Male Fisher 344 rats (150–200 g) were purchased from Charles River Laboratories (Fredrick, MD). Animals were allowed food and water ad libitum. All animals received humane care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences.
Isolation and culture of heaptocytes
Hepatocytes were isolated by adaptation of the calcium two step collagenase perfusion technique as previously described (15
). Hepatocytes were plated on collagen coated six-well plates (BD Biosciences, CA) at 250,000 cells/well. After the initial 2h attachment period, plating media was changed to either HGM complete with growth factors (+GF) HGF (supplemented at 40ng/ml) and EGF (supplemented at 20ng/ml) or without growth factors (−GF) and every 48 h thereafter. Cells were harvested on day 0 (2h plated), 2, 4, 6, 8, and 10 for RNA and protein.
Total RNA extraction
Total RNA was extracted from plated cells using the RNABee reagent (Invitrogen, Carlsbad, USA) according to the manufacturer’s protocol. The isolated RNA was treated with Turbo DNA-free (Ambion, Austin, TX) according to the manufacturer’s instructions. RNA was quantified by spectrophotometry at 260 nm, and purity was assessed by optical density 260/280 ratio. The RNA was stored at −80°C. The experiment was repeated in three rats and their mRNA was pooled for further processing in primary hepatocytes as well as PHx experiments.
Four micrograms RNA per sample was reverse transcribed using random hexamer to cDNA by using SuperScriptIII reverse transcriptase (Invitrogen, CA) according to the manufacture’s protocol. A no reverse transcriptase (RT) control was also included.
Real time reverse transcriptase polymerase chain reaction
The gene-specific primers used for rat were as follows. REST, cMyc, Klf4, Nanog (SuperArray Bioscience Corporation Cat. # PPR45101A, PPR45580A, PPR43919A, and PPR59663A, respectively).
- Oct4 forward: 5′- GGC GTT CTC TTT GCA AAG GTG TTC -3′
- Oct4 reverse: 5′- CTC GAA CCA CAT CCT TCT CT -3′
Expression levels of REST, Oct4, cMyc, and Klf4 were determined by quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) using SYBR green, and levels were normalized relative to expression of cyclophillin in each sample. Fold change in gene expression was calculated by using the 2(−ΔΔCt) method (18
). Reverse transcribed samples were amplified in parallel on an ABI prism 7000 SDS instrument (Applied Biosystems, Foster city, CA). qRT-PCR for each sample was performed in triplicate in a 20 μl reaction with 50ng of cDNA, 5 picomoles of each primer, and 1X SYBR green PCR mix (Applied Biosystems). The standard conditions for real time PCR were as follows: 2 minutes at 50°C, 10 minutes at 95°C followed by 40 cycles of 15 seconds denaturation at 95°C, and elongation at 60°C for 45 seconds. A dissociation curve analysis was performed at the end of every run. A no RT and a no template control were also included in every run.
A different set of primers designed in a common region with same sequence for rats and mice for comparison with MESC were as follows:
- REST forward: 5′ – AACCAT TTTCCCAGGAAAGTCTACAC
- REST Reverse: 5′ – AGTTCACATTTATACGGGCGTTCTC
- Oct4 forward: 5′- GAGGGCCAGGCAGGAGCACGAGTGG-3′
- Oct4 reverse: 5′- TTCTGCAGGGCTTTCATGTCCTGG -3′
- Klf4 forward: 5′- CTCAAGGCACACCTGCGAACTCACA-3′
- Klf4 reverse: 5′- TGTGTTTGCGGTAGTGCCTGGTCAGT -3′
- cMyc forward: 5′- AGTGTTCTCTGCCTCTGCCCGCGA-3′
- cMyc reverse: 5′- GTCGTAGTCGAGGTCATAGTTCCT -3′
Effect of Matrigel on expression of reprogramming factors
Primary hepatocytes were plated in 100 mm collagen coated plates at 2.4 million cells/plate. Matrigel (Cat. # 354234) was obtained from BD Biosciences, San Jose, CA. After the 2h attachment period hepatocytes were treated as follows: 1) HGM medium with growth factors and 0.233 mg/ml Matrigel (+MTG+GF), 2) HGM without growth factors and 0.233 mg/ml Matrigel (+MTG−GF), 3) HGM with growth factors but no Matrigel (−MTG+GF), and 4) HGM without growth factors and without Matrigel (−MTG−GF). Plates were harvested on days 2, 6, and 10 for RNA.
Semi-quantitative PCR for Matrigel experiment
Standard PCR was performed using 50ng cDNA and Amplitaq DNA polymerase (Applied Biosystems). PCR products were resolved on 2% agarose gels and visualized with ethidium bromide staining. The bands on gel were scanned for optical density using Image J software for quantitation purposes.
