Stromal CellCDerived Inducing Activity, Nurr1, and Signaling Molecules Synergist - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:10 |只看该作者 |倒序浏览 |打印

作者：Dong-Wook Kima,b,c, Sangmi Chunga,b, Mikyeong Hwanga,b, Andrew Ferreea,d, Hsing-Chen Tsaia,b, Jae-Joon Parka,b,c, Seungsoo Chungc, Taick Sang Namc, Un Jung Kange, Ole Isacsona,d, Kwang-Soo Kima,b作者单位：a Udall Parkinsons Disease Research Center of Excellence;b Molecular Neurobiology Laboratories; McLean Hospital/Harvard Medical School, Belmont, Massachusetts, USA;c Department of Physiology, Yonsei University College of Medicine, Seoul, Korea;d Neuroregeneration Laboratories; McLean Hospital/Harvar 0 t0 O. m: P3 G4 G
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      【摘要】0 d( f! ?; h$ T
   To induce differentiation of embryonic stem cells (ESCs) into specialized cell types for therapeutic purposes, it may be desirable to combine genetic manipulation and appropriate differentiation signals. We studied the induction of dopaminergic (DA) neurons from mouse ESCs by overexpressing the transcription factor Nurr1 and coculturing with PA6 stromal cells. Nurr1-expressing ESCs (N2 and N5) differentiated into a higher number of neurons (~twofold) than the na&iuml;ve ESCs (D3). In addition, N2/N5-derived cells contained a significantly higher proportion (>50%) of tyrosine hydroxylase (TH)  neurons than D3 (<30%) and an even greater proportion of TH  neurons (~90%) when treated with the signaling molecules sonic hedgehog, fibroblast growth factor 8, and ascorbic acid. N2/N5-derived cells express much higher levels of DA markers (e.g., TH, dopamine transporter, aromatic amino acid decarboxylase, and G protein¨Cregulated inwardly rectifying K  channel 2) and produce and release a higher level of dopamine, compared with D3-derived cells. Furthermore, the majority of generated neurons exhibited electrophysiological properties characteristic of midbrain DA neurons. Finally, transplantation experiments showed efficient in vivo integration/generation of TH  neurons after implantation into mouse striatum. Taken together, our results show that the combination of genetic manipulation(s) and in vitro cell differentiation conditions offers a reliable and effective induction of DA neurons from ESCs and may pave the way for future cell transplantation therapy in Parkinson¡¯s disease.
      【关键词】 Embryonic stem cells Stromal cell-derived inducing activity Nurr Dopaminergic neurons Parkinsons disease- R+ V4 l( Z2 h0 n
   INTRODUCTION

Embryonic stem cells (ESCs) are derived from the inner cell mass of mammalian blastocysts , ESCs are useful not only for analyzing critical steps of cell development but also as a potential source for cell replacement therapy.

A target for such an application is Parkinson¡¯s disease (PD) because it primarily involves the degenerative loss of a specific cell type, namely the midbrain dopaminergic (DA) neurons in the substantia nigra .; Y' _2 H1 V6 l6 z9 m

It is possible to successfully facilitate the differentiation of ESCs to certain lineages by genetic manipulation consisting of the specific activation of key fate-determining transcription factors . Whereas all these studies showed the potent ability of Nurr1 to induce the DA phenotype (typically TH expression), the maturity and function of the resulting DA cells were variable, suggesting that additional transcription factors and/or signaling molecules are necessary.
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At present it is not known whether Nurr1 overexpression and PA6 coculture may co-operatively facilitate the differentiation of ESCs into DA neurons. Whereas SDIA is known to accumulate on the surface of PA6 stromal cells, its molecular nature is not known . If SDIA shares a close relationship with the Nurr1 pathway, it may not have a synergistic effect on DA neuron induction using the Nurr1 overexpression and PA6 coculture method. Furthermore, questions remain about the synergistic effect of combining signaling molecules such as Shh and FGF8 with SDIA on the induction of DA neuronal phenotype. Our work centered on two main objectives: (a) to address questions about the combined roles of SDIA, signaling molecules, and Nurr1 on neurogenesis and/or DA neuron differentiation from ESCs; and (b) to establish an effective and reliable protocol for in vitro ESC differentiation to generate DA neurons suitable for transplantation. Toward these goals, we studied the in vitro behavior of Nurr1-overexpressing mESCs in the PA6 coculture method and the effect of signaling molecules on their differentiation. Through systematic analyses, we observed that a very high proportion of midbrain DA neurons (up to 90% of Tuj1  neurons) can be generated from Nurr1-overexpressing mESCs using the optimized PA6 coculture method within a short period of time (~14 days). Furthermore, transplantation studies using the resulting DA neurons demonstrate that they can stably integrate into the host striatum and express the DA phenotype.
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MATERIALS AND METHODS

