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发表于 2009-3-5 00:05 |只看该作者 |倒序浏览 |打印

作者：Sangmi Chunga,b, Byoung-Soo Shinb, Michelle Hwanga,b, Thomas Lardaroa,b, Un Jung Kangc, Ole Isacsona,d, Kwang-Soo Kima,b作者单位：a Udall Parkinsons Disease Research Center of Excellence, McLean Hospital/Harvard Medical School, Belmont, Massachusetts, USA;b Molecular Neurobiology Laboratories, McLean Hospital/Harvard Medical School, Belmont, Massachusetts, USA;c Department of Neurology, The University of Chicago, Chicago, Illi

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      【摘要】! z5 c* W# H/ Q( h" L
   Neural precursors (NPs) derived from ventral mesencephalon (VM) normally generate dopaminergic (DA) neurons in vivo but lose their potential to differentiate into DA neurons during mitogenic expansion in vitro, hampering their efficient use as a transplantable and experimental cell source. Because embryonic stem (ES) cell-derived NPs (ES NP) do not go through the same maturation process during in vitro expansion, we hypothesized that expanded ES NPs may maintain their potential to differentiate into DA neurons. To address this, we expanded NPs derived from mouse embryonic day-12.5 (E12.5) VM or ES cells and compared their developmental properties. Interestingly, expanded ES NPs fully sustain their ability to differentiate to the neuronal as well as to the DA fate. In sharp contrast, VM NPs almost completely lost their ability to become neurons and tyrosine hydroxylase-positive (TH ) neurons after expansion. Expanded ES NP-derived TH  neurons coexpressed additional DA markers such as dopa decarboxylase and DAT (dopamine transporter). Furthermore, they also expressed other midbrain DA markers, including Nurr1 and Pitx3, and released significant amounts of DA. We also found that these ES NPs can be cryopreserved without losing their proliferative and developmental potential. Finally, we tested the in vivo characteristics of the expanded NPs derived from J1 ES cells with low passage number. When transplanted into the mouse striatum, the expanded NPs as well as control NPs efficiently generated DA neurons expressing mature DA markers, with approximately 10% tumor formation in both cases. We conclude that ES NPs maintain their developmental potential during in vitro expansion, whereas mouse E12.5 VM NPs do not. 8 |7 }9 L# l; e" v. l. A, H6 s
      【关键词】 Differentiation Stem cell expansion Neural stem cell Embryonic stem cell
   INTRODUCTION
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Neural precursors (NPs) can be derived from various brain regions and stages during development and can generate all three cell types of the neural lineage (i.e., neurons, astrocytes, and oligodendrocytes) , once significantly expanded in vitro, these stem cells from fetal and adult brain show very limited ability to maintain their developmental potential for neurogenesis and/or differentiation to the specific neuronal fate. Thus, it is a critical issue to determine whether NP cells derived from different sources can maintain their developmental potential during mitogenic expansion.! ~% D  H3 X, J0 W. F% r
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Recently, many laboratories have reported that embryonic stem (ES) cells can efficiently differentiate into NP cells and then into DA neurons via optimal culture conditions and/or genetic manipulation , it is tempting to hypothesize that ES NPs maintain their potential to differentiate to the neuronal and/or DA fate during mitogenic expansion. To address this, we expanded NPs derived from embryonic day-12.5 (E12.5) VM or ES cells and compared their developmental properties in vitro. Here, we report that ES NPs, but not VM-derived NPs, maintain the potential to generate Tuj1  neurons as well as functional DA neurons after extensive in vitro expansion. These ES NP cells maintained their differentiation potential through multiple freeze-thaw cycles.

MATERIALS AND METHODS9 {9 B( V& ?# R$ x- Q/ ~
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ES Cell Culture and In Vitro Differentiation+ I, m% P6 O! U, @, w. |8 ?- e

Early passage J1 ES cells (with the passage number of 10) were obtained from Dr. Rudolf Jaenisch¡¯s laboratory (Massachusetts Institute of Technology, Lexington, MA). The mouse ES cell lines J1 and N2 were maintained as described previously . Briefly, undifferentiated ES cells were cultured on gelatin-coated dishes in Dulbecco¡¯s modified minimal essential medium (Life Technologies, Rockville, MD, http://www.invitrogen.com) supplemented with 2 mM glutamine (Life Technologies), 0.001% ß-mercaptoethanol (Life Technologies), 1x nonessential amino acids (Life Technologies), 10% donor horse serum (Sigma, St. Louis, http://www.sigmaaldrich.com), and 2,000 U/ml human recombinant leukemia inhibitory factor (LIF; R&D Systems, Minneapolis, http://www.rndsystems.com).
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ES cells were differentiated into embryoid bodies (EBs) on nonadherent bacterial dishes (FisherScientific, Pittsburgh, http://www1.fishersci.com) for 4 days in EB medium as described above without LIF and replacing horse serum with 10% fetal bovine serum (FBS; HyClone, Logan, UT, http://www.hyclone.com). EBs were then plated onto an adhesive tissue culture surface. After 24 hours of culture, selection of neuronal precursor cells was initiated in serum-free ITSFn (insulin, transferin, selenium, and fibronectin) medium . Cells were either fixed after 4 days of expansion for NP stage analysis or at 9 days after starting neuronal differentiation for analysis of neuronal phenotypes differentiation (ND) stage.1 }4 F" c8 X2 e

