Companies providing equipment, reagents and/or supplies: Abcam, Cambridge, MA; ACD Labs, Toronto, CA; Acris Antibodies, Inc), San Diego, CA; Advanced Bioscience Resources, Inc (ABR), Rockville, MD; Agilent Technologies, Santa Clara, CA; Alpco Diagnostics, Salem, NH; Applied Biosystems, Foster City, CA; BD Pharmingen, San Jose, CA; Becton Dickinson, Franklin Lakes, NJ; Bethyl Laboratories, Montgomery, TX; BioAssay Systems, Hayward, CA; Cambridge Isotope Laboratories, Tewksbury, MA; Biotime, Inc, Alameda, CA; Carl Zeiss Microscopy, Thornwood, NY; Carolina Liquid Chemistries, Corp., Winston-Salem, NC; Charles River Laboratories International, Inc, Wilmington, MA; Chenomx, Inc, Alberta, Canada; Cole-Parmer, Court Vernon Hills, IL; DiaPharma, West Chester Township, OH; Fisher Scientific, Pittsburgh, PA; Gatan, Inc, Pleasanton, CA; Illumina, San Diego, CA; Ingenuity, Redwood City, CA; Life Technologies Corp., Grand Island, NY; Leica, Washington, DC; LifeSpan Biosciences, Inc, Seattle, A; Molecular Devices, Sunnyvale, CA; Olympus Scientific Solutions Americas Corp., Waltham, MA; PhoenixSongs Biologicals (PSB), Branford, CT; Polysciences, Inc, Warrington, PA; Qiagen, Germantown, MD; R&D Systems, Minneapolis, MN; RayBiotech, Norcross, GA; Roche Diagnostics, Mannheim, Germany;Santa Cruz Biotechnology, Inc), Dallas, TX; Sigma-Aldrich, St. Louis, MO; Sofregen, Medford, MA; Takara, Otsu, Japan; Tousimis Research Corp., Rockville, MD; Triangle Research Labs (TRL), Research Triangle Park, NC; Umetrics, Umea, Sweden; Varian Medical Systems, Inc), Palo Alto, CA; Vector Laboratories, Burlingame, CA; VWR Scientific, Radnor, PA.

Ethical statements summarize how the animals were maintained. All animal experiments were performed in strict accordance with the approved Institutional Animal Care and Use Committees of North Carolina State University (NCSU) in Raleigh and at the University of North Carolina (UNC) in Chapel Hill and so have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. All procedures used in animal studies and those involving the use of human tissues were approved by The Institutional Animal Care and Use Committee (IACUC) and Institutional Review Board (IRB) committees at both institutions: the UNC and at the College of Veterinary Medicine at NCSU. The IACUC approval numbers are 16-316.0 and 17-225.0. Studies for human cells in this project were evaluated by the IRB committee and were provided an IRB approval number of 97-1063.

Human samples include information on each sample plus information general for all the samples and provided in detail in prior studies:^39,41,42

Fetal liver tissue was provided by an accredited agency (Advanced Biological Resources, San Francisco, CA) from fetuses between 18 and 22 weeks gestational age obtained by elective terminations of pregnancy. The research protocol was reviewed and approved by the IRB for Human Research Studies at UNC. All samples were screened for various pathogens and only those free of these were accepted for the research studies.

Postnatal livers were obtained from cadaveric neonatal, pediatric, and adult donors and were obtained through organ donation programs via UNOS. Those used for these studies were considered normal with no evidence of disease processes including following screening for pathogens. Informed consent was obtained from next of kin for the use of the livers for research purposes, protocols received IRB approval, and processing was compliant with Good Manufacturing Practice.

Pigs samples were from animals maintained at the facilities at the College of Veterinary Medicine at North Carolina State University (NCSU, Raleigh, NC). Some of them were used as hosts or as donors for cells. Surgeries, necropsies, and the collection of all biological fluids and tissues were performed at these facilities.