In Vitro Differentiation of ESCs
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PA6 cells were purchased from Riken (Tsukuba, Japan, http://www.riken.go.jp/engn) and were maintained in PA6 culture medium ¨Cminimum essential medium (Gibco, Rockville, MD, http://www.invitrogen.com) supplemented with 50 U/ml penicillin and 50 µg/ml streptomycin (PEST) (Gibco), and 10% fetal bovine serum (HyClone, Logan, UT, http://www.hyclone.com) . The culture medium was changed on day 4 and every other day thereafter.

Analyses of Dopamine

HPLC analyses of dopamine were performed after 24 hours of conditioning at day 14. The proteins from 0.2 ml of media from each well of a 12-well plate were precipitated by adding perchloric acid (PCA) and EDTA at final concentrations of 0.33 M and 0.17 mM, respectively. After aspiration of the residual media (0.6 ml), the cells were treated with 0.2 ml of 50 mM KCl in embryonic stem differentiation medium II for 30 minutes at 37¡ãC. Then the media were collected, and the proteins were precipitated by the addition of PCA and EDTA as described above. The mixture was centrifuged at 4¡ãC for 10 minutes at 14,000g, and the supernatant was used for HPLC analysis. Cells were allowed a 1-day recovery in fresh medium after which time they were washed, scraped, collected, and vortexed in a chilled (4¡ãC) 0.24 ml solution of 0.33 M PCA and 0.17 mM EDTA. After centrifugation at 14,000g for 10 minutes, intracellular fraction (supernatant) and cell pellet were separated for intracellular DA and protein analysis (data not shown), respectively. Samples were analyzed as described previously , 0.1 mM EDTA, 0.4 mM sodium octyl sulphate, and 9% methanol) was 0.8 ml/minute. The potentials of the guard cell and the first and the second electrodes in the analytical cell were set at 330, 0, and 310 mV, respectively. Dopamine was identified by retention time and quantified based on peak height using the EZChrom Chromatography Data System.
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Immunostaining5 `3 L! j& p* B) d+ Y! p& y
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After 14 days of differentiation on PA6 feeder layer, ESCs were fixed with 4% formaldehyde (Electron Microscopy Sciences, Hatfield, PA, http://www.emsdiasum.com) for 30 minutes, rinsed with phosphate-buffered saline (PBS), and then incubated with blocking buffer (PBS, 10% normal donkey serum  with a slight modification. We skipped fields where we could not find any cells.3 f0 G' A: U3 ^# L
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Semiquantitative Reverse Transcription¨C and Real-Time Polymerase Chain Reaction Analyses

After differentiation, total RNA from ESC-derived neurons was prepared using TriReagent (Sigma) followed by treatment with DNase I (Ambion, Austin, TX, http://www.ambion.com). cDNA was obtained using 5 µg of RNA with the SuperScriptTM first-strand synthesis system for reverse transcription¨Cpolymerase chain reaction (RT-PCR) (Invitrogen). The resulting cDNA was used as a template for the PCR reactions. To reduce the nonspecific signals, primers of each gene were designed using the MacVector software (Accelrys, San Diego, CA, http://www.accelrys.com). We selected primer sets that yielded specific products without the presence of nonspecific bands as evaluated by gel electrophoresis. The following primer sets were chosen for semiquantitative RT- and real-time PCR analyses.
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Actin: 5'-GGCATTGTGATGGACTCCGG-3'; 5'-TGCCACAGGATTCCATACCC-3' (358 bp)

TH: 5'-TTGGCTGACCGCACATTTG-3'; 5'-ACGAGAGGCATAGTTCCTGAGC-3' (336 bp): H9 s# j& A0 G- |- o& J/ {9 ?