NP cells were frozen after trypsinization by suspension in freezing media (90% serum and 10% dimethyl sulfoxide) at 1 x 107 cells per ml and placed in a styrofoam container at ¨C80¡ãC to ensure a gradual decrease in temperature. After 24 hours, frozen cells were moved to a liquid nitrogen tank. Frozen NPs were thawed in a 37¡ãC water bath followed by centrifugation to remove freezing media. The cells were then plated on PLO/FN-coated plates in N3bFGF media.8 Q; h8 r( s# l. B6 W
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VM Dissection and Expansion
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Timed-pregnant mice were purchased from Charles River Laboratories (Wilmington, MA, http://www.criver.com). On E12.5, the ventral-most part of mesencephalon was cut out and collected in NP media with 5% FBS. Mechanical trituration in phosphate-buffered saline (PBS) yielded single-cell suspension. Cells were plated on PLO/FN-coated plates and maintained in NP media. Expansion of VM precursors consisted of passaging cells once every week on to PLO/FN-coated plates in NP media. To differentiate VM precursors, 1.5 x 105 cells were plated onto PLO/FN-coated 24 wells with coverslips and expanded in the presence or absence of 500 ng/ml Shh-N (R&D Systems) and 100 ng/ml FGF-8 (R&D Systems) for 4 days. ND was induced by removal of bFGF in the absence or presence of 200 µM ascorbic acid (Sigma). Cells were fixed after 4 days of expansion for analysis of NP stage or after 9 days from the initiation of neuronal differentiation for analysis of ND stage.8 e% z- C# D9 f' z2 i9 O; A1 ?
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Immunocytochemistry

For immunofluorescence staining, cells were fixed for 30 minutes in 4% formaldehyde (Electron Microscopy Sciences, Ft. Washington, PA, http://www.emsdiasum.com), rinsed with PBS, and then incubated with blocking buffer (PBS, 10% normal donkey serum ) for 10 minutes. Cells were then incubated overnight at 4¡ãC with primary antibodies diluted in PBS containing 2% NDS. The following primary antibodies were used: mouse anti-nestin (Rat401, 1 µg/ml; Developmental Studies Hybridoma Bank, Iowa City, IA, http://www.uiowa.edu/~dshbwww), rabbit anti-ß-tubulin (1:2,000; Covance, Princeton, NJ, http://www.covance.com), sheep anti-tyrosine hydroxylase (TH, 1:200; Pel-Freez, Rogers, AK, http://www.pel-freez.com), sheep anti-aromatic L-amino acid decarboxylase (1: 200; Chemicon, Temecula, CA, http://www.chemicon.com), and rat anti-dopamine transporter (DAT, 1:2,000; Chemicon). After additional rinsing in PBS, the coverslips were incubated in fluorescent-labeled secondary antibodies (Cy2- or Rhodamine Red-X-labeled donkey immunoglobulin G; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, http://www.jacksonimmuno.com) in PBS with 2% NDS for 30 minutes at room temperature. After rinsing 3 x 10 minutes in PBS, sections were counterstained using 1 µg/ml DAPI (4,6-diamidino-2-phenylindole) and then mounted onto slides in Gel/Mount (Biømeda Corp., Foster City, CA, http://www.biomeda.com). Coverslips were examined using a Leica TCS/NT confocal microscope (Leica, Heerbrugg, Switzerland, http://www.leica.com) equipped with krypton, krypton/argon, and helium lasers.
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Cell Counting and Statistical Analysis

Cell density of DA neurons was determined by counting the numbers of TH  cells and the numbers of ß-tubulin  cells per field at x63 magnification using a Zeiss Axioplan I fluorescent microscope (Carl Zeiss, Thornwood, NY, http://www.zeiss.com). Ten visual fields were randomly selected and counted for each sample, and cell densities were calculated by dividing the numbers of TH  cells by that of ß-tubulin  cells. Numbers presented in figures represent the average percentage and SEM of TH  cells over ß-tubulin  cells from five samples per ES cell clone.
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For statistical analysis, we used Statview software (SAS, Cary, NC, http://www.statview.com) and performed analysis of variance (ANOVA) with an alpha level of 0.01 to determine possible statistical differences between group means. When significant differences were found, post hoc analysis was performed using Fisher¡¯s PLSD (protected least significant difference; alpha = 0.05).% J! |1 f- }/ ^0 G3 a0 o

