hES cells culture
Human ES (hES) cell lines, H1 (provided by WiCell Research Institute) and HSF-6 (University of California, San Francisco, CA, USA), were cultured on mitotically inactivated mouse embryonic fibroblasts (MEFs) in DMEM/F12 medium with 20% knockout serum replacement, penicillin (100 IU/mL), streptomycin (100 μg/mL), 1 mmol/L L-glutamine, 1% non-essential amino acids, 0.1 mmol/L β-mercaptoethanol, and 4 ng/mL basic fibroblast growth factor (all from Invitrogen, Carlsbad, CA, USA). For the maintenance of undifferentiated hES cells, cultures were passaged about once every week by mechanic dissection and enzymatic treatments and then small clusters were transferred on freshly prepared MEF feeder.
In vitro differentiation of ES cells
Neural differentiation of hES cells was induced by co-culture on MS5 stromal cells or MS5 cells stably over-expressing sonic hedgehog (MS5-SHH). MS5 stromal feeder cells were maintained in α-minimum essential medium containing 10% fetal bovine serum and 2 mmol/L L
-glutamine (Barberi et al. 2003
). The MS5-SHH stable cell line has been established by viral transduction using retroviral construct expressing human SHH N-terminus followed by blasticidine (10 μg/mL, Invitrogen) selection (Park et al. 2005
). Undifferentiated hES colonies were detached from MEF feeders by incubation with 200 U/mL collagenase IV (Invitrogen) for 15 min at 37 °C, followed by gentle dissociation into small clusters with pipet and then cells were resuspended in serum-free N2
medium with 0.2 mmol/L ascorbic acid (AA; Sigma-Aldrich, New London, NH, USA). The clusters on a layer of MS5 stromal cells were cultured for 7 days, and then passaged on freshly prepared feeder of MS5-SHH, and further cultured for 14 days. At the end of the co-culture, clusters of 20-300 cells in N2
supplemented with 20 ng/mL basic fibroblast growth factor (bFGF), 10 ng/mL epidermal growth factor (EGF), and AA were replated on poly-L
-ornithine/fibronectin (PLO/FN)-coated dishes. After 7 days of culture in N2
+ bFGF + EGF + AA, cells were transferred on PLO/FN-coated glass coverslips as clusters or a single cell. NP cells were frozen by suspension of small clusters in N2
+ bFGF + EGF + AA media containing 10% dimethyl sulfoxide and placed in a Styrofoam container at -80°C to ensure a gradual decrease in temperature. After 24 h, frozen cells were moved to a liquid nitrogen tank. Frozen NP cells were thawed in a 37°C water bath, and then plated on PLO/FN-coated plates in N2
+ bFGF + EGF + AA media. For neuronal differentiation, the NP cells were cultured by withdrawing bFGF and EGF from the media for 14 days or more.
Cells were fixed in 4% paraformaldehyde in 1× phosphate buffered saline (PBS), rinsed with PBS, and then incubated with blocking buffer [PBS/10% normal donkey serum (NDS)/0.1% Triton X-100] for 30 min at 22°C. Fixed cells were incubated overnight at 4°C with primary antibodies diluted in PBS containing 2% NDS. The following primary antibodies were used: mouse anti-mouse Nestin (1 : 500; Chemicon, Temecula, CA, USA; http://www.chemicon.com
), rabbit anti-β-tubulin (1 : 2000; Covance, Princeton, NJ, USA; http://www.covance.com
), rabbit anti-GFAP (1 : 250; DakoCytomation, Glostrup, Denmark, http://www.dakocytomation.com
), mouse anti-O4 (1 : 100; R&D Systems, Minneapolis, MN, USA; http://www.rndsystems.com
), sheep anti-myelin basic protein (1 : 200; Chemicon), rabbit anti-tyrosine hydroxylase (TH, 1 : 250; Pel-Freez, Rogers, AK, USA; http://www.pelfreez-bio.com
). After additional rinsing in PBS, the coverslips were incubated in the appropriate Alexa 488- and Alexa 594-labeled secondary antibodies (Invitrogen; http://www.invitrogen.com
) in PBS with 2% NDS for 60 min at 22°C. After rinsing 3 × 10 min in PBS, cells were counter-stained using 1.5 μg/mL 4′,6-diamidino-2-phenylindole and then mounted onto slides in Gel/Mount (Biomeda corp., Foster City, CA, USA; http://www.biomeda.com
). Stained cells were analyzed under Axioskope 2 plus
fluorescence microscope (Carl Zeiss, Oberkochen, Germany).
