Generation of NIH-3T3 cells overexpressing Wnt6
To generate NIH-3T3 feeder layers that overexpressed Wnt6 (Wnt6-3T3) and control NIH-3T3 feeder layers that overexpressed GFP only (GFP-3T3), we obtained the Wnt6 coding sequence from Dr. Eric Wexler (University of California, Los Angeles)[57] and inserted it into a pRRL.CAG.IRES.GFP lentiviral vector (from the UCLA Integrated Molecular Technologies Core)[58, 59]. The vector was packaged and transduced into NIH-3T3 cells (Howard Green, Harvard Medical School, Boston, MA, USA)[60] through viral infection as previously described by Caviness et al.[61]. The transduced 3T3s cells were then sorted on the basis of GFP fluorescence (Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research University of California, Los Angeles Flow Cytometry Core Resource). Low-, medium-, high-Wnt6-3T3 stable cell lines and their respective GFP-3T3 controls were selected for expansion. The similarity between the expression of GFP mRNA and Wnt6 mRNA was verified by qRT-PCR.
Human Corneoscleral Tissue
Human corneoscleral tissues from healthy donors (ages, 43 to 74 years) were obtained from the San Antonio Eye Bank (San Antonio, TX) with permissions for tissue collection, Saving Sight (Kansas City, MO), and CorneaGen (Seattle, WA). All tissues were preserved in Optisol-GS (Chiron Ophthalmics, Inc., Irvine, CA) with a death-to-preservation time less than 8 hours. Experiments using human tissues adhered to the tenets of the Declaration of Helsinki. The experimental protocol was exempted by the University of California Institutional Review Board (IRB#12–000363).
Isolation And Culture Of Human Lscs
Limbal epithelial cells (LECs), which included LSCs, were isolated as previously described[62]. Briefly, corneoscleral rims were digested with 2.4 U/mL Dispase II (Roche, Indianapolis, IN) in supplemental hormone epithelial medium (SHEM5) for 2 hours at 37°C. The limbal epithelial cell sheets were isolated and digested with 0.25% trypsin and 1 mM EDTA (Life technologies) for 5 minutes at 37°C to obtain a single-cell suspension. Single LECs were seeded at a density of 200 cells/cm2 on subconfluent Wnt6 NIH-3T3 mouse fibroblasts that overexpressed different levels of Wnt6[60]. Growth of the NIH-3T3 cells was arrested by treatment with 4 µg/mL of mitomycin C (Sigma-Aldrich; St Louis, MO) for 2 hours at 37°C.
SHEM5 consisted of DMEM/F12 medium (Gibco, Carlsbad, CA, USA) supplemented with 5% fetal bovine serum (FBS, Life Technologies), N2 Supplement (Life Technologies), 2 ng/mL of epidermal growth factor (EGF; Life Technologies), 8.4 ng/mL of cholera toxin (Sigma-Aldrich, St. Louis, MO), 0.5 µg/mL of hydrocortisone (Sigma-Aldrich), 0.5% of dimethyl sulfoxide (DMSO; Sigma-Aldrich), penicillin/streptomycin (Life Technologies), and gentamicin/amphotericin B (Life Technologies). The medium was changed every 2–3 days, and cells were cultured for 10 to 12 days. LECs from the same donor were subjected to different culture conditions in each experiment to minimize donor variation.
Colony-forming Efficiency And Cell Proliferation Rate
CFE was measured at the end of culture by dividing the number of colonies by the number of seeded LECs. The LSC colonies were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 10 minutes and stained with 0.5% rhodamine B (Sigma-Aldrich) for 15 minutes at room temperature. The proliferation rate was calculated by dividing the number of harvested cells by the number of seeded LECs, as previously described[56].
