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发表于 2009-3-5 00:14 |只看该作者 |倒序浏览 |打印

作者：Soojung Shina, Maisam Mitalipovaa,d, Scott Nogglec, Deanne Tibbittsa,d, Alison Venableb, Raj Raoa, Steven L. Sticea 9 b4 O" t3 h5 `( e7 ^

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      【摘要】) D& b6 l  h  G  a4 y: _- W
   Research on the cell fate determination of embryonic stem cells is of enormous interest given the therapeutic potential in regenerative cell therapy. Human embryonic stem cells (hESCs) have the ability to renew themselves and differentiate into all three germ layers. The main focus of this study was to examine factors affecting derivation and further proliferation of multipotent neuroepithelial (NEP) cells from hESCs. hESCs cultured in serum-deprived defined medium developed distinct tube structures and could be isolated either by dissociation or adherently. Dissociated cells survived to form colonies of cells characterized as NEP when conditioned medium from human hepatocellular carcinoma HepG2 cell line (MEDII) was added. However, cells isolated adherently developed an enriched population of NEP cells independent of MEDII medium. Further characterization suggested that they were NEP cells because they had a similar phenotype profile to in vivo NEP cells and expression SOX1, SOX2, and SOX3 genes. They were positive for Nestin, a neural intermediate filament protein, and Musashi-1, a neural RNA-binding protein, but few cells expressed further differentiation markers, such as PSNCAM, A2B5, MAPII, GFAP, or O4, or other lineage markers, such as muscle actin,  fetoprotein, or the pluripotent marker Oct4. Further differentiation of these putative NEP cells gave rise to a mixed population of progenitors that included A2B5-positive and PSNCAM-positive cells and postmitotic neurons and astrocytes. To proliferate and culture these derived NEP cells, ideal conditions were obtained using neurobasal medium supplemented with B27 and basic fibroblast growth factor in 5% oxygen. NEP cells were continuously propagated for longer than 6 months without losing their multipotent cell characteristics and maintained a stable chromosome number.
      【关键词】 Embryonic stem cell Differentiation Neuroepithelial stem cell Defined culture5 h( O5 u* t, n4 e
   INTRODUCTION
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After human embryonic stem cells (hESCs) were established . In addition, the supply of fetal neural tissue is limited because of ethical concerns.
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Neuronal stem cells derived from cancer cell lines have been considered a potential alternative cell source with unlimited capability for cell proliferation, but there is significant concern that cancer cells may be unstable and prone to tumorigenesis .: W" d9 Q7 I& m
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Neuroepithelial (NEP) stem cells are self-renewing multipotent cells that can differentiate into neurons, oligodendrocytes, and astrocytes .
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Neural developmental pathways can be delineated through ESC studies. Neuronal development in rodents is a well-documented stepwise process, much like hematopoietic stem cell differentiation. Mouse neurectoderm forms the earliest pluripotent neural stem cells, called NEP cells, which then differentiate further into neuronal-restricted precursor cells or glial-restricted precursor cells . These cells form neural rosettes and are Nestin and Musashi 1 positive.6 U& ^( M' v) v/ D

Nestin has been the primary antigen used as a marker of NEP cells . Therefore, expression of SOX genes can serve as conservative criteria for NEP characterization.
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A variety of methods have been used to derive NEP cells from ESCs  were cultured with conditioned medium from the human hepatocellular carcinoma HepG2 cell line (MEDII), they developed preferentially into neurectoderm. In this study, factors required for the neural differentiation of hESCs were examined and conditions allowing further proliferation were optimized. We show that adherent cultures of hESCs in serum-deprived medium without feeder layers gave rise to a rosette-enriched population. Characterization of this population showed that the cells were multipotent NEP cells with proper phenotype marker and SOX genes expression profiles and that they were able to differentiate further to both A2B5-positive and PSNCAM-positive precursor cells. Thus, this study demonstrates that derived NEP cells can be cultured more than 6 months in optimized conditions without the cells losing their capacity for neural and glial differentiation while maintaining a stable chromosome number.
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MATERIALS AND METHODS' F& A5 z% C- f5 x, b

Human Embryonic Stem Cell Culture
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Human ESC lines BG01 and BG02 used in this experiment were cultured on mouse embryonic fibroblasts (MEF) layer, inactivated by mitomycin C .
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Conditioned Medium Preparation

