Stem cell-derived polarized hepatocytes

Human stem cell-derived hepatocyte-like cells (HLCs) offer an attractive platform to study liver biology. Despite their numerous advantages, HLCs lack critical in vivo characteristics, including cell polarity. Here, we report a stem cell differentiation protocol that uses transwell filters to generate columnar polarized HLCs with clearly defined basolateral and apical membranes separated by tight junctions. We show that polarized HLCs secrete cargo directionally: Albumin, urea, and lipoproteins are secreted basolaterally, whereas bile acids are secreted apically. Further, we show that enterically transmitted hepatitis E virus (HEV) progeny particles are secreted basolaterally as quasi-enveloped particles and apically as naked virions, recapitulating essential steps of the natural infectious cycle in vivo. We also provide proof-of-concept that polarized HLCs can be used for pharmacokinetic and drug-drug interaction studies. This novel system provides a powerful tool to study hepatocyte biology, disease mechanisms, genetic variation, and drug metabolism in a more physiologically relevant setting.

A full description of the statistics including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.

For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings
For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated

Clearly defined error bars
State explicitly what error bars represent (e.g. SD, SE, CI) Our web collection on statistics for biologists may be useful.

Software and code
Policy information about availability of computer code Data collection NIS-Elements BR version 4.10.01 was used for image acquisition.

Data analysis
Graphpad PRISM version 5.0 was used for statistical analysis. Fiji version 2.0.0-rc-69/1.52p was used for image analysis. RNA-seq data analysis was performed using open source software. Specifically, Seqtk version 1.2 and fastx_toolkit version 0.0.14 were used to process sequences in FASTQ format. Sequencing reads were aligned to the reference genome using TopHat2 version 2.0.12 with Bowtie version 2.2.7. Aligned reads were counted using featureCounts from subread version 1.4.6. Statistical analyses were performed using the edgeR Bioconductor package version 3.12.1 in the statistical computing environment R (www.r-project.org).
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers upon request. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

April 2018
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: All studies must disclose on these points even when the disclosure is negative.

Sample size
Depending on the assay, we chose a sufficient number of biological replicates to produce data with errors at acceptably low level while avoiding making the study unnecessarily large, meaning high costs (for stem cell differentiation, RNA-seq analysis, and mass-spec analysis), logistical issues (sending biological samples to Singapore on dry ice while preserving the quality of the samples), and ethical issues (use of primary human hepatocytes).
Data exclusions No data were excluded from the analyses.

Replication
Experiments revealing differences in measured data were repeated 3 -4 times independently, as indicated in figure legends. For the RNA-seq analysis performed in Figure 2 and Supplemental Figure 8, experiments (including RNA extraction, library preparation, and next generation sequencing) were repeated independently twice. All results were successfully replicated.
Randomization The study includes no experiments dependent on allocation of samples/organisms/participants into experimental groups.

Blinding
The study includes no experiments dependent on group allocation and blinding is thus not relevant.
Reporting for specific materials, systems and methods  Widely used human pluripotent stem cells, including WA09 and HUES8 were obtained from WiCell and Huangfu's laboratory at MSKCC, respectively. These cells were routinely tested for expression levels of relevant pluripotent and cellular factors under in study (for example they express pluripotent markers, APOC3 knockout cells were null for APOC3, and CYP8B1 knockdown cells displayed reduced levels of CYP8B1 upon hepatic differentiation) by western and/or DNA/RNA analyses. For stability, seed stocks of all pluripotent cell lines were frozen at early passages (P25 for H9 and P18 for HUES8). Experiments were performed using cells within 10 passages after recovery from liquid nitrogen. For sterility, pluripotent cell lines were tested routinely for mycoplasma. For phenotyping, the expression of pluripotent biomarkers such as POU5F1, NANOG, SSEA4, etc were routinely checked against a baseline description of the cell line, using gene expression analysis. For pluripotency, pluripotent cells were tested routinely for trilineage differentiation using qRT-PCR for germ layer marker expression. S10-3 cells were subcloned from human hepatoma cell Huh7 and selected for their high efficiency to support hepatitis E virus replicaton. For mutation profile analysis, we performed amplicon sequencing and verified the mutation in KDR gene (1416A>T, Gln472His) in the S10-3 cells used in this study. For the stability, S10-3 cells have been expanded at early passages and frozen down using a seed lot system. Experiments were performed from the working lot, using cells within 5 passages after being recovered from liquid nitrogen. For sterility, S10-3 cells have been tested routinely for mycoplasma contamination. S10-3 cells have been routinely tested for expression levels of relevant factors (positive for CD24, CD133 and EpCAM, and negative for THY1) under this study by western and/or DNA/RNA analyses against a baseline description of the Huh7 cell line as reported previously (Hum Cell. 2018;31(3): 261-267).
Mycoplasma contamination All cell lines tested negative for mycoplasm contamination Commonly misidentified lines (See ICLAC register) No commonly misidentified lines were used