Liver tissue specimens suspended in DMEM were minced and digested with collagenase V (100 units/mL; Sigma-Aldrich, St. Louis, MO) at 37°C for 15 minutes, followed by filtering through a 40-μm nylon mesh to remove debris. Cells were

then collected by centrifugation (440×g for 20 minutes) at 4°C and either resuspended in fluorescence-activated cell-sorting (FACS) buffer (phosphate-buffered saline, 0.5% bovine serum albumin, and 0.1% NaN3) for cell-surface marker study or resuspended in R medium (Dulbecco’s phosphate-buffered saline, 2% fetal bovine serum, and 1 mM of ethylene diamine tetraacetic acid) for cell sorting. To determine the Lin−CD34+CD38−CD90+ population in liver tissue, 5-10 × 105 single liver cells from the unsorted sample, or from a sample previously sorted for CD45+, were incubated selleck with antihuman lineage cocktail 1/fluorescein isothiocyanate (FITC) (BD Immunocytometry Systems, San Jose, CA), anti-CD34-APC (allophycocyanin) anti-CD90-PE (phycoerythrin) (BD Pharmingen, San Diego, CA), and anti-CD38-PE/Cy7 (cyanin-7) (BioLegend, San Diego, CA) antibodies for 30 minutes at room temperature, followed by two washes with FACS

buffer. As a control, cells were also labeled with FITC, APC, PE, and PE/Cy7 isotype control antibodies (BioLegend). Cell-surface markers were then analyzed

selleck inhibitor by FACSCalibur (BD Immunocytometry Systems). Lin−CD34+ cells were isolated by magnetic cell sorting. Lin− liver cells were obtained by incubation with negative selection (two times) Idelalisib of human progenitor enrichment cocktail antibodies (anti-CD2, anti-CD3, anti-CD11b, anti-CD14, anti-CD16, anti-CD19, anti-CD24, anti-CD56, anti-CD66b, and anti–glycophorin A; StemCell Technologies, Vancouver, Canada), followed by magnetic separation. Sorted Lin− cells were then enriched for either CD34+ or CD45+ cells two times using a human CD34 or CD45 selection kit (StemCell Technologies). Cells (2,000-5,000) from Lin-depleted and CD34- or CD45-enriched cell populations from liver cell suspensions were seeded in complete methylcellulose (MethoCult GF+ H4435 or GF H4034; StemCell Technologies) in pretreated 35-mm dishes, following the manufacturer’s instructions, and incubated at 37°C, 5% CO2, and 95% humidity for 14-16 days. Hematopoietic colonies were then scored based on size, color, and morphology, with each colony containing at least 40 cells. Nonobese diabetic/severe combined immunodeficiency (NOD-SCID) mice (NOD.CB17-Prkdcscid/J; The Jackson Laboratory, Bar Harbor, ME), at 6-10 weeks of age, were irradiated with 2-3 Gy (Cs137, MDS Gammacell; MDS Nordion, Freiburg, Germany) 4 hours before transplantation.