Bile duct organoid

Abstract

Disclosed herein are improved pluripotent stem cell-derived biliary organoids, having extrahepatic cholangiocytes. In various embodiments, the biliary organoids can additionally include intrahepatic cholangiocytes. Also disclosed are methods of producing the biliary organoids having extrahepatic cholangiocytes. The disclosure also relates to methods of studying or treating a biliary-related disease or disorder using the described biliary organoids, including surgical methods such as in transplants, engraftments, ligations, and / or bililary bypass. The disclosure also includes surgical implants for biliary bypass. The disclosure additional describes methods of producing tubular organoids and cell culture devices for performing said methods.

Description

BILE DUCT ORGANOIDPRIORITY

[0001] The present application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63 / 631,073, BILE DUCT ORGANOID, filed on filed April 8, 2024, which is currently co-pending herewith and which is incorporated by reference in its entirety.FIELD

[0002] Aspects of the present disclosure generally relate to biliary, or bile duct, organoids prepared from human pluripotent stem cells, methods of preparation, and compositions including the same, as well as uses thereof.BACKGROUND

[0003] The biliary system plays an essential role in the transport of bile, a detergent-rich secretion critical for lipid digestion and absorption in the intestine. Bile, however, is inherently cytotoxic, and its misdirection or stagnation can trigger severe liver injury. The biliary tree is anatomically divided into intrahepatic (IHBD) and extrahepatic bile ducts (EHBD), with the latter spanning from the hepatic duct confluence to the common bile duct. Disorders affecting the EHBD, such as biliary atresia, Alagille syndrome, and chronic graft rejection after liver transplantation, frequently result in bile duct strictures or ductopenia, ultimately progressing to biliary cirrhosis and liver failure.

[0004] While palliative interventions, such as Roux-en-Y hepaticojejunostomy, offer temporary bile drainage, they are associated with high rates of cholangitis and recurrent biliary complications. Liver transplantation remains the only definitive therapy in advanced cases but is constrained by donor scarcity, surgical risks, and lifelong immunosuppression. Therefore, there is a pressing need for regenerative approaches capable of reconstructing functional EHBD architecture to preserve native liver function.SUMMARY

[0005] Various embodiments of the disclosure relate to in vitro methods of producing a human biliary organoid (HBO), the methods including: (a) culturing biliary progenitor cells derived from posterior foregut cells in biliary differentiation media (BDM); (b) activating an EGFsignaling pathway and a JAK-STAT signaling pathway in the cultured cells of step (a); (c) activating a JAK-STAT, Ras-MAPK, LIFR, and / or OSMR pathway in the treated cells of step (b); and (d) activating an EGF signaling pathway and a JAK-STAT, Ras-MAPK, LIFR, and / or OSMR signaling pathway in the treated cells of step (c); to provide a human biliary organoid including extrahepatic cholangiocytes.

[0006] In some embodiments, the HBO further includes intrahepatic cholangiocytes. In some embodiments, the HBO includes mature cholangiocyte cells, mesenchymal cells, and / or TROP2-expressing epithelial cells, and / or two or more distinct populations of cholangiocytes. In some embodiments, the cholangiocytes include intrahepatic bile duct (IHBD)-like cholangiocytes, gallbladder (GB)-like cholangiocytes, and common bile duct (CBD)-like cholangiocytes. In some embodiments, the HBO includes at least about 10%-50% mesenchymal cells and at least about 10%-50% cholangiocytes; optionally wherein the HBO includes at least about 15-35% mesenchymal cells and at least about 15%-35% cholangiocytes.

[0007] In some embodiments, the HBO further includes hepatic progenitors, stellate cells, endothelial cells, macrophages, and / or Kupffer cells. In some embodiments, the HBO further includes about 5%-60% hepatic progenitors, 1 %-25% stellate cells, l%-25% endothelial cells, 1%- 25% macrophages, and / or l%-25% Kupffer cells. In some embodiments, the HBO includes smooth muscle cells, peribiliary gland progenitors, glandular elements, tight junctions, and / or complex structures with cystic motifs communicating via a tubular assembly. In some embodiments, the HBO includes a TROP2-expressing cell population and / or a CK7-expressing cell population.

[0008] In some embodiments, the HBO cholangiocytes are enriched in one or more genes associated with one or more biological processes selected from bile acid metabolism, bile acid transport, bile salt transport, and bicarbonate transmembrane transporter activity. In some embodiments, the HBO has an elevated expression of one or more mature cholangiocyte marker, and / or reduced expression of one or more hepatoblast markers, relative to a human liver organoid (HLO). In some embodiments, the one or more cholangiocyte marker includes one or more of TROP2, cytokeratin 7 (CK7), cytokeratin 19 (CK19), aquaporin 1 (AQP1), multi-drug resistance- associated protein 3 (MRP 3), and bile salt export pump (BSEP),' and / or wherein the one or more hepatoblast marker includes one or more of alpha fetoprotein (AFP) and albumin (ALB). In some embodiments, the HBO expresses CD68, CD45, CD 11b, PEC AMI, CDH5, PDGFRA, VIM,ACTA2, HNF4A, DCN, PTN, CALCRL, CK7, CK19, EPCAM, SOX17, ZO1, a-SMA, FLT1, and / or vimentin.

[0009] In some embodiments, the HBO further includes a tubular assembly and / or a cystic structure. In some embodiments, the HBO includes a tubular assembly. In some embodiments, the HBO transports Rhodamine 123 (Rho 123) and / or bile into a luminal space of the HBO, and the capacity of Rho 123 and / or bile transportation into the luminal space is elevated as compared to a human liver organoid (HLO).

[0010] In some embodiments, the media in steps (a)-(d) includes biliary differentiation media. In some embodiments, the biliary differentiation media in one or more of steps (a)-(d) further includes Vitamin C; optionally wherein the biliary differentiation media in each of steps (a)-(d) further includes Vitamin C.

[0011] In some embodiments, the EGF signaling pathway in step (b) and / or step (d) is activated by EGF; the JAK-STAT signaling pathway in step (b) is activated by hGH; and / or the JAK-STAT, Ras-MAPK, LIFR, and / or OSMR signaling pathway in step (c) and / or step (d) is activated by IL-6 and / or OSM. In some embodiments, the JAK-STAT, Ras-MAPK, LIFR, and / or OSMR signaling pathway in step (c) and / or step (d) is activated by IL-6. In some embodiments, the EGF signaling pathway in step (b) and / or step (d) is activated by EGF; the JAK-STAT signaling pathway in step (b) is activated by hGH; and the JAK-STAT, Ras-MAPK, LIFR, and / or OSMR signaling pathway in step (c) and / or step (d) is activated by IL-6. In some embodiments, step (a) occurs for a first time period; step (b) occurs for a second time period; step (c) occurs for a third time period; and step (d) occurs for a fourth time period. In some embodiments, the first time period is from at least about 0.5-3 days, the second time period is from at least about 1-6 days, the third time period is from at least about 3-9 days; and the fourth time period is from at least about 1-6 days; optionally wherein the first time period is from at least about 1-2 days, the second time period is from at least about 2-4 days, the third time period is from at least about 4-7 days; and the fourth time period is from at least about 2-4 days. In some embodiments, the cells of step (d) self-assemble into the human biliary organoid.

[0012] In some embodiments, the biliary progenitor cells include a SOX17 / PDX1 coexpressing cell population. In some embodiments, the biliary progenitor cells include a cell population that is differentiated in vitro from posterior foregut cells to hepatoblasts, and then further differentiated to biliary progenitor cells via exposure to biliary differentiation media (BDM); optionally wherein the BDM further includes Vitamin C. In some embodiments, theexposure to biliary differentiation media (BDM) is for at least about 0.5 days to 5 days, or at least about 1 day to 3 days.

[0013] In some embodiments, the methods further include differentiating posterior foregut cells into biliary progenitor cells via in vitro expansion. In some embodiments, differentiating posterior foregut cells into biliary progenitor cells includes activating an EGF and an RA signaling pathway in cultured posterior foregut cells prior to differentiation into biliary progenitor cells. In some embodiments, the EGF and RA signaling pathway activation is for at least about 1 day to 4 days. In some embodiments, the EGF and RA signaling pathway activation is in combination with activation of an FGF signaling pathway and an HGF signaling pathway. In some embodiments, the EGF and RA signaling pathway activation is for at least about 1 day to 4 days, and wherein the EGF and RA signaling pathway activation follows at least about 2 days to 6 days of posterior foregut culturing with biliary progenitor differentiation conditions. In some embodiments, the biliary progenitor differentiation conditions include activating an FGF signaling pathway, and a Wnt signaling pathway; optionally wherein the culture media includes Vitamin C. In some embodiments, activating an EGF signaling pathway includes culturing with EGF, and wherein activating an RA signaling pathway includes culturing with RA. In some embodiments, the posterior foregut cells are differentiated by in vitro expansion from definitive endoderm. In some embodiments, the posterior foregut cells are differentiated from definitive endoderm by activation of a FGF pathway and activation of a Wnt pathway.

[0014] In some embodiments, the posterior foregut cells are differentiated by in vitro expansion from definitive endoderm derived from pluripotent stem cells; optionally wherein the pluripotent stem cells include embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs); optionally wherein the pluripotent stem cells include iPSCs. In some embodiments, the biliary progenitor cells are derived in vitro from induced pluripotent stem cells (iPSCs). In some embodiments, the iPSCs are engineered to lack or have reduced MHC class I / II expression. In some embodiments, the biliary progenitor cells are differentiated from definitive endoderm derived from pluripotent stem cells knocked out for beta-2 microglobulin (B2M) and class II transactivator (CIITA) (B2M / _CIITA’ ').

[0015] In some embodiments, the biliary progenitor cells are seeded, cultured, and differentiated into the human biliary organoid in a scaffold tray including: a culture plate with a horizontal surface; one or more channels attached to or protruding from the horizontal surface, wherein each channel has a vertical depth, longitudinal sidewalls, and open latitudinal ends, and avolume including a culture region; and a lengthwise segment in each of the one or more channels, wherein each lengthwise segment runs longitudinally through the respective channel; and wherein the lengthwise segment is removed from the organoid following culturing and differentiation, and wherein the organoid includes a lumen-containing tubular system. In some embodiments, the channels are coated with collagen, to provide a collagen-coated tubular organoid. In some embodiments, the cells are seeded on the scaffold tray at a cell density of at least about 5,000- 50,000, 7,500-20,000, or 8,500-15,000 spheroids per channel, and in a seeding volume of at least about 10-500 pL, 50-2500 pL, or 100-150 pL; optionally at a cell density of at least about 10,000 spheroids per channel and in a seeding area of at least about 100-150 pL. In some embodiments, the cells are seeded on the scaffold tray at a spheroid size of at least about 1-1000 pm2, 10-500 pm2, or 100-300 pm2; optionally at a spheroid size of at least about 100-300 pm2. In some embodiments, the method provides human biliary organoids of consistent size. In some embodiments, the HBO has a tubular diameter of at least about 0.1-20 mm, 0.5-15 mm, 1-10 mm, 5-10 mm, 6-7 mm, or 1-2 mm, and wherein the HBO has a length of at least about 0. 1-100 mm, 0.5-50 mm, 50-90 mm, 70-80 mm, 1-30 mm, or 10-20 mm; optionally wherein the HBO has a tubular diameter of at least about 1-2 mm and a length of at least about 10-20 mm.

[0016] Further embodiments of the disclosure include human biliary organoids (HBOs) including extrahepatic cholangiocytes. Additional embodiments of the disclosure include human biliary organoids (HBOs) including extrahepatic cholangiocytes, prepared by any of the methods as described above.

[0017] In some embodiments, the HBO is obtained by in vitro differentiation and expansion from definitive endoderm derived from pluripotent stem cells. In some embodiments, the HBO further includes intrahepatic cholangiocytes. In some embodiments, the HBO further includes mature cholangiocyte cells, mesenchymal cells, and / or TROP2-expressing epithelial cells, and / or two or more distinct populations of cholangiocytes. In some embodiments, the cholangiocytes include intrahepatic bile duct (IHBD)-like cholangiocytes, gallbladder (GB)-like cholangiocytes, and common bile duct (CBD)-like cholangiocytes. In some embodiments, the HBO includes 10%-50% mesenchymal cells and 10%-50% cholangiocytes; optionally 15-35% mesenchymal cells and 15%-35% cholangiocytes. In some embodiments, the HBO further includes hepatic progenitors, stellate cells, endothelial cells, macrophages, and / or Kupffer cells. In some embodiments, the HBO further includes 5%-40% hepatic progenitors, 1 %-25% stellate cells, l%-25% endothelial cells, l%-25% macrophages, and / or l%-25% Kupffer cells. In someembodiments, the HBO includes smooth muscle cells, peribiliary gland progenitors, glandular elements, tight junctions, and complex structures with cystic motifs communicating via the tubular assembly.

[0018] In some embodiments, the HBO includes a TROP2-expressing cell population and / or a CK7-expressing cell population. In some embodiments, the HBO cholangiocytes are enriched in genes associate with one or more biological processes selected from bile acid metabolism, bile acid transport, bile salt transport, and bicarbonate transmembrane transporter activity. In some embodiments, the HBO has an elevated expression of one or more mature cholangiocyte marker, and / or reduced expression of one or more hepatoblast markers, relative to a human liver organoid (HLO). In some embodiments, the one or more cholangiocyte marker includes one or more of TROP2, cytokeratin 7 (CK7), cytokeratin 19 (CK19), aquaporin 1 (AQP1), multi-drug resistance-associated protein 3 (MRP 3), and bile salt export pump (BSEP), and / or wherein the one or more hepatoblast marker includes one or more of alpha fetoprotein (AFP) and albumin (ALB). In some embodiments, elevated expression of one or more mature cholangiocyte marker, and / or reduced expression of one or more hepatoblast markers, includes a 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, or greater expression increase or decrease, respectively, relative to a human liver organoid (HLO). In some embodiments, the HBO expresses CD68, CD45, CDllb, PECAM1, CDH5, PDGFRA, VIM, ACTA2, HNF4A, DCN, PIN, CALCRL, CK7, CK19, EPCAM, SOX17, ZO1, ot-SMA, FLT1, and / or vimentin. In some embodiments, the HBO includes intrahepatic bile duct (IHBD)-like cholangiocytes expressing JAG1, HES1, SOX4 and / or BICC1, gallbladder (GB)-like cholangiocytes expressing CA4, SOX17, KRT7, ABCC3,' and common bile duct (CBD)-like cholangiocytes expressing TFF2, LYZ, and / or SLC28A3, and having lower expression of CA4 and SOX4 in relation to the IHBD-like cholangiocytes and GB-like cholangiocytes.

[0019] In some embodiments, the HBO further includes a tubular assembly and / or a cystic structure. In some embodiments, the HBO includes a tubular assembly. In some embodiments, the HBO transports Rhodamine 123 (Rho 123) and / or bile into a luminal space of the HBO, and wherein the capacity of Rho 123 and / or bile transportation into the luminal space is elevated as compared to a human liver organoid (HLO).

[0020] In some embodiments, the HBO is obtained by in vitro differentiation and expansion of biliary progenitor cells. In some embodiments, the biliary progenitor cells include a SOX17 / PDX1 co-expressing cell population. In some embodiments, the HBO ishypoimmunogenic. In some embodiments, the hypoimmunogenic HBO is engineered to lack or have reduced MHC class I / II expression. In some embodiments, the HBO is derived from iPSCs which are knocked out for beta-2 microglobulin (B2M) and class II transactivator (CIITA) (B2M' ‘ CIITA" ')- In some embodiments, the HBO expresses CD34 and / or CD68.

[0021] Additional embodiments of the disclosure include in vitro compositions including any of the HBOs as described above.

[0022] Further embodiments of the disclosure include methods of treating a cholangiopathic condition, the methods including: transplanting, into a subject having a cholangiopathic condition, a human biliary organoid (HBO), as described above, or a composition including the same, as described above, to provide in vivo engraftment of the human biliary organoid.

[0023] Further embodiments of the disclosure include methods of treating a cholangiopathic condition, the methods including: surgically performing a biliary bypass in a subject having a cholangiopathic condition, by engrafting a human biliary organoid (HBO), as described above, or a composition including the same, as described above, between the gallbladder and duodenum of the subject, wherein the bypass reconstructs continuity of an extrahepatic bile duct pathway, restores bile flow, and / or allows for bile drainage.

[0024] In some embodiments, the transplanting includes: transplanting the HBO at a base of a liver in the subject; and allowing the HBO to engraft. In some embodiments, the methods include ligating a bile duct, or performing a bile duct graft, in a subject.

[0025] In some embodiments, the HBO includes extrahepatic ductal features; optionally wherein the extrahepatic ductal features include one or more of hepatic ducts, gallbladder epithelium, and / or intrahepatic ducts. In some embodiments, the HBO, following engraftment, is innervated. In some embodiments, the HBO, following engraftment, includes a reconstructed bile duct including mature reconstructed biliary components and a mesenchymal population, and / or expresses c-Kit, S100, calretinin vimentin, CK7, and / or CK19. In some embodiments, the mesenchymal population is in close proximity to luminal CK7 and CK19 positive cells. In some embodiments, the HBO, following engraftment, further includes interstitial cells of Cajal-like cells within the mesenchyme of the reconstructed bile duct. In some embodiments, the engraftment reconstructs one or more extrahepatic bile duct pathway, and / or the engrafted HBO resists ischemic damage and maintains ductal integrity. In some embodiments, the HBO includes ahypoimmunogenic HBO; optionally wherein the HBO is engineered to lack or have reduced MHC class VII expression and wherein the subject has hypoxia.

[0026] In some embodiments, the disease is a cholangiopathic disease or condition. In some embodiments, the cholangiopathic disease or condition includes biliary stricture, hypoxia, jaundice, inflammation, bile obstruction, congenital anomaly, ischemic attack, immune-related pathology, transplantation-associated complication, surgical-associated complication, biliary injury and / or trauma, and / or iatrogenic condition; optionally wherein the bile obstruction is due to gallstone; and / or wherein the biliary injury and / or trauma is from intraoperative bile duct injury; optionally wherein the intraoperative bile duct injury occurs during cholecystectomy, hepatic resection, or surgery requiring mobilization or dissection near a biliary tree or bile duct; and / or wherein the iatrogenic condition is drug induced and / or endoscopy induced. In some embodiments, the cholangiopathic disease includes a drug-induced cholangiopathy.

[0027] In some embodiments, the subject, after engraftment, has resistance to hypoxia and / or reperfusion injury. In some embodiments, the subject is a mammal; optionally wherein the subject is a mouse or a human; optionally wherein the subject is a pediatric human subject. In some embodiments, the HBO is prepared from subject-derived cells; optionally from subject-derived induced pluripotent stem cells (iPSCs).

[0028] Embodiments of the disclosure also include methods for screening a compound or composition, wherein the compound or composition to be screened includes one or more exogenous agent, the method including: contacting the HBO as described above with the compound or composition; culturing the HBO with the compound or composition for a period of time; and assessing one or more effects of the compound or composition on the HBO, thereby screening the compound or composition. In some embodiments, the assessed effect includes therapeutic efficacy and / or toxicity of the compound or composition.

[0029] Embodiments of the disclosure additionally include the human biliary organoid as described above, or a composition including the same, for use in a disease model. Further embodiments include uses of the human biliary organoid as described above, or a composition including the same, in a disease model. In some embodiments, the disease is a cholangiopathic disease or condition. In some embodiments, the cholangiopathic disease or condition includes biliary stricture, hypoxiajaundice, inflammation, bile obstruction, congenital anomaly, ischemic attack, immune-related pathology, transplantation-associated complication, surgical-associated complication, biliary injury and / or trauma, and / or iatrogenic condition. In some embodiments, thechol angiopathic disease includes a drug-induced cholangiopathy. In some embodiments, the disease model is a hypoxia-induced inflammation disease model. In some embodiments, the HBO is hypoimmunogenic, and the hypoxia-induced inflammation is reduced.

[0030] Further embodiments of the disclosure include methods of forming an organoid including a lumen-containing tubular system, the method including: seeding progenitor cells in a scaffold tray including a culture plate with a horizontal surface; one or more channels attached to or protruding from the horizontal surface, wherein each channel has a vertical depth, longitudinal sidewalls, and open latitudinal ends, and a volume including a culture region; and a lengthwise segment in each of the one or more channels, wherein each lengthwise segment runs longitudinally through the respective channel; culturing and differentiating the seeded progenitor cells in the culture region for a period of time sufficient to develop a tubular system around the lengthwise segment; and removing the lengthwise segment from the organoid following culturing and differentiation, thus providing an organoid including a lumen-containing tubular system.

[0031] In some embodiments, the lengthwise segment extends beyond one or more of the open latitudinal ends. In some embodiments, the lengthwise segment includes a surgical filament. In some embodiments, each channel has a U-shaped or V-shaped bottom. In some embodiments, one or more channels are coated with collagen, to provide a collagen-coated tubular organoid.

[0032] Additional embodiments of the disclosure include scaffold tray systems for producing a tubular organoid, the system including: a culture plate with a horizontal surface; one or more channels attached to or protruding from the horizontal surface, wherein each channel has a vertical depth, longitudinal sidewalls, and open latitudinal ends, and wherein each channel contains a volume including a culture region; and a lengthwise segment in each of the one or more channels, wherein each lengthwise segment runs longitudinally through the respective channel.

[0033] In some embodiments, the lengthwise segment extends beyond one or more of the open latitudinal ends. In some embodiments, the lengthwise segment includes a surgical filament. In some embodiments, each channel has a U-shaped or V-shaped bottom. In some embodiments, one or more channels are coated with collagen. In some embodiments, the tray is 3D printed.

[0034] Further embodiments of the disclosure include kits including means for preparing a human biliary organoid as described herein, or for performing any of the methods as described herein. In some embodiments, the kits include any of the human biliary organoid as described herein, or any of the compositions as described herein.

[0035] In some embodiments, one or more of the kit components are provided in separate vials. In some embodiments, one or more of the kit components are pre-loaded onto one or more assay platform. In some embodiments, one or more of the kit components are pre-frozen. In some embodiments, the kits further include one or more the scaffold tray as described herein.

[0036] Additional embodiments of the disclosure include biliary implants including a lumen-containing tubular structure and extrahepatic cholangiocytes. In some embodiments, the biliary implants further include a human biliary organoid (HBO) as described herein, or a composition including the same, as described herein.

[0037] In some embodiments, the biliary implants include an HBO and / or extrahepatic cholangiocytes produced from induced pluripotent stem cells (iPSCs) derived from a subject. In some embodiments, the biliary implant is HLA matched to the subject. In some embodiments, the biliary implants, the subject has a cholangiopathic disease or condition.BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0039] Figure 1. Directed differentiation of human iPSCs into multicellular human biliary organoids (HBOs).

[0040] Fig. 1A. Schematic overview of the stepwise differentiation protocol used to generate HBOs from human pluripotent stem cells (PSCs), involving stage-specific modulation of developmental signaling pathways.

[0041] Fig. IB. Bright-field images capturing the morphological progression of HBOs over the course of differentiation. Scale bars, 200 pm.

[0042] Fig. 1C. Quantitative gene expression analysis comparing hepatocytic and cholangiocytic marker genes in HBOs.

[0043] Fig. ID. Confocal images of whole-mount immunofluorescence staining showing spatial localization of key cholangiocyte and structural markers within HBOs.

[0044] Fig. IE. Functional assessment of MDR1 transporter activity using Rhodamine 123 uptake assay in the presence or absence of Verapamil. Human liver organoids (HLOs) serve as controls to distinguish bile duct-specific transporter activity in HBOs.

[0045] Figure 2. Single-cell profiling of multicellular HBOs, showing composition and extrahepatic regionalization.

[0046] Fig. 2A. UMAP visualization of single-nucleus RNA sequencing data from differentiated HBOs, identifying seven distinct cellular clusters.

[0047] Fig. 2B. Heatmap displaying global gene expression patterns across the identified clusters, highlighting transcriptional heterogeneity.

[0048] Fig. 2C. Subclassification of cholangiocyte clusters into intrahepatic-like, gallbladder-like, and common bile duct-like populations based on region-specific identity markers.

[0049] Fig. 2D. Integrated UMAP embedding of HBO-derived and primary human bile duct datasets. Right panel shows annotated cell-type identities.

[0050] Fig. 2E. Expression of canonical markers for common bile duct (CBD), extrahepatic bile duct (EHBD), and intrahepatic bile duct (IHBD) across cholangiocyte subclusters in both HBO and primary datasets.

[0051] Fig. 2F. Confocal imaging of whole-mount immunofluorescence staining showing spatial distribution of EHBD- and IHBD-associated markers within HBOs.

[0052] Fig. 2G. Gene ontology enrichment analysis highlighting biological processes associated with HBO-derived cholangiocytes, including regional specification and secretory function.

[0053] Fig. 2H. Immunohistochemistry staining demonstrating the presence and organization of mesenchymal components in HBOs, including PDGFRp, a-SMA, and Vimentin, consistent with fibro-muscular elements of the EHBD.

[0054] Figure 3. Single-nucleus RNA sequencing (snRNA-seq) profiling of HBOs, showing lineage-defining markers.

[0055] Fig. 3A. UMAP feature plots reveals cellular heterogeneity and lineage composition through unsupervised clustering of single-nucleus RNA-seq data.

[0056] Fig. 3B. UMAP feature plots for cell types from B2M+ / +CIITA+ +as compared to B2M’ACIITA ' .

[0057] Figure 4. Generation of hypoxia-resilient and immunoengineered human biliary organoids via B2M / CIITA / iPSCs.

[0058] Fig. 4A. Bright-field images showing the morphological development of HBOs derived from B2 / CIITA7" human iPSCs using the established differentiation protocol.

[0059] Fig. 4B. Hematoxylin and eosin (H&E) staining and immunohistochemistry comparing the structural features of HBOs derived from B2M / CIITA / and B2M+ / +CIITA / iPSCs.

[0060] Fig. 4C. Bright-field and ReadyProbes™ viability imaging following exposure to hypoxic conditions, highlighting differential cell survival between immune-edited and wild-type HBOs.

[0061] Fig. 4D and Fig. 4E. Analysis of gene and protein expression levels of pro- inflammatory and anti-inflammatory cytokines in wild-type (B2M+ / +CIITA+ / +) versus immunoengineered (B2M / CIITA7") HBOs, indicating reduced inflammatory activation under stress conditions.

[0062] Figure 5. Validation of blood humanization in NOD.Cg- PrkdcscldI12rg,mlwj1 / SzJ mice.

[0063] Flow cytometry plots demonstrating successful engraftment of human hematopoietic cells, as indicated by the presence of human-specific CD45+cells, in four representative humanized mice.

