LPCs were first enriched by magnetic assisted cell sorting (MACS) (Miltenyi Biotec) and then isolated by fluorescence activated cell sorting (FACS). The mixed cell suspension from freshly prepared foetal liver was passed through a 40 um nylon mesh cell strainer (Becton Dickinson) and washed with cell separation buffer (CS buffer; phosphate buffered saline (PBS) containing 0.5% bovine serum albumin (BSA) and 2 mM EDTA (Sigma-Aldrich)). After centrifugation at 340g for 5 minutes at 4C, the cell pellet was resuspended in CS buffer. As foetal liver is the site of active haematopoiesis during development, haematopoietic cells are abundant and needed to be removed prior to isolation of LPCs. The filtered cell suspension was incubated in the dark at 4C for 15 minutes with CD235a microbeads and CD45 microbeads, washed with CS buffer, and depleted by MidiMACS LD columns and separation units. The effluent depleted of CD235a+ erythroid cells and CD45+ haematopoietic cells was collected and centrifuged at 340g for 5 minutes at 4C and the cell pellet was resuspended in CS buffer. FcR blocking reagent was added to block non-specific binding of antibody to Fc receptor-expressing cells prior to incubating the cell suspension with EpCAM (CD326) microbeads in the dark at 4C for 30 minutes. After washing with CS buffer, isolation of EpCAM+ cells was performed using a MidiMACS magnetic LS column separation system. The eluted EpCAM– fraction was collected, counted, and resuspended in WME+ in preparation for generation of conditioned medium. The retained EpCAM+ fraction was also collected, counted, and resuspended in WME+, and prepared for FACS isolation of LPCs. MACS-enriched EpCAM+ cells were labelled with fluorescent antibodies EpCAM-APC (BioLegend), CD29-PerCP-eFluor® 710 (eBioscience), CD49d-PE (BioLegend) and CD49e-FITC (BioLegend). Samples were sorted using a FACSAria I flow cytometer (Beckton Dickinson Immunocytometry Systems (BD), UK) equipped with 488nm and 633nm lasers running BD FACSDiva v5 Software. An electronic acquisition gate was applied to the Forward/Side scatter plot to exclude debris from intact material. FITC-conjugated anti-CD49e antibody, PE-conjugated anti-CD49d antibody, and PerCP-eFluor® 710-conjugated anti-CD29 antibody binding was detected following 488nm excitation at emission between 530/30nm, 585/42nm, and 695/40nm, respectively. APC-conjugated anti-EpCAM antibody binding was detected following 633nm excitation at emission between 660/20nm. Non-aggregated material was identified based on FSC Area versus Width plot profiles, before applying electronic gates for sorting. Cells that were triple positive for EpCAM, CD29, and CD49d but negative for CD49e were collected for microarray studies.
Growth protocol
hESCs were cultured and differentiated according to Hay, David C, Debiao Zhao, Judy Fletcher, Zoë A Hewitt, Doris McLean, Alai Urruticoechea-Uriguen, James R Black, et al. 2008. “Efficient Differentiation of Hepatocytes from Human Embryonic Stem Cells Exhibiting Markers Recapitulating Liver Development in Vivo.” Stem Cells (Dayton, Ohio) 26 (4): 894–902. doi:10.1634/stemcells.2007-0718.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from foetal cells using TriZol (Invitrogen) according to the manufacturer's protocol. Total RNA from adult hepatocytes was purchased from 3H Biomedical, Uppsala, Sweden. The Nanodrop instrument (Labtech UK) was used to quantify RNA and assess 260/280 and 260/230 ratios. RNA integrity was examined using a 2100 Bioanalyzer (Agilent UK). All samples used had RIN scores > 9, 260/280 ratios > 2, and 260/230 ratios > 1.9.
Label
biotin
Label protocol
RNA was amplified, fragmented, and biotin-labelled using the Ovation RNA Amplification System V2 and FL-Ovation cDNA Biotin Module (NuGEN Technologies) according to the manufacturer's protocol. 100ng of total RNA was reverse transcribed and amplified. 3.75ug of cDNA was fragmented and biotinylated.
Hybridization protocol
Labelled cDNA was hybridised to Affymetrix hgu133plus2 gene chip arrays using a protocol supplied by NuGEN.
Scan protocol
The arrays were washed, stained, and scanned using the standard Affymetrix protocol.
Description
Gene expression data from hESC at day 9 of differentiation to hepatocyte like cells
Data processing
All array data were normalised using the Microarray Suite version 5.0 (MAS 5.0) algorithm to a global scaling intensity of 100. This raw data was then normalised using the RMA algorithm in the bioconductor affy package (R version 2.15.1). A customs CDF was used to annotate the array (version 13 ENTREZ based cdf from http://brainarray.mbni.med.umich.edu/Brainarray/Database/CustomCDF/13.0.0/entrezg.asp).
