| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
EMBRYONIC STEM CELLS |
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
Key Words. Embryonic stem cells • Primate • Dopaminergic neuron • Differentiation • Sertoli cells • Coculture
Correspondence: Fengming Yue, Ph.D., Department of Anatomy and Organ Technology, Institute of Organ Transplants, Reconstructive Medicine and Tissue Engineering, Shinshu University Graduate School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Nagano, Japan Telephone: 81-263-372590; Fax: 81-263-373093; e-mail: yuefm{at}sch.md.shinshu-u.ac.jp
Received August 24, 2005;
accepted for publication April 5, 2006.
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
|---|
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
|---|
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 [5]. These unique properties of ES cells make them an excellent candidate as a source of functional differentiated cells for cell replacement therapies, provided that reliable means of inducing differentiation to specific cell types can be achieved. The generation of functional dopaminergic neurons from embryonic stem cells offers a promising cell replacement therapy for the treatment of Parkinson’s disease [6–8].
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 [7]. A second method requires only coculturing ES cells with a stromal cell line, stromal cell derived from skull bone marrow (PA6) [9]. This method has the advantage of simplicity and speed and can be used with cells from subhuman primates [10]. A third method, developed by Zeng et al. [11] and Park et al. [12] reported that the PA6 cell line robustly induced dopaminergic differentiation of human ES cells. However, unlike the observations of Zeng et al. [11] and Park et al. [12], BG01 human ES cell line did not generate significant numbers of tyrosine hydroxylase-positive (TH+) cells when cultured on PA6 stromal cells alone. Instead, the presence of additional factors, which included either the addition of glial cell line-derived neurotrophic factor (GDNF) or striatal astrocytes to the cocultures, was required not only for significant induction of TH immunoreactivity but for overall colony survival as well [13]. Perrier et al. [14] 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.
GDNF promotes the survival of the embryonic dopaminergic neurons of the midbrain, that is, exactly those neurons that degenerate in Parkinson’s disease [15]. Soon after that, GDNF also proved to be a very potent trophic factor for cranial and spinal cord motor neurons, brainstem noradrenergic neurons, basal forebrain cholinergic neurons, Purkinje cells, and certain groups of dorsal ganglion and sympathetic neurons [16, 17]. Thus, hopes were raised that this growth factor could be effective as a therapeutic agent in the treatment of several neurodegenerative diseases. Recently, mouse neurospheres were modified to produce GDNF [18]. These neurospheres promoted dopaminergic neuronal differentiation and increased the survival of transplanted dopamine neurons in 6-hydroxydopamine-lesioned animals. GDNF also enhanced differentiation of dopaminergic neurons by human embryonic stem cells that were cocultured with PA6 cells [11]. Buytaert-Hoefen et al. [13] 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 [19]. These cells can be transplanted into the brain to enhance regeneration and promote the survival of cografted tissues [20]. 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.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
|---|
Sertoli Cell Culture and Conditioned Medium
The Sertoli cells were isolated from 16–19-day-old Sprague-Dawley male rats as previously described [21]. The tunica albuginea was removed, and the testes were cut into pieces in Hanks’ balanced salt solution (HBSS) containing 0.25% trypsin. After 10 minutes, 5 mg/ml DNase I (Sigma) was added. The tissue pieces were incubated in a water bath and shaken at 60 cycles per minute at 37°C for 30 minutes to remove interstitial cells. After two washes in HBSS, tissue pieces were treated with HBSS containing 0.1% collagenase I and shaken at 60 cycles per minute at 37°C for 30 minutes to remove peritubular basement membrane. Cell aggregates were collected and washed in HBSS two times. The cells were resuspended at 1 x 107 cells per ml and cultured in DMEM:Ham’s F-12 medium (Gibco) with 0.1% insulin-transferrin-selenium (Sigma) at 37°C in 5% CO2-95% air for 48 hours. The Sertoli cell cultures were then washed twice and incubated for another 48 hours. The resulting pretreated Sertoli-enriched monocultures contained more than 95% Sertoli cells, as described previously [22]. The cells were gently trypsinized and centrifuged twice at 800 rpm for 2–3 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 –20°C after centrifugation at 5,000g for 15 minutes.