Porcine hosts used for the grafts were a mixture of six different breeds: a six-way cross consisting of Yorkshires, Large Whites, Landraces (from the sows), Durocs, Spots, and Pietrans (from the boars). This highly heterogeneous genetic background is desirable in that it parallels the heterogeneous genetic constitutions of human populations⁶⁵. The host animals were all females, ~6 weeks of age and ~15 kg.

Green fluorescent protein (GFP)⁺ transgenic pig donors were established carrying an H2B histone-eGFP transgene. The GFP⁺ donor animals were obtained by breeding a transgenic H2B-GFP boar with a wild-type gilt by standard artificial insemination⁶⁶. The model was developed via CRISPR-Cas9-mediated homology-directed repair (HDR) of IRES-pH2B-eGFP into the endogenous β-actin (ACTB) locus. The transgenic animals show ubiquitous expression of pH2B-eGFP in all tissues. Fusion of the GFP to H2B results in the localization of the GFP marker to the nucleosome and allows clear nuclear visualization as well as the study of chromosome dynamics. The founder line has been analyzed extensively and ubiquitously, and nuclear-localized expression has been confirmed. In addition, breeding has demonstrated transmission of the H2B-GFP to the next generation. All animals were healthy, and multiple pregnancies have been established with progeny showing the expected Mendelian ratio for the transmission of the pH2B-eGFP. The male offspring were genotyped at birth, and those that were positive for the transgene were humanely euthanized for tissue collection and isolation of donor cells.

Genotyping of porcine animals was done. For each donor and recipient animal, the swine leukocyte antigen class I (SLA-I) and class II (SLA-II) loci have been polymerase chain reaction (PCR) amplified using primers designed to amplify known alleles in these regions based on the PCR-sequence-specific-primer strategy¹⁸. The system consists of 47 discriminatory SLA-I primer sets amplifying the SLA-1, SLA-2, and SLA-3 loci, and 47 discriminatory SLA-II primer sets amplifying the DRB1, DQB1, and DQA loci⁶⁷. These primer sets have been developed to differentiate alleles by groups that share similar sequence motifs and have been shown easily and unambiguously to detect known SLA-I and SLA-II alleles. When used together, these primer sets effectively provided a haplotype for each animal that was tested, thus providing an assay to confirm easily a matched or mismatched haplotype in donor and recipient animals.

Surgeries on the pigs were done using anesthesia induced by administering a combination of ketamine/xylazine (2–3 mg/kg weight each) injected IV or 20 mg/kg ketamine plus 2 g/kg xylazine IM, and were maintained by isoflurane in oxygen administered via a closed-circuit gas anesthetic unit. In the grafts on the liver and general surgical procedures, the pigs were positioned in dorsal recumbency, and the ventral abdomen was clipped from xyphoid to pubis. The skin was aseptically prepared with alternating iodinated scrub and alcohol solutions. After entry into the surgery suite, preparation of the skin was repeated using a sterile technique, and the area was covered with a topical iodine solution before the application of sterile surgical drapes. The surgeons used an appropriate aseptic technique. A mid-ventral incision was made through the skin, through subcutaneous tissues and linea alba, starting at the xiphoid process and extending caudally 8–12 cm. The left hepatic division was exposed, and a 3 × 4.5 cm patch graft was applied to the ventral surface of the liver and containing a 1X hyaluronan hydrogel (~60 Pa) with embedded BTSC organoids and placed onto the backing containing 10X hyaluronan hydrogel (~760 Pa); the patch was placed in direct contact onto the surface of the liver capsule. The patch graft was sutured to the liver using 4–6 simple, interrupted sutures of 4–0 polypropylene. The exposed surface of the graft was then treated with 2 ml of 2X hyaluronan hydrogel (~200–300 Pa), a level of rigidity that was fluid enough to permit it to be painted or coated onto the outside of the graft; it served further to minimize adhesions from neighboring tissues. Following placement of the surgical graft, the linea alba was closed with a simple continuous suture using 0-PDS. The linea was blocked with 2 mg/kg 0.5% bupivacaine, IM. The subcutaneous tissues and skin were closed with continuous 2–0 PDS and 3–0 Monocryl sutures, respectively. Tissue adhesive was placed on the skin surface.

