Liver cells were isolated by collagenase digestion as follows: tissue (0.5-1cm3) was minced, rinsed 2x with DMEM (GIBCO) 1%FCS and incubated with the digestion solution (2.5 mg/ml collagenase D (Roche) + 0.1 mg/ml DNase I (Sigma) in EBSS (Hyclone, Thermoscientific), for 20-40 at 37°C. The digestion was stopped by adding cold DMEM 1%FCS and the suspension was then filtered through a 70 um Nylon cell strainer and spun 5 min at 300-400 g. The pellet was resuspended in DMEM 1%FCS and kept cold. Any material retained on the strainer was further digested for 10 min in Accutase (GIBCO) at 37C. Then, the digestion was stopped and the cells were collected as before. The different fractions (collagenase and accutase) were seeded and cultured as described in Experimental Procedures.

Expansion ratios were calculated from human liver cultures as follows: 3x10 3 cells were grown in our defined medium for 7 or 10 days. Then, the cultures were dissociated by incubation with TrypLE Express (GIBCO) until single cells. Cell numbers were counted by trypan blue exclusion at the indicated time points. From the basic formula of the exponential curve y(t) = y 0 x e (growth rate x t) (y = cell numbers at final time point; y 0 = cell numbers at initial time point; t = time) we derived the growth rate. Then, the doubling time was calculated as doubling time = ln(2)/growth rate for each time window analyzed.

Human liver cells were isolated according to standard protocol at the liver cell laboratory at the unit for transplantation surgery, CLINTEC, Karolinska Institute (). Cell suspensions were shipped overnight on ice. Suspensions were diluted in 2 volumes of cold Advanced DMEM/F12 (GIBCO) and washed 3 times in the same medium. Viable cells were counted with Trypan blue and split into 3 parts for EpCAM sorting, Percoll purification (see Hepatocyte Percoll purification) and direct seeding into matrigel. For sorting, liver cells were stained with 1:100 Anti-Human CD326 (EpCAM) Alexa Fluor® 488 (eBioscience) for 30 min at 4°C. Subsequently cells were washed and sorted on a MoFlo (Dako Cytomation) cell sorter. Sorted cells were spun down, resuspended in Matrigel and grown into organoids according to standard human liver organoid culture procedure (see Human liver organoid culture). After 14 days in culture the number of organoids larger than 100 μm in diameter was scored.

Human hepatocyte suspensions (see Isolation of EpCAM+ cell from primary human liver) were washed as described, spun down and resuspended in 35 ml Advanced DMEM/F12 (GIBCO) + 13.5 ml Percoll (GE healthcare, density 1.130 g/ml) + 1.5 ml 10x HBSS (GIBCO). Cells were pelleted at 100 g for 10 min and washed 3 times in Advanced DMEM/F12 (GIBCO). Viable cells were counted with Trypan blue and 10.000 viable cells per 50 ul drop were seeded into matrigel. Remaining cells were stained for EpCAM as described above (see Isolation of EpCAM+ cells from primary human liver) or seeded onto collagen coated tissue culture plates for subsequent determination of cytochrome 3A4 activity. To measure Cyp3a4 in primary hepatocytes, the seeded cells were cultured in Williams E medium (GIBCO) containing Hepatocyte plating supplement pack (GIBCO) for 4 days with daily medium changes. On day 0 and day 4 the cells were incubated with Luciferin-PFBE substrate (50 μM) and Cytochrome P450 activity was measured using the P450-Glo Assay Kit (Promega) according to manufacturer’s instructions and normalized to the number of cells in the plate. HepG2 cells cultured in the same medium served as controls.

DNA libraries for WGS analysis were generated from 1 μg of genomic DNA using standard protocols (Illumina). The libraries were sequenced with paired-end (2 × 100 bp) runs using Illumina HiSeq 2500 sequencers to a minimal depth of 30 x base coverage (average depth of ∼36.9 x base coverage). As reference sample, liver biopsies was sequenced to equal depth for the different donors. Sequence reads were mapped against human reference genome GRCh37 using Burrows-Wheeler Aligner (BWA) 0.7.5a with settings ‘bwa mem -c 100 -M’ resulting in sample-specific BAM files. To predict CNVs, BAM files were analyzed using Control-FREEC () and DELLY (). To obtain somatically acquired CNVs, we filtered called CNVs for occurrence in the reference samples (liver biopsies). Single nucleotide variants (SNVs) were multi-sampled called using the Genome Analysis Toolkit (GATK) v2.7.2 UnifiedGenotyper (). We only considered positions at autosomal chromosomes, which were covered at least 20x in all liver stem cell samples and corresponding biopsy from the same donor. Candidate somatic SNVs were further filtered using the following criteria: no evidence in reference samples; minimal alternative allele frequency of 0.3 to exclude sequencing artifacts and potential substitutions that occurred after the clonal step; a minimal GATK quality score of 100; no overlap with single nucleotide polymorphisms (SNPs) in the Single Nucleotide Polymorphism Database (dbSNP 137.b37); and no overlap with SNVs in the other tested individual ( Figure S2 ). Ultimately, SNVs with evidence in both clonal and subclonal cultures were considered as in vivo acquired somatic variation, and SNVs with evidence in only subclonal cultures were considered as variation accumulated during in vitro culturing.