Human hepatocellular carcinoma (HepG2) cells (ATCC HB-8065) were seeded at a density of 9.4 x 104 cells/cm2 and proliferated for 3 days in DMEM/F12 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin. To produce conditioned medium, cells were washed twice with phosphate-buffered saline (PBS), and DMEM/F12 medium without serum supplement was added at a ratio of 0.285 ml/cm2. In 3 days, conditioned medium was collected and stored at 4¡ãC for less than 5 weeks as MEDII.3 a/ a$ d1 t, O% w% z! N
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Antibodies and Immunocytochemistry
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Cells plated on polyornithine/laminin-coated permanox slides were washed in PBS and fixed with 4% paraformaldehyde/4% sucrose in PBS for 15 minutes. Fixed cells were washed two times with PBS before staining. Permeabilization and blocking was carried out in blocking buffer consisting of 0.1% Triton, 3% goat serum in Tris buffer for 40 minutes. For cell-surface antigen, permeabilization was excluded. Primary antibodies were applied in blocking buffer for 2 hours at room temperature and washed three times in blocking buffer before secondary antibody application. Secondary antibodies of goat anti-mouse Alexa-conjugated, goat anti-rabbit Alexa-conjugated (Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com) were diluted at 1:1,000 in blocking buffer and applied to cells for 40 minutes at room temperature. After two washes in PBS, 4',6'-diamidino-2-phenylindole was applied for nuclear staining for 10 minutes, and cells were observed under the fluorescence microscope. For flow cytometry application, cells were harvested by trypsinization and suspended in PBS to be fixed and stained using the same procedure coupled with serial centrifugation at 3,000 rpm and resuspension in PBS. For negative controls, first antibodies were omitted and the same staining procedure was followed. Primary antibodies and dilutions used included the following: mouse anti-Nestin (1:100; R&D Systems Inc., Minneapolis, http://www.rndsystems.com), rabbit anti-Nestin (1:200; Chemicon), rabbit anti-Musashi 1 (1:500; Chemicon), mouse anti-beta III tubulin (1:400; Sigma), rabbit anti-Tuj1 (1:500; Covance, Princeton, NJ, http://www.covance.com), mouse anti-Hu (1:50; Molecular Probes), mouse anti-muscle actin (1:50; DAKOCytomation, Glostrup, Denmark, http://www.dakocytomation.com), mouse anti¨C feto protein (1:50; DAKOCytomation), rabbit anti-GFAP (1: 50; Sigma), mouse anti-O4 (1:10; Chemicon), mouse anti-PSNCAM (1:400; Abcys, Paris, http://www.abcysonline.com), and mouse anti-A2B5 (1:100; a gift from Mayor Proschel).

Effect of ES, DN2, and MEDII Media on Differentiation of hESCs in a Three-Stage Process
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The differentiation procedure is outlined in Figure 1 and divided into three stages to assist in characterizing the progression of in vitro neural differentiation. After manual passage onto fresh feeder cells, hESCs were allowed to proliferate in ES medium for 7 days (stage 1). Cell differentiation was then induced with either DN2, MEDII, or ES medium for another 7 days (stage 2). DN2 medium is DMEM/F12-based medium supplemented with N2 (Gibco), L-glutamine, penicillin/streptomycin (P/S), and 4 ng/ml bFGF. MEDII medium for this study is DN2 medium supplemented at 50% (unless otherwise noted) with conditioned medium (described above). To understand and follow the differentiation steps applied here, phenotype marker expression was examined at the time intervals described in Figure 1. At stages 1, 2, and 3, populations were harvested and the markers Musashi-1, Nestin, and Oct-4 were observed. Immunocytochemical analysis was also performed on the adherent cell population. The cells at both stages were double-stained with Nestin and Oct-4 and observed under the fluorescence microscope for immunocytochemical examination associated with morphology. Groups that displayed phenotypic difference were then subjected to quantitative analysis for these same markers using flow cytometry. All experiments were replicated three times unless otherwise noted.( U; f7 M# Y) `1 }3 d% k, x+ ?4 L

Figure 1. Procedure for adherent derivation of human embryonic stem (ES) cells into neuroepithelial cells.

