cells as a feeder layer, neural differentiation was still induced compared with ES cells cultured alone on gelatin-coated dishes (Fig. 3A, lane 4; Fig. 3C). Together these data suggest that Sertoli cells secrete one or more soluble neural-inducing factors.% k# x3 Z  t3 }) k: q+ a' g

Based on the known ability to promote neuronal differentiation , GDNF seemed to be a likely candidate for the Sertoli-induced differentiation of primate ES cells. We confirmed the presence of GDNF expression in Sertoli cells by immunohistochemistry (Fig. 4A, 4B, lane 1) and in the conditioned medium by Western blot analysis (Fig. 4B, lane 2). We therefore tested whether GDNF promoted dopaminergic neuron differentiation of primate ES cells. First, we added GDNF to ES/Sertoli cell cocultures (Fig. 4E) and compared results with ES/Sertoli and ES/PA6 alone or with ES cells on a gelatin-coated substrate after 3 weeks in culture. We found that GDNF increased the number of TH  cells in coculture with Sertoli or PA6 cells (Fig. 4C, white bar vs. black bar in PA6 and Sertoli cell group) (TH  cells in Tuj III  neurons: Sertoli GDNF, 76% ¡À 12%; Sertoli alone, 60% ¡À 7%; PA6 GDNF, 56% ¡À 11%; PA6 alone, 31% ¡À 7%; gelatin-coated substrate GDNF, 8% ¡À 5%; gelatin-coated substrate alone, 6% ¡À 3%; n = 6,600). At the same time, we incubated the conditioned medium with GDNF-blocking antibodies and effectively removed GDNF as determined by Western blot (Fig. 4B, lane 3). The number of TH  cells supported by anti-GDNF-treated conditioned medium was reduced to 35% ¡À 6% (n = 6,000; Fig. 4C), which was significantly less than that induced by Sertoli cells (p  .05). This implies that GDNF is a component of the trophic activity of Sertoli cells and plays a significant role in the induction of neurons.7 K9 e+ J$ o! Q& Z* @! [% G5 y$ \2 H* Z

Figure 4. The role of GDNF in Sertoli-induced dopaminergic neuron differentiation. (A): Immunostaining of GDNF in Sertoli cells. (B): GDNF protein was detected by Western blot of lysed Sertoli cells (lane 1) and in conditioned medium (lane 2). However, it was not detected in conditioned medium blocked by anti-GDNF antibody (lane 3). (C): In embryonic stem (ES) cells cocultured with conditioned medium derived from Sertoli cells, the TH  cell percentage was reduced significantly when GDNF was blocked by antibody, whereas there were no significant changes in ES cells cultured on PA6 stromal cells and gelatin-coated dishes. (D¨CF): TH staining of ES cells cultured on Sertoli cells (D), on Sertoli cells treated with extraneous GDNF (E), and on blocked conditioned medium (F). Scale bars = 50 µm. Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; GDNF, glial cell line-derived neurotrophic factor; PA6, stromal cell derived from skull bone marrow; TH, tyrosine hydroxylase; Tuj, tubulin ß III.

Temporal Regulation of Primate Neural Differentiation

The time required for fetal development varies significantly among different mammalian species. We therefore examined temporal regulation of neuronal markers during Sertoli-induced neural differentiation in primate ES cells. The neural precursor marker Nestin appeared on day 2, another marker Nrp-1/Musashi1 was detected on day 4, and so forth in primate cells by RT-PCR analyses (Fig. 2F). Next, temporal expression profiles of NCAM (pan-neural) and Tuj III (neuronal) were analyzed by immunostaining. They developed after 1 and 2 days, respectively, in the primate cells. TH  neurons first appeared between day 7 and day 9 of the induction period, following the appearance of Tuj marker, whereas the onset of TH  cell generation in the ES colonies that cocultured with PA6 was on day 10.
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Implantation of Sertoli-Induced Neurons into the Mouse Brain8 I1 c& X! t4 V! ~9 E3 s

The data above indicate that the in vitro Sertoli-induced neural differentiation system may be a good source of mesencephalic dopaminergic neurons that could be used in cell transplantation therapy of Parkinson¡¯s disease. Therefore, we tested whether Sertoli-induced ES cells could be integrated into the mouse striatum after implantation. Immunohistochemical localization revealed presence of TH in the striatum of the intact side, whereas TH immunoreactivity was not found in the 6-OHDA-injected side (Fig. 5A). Implantation of Sertoli-induced neurons restored TH  areas in the graft 2 weeks after implantation (Fig. 5B). For at least 2 months, the implanted TH  neurons survived (Fig. 5C). Neural cell bodies and extended neurites in the tissue (Fig. 5D) were clearly observed.