[0064] Figure 6. In vivo engraftment and structural maturation of hypoimmune human biliary organoids (HBOs) in humanized mouse models.

[0065] Fig. 6A. Schematic of the transplantation protocol used to assess engraftment of HBOs in blood-humanized mice. Hematoxylin and eosin (H&E) staining of explanted tissues shows successful integration of B2M / CIITA / HBOs into host tissue.

[0066] Fig. 6B. Comparative H&E staining of engrafted HBO grafts alongside native human biliary tissues, including segmental bile ducts, hepatic ducts (HD), and gallbladder, demonstrating architectural resemblance.

[0067] Fig. 6C. Immunohistochemistry staining for CK7, CK19, and Vimentin reveals organized epithelial and mesenchymal components within the B2M / CIITA / HBO grafts.

[0068] Fig. 6D. Spatial expression patterns of CK7 and CK19 highlight epithelial heterogeneity and regionalization within the engrafted tissue.

[0069] Fig. 6E. Immunostaining for c-Kit, SI 00, and Calretinin indicates neural-like structures and innervation patterns within the mesenchymal layers of the HBO grafts.

[0070] Figure 7. Bioengineering of lumenized tubular biliary structures through organoid assembly.

[0071] Fig. 7A. Schematic illustrating the generation of tubular human biliary organoids (HBOs) by transferring biliary progenitor aggregates into 3D-printed tray wells centered around surgical Monocryl® filaments to guide tubular morphogenesis.

[0072] Fig. 7B. Time-course images documenting the progressive development and elongation of tubular HBO structures.

[0073] Fig. 7C and 7D. Hematoxylin and eosin (H&E) staining and EPCAM immunohistochemistry of tubular HBOs confirm epithelial organization along the tubular scaffold.

[0074] Fig. 7E, 7F, and 7G. Immunofluorescence staining for EPCAM, TROP2, a-SMA, CK7, and Vimentin reveals conserved multicellular architecture, including epithelial and mesenchymal compartments, consistent with conventional spherical HBOs.

[0075] Fig. 7H. Rhodamine 123 uptake assay demonstrates luminal continuity and functional bile acid transporter activity within the engineered tubular HBOs.

[0076] Figure 8. Bioengineering and optimization of extrahepatic bile duct continuity and pathway reconstruction using tubular engineered HBO bile duct grafts.

[0077] Fig. 8A. Schematic workflow outlining the stepwise generation of tubular HBO constructs for bile duct reconstruction.

[0078] Fig. 8B. 3D configuration of the custom-designed tray well plates used to guide tubular morphogenesis.

[0079] Fig. 8C. Optimization of input cell numbers for efficient and uniform spheroid formation prior to HBO assembly.

[0080] Fig. 8D. Quantification of spheroid numbers generated in various tray well plate formats.

[0081] Fig. 8E. Evaluation of tray well design parameters to enhance the efficiency and reproducibility of tubular HBO assembly.

[0082] Figure 9. Surgical reconstruction of extrahepatic bile duct continuity using engineered tubular human biliary organoid (HBO) grafts.

[0083] Fig. 9A and 9B. Intraoperative images illustrating the surgical procedure for interposing a tubular HBO graft between the mouse gallbladder and duodenum to reconstruct the extrahepatic bile duct pathway.

[0084] Fig. 9C and 9D. Macroscopic and high-magnification views of the anastomosed HBO graft following transplantation, showing successful integration with native tissue.

[0085] Fig. 9E. Anterograde dye injection after bile duct ligation (BDL) confirms luminal patency and functional flow through the HBO graft.

[0086] Fig. 9F. Evidence of jaundice resolution post-BDL surgery, demonstrating restored bile drainage via the HBO bypass conduit.DETAILED DESCRIPTION

[0087] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein.

[0088] The following description of various embodiments is exemplary and explanatory only and is not to be construed as limiting or restrictive in any way. Other embodiments, features, objects, and advantages of the present teachings will be apparent from the description and accompanying drawings, and from the claims.

[0089] The disclosure herein uses affirmative language to describe the numerous embodiments. The disclosure also includes embodiments in which subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, or procedures.

[0090] It should be understood that any use of subheadings herein are for organizational purposes, and should not be read to limit the application of those subheaded features to the various embodiments herein. Each and every feature described herein is applicable and usable in all the various embodiments discussed herein and that all features described herein can be used in any contemplated combination, regardless of the specific example embodiments that are described herein. It should further be noted that exemplary description of specific features are used, largely for informational purposes, and not in any way to limit the design, subfeature, and functionality of the specifically described feature.Overview

[0091] As described herein, embodiments of the disclosure relate to biliary, or bile duct organoids. Embodiments of the disclosure thus include extrahepatic biliary organoids, methods for their generation and preparation, and compositions including the same, as well as uses thereof.

[0092] Severe biliary stricture is a leading cause of liver failure in children, in most cases, requiring liver transplantation after palliative Roux-en-Y hepaticojejunostomy. Stem cell-derived tissue graft offers a hope to reconstruct bile ducts capable of native functions.

[0093] As described herein, a differentiation protocol has been developed directed toward extrahepatic biliary organoids, comprising cholangiocytes, peribiliary gland progenitors, fibroblast, and smooth muscle lineages. In accordance with various embodiments, these biliary organoids can be derived entirely from human pluripotent stem cells (PSCs). Single nucleus- RNAseq, immunoprofilings and functional transporter assays demonstrated the presence of extrahepatic cholangiocyte lineage, distinct from published intrahepatic cholangiocytes.

[0094] The disclosure additionally relates to gene-modified hypoimmune PSC-derived biliary organoids. In some embodiments, such hypoimmune biliary organoids are lacking B2M (beta-2-microglobulin) and CIITA (class II transactivator), and are highly engraftable after in vivo transplantation. These organoids unexpectedly were found to provide protection from hypoxia- induced cellular damage. Furthermore, the in situ anastomosis of self-assembled biliary organoids in a tubular form resulted in a human bile duct that can be anastomosed to native gallbladder, as presently demonstrated in rats. These results demonstrate a means for studying cholangiopathy and ultimately for tissue replacement therapy, with potential to evade HLA-mediated ductopenic rejection.

[0095] Further detail is provided in the sections that follow, including Examples 1-7.Definitions of Terms

[0096] Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. For purposes of the present disclosure, the following terms are explained below.

[0097] The disclosure herein uses affirmative language to describe the numerous embodiments. The disclosure also includes embodiments in which subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, or procedures.

[0098] As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” maymean one or more than one. Some embodiments of the disclosure may consist of or consist essentially of one or more elements, method steps, and / or methods of the disclosure. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein and that different embodiments may be combined.

[0099] By “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 10% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0100] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” For example, “x, y, and / or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” It is specifically contemplated that x, y, or z may be specifically excluded from an embodiment. As used herein “another” may mean at least a second or more.

[0101] The term “ones” means more than one.

[0102] As used herein, the term “plurality” may be 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

[0103] As used herein, the term “set of’ means one or more. For example, a set of items includes one or more items.

[0104] As used herein, the phrase “at least one of,” when used with a list of items, means different combinations of one or more of the listed items may be used and only one of the items in the list may be needed. The item may be a particular object, thing, step, operation, process, or category. In other words, “at least one of’ means any combination of items or number of items may be used from the list, but not all of the items in the list may be required. For example, without limitation, “at least one of item A, item B, or item C” means item A; item A and item B; item B; item A, item B, and item C; item B and item C; or item A and C. In some cases, “at least one of item A, item B, or item C” means, but is not limited to, two of item A, one of item B, and ten of item C; four of item B and seven of item C; or some other suitable combination.

[0105] As used herein, “substantially” means sufficient to work for the intended purpose. The term “substantially” thus allows for minor, insignificant variations from an absolute or perfect state, dimension, measurement, result, or the like such as would be expected by a person of ordinary skill in the field but that do not appreciably affect overall performance. When used withrespect to numerical values or parameters or characteristics that can be expressed as numerical values, “substantially” means within ten percent.

[0106] Throughout this specification, unless the context requires otherwise, the words “comprise”, “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By “consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of’ is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of’ indicates that the listed elements are required or mandatory, but that no other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0107] Reference throughout this specification to “one embodiment,” “an embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment,” “an additional embodiment,” or “a further embodiment” or combinations thereof means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in various embodiments.

[0108] The terms “individual”, “subject”, or “patient” as used herein have their plain and ordinary meaning as understood in light of the specification, and mean a human or a non-human mammal, e g., a dog, a cat, a mouse, a rat, a cow, a sheep, a pig, a goat, a non-human primate, or a bird, e.g., a chicken, as well as any other vertebrate or invertebrate. The term “mammal” is used in its usual biological sense. Thus, it specifically includes, but is not limited to, primates, including simians (chimpanzees, apes, monkeys) and humans, cattle, horses, sheep, goats, swine, rabbits, dogs, cats, rodents, rats, mice, guinea pigs, or the like. In some embodiments, the patient is a pediatric patient. In some embodiments, the patient is an adult patient.

[0109] As used herein, the terms “treatment,” “treating,” “treat,” and the like, with respect to a disease or condition, can refer to obtaining a desired pharmacologic and / or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptomthereof and / or can be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. For example, a treatment can include executing a protocol, which may include administering one or more drugs to a patient, in an effort to alleviate signs or symptoms of the disease. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis. Alleviation can occur prior to signs or symptoms of the disease or condition appearing, as well as after their appearance. Thus, “treating” or “treatment” may include “preventing” or “prevention” of disease or undesirable condition. In addition, “treating” or “treatment” does not require complete alleviation of signs or symptoms, does not require a cure, and specifically includes protocols that have only a marginal effect on the patient.

[0110] “Treatment,” as used herein, thus can cover any treatment of a disease in a subject, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease and / or relieving one or more disease symptoms. “Treatment” can also encompass delivery of an agent or administration of a therapy in order to provide for a pharmacologic effect, even in the absence of a disease or condition.

[0111] The term “therapeutically effective” or “therapeutically effective amount” as used throughout this application can refer to an amount effective to achieve a desired and / or beneficial effect, and / or anything that promotes or enhances the well-being of the subject with respect to the medical treatment of a condition. This includes, but is not limited to, a reduction in the frequency or severity of one or more signs or symptoms of a disease. An effective amount can be administered in one or more administrations. In the methods, a therapeutically effective amount is an amount appropriate to treat an indication. By treating an indication is meant achieving any desirable effect, such as one or more of palliate, ameliorate, stabilize, reverse, slow, or delay disease progression, increase the quality of life, or to prolong life. Such achievement can be measured by any suitable method, such as measurement of tumor size or blood cell count, or any other suitable measurement.

[0112] The terms “effective amount” or “effective dose” as used herein have their plain and ordinary meaning as understood in light of the specification, and can refer to that amount of a recited composition or compound that, results in an observable effect. Actual dosage levels of active ingredients in an active composition of the presently disclosed subject matter can be varied so as to administer an amount of the active composition or compound that, is effective to achievethe desired response for a particular subject and / or application. The selected dosage level wall depend upon a variety of factors including, but not limited to, the activity of the composition, formulation, route of administration, combination with other drugs or treatments, severity of the condition being treated, and the physical condition and prior medical history of the subject being treated. In some embodiments, a minimal dose is administered, and dose is escalated in the absence of dose-limiting toxicity to a minimally effective amount. Determination and adjustment of an effective dose, as well as evaluation of when and howto make such adjustments, are contemplated herein.

[0113] The term “disease state” as used herein, can generally refer to a condition that affects the structure or function of an organism. Disease states can include, for example, stages of a disease progression.

[0114] As used herein, the term “assessing” can include any form of measurement, and includes determining if an element is present or not. The terms “determining,” “measuring,” “evaluating,” “assessing” and “assaying” can be used interchangeably and can include quantitative and / or qualitative determinations.

[0115] As used herein, the terms “modulated” or “modulation,” or “regulated” or “regulation” and “differentially regulated” can refer to both up regulation (i.e., activation or stimulation, e.g., by agonizing or potentiating) and down regulation (i.e., inhibition or suppression, e.g., by antagonizing, decreasing or inhibiting), unless otherwise specified or clear from the context of a specific usage.

[0116] As used herein, the term “marker” or “biomarker” can refer to any measurable substance taken as a sample from a subject whose presence is indicative of some phenomenon. Non-limiting examples of such phenomenon can include a disease state, a condition, or exposure to a compound or environmental condition. In various embodiments described herein, biomarkers may be used for diagnostic purposes (e.g., to diagnose a disease state, a health state, an asymptomatic state, a symptomatic state, etc.). The term “biomarker” may be used interchangeably with the term “marker”. The term “marker” or “biomarker” can include a biological molecule, such as, for example, a nucleic acid, peptide, protein, hormone, and the like, whose presence or concentration can be detected and correlated with a known condition, such as a disease state. It can also be used to refer to a differentially expressed gene whose expression pattern can be utilized as part of a predictive, prognostic or diagnostic process in healthy conditions or a disease state, orwhich, alternatively, can be used in methods for identifying a useful treatment or prevention therapy.

[0117] As used herein, the term “cellular phenotype” can refer to any determinable, observable, and / or measurable characteristic associated with a cell population.

[0118] As used herein, a “model” can include one or more in vitro or in vivo disease models; a model can also include algorithms, one or more mathematical techniques, one or more machine learning algorithms, or a combination thereof. A model can be used in a process and / or applied to an assay, in accordance with various embodiments as disclosed herein.

[0119] As used herein, a “process” can include one or more steps involving one or more features of one or more model as disclosed herein.

[0120] The terms “function” and “functional” as used herein have their plain and ordinary meaning as understood in light of the specification, and can refer to a biological, enzymatic, or therapeutic function.

[0121] The term “inhibit” as used herein has its plain and ordinary meaning as understood in light of the specification, and can refer to the reduction or prevention of a biological activity. The reduction can be by a percentage that is, is about, is at least, is at least about, is not more than, or is not more than about, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or an amount that is within a range defined by any two of the aforementioned values. As used herein, the term “delay” has its plain and ordinary meaning as understood in light of the specification, and refers to a slowing, postponement, or deferment of a biological event, to a time which is later than would otherwise be expected. The delay can be a delay of a percentage that is, is about, is at least, is at least about, is not more than, or is not more than about, 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or an amount within a range defined by any two of the aforementioned values. The terms inhibit and delay may not necessarily indicate a 100% inhibition or delay, A partial inhibition or delay may be realized.

[0122] As used herein, the term “isolated” has its plain and ordinary meaning as understood in light of the specification, and can refer to a substance and / or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and / or in an experimental setting), and / or (2) produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities may be separated from equal to, about, at least, at least about, not more than, or not more than about, 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about95%, about 98%, about 99%, substantially 100%, or 100% of the other components with which they were initially associated (or ranges including and / or spanning the aforementioned values). In some embodiments, isolated agents are, are about, are at least, are at least about, are not more than, or are not more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, substantially 100%, or 100% pure (or ranges including and / or spanning the aforementioned values). As used herein, a substance that is “isolated” may be “pure” (e.g., substantially free of other components). As used herein, the term “isolated cell” can refer to a cell not contained in a multi -cellular organism or tissue.

[0123] As used herein, “in vivo” is given its plain and ordinary meaning as understood in light of the specification and can refer to the performance of a method inside living organisms, usually animals, mammals, including humans, and plants, as opposed to a tissue extract or dead organism.

[0124] As used herein, “ex vivo” is given its plain and ordinary meaning as understood in light of the specification and can refer to the performance of a method outside a living organism with little alteration of natural conditions.

[0125] As used herein, “in vitro” is given its plain and ordinary' meaning as understood in light of the specification and can refer to the performance of a method outside of biological conditions, e.g., in a petri dish or test tube.

[0126] The terms “nucleic acid” or “nucleic acid molecule” as used herein have their plain and ordinary meaning as understood in light of the specification, and can refer to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, those that appear in a cell naturally, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action. Nucleic acid molecules can be composed of monomers that are naturally-occurring nucleotides (such as DNA and RNA), or analogs of naturally-occurring nucleotides (e.g., enantiomeric forms of naturally-occurring nucleotides), or a combination of both. Modified nucleotides can have alterations in sugar moieties and / or in pyrimidine or purine base moieties. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars can be functionalized as ethers or esters. Moreover, the entire sugar moiety can be replaced with statically and electronically similar structures, such as azasugars and carbocyclic sugar analogs. Examples of modifications in a base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-knownheterocyclic substitutes. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodi selenoate, phosphoroanilothioate, phosphoramlidate, or phosphorami date. The term “nucleic acid molecule” also includes so-called “peptide nucleic acids,” which comprise naturally-occurring or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids can be either single stranded or double stranded. “Oligonucleotide” can be used interchangeable with nucleic acid and can refer to either double stranded or single stranded DNA or RNA. A nucleic acid or nucleic acids can be contained in a nucleic acid vector or nucleic acid construct (e.g. plasmid, virus, retrovirus, lentivirus, bacteriophage, cosmid, fosmid, phagemid, bacterial artificial chromosome (BAG), yeast artificial chromosome (YAC), or human artificial chromosome (HAG)) that can be used for amplification and / or expression of the nucleic acid or nucleic acids in various biological systems. Typically, the vector or construct will also contain elements including but not limited to promoters, enhancers, terminators, inducers, ribosome binding sites, translation initiation sites, start codons, stop codons, polyadenylation signals, origins of replication, cloning sites, multiple cloning sites, restriction enzyme sites, epitopes, reporter genes, selection markers, antibiotic selection markers, targeting sequences, peptide purification tags, or accessory genes, or any combination thereof.

[0127] A nucleic acid or nucleic acid molecule can comprise one or more sequences encoding different peptides, polypeptides, or proteins. These one or more sequences can be joined in the same nucleic acid or nucleic acid molecule adjacently, or with extra nucleic acids in between, e.g. linkers, repeats or restriction enzyme sites, or any other sequence that is, is about, is at least, is at least about, is not more than, or is not more than about, I, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases long, or any length in a range defined by any two of the aforementioned lengths. The term “downstream” on a nucleic acid as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a sequence being after the 3 ’-end of a previous sequence, on the strand containing the encoding sequence (sense strand) if the nucleic acid is double stranded. The term “upstream” on a nucleic acid as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a sequence being before the 5’- end of a subsequent sequence, on the strand containing the encoding sequence (sense strand) if the nucleic acid is double stranded. The term “grouped” on a nucleic acid as used herein has its plain and ordinary meaning as understood in light of the specification and refers to two or moresequences that occur in proximity either directly or with extra nucleic acids in between, e.g. linkers, repeats, or restriction enzyme sites, or any other sequence that is, is about, is at least, is at least about, is not more than, or is not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases long, or any length in a range defined by any two of the aforementioned lengths, but generally not with a sequence in between that encodes for a functioning or catalytic polypeptide, protein, or protein domain.

[0128] The nucleic acids described herein comprise nucleobases. Primary, canonical, natural, or unmodified bases are adenine, cytosine, guanine, thymine, and uracil. Other nucleobases include but are not limited to purines, pyrimidines, modified nucleobases, 5- methylcytosine, pseudouridine, dihydrouridine, inosine, 7-methylguanosine, hypoxanthine, xanthine, 5,6-dihydrouracil, 5-hydroxymethylcytosine, 5 -bromouracil, isoguanine, isocytosine, aminoallyl bases, dye-labeled bases, fluorescent bases, or biotin-labeled bases.

[0129] The terms “peptide”, “polypeptide”, and “protein” as used herein have their plain and ordinary meaning as understood in light of the specification and refer to macromolecules comprised of amino acids linked by peptide bonds. The numerous functions of peptides, polypeptides, and proteins are known in the art, and include but are not limited to enzymes, structure, transport, defense, hormones, or signaling. Peptides, polypeptides, and proteins are often, but not always, produced biologically by a ribosomal complex using a nucleic acid template, although chemical syntheses are also available. By manipulating the nucleic acid template, peptide, polypeptide, and protein mutations such as substitutions, deletions, truncations, additions, duplications, or fusions of more than one peptide, polypeptide, or protein can be performed. These fusions of more than one peptide, polypeptide, or protein can be joined in the same molecule adjacently, or with extra amino acids in between, e.g. linkers, repeats, epitopes, or tags, or any other sequence that is, is about, is at least, is at least about, is not more than, or is not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases long, or any length in a range defined by any two of the aforementioned lengths. The term “downstream” on a polypeptide as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a sequence being after the C-terminus of a previous sequence. The term “upstream” on a polypeptide as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a sequence being before the N- terminus of a subsequent sequence.

[0130] The term “purity” of any given substance, compound, or material as used herein has its plain and ordinary meaning as understood in light of the specification and can refer to the actual abundance of the substance, compound, or material relative to the expected abundance. For example, the substance, compound, or material may be at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure, including all decimals in between. Purity may be affected by unwanted impurities, including but not limited to nucleic acids, DNA, RNA, nucleotides, proteins, polypeptides, peptides, amino acids, lipids, cell membrane, cell debris, small molecules, degradation products, solvent, carrier, vehicle, or contaminants, or any combination thereof. In some embodiments, the substance, compound, or material is substantially free of host cell proteins, host cell nucleic acids, plasmid DNA, contaminating viruses, proteasomes, host cell culture components, process related components, mycoplasma, pyrogens, bacterial endotoxins, and adventitious agents. Purity' can be measured using technologies including but not limited to electrophoresis, SDS-PAGE, capillary electrophoresis, PCR, rtPCR, qPCR, chromatography, liquid chromatography, gas chromatography, thin layer chromatography, enzyme-linked immunosorbent assay (ELISA), spectroscopy, UV-visible spectrometry, infrared spectrometry, mass spectrometry, nuclear magnetic resonance, gravimetry, or titration, or any combination thereof.

[0131] The term “yield” of any given substance, compound, or material as used herein has its plain and ordinary meaning as understood in light of the specification and can refer to the actual overall amount of the substance, compound, or material relative to the expected overall amount. For example, the yield of the substance, compound, or material is, is about, is at least, is at least about, is not more than, or is not more than about 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the expected overall amount, including ail decimals in between. Yield may be affected by the efficiency of a reaction or process, unwanted side reactions, degradation, quality of the input substances, compounds, or materials, or loss of the desired substance, compound, or material during any step of the production.

[0132] As used herein, “pharmaceutically acceptable” has its plain and ordinary meaning as understood in light of the specification and refers to carriers, excipients, and / or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed or that have an acceptable level of toxicity. A “pharmaceutically acceptable” “diluent,” “excipient,” and / or “carrier” as used herein have their plain and ordinary meaning as understood in light of the specification and are intended to include any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with administration to humans, cats, dogs, or other vertebrate hosts. Typically, a pharmaceutically acceptable diluent, excipient, and / or carrier is a diluent, excipient, and / or carrier approved by a regulatory agency of a Federal, a state government, or other regulatory agency, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans as well as non-human mammals, such as cats and dogs. The term diluent, excipient, and / or “carrier” can refer to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Such pharmaceutical diluent, excipient, and / or carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. Water, saline solutions and aqueous dextrose and glycerol solutions can be employed as liquid diluents, excipients, and / or carriers, particularly for injectable solutions. Suitable pharmaceutical diluents and / or excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. A non-limiting example of a physiologically acceptable carrier is an aqueous pH buffered solution. The physiologically acceptable carrier may also comprise one or more of the following: antioxidants, such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, such as serum albumin, gelatin, immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids, carbohydrates such as glucose, mannose, or dextrins, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, salt-forming counterions such as sodium, and nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®. The composition, if desired, can also contain minor amounts of wetting, bulking, emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, sustained release formulations and the like. The formulation should suit the mode of administration.

[0133] Cryoprotectants are cell composition additives to improve efficiency and yield of low temperature cryopreservation by preventing formation of large ice crystals. Cryoprotectants include but are not limited to DMSO, ethylene glycol, glycerol, propylene glycol, trehalose, formamide, methyl-formamide, dimethyl-formamide, glycerol 3 -phosphate, proline, sorbitol, diethyl glycol, sucrose, triethylene glycol, polyvinyl alcohol, polyethylene glycol, or hydroxyethyl starch. Cryoprotectants can be used as part of a cry opreservation medium, which include other components such as nutrients (e.g. albumin, serum, bovine serum, fetal calf serum [FCS]) toenhance post-thawing survivability of the cells. In these cryopreservation media, at least one cryoprotectant may be found at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or any percentage within a range defined by any two of the aforementioned numbers.

[0134] Additional excipients with desirable properties include but are not limited to preservatives, adjuvants, stabilizers, solvents, buffers, diluents, solubilizing agents, detergents, surfactants, chelating agents, antioxidants, alcohols, ketones, aldehydes, ethylenediaminetetraacetic acid (EDTA), citric acid, salts, sodium chloride, sodium bicarbonate, sodium phosphate, sodium borate, sodium citrate, potassium chloride, potassium phosphate, magnesium sulfate sugars, dextrose, fructose, mannose, lactose, galactose, sucrose, sorbitol, cellulose, serum, amino acids, polysorbate 20, polysorbate 80, sodium deoxycholate, sodium taurodeoxy cholate, magnesium stearate, octylphenol ethoxylate, benzethonium chloride, thimerosal, gelatin, esters, ethers, 2-phenoxyethanol, urea, or vitamins, or any combination thereof. Some excipients may be in residual amounts or contaminants from the process of manufacturing, including but not limited to serum, albumin, ovalbumin, antibiotics, inactivating agents, formaldehyde, glutaraldehyde, P-propiolactone, gelatin, cell debris, nucleic acids, peptides, amino acids, or growth medium components or any combination thereof. The amount of the excipient may be found in composition at a percentage that is, is about, is at least, is at least about, is not more than, oris not more than about, 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% w / w or any percentage by weight in a range defined by any two of the aforementioned numbers.

[0135] The term “pharmaceutically acceptable salts” has its plain and ordinary meaning as understood in light of the specification and includes relatively non-toxic, inorganic and organic acid, or base addition salts of compositions or excipients, including without limitation, analgesic agents, therapeutic agents, other materials, and the like. Examples of pharmaceutically acceptable salts include those derived from mineral acids, such as hydrochloric acid and sulfuric acid, and those derived from organic acids, such as ethanesulfonic acid, benzenesulfonic acid, p- toluenesulfonic acid, and the like. Examples of suitable inorganic bases for the formation of salts include the hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium,calcium, magnesium, aluminum, zinc, and the like. Salts may also be formed with suitable organic bases, including those that are non-toxic and strong enough to form such salts. For example, the class of such organic bases may include but are not limited to mono-, di-, and trialkylamines, including methylamine, dimethylamine, and triethylamine; mono-, di-, or trihydroxyalkylamines including mono-, di-, and triethanolamine; amino acids, including glycine, arginine and lysine; guanidine; N-methylglucosamine; N-methylglucamine; L-glutamine; N-methylpiperazine; morpholine; ethylenediamine; N-benzylphenethylamine; trihydroxymethyl aminoethane.