Effect of ES, DN2, and MEDII Media on Differentiation of Stage 2 Cells in Adherent Cell Culture Without Feeder Cells
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To improve NEP cell derivation, a method using adherent differentiation was exploited. It was possible to isolate subpopulations of stage 2 cells that had infiltrated under the feeder layer to attach firmly on culture plates. To test the effect of ES, DN2, and MEDII media on this derivation method, the mouse feeder layer was physically removed from each group of stage 2 cells in calcium/magnesium-free PBS. The remaining cells were cultured another 3 days in respective media as described in Figure 1 (stage 3). At stage 3, populations were harvested from each group, and morphology and phenotype marker expression of Oct-4, Nestin, and Musashi 1 was observed as described before using flow cytometry and immunocytochemistry for Oct-4, Nestin, and Musashi-1.+ l' T8 u# ?! w/ {5 O0 R

Effect of MEDII Medium and Low Cell Density on Cell Survival of Stage 2 Differentiating Cells

The effect of MEDII medium was examined using single-cell passage of stage 2 cells in the medium supplemented with four different concentrations of MEDII. As shown in Figure 1, stage 2 MEDII-cultured cells were obtained. The resulting adherent cells were dissociated in 0.02 M EDTA containing PBS, and 104 cells/cm2 were then plated on polyornithine- and laminin-coated dishes in different concentrations of MEDII medium (0%, 25%, 50%, 100%). After 10 days of culture in respective media, cells were harvested and derivation efficiency (resulting cell number/starting cell number x 100) was determined over four replicates. In addition, TDT-mediated dUTP nick-end labeling (TUNEL) assay was performed at 6 and 24 hours after plating in 0% or 50% MEDII to determine levels of apoptosis in these cultures. The TUNEL assay was performed according to the manufacturer¡¯s instructions (Molecular Probes), and cells were subsequently analyzed by flow cytometry./ F; N) C6 Z4 j& s5 J

Characterization and Examination of Differentiation Capacity of Derived NEP Cells; e( j. r7 L8 i$ L- l
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Rosette-forming NEP cell populations from stage 3 cells derived in DN2 and MEDII media either by adherent feeder removal or by dissociated culture were characterized by immunocytochemistry. The markers included early neural stem cell markers (Nestin, Musashi1) for positive expression and mesodermal marker muscle actin, endodermal marker -fetoprotein, pluripotent marker Oct-4, and late-stage neuronal and glial markers (A2B5, PSNCAM, ß III tubulin, Hu, GFAP, O4) for negative expression. For terminal differentiation, NEP cells were cultured in neurobasal medium (Gibco) and supplemented with B27 (Gibco), L-glutamine, and penicillin/streptomycin without bFGF for 14 days. For oligodendrocyte differentiation, NEP cells were exposed to 5 µg/ml platelet-derived growth factor (Upstate, Lake Placid, NY, http://www.upstate.com) and 50 µM 3T3 (Sigma) for 6 days before terminal differentiation. Differentiated cells were characterized using the restricted progenitor markers PSNCAM, A2B5, and the postmitotic neural marker Hu, the neuron-specific tubulin, and ß-III tubulin, the oligodendrocyte marker O4, and the astrocyte-specific marker GFAP.# B$ N* q* J" `& l3 c7 t
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Effect of Medium, Supplement, Growth Factor, and Oxygen Conditions on Proliferation and Viability of Subcultures of Derived NEP Cells
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Effect of Culture Medium To obtain a more uniform subculture system, two different kinds of base media¡ªDMEM/F12 (D) and neurobasal medium (N)¡ªwere tested with supplements of N2-, B27-, or MEDII-conditioned media. Stage 3 NEP cells were allocated into four different media: DN2, NN2 (neurobasal medium supplemented with N2), NB27 (neurobasal medium supplemented with B27), and 50% MEDII in DN2 medium, as described above with the same supplement of L-glutamine, P/S, and 4 ng/ml bFGF. After 12 days of culture, cells were harvested and examined for morphology and viability using the Guava ViaCount (Guava Technologies, Hayward, CA, http://www.guavatechnologies.com) flow cytometry assay. Briefly, the Guava ViaCount reagent combines two different DNA dyes. One dye binds to the nucleus of every cell to give a total cell number, and the other dye binds differentially to only nonviable cells. The data collected include total cell number and viability of the sample.