Induction of Neural Differentiation of Primate ES Cells and EBs
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 [23]. Therefore, after trypsinization for 5 minutes at 37°C, partially dissociated ES cell clumps of 10–50 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.
|
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°C, washed with 0.01 M PBS, and incubated with primary antibodies at 4°C 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 ± standard deviation obtained from at least three independent experiments.
RNA Analysis
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°C for 1 minute, 55°C for 1 minute, 72°C for 1 minute; 30 cycles. The PCR cycle was preceded by an initial denaturation of 3 minutes at 94°C and followed by a final extension of 10 minutes at 72°C.
|
Dopamine Assay
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 –80°C until analyzed for dopamine by high-performance liquid chromatography (HPLC) with fluorescence detection as described previously [22]. Results were validated by elution with authentic substances.
Transfer Filter Assay
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°C, 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).
GDNF Antibody Blocking Experiments and Extraneous GDNF Treatment
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°C, 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.
Transplantation Experiments
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 [24], 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°C. 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).
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
|---|
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 [25] efficiently induced neural differentiation when cocultured with ES cells (data not shown). This is consistent with the previous report showing that skull bone marrow PA6 cells induce midbrain dopaminergic neurons from mouse and primate ES cells [9, 10].
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% ± 3%, n = 60; Fig. 1I). Within the colonies, 41% ± 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% ± 4% (n = 60) (Fig. 1I) and 34% ± 5% (n = 5,900), respectively (Fig. 1J).
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 <2% colonies, n = 50), or mesodermal markers, such as vascular endothelial growth factor receptors (Flk-1) (all <3% colonies, n = 50). This is consistent with a previous report showing that mesodermal markers PDGFR and Flk-1 are induced in ES cells cocultured with OP9 cells but not with other cocultured cells [26]. Thus, Sertoli cells can promote neural differentiation of cocultured ES cells without inducing endodermal and mesodermal differentiation. Consistent with the study by Kawasaki et al. [9], the present study showed that PA6 cells also cannot induce the differentiation of endodermal and mesodermal cells except for the neural differentiation. Primate ES cells plated on mouse embryonic fibroblasts (MEFs) continued to express undifferentiated ES cell marker Oct-4 and were devoid of neural markers. Less than 1% of cells were positive for NCAM and Tuj III.
Induction of Dopaminergic Neurons from Primate ES Cells
After 3 weeks of induction by Sertoli cells, immunocytochemical analyses of the Sertoli-induced neurons revealed that 90% ± 9% of the colonies contained TH+ neurons (n = 50; Fig. 2A). At the cellular level, 60% ± 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% ± 10% (n = 60) (Fig. 2A) TH+ colonies and 31% ± 7% (n = 6,100) TH+ cells among Tuj III+ neurons (Fig. 2B).
|
A definitive marker of neuronal fate is the generation of synapses. ES cell-derived neurons frequently contained aggregates of synaptic vesicles (50–100 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.
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% ± 7%), ChAT-positive neurons (cholinergic; 3% ± 2%), and serotonin (serotonergic; 0.6% ± 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% ± 10%, 10% ± 6%, and 3% ± 1%, respectively) at the cell level. Primate ES cells plated on MEFs were devoid of neural markers.
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).
|
Based on the known ability to promote neuronal differentiation [27, 28], 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% ± 12%; Sertoli alone, 60% ± 7%; PA6+GDNF, 56% ± 11%; PA6 alone, 31% ± 7%; gelatin-coated substrate+GDNF, 8% ± 5%; gelatin-coated substrate alone, 6% ± 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% ± 6% (n = 6,000; Fig. 4C), which was significantly less than that induced by Sertoli cells (p < .05; Fig. 4C, *, gray bar vs. white bar in Sertoli group), but still more than in colonies grown on gelatin alone (p < .05). GDNF antibody inhibited the TH+ neural induction activity of Sertoli cells, as shown by TH staining of ES/Sertoli cell cocultures (Fig. 4D) and ES cells cultured in conditioned medium blocked by GDNF antibody (Fig. 4F, correspondent to Fig. 4C, gray bar with asterisk). As a control, we added GDNF antibody into conditioned medium derived from PA6 cells. No significant change was found in the rate of TH+ cells covering total Tuj+ neurons (28% ± 9%; n = 5,000; Fig. 4C, white bar vs. gray bar in PA6 group), although PA6-conditioned medium could not elicit significant neural induction. The number of Tuj+ colonies was 23% ± 5% (n = 50) in conditioned medium and 20% ± 6% (n = 50) in GDNF antibody-treated conditioned medium. There was no significant difference between the two (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.