REST inhibition studies
shRNA for REST: we used a commercially available kit from Invivogen (catalog # ksirn4-gz21) to generate the plasmid containing shRNA targeted against REST. The shRNA vector employed also encodes a red-shifted variant of the jellyfish GFP. This plasmid is specifically designed for the cloning of small synthetic oligonucleotides that encode 2 complementary sequence of 21 nt, homologous to a segment of REST. The insert is cloned downstream of a human 7SK promoter. It is transcribed into a short dsRNA with a hairpin structure (shRNA) consisting of a 21 bp double stranded region corresponding to REST and a small loop formed by the spacer region. Sequences for REST shRNA insert:
- Forward: 5′- ACC TCTTGGTGAAGAGAGACAGATTCAAGAGATCTGTCTCTCTTCACCAAT T -3′
- Reverse: 5′ - CAAAAATTGGTGAAGAGAGACAGATCTCTTGAATCTGTCTCTCTTCACCAA G -3′
Primary hepatocytes were plated at a density of 1×106 cells per 100 mm dish or 0.25×106 cells per well (6-well plate) on day 1. After the 2 h attachment period, plating media was replaced with HGM complete without growth factors. On the 2nd day, hepatocytes were either transfected with shRNA for luciferase (C), or shRNA for REST (R). The transfection media was replaced with fresh HGM without growth factors after 6h. On the next day (day 3) media was changed to HGM with growth factors and thereafter replaced every 48h throughout the time course. Cells were harvested at day 0, 1, 2, 3, 4, and 5 after transfections for RNA and protein. MTT assay was done on days 2, 3, 4, and 5 as a marker of live cells. Tritiated Thymidine incorporation was measured on day 1–2 after transfections to assess proliferation of hepatocytes. TUNEL assay was done on 3 days after transfections to assess apoptosis in these cells.
Cell death was assessed by measuring the live mitochondrial activity using the TOX-1 in vitro toxicology assay kit (Sigma Aldrich corp., St. Louis, MO) according to the manufacturer’s protocol.
[3H]Thymidine was added to the medium on day 3 (24 h after transfections) at a concentration of 2.5 μCi/ml. The medium was removed after 24 h, and hepatocytes were fixed with ice-cold 5% trichloroacetic acid. Trichloroacetic acid was removed and the plates were washed in running tap water and air dried completely. 750 μl 0.33 M NaOH was added to each well for 30 minutes to solubilize the cells. The solution was transferred to a new tube, and 250 μl of 40% TCA/1.2 M HCl was added for precipitation. The tubes were centrifuged at 12000 g for 10 minutes, and the pellets were redissolved in 500 μl 0.33 M NaOH. A 200 μl aliquot was used to measure cpm/dpm in a Beckman LS6000IC scintillation counter (Beckman Coulter, CA), and 100 μl was used to determine optical density value of total DNA. Data are plotted as CPM/μg DNA.
The extent of apoptosis in hepatocytes was measured 3 days after transfection using TUNEL assay according to manufacturer’s protocol (ApopTag Peroxidase In Situ Apoptosis Detection Kit, S7100, Chemicon International). Brown stained apoptotic nuclei were counted in 5 different fields along with the total number of cells in the field for each group. Percent apoptotic nuclei were calculated and plotted.
Protein analysis by Western Blot
Protein levels in nuclear extracts were assessed by Western blot analysis by harvesting cells at different time points. Nuclear extracts pooled from 3 rats were prepared using NE-PER Nuclear and cytoplasmic extraction kit according to manufacturer’s protocol (Pierce Biotechnology Cat. # 78833, Rockford, IL). Nuclear extracts were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis in 4% to 12% NuPage Bis-Tris gels with 1× MOPS buffer (Invitrogen, Carlsbad, CA), then transferred to Immobilon-P membranes (Millipore, Bedford, MA) in NuPAGE transfer buffer containing 10% methanol. Membranes were stained with Ponceau S to verify equal loading of total protein and transfer efficiency. Membranes were probed with primary and secondary antibodies in Tris-buffered saline with Tween 20 containing 1.5% nonfat milk, then processed with SuperSignal West Pico chemiluminescence substrate (Pierce, Rockford, IL) and exposed to a X-ray film (Pierce, Rockford, IL). Horseradish peroxidase-conjugated secondary antibodies were used at a 1:50,000 dilution (Chemicon, Temecula, CA). Primary antibodies used were as follows: REST (07-579, Millipore, Temecula, CA), Oct4 (ab52014, Abcam Cambridge, MA), cMyc, Nanog, and Klf4 (sc-764, sc-33760, and sc-20691, respectively, Santa Cruz Biotechnology, Santa Cruz, CA). Since our model involves proliferation, Tata binding protein used as a loading control for nuclear extracts changed because of the treatment. Hence ponceau stain was used to verify equal loading of samples.
70% Partial Hepatectomy (PHx) on rats
Isoflurane inhalation (Baxter, IL) was used to anesthetize animals. Partial hepatectomy was performed as described previously (19
Immunohistochemisrty on rat tissues
Paraffin-embedded liver sections (4 μm thick) were used for immunohistochemical staining. Antigen retrieval was achieved by heating the slides in the microwave at high power in 1× citrate buffer for 10 minutes. The tissue sections were blocked in blue blocker for 20 minutes followed by incubation with primary antibody overnight at 4°C. The primary antibody was then linked to biotinylated secondary antibody followed by routine avidin-biotin complex method. Diaminobenzidine was used as the chromogen, which resulted in a brown reaction product. Primary antibodies used were as follows: NRSF (Millipore 07-579; 1:1000), Oct4 (ab27985; 1:250), KLF4 (SC-20691; 1:500), Nanog (ab80892; 1:700), and Myc (ab32072; 1:100).
Data are expressed as means ± S.E. Statistical differences were determined by one way analysis of variance (ANOVA) followed by Tukey’s honest significant difference test to determine which means were significantly different from each other or from controls using the JMP software (SAS Institute INC., Cary, NC). The criterion for significance was p < 0.05.