Subculture of NEP Cells NEP cells derived from either DN2 or MEDII were further propagated in NB27 with L-glutamine, P/S, 10 ng/ml LIF, and 20 ng/ml bFGF on polyornithine¨Ccoated and laminin-coated dishes. Cells were continuously passaged either by mechanical trituration or by trypsin (1 x 105/cm2) to be replated. After more than 6 months in culture, NEP cells were characterized as described above, metaphase spreads were prepared using standard protocols, and chromosomes were counted. Briefly, cells were treated with 0.02 µg/µl colcemide for 1.5 hours and harvested to be hydrated and fixed. Chromosomes were stained with Giemsa and then counted (15 cells).
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Effect of LIF and bFGF on Subcultured NEP Cells Two groups of cultured NEP cells, one less than 1 month and the other approximately 6 months in NB27 (described in previous section), were dissociated by 0.05% trypsin to obtain a single-cell suspension, and 50,000 cells/cm2 were plated in one of the subculture media on polyornithine- and laminin-coated dishes. Two concentrations of two growth factors (LIF, 0 or 10 ng/ml; bFGF, 0 or 20 ng/ml) in NB27 were applied to cells. Cells were harvested from each group, and nuclei were counted by flow cytometry on days 1 and 14. Plating efficiency rate was calculated as the ratio of cells harvested to cells plated on day 1. Proliferation was measured on day 14. For each replicate, counted nuclei from the four treatment groups were added to obtain an overall total. The total cell number within each group was then divided by the overall total cell number and expressed as a percent. This data conversion was carried out to reduce biological variation due to replicate preparation.

Effect of Oxygen Concentration on Subcultured NEP Cells To examine the effect of oxygen concentration on cell proliferation and viability, the subcultured NEP cells (described above) were dissociated by 0.05% trypsin, and 2 x 105 cells/cm2 were plated and propagated using the NEP subculture process, except one group was cultured at oxygen concentration of 20% and the other group was cultured at 5% O2. After 7 days of culture, cells were harvested to calculate total cell number and viable cell number, as described previously.# O( ^# B, c5 m0 `2 F
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Expression of SOX Genes in Freshly Derived and Long-Term Subcultured NEP Cells Along with their differentiation potential to neuron and glial cells and NEP marker expression, expression of SOX genes was examined both in freshly derived (early) and long-term subcultured (late) NEP cells. To examine expression of SOX genes, RNA was isolated from early and late NEP cells using Trizol. For reverse transcription¨Cpolymerase chain reaction (PCR), 2 µg of total RNA from each sample was treated with DNase (Promega, Madison, WI, http://www.promega.com). RNA 1 µg was converted to cDNA by using the Superscript III kit (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) using oligo dT as a primer, and 1 µg was prepared without reverse transcription to serve as control for exclusion of genomic amplification. ReadyMix REDTAQ (Sigma) was used, and 50 ng of cDNA was added for the PCR reaction for 35 cycles with denaturing at 95¡ãC for 30 seconds, annealing at 60¡ãC for 30 seconds, and elongation at 72¡ãC for 30 seconds. For SOX1, commercial primer and probe for real-time PCR were used, and 25 ng of cDNA was subjected to real-time PCR (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com) according to the manufacturer¡¯s instructions. After amplification, products were separated on 2% agarose gel and visualized using ethidium bromide (EtBr) staining under UV light. Primer sequences (forward and reverse), size of the product, and PCR condition were as follows: SOX2 (5'-AGT CTC CAA GCG ACG AAA AA-3' and 5'-GCA AGA AGC CTC TCC TTG AA-3', 141 bp); SOX3 (5'-GAG GGC TGA AAG TTT TGC TG-3' and 5'-CCC AGC CTA CAA AGG TGA AA-3', 131 bp); ß actin (4326315E, Applied Biosystems); SOX1 (Hs00534426 s1, Applied Biosystems).$ Y6 m1 ~4 G4 h2 X6 P# U% o

Statistical Analysis For each parameter, significance of main effects was determined using the GLM procedure of SAS 8.01. Significance of differences among individual treatment means was determined by the least-square means method. Differences were considered significant at p ; ?7 {% C: V8 ]' c- R
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RESULTS
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Effect of ES, DN2, and MEDII Media on Differentiation of ES Cells Cultured with Feeder Cells0 H' P# W" j% E* p1 G* P% I
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After 7 days of culture, hESCs in ES medium (stage 1) proliferated to form multicell layers. These cells expressed both Nestin (Fig. 2A) and Musashi-1 and the pluripotent marker Oct-4. When expression was quantitated for each phenotype marker using flow cytometry, 74.9%, 77.5%, and 88% of total cells were positive for Oct-4, Nestin, and Musashi-1, respectively (Table 1). These results showed that in ES medium, ESC transition to NEP cells occurred gradually, with intermediate stages expressing both Oct-4 and the Musashi-1, Nestin. This overlap in expression was observed using both flow cytometry and immunocytochemistry, including double-staining for both Nestin and Oct-4 (Fig. 2A).8 p" C% ~% k% S! j8 {7 `$ a