AADC: 5'-CCTACTGGCTGCTCGGACTAA-3'; 5'-GCGTACCAGGGACTCAAACTC-3' (715 bp)
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DAT: 5'-CAGAGAGGTGGAGCTCATC-3'; 5'-GGCAGATCTTCCAGACACC-3' (328 bp)
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Pitx3: 5'-CTCTCTGAAGAAGAAGCAGCG-3'; 5'-CCGAGGGCACCATGGAGGCAGC-3' (491 bp)
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Aldehyde dehydrogenase 2 (AHD2): 5'-CATTGAGAGTGGGAAGAAAGAAGG-3'; 5'-AGTGAAGAGTCCTGCTGCTAAACC-3' (232 bp)
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Calbindin: 5'-GCAGTCATCTCTGATCACAGC-3'; 5'-GAGGTCTGTGTACTCTGCTAG-3' (428 bp). p9 C: d" d1 s% T& a

Girk2: 5'-CCTTGTCTCCCATTTCCTTCTTTTC-3'; 5'-CTGTCTGCTTGTCCCATCTGAAC-3' (147 bp)6 S) v* Q! Z0 [# g
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Tryptophan hydroxylase (TPH): 5'-CTACACTCCAGAGCCAGACAC-3'; 5'-GACATCAAGGTCATACCGCAAC-3' (501 bp)% h) S: Z- w' K+ i' p: t( M

Glutamic acid decarboxylase (GAD): 5'-GGGTTTGAGGCACACATTGATAAG-3'; 5'-GCGGAAGAAGTTGACCTTGTCC-3' (279 bp)
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Choline acetyltransferase (ChAT): 5'-GCCAATCCATTCCCACTGAC-3'; 5'-CATCCAAGACAAAGAACTGG-3' (198 bp).
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For semiquantitative RT-PCR, PCR reactions were carried out with 1x IN Reaction Buffer (Epicentre Technologies, Madison, WI, http://www.epicentretechnology.com), 1.4 nM of each primer, and 2.5 units of Taq I DNA polymerase (Promega, Madison, WI, http://www.promega.com). Samples were amplified in an Eppendorf Thermocycler (Brinkmann Instruments, Westbury, NY, http://www.brinkmann.com) under the following conditions: denaturing step at 95¡ãC for 40 seconds, annealing step at 60¡ãC for 30 seconds, and amplification step at 72¡ãC for 1 minute for 20¨C35 cycles. cDNA templates were normalized based on the actin-specific signal, and PCR cycling was adjusted in such a way that each primer set amplified its corresponding gene product at its detection threshold to avoid saturation effects.
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We performed real-time PCR to quantify expression levels. The amplifications were performed in 25-µl volumes containing 0.5 µm of each primer, 0.5 X SYBR Green I (Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com), and 2 µl of 10-fold diluted cDNA using the DNA engine Opticon (MJ Research, Waltham, MA, http://www.mjr.com). The PCR reactions consisted of 50 cycles using the following temperature profile: 95¡ãC for 30 seconds, 60¡ãC for 30 seconds, 72¡ãC for 30 seconds, and 79¡ãC for 5 seconds. The melting temperature (Tm) of each PCR product was determined and turned out to be more than 79¡ãC. After each PCR cycle, the fluorescent signals were detected at 79¡ãC to melt primer dimers (Tm of all primer dimers used in this study was
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Electrophysiological Experiment
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Differentiated ESCs were examined using the whole-cell recording configuration of the conventional ¡®dialyzed¡¯ whole-cell patch-clamp technique. Differentiated cells were obtained by seeding ESCs onto 12-mm round coverslips and culturing for 14 days. Patch electrodes were fabricated from a borosilicate glass capillary (Sutter Instrument Company, San Rafael, CA, http://www.sutter.com) by using a vertical micropipette puller (Narishige, Tokyo, http://www.narishige.co.jp). The patch electrodes were fire-polished on a microforge (Narishige) and had resistances of 1¨C3 M when filled with the internal solution described below. The cell membrane capacitance and series resistance were compensated (typically > 80%) electronically using a patch-clamp amplifier (Axopatch-200A; Axon Instruments/Molecular Devices Corp., Union City, CA, http://www.moleculardevices.com). Current protocol generation and data acquisition were performed using pClamp 8.0 software on an IBM computer equipped with an analogue-to-digital converter (Digidata 1322A; Axon Instruments/Molecular Devices Corp.). Voltage traces were filtered at 2 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¡ãC¨C24¡ãC). For recording of membrane potential in current 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 126, KCl 5, CaCl2 2, MgCl2 1.2, glucose 14, and HEPES 10.5 (pH adjusted to 7.4 with NaOH).
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Transplantation Analysis

