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Acquisition of epithelial plasticity in human chronic liver disease – Nature

Ethics

Biopsy collection and processing of human samples were carried out under ethics approval by Addenbrookes Hospital REC 18/WM/0397. The study met all the UK criteria for the responsible use of human tissue. Every donor whose samples were used was offered the patient information sheet and provided informed consent. Healthy deceased transplant organ tissue and explants were taken under ethics approval by the National Research Ethics Service Committee East of England – Cambridge South (REC number REC 15/EE/152).

Tissue collection and freezing

Liver biopsies were done with ultrasound guidance using a 16 g end cut needle (Biopince). Two ultrasound-guided needle core liver biopsies of approximately 2 cm were obtained. Half of the second biopsy (1 cm) was placed in a cryo-vial and frozen immediately using liquid nitrogen. For healthy donors and explant tissue, a cube of approximately 1 cm3 was cut and frozen as above. For two healthy donors (Hl1 and HL3) and all end-stage patients, samples were taken from each of the three liver lobes (left, right and caudate), so these individuals contributed three samples to the dataset. Samples were then stored at −80 °C. Details of patient demographics and disease staging are included in Supplementary Tables 1 and 2.

Nucleus isolation

Frozen samples were transferred to a Dounce homogenizer and lysed in 1 ml lysis buffer (IGEPAL 0.1%, NaCl 10 mM, Tris-HCL pH 7.5 10 mM, MgCl2 3 mM in nuclease-free water supplemented with 0.2 U μl−1 RNasin plus). Lysis was done by performing five strokes with part A and 10–15 strokes with part B on ice, with 2 min incubation on ice between using parts A and B. After a further 2 min on ice, the sample was mixed using a P1000 by pipetting up and down ten times before a further 1 min on ice. The sample was then passed through a pre-wet 40 μm cell strainer, transferred to a 1.5 ml low-bind microfuge tube and centrifuged at 500g for 5 min at 4 °C. The pellet was resuspended in 1 ml wash buffer (Ultrapure BSA 1% in tissue-culture grade supplemented with 0.2  U  μl−1 RNasin plus) and centrifuged at 500g for 5 min at 4 °C. The pellet was resuspended in 400 μl wash buffer and transferred to a tube for FACS and kept on ice, and the sample was treated with 3 μM DAPI. FACS sorting was performed on an Influx or Aria Fusion cell sorter. Nuclei were defined by strict FSC (forward scatter) and SSC (side scatter) gating to remove debris and intact cells (larger events on the FSC). A strict singlet gate was applied and nuclei were sorted in high-purity mode with the sorter precooled. Then 20,000 DAPI-positive nuclei were sorted into a 1.5 ml microfuge tube containing 500 μl wash buffer and the tube was topped up and centrifuged at 500g for 5 min at 4 °C. The pellet was resuspended in 43 μl wash buffer and kept on ice until loading on the 10x chromium. As part of the protocol optimization, a series of lysis buffers and incubation times were tested and lysis was examined using Trypan blue and a cell counter, with efficient lysis showing more than 95% lysed cells before sorting. After sorting, nuclei were examined to ensure a single nuclei suspension of intact nuclei (nuclear membrane intact with minimal blebbing).

Single-nucleus RNA-seq

Single-nucleus RNA-seq libraries were prepared using the following: Chromium Single Cell 3′ Library and Gel Bead Kit v.3.1, Chromium Chip G Kit and Chromium Single Cell 3′ Reagent Kits v.3.1 User Guide (manual part CG000316 Rev A; 10x Genomics). One sample was run per lane of the 10x chip. For each sample, 16,000 nuclei were loaded on the Chromium instrument with the expectation of collecting gel–bead emulsions containing cell nuclei. RNA from the barcoded nuclei for each sample was subsequently reverse-transcribed in a C1000 Touch Thermal cycler (Bio-Rad) and all subsequent steps to generate single-nuclei libraries were done according to the manufacturer’s protocol with 19 PCR cycles in the cDNA amplification step. cDNA quality and quantity were measured using Agilent TapeStation 4200 (High Sensitivity 5000 ScreenTape) after which 25% of the material was used to prepare the gene-expression library. Library quality was confirmed with Agilent TapeStation 4200 (High Sensitivity D1000 ScreenTape to evaluate library sizes) and Qubit 4.0 Fluorometer (ThermoFisher Qubit dsDNA HS Assay Kit to evaluate the double-stranded DNA quantity). Each sample was normalized and pooled in equal molar concentrations. To confirm the concentration of the pool we performed qPCR using a KAPA Library Quantification Kit on QuantStudio 6 Flex before sequencing. The pool was sequenced on an Illumina NovaSeq6000 sequencer with the following parameters: 28 base pairs (bp), read 1; 10 bp, i5 index; 10 bp, i7 index; 90 bp, read 2.

