Reagents in these experiments were from Sigma, St. Louis, MO unless otherwise stated.
C57BL/6 mice (6-8 weeks old) were purchased from the Jackson Laboratory (Bar Harbor, ME) and used for all of the experiments. All care and use of animals was approved by the Institutional Animal Care and Use Committee at the University of North Carolina at Chapel Hill in accordance with the principles and procedures outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Hepatic Progenitor Cell Isolation
Hepatic progenitor cell containing fraction was isolated as previously described [11
Enrichment of Sca-1+-Hepatic Progenitor Cells
Sorting of liver cells was performed using a Sca-1 antibody conjugated to mini-magnetic beads (Miltenyi Biotec, Inc.; Auburn, CA, http://www.miltenyibiotec.com
) according to the manufacturer's instructions. Sca1+
cells were eluted with a purity of >99% by flow cytometry and >80% viability.
In-vitro cell culture
The Sca-1+-HPCs (sca-HPCs) were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Gibco; Carlsbad, California ) plus 10% fetal bovine serum (FBS), 20mM hepes, 10mM nicotinamide, 1 mM ascorbic acid 2-phosphate, 1 μM dexamethasone, 0.5 mg/L ITS (insulin-transferrin-selenium) solution, 30 mg/L proline, 100 mg/L antibiotic solution (Gibco), and 10 ng/mL epidermal growth factor (EGF) supplemented with conditioned media from previously unfractionated non-parenchymal cell population. The sca-HPCs were plated at a density of 8×104 cells/cm2. Tissue culture dishes were cultured in a 5% CO2 incubator at 37°C and the media was changed at regular time intervals.
Liver tissue was formaldehyde fixed and paraffin embedded. Five μm thick sections were cut and subjected to further immunohistochemical examination.
Histology and Immunohistochemical Characterization
Immunohistochemistry was performed by an indirect immunoperoxidase procedure for localization of select proteins. Primary antibody was against Sca-1 (BD Pharmingen™; San Jose, CA). The secondary antibody was from Vector Laboratories (Burlingame, CA) and the signal was detected using the ABC Elite and DAB kits (Vector Laboratories). All stained slides were viewed on the Nikon Microphot-FXA microscope equipped with an Optronics DEI 750 3-chip CCD camera and a Q Imaging Micropublisher CCD camera for digital image acquisition. Images were captured on an Apple Power Macintosh G3 computer utilizing Q Imaging software.
Fluroescent immunophenotyping and flow cytometry
Cells were stained for immunofluorescence using antibodies directly labeled with the relevant fluoroprobe. Unstained cells, cells stained with an irrelevant antibody and the same fluoroprobe or with the same antibody but no fluoroprobe were used as negative and isotype controls. Cells were suspended in room temperature HBSS containing 2% FBS and 2 mM HEPES buffer (SP buffer) at 5 × 106 cells per ml. Antibodies were primary conjugates against CD117, CD34 and CD45 (BD Biosciences; San Jose, CA). Cells were incubated for 90 minutes, centrifuged and resuspended in cold buffer at 5 × 106 per ml.
Isolation of Sca-1 negative cell fraction
The Sca-1 negative cell fraction was separated and confirmed using the Modular Flow (Mo Flo) cytometer from Cytomation Inc (Fort Collins, CO).
Reverse Transcription-Polymerase Chain Reaction
Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Inc.; Valencia, CA) and quality confirmed on an Agilent 2100 BioAnalyzer (Agilent Technologies Inc.; Palo Alto, CA). Two micrograms of total RNA, were reverse-transcribed using the RETROscript® Reverse Transcription Kit (Ambion; Austin, TX). Polymerase chain reaction (PCR) was performed as follows: 94°C, 30 seconds; 55-60°C, 1 minute; and 72°C, 1 minute for 40 cycles. Primer sequences for the genes are in the supplemental data
Western Blot Analysis
Cells were lysed in buffer containing 50mM Tris, 150mM NaCl, 1% Nonidet P40 (Roche; Indianapolis, IN) , 0.5% deoxycholate, 1mL protease inhibitor cocktail for every 100mL (5μg/mL aprotinin, leupeptin, pepstatin, and soybean trypsin inhibitor), and 1mL phosphatase inhibitor for every 100mL. Lysates were clarified by centrifugation at 14,000g × 2 min and stored at −20°C. Protein concentrations were determined by Bio-Rad protein assay. Total cellular proteins (50 μg/lane) were dissolved by SDS-PAGE, transferred to nitrocellulose membrane incubated with blocking buffer (5% nonfat dry milk in 1x Tris Buffered Saline with 0.05% Tween 20, pH 7.5), and probed with primary antibodies. After incubation with secondary antibodies, peroxidase activity was detected by enhanced chemiluminescence. Densitometric signals from Western blots were analyzed with NIH-ImageJ software (http://rsb.info.nih.gov/ij/
]. Protein levels were calculated in arbitrary units (AU) normalized with β-actin protein levels.
Cell Proliferation Assays
The 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyl tetrazolium bromide (MTT) assay quantifies cell number (Sigma; CGD-1) based on the MTT-Formazan assay [21
]. Cells were cultured on 96-well flat bottom plates in 150 μL of media. MTT equal to 10% culture volume in RPMI 1640 medium and the plates were kept at 37°C for 4 hrs in a humidified incubator. Media was removed and the cells were incubated with 150μL MTT solvent (0.1 N HCl in anhydrous isopropanol) until the purple formazan crystals were dissolved. The absorbance was read at 570 nm and 690nm using a microplate reader. The cell number was calculated from a standard absorbance curve [23