D3 and N2 ESC colonies were isolated from PA6 feeder layer at differentiation day 9 by incubation with 2.5 ml of papain solution (Papain Dissociation Kit, Worthington Biochemical Corporation, Freehold, NJ, http://www.worthington-biochem.com) for 5 minutes at 37¡ãC. The dissociated colonies were transferred to a 15-ml tube and then incubated for an additional 5 minutes at 37¡ãC followed by gentle trituration using a 1-ml tip and a 5-ml pipette. The cells were suspended at a density of 50,000 cells per µl. One microliter of the cell suspension was grafted into the right striatum (from the bregma: AP  0.05, L ¨C0.18, V ¨C0.30, IB 9) of C57/BL/6 mice (n = 10) (Charles River Laboratories, Wilmington, MA, http://www.criver.com). Prior to surgery, mice received an i.p. injection of preanesthesia (acepromazine . Cells were counted from blind-coded samples using an integrated Axioskop 2 microscope (Carl Zeiss, Thornwood, NY, http://www.zeiss.com) and StereoInvestigator image capture equipment and software (MBF Bioscience, Williston, VT, http://www.microbrightfield.com).

RESULTS
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Nurr1 Overexpression Promotes the Neuronal Differentiation of ESCs When Cocultured with PA6 Feeder Cells
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Using the five-stage in vitro differentiation method, it has been shown that exogenous expression of Nurr1 and treatment of signaling molecules (Shh, FGF8, and ascorbic acid) can synergistically increase the induction of DA neurons  generated a slight, but statistically significant (p
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Figure 1. Synergistic effects of Nurr1, signaling molecules, and SDIA on efficient generation of TH  neurons. (A): A schematic diagram for in vitro differentiation of ESCs on PA6 feeder layer. PA6 cells were plated on gelatin-coated culture dish and incubated overnight to make a confluent uniform feeder monolayer. The next day (day 0), the PA6 feeder monolayer was washed with embryonic stem differentiation medium, and D3 and N2 cells were added to initiate differentiation. ESCs were allowed to differentiate on the PA6 feeder cells for 14 days. During that differentiation period, signaling molecules such as Shh, FGF8, and ascorbic acid were added at different time intervals as follows: condition I, treatment with Shh and FGF8 during days 7¨C9 and ascorbic acid treatment during days 9¨C14; condition II, treatment with Shh and FGF8 during days 5¨C9 and ascorbic acid treatment during days 9¨C14; condition III, treatment with Shh and FGF8 during days 7¨C11 and ascorbic acid treatment during days 11¨C14. (B): Nurr1 expression during in vitro differentiation of D3 and N2 ESCs. Nurr1 gene expression was examined at day 0, 7, and 14 during in vitro differentiation of D3 and N2 ESCs by semiquantitative reverse transcription¨Cpolymerase chain reaction analysis. (C): Effect of transgene expression of Nurr1 on neurogenesis. ß-tubulin  neurons derived from ESCs were detected by staining with anti-Tuj1 antibodies. Positive cells were counted in 10 random fields per sample. Each group represents an average of three samples from independent experiments. Fisher¡¯s PLSD post hoc analysis was performed with a significance level of 0.05. *p
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Nurr1 Overexpression and Signaling Molecules Synergistically Facilitate the Differentiation of ESCs to the DA Fate When Cocultured with PA6 Feeder Cells% W5 ^( T% i" ]+ V* A0 Z: j

The expression of the DA phenotype amongst Tuj1  neurons was analyzed next. As shown in Figure 1D, approximately 28% of ß-tubulin  neurons were TH  in naïve D3 cells after the whole 14-day procedure. When signaling molecules were added to the D3 cells, the proportions of TH  neurons among total neurons were not significantly changed (30%¨C33% depending on treatment conditions). When N2 ESCs were differentiated on the PA6 feeder cells, a much higher proportion of Tuj1  neurons was TH  (~55%; p

