Illumina HiSeq 4000 paired end sequencing; Single-cell RNA sequencing of human liver development and hiPSC models
Single-cell RNA sequencing of human liver development and hiPSC models
A1ATD primary paper here: 10.1172/JCI43122. Related paper for additional information here: 10.1038/nature10424. Primary human adult liver tissue was obtained from deceased donors that have been rejected for transplantation. The tissue was stored at 4 C in organ preservation media after being removed from the patient, and subsequently washed with PBS+EDTA and incubated in HepatoZYME media (Thermo Fisher Scientific) supplemented with 0.2 Wnsch units/ml of Liberase DL (Sigma-Aldrich) and 1,000 U/ml DNase I (Sigma-Aldrich) on a shaker at 200 rpm, 37 C for 30 minutes to digest the ECM of the tissue. The tissue was passed through two cycles of the GentleMACS tissue dissociator (Miltenyi Biotech), after which PBS with 20% BSA was added to halt the enzymatic reaction. The hepatocytes were centrifuged at 50 x g, 4 C for 5 minutes, followed by a percoll gradient centrifugation to remove debris and dead cells and a red blood cell lysis step before final resuspension in HepatoZYME media. The non-parenchymal cell-containing supernatant was centrifuged sequentially at 300 x g and 650 x g, with both pellets being pooled. This fraction was cleaned using a debris removal kit (Miltenyi Biotech), followed by red blood cell lysis (Miltenyi Biotech) and resuspension in HepatoZYME media for barcoding and sequencing. Primary human adult cholangiocytes were collected by dissociating primary adult human liver, and passing the cell suspension through MACS beads to select EPCAM+ cells. These cells were resuspended in supplemented Williams E media for barcoding and sequencing. Primary human adult hepatocytes were collected from a collaborating lab using a method of enzymatic perfusion to isolate the cells. Subsequent clean-up steps were performed before barcoding and sequencing. Primary human fetal tissue was obtained from patients undergoing elective terminations (REC-96/085) in the first trimester. The liver was dissected from the abdominal cavity and placed into a solution containing Hanks Buffered Saline Solution (HBSS) supplemented with 1.07 Wnsch units/ml Liberase DH (Roche Applied Science) and 70 U/ml Hyaluronidase (Sigma-Aldrich), and placed on a microplate shaker at 37 C, 750 rpm, for 15 minutes. The sample was subsequently washed three times in HBSS using centrifugation at 400g for five minutes each time. The single cell suspension could then be sorted for EPCAM+/CD326+ cells using CD326 microbeads (Miltenyi Biotech), according to the manufacturers guidelines. The single cell suspension was resuspended in hepatoblast organoid media (HO-M) for barcoding and sequencing. Alternatively, primary human fetal tissue was digested and by a collaborating lab using enzymatic digestion and intermittent shaking. The resulting cell suspension was filtered and the red blood cells were lysed prior to resuspension for barcoding and sequencing. Many samples were sorted for CD45+ to separate the immune compartment for sequencing. Human induced pluripotent stem cells (hiPSC) were differentiated into hepatocyte-like cells, cholangiocyte-like cells, hepatic stellate-like cells, endothelial-like cells and macrophage-like cells following respective small molecule-driven differentiation protocols. These cells were detached from their plates in culture using common dissociation reagents including Accutase (Life Technologies), TrypLE Express (Life Technologies), Cell Dissociation Buffer (Thermo Fisher Scientific), and EDTA (Life Technologies) and resuspended in their respective medias for barcoding and sequencing. Hepatocyte-like cells were transduced with lentivirus at days 15 and 25 of the differentiation protocol and dissociated for sequencing on days 23-25 and 33 respectively. The cells were dissociated from culture using TryplE for 20 minutes at 37 C followed by accutase for 4 minutes at 37 C. The cells were centrifuged and resuspended in HepatoZYME media for barcoding and sequencing. Cell