FLASH imaging

FLASH was performed as described65. Samples were fixed overnight in 4% PFA at 4 °C. The sample was transferred to PBS and sliced using a vibratome to generate slices 500 μm thick. Depigmentation was performed by incubating samples in DMSO and H2O2 in PBS in a 1:1:4 (by volume) ratio overnight. The next day, samples were washed briefly in PBS and transferred to an antigen-retrieval solution. To prepare the antigen-retrieval solution, urea was dissolved in 200 mM boric acid to 250 g l−1. Zwittergent was then dissolved in the urea–borate solution to 80 g  l−1. Samples were incubated in 1 ml of the solution in a 2 ml microcentrifuge tube at room temperature for 1 h, then left overnight at 54 °C with gentle mixing on a thermo-mixer. The next day, samples were washed in PBT (0.2% Triton X-100 in PBS) three times for 1 h at room temperature before being moved to blocking buffer (1% BSA, 5% DMSO, 10% FCS and 0.2% Triton X-100) in PBS and incubated overnight at room temperature. Primary antibodies were then incubated in blocking buffer (dilution 1:100) for at least 2 nights at room temperature on a nutator. Samples were washed in PBT three times for 1 h per wash before fluorophore-conjugated secondary antibodies were added for two nights (dilution of 1:200) at room temperature on a nutator. Samples were then washed in PBS three times for 30 min per wash and passed through a dehydration series of 30%, 50%, 75% and then 2 × 100% methanol for at least 30 min in each solution, protected from light. Dehydrated samples were then gradually cleared by submerging in methyl salicylate diluted in methanol at 25%, 50%, 75% and 2 × 100% methyl salicylate for at least 30 min each in a glass dish protected from light. Cleared samples were then mounted on a glass slide in 100% methyl salicylate. Samples were imaged using an upright LSM 880 microscope, using 10× and 20× water immersion lenses.

Immunofluorescence staining of tissue slides

For all tissue-staining experiments, multiple tissue sections from at least four different patients of the relevant disease stage were analysed. Slides were dewaxed in HistoClear twice for 5 min before being washed in 100% ethanol for 5 min. Slides were then passed through a rehydration series for 5 min of 95%, 90%, 80% and 50% ethanol, then distilled water. Heat-mediated antigen retrieval was performed using 10 mM citrate (pH 6.2). The buffer was pre-warmed in a microwave until gently bubbling, before slides were submerged and heated for 15 min in the microwave on 50% power to maintain gentle bubbling. Slides were then cooled and washed twice briefly in PBS. Slides were incubated in blocking solution containing 1% (w/v) BSA, 5% (v/v) donkey serum and 0.1% (v/v) Triton X-100 for 30 min at room temperature in a humidified chamber. Primary antibodies were then diluted in blocking solution (all at 1:100 dilution except for anti-SOX4, which was used at 1:50) and incubated overnight at 4 °C in a humidified chamber. The next day, slides were washed three times for 15 min in PBS before fluorophore-conjugated secondary antibodies (1:500 dilution) plus DAPI were applied for 1 h at room temperature in the humidified chamber. Then, slides were washed three times for 15 min in PBS. Slides were mounted in one drop of DAKO fluorescent mounting medium. Slides were imaged using a Zeiss inverted 710 confocal microscope.