Semiquantitative Reverse Transcription-Polymerase Chain Reaction and Real-Time Polymerase Chain Reaction Analysis
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Total RNA from plated cells at different stages of the differentiation protocol was prepared using TRIzol Reagent (Sigma) followed by treatment with DNase I (Ambion, Austin, TX, http://www.ambion.com). For reverse transcription-polymerase chain reaction (RT-PCR) analysis, 5 µg of total RNA was transcribed into cDNA using the SuperScript Preamplification Kit (Life Technologies) and oligo (dT) primers. The cDNA was then analyzed in a PCR assay using the following primers: ß-actin: 5'-GGCATTGTGATGGACTCCGG-3'; 5'-TGCCA-CAGGATTCCATACCC-3' (358 bp); nestin: 5'-GGAGT-GTCGCTTAGAGGTGC-3'; 5'-TCCAGAAAGCCAAGAGA-AGC-3' (327 bp; ; ß-tubulin: 5'-AACTATGTAGGGGACTCAGACCTGC-3'; 5'-TCTCACACT-CTTTCCGCACGAC-3' (274 bp); TH: 5'-TCCTGCACTCCCT-GTCAGAG-3'; 5'-CCAAGAGCAGCCCATCAAAGG-3' (423 bp); DDC: 5'-CCTACTGGCTGCTCGGACTAA-3'; 5'-GCG-TACCAGGGACTCAAACTC-3' (715 bp); DAT: 5'-CA-GAGAGGTGGAGCTCATC-3'; 5'-GGCAGATCTTCCAGA-CACC-3' (328 bp); Pitx3: 5'-CTCTCTGAAGAAGA-AGCAGCG-3'; 5'-CCGAGGGCACCATGGAGGCAGC-3' (491 bp); Nurr1: 5'-CATGGACCTCACCAACACTG-3'; 5'-GAGA-CAGGTGTCTTCCTCTG-3' (383 bp)./ T  m  U9 L  H, G

PCR reactions were carried out with 1 x IN Reaction Buffer (Epicenter Technologies, Omaha, NE, http://www.epicentertechnology.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, 40 seconds; annealing step at 60¡ãC, 30 seconds; amplification step at 72¡ãC, 1 minute for 20 to 28 cycles and a final amplification step at 72¡ãC, 10 minutes. For semiquantitative PCR, cDNA templates were normalized by amplifying actin-specific transcripts, and levels of gene transcription were detected by adjusting PCR cycling and primer design in such a way that each primer set amplified its corresponding gene product at its detection threshold to avoid saturation effects.6 u: w+ V- W1 \2 O+ P( U

For quantitative analysis of the expression level of mRNAs, real-time PCR analyses using SYBR green I were performed using DNA engine Opticon (MJ Research, Waltham, MA, http://www.mjr.com). To reduce nonspecific signals, oligonucleotides amplifying small amplicons were designed using MacVector software (Oxford Molecular Ltd., Burlington, MA, http://www.accelrys.com). We selected primer sets amplifying the specific product without nonspecific bands. The following are the primer sets used for real-time PCR analysis: TH: 5'-TTGGCTGAC-CGCACATTTG-3'; 5'-ACGAGAGGCATAGTTCCTGAGC-3' (336 bp).* d- o. \  t' n  j5 s

The oligonucleotides used for RT-PCR were used to detect ß-actin, nestin, ß-tubulin, DDC, and DAT mRNA. Amplifications were performed in 25 µl containing 0.5 µM of each primer, 0.5x SYBR Green I (Molecular Probes, Inc., Eugene, OR, http://probes.invitrogen.com), and 2 µl of fivefold diluted cDNA. Forty PCR cycles were performed with the temperature profile of 95¡ãC for 30 seconds, 55¡ãC for 30 seconds, 72¡ãC for 30 seconds, and 79¡ãC for 5 seconds. The dissociation curve of each PCR product was determined to test the specificity of the fluorescent signals. The melting temperatures (Tm) of the PCR products were 85¡ãC, 85¡ãC, 85¡ãC, 85¡ãC, 84¡ãC, and 85¡ãC for ß-actin, nestin, ß-tubulin, TH, DDC, and DAT, respectively. After each PCR cycle, fluorescence was detected at 79¡ãC to melt primer dimers (the Tm of all primer dimers used in this study was
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Analysis of Catecholamines5 o9 I+ H" e8 C2 {% z
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Differentiated VM NPs and ES NPs (ND stage day 9) in six-well plates were treated with 200 µl of N3 medium supplemented with 50 mM KCl, and the media were collected after 30 minutes and concentrated solutions of perchloric acid (PCA) were added to a final concentration of 0.1 M PCA/0.1 mM EDTA. For measurement of catecholamine cell contents, cells were harvested in 0.1 M PCA/0.1 mM EDTA. These deproteinated samples were centrifuged, and supernatants were kept at ¨C80¡ãC until further analysis. Samples were further purified by using a 0.22-µm nylon filter (Osmonics, Inc., Trevose, PA, http://www.osmonics.com) followed by determination of cate-cholamine content by reverse-phase high-performance liquid chromatography (HPLC) using a Velosep RP-18 column (100 x 3.2 mm; Brownlee Labs, Shelton, CT, http://las.perkinelmer.com) and an ESA Coulochem II electrochemical detector (ESA, Inc., Chelmsford, MA, http://www.esainc.com) equipped with a model 5014 analytical cell as we described .' j& E7 |8 L0 c& l( O# I" r