culture maintenance, passaging and differentiation  Human iPS cells were maintained in defined culture conditions, using Essential 8 (e8) media (Gibco A1517001). The cells were incubated at 37 C and 5% CO2 with media changed daily. Typically, cells were maintained for two weeks before passaging. To passage the cells, PBS was used to wash followed by incubation at 37 C with 0.5 mM EDTA in PBS for 4 mins. The PBS + 0.5mM EDTA was aspirated, fresh e8 media was added, and a pipette was used to flush the wells to detach the cells and break them into 5-10 cell clumps. The cells were transferred into a tube and allowed to settle, thus eliminating the small clumps and single cells. The supernatant was aspirated, the pellet was resuspended in complete e8 media and plated, typically in a 1:12 ratio split.  The differentiation of hiPSCs to HLCs followed the protocol previously published by the Vallier lab. Prior to differentiation, 12-well plates were coated with 0.1% gelatin for an hour at 37 C followed by MEF media overnight at 37 C and washed with PBS before use. Confluent hiPSC wells were washed with PBS before adding Accutase cell dissociation reagent (ThermoFisher A1110501) and incubating at 37 C for 4 mins. After incubation, the Accutase was diluted at a 1:1 ratio with complete e8 media (Gibco A1517001) and the supernatant was pipetted gently to mechanically break any clumps into single cells. The resulting suspension was centrifuged at ~350 x g for 3 mins to pellet, resuspended in e8 media and counted. The hiPSCs were added to complete e8 media supplemented with Y27632 ROCK inhibitor (10 mM) and plated at a density of 50,000-60,000 cells/cm2 in 12-well plates. The media was changed the following day to complete e8 without ROCK inhibitor. On day 1 of differentiation (2 days after plating), CDM-PVA media supplemented with activin (100 ng/ml), FGF2 (80 ng/ml), BMP4 (10 ng/ml), LY294002 (10 mM), and CHIR99021 (3 M) was added to the cells to induce endoderm formation. This same media without CHIR99021 was used on day 2 of differentiation. On day 3, RPMI-1640 with nonessential amino acids (ThermoFisher 11140050) and B27 supplement (ThermoFisher 17504044) was supplemented with activin (100 ng/ml) and FGF2 (80 ng/ml). Foregut differentiation was initiated on day 4 and carried out until day 8 by changing media to complete RPMI media supplemented with activin (50 ng/ml). The hepatoblast and subsequent hepatocyte phenotype was induced by changing media to HepatoZYME-SFM (Life Technologies 17705-021) supplemented with nonessential amino acids, chemically defined lipid concentrate (ThermoFisher 11905031), L-glutamine (2 mM), insulin (14 ng/ml), transferrin (10 ng/ml), oncostatin M (OSM) (20 ng/ml) and hepatocyte growth factor (HGF) (50 ng/ml) from day 9 to day 33. Lentiviral Overexpression Protocol. In these transcription factor overexpression experiments, hiPSC-derived hepatocyte-like cells were transduced on day 15 of differentiation and assayed on day 23/25, or transduced on day 25 of differentiation and assayed on day 33. On the day of transduction, complete hepatocyte-like cell differentiation media using basal HepatoZYME-SFM (ThermoFisher 17705021) was supplemented with OSM (20 ng/ml) HGF (50 ng/ml) and polybrene (10 g/ml) and added to cells in a 12-well plate format. Lentiviral aliquots were thawed on ice and added to the desired treatment wells in sterile conditions. Plates were rocked gently to ensure even distribution of lentivirus on the adherent cells. The cells were incubated at 37 C for 24 hrs to allow the lentivirus to infect the cells to stably integrate its DNA into the host genome. After 24 hrs, the cells were washed with DPBS and fresh,  complete HepatoZYME-SFM with OSM (20 ng/ml) and HGF (50 ng/ml) was added to the cells. This washing procedure was repeated again at 48 hrs and 72 hrs post-transduction to ensure the removal of any remaining lentivirus. Conversion was performed following the 10X protocol (https://www.10xgenomics.com/) Chromium 10X platform. Constructed by core facility at Wellcome Sanger Institute.