Cell Cultures And Colony Forming Efficiency Imaging
Images of cell cultures and CFE were taken with the digital inverted Keyence BZ-X710 fluorescence microscope (Keyence, Osaka, Japan) and the image capture system BZ-X viewer. Quantitation of expression of markers such as p63α (p63αbright cells) was performed by using the BZ-X analyzer software (version 1.3.0.3) with the macro hybrid cell-count function. By specifying the mask area, the software extracted information about multiple parameters such as the intensity of fluorescence signals in different channels, cell counts, and target area measurements[62, 63].
Conditioned Media And Lsc Treatment
Conditioned media (CM) was obtained from 1x104/cm2 Wnt6-transduced NIH-3T3 cultured in DMEM supplemented with 10% BCS (Invitrogen) and 1% penicillin/streptomycin (Invitrogen) for 4 days. Following collection, the CM underwent centrifugation at 1000g for 10 minutes and was concentrated by using 30-kDa Amicon Ultra Centrifugal Filters (Sigma-Aldrich). Single-cell cultures of LSCs were washed once with 1X phosphate-buffered saline (PBS) and incubated with 2 mL of low-, medium-, and high-Wnt6-CM for specific time intervals. The LSCs were then washed twice with 1X PBS and harvested for analysis.
Western Blot
Cells were lysed with ice cold RIPA buffer (ThermoFisher Scientific, Carlsbad, CA), and proteins in the lysate were denatured in Sample Loading Buffer (Li-cor, Lincoln, Nebraska) with 10% β-mercaptoethanol (Sigma Aldrich) for 5 minutes at 95°C. Samples were loaded onto NuPAGE™ 4–12% Bis-Tris Protein Gels (ThermoFisher Scientific) and transferred onto Immobilon-FL PVDF membranes (Millipore, St. Louis, MO). Membranes were incubated for 1 hour at room temperature in Odyssey blocking buffer and then overnight with the following primary antibodies: rabbit phospho-β-catenin (Abcam, ab27798; dilution ratio, 1:500); rabbit phospho-CamKII (Abcam, ab32678; dilution ratio, 1:1000), rabbit phospho-RhoA (Abcam, ab41435; dilution ratio, 1:1000); mouse active β-catenin (Millipore, 05-665; dilution ratio, 1:1000); mouse CamKII (Santa Cruz Biotechnologies, Dallas, TX, sc-5306; dilution ratio, 1:200); mouse RhoA (Abcam, ab54835; dilution ratio, 1:1000); or mouse GAPDH (Millipore, MAB374; dilution ratio, 1:500). After overnight incubation, the membranes were washed 3 times for 10 minutes with TBS-Tween (0.1%) (ThermoFisher Scientific). Membranes were incubated at room temperature for 1 hour with the following secondary antibodies: IRDye 680LT Donkey anti-mouse IgG (Li-cor, 1:10000) and IRDye 800CW Donkey anti-rabbit IgG (Li-cor, 1:10000).
Rna Extraction And Qrt-pcr
Levels of Wnt6 mRNA were compared between human corneas, NIH-3T3 feeder cells, and low-, medium-, high-Wnt6-3T3 cells. Death-to-preservation time was < 6 hours for all tissues used for RNA extraction. RNA extraction was performed as previously described[20, 64]. Briefly, the cornea and limbal epithelia along with the immediate adjacent stroma were dissected from donor tissues and stored at -80°C until RNA extraction was performed. Homogenization of tissue samples was achieved by using a serrated homogenizer (Omni International, Marietta, GA). Total RNA was extracted (Qiagen RNeasy Mini Kit; Qiagen). The quantity and quality of total RNA were assessed by a spectrophotometer (NanoDrop 1000; NanoDrop, Wilmington, DE) and bioanalyzer (2100 Bioanalyzer; Agilent Technologies, Santa Clara, CA). RNA integrity number > 8 and minimal RNA degradation were required for subsequent experiments. Reverse transcription was performed by using the Superscript III Reverse Transcriptase (ThermoFisher Scientific). The relative abundance of transcripts was detected by qRT-PCR (KAPA SYBR FAST qPCR Master Mix; Stratagene, La Jolla, CA) and compared with transcript levels in NIH-3T3 cells and Wnt6-3T3 cells. GAPDH and 18S were used as internal controls to normalize the fluorescence level by using the 2(−ΔCt) formula. Three donors were used to compare Wnt6 mRNA in corneal tissue with that in NIH-3T3 cells. Three additional donors were used to compare Wnt6 mRNA level in corneal tissue with that in Wnt6-3T3 cells. Reactions were performed in triplicate. The primers used for qRT-PCR are listed in Supplemental Table 1.