Transplantation Analysis2 u" c( O3 @# K, ]5 @  k; a8 D
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J1 ES cell-derived NPs before and after 4 weeks of expansion were differentiated by removal of bFGF, trypsinized at day 3 of the differentiation stage ) followed by an i.p. injection of ketamine (60 mg/kg; PromAce) and xylazine (3 mg/kg; Phoenix Pharmaceuticals, Inc.). Transplantation was performed using a 22-gauge 10-µl Hamilton syringe (Hamilton Company, Reno, NV, http://www.hamiltoncompany.com) and a Kopf stereotaxic frame (David Kopf Instruments, Tujunga, CA, http://www.kopfinstruments.com). Buprenorphine (0.032 mg/kg, subcutaneous; Sigma) was given twice during 24 hours as postoperative analgesia. Four weeks after transplantation, mice were terminally anesthetized with an i.p. overdose of pentobarbital (150 mg/kg; Sigma). Mice were perfused intracardially with 100 ml of heparin saline (0.1% heparin in 0.9% saline) followed by 200 ml of paraformaldehyde (4% in PBS). Brains were postfixed for 8 hours, equilibrated in sucrose (20% in PBS), sectioned at 40 µm on a freezing microtome, and collected in PBS. For histological analysis, sections were stained with antibodies against TH (see above) and counterstained by Nissle staining. For measurement of graft volume and counting of total TH  cell number within grafts, every sixth section was stained with antibodies against TH (see above) and counterstained by Nissle staining. The stained sections were subjected to stereological analysis using an integrated Axioskop two microscope (Carl Zeiss) and StereoInvestigator image capture equipment and software (Microbright Field, Williston, VT, http://www.microbrightfield.com). TH  cell density was calculated by dividing the total TH  cell number by the graft volume.

RESULTS* e5 ^1 M* x9 p: |
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NP Cell Population Derived from VM and ES Cells Can Be Expanded In Vitro in the Presence of bFGF
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Previously, we have shown that N2 ES cells overexpressing the transcription factor Nurr1 efficiently generate DA neurons , was comparable before and after expansion in both cell populations. Real-time PCR analysis confirmed that consistent expression levels of nestin mRNAs were maintained after expansion among each group (Fig. 1G). These results show that both ES NP and VM NP cells maintain at least some aspects of their NP state upon expansion.$ w; p* Y0 z& s1 {  f/ z

Figure 1. Nestin-expressing NPs derived from VM and ES cells can be expanded in the presence of bFGF. (A): Expansion curve of VM-derived and N2 ES cell-derived NPs. Detection of nestin in VM-derived NPs without (control; B) and after expansion (4 weeks; C) by immunocytochemistry. N2 ES cell-derived NPs without (control; D) and after expansion (4 weeks; E). Representative fields using confocal microscopy at a x40 magnification are shown. Scale bar = 50 µm. (F): Semiquantitative RT-PCR analysis of neural stem cell markers. RNA samples were prepared from VM-derived and ES N2-derived NPs before (control) and after (4 weeks) expansion as described in Materials and Methods. cDNAs were prepared using oligo dT primers and then PCR-amplified using primers specific for ß-actin, nestin, and Bmi1. (G): Real-time PCR. Real-time PCR reactions were performed using SYBR green I. The standard curve was made using plasmid DNA containing the GAPDH gene. The expression of nestin was measured as described in Materials and Methods. The expression levels of ß-actin were used to normalize nestin gene expression. Relative expression levels were determined by setting those in unexpanded VM cells at 100. ANOVA revealed that there is no significant difference between groups (n = 4). Abbreviations: ANOVA, analysis of variance; ES, embryonic stem; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NP, neural precursor; PCR, polymerase chain reaction; RT-PCR, reverse transcription-polymerase chain reaction; VM, ventral mesencephalon.