Figure 2. (A¨CC): Phenotype marker expression of cells counter-stained with Oct-4 (green), Nestin (red), and 4',6'-diamidino-2-phenylindole (blue). (A): Stage 1 cells double stained both by Oct4 and Nestin. (B): Stage 3 cells developed in DN2 medium¨Cenriched rosette formation (MEDII-cultured cells were similar, so the data are not shown). (C): Stage 3 cells developed in embryonic stem (ES) medium. Bar = 100 µm. Stage 1 cells are ES cells that have proliferated for 7 days in ES medium. Stage 2 cells are stage 1 cells that have been further subjected to either ES or MEDII medium for 7 days. Stage 3 cells are stage 2 cells that have been further cultured for 3 days in respective medium with the feeder layer removed.
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Table 1. Phenotype marker expression changes over time6 E/ M1 f* }- Z% X; U

When the stage 1 cells were cultured for an additional week in either DN2, MEDII, or ES media (stage 2), resulting colony morphologies were compared and differences were observed between ES medium and DN2 or MEDII media. DN2- and MEDII-cultured stage 2 cells developed neural tube¨Clike structures (Fig. 3A), whereas ES medium¨Ccultured stage 2 cells failed to form these structures (Fig. 3B). When cells were examined under the microscope, nuclear staining indicated the distinct cell arrangement (neural tube¨Clike structures) developed in MEDII- and DN2-derived populations that was not seen in ES-derived populations. There was no morphological difference between DN2- and MEDII-derived stage 2 cells; therefore, quantitative data were obtained only for ES- and MEDII-derived stage 2 cells (Table 1). The pluripotent cell expression marker Oct-4 decreased in both groups from 74.9% (stage 1) to 32.6% and 18.8% for ES and MEDII stage 2 groups, respectively (p 1 l6 x2 e, \7 L9 Q2 `9 a5 j

Figure 3. (A, B): Phase-contrast image of stage 2 cells (A). Cells cultured in MEDII medium (DN2-cultured cells were similar, so the data are not shown). (B): Cells cultured in embryonic stem (ES) cell medium. (C¨CD): Phase-contrast image of stage 3 cells. (C): Neuroepithelial cells in adherent cell culture without feeder cells in MEDII medium (DN2-cultured cells were similar, so the data are not shown). (D): Cells cultured in ES medium. Bar = 100 µm. Stage 2 cells are stage 1 cells that have been further subjected to either ES or MEDII medium for 7 days. Stage 3 cells are stage 2 cells that been further cultured for 3 days in respective medium with the feeder layer removed.
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Effect of ES, DN2, and MEDII Media on Differentiation of Stage 2 Cells in Adherent Cell Culture Without Feeder Cells