For grafts on the pancreas, surgical procedures for pigs made use of a graft that can be any size that accommodates the dimensions of the pancreas. For exposure of the pancreas, stay sutures were placed in the descending duodenum. The graft was placed onto the head of the pancreas and adjacent to the duodenum with the soft hyaluronan (~60 Pa) hydrogel containing the organoids in direct contact with the pancreas, and with the more rigid hyaluronan hydrogel (~760 Pa) within the silk backing. We used sutures at the four corners (4–0 Prolene); the first two corner sutures were placed in the descending duodenum; the other 2 were placed in the mesentery and avoiding the pancreatic parenchyma (to minimize any propensity for autolysis by the pancreas). The serosal surface of the graft was then covered with 2X hyaluronan (~200–300 Pa) hydrogel to minimize adhesions. The abdomen was closed with continuous absorbable (PDS) sutures in the linea, subcutaneous and subcuticular layers. Tissue adhesive was used on the skin to avoid skin sutures.

Immunosuppression was required since the transplants from the transgenic pigs to the wild-type recipients were allogeneic. The immune-suppression protocols used were ones established by others^68,69. All pigs received oral dosages of the immunosuppressive drugs, Tacrolimus (0.5 mg/kg) and Mycophenolate (500 mg) twice daily, beginning 24 h prior to surgery. The drugs were given continuously for the entire experimental period. These could be given to the animals easily if mixed with their favorite foods.

Isolation of normal tissues was done from newborn piglets and from older, 12-week-old pigs. Newborn piglets weighed ~10 lbs; the older pigs, 12 weeks of age, were ~50–60 lbs. Pigs were sacrificed by penetrating captive bolt euthanasia followed by jugular exsanguination. The method meets the recommended guidelines of the American Veterinary Medical Association for euthanasia in pigs⁶⁵. A listing of donors used for biliary tissues from piglets or adult pigs is given in Supplementary Table 4.

All media were sterile filtered (0.22 µm filter) and kept in the dark at 4 °C before use. Basal medium and fetal bovine serum (FBS) were purchased from GIBCO/Invitrogen. All growth factors were purchased from R&D Systems. All other reagents, except those noted, were obtained from Sigma.

Cell wash buffers consisted of 500 mls of basal medium (e.g. RPMI 1640; Gibco # 11875-093) were supplemented with 0.5 g of serum albumin (Sigma, # A8896-5G, fatty-acid-free), 10⁻⁹ M selenium, and 5 mls of antibiotics (Gibco #35240-062, AAS). It was used for washing tissues and cells during processing.

Collagenase buffer consisted of 100 mls of cell wash supplemented with collagenase (Sigma # C5138) with a final concentration of 600 U/ml (R1451 25 mg) for biliary tree (ducts) tissue and 300 U/ml (12.5 mg) for organs (e.g. liver).

Kubota’s Medium, a wholly defined, serum-free medium designed originally for hepatoblasts⁷⁰, and then found successful for the maintenance of biliary tree stem cells, hepatic stem cells, and pancreatic stem cells and progenitors (and later found successful in general for all endodermal stem/progenitors assessed), was used to prepare cell suspensions, organoids, and hyaluronan hydrogels. This medium consists of any basal medium (here being RPMI 1640) with no copper, low calcium (0.3 mM), 1 nM selenium, 0.1% serum albumin (purified, fatty-acid-free; fraction V), 4.5 mM nicotinamide, 0.1 nM zinc sulfate heptahydrate, 5 µg/ml transferrin/Fe, 5 µg/ml insulin, 10 µg/ml high-density lipoprotein, and a mixture of purified free fatty acids that are presented complexed with fatty-acid-free, highly purified albumin. The free fatty acid mixture was comprised of palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid. Kubota’s medium is available commercially from PhoenixSongs Biologicals (Branford, CT).