Figure 5. Integration of Sertoli-induced dopaminergic neurons in the nude mouse striatum. (A): TH immunohistochemical localization revealed that injection of 6-hydroxydopamine depleted TH immunoreactivity from the injected side of striatum 1 week after drug treatment, and TH  cells remained in the striatum of the intact side. (B, C): Surviving TH  neurons (red) in the striatum 3 weeks (B) and 2 months (C) after grafting. Implantation primate embryonic stem cells were labeled with HNA (green) (D): Arrows and arrowheads indicate cell bodies and neurites of TH  neurons, respectively. Scale bars (bars represent different scales) = 50 µm. Abbreviations: HNA, human nucleolar antigen; TH, tyrosine hydroxylase.; I$ R& v& N. e2 L9 j( j  }9 W
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DISCUSSION

Dopaminergic Neuron Differentiation Induced by Sertoli Cells  o: ^3 n. K0 o; \/ |. y- N. Z1 l+ C
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Recently, dopaminergic neurons were generated from neural precursor cells amplified from EBs. One group . In this study, we developed a new method to induce dopaminergic neuron differentiation from ES cells by coculture with Sertoli cells. The protocol is technically simple, rapid, and efficient. In contrast to the previous protocols, this method does not require growth serum, the formation of EBs, selection of neural precursor cells, retinoic acid, growth factor, or other special treatment. Thus, our method is more suited to detailed analyses of differentiation and transplantation therapy.
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The differentiation of midbrain dopamine neurons from primate ES cells is highly efficient, with up to 60% of all neurons expressing TH, the rate-limiting enzyme in the synthesis of dopamine. Except for TH expression, the markers associated with the mature dopaminergic neuronal phenotype (TH, DAT, and AADC) and transcription factors Nurr1 and Lmx1b were expressed. The growth factor receptors TrkA, TrkB, and TrkC were also present. The reason why Nurr1 was expressed in undifferentiated primate ES cells is unclear. However, it has been reported that undifferentiated BG01 human ES cells expressed Nurr1 , and we cannot exclude the possibility that a small number of differentiated cells were present within the human ES cell colonies. Negative expression of DBH and positive expression of TH, Pax2, En1, and AADC by immunostaining and RT-PCR supported the conclusion that the differentiated neurons induced by Sertoli cells are dopaminergic rather than noradrenergic. In addition to dopaminergic markers, cholinergic (ChAT and VAChT), GABAergic (GAD), and serotonergic (serotonin) markers were detected in the induced neurons, indicating the potential for generation of multiple neuronal types by this method. Besides, the dopaminergic phenotype is confirmed by functional KCl-evoked dopamine release. Thus, this methodology should provide a convenient experimental system for many aspects of neuroscience research, including neural development, neuropharmacology, and electrophysiology.
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Possible Roles of Sertoli Cells in the Differentiation of ES Cells into Dopaminergic Neurons
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Sertoli cells have an active role in the promotion of neural differentiation of ES cells. The mechanism of dopaminergic neuron induction in cocultures of ES cells with Sertoli cells remains to be understood. First, whether direct physical contact between ES cells and Sertoli cells was essential for the induction was tested through filter membrane to separate ES cells with Sertoli cells. The result showed that Sertoli cells were still able to induce significant neural differentiation of ES cells, indicating that Sertoli cells produce soluble inducing factors. However, Sertoli-conditioned medium could not elicit significant induction. It suggested two possibilities as to the molecular nature of neuron-inducing activity by Sertoli cells. One is that Sertoli cells secrete two different neuron-inducing factors, a cell surface-anchored factor and a labile soluble factor. Another might be that the neuron-inducing activity is mediated by secreted factors. At present, we cannot exclude either possibility.
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Some factors have been implicated in the regulation of dopaminergic differentiation  that GDNF is required but not necessary for the induction of dopaminergic neurons.