Figure 2. TH  neurons generated by N2 cells under condition II coexpress other markers specific for midbrain DA neurons. (A): Detection of ß-tubulin and TH  cells by immunocytochemistry in differentiated D3 and N2  cells. (B): TH  neurons coexpress midbrain DA markers such as AADC and DAT, but not GABA and 5-HT. Embryonic stem cell¨Cderived neurons were stained with anti-TH, anti-AADC (dopaminergic, serotonergic), anti-DAT (dopaminergic), anti-GABA (GABAergic), and anti¨C5-HT (serotonergic) antibodies. Abbreviations: AADC, aromatic L-amino acid decarboxylase; DA, dopaminergic; DAT, dopamine transporter; GABA, -aminobutyric acid; HT, hydroxytryptamine; SM, signaling molecule; TH, tyrosine hydroxylase.6 n" u, }4 h) O1 N5 T' j  h" e
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TH  Neurons Coexpress the Other Midbrain DA Markers
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TH  cells exist in many neuronal phenotypes, including DA and noradrenergic (NA) neurons. Therefore, we determined whether TH  neurons generated by the optimized in vitro condition II have the midbrain DA neuron phenotype. The in vitro differentiated N2 cells under condition II were costained using antibodies against TH, AADC, DAT, -aminobutyric acid (GABA), and 5-HT. AADC is an enzyme that is involved in the last step of dopamine and serotonin biosynthesis , was marginally increased. Other DA markers, like calbindin and Pitx3, were also nonsignificantly upregulated (Fig. 3B).; e: {1 q, r/ o

Figure 3. Semiquantitative RT- and real-time PCR analyses for DA neuron markers and other neurotransmitter markers. Semiquantitative RT- (A) and real-time PCR analyses (B, C) of midbrain DA markers (A, B) and neurotransmitter markers (A, C) after in vitro differentiation of naïve D3, D3 SM(II), N2, and N2 SM(II) conditions. The expression levels of each gene were normalized to that of actin. Similar patterns were obtained when we used another gene, GAPDH, for normalization (data not shown). In (B, C), relative expression levels of each gene were determined in relation to those from naïve D3 cells. Each group represents an average of two samples from independent experiments. Fisher¡¯s PLSD post hoc analysis was performed with a significance level of 0.05. *p 7 a6 A$ G3 L: p, K

We next examined gene regulation of various other neurotransmitter markers (Fig. 3C). Expression of NA marker, dopamine ß-hydroxylase, was not detected in either D3 or N2 clones (data not shown). Therefore, these TH  cells exhibit the DA, not the NA, phenotype. In contrast to the upregulation of DA marker genes, the expression of the GABAergic and cholinergic (GAD and ChAT, respectively) was only minimally changed. Thus, Nurr1 overexpression and signaling molecules seem to specifically facilitate the differentiation of ESCs to the DA fate when cocultured with PA6 feeder cells. In support of this, real-time PCR analysis demonstrated that the DA-specific markers are specifically upregulated in the N5 clone as well (data not shown). However, the expression of a serotonergic neuronal marker, TPH, was upregulated by approximately 2.5-fold.% T, a4 f; [7 f3 H* W3 A+ W# l
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In Vitro Differentiated Cells Exhibit Active Membrane Properties Characteristic of DA Neurons
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The electrophysiological properties of neurons differentiated from N2 SM (II) cells were investigated using the conventional "dialyzed" whole-cell recording technique. The resting membrane potentials of the differentiated neurons ranged from ¨C 48 to ¨C57 mV (average, ¨C55.3 ¡À 2.3 mV). Recordings in the current-clamp configuration allowed us to determine the active membrane characteristics of these neurons. Prolonged depolarizing current injections (Fig. 4A) demonstrated the capability to fire fast action potentials and action potential series (action potential amplitude ranging from 32 to 47 mV . These time-dependent inward rectifications were observed in five out of seven cells tested, indicating that a majority of the neurons have the electrophysiological property of DA neurons.