[0136] Proper formulation is dependent upon the route of administration chosen. Techniques for formulation and administration of the compounds described herein are known to those skilled in the art. Multiple techniques of administering a compound exist in the art including, but not limited to, enteral, oral, rectal, topical, sublingual, buccal, intraaural, epidural, epicutaneous, aerosol, parenteral delivery, including intramuscular, subcutaneous, intra-arterial, intravenous, intraportal, intra-articular, intradermal, peritoneal, intramedullary injections, intrathecal, direct intraventricular, intraperitoneal, intranasal or intraocular injections. Pharmaceutical compositions will generally be tailored to the specific intended route of administration.

[0137] As used herein, a “carrier” has its plain and ordinary meaning as understood in light of the specification and refers to a compound, particle, solid, semi-solid, liquid, or diluent that facilitates the passage, delivery and / or incorporation of a compound to cells, tissues and / or bodily organs.

[0138] As used herein, a “diluent” has its plain and ordinary meaning as understood in light of the specification and refers to an ingredient in a pharmaceutical composition that lacks pharmacological activity but may be pharmaceutically necessary or desirable. For example, a diluent may be used to increase the bulk of a potent drug whose mass is too small for manufacture and / or administration. It may also be a liquid for the dissolution of a drug to be administered by injection, ingestion or inhalation. A common form of diluent in the art is a buffered aqueous solution such as, without limitation, phosphate buffered saline that mimics the composition of human blood.

[0139] The term “% w / w” or “% wt / wt” as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a percentage expressed in terms of the weight of the ingredient or agent over the total weight of the composition multiplied by 100. The term “% v / v” or “% vol / vol” as used herein has its plain and ordinary meaning as understood inthe light of the specification and refers to a percentage expressed in terms of the liquid volume of the compound, substance, ingredient, or agent over the total liquid volume of the composition multiplied by 100.

[0140] The term “basement membrane matrix” or “extracellular matrix” as used herein has its plain and ordinary meaning in light of the specification and refers to any biological or synthetic compound, substance, or composition that enhances cell attachment and / or growth. Any extracellular matrix, as well as any mimetic or derivative thereof, known in the art can be used for the methods disclosed herein. Some examples of extracellular matrices, or mimetics or derivative thereof, include but are not limited to cell-based feeder layers, polymers, proteins, polypeptides, nucleic acids, sugars, lipids, poly-lysine, poly-omithine, collagen, collagen IV, gelatin, fibronectin, vitronectin, laminin, laminin-511 elastin, tenascin, heparan sulfate, entactin, nidogen, osteopontin, perl ecan, fibrin, basement membrane, Matrigel®, hydrogel, PEI, WGA, or hyaluronic acid, or any combination thereof. A common basement membrane matrix that is used in laboratories are those isolated from murine Engelbreth-Holm- Swarm (EHS) sarcoma cells. However, these basement membrane matrices are derived from non-human animals and therefore contain xenogeneic components that prevent its use towards humans. They are also not defined, which can lead to variability in manufacturing, as well as potentially harbor pathogens. Accordingly, in some embodiments, the methods for culturing cells may involve the use of synthetic and / or defined alternatives to these xenogeneic basement membrane matrices. The use of non -xenogeneic basement membrane matrices or mimetics or derivatives thereof enables manufacturing of biological products better suited for human use.

[0141] The terms “passage” and “passaging” as used herein have their plain and ordinary meaning as understood in light of the specification, and refer to the conventional approaches performed in biological cell culture methods to maintain a viable population of cells for prolonged periods of time. As cells are generally proliferative in cell culture, they undergo multiple cycles of mitosis until occupying the available space, which is typically a surface of a cell culture container (e g., a plate, dish, or flask) submerged under culture medium. For example, the cells may grow out as a monolayer on a cell culture container surface. If the growing cells occupy the entire available space of surface, they cannot proliferate further and may exhibit senescent behavior. In order to continue growth of the cells, which may be performed to maintain the viability and proliferative nature of the cells and / or to expand the number of cells for downstream purposes, the cells may be passaged by taking a fraction of the cells and seeding this fraction onto a fresh surface(e.g., of a cell culture container) in culture medium. This fraction of the cells will continue to proliferate and multiply until they occupy the available space of the new surface, upon which this passaging can be repeated successively.

[0142] The term “exogenous” as used herein has its plain and ordinary meaning as understood in light of the specification and refers to external factors that originate outside of a biological specimen (e.g., a cell, population of cells, organoid, etc.), as opposed to being naturally occurring and / or produced by the biological specimen itself. As used herein, exogenous components, reagents, and / or conditions, are components, reagents, and / or conditions that are added to compositions described herein, although this does not necessarily preclude the possibility of the same components, reagents, and / or conditions also being present through a function endogenous to a biological specimen.

[0143] The terms “liver organoid” and “hepatocyte organoid” are used interchangeably herein, and refer to populations of cells differentiated in vitro to form self-organizing structures, which generally are three-dimensional (3D), and include one or more functional cell types. Liver organoids differ from naturally occurring liver tissue in a number of ways. For example, as compared with naturally occurring liver tissue, liver organoids can have a structure having a single lumen and generally a spherical shape, and can include a basement membrane which is unnatural. The single lumen of a liver organoid contains 3D tissues but generally does not make any hepatic lobular structure nor cord-like structure, as with naturally occurring liver tissue. Liver organoids also generally do not contain extrahepatic cholangiocytes, peribiliary progenitors, hematopoietic tissue, and / or acquired immune cell subsets, such as T cell lineages. Further, as compared with naturally occurring liver tissue, liver organoids can have different efflux mechanisms, as a liver organoid can have a three-dimensional structure with a luminal structure but no ejection mechanism. In addition, liver organoids generally cannot receive dietary inputs, as they lack a gut and connected vascular channel. Organoids differ from embryonic bodies (EBs) in that organoids are composed of a majority of endoderm-derivatives (more than 50%).

[0144] Liver organoids can be derived from pluripotent stem cells (PSCs), including at least embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). Liver organoids may also be formed from liver-derived stem cells. In general, liver organoids can self-organize through cell sorting and spatially restricted lineage commitment in a manner similar to that which occurs in vivo, but as directed in vitro by thoughtful introduction of exogenous and / or endogenousdifferentiating factors and / or conditions as described herein, optionally through one or more directed steps, optionally involving introduction of one or more components.

[0145] The term “biliary organoid”, or “human biliary organoid”, or “bile duct organoid”, as used herein refers to three-dimensional organoids which develop in vitro from biliary progenitor cells to include, in various embodiments, extrahepatic cholangiocytes. Biliary organoids also generally have a tubular assembly (as compared to, liver organoids, which generally have a single lumen); alternatively, biliary organoids can develop to have a cystic structure. Biliary organoids also generally can further include intrahepatic cholangiocytes, mature cholangiocyte cells, mesenchymal cells, and / or TROP2-expressing epithelial cells. Other cell types, such as hepatic progenitors, stellate cells, endothelial cells, macrophages, Kupffer cells, and others, may be present in a biliary organoid. In some embodiments, biliary organoids may be generated through addition of exogenous components during culturing, to activate one or more pathways which lead to differentiation to develop a biliary character.

[0146] The term “tissue culture surface” as used herein has its plain and ordinary meaning as understood in light of the specification and refers to a substrate surface on which cells may aggregate and / or adhere to facilitate cell growth, differentiation, and / or function.

[0147] The term “engineered” as used herein refers to an entity that is generated by the hand of man, including a cell, nucleic acid, polypeptide, vector, and so forth. In at least some cases, an engineered entity is synthetic and comprises elements that are not naturally present or configured in the manner in which it is utilized in the disclosure. In certain embodiments, a construct and / or vector is engineered through recombinant nucleic acid technologies, and a cell is engineered through transfection or transduction of an engineered vector. Cells may be engineered to express heterologous proteins that are not naturally expressed by the cells, either because the heterologous proteins are recombinant or synthetic or because the cells do not naturally express the proteins.

[0148] The term “tubular” as used herein refers to a structure present in the disclosed biliary organoids, with a network, or assembly of hollow, branched features. For example, a biliary organoid can have epithelial invagination which protrudes from an organoid structure, region, or sub-region, such as from a region in a biliary organoid. In some embodiments, the biliary organoid from which epithelial invagination protrudes can be circular in shape. Invaginated tubular morphogenesis (growth) can originate from single lumen organoids or from fusion-driven tubular assembly.Extrahepatic Biliary Ducts

[0149] A critical barrier to successful biliary reconstruction is the ductopenic reaction following transplantation, often exacerbated by ischemia-reperfusion injury. The biliary epithelium is especially vulnerable to warm ischemia, which can lead to extensive cholangiocyte loss and progressive cholestasis. Furthermore, allogeneic transplanted grafts remain susceptible to HLA mismatch-driven immune rejection and poor engraftment. These challenges underscore the need for an immunologically compatible, multicellular, and engraftable biliary tissue capable of resisting ischemic damage and maintaining ductal integrity.

[0150] Recent advances in pluripotent stem cell (PSC) organoid technologies have enabled directed generation of functionally ciliated cholangiocyte organoids by dictating molecular developmental signals. However, existing models primarily recapitulate the intrahepatic biliary epithelium and fail to capture the complex, multicellular composition of the extrahepatic bile ducts (EHBDs). The extrahepatic bile duct, including the gallbladder and common bile duct, is structurally more intricate, featuring not only cholangiocytes but also smooth muscle layers, peribiliary glands (PBGs), and periductal stromal components derived from mesenchyme. These mesenchymal and muscular elements contribute to bile duct contractility, mechanical resilience, and tissue remodeling in response to injury, such as in cholestasis. Notably, PBGs harbor specialized cell types, such as mucus-secreting cells, tuft cells, and putative stem / progenitor populations that may play roles in epithelial regeneration and innate defense. Thus, an effective regenerative model ideally can reconstitute this multicellular niche to truly restore EHBD function.

[0151] During embryogenesis, the extrahepatic biliary system arises from the ventral foregut endoderm that evaginates to form the hepatic diverticulum around week 4 of human development. Unlike the intrahepatic biliary network, which forms through ductal plate remodeling and is heavily reliant on Notch and TGF-|3 signaling, EHBD development is governed by distinct cues. Notch signaling, particularly via HES1, mediates the binary fate decisions between biliary and pancreatic lineages within hepatopancreatic progenitors. FGF10, expressed by adjacent mesenchyme, plays a key role in specifying and expanding the EHBD epithelium, including the formation of PBGs. Meanwhile, the surrounding splanchnic mesoderm contributes smooth muscle and stromal components through epithelial-mesenchymal cross-talk involving BMP signaling.

[0152] As described herein, a directed differentiation protocol has been established to generate caudalized liver bud progenitors from human induced pluripotent stem cells (iPSCs),enriched for EHBD lineage competency. These progenitors give rise to human biliary organoids (HBOs) that exhibit epithelial diversity, including multiple cholangiocyte subsets, PBG-like glandular elements, and stromal components such as smooth muscle cells. By incorporating MHC class I / II deletion, HBO graft engraftability was enhanced in immune-humanized rodent models. Furthermore, biofabricated tubular HBO grafts were engineered that successfully anastomose with native bile ducts in a rodent model of biliary stricture. This multicellular and immunocompatible organoid platform presents new opportunities to model EHBD disorders and develop strategies for bile duct reconstruction.Human Biliary Organoids

[0153] The present study represents the first disclosure of a directed differentiation protocol to generate extrahepatic biliary organoids entirely from human pluripotent stem cells, termed herein as human biliary organoids, or bile duct organoids. Single-nucleus RNA sequencing and immunoprofiling confirmed the formation of lumenized cholangiocyte monolayers, surrounded by supportive periductal stromal components, including glandular progenitors and smooth muscle cells. Functional assays further validated the biliary physiology of the organoid epithelium. Following transplantation into mouse models, the organoids underwent further maturation and reassembled into patterned ductal structures that closely resembled native extrahepatic bile ducts. This highlights the self-organizing potential of the bioengineered organoids and their ability to respond to in vivo cues for continued development and structural refinement.

[0154] To enable immune-compatible allogeneic transplantation, genetic deletions of P2- microglobulin (B2M) and class II transactivator (CIITA) were introduced into the iPSC lines, generating hypoimmunogenic HBOs. This strategy not only promoted robust engraftment in blood-humanized mice without the need for long-term immunosuppression but also conferred unexpected resistance to hypoxia / reperfusion injury, a major contributor to bile duct graft failure in clinical settings. These findings demonstrate the dual benefit of immune evasion and stress tolerance in genetically engineered HBOs.

[0155] Leveraging a guided self-assembly strategy within 3D-printed tray culture plates, lumenized tubular HBO grafts suitable for surgical implantation were further engineered. When interposed between the gallbladder and duodenum in a rat model of extrahepatic biliary injury, these grafts successfully restored bile flow and reconstructed the continuity of the extrahepatic biliary tract.

[0156] Collectively, this work establishes a blueprint for allogeneic bile duct bioengineering, offering an alternative to liver transplantation for a spectrum of conditions, such as, for example cholangiopathies (including pediatric cholangiopathies) and biliary tract disorders. The development of robust, multicellular extrahepatic biliary organoids from patient-specific iPSCs also opens new avenues for modeling disease pathogenesis and advancing personalized regenerative therapies for the biliary system.

[0157] In accordance with various embodiments, human biliary organoids (HBOs), also referred to herein as bile duct organoids, can be derived from progenitor cells, such as, for example, induced pluripotent stem cells (iPSCs), or from patient-derived iPSCs, where the patient can be healthy or having a diseased condition, and are identical in genetic content to the respective patient. They express most cholangiocyte markers that are expressed in the pre-natal stages of development. Furthermore, they are clonal and therefore react similarly to external stimuli and biochemical perturbations. These HBOs are highly scalable and tractable, allowing screening approaches to test a vast array of drugs and small molecules.

[0158] HBOs are easy to work with as model systems and have very low variation across batches. Large batches of HBOs can be generated within a couple of weeks. Leveraging these qualities, several drugs can be tested within a short span of time to identify pathways involved in biliary diseases and disorders. In contrast, breeding model organisms such as mice and rats takes months of work and planning, and the chance of getting the desired genotype is relatively low. Furthermore, model organisms show high variations in responses to biochemical perturbations over generations. These rodents also run the risk of losing the desired genotype when bred over long periods of time, and also require complex training and procedures to model diseases and evaluate the efficacy of treatments. Compared to model organisms, genetic modifications are much easier in iPSC cell lines, and they can be maintained easily over longer periods before differentiation into organoids.

[0159] Accordingly, the disclosure describes the formation of a human biliary organoid, by culturing biliary progenitor cells derived from posterior foregut cells in vitro in an appropriate media (such as, for example, biliary differentiation media (BDM); optionally in BDM which further includes Vitamin C), in combination with various exogenous components to direct differentiation, via in vitro differentiation and expansion, as described herein. When the biliary progenitor cells are differentiated in this fashion, the HBOs are formed by self-assembly into three- dimensional organoid systems, e.g. artificial organoids, having multiple cell types, includingextrahepatic cholangiocytes. In some embodiments, the HBOs include intrahepatic cholangiocytes and extrahepatic cholangiocytes.

[0160] These HBOs include extrahepatic cholangiocytes and exhibit characteristics of extrahepatic bile ducts, including the genetic, functional, and cellular marker characteristics of extrahepatic bile ducts. In accordance with various embodiments, the HBOs additionally include intrahepatic cholangiocytes and can exhibit characteristics of intrahepatic bile ducts, including the genetic, functional, and cellular marker characteristics of intrahepatic bile ducts.

[0161] In accordance with various embodiments, HBOs prepared as described herein can include additional cell types, such as mature cholangiocyte cells, mesenchymal cells, and / or TROP2-expressing epithelial cells. In some embodiments, HBOs prepared as described herein can include mature cholangiocyte cells, mesenchymal cells, and TROP2-expressing epithelial cells. For example, the presently described HBOs can include at least about 10%-50% mesenchymal cells and at least about 10%-50% cholangiocytes, or 15-35% mesenchymal cells and 15%-35% cholangiocytes. Additional cell types can be present, including hepatic progenitors, stellate cells, endothelial cells, macrophages, and Kupffer cells. For example, the presently described HBOs can include at least about 5%-60% hepatic progenitors, at least about l%-25% stellate cells, at least about l%-25% endothelial cells, at least about l%-25% macrophages, and / or at least about 1%- 25% Kupffer cells. One skilled in the art will appreciate that there can be some natural variation and that any combination of cell population percentage numbers in these ranges are contemplated in accordance with the disclosure. One skilled in the art will further appreciate that any intermediate value, or combination of intermediate values, within the ranges provided herein are within the scope of the disclosure. In some embodiments, each of the aforementioned cell types, namely hepatic progenitors, stellate cells, endothelial cells, macrophages, and Kupffer cells, can be present. Further, two or more distinct populations of cholangiocytes can be present and observable in the presently described HBOs. These distinct cholangiocyte populations can include intrahepatic bile duct (IHBD)-like cholangiocytes, gallbladder (GB)-like cholangiocytes, and common bile duct (CBD)-like cholangiocytes. In some embodiments, each of IHBD-like, GB-like, and CBD-like cholangiocytes can be present. The HBOs prepared as described herein can additionally include smooth muscle cells, peribiliary gland progenitors, glandular elements, tight junctions, and / or complex structures with cystic motifs communicating via a tubular assembly.

[0162] The cell types present in the HBOs prepared as described herein can have characteristic gene expression, demonstrating evidence of specific cell types and functions,wherein the gene expression can distinguish the HBOs prepared as described herein from other organoid types. For example, the presently described HBOs can include a TROP2-expressing cell population and / or a CK7-expressing cell population; in various embodiments, both a TROP2- expressing cell population and a CK7-expressing cell population can be present. The presently described HBOs can further include cholangiocytes enriched in genes associated with one or more biological processes selected from bile acid metabolism, bile acid transport, bile salt transport, and bicarbonate transmembrane transporter activity.

[0163] In various embodiments, HBOs prepared as described herein can have elevated expression of one or more mature cholangiocyte marker, and / or reduced expression of one or more hepatoblast markers, relative to a human liver organoid (HLO). For example, the HBOs can have elevated expression of one or more cholangiocyte marker including one or more of TROP2, cytokeratin 7 (CK7), cytokeratin 19 (CK19), aquaporin 1 (AQP1), multi-drug resistance-associated protein 3 (MRP 3), and bile salt export pump (BSEP), relative to a human liver organoid (HLO); and / or the HBOs can have elevated expression of one or more hepatoblast marker including one or more of alpha fetoprotein (AFP) and albumin (ALB), relative to a human liver organoid (HLO). The HBOs can additionally expresses TROP2 and / or CK7; in some embodiments, the HBOs can express both TROP2 and CK7. In various embodiments, elevated expression of one or more mature cholangiocyte marker, and / or reduced expression of one or more hepatoblast markers, relative to a human liver organoid (HLO), can encompass a 1.5-fold, or greater, expression increase or decrease, respectively. For example, elevated expression of one or more mature cholangiocyte marker, and / or reduced expression of one or more hepatoblast markers, relative to a human liver organoid (HLO), can include a 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, or greater, expression increase or decrease, respectively. Different expression increases or decreases can correspond with different genes. For example, CK7 has been measured herein at a 20-fold expression increase and in accordance with various embodiments can have an elevated expression, relative to an HLO, of 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, or higher; similarly, CK19 has been measured herein at a 15-fold expression increase and in accordance with various embodiments can have an elevated expression, relative to an HLO, of 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, or higher; similarly, AQP1 has been measured herein at a 15-fold expression increase and in accordance with various embodiments can have an elevated expression, relative to an HLO, of 2-fold, 5-fold, 10-fold, 15- fold, 20-fold, or higher.

[0164] In various embodiments, the HBOs can expresses one or more additional markers, such as (1)68, CD45, GDI lb, PECAM1, CDH5, PDGFRA, VIM, ACTA2, HNF4A, DCN, PIN, CALCRL, CK7, CK19, EPCAM, SOX17, ZO1, a-SMA, FLT1, and / or vimentin. In various embodiments, the HBOs can include IBHD-like cholangiocytes expressing JAG1, HES1, SOX4 and / or BICC1, GB-like cholangiocytes expressing CA4, SOX17, KRT7, ABCC3 and CBD-like cholangiocytes expressing TFF2, LYZ, and / or SLC28A3, and having lower expression of CA4 and SOX4 in relation to the IHBD-like cholangiocytes and GB-like cholangiocytes.

[0165] The cell types present in the HBOs prepared as described herein can have characteristic physical features, indicating functionality, wherein the physical features can distinguish the HBOs prepared as described herein from other organoid types. For example, the presently described HBOs can include a stochastic or complex tubular assembly and / or a cystic structure. In particular embodiments, the HBOs include a tubular assembly. In accordance with various embodiments as described herein, the HBOs have capability to transport Rho 123 and / or bile into a luminal space of the HBO, demonstrating luminal continuity and functional bile acid transporter activity within the HBOs. For example, the capacity of Rho 123 and / or bile transportation into the luminal space can be elevated as compared to a human liver organoid (HLO). For example, elevated capacity of Rho 123 and / or bile transportation into the luminal space, relative to a human liver organoid (HLO), can include a 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, or greater, transportation increase or decrease, respectively.

[0166] The biliary organoid can have various beneficial qualities and uses. For example, the biliary organoids, and compositions thereof, can be used in treating a cholangiopathic condition, such as by transplanting, into a subject having a cholangiopathic condition, a human biliary organoid (HBO), as described above, or a composition including the same, as described above, to provide in vivo engraftment of the human biliary organoid, the biliary organoids, and compositions thereof, can be used in treating a cholangiopathic condition, such as by surgically performing a biliary bypass in a subject having a cholangiopathic condition, by engrafting a human biliary organoid (HBO), as described above, or a composition including the same, as described above, between the gallbladder and duodenum of the subject, wherein the bypass reconstructs continuity of an extrahepatic bile duct pathway, restores bile flow, and / or allows for bile drainage. Transplantation can include, for example, transplanting the HBO at a base of a liver in the subject; and allowing the HBO to engraft; and / or ligating a bile duct; and / or performing a bile duct graft, in a subject. One skilled in the art would appreciate that various methods of surgically andtherapeutically using the biliary organoid as described herein are within the scope of, and contemplated by, the present disclosure.

[0167] The biliary organoid, following transplantation, can include extrahepatic ductal features; optionally wherein the extrahepatic ductal features include one or more of hepatic ducts, gallbladder epithelium, and / or intrahepatic ducts. The biliary organoid, following transplantation, can be innervated and / or can include a reconstructed bile duct including mature reconstructed biliary components and a mesenchymal population, and / or expresses c-Kit, SI 00, calretinin vimentin, CK7, and / or CK19. The mesenchymal population may be in close proximity to luminal CK7 and CK19 positive cells. The biliary organoid, following engraftment, can further include interstitial cells of Cajal-like cells within the mesenchyme of the reconstructed bile duct. Such engraftment can serve to reconstruct one or more extrahepatic bile duct pathway, and / or the engrafted biliary organoid resists ischemic damage and maintains ductal integrity. The biliary organoid can be a hypoimmunogenic biliary organoid; for example, the biliary organoid can be engineered to lack or have reduced MHC class I / II expression, which is particularly useful when treating a subject having hypoxia, or in preventing occurrence or recurrence of hypoxia in a subject. In such patients, the subject, after engraftment, can have resistance to hypoxia and / or reperfusion injury. In some embodiments, the subject is a mammal; optionally wherein the subject is a mouse or a human; optionally wherein the subject is a pediatric human subject. In some embodiments, the HBO can be prepared from subject-derived cells; optionally from subject- derived induced pluripotent stem cells (iPSCs).

[0168] The biliary organoids can also be used in methods for screening a compound or composition, wherein the compound or composition to be screened includes one or more exogenous agent. The biliary organoid can be contacted with the compound or composition, and cultured with the compound or composition for a period of time; after which point one or more effects of the compound or composition on the HBO are assessed, thereby screening the compound or composition. The assessed effect can include therapeutic efficacy and / or toxicity of the compound or composition; however, other features and parameters may be assessed to determine various properties of the compound or composition, as appropriate.

[0169] The human biliary organoid can also be used in disease model. In particular, biliary organoids can be used in a hypoxia-induced inflammation disease model. A hypoimmunogenic biliary organoid can be used in a disease model, wherein the hypoxia-induced inflammation is reduced.Methods of Producing Biliary Organoids

[0170] Methods of producing organoids, such as liver organoids, from posterior foregut cells have been explored previously in, for example, Ouchi et al. “Modeling Steatobepatitis in Humans with Pluripotent Stem Cell-Derived Organoids” Cell Metabolism (2019) 30(2):374~384; Shinozawa et al. “High-Fidelity Drug- Induced Liver Injury Screen Using Human Pluripotent Stem Cell Derived Organoids” Gastroenterology (2021) 160(3) 831-846; PCX Publications WO 2018 / 085615, WO 2018 / 191673, WO 2018 / 226267, WO 2019 / 126626, WO 2020 / 023245, WO 2020 / 069285, WO 2020 / 243613, WO 2021 / 030373, and WO 2021 / 262676, each of which is hereby expressly incorporated by references in its entirety.

[0171] Embodiments of methods for producing biliary organoids are provided herein. In some embodiments, the methods include a) culturing biliary progenitor cells derived from posterior foregut cells in biliary differentiation media (BDM); b) activating an EGF signaling pathway and a JAK-STAT signaling pathway in the cultured cells of step (a); c) activating / inhibiting a JAK-STAT, Ras-MAPK, LIFR, and / or OSMR signaling pathway in the treated cells of step (b); and d) activating / inhibiting an EGF signaling pathway and a JAK-STAT, Ras-MAPK, LIFR, and / or OSMR signaling pathway in the treated cells of step (c). In some embodiments, activating an EGF signaling pathway and a JAK-STAT signaling pathway in the cultured hepatoblasts of step (a) includes providing the cells with EGF and hGH. In some embodiments, activating an EGF signaling pathway and a JAK-STAT and / or Ras-MAPK signaling pathway in the cultured cells of step (a) includes providing the cells with EGF and hGH. In some embodiments, activating / inhibiting a JAK-STAT, Ras-MAPK, LIFR, and / or OSMR signaling pathway in the treated cells of step (b) includes providing the cells with IL-6 and / or OSM. In some embodiments, activating / inhibiting an EGF signaling pathway and a JAK-STAT, Ras-MAPK, LIFR, and / or OSMR signaling pathway in the treated cells of step (c) includes providing the cells with EGF in combination with IL-6 and / or OSM. In some embodiments, the EGF signaling pathway in step (b) and / or step (d) is activated by EGF; the JAK-STAT signaling pathway in step (b) is activated by hGH; and / or the JAK-STAT, Ras-MAPK, LIFR, and / or OSMR signaling pathway in step (c) and / or step (d) is activated by IL-6 and / or OSM; optionally wherein the JAK-STAT, Ras-MAPK, LIFR, and / or OSMR signaling pathway in step (c) and / or step (d) is activated by IL-6. In various embodiments, the culturing and activating steps are carried out in biliary differentiation media (BDM); optionally in BDM which further includes Vitamin C.