NPs Derived from ES Cells but Not from VM Cells Efficiently Differentiate to Tuj1  Neurons and TH  DA Neurons after Mitogen-Induced Expansion9 p+ b8 F# a; A9 m, G) m: _
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To analyze their developmental potential, we differentiated expanded NPs in vitro into mature neurons. As previously reported, NPs derived from both unexpanded ES cells and VM cells readily differentiated into neurons and TH  neurons (Fig. 2A, 2C, 2G, 2I, 2M, 2O, 2S, 2U). After 4 weeks of expansion, VM-derived NPs almost completely lost their potential to differentiate into neurons (Fig. 2G, 2H) or into TH  neurons (Fig. 2M, 2N). In sharp contrast, N2 ES cell-derived NPs differentiated into neurons (Fig. 2I, 2J) and TH  neurons (Fig. 2O, 2P) with the same efficiency after 4 weeks of expansion. To test whether this dramatic difference was artificially affected by overexpression of Nurr1 by N2 ES cells, we tested the wild-type J1 ES cell line. As shown in Figure 2L and 2R, NPs derived from the wild-type J1 ES cells also maintained their developmental potential after the same 4 weeks of expansion. These results show that ES cell-derived NPs dramatically differ from VM-derived NPs in their developmental potential after extensive expansion although they appear similar before expansion.1 n6 a- V8 O! N. V! }: v
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Figure 2. ES cell-derived NPs maintain their potential to become DA neurons during expansion in the presence of bFGF, whereas VM-derived NPs lose their potential after expansion in bFGF. (A) Unexpanded VM-derived cells with DAPI staining, (B) expanded VM-derived cells with DAPI staining, (C) unexpanded N2 ES-derived cells with DAPI staining, (D) expanded N2 ES-derived cells with DAPI staining, (E) unexpanded J1 ES-derived cells with DAPI staining, (F) expanded J1 ES-derived cells with DAPI staining, (G) unexpanded VM-derived cells with ß-tubulin staining, (H) expanded VM-derived cells with ß-tubulin staining, (I) unexpanded N2 ES-derived cells with ß-tubulin staining, (J) expanded N2 ES-derived cells with ß-tubulin staining, (K) unexpanded J1 ES-derived cells with ß-tubulin staining, (L) expanded J1 ES-derived cells with ß-tubulin staining, (M) unexpanded VM-derived cells with TH staining, (N) expanded VM-derived cells with TH staining, (O) unexpanded N2 ES-derived cells with TH staining, (P) expanded N2 E2-derived cells with TH staining, (Q) unexpanded J1 ES-derived cells with TH staining, (R) expanded J1 ES-derived cells with TH staining, (S) unexpanded VM-derived cells with merged image, (T) expanded VM-derived cells with merged image, (U) expanded N2 ES-derived cells with merged image, (V) expanded N2 ES-derived cells with merged image, (W) unexpanded J1 ES-derived cells with merged image, and (X) expanded J1 ES-derived cells with merged image. NPs with (4 weeks) or without (control) expansion were fully differentiated into neurons by removal of bFGF for 9 days (ND stage) and fixed for immunocytochemistry analysis. Shown are representative fields using confocal microscopy at x40 magnification. Scale bar = 50 µm. (Y): The number of ß-tubulin  cells from 10 random fields per sample from in vitro differentiated VM-, N2 ES-, and J1 ES-derived NPs were counted, and the total numbers of cells are shown. Each group represents an average of three samples from each independent experiment. ANOVA revealed F = 17.224, p $ z7 s# n% R9 s5 _
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For quantitative analysis, we performed cell counting as described in Materials and Methods (). To ensure an accurate quantitative analysis of cell numbers, we designed a cell-counting method using a grid of 10 random fields, which was applied to multiple coverslips per analysis. After in vitro differentiation, unexpanded VM NPs, expanded VM NPs, unexpanded N2-ES NPs, expanded N2-ES NPs, unexpanded J1-ES NPs, and expanded J1-ES NPs contained 218 ¡À 38, 48 ¡À 11, 321 ¡À 43, 335 ¡À 25, 350 ¡À 12, and 330 ¡À 10 ß-tubulin  neurons in 10 fields, respectively (average cell number ¡À SEM; Fig. 2Y). We also counted the number of TH  neurons, and the above cells contained 5.9% ¡À 0.7%, 1.1% ¡À 1.1%, 24.7% ¡À 2.2%, 21.6% ¡À 1.1%, 22.7% ¡À 2.4%, and 20.0% ¡À 0.6% TH /ß-tubulin  neurons, respectively, and 1.24% ¡À 0.24%, 0.04% ¡À 0.04%, 6.95% ¡À 1.78%, 6.45% ¡À 0.78%, 6.61% ¡À 1.07%, and 5.43% ¡À 0.87% TH /total cells, respectively (average percentage ¡À SEM; Fig. 2Z).
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In Vitro Expanded NPs Derived from ES Cells Differentiate into Functional Midbrain-Like DA Neurons
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Next, we performed further immunocytochemistry analyses to test whether TH  neurons generated from expanded NPs express other DA neuronal markers. After in vitro differentiation, both unexpanded (control) and 4-week expanded N2 cells expressed another DA neuronal marker, DDC, with almost a complete coexpression pattern with TH (Fig. 3A¨C3F), and some of them also expressed the late DA marker, DAT (Fig. 3G¨C3L). In addition, J1 ES cell-derived NPs prominently generated DA neurons, many of which coexpress DDC (Fig. 3M¨C3R) and DAT (Fig. 3S¨C3X) before and after expansion.5 H' |" o4 A: B9 D% y# `