From these results, we can conclude that Sertoli cells may stimulate dopaminergic differentiation by a complex combination of growth factors or other factors, including other unidentified components. Among these, GDNF plays some role, but not a decisive role. It is also possible that the supporting environment provided by Sertoli cells, or an interaction between Sertoli cells and primate ES cells, plays a role in their neuron-inducing activity.

In addition, isolated Sertoli cells enable survival and function of cografted foreign dopaminergic neurons in rodent models of Parkinson¡¯s disease. They also promote regeneration of damaged striatal dopaminergic circuitry in those same Parkinson¡¯s disease models . In our study, significant TH  cells were found in the degenerative striatum when differentiated primate ES cells were cotransplanted with Sertoli cells. Moreover, 2-month survival of TH  neurons derived from ES cells was observed. It is likely that the nutritive support of the Sertoli cells is responsible for this enhanced TH  cell survival.

Temporal Regulation of Primate Neural Differentiation

The genesis of midbrain TH  neurons occurs during the organogenetic period. TH  neurons first appear in the murine midbrain on E10.5¨CE11.5 . In contrast, in vitro induction by Sertoli cells of TH  neurons from primate ES cells took only 7¨C9 days. It showed that primate TH  neurons differentiate from embryonic cells much faster in vitro than in vivo. Possibly, the speed of cell differentiation is actively reduced in primate embryos to allow sufficient numbers of precursor cells to accumulate for construction of the large primate brain. Besides, the speed of dopaminergic neuron differentiation induced by Sertoli cells is faster than that by PA6. Regardless of the reason, in practice, the Sertoli-induced protocol carries the advantage of requiring less time to produce dopaminergic neurons.* @5 |$ v' G5 z% [5 y2 C

Application of Dopaminergic Neurons Derived from Primate ES Cells6 J- T7 q5 W, F9 B6 O4 n6 K8 r

In the present study, we showed that primate cells can successfully differentiate into neural cells by coculture of ES cells with Sertoli cells. This is a promising first step toward the use of ES cells for replacement therapy in Parkinson¡¯s disease. Besides long-term survival of Sertoli-induced neurons, safety issues, and functional consequences, such as motor recovery, have to be addressed before the clinical utility of Sertoli cells becomes viable. First, inappropriate cells may be included along with the midbrain dopamine neurons. For example, tumor formation is a problem associated with ES cell grafting in models of Parkinson¡¯s disease. It maybe beneficial to select only postmitotic neurons by eliminating dividing cells with mitomycin C before grafting, as we did in this study. However, additional long-term data are needed to show that ES-derived cells do not divide in vivo. Second, positive selection strategies could also be developed to further increase the safety and purity of the dopaminergic neurons pool for transplantation. Sorting by flow cytometry or separating with magnetic beads should be feasible once appropriate surface antigens for early dopaminergic neurons become available. Third, we need to perform function studies after ES cells are transplanted into lesioned striatum. In the study of Takagi et al. . Fourth, we must test whether human ES cells differentiate into dopaminergic neurons in a manner similar to monkey ES cells. Considering the close phylogenetic relationship between humans and monkeys, we expect that the Sertoli-inducing method should be applicable to the generation of dopaminergic neurons from human ES cells also.
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In conclusion, we are still far from an established in vitro or in vivo source of dopamine neurons to combat Parkinson¡¯s disease. However, Sertoli-derived dopaminergic neuron differentiation provides a promising method for therapeutic application and basic neuroscience research.

DISCLOSURES9 w& F  @# t6 O/ J3 W8 k

The authors indicate no potential conflicts of interest.
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ACKNOWLEDGMENTS

We thank Prof. Tetsuji Moniizumi (Second Department of Anatomy, Shinshu University, Matsumoto, Japan) for Stereotactic device and Dr. Huanying Zhao (Bejing Institute for Neurosciences, Capital University of Medical Science, Beijing, China) for technical assistance. We also thank Dr. Kametani Kiyokazo and Ms. Suzuki Kayo (Research Center for Instrumental Analysis of Shinshu University, Matsumoto, Japan) for excellent technical assistance. This work was supported by grant-in-aid for the Twenty-first Center of Exellence program from the Ministry of Education.
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