Hyaluronans (HAs) used included the soluble, long-chain forms of HA (Sigma Catalog #52747) and were used in the stabilization of organoid cultures and in cryopreservation of cell suspensions^71,72. Those used to make the hydrogels, thiol-modified HAs, were obtained from Glycosan Biosciences, formerly a subsidiary of Lineage Cell Therapeutics (Alameda, CA), and are now offered in non-clinical grade form from Advanced Biomatrix (Carlsbad, CA) and in the clinical grade form from Sentrex Animal Care (Salt Lake City, UT) (G. Prestwich, personal communications). The components for these thiol-modified HAs were made by a proprietary bacterial-fermentation process using Bacillus subtilis as the host in an ISO 9001:2000 process (www.biopolymer.novozymes.com/). The components were produced by Novozymes under the trade name, HyaCare®, and are 100% free of animal-derived materials and residual organic solvent. No animal-derived ingredients are used in the production; there are very low protein levels and no endotoxins. The production follows the standards set by the European Pharmacopoeia. The HA hydrogels were prepared using Glycosil (HyStem® HAs, ESI BIO-CG313), the thiol-modified HAs that can be triggered to form disulfide bridges in the presence of oxygen, or by forming thio-ether linkages using polyethylene glycol diacrylate (PEGDA). Glycosil® is reconstituted as a 1% solution of thiolated HA in 1% phosphate-buffered saline (PBS) using degassed water, or, in our case, in serum-free Kubota’s Medium. Upon reconstitution, it remains liquid for several hours but can undergo some gelation if exposed to oxygen. More precise gelation occurs with no temperature or pH changes if Glycosil is treated with a cross-linker such as PEGDA causing gelation to occur within a few minutes^73,74,75,76.

The level of cross-linking is the primary contributor to the level of stiffness (viscoelasticity) or rigidity and can be controlled by adjusting the ratio of the thiol-modified hyaluronans to PEGDA⁷⁶. In prior studies, hepatic stem cell populations were tested in HA hydrogels of varying levels of rigidity and were found to remain as stem cells, both antigenically and functionally (e.g. with respect to the ability to migrate and to the expression of stem cell-associated genes such as pluripotency genes and matrix metalloproteinases), only if the level of rigidity was less than ~100 Pa⁷⁶. We used this finding to design grafts with a soft layer (~100 Pa or less) in which to embed organoids of BTSCs and with more rigid layers (~700 Pa) of hyaluronan hydrogels in the backing to form a barrier to migration in directions other than the target tissue as well as ones with intermediate levels of rigidity (~200–300 Pa) to minimize adhesions.

Macro-scale rheological properties of hyaluronan hydrogels were determined using a stress-controlled cone-and-plate rheometer (TA Instruments, AR-G2, 40 mm cone diameter, 1° angle). Gels actively polymerized on the rheometer while oscillating at 1 rad/s frequency and 0.6 Pa stress amplitude with the modulus monitored continuously to query for sufficient completion of the cross-linking reactions. Once equilibrated, the hydrogels were subjected to an oscillatory frequency sweep (stress amplitude: 0.6 Pa, frequency range: 0.01–100 Hz).