Figure 3. The potential to differentiate into other dopaminergic phenotypes is also maintained in ES cell-derived NPs before and after expansion. (A) Unexpanded N2 ES-derived cells with TH staining, (B) expanded N2 ES-derived cells with TH staining, (C) unexpanded N2 ES-derived cells with DDC staining, (D) expanded N2 ES-derived cells with DDC staining, (E) unexpanded N2 ES-derived cells with merged image, (F) expanded N2 ES-derived cells with merged image, (G) unexpanded N2 ES-derived cells with TH staining, (H) expanded N2 ES-derived cells with TH staining, (I) unexpanded N2 ES-derived cells with DAT staining, (J) expanded N2 ES-derived cells with DAT staining, (K) unexpanded N2 ES-derived cells with merged image, (L) expanded N2 ES-derived cells with merged image, (M) unexpanded J1 ES-derived cells with TH staining, (N) expanded J1 ES-derived cells with TH staining, (O) unexpanded J1 ES-derived cells with DDC staining, (P) expanded J1 ES-derived cells with DDC staining, (Q) unexpanded J1 ES-derived cells with merged image, (R) expanded J1 ES-derived cells with merged image, (S) unexpanded J1 ES-derived cells with TH staining, (T) expanded J1 ES-derived cells with TH staining, (U) unexpanded J1 ES-derived cells with DAT staining, (V) expanded J1 ES-derived cells with DAT staining, (W) unexpanded J1 ES-derived cells with merged image, and (X) expanded J1 ES-derived cells with merged image. Shown are representative fields using confocal microscopy at x40 magnification. Scale bar = 50 µm. Abbreviations: DAT, dopamine transporter; DDC, dopa decarboxylase; ES, embryonic stem; ND, neuronal phenotypes differentiation; NP, neural precursor; TH, tyrosine hydroxylase.
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We next analyzed DA marker gene expression by semi-quantitative RT-PCR. mRNA expression of the neuronal marker ß-tubulin gene was comparable before and after expansion of N2 and J1 cells (Fig. 4A). In contrast, expression of ß-tubulin mRNA was almost undetectable in the case of expanded VM NP cells, which is consistent with immunocytochemistry analysis (Fig. 2). mRNA expression of midbrain DA markers such as TH, DDC, DAT, Pitx3, and Nurr1 was well maintained after expansion of N2 and J1 cells, whereas VM cells almost completely lost their expression after expansion (Fig. 4A). As shown in Figure 5B, all of these gene expression data were quantitatively confirmed by real-time PCR analyses (Fig. 4B).
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Figure 4. Analysis of mRNA expression. (A): Semiquantitative RT-PCR analysis of midbrain DA markers. RNA samples were prepared from in vitro differentiated (ND stage day 9) VM-derived, N2 ES-derived and J1 ES-derived cells with (4 weeks) or without (control) expansion of NPs. cDNAs were prepared using oligo dT primers and then PCR-amplified using primers specific for various DA markers. To avoid a saturation effect, the numbers of PCR cycles were experimentally determined for each cDNA to maintain amplification of each specific transcript in the linear range. (B): Real-time PCR. Real-time PCR reactions were performed using SYBR green I. The standard curve was constructed using plasmid DNA containing the GAPDH gene. The expression of ß-tubulin, TH, DDC, DAT, and ß-actin was measured as described in Materials and Methods. ß-Actin expression levels were used to normalize that of each gene. Relative expression levels were determined by setting those in unexpanded VM cells at 100. Fisher¡¯s PLSD post hoc analysis was performed with a significance level of .05. Asterisk indicates significant difference from unexpanded (control) samples. Abbreviations: DA, dopaminergic; DAT, dopamine transporter; DDC, dopa decarboxylase; ES, embryonic stem; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ND, neuronal phenotypes differentiation; NP, neural precursor; PLSD, protected least significant difference; RT-PCR, reverse transcription-polymerase chain reaction; TH, tyrosine hydroxylase; VM, ventral mesencephalon.3 h* k- e$ K$ N' W
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Figure 5. Analysis of dopamine release. In vitro differentiated VM NPs and ES NPs (ND stage day 9) were challenged with 50 mM KCl, and the media were assayed for DA levels by reverse-phase HPLC. Each group represents an average of three to four samples from independent experiments. ANOVA revealed F = 27.256, p = .0001. Fisher¡¯s PLSD post hoc analysis was performed with a significance level of .05. Asterisk indicates significant difference from all other samples at p 6 K$ J- S) F& l/ m- j  I8 F! j