Organoid derivation

Tissue was stored at 4 °C in basal medium (Advanced DMEM/F12, 1% Glutamax, 1% HEPES, 1% penicillin-streptomycin) after retrieval and derivation was attempted within 24 h of tissue storage. Tissue was minced with a scalpel or scissors to small pieces of less than 1 mm3 in basal medium. The minced tissue was transferred to a 50 ml conical tube with enough digestion medium (collagenase D 2.5 mg ml−1 and DNAse I 0.1 mg ml−1 in HBSS) to fully cover it and placed in a water-bath at 37 °C for 70 min with pipetting to mix every 10 min. Cold wash medium (DMEM, 1% Glutamax, 1% FBS, 1% pen-strep) was added to stop the digestion and the sample was centrifuged at 400g for 4 min. The pellet was resuspended in 5 ml wash medium and centrifuged again as before. The resulting pellet was then resuspended in growth-factor-reduced Matrigel and plated in 50-μl domes on a 24-well plate. The plate was incubated at 37 °C for 15 min before 500 μl isolation medium was added (Advanced DMEM/F12, 1% Glutamax, 1% HEPES, 1% pen-strep, 1% B27 without vitamin A, 1% N2 supplement, 10% conditional RSPO medium, 30% WNT-conditioned medium, 25 ng ml−1 Noggin, 100 ng ml−1 FGF10, 25 ng ml−1 HGF, 50 ng ml−1 EGF, 10 mM nicotinamide 0.4 M, 10 nM gastrin, 1 mM N-acetyl cysteine, 10 μM FSK, 5 μM A8301, Noggin, 10 μM Y27632). Details of patient demographics are included in Supplementary Table 4.

Organoid culturing

After organoid derivation, the medium was changed from isolation medium to expansion medium (isolation medium without Y27632, Noggin and WNT-conditioned medium). Organoids were typically passaged every 7–10 days and the medium was changed every 2–3 days. For splitting organoids, the medium was replaced with 500 μl Cell Recovery solution (Corning). The Matrigel dome was scraped and collected using a P1000 and incubated on ice for 20 min. This was then spun at 400g for 4 min and the pellet was resuspended in basal medium using a P1000 to break up the organoids, before being centrifuged as before. The pellet was resuspended in an appropriate volume of Matrigel and plated in 50-μl domes in a 24-well plate. The plate was incubated at 37 °C for 15 min before 500 μl expansion medium was added.

Differentiation of organoids

Cholangiocyte organoids were split into expansion medium with the addition of 25 ng ml−1 BMP7 for 5 days (medium renewed every 2–3 days). Organoids were then passaged as above and plated into differentiation medium for additional 10 days and renewed every 2–3 days (Advanced DMEM/F12, 1% Glutamax, 1% HEPES, 1% pen-strep, 1% B27 without vitamin A, 1% N2 supplement, 25 ng ml−1 HGF, 50 ng ml−1 EGF, 10 nM Gastrin, 1mM N-acetyl cysteine, 0.5 μM A8301, 100 ng ml−1 FGF19, 10 μM DAPT, 3 μM dexamethasone, 25 ng ml−1 BMP7).

In vitro treatments

Cholangiocyte organoids were treated with expansion medium and BMP7 for 5 days. They were then passaged directly into differentiation medium (as above) with the addition of the small molecule of interest per condition (10 μM LY294002, 20 nM copanlisib, 1 μM MK-2206, 100 nM rapamycin, 10 μM MHY1485) for a total of 10 days. The medium was renewed every 2–3 days. For the time-course experiment, inhibitors were applied only at the time point indicated in the figure. For experiments in which organoids were cultured in increased collagen, the cell pellet was resuspended in a 50:50 mix of Matrigel and collagen I with NaOH added to neutralize the collagen before resuspending the cells. Organoids were then cultured as described above.

Immunofluorescence staining of organoids

Organoids that were planned for immunofluorescence staining were plated after splitting in a µ-Slide 8 Well High Glass Bottom (Ibidi) for better imaging quality. For staining on 3D organoid cultures, cells were washed with PBS once and then incubated with 4% PFA–PBS for 20 min at room temperature. After incubation, cells were washed three times with PBS and stored in PBS at 4 °C for up to a month. For intracellular epitopes, organoids were permeabilized using a solution of 10% donkey serum in PBS plus 0.3% Triton X-100 for at least 3 h. Cells were incubated with the primary antibody (1:100 dilution) in 1% donkey serum plus 0.1% Triton X-100 at 4 °C overnight. Cells were washed with PBS three times at room temperature for 1 h per wash. Then, cells were incubated with secondary antibody diluted 1:1000 in 1% donkey serum plus 0.1% Triton X-100 at 4 °C overnight. Cells were washed with PBS three times at room temperature for 1 h per wash. Cells were stained with Hoechst dye at 1:10,000 dilution in PBS for 30 min and washed twice. Cells were stored in PBS at 4 °C for up to a month. A Zeiss LSM 710 confocal microscope was used for imaging.