[0172] In some embodiments, the JAK-STAT, Ras-MAPK, LIFR, and / or OSMR signaling pathway in step (c) and / or step (d) is activated by an IL-6 family cytokine. In some embodiments, the JAK-STAT, Ras-MAPK, LIFR, and / or OSMR signaling pathway in step (c) and / or step (d) is activated by an IL-6 family cytokine (e.g. IL-6), Oncostatin M (OSM), leukemia inhibitory factor (LIF), cardiotrophin-1, ciliary neurotrophic factor (CTNF), and / or cardiotrophin-like cytokine (CLC).

[0173] In various embodiments, the biliary progenitor cells can include hepatoblasts. In various embodiments, the biliary progenitor cells can include a SOX17 / PDX1 co-expressing cell population. In various embodiments, the biliary progenitor cells can include posterior foregut cells and / or hepatoblasts which have been exposed to biliary differentiation media (BDM). For example, in various embodiments, the biliary progenitor cells can include a cell population that is differentiated in vitro from posterior foregut cells to hepatoblasts, and then further differentiated to biliary progenitor cells via exposure to biliary differentiation media (BDM); the BDM can optionally further include Vitamin C. In some methods, the exposure of posterior foregut cells and / or hepatoblasts to biliary differentiation media (BDM) is for at least about 0.5 days to 5 days, or at least about 1 day to 3 days, in order to produce biliary progenitor cells.

[0174] In some embodiments, step (a) occurs for a first time period; step (b) occurs for a second time period; step (c) occurs for a third time period; and step (d) occurs for a fourth time period. In particular, the first time period is from at least about 0.5-3 days, the second time period is from at least about 1-6 days, the third time period is from at least about 3-9 days; and the fourth time period is from at least about 1-6 days; optionally wherein the first time period is from at least about 1-2 days, the second time period is from at least about 2-4 days, the third time period is from at least about 4-7 days; and the fourth time period is from at least about 2-4 days. In some embodiments, the first time period is from at least about 1 day, the second time period is from at least about 3 days, the third time period is from at least about 5 days; and the fourth time period is from at least about 3 days.

[0175] In some embodiments, the methods further include differentiating posterior foregut cells into biliary progenitor cells. In some embodiments, differentiating posterior foregut cells into biliary progenitor cells includes activating an EGF and an RA signaling pathway in cultured posterior foregut cells prior to differentiation into biliary progenitor cells. In some embodiments, the culturing is in biliary differentiation media, optionally in the presence of Vitamin C. In some embodiments, the EGF and RA signaling pathway activation is for at least about 1 day to 4 days.In some embodiments, the EGF and RA signaling pathway activation is in combination with activation of an FGF signaling pathway and an HGF signaling pathway. In some embodiments, the EGF and RA signaling pathway activation is for at least about 1 day to 4 days, and the EGF and RA signaling pathway activation follows at least about 2 days to 6 days of posterior foregut culturing with biliary progenitor differentiation conditions. In some embodiments, the biliary progenitor differentiation conditions can include activating an FGF signaling pathway, and a WNT signaling pathway, and wherein the culture media comprises Vitamin C. In some embodiments, activating an EGF signaling pathway comprises culturing with EGF, and / or activating an RA signaling pathway comprises culturing with RA; optionally using 1-1000 ng / mL EGF and 0.01- lOO uM RA.

[0176] In some embodiments, the posterior foregut cells are differentiated by in vitro expansion from definitive endoderm. In some embodiments, the posterior foregut cells can be differentiated from definitive endoderm by activation of a FGF pathway and activation of a Wnt pathway; optionally wherein the differentiation further involves activation of a VEGF pathway. For example, the FGF pathway can be activated via administration of FGF4, and the Wnt pathway can be activated by administration of a GSK-3 inhibitor, such as CHIR.

[0177] In some embodiments, methods of differentiating DE to posterior foregut cells further include activating an FGF signaling pathway and a Wnt signaling pathway in definitive endoderm cells (DE), and optionally additionally activating a VEGF signaling pathway, followed by, activating an EGF signaling pathway and a retinoic acid (RA) signaling pathway, and optionally activating the FGF signaling pathway and an HGF signaling pathway, thereby differentiating the DE to posterior foregut cells.

[0178] In some embodiments, the posterior foregut cells can be differentiated by in vitro expansion from definitive endoderm derived from pluripotent stem cells; optionally wherein the pluripotent stem cells comprise embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs); optionally wherein the pluripotent stem cells comprise iPSCs.

[0179] In some embodiments, the DE has been derived from pluripotent stem cells. In some embodiments, the pluripotent stem cells are embryonic stem cells and / or induced pluripotent stem cells. In some embodiments, the fifth period of time is, is about, is at least, is at least about, is not more than, or is not more than about, 0.5, 1, 2, 3, or 4 days, or a range defined by any two of the preceding values, for example 0.5-4, 1-4, 0.5-2, or 3-4 days. In some embodiments, the sixth period of time is, is about, is at least, is at least about, is not more than, or is not more than about0.5, 1 , or 2 days. In some embodiments, the biliary organoid, DE, and / or pluripotent stem cells are derived from a patient.

[0180] In some embodiments of the methods of making biliary organoids, the FGF signaling pathway activator is selected from the group consisting of FGF1, FGF2, FGF3, FGF4, FGF4, FGF 5, FGF6, FGF7, FGF8, FGF8, FGF9, FGF 10, FGF 11, FGF 12, FGF13, FGF 14, FGF 15, FGF 16, FGF 17, FGF 18, FGF 19, FGF20, FGF21 , FGF22, and FGF23. in some embodiments, the FGF signaling pathway activator is FGF4. In some embodiments, the FGF signaling pathway activator is provided at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 ng / mL, or any concentration within a range defined by any two of the aforementioned concentrations, including 0.1-1000 ng / mL, 100-1000 ng / mL, 100-500 ng / mL, 500-1000 ng / mL, 250-750 ng / mL, or 400-600 ng / mL, In some embodiments, the FGF signaling pathway activator is contacted at a concentration of 500 ng / mL or about 500 ng / mL.

[0181] In some embodiments of the methods of making biliary organoids, the Wnt signaling pathway activator is selected from the group consisting of Wntl, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, WntlOa, WntlOb, Wntl l, Wntl6, BML 284, IQ-1, WAY 262611, CHIR99021, CHIR98014, AZD2858, BIO, AR-A014418, SB 216763, SB 415286, aloisine, indirubin, alsterpaullone, kenpaullone, lithium chloride, TDZD 8, and TWS119. In some embodiments, the Wnt signaling pathway activator is CHIR9902L In some embodiments, the Wnt signaling pathway activator is provided at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, or 3.5 mM, or any concentration within a range defined by any two of the aforementioned concentrations, including 0.5-3.5 mM, 0.5-2 mM, 2-3.5 mM, 1-3 mM, or 1.5-2.5 mM. In some embodiments, the Wnt signaling pathway activator is provided at a concentration of 2 mM or about 2 mM. In some embodiments, the Wnt signaling pathway activator is provided at a concentration of about 0.1-1000 ng / mL.

[0182] In some embodiments of the methods of making biliary organoids, the RA signaling pathway activator is selected from the group consisting of retinoic acid, all-trans retinoic acid, 9- eis retinoic acid, CD437, EC23, BS 493, TTNPB, and AMS 80. In some embodiments, the RA signaling pathway activator is RA. In some embodiments, the RA signaling pathway activator isprovided at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.9, or 3 mM, or any concentration within a range defined by any two of the aforementioned concentrations, including 1-3 mM, 1-2 mM, 2-3 mM, or 1.5-2.5 mM. In some embodiments, the RA signaling pathway activator is provided at a concentration of 2 mM or about 2 mM. In some embodiments, the RA signaling pathway activator is provided at a concentration of about 0.1-1000 ng / mL.

[0183] In some embodiments of the methods of making biliary organoids, the EGF signaling pathway activator is EGF. In some embodiments, the EGF signaling pathway activator is provided at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.9, or 3 mM, or any concentration within a range defined by any two of the aforementioned concentrations, including 1-3 mM, 1-2 mM, 2-3 mM, or 1.5-2.5 mM. In some embodiments, the EGF signaling pathway activator is provided at a concentration of 2 mM or about 2 mM. In some embodiments, the EGF signaling pathway activator is provided at a concentration of about 0.1- 1000 ng / mL.

[0184] In some embodiments of the methods of making biliary organoids, the JAK-STAT signaling pathway activator is selected from the group consisting of hGH and IL-6. In some embodiments, the JAK-STAT signaling pathway activator is hGH. In some embodiments, the JAK-STAT signaling pathway activator is IL-6. In some embodiments, the JAK-STAT signaling pathway activator in step (b) is hGH. In some embodiments, the JAK-STAT signaling pathway activator in step (c) and step (d) is IL-6. In some embodiments, the JAK-STAT signaling pathway activator is provided at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.9, or 3 mM, or any concentration within a range defined by any two of the aforementioned concentrations, including 1-3 mM, 1-2 mM, 2-3 mM, or 1.5-2.5 mM. In some embodiments, the JAK-STAT signaling pathway activator is provided at a concentration of 2 mM or about 2 mM. In some embodiments, the JAK-STAT signaling pathway activator is provided at a concentration of about 0.1-1000 ng / mL.

[0185] In some embodiments of the methods of making biliary organoids, the JAK-STAT, Ras-MAPK, LIFR, and / or OSMR signaling pathway activator can be an IL-6 family cytokine (e g. IL-6), Oncostatin M (OSM), leukemia inhibitory factor (LIF), cardiotrophin-1, ciliaryneurotrophic factor (CTNF), and / or cardiotrophin-like cytokine (CLC). In some embodiments, the JAK-STAT, Ras-MAPK, LIFR, and / or OSMR signaling pathway activator in step (c) and step (d) is IL-6. In some embodiments, the JAK-STAT, Ras-MAPK, LIFR, and / or OSMR signaling pathway activator in step (c) and step (d) is OSM. In some embodiments, the JAK-STAT, Ras- MAPK, LIFR, and / or OSMR signaling pathway activator is provided at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.9, or 3 mM, or any concentration within a range defined by any two of the aforementioned concentrations, including 1-3 mM, 1-2 mM, 2- 3 mM, or 1.5-2.5 mM. In some embodiments, the JAK-STAT, Ras-MAPK, LIFR, and / or OSMR signaling pathway activator is provided at a concentration of 2 mM or about 2 mM. In some embodiments, the JAK-STAT, Ras-MAPK, LIFR, and / or OSMR signaling pathway activator is provided at a concentration of about 0.1-1000 ng / mL.

[0186] In various embodiments, the biliary organoids can be prepared to have a lumencontaining tubular system. This preparation method is based on the newly designed scaffold tray described herein, which has surprisingly proven highly beneficial in forming tubular organoids. The methods involve seeding progenitor cells in a scaffold tray, where the scaffold tray includes a culture plate with a horizontal surface, one or more channels having a vertical depth, wherein each channel has longitudinal sidewalls and open latitudinal ends, and a volume comprising a culture region, and a lengthwise segment in each of the one or more channels, wherein each lengthwise segment runs longitudinally through the respective channel. In some embodiments, the lengthwise segment extends beyond one or more of the open latitudinal ends. The seeded progenitor cells can then be cultured and differentiated in the culture region for a period of time sufficient to develop a tubular system around the lengthwise segment. The lengthwise segment can then be removed from the organoid following culturing and differentiation, thus providing an organoid comprising a lumen-containing tubular system. As described herein, particular embodiments of the scaffold tray include a surgical filament as the lengthwise segment. However, the lengthwise segment can be made from any material that can co-exist with the cells without harming viability. In exemplary embodiments, each channel can have a U-shaped or V-shaped bottom, which serves as a culture region; this bottom culture region supports aggregate fusion with the lengthwise segment, such as with a surgical filament. In addition, the channels can be coated with collagen, to provide a collagen-coated tubular organoid.

[0187] The progenitor cells can be selected as appropriate according to the desired organoid to be produced. For example, for the presently described biliary organoids, the progenitor cells are biliary progenitors. One skilled in the art will appreciate the appropriate cells to use at the seeding stage using the above-described scaffold tray and culture system.

[0188] In various embodiments involving preparation of organoids including a lumencontaining tubular system, based on the newly designed scaffold tray described herein, the cells can be seeded on the scaffold tray at a cell density of at least about 5,000-50,000, 7,500-20,000, or 8,500-15,000 spheroids per channel, and in a seeding volume of at least about 10-500 pL, 50- 2500 pL, or 100-150 pL. In particular embodiments, the cells are seeded at a cell density of at least about 10,000 spheroids per channel and in a seeding area of at least about 100-150 pL. In some embodiments, the cells can be seeded on the scaffold tray at a spheroid size of at least about 1- 1000 pm2, 10-500 pm2, or 100-300 pm2. In particular embodiments, the cells are seeded at a spheroid size of at least about 100-300 pm2. In some embodiments, the resultant organoid (e g. human biliary organoid, or other organoid type) can have a tubular diameter of at least about 0.1- 20 mm, 0.5-15 mm, 1-10 mm, 5-10 mm, 6-7 mm, or 1-2 mm, and wherein the organoid has a length of at least about 0.1-100 mm, 0.5-50 mm, 50-90 mm, 70-80 mm, 1-30 mm, or 10-20 mm. In particular embodiments relate to an HBO having a tubular diameter of at least about 1-2 mm and a length of at least about 10-20 mm. The ranges provided above are generalizations, and every value within a range, and combination of values of different ranges listed above, are encompassed by the disclosure. One skilled in the art will appreciate that there can be some variability in these ranges, which can be adapted based on the desired organoid to be produced and based on the parameters of the specific culture. One of the benefits of this method is the ability to produce organoids, such as human biliary organoids (or other organoid types), of consistent size, and having a tubular diameter of consistent size as well. The tubular diameter can vary with the diameter of the lengthwise segment which is removed following organoid formation. Thus, the size of the channel and the lengthwise segment can be particularly selected in order to achieve a desired organoid size and tubular diameter, allowing for bespoke organoid design, wherein the organoid has custom shape and size.

[0189] Further embodiments of the disclosure are directed to the scaffold tray itself, i.e. a culture plate with a horizontal surface; one or more channels attached to or protruding from the horizontal surface, wherein each channel has a vertical depth, longitudinal sidewalls, and open latitudinal ends, and wherein each channel contains a volume comprising a culture region, and alengthwise segment in each of the one or more channels, wherein each lengthwise segment runs longitudinally through the respective channel. In some embodiments, the lengthwise segment extends beyond one or more of the open latitudinal ends. As described above, particular embodiments of the scaffold tray include a surgical filament as the lengthwise segment; other biocompatible materials can be used as well, as would be appreciated by one skilled in the art. In exemplary embodiments, each channel can have a U-shaped or V-shaped bottom and can be coated with collagen. Scaffold trays in accordance with the disclosure can be custom designed (e.g. for size and shape parameters) and can be 3D printed. Exemplary scaffold tray systems, or portions thereof, in accordance with the disclosure are shown in Figures 8B, 8D, and 8E. One skilled in the art will appreciate that these are exemplary only and that other designs, shapes, sizes, etc., can be contemplated and used in accordance with the disclosure.

[0190] One skilled in the art will appreciate that the above-described scaffold tray system need not be limited to biliary organoids and that the scaffold tray and methods of forming an organoid thereon, wherein the organoid is produced to have a lumen-containing tubular system, can be applied to other organoid types as well. For example, various types of spheroids can be seeded in the scaffold tray and differentiated in appropriate media and with appropriate components to produce the respective organoid types, wherein the resulting organoid will be produced to have a lumen-containing tubulary system. Such methods are encompassed by the present disclosure.Biliary Diseases and Disorders

[0191] The biliary organoids of the disclosure can be used in treatment and / or studying or modeling biliary-related diseases and disorders, and / or cholangiopathic conditions, for which their extrahepatic and intrahepatic cholangiocyte character and functionality is particularly advantageous and renders them applicable to a wide range of conditions. In some embodiments, the methods include administering any of the biliary organoids or cholangiocytes disclosed herein, for treating biliary-related diseases and disorders, and / or cholangiopathic conditions. In some embodiments, the disclosure relates to uses of the biliary organoids or cholangiocytes disclosed herein in treating biliary-related diseases and disorders, and / or cholangiopathic conditions. Also disclosed herein are the biliary organoids or cholangiocytes disclosed herein for use in the manufacture of a medicament for the treatment of biliary-related diseases or disorders and / or cholangiopathic conditions. Also disclosed herein are the biliary organoids or cholangiocytes disclosed herein for use in the treatment of biliary-related diseases or disorders, and / orchol angiopathic conditions, in a subject in need thereof. The biliary organoids of the disclosure provide an alternative to liver transplantation for wide range of conditions, including those described below; in particular, biliary organoids can be used as alternative methods for treating cholangiopathies (including pediatric cholangiopathies) and biliary tract disorders. The biliary organoids as described herein have the particular advantage of optionally being formed from patient-specific cells, such as patient-derived pluripotent stem cells, e.g. patient-derived induced pluripotent stem cells (iPSCs).

[0192] Exemplary biliary-related diseases and disorders, and / or cholangiopathic conditions, relevant to the disclosure, which can be treatable via the biliary organoids as described herein, can include conditions such as biliary stricture, hypoxia, jaundice, inflammation, bile obstruction (such as, for example, due to gallstone), congenital anomalies, ischemic attack, immune-related pathology, transplantation-associated complications, surgical-associated complications, injury, trauma, and / or iatrogenic conditions (such as, for example, drug induced and / or endoscopy induced conditions), and the like. For example, biliary injury and / or trauma can occur from intraoperative bile duct injury, such as during cholecystectomy, hepatic resections for other purposes, or other surgeries that require mobilization or dissection near the biliary tree or bile duct (i.e. Whipple pancreaticoduodenectomy). One skilled in the art will appreciate that other biliary-related diseases and disorders are contemplated as being relevant to the disclosure, and can be treated, surgically or otherwise, with the biliary organoids as disclosed herein.

[0193] The presently described HBOs can be used in various methods of treating biliary- related diseases and disorders, and / or cholangiopathic conditions. For example, in some embodiments, an HBO of the disclosure (or composition thereof) can be transplanted into a subject having a cholangiopathic condition, to provide in vivo engraftment of the HBO. In some embodiments of the methods, the HBO can be transplanted at a base of a liver in the subject and then allowed to engraft. Further exemplary methods include treating biliary-related diseases and disorders, and / or cholangiopathic conditions, by surgically performing a biliary bypass in a subject having a cholangiopathic condition, by engrafting an HBO of the disclosure (or composition thereof), between the gallbladder and duodenum of the subject, wherein the bypass reconstructs the extrahepatic bile duct pathway, thereby allowing for, and restoring, bile drainage in the subject. Various methods of treatment using the HBOs involve ligating at least a portion of a bile duct in a subject.

[0194] For example, the biliary organoid can be transplanted into a subject having hypoxia and / or jaundice, where the transplanted biliary organoids engraft onto a bile duct of the subject. Following transplantation, the subject can have reconstruction of one or more extrahepatic bile duct pathway, resistance to ischemic damage, and maintenance of ductal integrity.

[0195] The HBOs of the disclosure are particularly useful in these methods, in embodiments wherein the HBO includes extrahepatic ductal features, either before or after engraftment. In particular, HBOs having extrahepatic ductal features including hepatic ducts, gallbladder epithelium, and / or intrahepatic ducts can have good success in various methods of treatment.

[0196] In various uses and / or methods of treatment, the engrafted HBO is innervated. In addition, the HBO, following engraftment, can include a reconstructed bile duct comprising mature reconstructed biliary components and a mesenchymal population, and / or expresses c-Kit, SI 00, calretinin vimentin, CK7, and / or CK19; for example, the mesenchymal population can be located in close proximity to luminal CK7 and CK19 positive cells. Further, the HBO, following engraftment, can additionally include interstitial cells of Cajal-like cells within the mesenchyme of the reconstructed bile duct.

[0197] In various uses and / or methods of treatment, the engraftment can reconstruct, wholly or partially, an extrahepatic bile duct pathway. Following engraftment, the HBO can be demonstrated to resist ischemic damage and maintain ductal integrity.

[0198] In particular embodiments of the disclosure, the HBO can be a hypoimmunogenic HBO. For example, the HBO can be engineered to lack or have reduced MHC class I / II expression. This can be particularly relevant when the subject has hypoxia. In various embodiments, a subject being treated for a cholangiopathic disease or condition, after engraftment by the HBO, has resistance to hypoxia and / or reperfusion injury.

[0199] In some embodiments, the subject to be treated is a mammal; optionally wherein the subject is a mouse or a human; optionally wherein the subject is a pediatric human subject. In particular embodiments, the HBO is prepared from patient derived cells; optionally from subject- derived induced pluripotent stem cells (iPSCs). In this way, the subject can be treated with a composition (e.g. a biliary organoid) which is HLA matched to the subject.

[0200] The disclosure also includes biliary implants comprising a lumen-containing tubular structure and extrahepatic cholangiocytes. The biliary implants produced in accordance with the disclosure can be used in various surgical methods, including, for example, biliarytransplants, biliary grafts, biliary ligations, biliary bypass, and the like. For example, the biliary implants can include the biliary organoids produced as described herein, or compositions including the same. In the biliary implants, the HBO and / or extrahepatic cholangiocytes can be produced from induced pluripotent stem cells (iPSCs) derived from a subject. Accordingly, the implant can be HLA matched to a subject needing treatment. These biliary implants can be used in treating a subject having a cholangiopathic disease or condition.

[0201] The biliary organoids as described herein can be used an in vitro human model system for studying cholangiocyte function and developmental divergence, studying biliary- related disease, identifying and / or screening for therapeutic targets, and / or identifying therapeutic compounds and / or compositions effective in treating a biliary-related disease or disorder. Accordingly, the biliary organoids of the disclosure can allow for new developments in bile duct disease treatment and study.

[0202] For example, HBOs prepared by the methods as described herein can be used for screening compounds or compositions, wherein the compound or composition to be screened includes one or more exogenous agent, by contacting the HBO with the compound or composition, culturing the HBO with the compound or composition for a period of time, and then assessing one or more effects of the compound or composition on the HBO, thereby screening the compound or composition. The assessed effect can include, for example, therapeutic efficacy and / or toxicity of the compound or composition.

[0203] For example, biliary organoids prepared by the methods as described herein can be used in a disease model, such as a model for a cholangiopathic disease or condition. The cholangiopathic disease or condition to be modeled can be biliary stricture, hypoxia, jaundice, inflammation, bile obstruction, congenital anomaly, ischemic attack, immune-related pathology, transplantation-associated complication, surgical-associated complication, biliary injury and / or trauma, and / or an iatrogenic condition. In particular embodiments, the disease model can be a drug-induced cholangiopathy disease model, or a hypoxia-induced inflammation disease model. The latter disease model can be particularly useful when the biliary organoid is prepared to be hypoimmunogenic, and the hypoxia-induced inflammation is reduced.

[0204] The biliary organoids of the disclosure can be used in treatment and / or studying or modeling liver-related diseases and disorders, such as liver dysfunction and / or failure (e.g. hyperammonemia and / or hyperbilirubinemia, and the like), hepatitis (e.g. hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis G, hepatitis TT, and / or autoimmune hepatitis, and thelike), viral hepatitis, cholangitis, fibrosis, hepatic encephalopathy, hepatic porphyria, cirrhosis, cancer, drug-induced cholestasis, metabolic disease (e.g. metabolic dysfunction-associated liver disease (MASLD), MetALD, nonalcoholic fatty liver disease (NAFLD), metabolic dysfunction- associated steatohepatitis (MASH), and the like), autoimmune liver disease, Wilson’s disease, metabolic-associated fatty liver disease, hyperammonemia, hyperbilirubinemia, Crigler-Najjar Syndrome, urea cycle disorders, Wolman disease, hepatic cancer, hepatoblastoma, drug-induced liver injury (DILI), glycogen storage disease, hemorrhagic disease, hepatic cyst, and / or alcohol- associated liver disease. One skilled in the art will appreciate other liver-related diseases and conditions for which the liver organoids disclosed herein could have relevance.Stem Cells

[0205] The term “totipotent stem cells” (also known as omnipotent stem cells) as used herein has its plain and ordinary meaning as understood in light of the specification and are stem cells that can differentiate into embryonic and extra-embryonic cell types. Such cells can construct a complete, viable organism. These cells are produced from the fusion of an egg and sperm cell. Cells produced by the first few divisions of the fertilized egg are also totipotent.

[0206] The term “embryonic stem cells (ESCs),” also commonly abbreviated as ES cells, as used herein has its plain and ordinary meaning as understood in light of the specification and refers to cells that are pluripotent and derived from the inner cell mass of the blastocyst, an early- stage embryo. For purpose of the present disclosure, the term "ESCs" is used broadly sometimes to encompass the embryonic germ cells as well.

[0207] The term “pluripotent stem cells (PSCs)” as used herein has its plain and ordinary1meaning as understood in light of the specification and encompasses any cells that can differentiate into nearly all cell types of the body, i.e., cells derived from any of the three germ layers (germinal epithelium), including endoderm (interior stomach lining, gastrointestinal tract, the lungs), mesoderm (muscle, bone, blood, urogenital), and ectoderm (epidermal tissues and nervous system), PSCs can be the descendants of inner cell mass cells of the preimplantation blastocyst or obtained through induction of a non-pluripotent cell, such as an adult somatic cell, by forcing the expression of certain genes. Pluripotent stem cells can be derived from any suitable source. Examples of sources of pluripotent stem cells include mammalian sources, including human, rodent, porcine, and bovine.