An important physiological aspect of authentic DA neuron phenotypes is the ability to synthesize DA and release it in response to membrane depolarization. Having observed specific midbrain DA marker expression after in vitro differentiation of expanded N2 NP cells, we next tested whether DA could be released after membrane depolarization. In vitro differentiated cells (day 9 of the five-stage protocol) from unexpanded and expanded NP cells were treated with 50 mM KCl, and the released DA in the media was analyzed by HPLC. In response to membrane depolarization from unexpanded VM, expanded VM, unexpanded N2, and expanded N2 cells, 2.70 ¡À 0.22, 0.00 ¡À 0.00, 3.59 ¡À 0.41, and 3.29 ¡À 0.42 pg DA/µg cellular proteins were released, respectively (Fig. 5). Taken together, neurons derived from expanded N2 cells maintain the ability to differentiate into midbrain-like DA neurons and to produce and release DA in response to membrane depolarization whereas VM NP cells completely lost these properties after expansion.
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Expanded ES Cell-Derived NPs Efficiently Generate DA Neurons After In Vivo Transplantation into Mouse Striatum% U  L. n6 Y2 ?: z* m8 O: ^% q
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To characterize the developmental potential of ES cell-derived NP cells in vivo, we differentiated unexpanded and expanded J1 ES NPs in vitro, trypsinized them at day 3 of the stage V protocol, and transplanted them into normal mouse striatum. Early passage J1 ES cells were used for transplantation analysis to avoid high-frequency tumor formation from high-passage N2 ES cells ). Four weeks after transplantation, the animals were sacrificed and the grafts were analyzed for survival and expression of phenotypic markers. J1 ES cell-derived NPs generated mostly well-contained TH  grafts, but occasional tumor formation was observed in approximately 10% of transplanted mice per each group. This result suggests that 4-week expansion does not apparently increase teratoma/tumor formation for the low-passage J1 ES cells. As shown in Figure 6A and 6B, both unexpanded and expanded J1 NP cells efficiently generated abundant TH  neurons in the host brain. TH  cell bodies appear to be derived from transplanted cells, because sham-treated side (contralateral to the transplanted side) shows only TH fibers projecting from substantia nigra but never TH cell bodies as in the grafts. No significant differences in the density of TH  cells in the grafts were observed between unexpanded and expanded NP cell grafts (Fig. 6C; 3,196.5 ¡À 739.7 and 3,305.5 ¡À 453.1, respectively). We next examined whether these TH  neurons generated in vivo also express other markers of DA neurons. Triple immunocytochemistry analysis demonstrated that virtually all of TH  neurons coexpressed DAT and DDC in grafts of unexpanded (Fig. 6D¨C6G) and expanded (Fig. 6H¨C6K) NP cells.
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Figure 6. Expanded J1 ES cell-derived NPs maintain their potential to differentiate into DA neurons in vivo. Representative images of TH immunohistochemistry in grafts from unexpanded (A) and 4-week expanded (B) cells in naïve mouse striatum. Scale bar =100 µm. (C): Total number of TH  cells per cubic millimeter of graft volume. No significant difference between groups was found by ANOVA (n = 4 for unexpanded and n = 6 for expanded cells). Representative confocal images of TH, DAT, and DDC immuno-histochemistry on grafts of unexpanded, (D) grafts from unexpanded J1 ES-derived cells with TH staining, (E) grafts from unexpanded J1 ES-derived cells with DAT staining, (F) grafts from unexpanded J1 ES-derived cells with DDC staining, (G) grafts from unexpanded J1 ES-derived cells with merged image, (H) grafts from expanded J1 ES-derived cells with TH staining, (I) grafts from expanded ES-derived cells with DAT staining, (J) grafts from expanded J1 ES-derived cells with DDC staining, and (K) grafts from expanded J1 ES-derived cells with merged image. Scale bar = 50 µm. Abbreviations: ANOVA, analysis of variance; DA, dopaminergic; DAT, dopamine transporter; DDC, dopa decarboxylase; ES, embryonic stem; NP, neural precursor; TH, tyrosine hydroxylase.
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Long-Term Storage and Freeze-Thaw Cycle Do Not Abrogate the Developmental Potential of Expanded ES-Derived NP Cells
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The above data demonstrate that expanded NP cells from ES cells do not lose their developmental potential for neurogenesis and lineage-specific differentiation to DA neurons. These results suggest that these cells may serve as a convenient and unlimited cell source for many basic and therapeutic approaches. One prerequisite for this application is that these expanded cells can be stably stored in liquid nitrogen without losing their developmental and proliferative potential. We tested whether expanded NPs from ES cells maintain their differentiation potential after a freeze-thaw cycle. We froze N2 ES-derived NP cells in liquid nitrogen after a 4-week expansion and subsequently thawed and subjected them to an additional 4 days of expansion in the presence of bFGF followed by in vitro differentiation. As shown in Figure 7, generation of Tuj1  cells (Fig. 7B, 7F) and TH  neurons (Fig. 7C, 7G) was as efficient as those without the freeze-thaw cycle. These thawed NP cells could also be expanded further, demonstrating that they also fully sustain the proliferative capacity. The same pattern was observed after the freeze-thaw cycle of expanded NP cells from J1 ES cells. Furthermore, we found that these ES NP cells could be stored up to 1 year in liquid nitrogen without losing their differentiation potential to TuJ1  cells and TH  neurons (data not shown).9 X$ Z* U$ |8 o