Preparation of cells was done from extrahepatic biliary tree tissue (gall bladder, common duct, hepatic ducts) obtained from pigs [or from human tissue]. For the tissues used to generate organoids of normal cells, tissues were pounded with a sterilized, stainless-steel mallet to eliminate the parenchymal cells, carefully keeping the linkage of the intra-hepatic and extrahepatic bile ducts. The biliary tree was then washed with the “cell wash” buffer comprised of a sterile, serum-free basal medium supplemented with antibiotics, 0.1% serum albumin, and 1 nM selenium (10^–9 M). It was then mechanically dissociated with crossed scalpels, and the aggregates enzymatically dispersed into a cell suspension in RPMI-1640 supplemented with 0.1% bovine serum albumin (BSA) [or for human tissues, human serum albumin, HAS], 1 nM selenium, 300 U/ml type IV collagenase, 0.3 mg/ml deoxyribonuclease (DNAse) and antibiotics. Digestion was done at 32 °C with frequent agitation for 30–60 min. Most tissues required two rounds of digestions followed by centrifugation at 1100 rpm at 4 °C. Cell pellets were combined and re-suspended in cell wash. The cell suspension was centrifuged at 30×g for 5 min at 4 °C to remove red blood cells. The cell pellets were again re-suspended in cell wash and filtered through a 40 µm nylon cell strainer (Becton Dickenson Falcon #352340) with fresh cell wash. The cell numbers were determined, and viability was assessed using Trypan Blue. Cell viability above 90–95% was observed routinely.

Formation of organoids was done from cell suspensions added to multiwell, flat-bottom cell culture plates (Corning #353043) in serum-free Kubota’s medium and incubated for ~an hour at 37 °C to facilitate attachment of mature mesenchymal cells (e.g. mature stroma, mature stellate cells). Mature mesenchymal cells attached to the dishes within ~15 min even though the medium was serum-free. The immature cells remaining in suspension and were transferred to another dish and again incubated for up to an hour. This was repeated several times to ensure the depletion of a significant fraction of the mature mesenchymal cells. After depletion of mature mesenchymal cells, the remaining floating cells were seeded at ~2 × 10⁵ cells per well in serum-free Kubota’s medium in Corning’s ultralow attachment dishes (Corning #3471) and were incubated overnight at 37 °C in a CO₂ incubator. Organoids comprised of the biliary tree stem cells (BTSCs) partnered with early lineage stage mesenchymal cells (ELMSCs) formed overnight. The ELMSCs were found by their expression of specific surface antigens to be angioblasts (CD117⁺, CD31⁻, VEGFr⁺) and precursors to endothelia (CD31⁺, VEGFr⁺) and to stellate cells (ICAM-1⁺, CD146⁺). More extensive characterizations of these ELSMCs were done, and the findings have been published previously^77,78. The organoid cultures survived for weeks in Kubota’s medium, especially if the medium was supplemented (0.1%) with soluble forms of hyaluronans (Sigma).

The cells could also be cryopreserved as described below. From each gram of pig biliary tree tissue, we obtained ~1.5 × 10⁷ cells. We used ~3–6 × 10⁵ cells per well of a 6-well, ultra-low attachment plate and incubated in the serum-free Kubota’s medium. The cells produced, on average, 6000–20,000 small organoids (~50–100 cells/organoid/well). For the grafts, we used at least 100,000 organoids (>10⁷ cells). Depending on the size of the backing, we were able to increase the number of organoids in the grafts up to >10⁸ organoids (i.e. ~10⁹ cells total) or more embedded in ~1 ml of the soft hyaluronan hydrogel on a 3 cm × 4.5 cm backing.

Cryopreservation of stem/progenitors was most successfully done by cryopreservation of isolated cells or small cell aggregates that had been subjected to the panning procedures to minimize the numbers of mature mesenchymal cells. One can cryopreserve the mix of stem/progenitors and early lineage stage mesenchymal cells in Cryostor10, an isotonic cryopreservation buffer containing antifreeze factors, dextran, and DMSO (Bioliife, Seattle, WA). The viability of the cells was improved further with supplementation with 0.1% HAs (Sigma #52747)^71,72. The highest viability of the organoids was when prepared from freshly thawed, cryopreserved cell suspensions; lower viabilities were observed with cryopreserved, fully formed organoids. Cryopreservation was done using CryoMed™ Controlled-Rate Freezers. The viability on thawing was greater than 90% for those cryopreserved as a cell suspension and then, upon thawing, used to form organoids^71,72.