Immunocytochemistry And Quantitative Analysis
Cultured colonies were incubated for 2 hours in SHEM5 growth medium supplemented with 2.4 U/mL Dispase II (Roche) at 37oC. Single cells were obtained with 0.25% trypsin and 1 mM EDTA (Gibco) for 7 minutes at 37oC. Harvested cells underwent cytospinning onto slides by a cytocentrifuge (Cytofuge; ThermoFisher Scientific), and these slides were stored at -80oC until further use.
Slides were fixed with 4% paraformaldehyde at room temperature for 15 minutes, washed 3 times with PBS, blocked and permeabilized with 1X PBS containing 1% bovine serum albumin (BSA) and 0.5% Triton X-100 (Sigma-Aldrich) for 30 minutes at room temperature. Slides were incubated with primary antibodies in 1X PBS containing 1% BSA and 0.1% Triton X-100 overnight at 4oC in a moist chamber. Primary antibodies used were the following: anti-K12 (Santa Cruz Biotechnologies, sc-25722; dilution ratio, 1:100); anti-K14 (ThermoFisher Scientific, MS-115-R7); and anti-p63α (Cell Signaling Technologies, 4892; dilution ratio, 1:100). After incubation, cells were washed with 1X PBS, incubated with the following secondary antibodies: goat anti-mouse IgG conjugated to AlexaFluor 488 (ThermoFisher Scientific, A11029, 1:500) or goat anti-rabbit IgG conjugated to AlexaFluor 546 (ThermoFisher Scientific, A11035, 1:500) at 20°C for 1 hour. Nuclei were labeled with Hoechst 33342 (Invitrogen, 33342, 4 µg/mL) at 20°C for 15 minutes and mounted in Fluoromount medium (Sigma-Aldrich). Images were taken with a Keyence BZ-X710 inverted microscope (Osaka, Japan), and characteristics such as fluorescence intensity, number of cells, and size were evaluated with the BZ-X analyzer software (version 1.3.0.3) as described above.
Mrna In Situ Hybridization Using Rnascope Assays
The RNAscope V2 Fluorescent Assay (ACD Biosystems, Newark, CA) was performed according to the ACD Biosystems protocol for fresh-frozen tissue. Corneal sections from 3 donors were hybridized with the Wnt6 mRNA probe. Assays were performed in 2 independent experiments for each donor. To confirm the mRNA integrity of the tissues, positive control probes targeting human housekeeping genes Polr2a, PPIB, and HPRT were visualized (Supplemental Fig. 1). The probes were amplified according to the manufacturer’s instructions and labeled with the fluorophore Opal 520 nm (Akoya Biosciences, FP1487001KT, 1:250), Opal 570 nm (Akoya Biosciences, FP1488001KT, 1:1500), and Opal 690 nm (Akoya Biosciences, SKU FP1497001KT, 1:1500). The Olympus FluoView FV1000 was used to visualize the fluorescence in situ hybridization signals. Quantitation of the dots among the limbal layers was performed with Imaris software and the surface and dots functions (V. 9.7.0, Imaris, Oxford Instruments, Oxon, UK).
Statistical analysis
The Student’s t test was used to analyze data from at least 4 independent experiments in which 4 different donor tissues were used for cell culture. The mean ± standard error of the mean (SEM) are reported in each bar graph. Statistical significance was established with P values < 0.05.