Figure 7. ES cell-derived NPs maintain their ability to become DA neurons after the freeze-thaw cycle. (A) Control cells with Hoechst staining, (B) control cells with ß-tubulin staining, (C) control cells with TH staining, (D) control cells with merged image, (E) cells after freeze-thaw cycle with Hoechst staining, (F) cells after freeze-thaw cycle with ß-tubulin staining, (G) cells after freeze-thaw cycle with TH staining, and (H) cells after freeze-thaw cycle with merged image. Shown are representative fields using confocal microscopy at a x40 magnification. Scale bar = 50 µm. Abbreviations: bFGF, basic fibroblast growth factor; DA, dopaminergic; ES, embryonic stem; NP, neural precursor; TH, tyrosine hydroxylase.9 y! P, |  |5 ~7 D( u" m
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DISCUSSION

We systematically analyzed and compared the developmental potential of fetal VM- and ES cell-derived NPs before and after 4-week expansion in vitro in the presence of mitogens. To perform quantitative comparisons between NPs from different sources, we used an adherent culture system. NPs can be cultured in vitro using different methods, such as adherent culture or neurosphere cultures, which have pros and cons. Neurosphere culture can preserve cell-cell interaction better, which may play an important role in the behavior of NPs . However, it is not easy to efficiently quantify all the cells within the spheres even after attaching to the substrate. Thus, we compared ES-derived NPs and fetal VM-derived NPs using the same culture method, one that is easier for quantification and could clearly demonstrate differences in terms of their differentiation potential during mitogenic expansion. Our results demonstrate that ES NPs do not lose their potential to differentiate into mature neurons, including DA neurons, after subsequent expansion by passaging in vitro. This was in sharp contrast to VM-derived NPs, which almost completely lost their potential for both neurogenesis and DA differentiation after expansion in vitro. Salient features about in vitro expanded NP cells derived from ES cells are as follows. First, NP cells derived from ES cells (both the wild-type J1 and the genetically engineered N2) could be exponentially expanded in vitro with a 1,000-fold increase in numbers after 4 weeks, whereas VM-derived NP cells were expanded at a much lower efficiency (10-fold increase after 4 weeks). Second, NP cells from both J1 and N2 ES cells could efficiently generate neurons and TH  neurons after expansion. In addition, ES NPs spontaneously differentiated into neurons after expansion by removal of bFGF with high efficiency, which is again different from fetal tissue-derived NPs. Expanded ES NPs also showed maintenance of differentiation potential in vivo after transplantation into mice striatum. Furthermore, ES NPs proliferate robustly in the presence of mitogen and can be easily kept by cryopreservation without losing their developmental potential, which make them an even more convenient cell source.

Importantly, our in vivo transplantation studies showed that expansion of NPs of the low-passage J1 ES cells did not apparently increase the incidence of tumor formation after 4 weeks of expansion. However, it has been also reported that long-term-passaged fetal tissue-derived NPs showed increased tumor formation accompanied by changes in cell behavior such as faster proliferation kinetics . Thus, it would be interesting to see whether further expansion of ES NPs (e.g., more than several months) may lead to increased tumor formation. If that is the case, even though expansion of ES NPs could provide unlimited number of NPs with stable developmental potential, changes in cellular and molecular characteristics should be carefully monitored, if long-term expansion is attempted for cell replacement therapy.$ ^$ Z7 \4 z3 ^3 Z' J( M
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In line with our current observation, there are previous reports of the intrinsic differences between NPs derived from fetal tissue or ES cells. Hitoshi et al. . Thus, it is possible that ES NPs may have intrinsic mechanism(s) to counteract the stress caused by high oxygen condition compared with VM NPs." @/ w, x5 r' U) B! e# }
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In summary, this study shows that ES NPs, but not VM-derived NPs, can be greatly expanded as a population (1,000-fold in 4 weeks) into transplantable cells without compromising their developmental potential. Expanded NPs from J1 ES cells with low passage number did not increase tumor/teratoma formation and maintained the developmental potential to generate midbrain-like DA neurons and to produce DA in vitro as well as in vivo. Furthermore, these ES-derived NPs can be easily kept by cryopreservation without losing their developmental potential, which make them a very convenient and readily available cell source for drug screening, developmental stem cell studies, and transplantation studies.

CONCLUSION
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By using the same culture and in vitro differentiation methods, we compared the differentiation potential of fetal VM-derived NPs and ES-derived NPs during mitogen-induced expansion. ES NPs fully maintained their potential to spontaneously generate neurons, including DA neurons, whereas VM NPs did not. ES NPs also maintain their proliferative and developmental potential during cryopreservation and the freeze-thaw cycle, thus providing a useful source for cell replacement therapy for neurodegenerative disorders as well as biological and drug discovery studies.+ X8 q3 c1 K* E3 g" ?- @

DISCLOSURES

The authors indicate no potential conflicts of interest.

ACKNOWLEDGMENTS2 [# n, n1 q# w6 M8 M/ f2 X0 Y; B
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This work was supported by Udall Parkinson¡¯s Disease Center of Excellence grants P50 NS39793, MH48866, DAMD-17-01-1-0762, and DAMD-17-01-1-0763 and the postdoctoral fellowship program of Korea Science & Engineering Foundation (B.-S.S.).% i5 B! c  k' j# X/ ~4 J: D& [# {
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