Similar to results from stage 2 cells, we found differences for stage 3 cells cultured in ES medium compared with cells cultured in MEDII or DN2 media after feeder cell removal. After feeder cell removal, cell culture gave rise to enriched rosette formation in MEDII or DN2 media, characteristic of NEP cell formation (Fig. 3C), but ES medium¨Cderived cell culture resulted in cells with large nucleus-to-cytoplasmic ratios, characteristic of ESCs (Fig. 3D). Both MEDII and DN2 groups developed a similar differentiation pattern with distinct structure of neural tube¨Clike formation  and further rosette-enriched populations.
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In addition to microscopic examination, quantitative data obtained from whole populations indicated differences between cell populations. When cells were differentiated in MEDII medium, the percent of cells expressing Oct-4 was decreased dramatically (74.9% at stage 1 versus 17.4% at stage 3). Furthermore, stage 3 MEDII-cultured cell populations with rosette structures showed expression of Nestin and Musashi-1, markers found in early neural stem cells. However, most stage 3 ES medium¨Ccultured cell populations retained their Oct-4 expression even after spontaneous differentiation (74% at stage 1 versus 62.8% at stage 3). In accordance with the flow cytometry results, immunocytochemistry demonstrated that for cells cultured in ES medium, stage 3 cell populations were positive for both Nestin and Oct-4 (Fig. 2C), whereas rosette-forming stage 3 cells cultured in MEDII medium had only increased Nestin staining without Oct-4 expression (Nestin /Oct-4¨C Fig. 2B). These results indicate that in adherent cell cultures without feeder cells, DN2 and MEDII medium promote differentiation to NEP cells whereas ES medium does not.
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Effect of MEDII Medium and Low Cell Density on Cell Survival of Stage 2 Differentiating Cells (Tube-Like Structure¨CForming Cells)" ~, y7 \) N! a. _
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To obtain enriched populations of the desired cells (Nestin /Oct-4¨C), we attempted single-cell passaging to propagate the differentiating cells in various concentrations of MEDII. A 50% MEDII medium was used based on previous mESC MEDII neural differentiation studies . However, no previous reports have tested different concentrations of MEDII on single-cell or clonal propagation of NEP cells.3 D8 D$ u- F! Q9 E; }0 m" o) d4 Z" X

In an attempt to propagate stage 2 cleaner populations, these cells were single-passaged in one of four concentrations of MEDII serum¨Cdeprived medium in feederless cultures (Table 2). Regardless of treatment, significant cell death was observed; without MEDII, few cells survived and/or propagated (1.9% ¡À 1.2% cell survival). However, when these cultures were supplemented with as little as 25% MEDII-conditioned medium, there was a tenfold increase in surviving colony-forming cells (22.3% cell survival). Cell survival and cell propagation were further improved and optimized at the 50% MEDII level, with 40,200 (40.2%) of the original cells surviving or propagating over the 5 days in culture. Although MEDII treatment significantly increased the number of cells at 10 days of culture, it was obvious that most cells passaged in this manner were lost during the first 24 hours of culture. Therefore, a TUNEL assay was used to determine if these cells were undergoing apoptosis. At 6 hours of culture, 25% of both the 0% MEDII and 50% MEDII single-passaged cell cultures had undergone apoptosis. The apoptotic population increased to 36% and 38% for 0% and 50% MEDII groups, respectively, by 24 hours of culture.

Table 2. Effect of MEDII supplement on percent cell survival of dissociated stage 2a cells in serum-deprived and feeder cell¨Cdeprived culture conditions (means ¡À S.E.)9 i$ v7 p/ c" b0 Z( C8 Q
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Characterization and Examination of Differentiation Capacity of Derived NEP Cells

Rosette-forming NEP cells were enriched in DN2 and MEDII stage 3 groups and from clonally passaged cells. To characterize NEP cells, rosette structures were examined by using a combination of positive and negative markers. Nearly 100% of rosette-forming cells were positive for the early NEP markers Nestin and Musashi 1 (Figs. 4A, 4B) and negative for later stages of differentiation markers A2B5, PSNCAM, ß III tubulin, Hu, GFAP, and O4. In addition, they did not express the mesodermal marker muscle actin, the endodermal marker  fetoprotein, or the pluripotent marker Oct4. Removal of FGF and LIF from the culture medium resulted in further differentiation of NEP cells to form intermediate precursors staining positive for A2B5 or PSNCAM (Figs. 4C, 4D). After 14 days of culture in neurobasal medium supplemented with B27 without bFGF, terminally differentiated cell cultures contained neurons positive for Hu and Tuj1 (Fig. 4E), astrocytes stained with GFAP (Fig. 4F), and oligodendrocytes stained with O4 (Fig. 4G).