|
Implantation of Sertoli-Induced Neurons into the Mouse Brain
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.
|
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
|---|
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 [11], 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.
Possible Roles of Sertoli Cells in the Differentiation of ES Cells into Dopaminergic Neurons
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.
Some factors have been implicated in the regulation of dopaminergic differentiation [29], such as FGF8, Shh, interleukin (IL) 1, IL11, GDNF, and neutralizing antibodies of FGF8 and Shh. Among them, GDNF has the most potent neuroprotective and trophic effects on dopamine neurons in many model systems [15, 30, 31]. However, it is a large protein and has to be delivered directly to the brain rather than given peripherally. When successfully delivered, GDNF supports the survival and outgrowth of dopamine neurons following transplantation [27]. In addition, GDNF added to cell suspensions of embryonic ventral mesencephalic tissue improves the survival of dopamine neurons following grafting into the degenerative striatum [28, 32]. Other studies have shown that intermittent injections of GDNF in the vicinity of intrastriatal nigral cell suspension grafts have similar effects on improving the survival and/or fiber outgrowth of transplanted dopamine neurons [33, 34]. In a recent study [23], neurospheres modified to produce GDNF increased the survival of transplanted dopamine neurons in 6-OHDA-lesioned animals. GDNF is also capable of promoting differentiation of mesencephalic neurospheres towards the neuronal lineage, and more importantly, towards the dopaminergic development indicated by expression of NurrI and Ptx3. Buytaert-Hoefen et al. [13] proved that significant differentiation of dopaminergic neurons were not induced when cultured on PA6 stromal cells alone except for the presence of GDNF or striatal astrocytes. Sertoli cells secrete GDNF and promote the survival of transplanted dopaminergic neurons. In the present study, we showed that GDNF plays a role in dopaminergic neuron differentiation when primate ES cells were cocultured with Sertoli cells. In our study, extraneous GDNF induced the differentiation of dopaminergic neurons. Sertoli-induced neural differentiation of ES cells was partly suppressed by a low-dose of anti-GDNF antibody. However, blocking GDNF did not completely inhibit the neural differentiation. We cannot be certain that GDNF activity was completely blocked, although it was not found by Western blot. Therefore, the diminished neuronal differentiation that occurred could have been in response to the remaining, unblocked GDNF. Alternatively, if all of GDNF was blocked, then one or more other factors were present in the conditioned medium and promoted differentiation at a reduced rate. If these factors exist, they are more effective in the presence of GDNF. This confirmed the conclusion of Buytaert-Hoefen et al. [13] 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 [20]. 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–E11.5 [35]. In Macaca, TH+ neurons first appear approximately 5 weeks after implantation [36]. In contrast, in vitro induction by Sertoli cells of TH+ neurons from primate ES cells took only 7–9 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.
Application of Dopaminergic Neurons Derived from Primate ES Cells
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. [37], posture and motility were the symptoms showing the most marked improvement. These results are comparable with clinical report [38] demonstrating improvement in rigidity and hypokinesia. A recovery in rotation behavior was showed in animals grafted with Nurr 1 ES cells. In addition, a significant improvement in the adjusting step, cylinder, and paw-reaching tests were observed [6]. 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.
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.
|
DISCLOSURES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
|---|
|
ACKNOWLEDGMENTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
|---|
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  Z. He, J. Jiang, M.-C. Hofmann, and M. Dym Gfra1 Silencing in Mouse Spermatogonial Stem Cells Results in Their Differentiation Via the Inactivation of RET Tyrosine Kinase Biol Reprod, October 1, 2007; 77(4): 723 - 733. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||