Figure 4. Electrophysiological properties of neurons differentiated from N2 SM (II) cells. (A): Current-clamp recordings during prolonged depolarizing current injections. Top traces represent current injections, whereas bottom traces indicate voltage recordings. Depolarizing current injections elicited fast action potentials. (B): Voltage-dependent membrane currents. Depolarizing voltage steps (top traces) elicited outward K  currents and fast inward Na  currents. (C): Current-clamp recordings during prolonged hyperpolarizing current injections. The neurons display the time-dependent anomalous rectification characteristic of dopaminergic neurons after a hyperpolarizing pulse. Abbreviation: SM, signaling molecule.
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Nurr1 Overexpression and Signaling Molecules Greatly Facilitate the Production and Release of DA on PA6 Feeder Cells# E1 M7 \/ i' I, p+ r- O% H+ z% [
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An important physiological aspect of in vitro differentiated DA neurons is their ability to produce and release DA in response to membrane depolarization. To test this, ESCs were differentiated for 14 days under our optimized conditions and analyzed for the production and release of DA. The 24 hour¨Cconditioned media were prepared at day 14, and then cells were treated with 50 mM KCl. After a 1-day recovery in fresh medium, cells were harvested and intracellular DA levels were measured as described in the Materials and Methods section. For these samples, DA levels were assayed by reverse-phase HPLC and expressed as pg per 20 µl medium/solution. As shown in Figure 5, DA released into conditioned media and in response to membrane depolarization were 15- and four fold higher in N2 SM (II) (48 pg/20 µl and 24 pg/20 µl, respectively) than in D3 cells (3.25 pg/20 µl and 5.75 pg/20 µl, respectively). The intracellular DA level was also 18-fold higher in N2 SM (II) cells (99.5 pg/20 µl) compared with that in D3 cells (5.5 pg/20 µl).

Figure 5. Analyses of dopamine synthesis and release. Results of HPLC analyses of dopaminergic (DA) levels in 24 hour¨Cconditioned media, 50 mM KCl-challenged media, and intracellular fractions of D3 and N2 SM (II) cells are shown. Each group represents an average of six samples. Fisher¡¯s PLSD post hoc analysis was performed with a significance level of 0.05. *p 9 Q+ E  `" k5 H7 P, R8 j- [* ?

Efficient In Vivo Integration and Generation of TH  Neurons after Implantation of N2 Cells Differentiated under Condition II into the Mouse Striatum

The in vitro data above indicate that the combined condition of Nurr1, signaling molecules, and SDIA may provide an optimal method for efficiently generating midbrain DA neurons for the future cell transplantation therapy to treat PD. Toward this long-term goal, we next investigated whether ESCs differentiated under this condition could efficiently integrate or generate TH  neurons after implantation. Morizane et al., using naïve ESCs, grafted cells that had been cultured on PA6 for 12 days . When the cells were grafted as individual cell suspensions into mouse striatum on day 12, the number of surviving cells was much lower than when the cells were grafted as nondissociated cell clumps. This may be due to damage to the neurites when the cells are dissociated prior to transplantation. Because cell suspension has the advantage of quantification and reproducibility of grafting conditions, we retested the transplantation condition for individual cell suspension. First, we examined when the ESCs enter the postmitotic phase during differentiation (Fig. 6A). After differentiation for 7¨C10 days, ESC colonies were all isolated from the feeder layers by treatment with papain for 5 minutes at 37¡ãC, dissociated into a single cell suspension in 5 ml medium, and counted. As shown in Figure 5A, ESCs stopped growing after 8¨C9 days. N2 cells entered postmitotic phase 1 day sooner than D3 cells, indicating earlier neuronal maturation. Next, we examined cell viability after isolation by papain digestion (Fig. 6B). Cells were isolated from feeder layers after 7¨C10 days of differentiation, replated onto poly-L-ornithine/fibronectin plates, and then counted for cell viability after 12 hours. The viability of D3 cells decreased after 9 days of induction. This compares favorably with N2 cells whose viability started to decrease at day 7 and even more after the ninth day. Finally, TH  cell number was examined during differentiation (Fig. 6C). The number of TH  cells in D3 cells showed a mild increase through differentiation, whereas a dramatic increase in N2 cells was observed after 9 days of incubation under condition II. Based on these observations, we transplanted ESCs after 9 days of differentiation, when those parameters (i.e., total cell number, survival, and TH  cell numbers) are suboptimal. Four weeks after transplantation, the recipient mice were sacrificed, fixed by perfusion, and then analyzed for in vivo generation and integration of TH  neurons. In a graft of N2 cells differentiated under condition II, we could see many TH  cell bodies (Figs. 7C, 7D). In contrast, few TH  cell bodies were detected in control grafts of D3 cells (Figs. 7A, 7B). When we counted the total number of TH  cells per graft area (mm3) by staining for TH/Nissl, we found an average of 312 neurons for N2 cells under condition II and 28 for the naïve D3 cells (Fig. 7E). Costaining with antibodies against TH and NeuN (Figs. 7F¨C7K) was consistent with the TH/Nissl results, showing a much higher number of TH  cells with N2 cells (Fig. 7J) than with D3 cells (Fig. 7G). These results indicate that TH  cells derived from N2 cells under condition II are more efficiently generated and maintained in vivo.4 q; P2 S) k$ S; ]5 U  c$ e* g& z5 M
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Figure 6. Evaluation of conditions for transplantation. (A): Embryonic stem cells (ESCs) stop growing after approximately 8¨C9 days of differentiation. ESCs (1.05 x 104) were plated on PA6 feeder layer of 6-cm culture dish at differentiation day 0. After differentiation for the indicated times, ESC colonies were isolated from the feeder layer by incubation with papain (1.5 ml/6 cm dish) for 5 minutes, dissociated into single cells, suspended in 5 ml of medium, and counted. (B): Cell viability after isolation using papain treatment. At the indicated days, ESC colonies were isolated from the feeder layer by incubation with papain (1.5 ml/6 cm dish) for 5 minutes, dissociated into single cells suspensions, replated onto poly-L-ornithine/fibronectin plates, and counted after 12 hours. Starting cell numbers were expressed as 100%. (C): The number of tyrosine hydroxylase (TH) cells increased dramatically on induction day 9. At the indicated days, ESC colonies were isolated from the feeder layer by incubation with papain (1.5 ml/6 cm dish) for 5 minutes, dissociated into single cells, replated onto poly-L-ornithine/fibronectin plates, and stained with anti-TH antibodies after 12 hours.