[0208] The term “induced pluripotent stem cells (iPSCs),” also commonly abbreviated as iPS cells, as used herein has its plain and ordinary meaning as understood in light of thespecifi cation and refers to a type of pluripotent stem cells artificially derived from a normally non- pluripotent cell, such as an adult somatic cell, by inducing a "forced" expression of certain genes. hiPSC refers to human iPSCs. In some methods known in the art, iPSCs may be derived by transfection of certain stem cell-associated genes into non-pluripotent cells, such as adult fibroblasts. Transfection may be achieved through viral transduction using viruses such as retroviruses or lentiviruses. Transfected genes may include the master transcriptional regulators Oct-3 / 4 (PUU5F1) and Sox2, although other genes may enhance the efficiency of induction. After 3-4 weeks, small numbers of transfected cells begin to become morphologically and biochemically similar to pluripotent stem cells, and are typically isolated through morphological selection, doubling time, or through a reporter gene and antibiotic selection. As used herein, iPSCs include first generation iPSCs, second generation iPSCs in mice, and human induced pluripotent stem cells. In some methods, a retroviral system is used to transform human fibroblasts into pluripotent stem cells using four pivotal genes: Oct3 / 4, Sox2, Klf4, and c-Myc. In other methods, a lentiviral system is used to transform somatic cells with GCT4, SOX2, NANOG, and LIN28. Genes whose expression are induced in iPSCs include but are not limited to Oct-3 / 4 (POU5F1); certain members of the Sox gene family (e.g., Soxl, Sox2, Sox3, and Soxl5); certain members of the Klf family (e.g., Kill, Klf2, Klf4, and Klf5), certain members of the Mye family (e.g., C-myc, L-myc, and N- myc), Nanog, LIN28, Tert, Fbxl5, ERas, EC ATI 5-1, ECAT 15-2, Tell, b-Catenm, EC ATI, Esgi, Dnmt3L, EC ATS, Gdf3, Fthll7, Sall4, Rexl, UTF1, Stella, Stat3, Grb2, Prdml4, Nr5al, Nr5a2, or E-cadherin, or any combination thereof.

[0209] The term “precursor cell” as used herein has its plain and ordinary meaning as understood in light of the specification and encompasses any cells that can be used in methods described herein, through which one or more precursor cells acquire the ability to renew itself or differentiate into one or more specialized cell types. In some embodiments, a precursor cell is pluripotent or has the capacity to becoming pluripotent. In some embodiments, the precursor cells are subjected to the treatment of external factors (e.g., growth factors) to acquire pluripotency. In some embodiments, a precursor cell can be a totipotent (or omnipotent) stem cell; a pluripotent stem cell (induced or non-induced); a multipotent stem cell; an oligopotent stem cells and a unipotent stem cell. In some embodiments, a precursor cell can be from an embryo, an infant, a child, or an adult. In some embodiments, a precursor cell can be a somatic cell subject to treatment such that pluripotency is conferred via genetic manipulation or protein / peptide treatment.Precursor cells include embryonic stem cells (ESC), embryonic carcinoma cells (ECs), and epiblast stem cells (EpiSC).

[0210] In some embodiments, one step can include obtaining stem cells that are pluripotent or can be induced to become pluripotent. In some embodiments, pluripotent stem cells are derived from embryonic stem cells, which are in turn derived from totipotent cells of the early mammalian embryo and are capable of unlimited, undifferentiated proliferation in vitro. Embryonic stem cells are pluripotent stem cells derived from the inner cell mass of the blastocyst, an early-stage embryo. Methods for deriving embryonic stem cells from blastocytes are well known in the art. It would be understood by one of skill in the art that the methods and systems described herein are applicable to any stem cells.

[0211] Additional stem cells that can be used in embodiments in accordance with the present disclosure include but are not limited to those provided by or described in the database hosted by the National Stem Cell Bank (NSCB), Human Embryonic Stem Cell Research Center at the University of California, San Francisco (UCSF); WISC cell Bank at the Wi Cell Research Institute; the University of Wisconsin Stem Cell and Regenerative Medicine Center (IJW- SCRMC); Novocell, Inc. (San Diego, Calif.); Cellartis AB (Goteborg, Sweden); ES Cell International Pte Ltd (Singapore); Techmon at the Israel Institute of Technology (Haifa, Israel); and the Stem Cell Database hosted by Princeton University and the University of Pennsylvania. Exemplary embryonic stem cells that can be used in embodiments in accordance with the present disclosure include but are not limited to SA01 (SA001); SA02 (SA002); ESDI (HES-1); ES02 (HES-2); ES03 (HES-3); ES04 (HES-4); ES05 (HES-5); ES06 (HES-6); BG01 (BGN-01); BG02 (BGN-02); BG03 (BGN-03); TE03 (13); TE04 (14); TE06 (16); UCO1 (HSF1); UC06 (HSF6); WA01 (HI); WA07 (H7); WA09 (H9); WA13 (HI 3); WA14 (HI 4). Exemplary human pluripotent cell lines include but are not limited to TkDA3-4, 1231 A3, 317-D6, 317-A4, CDH1, 5-T-3, 3-34- 1, NAFLD27, NAFLD77, NAFLD150, WD90, WD91, WD92, 1.20012. C213, 1383D6, FF, or 317-12 cells.

[0212] In developmental biology, cellular differentiation is the process by which a less specialized cell becomes a more specialized cell type. As used herein, the term “directed differentiation” describes a process through which a less specialized cell becomes a particular specialized target cell type. The particularity of the specialized target cell type can be determined by any applicable methods that can be used to define or alter the destiny of the initial cellExemplary methods include but are not limited to genetic manipulation, chemical treatment, protein treatment, and nucleic acid treatment.

[0213] In some embodiments, an adenovirus can be used to transport the requisite four genes, resulting in iPSCs substantially identical to embryonic stem cells. Since the adenovirus does not combine any of its own genes with the targeted host, the danger of creating tumors is eliminated, in some embodiments, non-viral based technologies are employed to generate iPSCs. In some embodiments, reprogramming can be accomplished via plasmid without any virus transfection system at all, although at very low efficiencies. In other embodiments, direct deliver}' of proteins is used to generate iPSCs, thus eliminating the need for viruses or genetic modification. In some embodiment, generation of mouse iPSCs is possible using a similar methodology: a repeated treatment of the cells with certain proteins channeled into the cells via poly-arginine anchors was sufficient to induce pluripotency. In some embodiments, the expression of pluripotency induction genes can also be increased by treating somatic cells with FGF2 under low oxygen conditions.

[0214] The term “feeder cell” as used herein has its plain and ordinary meaning as understood in light of the specification and refers to cells that support the growth of pluripotent stem cells, such as by secreting growth factors into the medium or displaying on the cell surface. Feeder cells are generally adherent cells and may be growth arrested. For example, feeder cells are growth-arrested by irradiation (e.g. gamma rays), mitomycin-C treatment, electric pulses, or mild chemical fixation (e g. with formaldehyde or glutaraldehyde). However, feeder cells do not necessarily have to be growth arrested. Feeder cells may serve purposes such as secreting growth factors, displaying growth factors on the cell surface, detoxifying the culture medium, or synthesizing extracellular matrix proteins. In some embodiments, the feeder cells are allogeneic or xenogeneic to the supported target stem cell, which may have implications m downstream applications. In some embodiments, the feeder cells are mouse cells. In some embodiments, the feeder cells are human cells. In some embodiments, the feeder cells are mouse fibroblasts, mouse embryonic fibroblasts, mouse STO cells, mouse 3T3 cells, mouse SNL 76 / 7 cells, human fibroblasts, human foreskin fibroblasts, human dermal fibroblasts, human adipose mesenchymal cells, human bone marrow mesenchymal cells, human amniotic mesenchymal cells, human amniotic epithelial cells, human umbilical cord mesenchymal cells, human fetal muscle cells, human fetal fibroblasts, or human adult fallopian tube epithelial cells. In some embodiments, conditioned medium prepared from feeder cells is used in lieu of feeder cell co-culture or incombination with feeder cell co-culture. In some embodiments, feeder cells are not used during the proliferation of the target stem cells.Differentiation of PSCs

[0215] Known methods for producing definitive endoderm from pluripotent cells (e.g., iPSCs or ESCs) are applicable to the methods described herein. In some embodiments, pluripotent cells are derived from a morula. In some embodiments, pluripotent stem cells are stem cells. Stem cells used in these methods can include, but are not limited to, embryonic stem cells or induced pluripotent stem cells. Embryonic stem cells can be derived from the embryonic inner cell mass or from the embryonic gonadal ridges. Embryonic stem cells or germ cells can originate from a variety of animal species including, but not limited to, various mammalian species including humans. In some embodiments, human embryonic stem cells are used to produce definitive endoderm. In some embodiments, human embryonic germ cells are used to produce definitive endoderm. In some embodiments, iPSCs are used to produce definitive endoderm, in some embodiments, human iPSCs (hiPSCs) are used to produce definitive endoderm.

[0216] In some embodiments, PSCs, such as ESCs and iPSCs, undergo directed differentiation into embryonic germ layer cells, organ tissue progenitor cells, and then into tissue such as liver tissue or any other biological tissue. In some embodiments, the directed differentiation is done in a stepwise manner to obtain each of the differentiated cell types where molecules (e.g. growth factors, ligands, agonists, antagonists) are added sequentially as differentiation progresses. In some embodiments, the directed differentiation is done in a nonstepwise manner where molecules (e.g. growth factors, ligands, agonists, antagonists) are added at the same time. In some embodiments, directed differentiation is achieved by selectively activating certain signaling pathways in the PSCs or any downstream cells.

[0217] In some embodiments, the embryonic stem cells or germ cells or iPSCs are treated with one or more small molecule compounds, activators, inhibitors, or growth factors for a time that is, is about, is at least, is at least about, is not more than, or is not more than about, 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 120 hours, 150 hours, 180 hours, 240 hours, 300 hours or any time within a range defined by any two of the aforementioned times, for example 6 hours to 300 hours, 24 hours to 120 hours, 48 hours to 96 hours, 6 hours to 72 hours, or 24 hours to 300 hours, in some embodiments, more than one small molecule compounds, activators, inhibitors, or growth factors are added. In these cases, themore than one small molecule compounds, activators, inhibitors, or growth factors can be added simultaneously or separately.

[0218] In some embodiments, the embryonic stem cells or germ cells or iPSCs are treated with one or more small molecule compounds, activators, inhibitors, or growth factors at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 10 ng / mL, 20 ng / mL, 50 ng / mL, 75 ng / mL, 100 ng / mL, 120 ng / mL, 150 ng / mL, 200 ng / mL, 500 ng / mL, 1000 ng / mL, 1200 ng / mL, 1500 ng / mL, 2000 ng / mL, 5000 ng / mL, 7000 ng / niL, 10000 ng / mL, or 15000 ng / mL, or any concentration that is within a range defined by any two of the aforementioned concentrations, for example, 10 ng / mL to 15000 ng / mL, 100 ng / mL to 5000 ng / mL, 500 ng / mL to 2000 ng / mL, 10 ng / mL to 2000 ng / mL, or 1000 ng / mL to 15000 ng / mL. In some embodiments, concentration of the one or more small molecule compounds, activators, inhibitors, or growth factors is maintained at a constant level throughout the treatment. In some embodiments, concentration of the one or more small molecule compounds, activators, inhibitors, or growth factors is varied during the course of the treatment. In some embodiments, more than one small molecule compounds, activators, inhibitors, or growth factors are added. In these cases, the more than one small molecule compounds, activators, inhibitors, or growth factors can differ in concentrations.

[0219] In some embodiments, the ESCs or iPSCs, or the ESCs, germ cells, or iPSCs are cultured in growth media that supports the growth of stem cells. In some embodiments, the ESCs or iPSCs, or the ESCs, germ cells, or iPSCs, are cultured in stem cell growth media. In some embodiments, the stem cell growth media is RPMI 1640, DMEM, DMEM / F12, or Advanced DMEM / F12. In some embodiments, the stem cell growth media comprises fetal bovine serum (FBS). In some embodiments, the stem cell growth media comprises FBS at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0,6%, 0.7%, 0.8%, 0,9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, or any percentage within a range defined by any two of the aforementioned concentrations, for example 0% to 20%, 0.2% to 10%, 2% to 5%, 0% to 5%, or 2% to 20%. In some embodiments, the stem cell growth media does not contain xenogeneic components. In some embodiments, the growth media comprises one or more small molecule compounds, activators, inhibitors, or growth factors.

[0220] In some embodiments, populations of cells enriched in definitive endoderm cells are used. In some embodiments, the definitive endoderm cells are isolated or substantially purified.In some embodiments, the isolated or substantially purified definitive endoderm cells express one or more (e.g. at least 1, 3) of SOX17, F0XA2, or CXRC4 markers to a greater extent than one or more (e.g. at least 1, 3, 5) of GCT4, AFP, I'M, SPARC, or SGX7 markers.

[0221] In some embodiments, pluripotent stem cells are prepared from somatic cells. In some embodiments, pluripotent stem cells are prepared from biological tissue obtained from a biopsy. In some embodiments, the pluripotent stem cells are cryopreserved. In some embodiments, the somatic cells are cryopreserved. In some embodiments, pluripotent stem cells are prepared from PBMCs. In some embodiments, human PSCs are prepared from human PBMCs. In some embodiments, pluripotent stem cells are prepared from cryopreserved PBMCs. In some embodiments, PBMCs are grown on a feeder cell substrate. In some embodiments, PBMCs are grown on a mouse embryonic fibroblast (MEF) feeder cell substrate. In some embodiments, PBMCs are grown on an irradiated MEF feeder cell substrate. In some embodiments, iPSCs are grown on an irradiated MEF feeder cell substrate. In some embodiments, iPSCs are grown in RPMI 1640, DMEM, DMEM / F12, mTeSR 1, or mTeSR Plus media.

[0222] In some embodiments, iPSCs are expanded in cell culture. In some embodiments, iPSCs are expanded in an extracellular matrix, or mimetic or derivative thereof. In some embodiments, the extracellular matrix, or mimetic or derivative thereof, comprises polymers, proteins, polypeptides, nucleic acids, sugars, lipids, poly-lysine, poly-ornithine, collagen, gelatin, fibronectin, vitronectin, laminin, elastin, tenascin, heparan sulfate, entactin, nidogen, osteopontin, basement membrane, Matrigel, Geltrex, hydrogel, PEI, WGA, or hyaluronic acid, or any combination thereof. In some embodiments, PSCs are expanded in Matrigel, Geltrex, or 1% gelatin, or any combination thereof. In some embodiments, iPSCs are expanded in Matrigel. In some embodiments, the iPSCs are expanded in cell culture while inhibiting a ROCK signaling pathway. In some embodiments, the iPSCs are expanded in cell culture comprising a ROCK inhibitor (e.g. Y-27632).

[0223] In some embodiments, inhibiting one or more signaling pathways, e.g. via providing proteins, activators, or inhibitors of the FGF, Wnt, BMP, TGF-b, EGF, PI3K, ROCK, cAMP, VEGF, Notch, HH, and / or retinoic acid (RA) pathways, or any combination thereof, can be used to mimic development in culture to obtain various cell types used herein that are differentiated from pluripotent stem cells. In some embodiments, cellular constituents associated with the FGF, Wnt, BMP, TGF-b, EGF, PI3K, ROCK, cAMP, VEGF, Notch, HH, and / or retinoic acid (RA) signaling pathways, for example, natural inhibitors, antagonists, activators, or agonistsof the pathways can be used to result in inhibition or activation of the FGF, Wnt, BMP, TGF-b, EGF, PI3K, ROCK, cAMP, VEGF, Notch, HH, and / or retinoic acid (RA) signaling pathways. In some embodiments, siRNA and / or shRNA targeting cellular constituents associated with the FGF, Wnt, BMP, TGF-b, EGF, PI3K, ROCK, cAMP, VEGF, Notch, HH, and / or retinoic acid (RA) signaling pathways are used to inhibit or activate these pathways. Furthermore, the methods disclosed herein may also involve the use of an EGF pathway activator acting as a mitogen, which promotes proliferation and growth of desired cell populations.

[0224] In some embodiments, stem cells are treated with one or more growth factors to differentiate to definitive endoderm cells. Such growth factors can include growth factors from the TGF-beta superfamily. In some embodiments, the one or more growth factors comprise the Nodal / Activin and / or the BMP subgroups of the TGF-beta superfamily of growth factors. In some embodiments, the one or more growth factors are selected from the group consisting of Nodal, Activin A, Activin B, BMP4, Wnt3a or combinations of any of these growth factors. In some embodiments, the stem cells are contacted with Activin A. In some embodiments, the stem cells are contacted with Activin A and BMP4.

[0225] In some embodiments, activin-induced definitive endoderm (DE) can further undergo anterior endoderm pattering, foregut specification and morphogenesis, dependent on FGF, Wnt, or retinoic acid, or any combination thereof, or on FGF, Wnt, BMP, or retinoic acid, or any combination thereof, and a culture system that promotes cholangiocyte growth, morphogenesis and cytodifferentiation. In some embodiments, human PSCs are efficiently directed to differentiate in vitro into appropriate epithelium and mesenchyme, it will be understood that molecules such as growth factors can be added to any stage of the development to promote a particular type of hepatic tissue formation.

[0226] It will be understood by one of skill in the art that altering the concentration, expression or function of one or more Wnt signaling proteins in combination with altering the concentration, expression, or function of one or more FGF proteins can give rise to directed differentiation in accordance with the present disclosure. In some embodiments, cellular constituents associated with the FGF, Wnt, or retinoic acid (RA) signaling pathways, or with the FGF, Wnt, BMP, or retinoic acid (RA) signaling pathways, for example, natural inhibitors, antagonists, activators, or agonists of the pathways can be used to result in inhibition or activation of the FGF, Wnt, or retinoic acid signaling pathways, or of the FGF, Wnt, BMP, or retinoic acid signaling pathways. In some embodiments, siRNA and / or shRNA targeting cellular constituentsassociated with the FGF, Wnt, or retinoic acid signaling pathways, or the FGF, Wnt, BMP, or retinoic acid signaling pathways, are used to inhibit or activate these pathways.

[0227] In some embodiments, pluripotent stem cells, definitive endoderm, posterior foregut spheroids, or downstream liver cell types are contacted with a Wnt signaling pathway activator or Wnt signaling pathway inhibitor. In some embodiments, the Wnt signaling pathway activator comprises a Wnt protein, in some embodiments, the Wnt protein comprises a recombinant Wnt protein. In some embodiments, the Wnt signaling pathway activator comprises Wntl, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, WntSa, WntSb, Wnt6, Wnt7a, Wnt7b, Wnt8a, WntSb, Wnt9a, Wnt9b, WntlOa, WntlOb, Wntl l Wntl6, BML 284, IQ-1, WAY 262611, or any combination thereof. In some embodiments, the Wnt signaling pathway activator comprises a GSK3 signaling pathway inhibitor. In some embodiments, the Wnt signaling pathway activator comprises CHIR99Q21, CfflR 98014, AZD2858, BIO, AR-A014418, SB 216763, SB 415286, aloisine, indirubin, alsterpaullone, kenpauilone, lithium chloride, TDZD 8, or TWS119, or any combination thereof. In some embodiments, the Wnt signaling pathway inhibitor comprises C59, PNU 74654, KY-02111, PRI-724, FH-535, DIF-1, or XAV939, or any combination thereof. In some embodiments, the cells are not treated with a Wnt signaling pathway activator or Wnt signaling pathway inhibitor. The Wnt signaling pathway activator or Wnt signaling pathway inhibitor provided herein may be used in combination with any of the other growth factors, signaling pathway activators, or signaling pathway inhibitors provided herein.

[0228] In some embodiments, pluripotent stem cells, definitive endoderm, posterior foregut spheroids, or downstream liver cell types are contacted with an FGF signaling pathway activator. In some embodiments, the FGF signaling pathway activator comprises an FGF protein. In some embodiments, the FGF protein comprises a recombinant FGF protein. In some embodiments, the FGF signaling pathway activator comprises one or more of FGF1 , FGF2, FGF3, FGF4, FGF4, FGF 5, FGF6, FGF7, FGF 8, FGF8, FGF9, FGF 10, FGF11, FGF 12, FGF 13, FGF 14, FGF 15 (FGF 19, FGF15 / FGF19), FGF 16, FGF 17, FGF 18, FGF20, FGF21, FGF22, or FGF23. In some embodiments, the cells are not treated with an FGF signaling pathway activator. The FGF signaling pathway activator provided herein may be used in combination with any of the other growth factors, signaling pathway activators, or signaling pathway inhibitors provided herein.

[0229] In some embodiments, pluripotent stem cells, definitive endoderm, posterior foregut spheroids, or downstream liver cell types are contacted with a retinoic acid signalingpathway activator or retinoic acid signaling pathway inhibitor. In some embodiments, the retinoic acid signaling pathway activator comprises retinoic acid, all-trans retinoic acid, 9-cis retinoic acid, CD437, EC23, BS 493, TTNPB, or AM580, or any combination thereof, in some embodiments, the retinoic acid signaling pathway inhibitor comprises guggul sterone. In some embodiments, the cells are not treated with a retinoic acid signaling pathway activator or retinoic acid signaling pathway inhibitor. The retinoic acid signaling pathway activator or retinoic acid signaling pathway inhibitor provided herein may be used in combination with any of the other growth factors, signaling pathway activators, or signaling pathway inhibitors provided herein.

[0230] In some embodiments, pluripotent stem cells are converted into liver cell types via a “one step” process. For example, one or more molecules that can differentiate pluripotent stem cells into DE culture (e.g., Activin A) are combined with additional molecules that can promote directed differentiation of DE culture (e.g., FGF4, CHIR99021, RA; or e.g., FGF4, Wnt, Noggin, RA) to directly treat pluripotent stem cells.

[0231] In some embodiments, iPSCs are expanded in cell culture. In some embodiments, pluripotent stem cells are expanded in a basement membrane matrix. In some embodiments, iPSCs are expanded in Matrigel, In some embodiments, the iPSCs are expanded in cell culture comprising a ROCK inhibitor (e.g. Y-27632). In some embodiments, the iPSCs are differentiated into definitive endoderm cells. In the iPSCs are differentiated into definitive endoderm cells bycontacting the iPSCs with Activin A, BMP4, or both. In some embodiments, the iPSCs are contacted with a concentration of Activin A that is, is about, is at least, is at least about, is not more than, or is not more than about, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL, or any concentration of Activin A within a range defined by any two of the aforementioned concentrations, for example, 10 to 200 ng / mL, 10 to 100 ng / mL, 100 to 200 ng / mL, or 50 to 150 ng / mL. In some embodiments, the pluripotent stem cells are contacted with Activin A at a concentration of 100 ng / mL or about 100 ng / mL. In some embodiments, the iPSCs are contacted with a concentration of BMP4 that is, is about, is at least, is at least about, is not more than, or is not more than about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 ng / mL, or any concentration of BMP4 within a range defined by any two of the aforementioned concentrations, for example, 1 to 200 ng / mL, 1 to 100 ng / mL, 25 to 200 ng / mL, 1 to 80 ng / mL, or 25 to 100 ng / mL, In some embodiments, the pluripotent stem cells are contacted with BMP4 at a concentration of 50 ng / mL or about 50 ng / mL.

[0232] In some embodiments, for any of the small molecule compounds, pathway activators, pathway inhibitors, or growth factors, the cells are contacted for a time that is, is about, is at least, is at least about, is not more than, or is not more than about, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 120 hours, 150 hours, 180 hours, 240 hours, 300 hours or any time within a range defined by any two of the aforementioned times, for example 1 hour to 300 hours, 24 hours to 120 hours, 48 hours to 96 hours, 6 hours to 72 hours, or 24 hours to 300 hours. In some embodiments, more than one small molecule compounds, activators, inhibitors, or growth factors are added. In these cases, the more than one small molecule compounds, activators, inhibitors, or growth factors can be added simultaneously or separately.

[0233] In some embodiments, the PSCs are differentiated into definitive endoderm cells. In some embodiments, the PSCs are differentiated into posterior foregut cells, in some embodiments, the PSCs are differentiated into a liver organoid.

[0234] In some embodiments, any of the cells disclosed herein may be cryopreserved for later use. The cells can be cryopreserved according to methods generally known in the art, optionally including one or more cryoprotectants.

[0235] Cryoprotectants are cell composition additives to improve efficiency and yield of low temperature cryopreservation by preventing formation of large ice crystals. Cryoprotectants include but are not limited to DMSO, ethylene glycol, glycerol, propylene glycol, trehalose, formamide, methyl-formamide, dimethyl-formamide, glycerol 3 -phosphate, proline, sorbitol, diethyl glycol, sucrose, tri ethylene glycol, polyvinyl alcohol, polyethylene glycol, or hydroxy ethyl starch. Cryoprotectants can be used as part of a cryopreservation medium, which include other components such as nutrients (e.g. albumin, serum, bovine serum, fetal calf serum (FCS)) to enhance post-thawing survivability of the cells, in these cryopreservation media, at least one cryoprotectant may be found at a concentration that is, is about, is at least, is at least about, is not more than, or is not more than about, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or any percentage within a range defined by any two of the aforementioned numbers.Gene Editing

[0236] Embodiments of the disclosure can include PSCs, iPSCs, definitive endoderm cells, posterior foregut spheroids, or organoids which have been or which can be genetically modifiedor edited according to methods known in the art. For example, gene editing using CRTSPR nucleases such as Cas9 are explored in PCT Publications WO 2013 / 176772, WO 2014 / 093595, WO 2014 / 093622, WO 2014 / 093655, WO 2014 / 093712, WO 2014 / 093661, WO 2014 / 204728, WO 2014 / 204729, WO 2015 / 071474, WO 2016 / 115326, WO 2016 / 141224, WO 2017 / 023803, and WO 2017 / 070633, each of which is hereby expressly incorporated by reference in its entirety.Pharmaceutical Compositions

[0237] Embodiments of the disclosure can include pharmaceutical compositions. Such pharmaceutical compositions can include one or more additional pharmaceutically acceptable components, which can include carriers, excipients, and / or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed or that have an acceptable level of toxicity. A “pharmaceutically acceptable” “diluent,” “excipient,” and / or “carrier” as used herein have their plain and ordinary meaning as understood in light of the specification and are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with administration to humans, cats, dogs, or other vertebrate hosts. Typically, a pharmaceutically acceptable diluent, excipient, and / or carrier is a diluent, excipient, and / or earner approved by a regulatory agency of a Federal, a state government, or other regulatory agency, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans as well as non-human mammals, such as cats and dogs. The term diluent, excipient, and / or “carrier” can refer to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Such pharmaceutical diluent, excipient, and / or earners can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. Water, saline solutions and aqueous dextrose and glycerol solutions can be employed as liquid diluents, excipients, and / or carriers, particularly for injectable solutions. Suitable pharmaceutical diluents and / or excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. A non-limiting example of a physiologically acceptable carrier is an aqueous pH buffered solution. The physiologically acceptable carrier may also comprise one or more of the following: antioxidants, such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, such as serum albumin, gelatin, immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, ammo acids, carbohydrates such as glucose, mannose, or dextrins, chelatingagents such as EDTA, sugar alcohols such as mannitol or sorbitol, salt-forming counterions such as sodium, and nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®. The composition, if desired, can also contain minor amounts of wetting, bulking, emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, sustained release formulations and the like. The formulation should suit the mode of administration.