Tissues and organoids were fixed o/n with formalin or 4% PFA respectively, and stained washed and transferred to tissue cassettes and paraffin blocks using standard methods. Tissue sections (4 μM) were prepared and stained with antibodies, H&E or PAS using standard techniques. The antibodies and dilutions used are listed in Table S5 . Stained tissues were counterstained with Mayer’s Hematoxylin. Pictures were taken with a Nikon E600 camera and a Leica DFDC500 microscope (Leica). For whole mount immunofluorescence staining, organoids were processed as described in Barker et al., (). Nuclei were stained with Hoechst33342 (Molecular Probes). Immunofluorescence images were acquired using a confocal microscope (Leica, SP5). Images were analyzed and processed using Leica LAS AF Lite software (Leica SP5 confocal). All phase contrast pictures were acquired using a Leica DMIL microscope and a DFC420C camera.

RNA was extracted from organoid cultures or freshly isolated tissue using the RNeasy Mini RNA Extraction Kit (QIAGEN), and reverse-transcribed using reverse-transcribed using Moloney Murine Leukemia Virus reverse transcriptase (Promega). All targets were amplified (40 cycles) using gene-specific primers and MiIQ syber green (Bio-Rad). Data were analyzed using BioRad CFX manager. For Figure S7 , cDNA was amplified in a thermal cycler (GeneAmp PCR System 9700; Applied Biosystems, London, UK) as previously described (). Primers used are listed in Table S4

Functional Hepatocyte Studies

To assess glycogen storage and LDL uptake, liver organoids grown in EM or DM for 11 days were stained by Periodic acid-Schiff (PAS, Sigma) and DiI-Ac-LDL (Biomedical Technologies), respectively, following manufacturer’s instructions. To determine albumin and A1AT secretion, liver organoids were differentiated as described. Culture medium was changed every 3-4 days and culture supernatant was collected was collected 24h after the last medium change. HepG2 (ATCC number 77400) and HEK293T (ATCC number CRL-3216) cells were cultured for 24h in the same medium without growth factors and were used as positive and negative control respectively. The amount of albumin and A1AT in culture supernatant was determined using a human specific Albumin or human specific A1AT ELISA kit (both from Assay Pro). To measure Cyp3a activity the cultures were differentiated as described and the day of the experiment the cells were removed from the matrigel and cultured with the Luciferin-PFBE substrate (50 μM) in Hepatozyme medium supplemented with 10% FBS (GIBCO). As controls, HepG2 and HEK293Tcells were cultured for 24h in DMEM 10%FBS and the day of the experiment transferred to Hepatozyme medium supplemented with 10% FBS (GIBCO) and Luciferin-PFBE substrate (50 μM). Cytochrome P450 activity was measured 8h later using the P450-Glo Assay Kit (Promega) according to manufacturer’s instructions.

Concentrations of midazolam, 1-hydroxymidazolam (1-OH-M) and 1-hydroxymidazolam-glucuronide (1-OH-MG) were determined in 50 μl using LC-MS/MS. Analysis was carried out at the Clinical Pharmaceutical and Toxicological Laboratory of the Department of Clinical Pharmacy of the University Medical Center Utrecht, the Netherlands. All experiments were performed on a Thermo Fisher Scientific (Waltham, MA) triple quadrupole Quantum Access LC-MS/MS system with a Surveyor MS pump and a Surveyor Plus autosampler with an integrated column oven. Analytes were detected via MS/MS, with an electrospray ionization-interface in selected reaction monitoring-mode, by their parent and product ions. The method showed linearity over the range of 0.02 – 1.50 mg/L for MDZ and OHM and over the range of 0.10 – 10.0 mg/L for HMG. The analytical accuracy and precision were within the maximum tolerated bias and CV (20% for LLOQ, 15% for the other concentrations). Since a 1-OH-MG standard was not available, a Gold Standard was used. The Gold Standard consisted of the urine from two adult intensive care patients with a high dose of intravenous midazolam and good renal function. Total bile acids were measured on an AU5811 routine chemistry analyzer (Beckman Coulter, Brea, California) with an enzymatic colorimetric assay (Sentinel Diagnostics, Milano, Italy). Ammonia elimination was analyzed as follows: organoid cultures were expanded and differentiated in DM medium for 8 days. On day 8 CAG was added to the medium and 3 days later the organoids were remouved from the matrigel, washed with Williams’ medium and subsequently incubated with 1 ml of test medium (Williams’ E medium (Lonza, Basel, Switzerland) with 10% fetal bovine serum (Lonza), 5 μg / mL insulin (Sigma, St. Louis, U.S.), 50 μM hydrocortisone hemisuccinate (Sigma), 2mM glutamine (Lonza), 50 U / mL penicilline and 50 μg / mL streptomycin (penicilline/streptomycine mix (Lonza), 1.5 mM NH4Cl (Sigma), 2.27 mM D-galactose (Sigma), 2 mM L-lactate (Sigma) and 2 mM ornithine hydrochloride (Sigma)). Then 0.25 ml samples were taken after 45 min, 7 and 24 hr and stored at −20°C for further analysis. Subsequently, all cultures were washed twice with PBS, trypsinized and cell number was counted by tripan blue exclusion.

Concentrations of ammonia were assessed in all samples by using the Ammonia (rapid) kit (Megazyme International, Wicklow, Ireland). The rates of ammonia elimination were established by calculating the changes in absolute molecular amounts of ammonia in the medium and corrected for time and cell number.