Figure 4. (A, B): Rosette-forming neuroepithelial cells stained with Nestin (A) or Musashi (B). (C, D): Intermediate precursor cells after removal of basic fibroblast growth factor (bFGF) and leukocyte inhibitory factor from the culture medium. (C): Cells stained for A2B5 (red) and 4',6'-diamidino-2-phenylindole (DAPI) (blue). (D): Cells stained for PSNCAM (red) and DAPI (blue). (E¨CG): Terminally differentiated neurons and astrocytes after 14 days of culture in neurobasal medium supplemented with B27 and L-glutamine, without bFGF. (E): Neurons double stained for Hu C/D (green), Tuj1 (red), and DAPI (blue). (F): Astrocyte stained with GFAP (red) and DAPI (blue).
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Figure 4. cont. (G): Oligodendrocyte stained with O4 (green) and DAPI (blue). (H): Long-term (10 months) cultured neuroepithelial cells stained with Nestin (green), Musashi (red), and DAPI (blue). Bar = 100 µm.
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Effect of Medium, Supplement, Growth Factor, and Oxygen Conditions on Proliferation and Viability of Subcultures of Derived NEP Cells
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Effect of Culture Medium The effects of base media and supplements on survival of stage 3 NEP cells were determined to establish the most effective subculture conditions. A higher percentage of cells cultured in NN2 survived compared with cells cultured in DN2 (33.8% DN2 versus 75.4% NN2, p  .05).

Subculture of NEP Cells These derived NEP cells have been cultured for more than 6 months without losing this characteristic and maintained a normal chromosomal number. Cells retained expression of Nestin and Musashi-1 (Fig. 4H), and when terminally differentiated in medium lacking bFGF and LIF, the cell population included both neurons and glial cells (data not shown). To further characterize freshly derived and subcultured NEP cells, we examined SOX1, SOX2, and SOX3 gene expressions in Oct-4 ¨Cnegative early and late NEP cells. Both groups expressed SOX2 and SOX3 (Fig. 5A). By using real-time PCR, the SOX1 gene was amplified and the amplicon was visualized by EtBr staining. The SOX1 gene was also expressed in both cell groups (Fig. 5B). When subcultured NEP cell metaphase spreads were visualized by Giemsa staining, all 15 samples examined were stable with 46 XY chromosome numbers.6 |3 Y& }3 A5 }$ e4 x/ J! s

Figure 5. Both early and late neuroepithelial cells express SOX1, SOX2, and SOX3. Reverse transcription¨Cpolymerase chain reaction analysis of the expression of SOX1 (B), SOX2, and SOX3 (A). Panels show 2% agarose gels stained with ethidium bromide. Genomic contamination was monitored by sample prepared without reverse transcription (¨C). For size marker, 1-kb DNA ladder was used. The size of SOX2 is 141, and SOX3 is 131.

Effect of LIF and bFGF NEP cells propagated in NB27 for approximately 1 or 6 months were subjected to different concentrations of LIF and bFGF, and cell survival as well as cell proliferation was determined at 14 days (Table 3). For early NEP cells (1 month), the addition of LIF, bFGF, or LIF bFGF had no effect on plating efficiency, which was only approximately 50%, indicating a relatively high rate of cell death. In contrast, the presence of bFGF increased cell proliferation more than fourfold (8.9% versus 38.5%; p / G! v1 q% A) ^. F( Q" r
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Table 3. Effect of basic fibroblast growth factor (bFGF) and leukemia inhibitory factor (LIF) supplementation on plating efficiency and proliferation of neuroepithelial cells (means ¡À S.E.)
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Effect of Oxygen Concentration After 7 days of culture in NB27 medium, total NEP cell number was approximately 25% greater in 5% oxygen compared with 20% oxygen (p
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Table 4. Effect of oxygen (O2) concentration on viability and proliferation of neuroepithelial cells (means ¡À S.E.)* a5 ^& Q! G2 M5 y! E7 K) k
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The overall objective of these experiments was to obtain efficient neural differentiation of hESCs and to develop a defined medium that would be supportive of NEP cells and allow enzymatic passage, thereby facilitating more controlled and refined future studies. In contrast to previous reports, we used both immunocytochemistry and flow cytometry analysis to obtain both quantitative and morphological information on NEP formation at various stages of in vitro differentiation and culture conditions. Most studies investigating mouse and human ESC differentiation to neural progenitors have used methods involving cell aggregation or EB formation. EB formation in serum-containing medium included cells differentiated into NEP cells , and removal of the feeder cell layer produced homogenous rosette formation from homogenous spread of cells in adherent culture conditions.7 X6 s/ y; L/ p# k; v
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In an attempt to follow the spatial and temporal differentiation of ESCs to neural lineages, we divided the process into three stages. We found that Oct-4 expression gradually decreased with the onset of expression of Nestin and Musashi-1, markers associated with NEP cells. At an initial stage (stage 1), when cells were allowed to proliferate in ES medium, most cells were positive for both pluripotent and NEP cell markers. This Oct4 and Nestin double staining has not been reported in other species. However, in mESCs, an intermediate cell status was reported as primitive ectoderm-like cells .