Patch grafts are novel methods established rapidly to transplant large numbers (e.g. >10⁸th) of organoids into solid organs and to have the organoids fully integrate within the organs within a week and mature into adult cells within another week. Further details of the logic, strategies, and methods for patch grafting of organoids into solid organs are given in our prior publications^54,55. Grafts were formed using a backing, Contour Seri-silk (Sofregen, Medford, MA), onto which were placed the stem/progenitor organoids embedded in soft hyaluronan hydrogels (~50–100 Pa). These were readily prepared ahead of time and maintained in a culture dish in an incubator overnight. The lack of success with attempts to cryopreserve organoids within soft hydrogels meant that embedding the organoids in the soft hydrogel had to be done just prior to surgery to attach the grafts.

Update: We have learned that Contour Seri-silk is no longer available from the manufacturer. Amnion-based matrix materials, such as ones from Vivex Biologics (Miami, FL), should provide an appropriate substitute and is an FDA-approved implant material for clinical or non-clinical purposes. These should provide adequate mechanical support for the grafts and are hypothesized to be reasonably neutral in effects on donor organoids; this is important to ensure that the organoids retain their stem cell traits enabling them to express matrix metalloproteinases (MMPs) required for engraftment and migration⁵⁴. Clinical uses of such amnion-derived matrices have been discussed by others in a recent review⁷⁹. It is assumed that the more rigid hyaluronan hydrogel (~700 Pa) will not be required given the complex matrix chemistry within the amnion that should serve as the barrier. However, it is hypothesized that after tethering to the target site, an amnion matrix barrier might still require coating with hyaluronans on both sides and with the hyaluronan prepared with serum-free Kubota’s Medium and at an intermediate level of rigidity (~200–300 Pa). The coating on the serosal side at the time of surgery should minimize adhesions to the neighboring tissues.

For necropsy procedures, all animals were humanely euthanized at the designated time point by sedation with Ketamine/Xylazine, and isofluorane anesthesia, followed by an intravenous injection of a lethal dose of sodium pentobarbital. Upon confirmation of death, the carcass was carefully dissected, and the target organs were removed, and placed in chilled Kubota’s Medium for transportation to the lab. In addition to the liver, the lungs, heart, kidney, and spleen were collected and fixed in 10% neutral formalin.

Histology was done on portions of the freshly dissected tissues from the pigs or the grafts on the liver or pancreas were fixed with 4% paraformaldehyde (PFA) and then paraffin-embedded. Five-micron sections were cut and stained with hematoxylin-eosin for routine histological analyses. The tissues obtained from neonatal pigs were prepared also as samples (liver, pancreas, biliary tree and duodenum) in large paraffin blocks to facilitate analyses of the connections throughout the biliary tree and pancreatic duct system.

Immunohistochemistry (IHC) was used for analyses of various markers and traits. For immunofluorescent staining, 5 µm frozen sections or cultured cells were fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature, rinsed with PBS, blocking with 10% goat serum in PBS for 2 h, and rinsed. Fixed cells were incubated with primary antibodies at 4 °C for 14 h, washed, incubated for 1 h with labeled isotype-specific secondary antibodies, washed, and counterstained with 4´,6-diamidino-2-phenylindole (DAPI) for visualization of cell nuclei and viewed using Leica DMIRB inverted microscope (Leica, Houston, TX) or a Zeiss ApoTome Axiovert 200 M (Carl Zeiss Inc, Thornwood, NY).