[0238] Additional excipients with desirable properties include but are not limited to preservatives, adjuvants, stabilizers, solvents, buffers, diluents, solubilizing agents, detergents, surfactants, chelating agents, antioxidants, alcohols, ketones, aldehydes, ethylenediaminetetraacetic acid (EDTA), citric acid, salts, sodium chloride, sodium bicarbonate, sodium phosphate, sodium borate, sodium citrate, potassium chloride, potassium phosphate, magnesium sulfate sugars, dextrose, fructose, mannose, lactose, galactose, sucrose, sorbitol, cellulose, serum, amino acids, polysorbate 20, polysorbate 80, sodium deoxycholate, sodium taurodeoxy cholate, magnesium stearate, octylphenol ethoxylate, benzethonium chloride, thimerosal, gelatin, esters, ethers, 2-phenoxyethanol, urea, or vitamins, or any combination thereof. Some excipients may be in residual amounts or contaminants from the process of manufacturing, including but not limited to serum, albumin, ovalbumin, antibiotics, inactivating agents, formaldehyde, glutaraldehyde, b-propiolactone, gelatin, cell debris, nucleic acids, peptides, ammo acids, or growth medium components or any combination thereof. The amount of the excipient may be found in composition at a percentage that is, is about, is at least, is at least about, is not more than, oris not more than about, 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% w / w or any percentage by weight in a range defined by any two of the aforementioned numbers.

[0239] Pharmaceutical compositions can include one or more “pharmaceutically acceptable salts”, which can include relatively non-toxic, inorganic and organic acid, or base addition salts of compositions or excipients, including without limitation, analgesic agents, therapeutic agents, other materials, and the like. Examples of pharmaceutically acceptable salts include those derived from mineral acids, such as hydrochloric acid and sulfuric acid, and those derived from organic acids, such as ethanesulfonic acid, benzenesulfonic acid, p- toluenesulfonic acid, and the like. Examples of suitable inorganic bases for the formation of salts include the hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium, calcium,magnesium, aluminum, zinc, and the like. Salts may also be formed with suitable organic bases, including those that are non-toxic and strong enough to form such salts. For example, the class of such organic bases may include but are not limited to mono-, di-, and trialkylamines, including methylamine, dimethylamine, and triethylamine; mono-, di-, or trihydroxyalkylamines including mono-, di-, and triethanolamine; ammo acids, including glycine, arginine and lysine; guanidine; N-methylglucosamine; N-methylglucamine; L-glutamine; N-methylpiperazine; morpholine; ethylenediamine; N-benzylphenethylamine; trihydroxymethyl ammoethane.

[0240] Proper formulation is dependent upon the route of administration chosen. Techniques for formulation and administration of the compounds described herein are known to those skilled in the art. Multiple techniques of administering a compound exist in the art including, but not limited to, enteral, oral, rectal, topical, sublingual, buccal, intraaural, epidural, epicutaneous, aerosol, parenteral delivery, including intramuscular, subcutaneous, intra-arterial, intravenous, intraportal, intra-articular, intradermal, peritoneal, intramedullary injections, intrathecal, direct intraventricular, intraperitoneal, intranasal or intraocular injections. Pharmaceutical compositions will generally be tailored to the specific intended route of administration.

[0241] As used herein, a “carrier” has its plain and ordinary meaning as understood in light of the specification and can refer to a compound, particle, solid, semi-solid, liquid, or diluent that facilitates the passage, delivery and / or incorporation of a compound to cells, tissues and / or bodily organs.

[0242] As used herein, a “diluent” has its plain and ordinary meaning as understood in light of the specification and can refer to an ingredient in a pharmaceutical composition that lacks pharmacological activity but may be pharmaceutically necessary or desirable. For example, a diluent may be used to increase the bulk of a potent drug whose mass is too small for manufacture and / or administration. It may also be a liquid for the dissolution of a drug to be administered by injection, ingestion or inhalation. A common form of diluent in the art is a buffered aqueous solution such as, without limitation, phosphate buffered saline that mimics the composition of human blood.Dosage and Administration Routes

[0243] Embodiments of the disclosure can include methods of administering or treating an animal, which can involve administering an amount of at least one treatment, that is effective to treat the disease, condition, or disorder that the organism has, or is suspected of having, or issusceptible to, or to bring about a desired physiological effect. In some embodiments, the disease, condition, or disorder can be a liver-related disease or disorder.

[0244] In some embodiments, at least one treatment can include a composition or pharmaceutical composition, which can be administered to an animal (e.g., mammals, primates, monkeys, or humans) in an amount of about 0.005 to about 50 mg / kg body weight, about 0.01 to about 15 mg / kg body weight, about 0.1 to about 10 mg / kg body weight, about 0.5 to about 7 mg / kg body weight, about 0.005 mg / kg, about 0.01 mg / kg, about 0.05 mg / kg, about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 3 mg / kg, about 5 mg / kg, about 5.5 mg / kg, about 6 mg / kg, about 6.5 mg / kg, about 7 mg / kg, about 7.5 mg / kg, about 8 mg / kg, about 10 mg / kg, about 12 mg / kg, or about 15 mg / kg. In regard to some conditions, the dosage can be about 0.5 mg / kg human body weight or about 6.5 mg / kg human body weight. In some instances, some subjects (e.g., mammals, mice, rabbits, feline, porcine, or canine) can be administered a dosage of about 0.005 to about 50 mg / kg body weight, about 0.01 to about 15 mg / kg body weight, about 0.1 to about 10 mg / kg body weight, about 0.5 to about 7 mg / kg body weight, about 0.005 mg / kg, about 0.01 mg / kg, about 0.05 mg / kg, about 0.1 mg / kg, about 1 mg / kg, about 5 mg / kg, about 10 mg / kg, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 80 mg / kg, about 100 mg / kg, or about 150 mg / kg. Of course, those skilled in the art will appreciate that it is possible to employ many concentrations in the methods of the present disclosure, and using, in part, the guidance provided herein, will be able to adjust and test any number of concentrations in order to find one that achieves the desired result in a given circumstance. In some embodiments, a dose or a therapeutically effective dose of a compound disclosed herein will be that which is sufficient to achieve a plasma concentration of the compound or its active metabolite(s) within a range set forth herein, e.g., about 1-10 nM, 10- 100 nM, 0.1-1 pM, 1-10 pM, 10-100 pM, 100-200 pM, 200-500 pM, or even 500-1000 pM, preferably about 1-10 nM, 10-100 nM, or 0.1-1 pM.

[0245] In other embodiments, a treatment can be administered in combination with one or more other therapeutic agents for a given disease, condition, or disorder.

[0246] The compounds and pharmaceutical compositions are preferably prepared and administered in dose units. Solid dose units are tablets, capsules and suppositories. For treatment of a subject, depending on activity of the compound, manner of administration, nature and severity of the disease or disorder, age and body weight of the subject, different daily doses can be used.

[0247] Under certain circumstances, however, higher or lower daily doses can be appropriate. The administration of the daily dose can be carried out both by single administrationin the form of an individual dose unit or else several smaller dose units and also by multiple administrations of subdivided doses at specific intervals.

[0248] A treatment can be administered locally or systemically in a therapeutically effective dose. Amounts effective for this use will, of course, depend on the severity of the disease or disorder and the weight and general state of the subject. Typically, dosages used in vitro can provide useful guidance in the amounts useful for in situ administration of the pharmaceutical composition, and animal models can be used to determine effective dosages for treatment of particular disorders.

[0249] Various considerations are described, e. g. , in Langer, 1990, Science, 249: 1527; Goodman and Gilman's (eds.), 1990, Id., each of which is herein incorporated by reference and for all purposes. Dosages for parenteral administration of active pharmaceutical agents can be converted into corresponding dosages for oral administration by multiplying parenteral dosages by appropriate conversion factors. As to general applications, the parenteral dosage in mg / mL times 1.8 = the corresponding oral dosage in milligrams (“mg”). As to oncology applications, the parenteral dosage in mg / mL times 1.6 = the corresponding oral dosage in mg. An average adult weighs about 70 kg. See e.g., Miller-Keane, 1992, Encyclopedia & Dictionary of Medicine, Nursing & Allied Health, 5th Ed., (W. B. Saunders Co.), pp.1708 and 1651.

[0250] It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

[0251] In some embodiments, the administration can include a unit dose of one or more treatments in combination with a pharmaceutically acceptable carrier and, in addition, can include other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, and excipients. In certain embodiments, the carrier, vehicle or excipient can facilitate administration, delivery and / or improve preservation of the composition. In other embodiments, the one or more carriers, include but are not limited to, saline solutions such as normal saline, Ringer's solution, PBS (phosphate- buffered saline), and generally mixtures of various salts including potassium and phosphate salts with or without sugar additives such as glucose. Carriers can include aqueous and non-aqueous sterile injection solutions that can contain antioxidants, buffers, bacteriostats, bactericidal antibiotics, and solutes that render the formulation isotonic with the bodily fluids of the intended recipient; and aqueous and non-aqueous sterile suspensions, which can include suspending agentsand thickening agents. In other embodiments, the one or more excipients can include, but are not limited to water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. Nontoxic auxiliary substances, such as wetting agents, buffers, or emulsifiers may also be added to the composition. Oral formulations can include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate.

[0252] The quantity of active component in a unit dose preparation can be varied or adjusted from 0.1 mg to 10000 mg, more typically 1.0 mg to 1000 mg, most typically 10 mg to 500 mg, according to the particular application and the potency of the active component. The composition can, if desired, also contain other compatible therapeutic agents.

[0253] A treatment can be administered to subjects by any number of suitable administration routes or formulations. The treatment, such as an immunotherapy, can also be used to treat subjects for a variety of diseases. Subjects include but are not limited to mammals, primates, monkeys (e.g., macaque, rhesus macaque, or pig tail macaque), humans, canine, feline, bovine, porcine, avian (e.g., chicken), mice, rabbits, and rats. In particular embodiments described herein, the subject is a human.

[0254] The route of administration of the compounds of the treatments described herein can be of any suitable route. Administration routes can be, but are not limited to the oral route, the parenteral route, the cutaneous route, the nasal route, the rectal route, the vaginal route, and the ocular route. In other embodiments, administration routes can be parenteral administration, a mucosal administration, intravenous administration, subcutaneous administration, topical administration, intradermal administration, oral administration, sublingual administration, intranasal administration, or intramuscular administration. The choice of administration route can depend on the compound identity (e.g., the physical and chemical properties of the compound) as well as the age and weight of the animal, the particular disease (e.g., type of cancer), and the severity of the disease (e.g., stage or severity of cancer). Of course, combinations of administration routes can be administered, as desired.

[0255] Some embodiments of the disclosure include a method for providing a subject with a treatment which comprises one or more administrations of one or more compositions; the compositions may be the same or different if there is more than one administration.Toxicity

[0256] The ratio between toxicity and therapeutic effect for a particular treatment is its therapeutic index and can be expressed as the ratio between LD50 (the amount of compound lethal in 50% of the population) and ED50 (the amount of compound effective in 50% of the population). Compounds that exhibit high therapeutic indices are preferred. Therapeutic index data obtained from in vitro assays, cell culture assays and / or animal studies can be used in formulating a range of dosages for use in humans. The dosage of such compounds preferably lies within a range of plasma concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. See, e g. Fingl et al., In: THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, Ch.l, p.l, 1975. The exact formulation, route of administration, and dosage can be chosen by the individual practitioner in view of the patient’s condition and the particular method in which the compound is used. For in vitro formulations, the exact formulation and dosage can be chosen by the individual practitioner in view of the patient’s condition and the particular method in which the compound is used.Compositions

[0257] In some embodiments, also provided herein are compositions for performing any of the methods disclosed herein. In some embodiments, also provided herein are compositions produced according to processes provided in any of the methods disclosed herein. It is expressly contemplated that, in certain embodiments, any method or composition described herein can be implemented with respect to any other method or composition described herein and that different embodiments may be combined.

[0258] In some embodiments, provided herein are cell compositions in the form of a three- dimensional artificial biliary organoid, comprising extrahepatic cholangiocytes. In some embodiments, the cell compositions further include intrahepatic cholangiocytes and extrahepatic cholangiocytes. In some embodiments, provided herein are ex vivo compositions comprising a three-dimensional biliary organoid, comprising extrahepatic cholangiocytes. In some embodiments, the ex vivo compositions further include intrahepatic cholangiocytes and extrahepatic cholangiocytes.

[0259] In some embodiments, provided herein are compositions, such as cell compositions and / or biliary organoids, that further comprise cholangiocytes, mesenchymal cells, and a stochastic tubular assembly. In some embodiments, provided herein are ex vivo compositions including a three-dimensional biliary organoid, comprising cholangiocytes, mesenchymal cells, and astochastic tubular assembly. Tn some embodiments, provided herein are compositions, such as cell compositions and / or biliary organoids, that further comprise comprise intrahepatic bile duct (IHBD)-like cholangiocytes, gallbladder (GB)-like cholangiocytes, and common bile duct (CBD)- like cholangiocytes. In some embodiments, provided herein are compositions, such as cell compositions and / or biliary organoids, that further comprise hepatic progenitors, stellate cells, endothelial cells, macrophages, and / or Kupffer cells. In some embodiments, the hepatocytes selfassemble into the three-dimensional artificial biliar organoid. In some embodiments, the three- dimensional artificial biliary organoid includes a structure with a stochastic, or complex tubular assembly, rather than a single lumen as in a liver organoid.

[0260] In some embodiments compositions provided herein may comprise cell populations differentiated from pluripotent stem cells. In some embodiments compositions provided herein may comprise cell populations differentiated from induced pluripotent stem cells (iPSCs). In some embodiments, compositions provided herein comprise exogenously added and / or transgenically produced ascorbate (vitamin C). In some embodiments, provided herein are compositions comprising exogenously provided ascorbate (vitamin C)at a concentration of about 0.1 mg / L, 0.2 mg / L, 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, 1 mg / L, 1.1 mg / L, 1.2 mg / L, 1.3 mg / L, 1.4 mg / L, 1.5 mg / L, 1.6 mg / L, 1.7 mg / L, 1.8 mg / L, 1.9 mg / L, 2 mg / L, 2.1 mg / L, 2.2 mg / L, 2.3 mg / L, 2.4 mg / L, 2.5 mg / L, 2.6 mg / L, 2.7 mg / L, 2.8 mg / L, 2.9 mg / L, or 3 mg / L. In some embodiments, provided herein are compositions comprising exogenously provided ascorbate (vitamin C) at a concentration of about 1 mg / L.

[0261] In some embodiments, compositions provided herein are in vitro compositions, created outside of a multicellular living organism. In some embodiments, compositions provided herein may be introduced into a multicellular living organism. In some embodiments, compositions provided herein comprise exogenously provided components, reagents, and / or conditions. In some embodiments, compositions provided herein comprise exogenously provided components, reagents, and / or conditions that mimic in vivo characteristics desirable for inducing specific cellular differentiation and / or organoid organization.

[0262] In some embodiments, provided herein are compositions comprising a tissue culture surface that is coated with a basement membrane matrix or component thereof. In some embodiments, a basement membrane matrix or component thereof does not comprise non-human animal components. In some embodiments, a basement membrane matrix or component thereof does not comprise non-human animal components such that the basement membrane matrix orcomponent thereof is xenogeneic to humans. In some embodiments, a basement membrane matrix or component thereof is not isolated from murine Engelbreth-Holm- Swarm (EHS) sarcoma cells, is not Matrigel®, is not Cultrex®, and / or is not Geltrex®. In some embodiments, a basement membrane matrix or component thereof comprises human laminin, collagen IV, entactin, perlecan, fibrin, and / or hydrogel.

[0263] In some embodiments, provided herein are compositions that include an exogenous TGF-b pathway inhibitor. In some embodiments, an exogenous TGF-b pathway inhibitor comprises, consists essentially of, or consists of A83-01, RepSox, LY365947, and / or SB431542. In some embodiments, an exogenous TGF-b pathway inhibitor comprises, consists essentially of, or consists of TGF-b pathway inhibitor A83-01. In some embodiments, a composition comprises a TGF-b pathway inhibitor at a concentration of, or of about, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nM, or any concentration within a range defined by any two of the aforementioned concentrations. In some embodiments, a composition comprises a TGF-b pathway inhibitor at a concentration of, or of about, 500 nM.

[0264] In some embodiments, provided herein are compositions that include an exogenous FGF pathway activator. In some embodiments, a composition comprises an exogenous FGF pathway activator that comprises, consists essentially of, or consists of FGF1, FGF2, FGF3, FGF4, FGF4, FGF5, FGF6, FGF7, FGF8, FGF8, FGF9, FGF 10, FGF11, FGF 12, FGF 13, FGF 14, FGF 15, FGF 16, FGF 17, FGF 18, FGF 19, FGF20, FGF21, FGF22, and / or FGF23. In some embodiments, an exogenous FGF pathway activator comprises, consists essentially of, or consists of FGF2. In some embodiments, a composition comprises a FGF pathway activator at a concentration of, or of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ng / mL, or any concentration within a range defined by any two of the aforementioned concentrations. In some embodiments, a composition comprises a FGF pathway activator at a concentration of, or of about 5 ng / mL.

[0265] In some embodiments, provided herein are compositions that include an exogenous Wnt pathway activator. In some embodiments, a composition comprises an exogenous Wnt pathway activator that comprises, consists essentially of, or consists ofWntl, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, WntlOa, WntlOb, Wntl l, Wntl6, BML 284, IQ-1, WAY 262611, CHIR99021, CHIR 98014, AZD2858, BIO, AR-A014418, SB 216763, SB 415286, aloisine, indirubin, alsterpaullone, kenpaullone, lithium chloride, TDZD 8, and / or TWS119. In some embodiments, a composition comprises an exogenous Wnt pathway activator that comprises, consists essentially of, or consists ofCHIR99021. In some embodiments, a composition comprises a Wnt pathway activator at a concentration of, or of about, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8 pM, or any concentration within a range defined by any two of the aforementioned concentrations. In some embodiments, In some embodiments, a composition comprises a Wnt pathway activator at a concentration of, or of about, 3 pM.

[0266] In some embodiments, provided herein are compositions that include an exogenous VEGF pathway activator. In some embodiments, a composition comprises an exogenous VEGF pathway activator that comprises, consists essentially of, or consists of VEGF and / or GS4012. In some embodiments, a composition comprises an exogenous VEGF pathway activator that comprises, consists essentially of, or consists of VEGF. In some embodiments, a composition comprises a VEGF pathway activator at a concentration of, or of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / mL, or any concentration within a range defined by any two of the aforementioned concentrations. In some embodiments, a composition comprises a VEGF pathway activator at a concentration of, or of about 10 ng / mL.

[0267] In some embodiments, provided herein are compositions that include an exogenous EGF. In some embodiments, provided herein are compositions that do not include an exogenous EGF. In some embodiments, provided herein are compositions comprising EGF at a concentration of, or of about, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ng / mL, or any concentration within a range defined by any two of the aforementioned concentrations. In some embodiments, provided herein are compositions comprising EGF at a concentration of, or of about, 20 ng / mL.

[0268] In some embodiments, provided herein are compositions that include exogenous and / or transgenically produced ascorbic acid. In some embodiments, provided herein are compositions that do not include exogenous and / or transgenically produced ascorbic acid. In some embodiments, provided herein are compositions comprising ascorbic acid at a concentration of, or of about, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 pg / mL or any concentration within a range defined by any two of the aforementioned concentrations. In some embodiments, provided herein are compositions comprising ascorbic acid at a concentration of, or of about, 50 pg / mL.

[0269] In some embodiments, provided herein are compositions that include a ROCK inhibitor. In some embodiments, provided herein are compositions that do not include a ROCK inhibitor. In some embodiments, a ROCK inhibitor comprises, consists essentially of, or consists of Y-27632. In some embodiments, provided herein are compositions comprising a ROCKinhibitor at a concentration of, or of about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 pM, or any concentration within a range defined by any two of the aforementioned concentrations. In some embodiments, provided herein are compositions comprising a ROCK inhibitor at a concentration of, or of about, 10 pM.

[0270] In some embodiments, provided herein are compositions comprising liver organoids that have and / or that are being differentiated from stem cells. In some embodiments, provided herein are compositions comprising liver organoids that have and / or that are being differentiated from induced pluripotent stem cells. In some embodiments, provided herein are compositions comprising liver organoids comprising cells that have been passaged 1 time, 2 times, or 3 times. In some embodiments, provided herein are compositions comprising liver organoids comprising cells that have been passaged less than 4 times.

[0271] In some embodiments, provided herein are compositions comprising A83-01, FGF2, CHIR99021, VEGF, and / or Y-27632, optionally further comprising iPSCs, PSCs, and / or posterior foregut cells and / or posterior foregut endoderm cells.

[0272] In some embodiments, provided herein are compositions comprising: a) posterior foregut cells and / or posterior foregut endoderm cells, biliary organoids, and / or biliary progenitor cells, and b) a medium, wherein the medium comprises biliary differentiation medium (as described herein) and is optionally supplemented with Vitamin C. The composition optionally can additionally comprise a retinoic acid pathway activator, and / or a cMET tyrosine kinase receptor agonist. In some embodiments, compositions provided herein comprise a cMET tyrosine kinase receptor agonist that comprises, consists essentially of, or consists of hepatocyte growth factor (HGF), PG-001, fosgonimeton, tereval efim, recombinant InlB321 protein, and / or an agonist c-Met antibody (e.g., LMH85).

[0273] In some embodiments, provided herein are compositions comprising an IL-6 family cytokine. In some embodiments, an IL-6 family cytokine comprises, consists essentially of, or consists of IL-6, Oncostatin M (OSM), leukemia inhibitory factor (LIF), cardiotrophin- 1 , ciliary neurotrophic factor (CTNF), and / or cardiotrophin-like cytokine (CLC).

[0274] In some embodiments, provided herein are compositions comprising a biliary culture and differentiation media supplemented with EGF, hGH, IL-6 and / or OSM. In some embodiments, provided herein are compositions comprising a biliary culture and differentiation media supplemented with EGF and hGH. In some embodiments, provided herein are compositions comprising a biliary culture and differentiation media supplemented with EGF and IL-6. In someembodiments, provided herein are compositions comprising a biliary culture and differentiation media supplemented with EGF and OSM. In some embodiments, provided herein are compositions comprising a biliary culture and differentiation media supplemented with IL-6. In some embodiments, provided herein are compositions comprising a biliary culture and differentiation media supplemented with OSM. In some embodiments, provided herein are compositions comprising a hepatocyte culture media supplemented with EGF. In some embodiments, provided herein are compositions comprising a hepatocyte culture media supplemented with hGH. In some embodiments, provided herein are compositions comprising a hepatocyte culture media supplemented with IL-6. In some embodiments, provided herein are compositions comprising a hepatocyte culture media supplemented with OSM.

[0275] In some embodiments, compositions comprise OSM. In some embodiments, compositions comprise OSM at a concentration of, or of about, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ng / mL, or any concentration within a range defined by any two of the aforementioned concentrations. In some embodiments, compositions comprise OSM at a concentration of, or of about 20 ng / mL.Kits

[0276] In some embodiments, also disclosed herein are kits providing means for performing any of the methods described herein. In some embodiments, also disclosed herein are kits comprising any of the compositions or means of producing the compositions described herein.

[0277] In some embodiments, a kit can be prepared from readily available components and reagents. For example, such kits can comprise any one or more of the following components and / or reagents: enzymes, reaction tubes, buffers, detergent, primers, probes, antibodies, cell culture media, differentiation induction reagents, amino acid mixtures / supplements, engineered constructs and / or polynucleotides, transcription induction agents, biliary differentiation medium (BDM), Vitamin C, retinoic acid pathway activators, corticosteroids, cMET tyrosine kinase receptor agonists, IL-6 family cytokines, TGF-b pathway inhibitors, FGF pathway activators, Wnt pathway activators, EGF pathway activators, OSM pathway activators, JAK-STAT pathway activators, VEGF pathway activators, ROCK inhibitors, organoids, and / or cells. In some embodiments, components and reagents may be packaged together in any combination, and / or may be packaged individually. In some embodiments, kits may include components and reagents concentrated above the working concentrations disclosed herein, or at the working concentrations provided herein. In some embodiments, individual components may also be provided in a kit in concentrated amounts;in some aspects, a component is provided individually in the same concentration as it would be in a solution with other components. In some embodiments, concentrations of components may be provided as lx, 2x, 5x, lOx, or 20x or more. In some embodiments, a kit may comprise components, which may be individually packaged or placed in a container, such as a tube, bottle, vial, syringe, or other suitable container means.

[0278] In some embodiments, a kit can additionally include one or more scaffold trays as described herein, i.e. a culture plate with a horizontal surface; one or more channels having a vertical depth, wherein each channel has longitudinal sidewalls and open latitudinal ends, and a volume comprising a culture region, and a lengthwise segment in each of the one or more channels, wherein each lengthwise segment runs longitudinally through the respective channel. In some embodiments, the lengthwise segment extends beyond one or more of the open latitudinal ends. Particular embodiments of the scaffold tray include a surgical filament as the lengthwise segment; other biocompatible materials can be used as well. In exemplary embodiments, each channel can have a U-shaped or V-shaped bottom and can be coated with collagen.

[0279] In some embodiments, the kits can include one or more biliary implants including a lumen-containing tubular structure and extrahepatic cholangiocytes. For example, the biliary implants can include the biliary organoids produced as described herein, or compositions including the same.

[0280] In some embodiments, a kit is housed in a container. Kits may further comprise instructions for using the kit for assessing expression and / or differentiation of cells. Agents in a kit for measuring expression and / or determining differentiation may comprise a plurality of PCR probes and / or primers for qRT-PCR and / or a plurality of antibody or fragments thereof for assessing expression of biomarkers appropriate for classifying cell states.

[0281] In some embodiments, kits are created using and comply with good manufacturing practice (GMP).