At stage 3, we found that removal of LIF, nonessential amino acids, KSR, and undefined factors in serum forced ESCs to choose a neurectodermal fate. Rosette formation was not promoted when cells were cultured in ES medium with these factors included. Instead, cells left in ES medium retained their Oct-4 expression and delayed progression to a more differentiated state. This finding is similar to that seen with spontaneous differentiation. For example, Reubinoff et al. . Our results indicated that the total cell number expressing Oct4 was higher for stage 3 than for stage 2 for the ES medium group. This surprising result may be due the techniques used rather than a change in Oct4 expression in this group. During feeder cell removal, cells were separated into two populations, one removed with the feeder layer and the other remaining to proliferate further in ES medium. It is likely that spontaneously differentiating Oct4-negative cells were removed, leaving Oct4-retaining cells behind in the ES medium group.9 F! J$ j% K% I9 }6 @; g
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MEDII added to DN2 medium did not improve tube-like structure formation (stage 2) or subsequent progression to stage 3 adherent colonies. The effect of MEDII was distinct, however, on low-cell-density NEP cell derivation. When tube structure¨Cforming cells were dissociated and passaged in DN2, more than 98% of cells died. This finding is similar to results obtained with mouse cells. Tropepe et al. .
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We also tested the effects of the growth factors LIF and bFGF on subculture of NEP cells. Mouse neural stem cells have been shown to be dependent on bFGF  reported that LIF had no effect on proliferation of derived NEP after 14 days of culture. However, we found that after 6 months, culture in LIF-containing medium increased cell responsiveness and cell proliferation was improved.! o) ]* Y* m  @1 O( T" ~- S

Physiological oxygen concentration does not exceed 5%; however, in conventional cell culture, oxygen concentration is maintained at 20%. In rat CNS stem cell culture, it has been reported that reduced oxygen concentration helped to improve cell proliferation and to reduce apoptosis . We tested whether reduced oxygen concentration produces the same advantage on the growth of NEP cells derived from hESCs. In agreement with this previous study, low oxygen concentration improved cell proliferation rates approximately 25% after 1 week of culture. Because there was no difference in viability as measured by flow cytometry, the increased cell numbers do not seem to be due to increased initial cell survival.8 f1 k0 i$ x6 q% G  _
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In this study, SOX genes were used to further characterize derived NEP cells and long-term cultured NEP cells. Among characterization markers, Nestin and Musashi1 have been primary phenotype markers for these cells , and we observed that proliferating hESCs expressed SOX2 and SOX3 (unpublished data). In this study we showed that NEP cell cultures expressed all three SOX genes. Both early and late NEP cells expressed SOX1, SOX2, and SOX3, and there was no difference in expression between the two populations. These results indicate that expression of SOX genes in the absence of Oct4 can be used as further verification for NEP cells.

CONCLUSION
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In this study, we showed that NEP cells can be derived from hESCs efficiently by adherent differentiation in defined medium. Derived NEP cells were broadly characterized with phenotype markers and expression of SOX genes; in addition, differentiation capacity was similar to that of in vivo purified human NEP cells . Further NEP cell subculture conditions were optimized, and cells were propagated successfully for more than 6 months without loss of differentiation potential or stable chromosome number. Our efficient derivation and proliferation of NEP cells demonstrates that this system can serve as an in vitro model for the examination of human neural development. A defined culture system would be ideal for further studies of effects of extrinsic factors on neuronal cell fate decision. In addition, long-term cultured NEP cells may be good candidates for replacement cell therapy, with little possibility of pluripotent cell contamination.9 ]  f+ k1 B9 K9 Y& |# e2 m$ |
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ACKNOWLEDGMENTS5 {5 V4 s& q& Q" [) m

We wish to thank Deb Weiler for preparing feeder layers; Karen Jones and Olivia Wei, Kate Hodges and Allison Adam for flow cytometry support; and Mary Anne Della-Fera for manuscript preparation. This work was supported in part by BresaGen and hESC Supplement to R21NS44208 (NIH).6 p% O! I. S( c$ `. e0 O+ [7 s
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DISCLOSURES3 V# p6 V  p4 J, u
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S.L.S., M.M., and D.T. have acted as consultants for Bresagen within the last 2 years.
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