Quantitative real-time RT-PCR analysis
Total RNAs were prepared from in vitro differentiated hES-NP cells using TriReagent (Sigma) followed by treatment with DNase I (Ambion, Austin, TX, USA). Two micrograms of total RNA were reverse-transcribed into cDNA using oligo (dT) primers, according to the SuperScript Preamplification Kit (Invitrogen). The cDNA was then analyzed by PCR using the following primers:
Glyceraldehyde-3-phosphate dehydrogenase: 5′-TGACATCAAGAAGGTGGTGAAGC-3′,5′-CCCTGTTGCTGTAGCCGTATTC-3′, Nestin: 5′-CAGCGTTGGAACAGAGGTTGG-3′,5′-TGGCACAGGTGTCTCAAGGGTAC-3′, β-tubulin III: 5′-CAACAGCACGGCCATCCAGG-3′,5′-CTTGGGGCCCTGGGCCTCCGA-3′, TH: 5′-GAGTACACCGCCGAGGAGATTG-3′, 5′-GCGGATATACTGGGTGCACTGG-3, Engrail-1: 5′-GCA ACCCGGCTATCCTACTTATG-3′,5′-ATGTAGCGGTTTGCCTGGAAC-3′, Nurr1: 5′-TTCTCCTTTAAGCAATCGCCC-3′, 5′-AAGCCTTTGCAGCCCTCACAG-3′, Pitx3: 5′-GGAATGGTCACCCTGACATGAG-3′, 5′-TGAAGGCGAACGGGAAGGTCT-3′, aromatic L-amino acid decarboxylase: 5′-CTCGGACCAAAGTGATCCAT-3′,5′-GTCTCTCTCCAGGGCTTCCT-3′, GFAP: 5′-GGCACGTGCGGGAGGCGGCC-3′, 5′-TCTCATCACATCCTTGTGC-5′, Myelin Basic Protein: 5′-AAGGACTCACACCACCCGGC-3′, 5′-TTTCAGCGTCTAGCCATGGG-3′, O4 : 5′-CTACTGCTCTGGGTCCCAGG-3′,5′-CTGCCACTGAACCGAGATGG-3′.
PCR reactions were carried out in a PCR Reaction Buffer (Promega, Madison, WI, USA) containing 1.4 nmol/L of each primer and 1.25 U Taq I DNA polymerase (Promega). Samples were amplified in a Thermal Cycler (Eppendorf, Westbury, NY, USA) and a Continuous Fluorescence Detector (MJ Research, Waltham, MA, USA) using DNA Engine Opticon software under the following conditions: denaturing step at 94°C, 30 s; annealing step at 55°C, 30 s; extension step at 72°C, 30 s for 50 cycles and a final extension step at 72°C, 10 min. Relative expression of mRNAs was assessed by normalizing the levels of cDNA to the signal from glyceraldehyde-3-phosphate dehydrogenase mRNA.
Cell counting and statistical analysis
Cell density of three neural lineages (neurons, astrocytes, and oligodendrocytes) and DA neurons was determined by counting the numbers of β-tubulin+,GFAP+,O4+, and TH+ cells per field at ×100 magnification using an Axioskope 2 plus fluorescence microscope (Carl Zeiss). Seven visual fields were randomly selected and counted for each sample. Numbers presented in figures represent the average percentage and SEM of TH+ cells over β-tubulin+ from three samples per each hES-NP cell passage.
All statistical analyses were conducted using SAS v 9.1 (SAS Institute, Cary, NC, USA) for Windows 2000 Professional. The analysis was conducted on these means using a mixed models ANOVAprocedure (SAS) to determine possible statistical differences between group means.
Analysis of DA release
Differentiated hES-NP cells were treated with 200 μL of serum-free N2
medium supplemented with 56 mmol/L KCl in six-well plates. Their media were collected after 30 min and concentrated solutions of perchloric acid (PCA) were added to a final concentration of 0.1 mol/L PCA/0.1 mmol/L EDTA. These deproteinated samples were centrifuged, and their supernatants were kept at -80°C until further analysis. Samples were further purified by using a 0.22 μm nylon filter (Osmonics, Inc., Trevose, PA, USA; http://www.osmonics.com
). The DA content of the supernatants was measured by reverse-phase HPLC using a Velosep RP-18 column (100 × 3.2 mm; Brownlee Labs, Shelton, CT, USA; http://las.perkinelmer.com
) and an ESA Coulochem II electrochemical detector (ESA, Inc., Chelmsford, MA, USA; http://www.esainc.com
) equipped with model 5014 analytical cell as we previously described (Wachtel et al., 1997
). The mobile phase was composed of 0.1 mol/L sodium phosphate buffer (pH 2.65), 0.1 mmol/L EDTA, 0.4 mmol/L sodium octyl sulfate, and 9% (vol/vol) methanol and operated at a flow rate of 0.8 mL/min. The potential of the guard cell was set at 330 mV. The potential of the first electrode in the analytical cell was set at 0 mV and the second at 310 mV. L
-DOPA, DA, dihydroxyphenyl acetic acid, and homovanillic acid were identified by retention time and quantified by their peak using an EZChrom Chromatography Data System (ESA, Inc.). The limit of detection for all compounds was <1 pg. DA content of each sample was normalized with the amount of total cellular proteins. For protein measurement, the cells were harvested in 0.1 mol/L PCA/0.1 mmol/L EDTA, precipitated, resuspended in 0.2% Triton X-100/10 mmol/L potassium phosphate buffer (pH 7) and sonicated. The protein content was measured by the Bradford method (Bio-Rad Laboratories, Hercules, CA, USA; http://www.bio-rad.com