[0282] Having described the embodiments in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing from the scope of the embodiments defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.EXAMPLES

[0283] The following non-limiting examples are provided to further illustrate embodiments disclosed herein. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches that have been found to function well in the practice of the embodiments, and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the embodiments disclosed herein.EXAMPLE 1Materials and MethodsMaintenance of PSCs

[0284] Human PSC lines were maintained as described and as known in the art. Undifferentiated human PSCs were maintained on feeder-free conditions in mTeSRl medium (StemCell technologies) or Stem Fit medium (Ajinomoto) on plates coated 0.5 mg / ml iMatrix (Nippi inc., Japan) at 1 / 200 dilution at 37°C in an incubator with 5% CO2, 95% air at 37 °C. Human PSC clones (the parental PSC line without any genetic modification, passage number 6 to 20), GFP-knocked-in human PSCs on a safe harbor site on Chr 13 (passage number 7 to 20) with or without human leukocyte antigen class I and II double knockout induced by genetic knockout of B2M and CIIA genes, were used in this study (reference iPSC lines are made at CCHMC PSCF core, and gene knock out lines are provided by RxCell, Inc.). Cell lines have been tested for mycoplasma contamination and chromosomal aberration regularly.Differentiation of PSCs into anterior and posterior gut spheroid (AFG and PFG)

[0285] PSCs were detached by Accutase (Thermo Fisher Scientific Inc., MA, USA) and were seeded on Laminin coated 24-well tissue culture plate with 2>< 105cells / well with mTeSR with 10 pM Y-27632. The medium was changed to RPMI 1640 medium (Life Technologies) containing 100 ng / mL Activin A (R&D Systems) and 50 ng / mL bone morphogenetic protein 4 (BMP4; R&D Systems) at day 1, 100 ng / mL Activin A and 0.2% fetal calf serum (FCS; Thermo Fisher Scientific Inc.) at day 2, and 100 ng / mL Activin A and 2% FCS at day 3. For 4-6 days, cells were cultured in gut growth medium (Advanced DMEM / F12 (Thermo Fisher Scientific) with 15 mM HEPES, 2 mM 1-glutamine, penicillin-streptomycin, B27 (Life Technologies) and N2 (Gibco)) supplemented with 200 ng / ml noggin (NOG; R&D Systems), 500 ng / ml fibroblastgrowth factor 4 (FGF4; R&D Systems) and 2 pM CHIR99021 (Stemgent) for AFG induction and supplemented with 500 ng / ml FGF 4 and 3 pM CHIR99021 for PFG induction. Cultures for cell differentiation were maintained at 37°C in an atmosphere of 5% CO2, 95% air and the medium was replaced daily.

[0286] On day 7, the cells were dissociated into a single cell suspension using Accutase treatment. Then, cells were gently centrifuged at 800 rpm for 3 min. This single cell suspension was then mixed with 50% Matrigel (Corning Cat# 356237) and 50% EP media [for PFG, advanced DMEM / F12 with 2% B27, 1% N2, 10 mM HEPES, 1% Glutamax, 1% Pen / Strep, 5 ng / mL fibroblast growth factor 2 (FGF2), 10 ng / mL vascular endothelial growth factor (VEGF), 20 ng / mL epidermal growth factor (EGF), 3 pM CHIR99021, 0.5 pM A83-01, and 50 pg / mL ascorbic acid; for AGF, advanced DMEM / F12 with 2% B27, 1% N2, 10 mM HEPES, 1% Glutamax, 1% Pen / Strep, 5 ng / mL fibroblast growth factor 2 (FGF2), 10 ng / mL vascular endothelial growth factor (VEGF), 20 ng / mL epidermal growth factor (EGF), 2 mp CHIR99021, 0.5 mp A83-01, and 50 pg / mL ascorbic acid, 200 ng / ml noggin] for a final concentration of 3.3 x 106cells / mL, and plated as 60 pl drops in a 24-well plate (VWR® Tissue Culture Plate, 24 Wells, Surface Treated, Sterile, 10062-896, VWR International, LLC). These cells were fed with EP media every 48 hrs for 4 days to generate organoids.Human Biliary Organoid Induction

[0287] The HBOs were induced from PFG cell lineages. On day 11 and 12, cells were incubated with RPMI / B27 medium without methionine [RPMI 1640 basal medium deprived of methionine (Gibco™, 12857552, Thermo Fisher) supplemented with 1% L-Glutamine (Gibco™, 11500626, Thermo Fisher), 1% Penicillin / Streptomycin (Gibco™, 11548876, Thermo Fisher) and l *serum-free B27 supplement (Gibco™, 11530536, Thermo Fisher)] supplemented with 30 ng / mL FGF4, 25 ng / mL HGF, 50 ng / mL EGF and ImM retinoic acid.

[0288] On day 13, 3D-cultured cells were collected and centrifuged at 800 rpm for 3 min. Gently aspirate the medium and resuspend the cells in l-5mL of Biliary Differentiation Medium (BDM) [mix 1 : 1 of Phenol red free Williams’E culture medium (Gibco™, 10137414, Thermo Fisher): Ham’s F-12 Nutrient Mix (Gibco™, 15172529, Thermo Fisher), 10'3M linoleic acid- Albumin (L9530, Merck), 2.5* 1 O’6M 3,30,5-Triiodo-L-thyronine sodium salt (T2752, Merck), 0.2UI Insulin (Umuline), 1.25*10'4M human apo-transferrin (3188-AT, R and D), ImM sodium pyruvate (Gibco™, 12539059, Thermo Fisher), 1% L-Glutamine (Gibco™, 11500626, Thermo Fisher) and l% Penicillin / Streptomycin (Gibco™, 11548876, Thermo Fisher)] supplemented with6* 1 O'4M Vitamin C, where the Vitamin C is provided to alleviate oxidant stress. The cholangiocyte organoid suspension was then mixed with 50% Matrigel (Corning Cat# 356237) and 50% BDM.

[0289] On day 14 to 16, refresh the medium daily with BDM supplemented with 50ng / mL human Growth Hormone (GH), 25 ng / mL EGF and 6x 10'4M Vitamin C.

[0290] On day 17 to 21, refresh the medium daily with BDM supplemented with lOng / mL IL-6, and 6* 10'4M Vitamin C.

[0291] On day 22, to further expand 3D-structural cholangiocyte organoid, Matrigel- embedded cholangiocyte organoids were collected and centrifuged at 800 rpm for 3 min. The culture medium was carefully aspirated, and the cells were suspended in an appropriate volume of a freshly prepared 50% (vol / vol) ice cold BDM / Matrigel mix supplemented with 20ng / mL EGF, lOpM Y27632 and 6* 10'4M Vitamin C, and add BDM supplemented with 20ng / mL EGF, lOpM RI and 6* 1 O’4M Vitamin C.

[0292] From day 23 to the end of the differentiation, the medium was replaced every days with fresh BDM supplemented with lOng / mL IL-6, 6* 10'4M Vitamin C and 20ng / mL EGF.Development of tray culture plate

[0293] The tray culture plate was created to bioengineer tubular biliary structures by using a specifically designed microenvironment that forces tubular morphology. The tray culture plates consist of long, deep channels that when seeded with a high density of spheroids will promote tubular fusion. These channels were created using desired dimensions and the tray culture plate was sized to fit within a Falcon 6-well Clear Multiwell Plate (Corning Cat#353046, NY, USA). The channels are 20 mm long with a 2 mm diameter that opens to the surface of the tray culture plate, creating an overall U-shaped channel. A diameter of 2 mm was selected after completing trials using 2, 1.75, 1.5, and 1.25 mm diameters. It was observed that utilizing the smaller diameters led to a lack of consistent tubularization with viable organoids.

[0294] The tray culture plate was designed in SolidWorks 2022 after which a negative mold was created. The mold was created using stereolithography 3D printing with a Form 3 (Formlabs, MA, USA) using Clear V4 Resin (Formlabs, MA, USA). SylgardTM 184 (Electron Microscopy Sciences, PA, USA) was then poured into the mold. After allowing to cure, the tray culture plate was removed from the mold. A 5-0 Vicryl Suture (Ethicon, Cat#J500G, NJ, USA) was pushed through the side of the tray culture plate, run longitudinally though the center of the channel, and exiting at the opposing end of the tray culture plate.Bioengineering of tubular HBQ with tray culture plate

[0295] On day 7, the cells were single cell dissociated with Accutase and centrifuged at 1500 rpm for 3 min. to form a cell pellet. The culture medium was carefully aspirated, and the cell pellet was resuspended using EP media with 10 pM Y-27632, for a final concentration of 2.4* 106cells / well and plate at 2 mL per well in a 24-well plate (AggreWell 400 Microwell Culture Plates, STEMCELL Technologies, BC, Canada). Cells were left undisturbed for 48 hours to generate spheroids.

[0296] On day 9, the spheroids were gently collected and centrifuged at 1500 rpm for 1 min. Culture medium was aspirated and the spheroids were resuspended at 10,000 spheroids per 100 pL of 50% Matrigel and 50% EP media solution. The solution is then seeded into the tray culture plate at 100 pL per channel, ensuring that the suture is fully covered. Finally, the tray culture plate system is fed with EP media at a volume of 4 mL / well. Tray culture plates are then left undisturbed for 48 hours. HBO induction is then continued as described.

[0297] From day 23 of culture, the tubular HBOs reach a level of stiffness where they can be removed from the tray culture plate. At this point, the tubular HBOs can be picked up and manipulated by a pair of tweezers without losing structural integrity. In preparation for transplantation, the stiffness of the tubular HBOs can be further increased by encapsulation in a collagen matrix. To prepare one ml of Collagen solution, components were combined in this order; 600ul of collagen (ADVANCED BIOMATRIX, 5133), lOOul ofDMEM 10X (Gibco, 31600026), 20ul of sodium bicarbonate (NaHCo3, 25080094) to neutralize the pH, 280 ul of distilled water (Gibco, 15230147). The collagen solution was maintained on ice to prevent gelation. Next, the collagen solution was mixed with Matrigel (Corning Cat# 356237) in a ratio of 1 : 1. The final solution of CollagemMatrigel was used to prepare the tubular HBO and plated at 50 pL per channel.

[0298] The tubular HBO was then inserted into the channel and using a pair of tweezers, lightly pushed into the solution. The tubular HBO was then covered with 50 pL of the collagen, Matrigel solution. If completed carefully, the viscous collagen, Matrigel solution will not overflow. The tubular HBOs can then be maintained until transplantation, days 26-31.Characterization of tubular HBO

[0299] After 48 hours in the tray culture plate, tubularization can be visualized. The tubular HBO is then opaque and can be seen condensing inward onto the suture; as this happens, theorganoid begins to move away from the channel walls. One week post seeding in the scaffold tray, a stiff tubular organoid was present.

[0300] From day 23 of culture, analysis of tubular HBO was completed. For whole mount immunostaining with confocal imaging, samples were fixed overnight using 4% paraformaldehyde (PF A, Thermo Fisher Scientific Inc., MA, USA). The samples were then stained for alpha-Smooth muscle actin (aSMA, Abeam, UK), Cluster of Differentiation 68 (CD68, Abeam, UK), Cystic fibrosis transmembrane conductor regulator (CFTR, R&D Systems, MN, USA), Cytokeratin 7 (CK7, Abeam, UK), Epithelial Cellular Adhesion Molecule (EpCAM, R&D Systems, MN, USA), and Platelet-derived growth factor receptor alpha (PDGFRa, Abeam, UK).

[0301] For immunohistochemical analysis, the samples were fixed overnight using 4% PFA. The samples then underwent two 24 hour washes with Gibco DPBS, no calcium, no magnesium (Thermo Fisher Scientific Inc., MA, USA). The samples were then sent to the Integrated Pathology Research Facility (Cincinnati Children’s Hospital Medical Center, OH, USA) for paraffin embedding, sectioning, and antibody staining.Animal surgery procedures

[0302] The mice were optimally anesthetized using isoflurane inhalation, and then were placed on a 37°C thermostatic heating plate. Following the abdominal skin sterilization, the approximately 2-cm transverse skin incision was placed at the upper abdomen and stay suture was further placed to enlarge the surgical field.

[0303] Under the 7.5-16 x microscope, first, common bile duct was exposed doing the caudal movement of the gut and the bile duct ligation was performed using 8-0 Ethilon ® at the level just above the pancreatic duct branch. To mobilize and expose the gallbladder (GB), a round ligament was dissected so that the whole liver moved down; and subsequently, the left and right lobes of the liver were lifted with a moistened and warmed cotton swab so that GB was clearly visible. To fix the GB aiming for the precise anastomosis, the back side of GB was glued onto the duodenum using GLUture ®. Next, the apex of GB was opened and then the collagen-coated tubular HBO was gently inserted into the cavity of GB, and immediately glued GB and organoid as a GB-organoid anastomosis. Further, the 5mm-longitudinal incision was made at the anti mesenteric side of duodenum and organoid was inserted into the lumen of duodenum. Using 8-0 Ethilon ®, the interrupted suture to anastomose organoid and duodenum was performed. The coating GB, organoid, duodenum with GLUture ® and TISEEL ® was finally done.

[0304] Then, warm 0.9% NaCl solution was applied to the peritoneal cavity enough, and the abdominal organs were placed back in their physiological positions. The peritoneum and skin were finally closed using simple continuous suture with 4-0 Vicryl suture. The mice were then placed in a warm incubator. In the control group, the bile duct was separated, and the abdominal cavity was immediately closed.Animals

[0305] All animal experiments were conducted with the approval of the Institutional Review Board (IRB) and Institutional Animal Care and Use Committee (IACUC) of the Cincinnati Children's Hospital Medical Center. Adult II2rg-deficient, Rag 1 -deficient RRG (SD / Crl) rats (breeding pairs, 9-12 weeks old) were housed in standard rat cages with paper bedding and maintained at a temperature of 20-24 °C and relative humidity of 45-55%, under a 12 h: 12 h light:dark cycle. All animals had ad libitum access to dox chow before study. All animals were treated in accordance with the guidelines and regulations of the institution.Quantification and statistical analysis

[0306] Statistical analyses were mainly performed using R software v4.2.0 with unpaired two-tailed Student’s t-test, one-way ANOVA and post hoc Tukey's test, or Welch’s t-test. Statistical analyses for non-normally distributed measurements were performed using nonparametric Kruskal-Wallis and post hoc Dunn-Holland-Wolfe test. For comparisons between unpaired groups, when groups were independent and the variances were unequal, non-parametric Brunner-Munzel test was performed, unless noted otherwise. P values < 0.05 were considered statistically significant, n value refers to biologically independent replicates. The image analyses were non-blinded. Statistical parameters are found in the figures and Figure legends where ns = P > 0.05, * = P < 0.05, ** = p < 0.01, *** = P < 0.001, and **** = P < 0.0001. Using G*Power software, for each experiment, we determined the minimum sample size to collect the data for using the preliminary effect sizes, a = 0.05 and power = 0.8. For each experimental data, a post hoc power analysis was also conducted to determine whether our design had enough power. There was sufficient power (power > 0.8) for all our experiments.EXAMPLE 2Directed differentiation of human iPSCs into human biliary organoids

[0307] The extrahepatic biliary system develops from the caudal liver bud, whereas the intrahepatic system develops from the cranial liver bud during embryogenesis. Both systems areinitially derived from definitive endoderm. Therefore, healthy iPSC lines were utilized to direct differentiation into definitive endoderm (DE). This definitive endoderm was then further directed towards posterior foregut cells by FGF4 and GSK-3 inhibitor CHIR.

[0308] Prior work in creating boundary hepato-biliary-pancreatic organoids has demonstrated that such posterior foregut derived cells exhibited biliary characteristics. As described herein, it was found that these posterior foregut derive cells can eventually develop into extrahepatic biliary system. These posteriorized cells analogous to caudal liver bud were developed into hepatoblasts. As described herein, at the hepatoblast stage, the protocol was switched to primarily utilize biliary differentiation media and ultimately resulted in human biliary organoids (HBO, Fig. 1A). Posterior foregut derived organoids using the HBO protocol in biliary differentiation media exhibited a stochastic tubular assembly (Fig. IB); this is markedly different from established HLOs, which develop with predominantly single lumen morphology.

[0309] To further evaluate the lumenized morphology of the HBOs, a separate experiment was conducted to determine the effects of epidermal growth factor (EGF) and retinoic acid (RA). Two days prior to the hepatoblast stage, differing concentrations of EGF and RA were observed. Based on these findings, studies moved forward with a 100 ng / mL EGF and 1 uM RA addition to the HBO protocol.

[0310] By day 26 of differentiation, foregut derived organoids expressed mature cholangiocyte markers cytokeratin 7 (CK7) and cytokeratin 19 (CK19). along with aquaporin 1 (AQP1), multi-drug resistance-associated protein 3 (MRP 3), and bile salt export pump (BSEP) at a relatively higher rate of expression relative to HLOs. Conversely, HLOs had a relatively higher expression of hepatoblast markers alpha fetoprotein (AFP) and albumin (ALB) in higher expression compared to HBOs (Fig.lC).

[0311] The cholangiocytic nature of HBOs was confirmed on whole mount confocal immunostaining (Fig. ID). HBOs demonstrated complex structures consisting cystic motifs communicating via tubular structures. These were predominantly stained for TROP2, a known intra and extrahepatic cholangiocyte marker. This was not seen as uniformly in HLOs. CK7 was also positive in HBOs but markedly decreased / absent in HLOs, which did show positivity for SOX9, an cholangiocyte and periportal hepatocyte marker. Conversely, HBOs did not stain for ASGR.

[0312] To confirm HBO functionality, it was explored as to whether HBO has a MDR1- dependent secretory function with Rhodamine 123 (Rhol23), a substrate of MDRL As expected,HBOs show a higher capacity of Rho 123 transportation into the luminal space, as compared with HLOs (Figs. IE and IF). On the other hand, the accumulation of Rho 123 was blocked with verapamil, a MDR1 antagonist (Figs. IE and IF). These results demonstrate that posterior foregut cells gain intrahepatic and extrahepatic cholangiocyte character in HBOs.EXAMPLE 3Single cell profiling of multicellular biliary organoids with extrahepatic features

[0313] Having directed differentiation of iPSCs into biliary organoids, the study next sought to characterize the multicellularity of the HBOs using single nuclear RNA sequencing (snRNA-seq). Clustering of the integrated biliary organoids identified distinct subpopulations of hepatic progenitors (20%), mesenchymal cells (25%), stellate cells (9%), endothelial cells (11%), cholangiocytes (28%), and Kupffer cells (6%) (Figs. 2A and 2B). Key markers allowed for segregation of each cluster, including CD45 for lymphocytes, HNF4A for hepatic progenitors, DCN for mesenchyme / fibroblast progenitors, PIN for stellate cells, CALCRL for endothelial cells, CK7, CK19 and EPCAM for cholangiocytes, and FLT1 for Kupffer cells. The cholangiocyte cluster was further subset into distinct regionalized cholangiocytes mainly: intrahepatic bile duct (IHBD)- like, gallbladder (GB)-like, and common bile duct (CBD)-like cholangiocytes. In this biliary organoid system, intrahepatic duct-like cells are characterized by relatively higher average expression of SOX4 and BICCl, whereas gallbladder-like cholangiocytes have a relatively higher expression of CA 4 and SOX17, and CBD-like cholangiocytes had comparatively greater expression of TFF2 and LYZ, and lower expression of CA4 and SOX4 in relation to intrahepatic and gallbladder-like cholangiocytes (Fig. 2C).

[0314] It was noted that cell types segregate into cholangiocytes, and hepatic progenitors, as well as non-parenchymal populations including stellate cells, macrophages and endothelial cells (Fig. 2D) Tissue resident liver macrophages markers are enriched in CD68, CD45 (PTPRC) and CDllb (ITGAM). PECAM1 (CD31) and CDH5 are also enriched in small numbers of cells, signifying the presence of endothelial markers. Mesenchymal markers were also demonstrated, as shown by PDGFRA, VIM, and ACTA2 (a-SMA) (Fig. 3 /

[0315] The human biliary organoid system was then compared to a published single cell RNA sequencing dataset of primary human cholangiocytes from the intrahepatic bile ducts (IHD), common bile duct (CBD), and gallbladder (GB) (Fig. 2D). This multi-source primary human cholangiocyte dataset was integrated with the snRNA sequencing data for the HBO that wasgenerated. The integrated data analysis revealed two distinct cholangiocyte populations within the HBO system (Fig. 2D, inset), namely HBO Cholangiocytes (HBO.C) and HBO Cholangiocytes 2 (HB0.C2), which were very similar to the primary cholangiocyte populations and therefore clustered with these samples. The HBO.C population was enriched for genes expressed relatively more in intrahepatic bile duct (IHD) cholangiocytes - particularly intrahepatic bile duct markers - such as S0X4, JAG I, and HES1. On the other hand, HB0.C2 was somewhat more difficult to distinguish, since it was enriched for genes expressed in both common bile duct (LYZ, TFF2 and SLC28A3) and gallbladder (KRT7 and A BCC 3). although the expression was markedly not significantly increased when compared to that of HBO.C (Fig. 2E).

[0316] Complex regionalized biliary organoids demonstrated extrahepatic features. CK7 was found to be stained at branched outpouching of tubular structures with SOX9, an intrahepatic marker (Fig. 2F), wherein the branched outpouching involves epithelial invagination protruding from the circular shaped HBO. Additionally SOX17 - a marker of gallbladder and extrahepatic biliary epithelial cells - was broadly positive (Fig. 2G). This epithelial lining was surrounded by ZO1, demonstrating continuous tight junctions. A gene-set enrichment analysis revealed that the HBO cholangiocyte sub-population was significantly enriched in expected biological processes, including bile acid metabolism, bile acid and bile salt transport, and bicarbonate transmembrane transporter activity, which are hallmark functions that are generally attributed to cholangiocytes (Fig. 2G). HBOs also demonstrated a-SMA positivity, which is a marker for hepatic stellate cells, with a small presence of PDGFR-0. Further immunostaining demonstrated the non-epithelial lineages as marked by vimentin and a-SMA (Fig. 2H). These results demonstrate that HBOs harbor diverse regionalized cholangiocytes as well as non-epithelial lineages.EXAMPLE 4Development of hypoxia-resilient B2M~ / ~ CIITA ^ human biliary organoids

[0317] After establishing human biliary organoid systems, previously established iPSC lines knocked out for beta-2 microglobulin (B2M) and class II transactivator (CIITA), herein referred to as B2M' ' CIITA- ', were utilized. These cells have no MHC Class I / II expression.

[0318] When comparing B2M' ' CIITA" to B2M+ / +CIITA+ +at day 23 of differentiation, brightfield imaging demonstrates similar morphology (Fig. 4A). Both lines demonstrate CK7 staining, and the B2M' ' CIITA' ' line demonstrates lack of HLA class I expression (Fig. 4B). Brightfield imaging of B2M' ' CIITA' ' double knockout iPSC-derived organoids demonstratedecreased fibrotic changes after exposure to hypoxic conditions for 72 hours when compared to wild-type B2M+ / +CIITA+ +iPSC derived organoids (Fig. 4C, left-most panel). Live-dead analysis demonstrated increased cell death in response to hypoxia in wild-type B2M+ +CIITA+ / +iPSC derived organoids (Fig. 4C, middle and right panels). Although both wild-type and B2M- -CIITA- ' double knockout derived organoids had significantly increased cell death, the change in cell death as marked by bioluminescent intensity units from normoxic conditions to hypoxic conditions in the wild-type group was 9.12, compared to only 0.74 in the double knockout iPSC derived organoids.

[0319] Hypoxia induced inflammation was found to be alleviated. This was confirmed by the differential gene expression of pro- and anti-inflammatory cytokines between wild-type B2M+ / +CIITA+ +iPSC-derived organoids and B2M- CIITA- -double knockout iPSC-derived organoids in hypoxic conditions for double knockout derived organoids and a relatively decreased protein secretion of pro-inflammatory cytokines IL-2 and IFN-gamma (Figs. 4D and 4E).EXAMPLE 5In vivo engraftment and development of B2M" CIITA '' biliary organoids in humanized mouse

[0320] To verify the ability of HBOs to engraft into tissue, HBOs were harvested at day 26-28 and transplanted into the murine subrenal capsule. Before HBO transplant, blood humanization status was confirmed by FACS analysis, confirming 40-70% of CD45 positive cells were originated from human after the humanization (Fig. 5).

[0321] Organoids were harvested 6 weeks after implantation and demonstrated hyperplastic growth, suggestive of engraftment on gross pathology (Fig. 6A). On HE staining, organoids transplanted into humanized NSG-S mice demonstrated engraftment. These engrafted organoids exhibited histological characteristics similar to extrahepatic ductal systems, including hepatic ducts, gallbladder epithelium, and also larger intrahepatic ducts (Fig. 6B).

[0322] On immunohistochemical staining, B2M- -CIITA- -iPSC derived organoids transplanted into humanized NSG-S mice demonstrated engraftment as demonstrated on HE staining and robust CK7 and CK19 staining, which was indicative of mature reconstructed biliary components 6 weeks after transplantation (Fig. 6C). HBO derived from B2M- -CIITA- -also demonstrated Vimentin positivity, indicating a robust mesenchymal population engrafted in the HBO in close proximity to the luminal CK7 and CK19 positive cells. Such a mesenchymal population serves to provide support to the engrafted biliary organoid system. In addition to amesenchymal population, there was also evidence of CD34 / CD68 positivity in B2M / _CIITA' ' HBOs. Wild type B2M+ / +CIITA+ / +iPSC derived organoids also had CK7 and CK19 positivity albeit in a more variable fashion (Fig. 6D). Interestingly, c-Kit, SI 00 and Calretinin were expressed in mesothelial cells around CK7 expressing a bile duct-like structure (Fig. 6E). These results indicate the presence of interstitial cells of Cajal-like cells within the mesenchymal layer of reconstructed bile duct.EXAMPLE 6Bioengineering of luminal tubular biliary system by organoid assembly

[0323] Next, HBOs were utilized to engineer a lumen-containing tubular biliary system. First, day 7 single cell solutions of posterior foregut cells were plated onto AggreWell culture plates and cultured for 48 hrs, resulting in spheroids (Fig.7A, left panel). These were then plated onto custom-designed tray culture plates (Fig. 7A, right panel).

[0324] Additional experiments were performed to determine if spheroid size and seeding density affected tubular assembly (Fig. 8). Using these findings, spheroids were used with a larger area at a seeding density of approximately 10,000 spheroids per channel of the tray culture system.

