Rhesus monkey (Macaca mulatta) ESC lines were obtained from the Oregon National Primate Research Center, Beaverton, OR. Oregon Rhesus Macaque Embryonic Stem (ORMES) -6 and -7 lines were maintained on feeder layers of mouse embryonic fibroblast (mEF) cells that had been mitotically inactivated. Cell lines were maintained in Dulbecco's Modified Eagle/F-12 medium (DMEM/F12, 11320-033, Invitrogen, Carlsbad, CA) supplemented with 15% fetal bovine serum (Hyclone, Logan, UT), 1% minimum essential medium (MEM) non-essential amino acids, 1mM L-glutamine, 0.1mM β-mercaptoethanol, and 0.1% gentamycin in 60mm cell culture dishes (Mitalipov et al., 2006
). Cultures were incubated at 37° C in 5%CO2
. Components for cell culture medium were purchased from Sigma (St. Louis, MO) unless otherwise noted. All experiments and analyses were repeated 5 times for each cell line with colonies that were cultured in separate dishes so that N=5 for each analysis for ORMES-6 and -7.
Approximately 40 random colonies per dish for both ORMES-6 and -7 were transferred to fresh feeder layers each time ESC colonies were passaged, which occurred every 3 to 4 days for ORMES-6 and every 7 days for ORMES-7. Colonies for passage were taken from a pre-marked, wedge-shaped section of each dish to assure a random sample. Approximately 18 to 24 hours after the initial passage, cells were cultured in DMEM/F12 medium (modified as above) with 0, 0.01%, 0.1%, or 1.0% (v/v) ethanol and with or without 150 pg/ml estradiol (Sigma E2758). Media changes were performed daily and experimental treatments continued for a total of 5 weeks.
To determine the level of ethanol that was sustained between media changes, samples of culture media were taken at 0, 12 and 24 hours from dishes with only MEF cells and with both MEF and ORMES cells. Samples were stored frozen until assayed by a gas chromatography volatile screen (University of California Medical Center Clinical Laboratory). For all treatment levels, ethanol decreased in a linear manner to 40% of the initial dose by 24 hrs of culture.
Colony number and differential morphology counts were assessed after 4 weeks of ethanol treatment by manual scoring of colonies. Imaging of representative colony morphology types was performed using a Nikon Eclipse TE300 microscope (Nikon, Tokyo, Japan) and MicroPublisher camera (QImaging, Burnaby, BC). A repeated measures ANOVA with a Tukey's post-test using Prism 4 for Mac (GraphPad software, Inc., La Jolla, CA) to determine differences among treatments.
Standard stem cell markers for all colonies were assessed by immunofluorescence after 4 weeks of culture in ethanol-containing medium. At 4 weeks, 24 colonies from each 60 mm plate were harvested as described above for passaging; three colonies were put in each well of 8-well slides (Nalge Nunc International, Rochester, NY). The slides were maintained under the same conditions and treatments as the corresponding 60mm dishes for 3 days before being fixed with 4% paraformaldehyde. The fixed slides were stored filled with Dulbecco's phosphate buffered saline (DPBS) at 4°C until stained. All staining incubations were for 40 minutes at room temperature. As a control, one well of each slide was excluded from immunofluorescence staining and was maintained in 100 mM 2-amino-2-hydroxymethyl-1,3-propanediol (TRIS; Fisher Scientific, Fair Lawn, NY), pH 8.2, until the end of the staining procedure. Wells for immunofluorescence labeling were permeabilized with 0.2% Triton X-100, 0.1% Tween-20 in Dulbecco's phosphate-buffered saline (DPBS, Sigma), then blocked with 2% normal goat serum (NGS; Sigma G6767) in DPBS with 0.05%Tween-20 (T-DPBS). Primary antibodies for OCT-3/4, SSEA-4, SSEA-1, TRA-1-60 and TRA-1-81 (Santa Cruz Biotechnology, Santa Cruz, CA) were diluted 1:100 in T-DPBS then applied to the appropriate wells. Cy3-conjugated secondary antibodies to mouse IgG and IgM (Jackson Laboratories, Bar Harbor, ME) were diluted 1:50 in T-DPBS and applied to the appropriate wells after washing 3 times with T-DPBS. Cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, Invitrogen) added to the diluted secondary antibodies. During the secondary antibody incubation, the reserved well of each slide was stained using an alkaline phosphatase detection kit (SCR004, Millipore, Billerica, MA). After a final washing with DPBS, the slides were mounted with FluoroGel (Electron Microscopy Sciences, Hatfield, PA) and sealed with nail polish. Imaging was performed using a Delta Vision microscope (Applied Precision, Issaquah, WA).