Figure 7. Efficient in vivo generation of TH  neurons post transplantation in the mouse striatum from N2 cells subjected to in vitro differentiation condition II. Naïve D3 cells and Nurr1-overexpressing N2 cells  were transplanted into mice striatum at in vitro differentiation day 9, and the animals were sacrificed 4 weeks later for histological analyses. Grafts of D3 (A, B) and N2 (C, D) cells in naïve mouse striatum were analyzed by TH/Nissl staining. The number of TH  cells per graft area (mm2) is shown (E). Images for panels (F¨CH) and (I¨CK) represent TH/NeuN staining of grafts of D3 and N2 cells, respectively. Each group represents an average of 6¨C8 animals. Tumor growth was observed in one grafted animal in each group, which was not included in further analysis. Fisher¡¯s PLSD post hoc analysis was performed with a significance level of 0.05. *p 5 A7 A# |1 `; h/ D. P7 F
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DISCUSSION

In this study, we showed that coculturing Nurr1-overexpressing ESCs (N2 or N5) with PA6 stromal feeder cells leads to the generation of a high proportion of TH  neurons (~55% of Tuj1  neurons; Fig. 1D). Surprisingly, when N2 cells were treated with Shh and FGF8 during days 5¨C9 followed by ascorbic acid treatment, the proportion of TH  neurons was further enhanced up to ~90% of Tuj1  neurons, whereas naïve D3 cells did not show any cooperative effect with signaling molecules on the PA6 stromal cell feeder layer. Importantly, these ESC-derived TH  neurons were found to coexpress AADC and DAT, but not GABA or 5-HT (Fig. 2B), suggesting that they have the midbrain DA neuronal phenotype.
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Lee et al. . Taken together, combined with a higher yield of neurons only in PA6 coculture method, our optimized condition II represents a very effective protocol for efficient generation of DA neurons in a short period of time.) D3 x/ R7 k$ @0 p5 e9 J8 T2 E: R
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Our coimmunostaining and gene expression analysis indicate that those TH  neurons generated from our optimal in vitro differentiation have midbrain DA neuronal properties. First, all TH  neurons tested were found to be AADC , suggesting that they have the ability to synthesize DA. Some of these neurons also coexpressed DAT, whereas none of them express other neurotransmitter markers such as 5-HT and GABA. Absence of GABA is characteristic of midbrain DA neurons, whereas olfactory DA neurons coexpress TH and GABA. Analyses of mRNAs from these neurons by semiquantitative and real-time PCR revealed a robust upregulation of DAT, AADC, and Girk2 mRNAs (Figs. 3A, 3B). However, other midbrain DA markers such as AHD2 and Pitx3 were only minimally upregulated, suggesting that additional factor(s)/signal(s) may be necessary for full maturation of these DA neurons. In this context, it is of great interest that Pitx3-overexpressing ESC lines can generate a significantly higher proportion of AHD2 /TH  neurons .: L+ m7 Y) a1 t4 C