Collagen and haematoxylin-and-eosin staining

Collagen (Picro Sirius Red) staining and haematoxylin-and-eosin staining was done by the Department of Pathology at Addenbrookes Hospital in Cambridge according to their local protocol.

Statistical analysis of qPCR

Unpaired t-tests were used to perform statistical analysis on the qPCR, comparing uICOs and dICOs. One-way ANOVA adjusted for multiple comparisons was used to analyse in vitro treatments of organoids and patient insulin serum level. P values are indicated in the figure legends.

Computational methods

Sample quantification

The samples were mapped and the expression levels summarized using 10x Genomics CellRanger v.5.0.0 (ref. 66) against version GRCh38.p13 of the H. sapiens genome. To accommodate the characteristics of single-nucleus data, that is, a higher proportion of reads mapped to introns, the option ‘–include-introns’ was enabled.

Quality control

Seurat (v.4.0.3)67 objects were created considering genes expressed in more than three cells, and cells with more than 200 features expressed. Barcodes (nuclei) were excluded that had less than 1,000 or less than 800 features, or for which more than 10% of counts mapped to mitochondrial or ribosomal genes. To remove potential doublets, nuclei with more than 50,000 counts were also removed; the nCount, nFeature, %MT and %RP distributions per patient were visualized. After filtering, mitochondrial and ribosomal protein-coding genes were removed from the dataset, resulting in a dataset of 99,809 cells and 31,257 features across 47 samples.

Preprocessing

The preprocessing of raw count matrices was performed using Seurat (v.4.0.3)67. Gene-expression values were normalized for library size using sctransform68. Principal component analysis was carried out using the top 3,000 highly variable genes. Neighbours were identified using the first 50 principal components and clustering was done using the Louvain algorithm with the 20 nearest neighbours per cell. UMAP projections were calculated using ‘RunUMAP(n.neighbors = 20, min.dist = 0.3)’. The clustering parameters used were identified by evaluating the resulting cluster stability using ClustAssess69

Annotation of cells

Expression of cell-type marker genes (Supplementary Table 3 and Extended Data Fig. 3e) was used to assign cell-type labels.

Data integration

For hepatocyte and cholangiocyte cells, some sample-specific segregation was observed within each disease stage. To alleviate potential batch effects, the data were integrated using Harmony40 with default parameters except θ (the diversity clustering penalty parameter), which was minimized such that within each disease stage, all recovered clusters included cells from each patient.

Differential expression analysis

Genes differentially expressed between cell groups were identified using the Seurat FindMarkers function. Differentially expressed genes were called on: abs(log2FC) > 0.5, Benjamini–Hochberg corrected P < 0.05 and a minimum of 25% of cells expressing the gene in the higher-expression group. GSEA of differentially expressed genes was carried out using gprofiler2 (v.0.2.0)70 using all genes detected in the compared cell groups as the background set. Enrichment was tested on the standard Gene Ontology terms, KEGG and Reactome pathway databases, and the microRNA and TF regulatory features. The Benjamini–Hochberg correction for multiple testing was applied to GSEA P values.

Cells labelled as positive for one or multiple genes are those with SCtransform-normalized expression greater than 0, per gene. The proportion of biphenotypic cells across each disease stage was compared using Welch’s t-test. Loss of zonation through disease was assessed by comparing the correlation between pairs of periportal and pericentral markers, which were then contrasted using Welch’s t-test.

RNA velocity

Velocyto (v.0.17.17), and velocyto.R (v.0.6) were used to estimate RNA velocity on the basis of the prevalence of spliced and unspliced mRNA71. Velocyto run10× was run using GRCh38.p13 annotation and repeat mask. The dataset was randomly downsampled to 20,000 cells, and cell distance was calculated as 1 minus correlation in the first 50 principal components. RNA velocity was estimated using ‘gene.relative.velocity.estimates(deltaT = 1, kCells = 20, cell.dist = D, fit.quantile = 0.2)’.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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