Colony Area Measurements
Colony size was measured at 4 weeks after the start of treatment. Images of stem cell colonies were taken at 72 hours after passage through the ocular of a Nikon SMZ645 stereomicroscope (Nikon, Tokyo, Japan) using a digital camera (Kodak model # Z712, Rochester, NY). The area of each colony in the image was determined with ImageJ (NIH, Bethesda, MD). A repeated measures ANOVA with a Tukey's post-test using Prism 4 for Mac (GraphPad software, Inc., La Jolla, CA) to determine differences among treatments.
Labeling of fixed, intact ESC colonies with aberrant morphology
The colonies with aberrant morphology appeared very dense and thick, which can be problematic when trying to determine the cellular structure inside of these colonies. The cells that were inside of these large colonies were investigated by evaluating Oct3/4 staining as well as proliferation (EdU) and apoptosis (active caspase 3) on fixed aberrant colonies. At 4 weeks of ethanol treatment, additional morphologically aberrant colonies from the 1.0% ethanol treatment plate for ORMES -6 and -7 were harvested as above and were put onto a Cell Well insert (Nunc # 137443, Thermo Fisher, Rochester, NY) that had been seeded 24 hours in advance with Mitomycin C treated mEF cells The medium both above and below the insert was changed daily for 3 days before treatment with EdU reagent (C10337, Invitrogen, Carlsbad, CA) prepared per the kit instructions. The culture inserts were incubated with the EdU reagent for 1 hour, fixed with 4% paraformaldehyde in PBS for 20 min and stored at 4°C in PBS until embedding. Membranes were removed from the inserts with a scalpel, processed and embedded in paraffin blocks for sectioning. 5μm sections were cut, placed on SuperFrost slides (Fisher Scientific, Pittsburg, PA) and dried overnight at 37°C. After deparaffinization, the slides were incubated in 10 mM sodium citrate at 95°C for 40 minutes and allowed to cool in the same solution to room temperature. The slides were transferred to 3% BSA in DPBS and held at 4°C overnight before EdU labeling per the kit protocol. After EdU labeling, the slides were washed with 3 changes of DPBS with 0.05% Tween 20 (DPBS/T) and then blocked for 40 minutes at room temperature in 10% NGS in DPBS/T. The individual sections on each slide were circled with a PAP Pen (Ted Pella, Redding, CA) placed in a humid chamber and incubated for 40 minutes at room temperature with polyclonal rabbit anti-active Caspase3 (AbCam, Cambridge, MA) at a 1:100 dilution in DPBS/T with 2% NGS. Slides were washed × 3 with DPBS/T and incubated 40 min with 1:100 Alexa635-conjugated anti-rabbit IgG (Invitrogen, Carlsbad, CA) and 2 μg/ml DAPI in DPBS/T with 2% NGS. After the final incubation the slides were washed as before and mounted in FluoroGel (Electron Microscopy Sciences, Hatfield, PA). A different set of colony sections were labeled with monoclonal mouse anti-Oct3/4 instead of EdU, but also labeled with Caspase 3 and DAPI as described above. Images were captured using the DeltaVision Restoration Microscopy System (Applied Precision, Issaquah, WA) equipped with a 20x/0.70 water immersion lens and analyzed with PhotoShop (Adobe Systems, San Jose, CA).