An important physiological criterion of a functional DA neuron is the ability to release synthesized DA in response to membrane depolarization. When compared with naïve D3 cells, these N2-derived neurons not only expressed higher levels of intracellular DA (> 18-fold) but also efficiently released it into the medium either spontaneously (> 14-fold) or in response to depolarization induced by high KCl (> fourfold) (Fig. 5). Thus, N2 cells under our optimized condition II have the ability to efficiently differentiate into functional midbrain DA neurons in vitro. Furthermore, differentiated cells exhibit typical excitable properties of neuronal membrane (e.g., firing of action potentials and existence of fast inward Na  current) (Figs. 4A, 4B). In addition, the majority of differentiated cells subjected to hyperpolarizing voltage steps showed anomalous rectification (Fig. 4C), which is the electrophysiological characteristic of mesencephalic DA neurons .
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Interestingly, a majority of TH¨C neurons generated from N2 cells were 5-HT  (data not shown; Fig. 2B). In addition, our semiquantitative and real-time PCR analyses showed a threefold upregulation of TPH mRNA levels, a key enzyme involved in serotonin synthesis. In agreement with this, in vitro differentiated cells from N2 cells were able to produce and release significantly higher levels of serotonin than D3 ESCs (data not shown). Thus, it is likely that some factors/signals regulating the development of DA and 5-HT neurons overlap in our in vitro differentiation system, as is the case in the in vivo situation .

Finally, with the long-term goal of translating our in vitro studies to the cell replacement therapy of PD, we transplanted DA neurons generated from our optimized condition. Toward this goal, we first defined the optimal in vitro differentiation period by assessing cell proliferation, viability, and generation of TH  neurons under our differentiation condition II. We determined that the optimal time for transplantation is the ninth day of N2 cell differentiation, when the total cell number reaches the maximum with high cell survival rate. At this time, the number of TH  neurons is suboptimal and neurites have not been formed extensively (Fig. 6), which may reduce the damage resulting from the dissociation procedure prior to transplantation. When 50,000 cells were harvested at day 9 and transplanted into the striatum of mice, we observed an approximately 10-fold increase in the number of TH  neurons of N2 cell grafts compared with D3 cell grafts.

In conclusion, these results suggest that DA neurons generated under in vitro conditions described in the present protocol may be useful as a cell source for in vivo cell transplantation studies. Two research groups have shown that the hESCs can differentiate into neuronal cell types in vitro . Therefore, it is of great interest to determine whether the optimized protocol we describe here can also be applied to improve the generation of DA neurons from these hESCs.

SUMMARY
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The present study sought to optimize the differentiation of ESCs to the DA cell fate by combining genetic manipulation and in vitro culture conditions. When Nurr1-overexpressing ESCs were cocultured with PA6 stromal cells and treated with signaling molecules (i.e., Shh, FGF8, ascorbic acid), we found that ESCs generate a significantly higher number of neurons (~twofold) and that the majority (up to 90%) of them become TH . Optimal induction of the DA fate was also confirmed by real-time PCR analysis of mRNA expression, characterization of electrophysiological properties, and the analysis of the production and release of DA. Furthermore, transplantation experiments showed that TH  neurons generated by our optimal condition can efficiently integrate into mouse striatum after implantation. Thus, this study shows that combination of genetic manipulation(s) and in vitro cell differentiation conditions offers a reliable and effective induction of DA neurons from ESCs and may serve as a platform for future cell transplantation therapy in PD.

DISCLOSURES
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The authors indicate no potential conflicts of interest.

ACKNOWLEDGMENTS

This work was supported by (P50)NS39793, MH48866, NS044439, NS32080, DAMD-17-01-1-0763, and DAMD-17-01-1-0762. This work was also supported by a grant (code: SC2140) from the Stem Cell Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology, Republic of Korea. The authors thank Robert H. Kennedy for his technical assistance in HPLC analyses and Oliver Cooper for analyses of grafted mouse brain sections.) i1 i* s. G8 T% e1 F6 N5 n5 a" h
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Abstract# ^4 `2 [" |1 O0 a0 {: R0 E2 ], k% s2 ]

Introduction
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Materials and Methods
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Results
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Discussion1 U6 }# P$ |0 W

Summary

Disclosures
      【参考文献】
* The two articles cited in references 45 and 46 have been retracted by Science.( Y8 H- @( q. f" {

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