For IHC, the tissues were fixed in 4% PFA overnight and stored in 70% ethanol. They were embedded in paraffin and cut into 5 μm sections. After deparaffinization, antigen retrieval was performed with sodium citrate buffer (pH 6.0) or ethylenediaminetetraacetic acid (EDTA) buffer (pH 8.0) in a steamer for 20 min. Endogenous peroxidases were blocked by incubation for 15 min in 3% H₂O₂. Sections were incubated for 30 min at room temperature with ImmPRESS peroxidase staining kits and 3,3’-diaminobenzidine substrates (Vector Laboratories). Sections were counterstained with hematoxylin. Antibodies used are listed in Supplementary Table 1.

Purification of cell populations for genetic studies was important for various analyses. The purity of the subpopulations being isolated was dictated by a combination of strategies for the isolation of the cells. The adult hepatocytes were freshly isolated from neonatal porcine livers versus from neonatal, pediatric, and adult human livers by standard collagenase digestion and percoll fractionation⁸⁰. The isolated adult hepatocyte fractions (those with average diameters above 17 µm) were rinsed in RPMI1640 supplemented with 2% serum to inactivate the enzymes used in isolation of the cells; then treated with multiple rounds of rinsing with serum-free RPMI 1640 medium; and finally, snap frozen using liquid nitrogen. The cells were not cultured.

The subpopulations of stem/progenitors, both human and porcine ones, were prepared from freshly isolated cells from the fetal or neonatal biliary tree (biliary tree stem cells), from fetal or neonatal livers (hepatic stem cells and hepatoblasts), or fetal or neonatal pancreases (pancreatic stem cells and ductal progenitors); purified by a combination of immunoselection for surface antigens, enabling the isolation of separate cellular sub-populations^39,41,50,54. Supplementary Table 5 summarizes the markers of the subpopulations that have been characterized extensively, especially in past studies^{31,32,33,38,39,40,41,42,43,49,50}.

The immunoselected cells were suspended in serum-free Kubota’s Medium⁸¹, designed for endodermal stem cells, and seeded onto low attachment culture dishes for 6–12 h enabling the formation of organoids. The organoids that formed in that 6–12 h comprised the endodermal stem cells partnered with early lineage stage mesenchymal cells: angioblasts and precursors to endothelial and stellate cells (note: the mature mesenchymal cells were minimized by the immunoselection process as indicated by the depletion of cells for surface antigens for the mature hemopoietic and mesenchymal cells).

RNA-sequencing and gene expression analysis included RNA purified using Qiagen RNeasy Kit from the porcine (or human) adult liver, pancreas, gallbladder, and biliary tree tissue and from isolated cell suspensions of hepatocytes (AHeps), hepatic stem cells (HpSCs), biliary tree stem cells (BTSCs), each from three different donors for human cells, and five different donors for pig cells (Supplementary Tables 6 and 7). RNA-seq data collection and quality control analyses were specifically depicted in our previous study⁵². The LIMMA (version 3.50.1) pipeline was used to identify differentially expressed genes (DEGs). Gene expression profiles were compared using Pearson’s and Spearman’s correlation analyses. Principal component analysis (PCA) and hierarchical clustering were performed in R (R version 4.1.0).

Quantitative reverse transcription and polymerase chain reaction were done using total RNA from cells. Total RNA was extracted from the cells using Trizol (Invitrogen, Carlsbad, CA). First-strand cDNA synthesized using the Prime script 1st strand cDNA synthesis kit (Takara, Otsu, Japan) was used as a template for PCR amplification. Quantitative analyses of mRNA levels were performed using Faststart Universal Probe Master (R oche Diagnostics, Mannheim, Germany) with ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA). Primers were designed with the Universal Probe Library Assay Design Center (Roche Applied Science). Primer sequences are listed in Supplementary Table 4a. The primers were annealed at 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of 95 °C (15 s) and 60 °C (1 min). Expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used generally as a standard.

Statistical analysis

Statistically significant differences between samples are calculated by using Student’s 2-tailed t-test and results are presented as the mean ± SD. P values of <0.05 were considered statistically significant.

Reporting summary

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

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• Source: https://www.nature.com/articles/s41536-023-00303-5