[0325] After approximately one week of culture (day 16 of total protocol), a stiff tubular organoid was present, as seen by condensation inwards and away from culture plate channel walls (Fig. 7B), wherein stiffness was characterized visually and / or by the organoid being sufficiently mechanically stable to be picked by surgical biceps and tweezer. In comparison with the previously described HBO protocol, bioinspired HBOs presented an increased consistency in large tubular organoid formation. Whole mount staining revealed an intact tubular biliary organoid, approximately 2 cm long (Fig. 7B, right panel and Fig. 7C), as evidenced by immunofluorescent staining for Vimentin, marking mesenchymal cells, and EPC AM, marking biliary epithelium. The multicellular nature of the tubular biliary organoid is further evidenced by presence of CK7 and alpha-SMA (Fig. 7D). The rhodamine uptake assay confirmed luminal uptake - and thereby functionality - of the tubular biliary organoid. The luminal character of the complex tubular organoid was confirmed by colored dye time-lapse (Fig. 7E). Similar to the individual organoid data, this was again dependent on verapamil, indicating a functional MDR2 receptor mechanism active in the tubular biliary organoid (Fig. 7F).EXAMPLE 7Surgical reconstruction of extrahepatic bile duct pathways by engineered biliary organoid graft

[0326] Once the luminal tubular biliary organoid was established, this was utilized to create a cholecystoduodenal interposition graft. A collagen-coated tubular day 25 organoid with internal suture thread was juxtaposed into the surgical field and internal suture was progressively removed from the organoid (Fig. 9A, panels 1-4). The gallbladder was then fixed into position (Fig. 9B, panel 2), followed by surgical glue (GLUture) fixation of the organoid to an intentionally created cholecystostomy (Fig. 9B, panel 3,4). Finally, a 0.5 mm longitudinal duodenostomy was created and this was suture-anastomosed to the organoid (Fig. 9B, panel 5,6 and Fig. 9C).

[0327] Dye injection via the gallbladder was performed in a bile duct ligation (BDL) experiment with no interposition cholecystoduodenal bypass and no dye was seen making it into duodenum (Fig. 9E, left panel). However, in the interposition graft with cholecystoduodenal bypass, dye is visible in the organoid system to demonstrate potency. This was further confirmed by evaluating jaundice in animals undergoing BDL alone compared to those who underwent BDL with cholecystoduodenal bypass. It was found that 80% of animals undergoing BDL alone were jaundiced, whereas 20% undergoing BDL and bypass were jaundiced (Welch’s t-test p=0.03) (Fig. 9F).

[0328] The various methods and techniques described above provide a number of ways to carry out the embodiments disclosed herein. Of course, it is to be understood that not necessarily all objectives or advantages described can be achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods can be performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as taught or suggested herein. A variety of alternatives are mentioned herein. It is to be understood that some preferred embodiments specifically include one, another, or several features, while others specifically exclude one, another, or several features, while still others mitigate a particular feature by inclusion of one, another, or several advantageous features.

[0329] Furthermore, the skilled artisan will recognize the applicability of various features from different embodiments. Similarly, the various elements, features and steps discussed above, as well as other known equivalents for each such element, feature or step, can be employed in various combinations by one of ordinary skill in this art to perform methods in accordance with the principles described herein. Among the various elements, features, and steps some will be specifically included and others specifically excluded in diverse embodiments.

[0330] Although the application has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the embodiments disclosed herein extend beyond the specifically disclosed embodiments to other alternative embodiments and / or uses and modifications and equivalents thereof.

[0331] In some embodiments, the numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the application are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the application are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable.

[0332] In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment of the application (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (for example, “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the application and does not pose a limitation on the scope of the application otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the application.

[0333] Preferred embodiments of this application are described herein. Variations on those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. It is contemplated that skilled artisans can employ such variations as appropriate, and the application can be practiced otherwise than specifically described herein. Accordingly, many embodiments of this application include all modifications and equivalents ofthe subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the application unless otherwise indicated herein or otherwise clearly contradicted by context.

[0334] All patents, patent applications, publications of patent applications, and other material, such as articles, books, specifications, publications, documents, things, and / or the like, referenced herein are hereby incorporated herein by this reference in their entirety for all purposes, excepting any prosecution file history associated with same, any of same that is inconsistent with or in conflict with the present document, or any of same that may have a limiting affect as to the broadest scope of the claims now or later associated with the present document. By way of example, should there be any inconsistency or conflict between the description, definition, and / or the use of a term associated with any of the incorporated material and that associated with the present document, the description, definition, and / or the use of the term in the present document shall prevail.

[0335] In closing, it is to be understood that the embodiments of the application disclosed herein are illustrative of the principles of the embodiments disclosed herein. Other modifications that can be employed can be within the scope of the application. Thus, by way of example, but not of limitation, alternative configurations of the embodiments of the application can be utilized in accordance with the teachings herein. Accordingly, embodiments of the present application are not limited to that precisely as shown and described.Recitation of Embodiments

[0336] Exemplary embodiments of the present disclosure are provided in the following numbered embodiments.Embodiment 1. An in vitro method of producing a human biliary organoid (HBO), the method comprising:(a) culturing biliary progenitor cells derived from posterior foregut cells in biliary differentiation media (BDM);(b) activating an EGF signaling pathway and a JAK-STAT signaling pathway in the cultured cells of step (a);(c) activating a JAK-STAT, Ras-MAPK, LIFR, and / or OSMR pathway in the treated cells of step (b); and(d) activating an EGF signaling pathway and a JAK-STAT, Ras-MAPK, LIFR, and / or OSMR signaling pathway in the treated cells of step (c);to provide a human biliary organoid comprising extrahepatic cholangiocytes.Embodiment 2. The method of embodiment 1, wherein the HBO further comprises intrahepatic cholangiocytes.Embodiment 3. The method of embodiment 1 or embodiment 2, wherein the HBO comprises mature cholangiocyte cells, mesenchymal cells, and / or TROP2-expressing epithelial cells, and / or two or more distinct populations of cholangiocytes.Embodiment 4. The method of any preceding embodiment, wherein the cholangiocytes comprise intrahepatic bile duct (IHBD)-like cholangiocytes, gallbladder (GB)-like cholangiocytes, and common bile duct (CBD)-like cholangiocytes.Embodiment 5. The method of any preceding embodiment, wherein the HBO comprises at least about 10%-50% mesenchymal cells and at least about 10%-50% cholangiocytes; optionally wherein the HBO comprises at least about 15-35% mesenchymal cells and at least about 15%-35% cholangiocytes.Embodiment 6. The method of any preceding embodiment, wherein the HBO further comprises hepatic progenitors, stellate cells, endothelial cells, macrophages, and / or Kupffer cells.Embodiment 7. The method of any preceding embodiment, wherein the HBO further comprises about 5%-60% hepatic progenitors, l%-25% stellate cells, l%-25% endothelial cells, l%-25% macrophages, and / or l%-25% Kupffer cells.Embodiment 8. The method of any preceding embodiment, wherein the HBO comprises smooth muscle cells, peribiliary gland progenitors, glandular elements, tight junctions, and / or complex structures with cystic motifs communicating via a tubular assembly.Embodiment 9. The method of any preceding embodiment, wherein the HBO comprises a TROP2-expressing cell population and / or a CK7-expressing cell population.Embodiment 10. The method of any preceding embodiment, wherein the HBO cholangiocytes are enriched in one or more genes associated with one or more biological processes selected from bile acid metabolism, bile acid transport, bile salt transport, and bicarbonate transmembrane transporter activity.Embodiment 11. The method of any preceding embodiment, wherein the HBO has an elevated expression of one or more mature cholangiocyte marker, and / or reduced expression of one or more hepatoblast markers, relative to a human liver organoid (HLO).Embodiment 12. The method of embodiment 11, wherein the one or more cholangiocyte marker comprises one or more of TROP2, cytokeratin 7 (CK7), cytokeratin 19 (CK19), aquaporin 1(AQPl), multi-drug resistance-associated protein 3 (MRP3), and bile salt export pump (BSEP), and / or wherein the one or more hepatoblast marker comprises one or more of alpha fetoprotein (AFP) and albumin (ALB).Embodiment 13. The method of any preceding embodiment, wherein the HBO expresses CD68, CD45, CDllb, PECAM1, CDH5, PDGFRA, VIM, ACTA2, HNF4A, DCN, PTN, CALCRL, CK7, CK19, EPC AM, S0X17, ZO1, a-SMA, FLTI, and / or vimentin.Embodiment 14. The method of any preceding embodiment, wherein the HBO further comprises a tubular assembly and / or a cystic structure.Embodiment 15. The method of embodiment 14, wherein the HBO comprises a tubular assembly.Embodiment 16. The method of any preceding embodiment, wherein the HBO transports Rhodamine 123 (Rho 123) and / or bile into a luminal space of the HBO, and wherein the capacity of Rho 123 and / or bile transportation into the luminal space is elevated as compared to a human liver organoid (HLO).Embodiment 17. The method of any preceding embodiment, wherein the media in steps (a)-(d) comprises biliary differentiation media.Embodiment 18. The method of any preceding embodiment, wherein the biliary differentiation media in one or more of steps (a)-(d) further comprises Vitamin C; optionally wherein the biliary differentiation media in each of steps (a)-(d) further comprises Vitamin C.Embodiment 19. The method of any preceding embodiment, wherein the EGF signaling pathway in step (b) and / or step (d) is activated by EGF; wherein the JAK-STAT signaling pathway in step (b) is activated by hGH; and / or wherein the JAK-STAT, Ras-MAPK, LIFR, and / or OSMR signaling pathway in step (c) and / or step (d) is activated by IL-6 and / or OSM; optionally wherein the JAK-STAT, Ras-MAPK, LIFR, and / or OSMR signaling pathway in step (c) and / or step (d) is activated by IL-6.Embodiment 20. The method of any preceding embodiment, wherein step (a) occurs for a first time period; step (b) occurs for a second time period; step (c) occurs for a third time period; and step (d) occurs for a fourth time period.Embodiment 21. The method of embodiment 20, wherein the first time period is from at least about 0.5-3 days, the second time period is from at least about 1-6 days, the third time period is from at least about 3-9 days; and the fourth time period is from at least about 1-6 days; optionally wherein the first time period is from at least about 1-2 days, the second time period is from at leastabout 2-4 days, the third time period is from at least about 4-7 days; and the fourth time period is from at least about 2-4 days.Embodiment 22. The method of any preceding embodiment, wherein the cells of step (d) selfassemble into the human biliary organoid.Embodiment 23. The method of any preceding embodiment, wherein the biliary progenitor cells comprise a S0X17 / PDX1 co-expressing cell population.Embodiment 24. The method of any preceding embodiment, wherein the biliary progenitor cells comprise a cell population that is differentiated in vitro from posterior foregut cells to hepatoblasts, and then further differentiated to biliary progenitor cells via exposure to biliary differentiation media (BDM); optionally wherein the BDM further comprises Vitamin C.Embodiment 25. The method of embodiment 24, wherein the exposure to biliary differentiation media (BDM) is for at least about 0.5 days to 5 days, or at least about 1 day to 3 days.Embodiment 26. The method of any preceding embodiment, further comprising differentiating posterior foregut cells into biliary progenitor cells via in vitro expansion.Embodiment 27. The method of embodiment 26, wherein differentiating posterior foregut cells into biliary progenitor cells comprises activating an EGF and an RA signaling pathway in cultured posterior foregut cells prior to differentiation into biliary progenitor cells; optionally wherein the EGF and RA signaling pathway activation is for at least about 1 day to 4 days; optionally wherein the EGF and RA signaling pathway activation is in combination with activation of an FGF signaling pathway and an HGF signaling pathway; optionally wherein the EGF and RA signaling pathway activation is for at least about 1 day to 4 days, and wherein the EGF and RA signaling pathway activation follows at least about 2 days to 6 days of posterior foregut culturing with biliary progenitor differentiation conditions.Embodiment 28. The method of embodiment 27, wherein the biliary progenitor differentiation conditions comprise activating an FGF signaling pathway, and a Wnt signaling pathway; optionally wherein the culture media comprises Vitamin C.Embodiment 29. The method of any one of embodiments 24-28, wherein activating an EGF signaling pathway comprises culturing with EGF, and wherein activating an RA signaling pathway comprises culturing with RA.Embodiment 30. The method of any one of embodiments 24-29, wherein the posterior foregut cells are differentiated by in vitro expansion from definitive endoderm.Embodiment 31 . The method of embodiment 30, wherein the posterior foregut cells are differentiated from definitive endoderm by activation of a FGF pathway and activation of a Wnt pathway.Embodiment 32. The method of any one of embodiments 24-31, wherein the posterior foregut cells are differentiated by in vitro expansion from definitive endoderm derived from pluripotent stem cells; optionally wherein the pluripotent stem cells comprise embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs); optionally wherein the pluripotent stem cells comprise iPSCs.Embodiment 33. The method of any preceding embodiment, wherein the biliary progenitor cells are derived in vitro from induced pluripotent stem cells (iPSCs).Embodiment 34. The method of embodiment 33, wherein the iPSCs are engineered to lack or have reduced MHC class VII expression.Embodiment 35. The method of embodiment 34, wherein the biliary progenitor cells are differentiated from definitive endoderm derived from pluripotent stem cells knocked out for beta- 2 microglobulin (B2M) and class II transactivator (CIITA) (B2M‘ ' CIITAA)Embodiment 36. The method of any preceding embodiment, wherein the biliary progenitor cells are seeded, cultured, and differentiated into the human biliary organoid in a scaffold tray comprising: a culture plate with a horizontal surface; one or more channels attached to or protruding from the horizontal surface, wherein each channel has a vertical depth, longitudinal sidewalls, and open latitudinal ends, and a volume comprising a culture region; and a lengthwise segment in each of the one or more channels, wherein each lengthwise segment runs longitudinally through the respective channel; and wherein the lengthwise segment is removed from the organoid following culturing and differentiation, and wherein the organoid comprises a lumen-containing tubular system. Embodiment 37. The method of embodiment 36, wherein the channels are coated with collagen, to provide a collagen-coated tubular organoid.Embodiment 38. The method of any one of embodiments 36-37, wherein the cells are seeded on the scaffold tray at a cell density of at least about 5,000-50,000, 7,500-20,000, or 8,500-15,000 spheroids per channel, and in a seeding volume of at least about 10-500 pL, 50-2500 pL, or 100-150 pL; optionally at a cell density of at least about 10,000 spheroids per channel and in a seeding area of at least about 100-150 pL.Embodiment 39. The method of any one of embodiments 36-38, wherein the cells are seeded on the scaffold tray at a spheroid size of at least about 1-1000 pm2, 10-500 pm2, or 100-300 pm2; optionally at a spheroid size of at least about 100-300 pm2.Embodiment 40. The method of any one of embodiments 36-39, where the method provides human biliary organoids of consistent size.Embodiment 41. The method of any one of embodiments 36-40, wherein the HBO has a tubular diameter of at least about 0.1-20 mm, 0.5-15 mm, 1-10 mm, 5-10 mm, 6-7 mm, or 1-2 mm, and wherein the HBO has a length of at least about 0.1-100 mm, 0.5-50 mm, 50-90 mm, 70-80 mm, 1- 30 mm, or 10-20 mm; optionally wherein the HBO has a tubular diameter of at least about 1-2 mm and a length of at least about 10-20 mm.Embodiment 42. A human biliary organoid (HBO) comprising extrahepatic cholangiocytes.Embodiment 43. A human biliary organoid (HBO) comprising extrahepatic cholangiocytes, prepared by the method of any of embodiments 1-41.Embodiment 44. The human biliary organoid of any one of embodiments 42 or 43, wherein the HBO is obtained by in vitro differentiation and expansion from definitive endoderm derived from pluripotent stem cells.Embodiment 45. The human biliary organoid of any one of embodiments 42-44, further comprising intrahepatic cholangiocytes.Embodiment 46. The human biliary organoid of any one of embodiments 42-44, wherein the HBO further comprises mature cholangiocyte cells, mesenchymal cells, and / or TROP2-expressing epithelial cells, and / or two or more distinct populations of cholangiocytes.Embodiment 47. The human biliary organoid of any one of embodiments 42-46, wherein the cholangiocytes comprise intrahepatic bile duct (IHBD)-like cholangiocytes, gallbladder (GB)-like cholangiocytes, and common bile duct (CBD)-like cholangiocytes.Embodiment 48. The human biliary organoid of any one of embodiments 42-47, wherein the HBO comprises 10%-50% mesenchymal cells and 10%-50% cholangiocytes; optionally 15-35% mesenchymal cells and 15%-35% cholangiocytes.Embodiment 49. The human biliary organoid of any one of embodiments 42-48, wherein the HBO further comprises hepatic progenitors, stellate cells, endothelial cells, macrophages, and / or Kupffer cells.Embodiment 50. The human biliary organoid of embodiment 42-49, wherein the HBO further comprises 5%-40% hepatic progenitors, l%-25% stellate cells, l%-25% endothelial cells, 1%- 25% macrophages, and / or l%-25% Kupffer cells.Embodiment 51. The human biliary organoid of any one of embodiments 42-50, wherein the HBO comprises smooth muscle cells, peribiliary gland progenitors, glandular elements, tight junctions, and complex structures with cystic motifs communicating via the tubular assembly.Embodiment 52. The human biliary organoid of any one of embodiments 42-51, wherein the HBO comprises a TROP2-expressing cell population and / or a CK7-expressing cell population.Embodiment 53. The human biliary organoid of any one of embodiments 42-52, wherein the HBO cholangiocytes are enriched in genes associate with one or more biological processes selected from bile acid metabolism, bile acid transport, bile salt transport, and bicarbonate transmembrane transporter activity.Embodiment 54. The human biliary organoid of any one of embodiments 42-53, wherein the HBO has an elevated expression of one or more mature cholangiocyte marker, and / or reduced expression of one or more hepatoblast markers, relative to a human liver organoid (HLO).Embodiment 55. The human biliary organoid of embodiment 42-54, wherein the one or more cholangiocyte marker comprise one or more of TROP2, cytokeratin 7 (CK7), cytokeratin 19 (CK19), aquaporin 1 (AQP1), multi-drug resistance-associated protein 3 (MRP3), and bile salt export pump (BSEP),' and / or wherein the one or more hepatoblast marker comprises one or more of alpha fetoprotein (AFP) and albumin (ALB)Embodiment 56. The human biliary organoid of any one of embodiments 42-55, wherein elevated expression of one or more mature cholangiocyte marker, and / or reduced expression of one or more hepatoblast markers, comprises a 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, or greater expression increase or decrease, respectively, relative to a human liver organoid (HLO).Embodiment 57. The human biliary organoid of any one of embodiments 42-56, wherein the HBO expresses CD68, CD45, GDI lb, PECAM1, CDH5, PDGFRA, VIM, ACTA2, HNF4A, DCN, PIN, CALCRL, CK7, CK19, EPCAM, SOX17, ZO1, a-SMA, FLT1, and / or vimentin.Embodiment 58. The human biliary organoid of any one of embodiments 42-57, wherein the HBO comprises intrahepatic bile duct (IHBD)-like cholangiocytes expressing JAG I, HES1, SOX4 and / or BICCP, gallbladder (GB)-like cholangiocytes expressing CA4, SOX17, KRT7, ABCC3,' and common bile duct (CBD)-like cholangiocytes expressing TFF2, LYZ, and / or SLC28A3, and havinglower expression of CA4 and S0X4 in relation to the IHBD-like cholangiocytes and GB-like cholangiocytes.Embodiment 59. The human biliary organoid of any one of embodiments 42-58, wherein the HBO further comprises a tubular assembly and / or a cystic structure.Embodiment 60. The human biliary organoid of embodiment 59, wherein the HBO comprises a tubular assembly.Embodiment 61. The human biliary organoid of any one of embodiments 42-60, wherein the HBO transports Rhodamine 123 (Rho 123) and / or bile into a luminal space of the HBO, and wherein the capacity of Rho 123 and / or bile transportation into the luminal space is elevated as compared to a human liver organoid (HLO).Embodiment 62. The human biliary organoid of any one of embodiments 42-61, wherein the HBO is obtained by in vitro differentiation and expansion of biliary progenitor cells.Embodiment 63. The human biliary organoid of any one of embodiments 42-62, wherein the biliary progenitor cells comprise a SOX17ZPDX1 co-expressing cell population.Embodiment 64. The human biliary organoid of any one of embodiments 42-63, wherein the HBO is hypoimmunogenic.Embodiment 65. The human biliary organoid of embodiment 64, wherein the hypoimmunogenic HBO is engineered to lack or have reduced MHC class I / II expression.Embodiment 66. The human biliary organoid of embodiment 65, wherein the HBO is derived from iPSCs which are knocked out for beta-2 microglobulin (B2M) and class II transactivator (CIITA) (B2M ■ ■ CIITA7).Embodiment 67. The human biliary organoid of embodiment 66, wherein the HBO expresses CD34 and / or CD68.Embodiment 68. An in vitro composition comprising the HBO of any of embodiments 42-67.Embodiment 69. A method of treating a cholangiopathic condition, the method comprising: transplanting, into a subject having a cholangiopathic condition, a human biliary organoid (HBO) according to any one of embodiments 42-67, or the composition of embodiment 68, to provide in vivo engraftment of the human biliary organoid.Embodiment 70. A method of treating a cholangiopathic condition, the method comprising: surgically performing a biliary bypass in a subject having a cholangiopathic condition, by engrafting a human biliary organoid (HBO) according to any one of embodiments 42-67, or the composition of embodiment 68, between the gallbladder and duodenum of the subject, whereinthe bypass reconstructs continuity of an extrahepatic bile duct pathway, restores bile flow, and / or allows for bile drainage.Embodiment 71. The method of embodiment 69, wherein the transplanting comprises: transplanting the HBO at a base of a liver in the subject; and allowing the HBO to engraft.Embodiment 72. The method of embodiment 69 or 70, wherein the method comprises ligating a bile duct, or performing a bile duct graft, in a subject.Embodiment 73. The method of any one of embodiments 69-72, wherein the HBO comprises extrahepatic ductal features; optionally wherein the extrahepatic ductal features comprise one or more of hepatic ducts, gallbladder epithelium, and / or intrahepatic ducts.Embodiment 74. The method of any one of embodiments 69-73, wherein the HBO, following engraftment, is innervated.Embodiment 75. The method of any one of embodiments 69-74, wherein the HBO, following engraftment, comprises a reconstructed bile duct comprising mature reconstructed biliary components and a mesenchymal population, and / or expresses c-Kit, SI 00, calretinin vimentin, CK7, and / or CK19.Embodiment 76. The method of embodiment 75, wherein the mesenchymal population is in close proximity to luminal CK7 and CK19 positive cells.Embodiment 77. The method of any one of embodiments 75-76, wherein the HBO, following engraftment, further comprises interstitial cells of Cajal-like cells within the mesenchyme of the reconstructed bile duct.Embodiment 78. The method of any one of embodiments 69-77, wherein the engraftment reconstructs one or more extrahepatic bile duct pathway, and / or wherein the engrafted HBO resists ischemic damage and maintains ductal integrity.Embodiment 79. The method of any one of embodiments 69-78, wherein the HBO comprises a hypoimmunogenic HBO; optionally wherein the HBO is engineered to lack or have reduced MHC class Eli expression and wherein the subject has hypoxia.Embodiment 80. The method of any one of embodiments 69-79, wherein the disease is a cholangiopathic disease or condition.Embodiment 81. The method of any one of embodiments 69-80, wherein the cholangiopathic disease or condition comprises biliary stricture, hypoxiajaundice, inflammation, bile obstruction, congenital anomaly, ischemic attack, immune-related pathology, transplantation-associatedcomplication, surgical-associated complication, biliary injury and / or trauma, and / or iatrogenic condition; optionally wherein the bile obstruction is due to gallstone; and / or wherein the biliary injury and / or trauma is from intraoperative bile duct injury; optionally wherein the intraoperative bile duct injury occurs during cholecystectomy, hepatic resection, or surgery requiring mobilization or dissection near a biliary tree or bile duct; and / or wherein the iatrogenic condition is drug induced and / or endoscopy induced.Embodiment 82. The method of any one of embodiments 69-81, wherein the cholangiopathic disease comprises a drug-induced cholangiopathy.Embodiment 83. The method of any one of embodiments 69-82, wherein the subject, after engraftment, has resistance to hypoxia and / or reperfusion injury.Embodiment 84. The method of embodiment 83, wherein the subject is a mammal; optionally wherein the subject is a mouse or a human; optionally wherein the subject is a pediatric human subject.Embodiment 85. The method of any one of embodiments 69-84, wherein the HBO is prepared from subject-derived cells; optionally from subject-derived induced pluripotent stem cells (iPSCs). Embodiment 86. A method for screening a compound or composition, wherein the compound or composition to be screened comprises one or more exogenous agent, the method comprising: contacting the HBO of any of embodiments 42-67 with the compound or composition; culturing the HBO with the compound or composition for a period of time; and assessing one or more effects of the compound or composition on the HBO, thereby screening the compound or composition.Embodiment 87. The method of embodiment 86, wherein the assessed effect comprises therapeutic efficacy and / or toxicity of the compound or composition.Embodiment 88. The human biliary organoid of any one of embodiments 42-67, or the composition of embodiment 68, for use in a disease model.Embodiment 89. Use of the human biliary organoid of any one of embodiments 42-67, or the composition of embodiment 68, in a disease model.Embodiment 90. The use of embodiment 89, wherein the disease is a cholangiopathic disease or condition.Embodiment 91. The use of embodiment 90, wherein the cholangiopathic disease or condition comprises biliary stricture, hypoxiajaundice, inflammation, bile obstruction, congenital anomaly,ischemic attack, immune-related pathology, transplantation-associated complication, surgical- associated complication, biliary injury and / or trauma, and / or iatrogenic condition.Embodiment 92. The use of embodiment 90, wherein the cholangiopathic disease comprises a drug-induced cholangiopathy.Embodiment 93. The use of embodiment 90, wherein the disease model is a hypoxia-induced inflammation disease model.Embodiment 94. The use of embodiment 93, wherein the HBO is hypoimmunogenic, and wherein the hypoxia-induced inflammation is reduced.Embodiment 95. A method of forming an organoid comprising a lumen-containing tubular system, the method comprising: seeding progenitor cells in a scaffold tray comprising: a culture plate with a horizontal surface; one or more channels attached to or protruding from the horizontal surface, wherein each channel has a vertical depth, longitudinal sidewalls, and open latitudinal ends, and a volume comprising a culture region; and a lengthwise segment in each of the one or more channels, wherein each lengthwise segment runs longitudinally through the respective channel; culturing and differentiating the seeded progenitor cells in the culture region for a period of time sufficient to develop a tubular system around the lengthwise segment; and removing the lengthwise segment from the organoid following culturing and differentiation, thus providing an organoid comprising a lumen-containing tubular system. Embodiment 96. The method of embodiment 95, wherein the lengthwise segment extends beyond one or more of the open latitudinal ends.Embodiment 97. The method of embodiment 95 or 96, wherein the lengthwise segment comprises a surgical filament.Embodiment 98. The method of any one of embodiments 95-97, wherein each channel has a El- shaped or V-shaped bottom.Embodiment 99. The method of embodiment 95-98, wherein one or more channels are coated with collagen, to provide a collagen-coated tubular organoid.Embodiment 100. A scaffold tray system for producing a tubular organoid, the system comprising: a culture plate with a horizontal surface;one or more channels attached to or protruding from the horizontal surface, wherein each channel has a vertical depth, longitudinal sidewalls, and open latitudinal ends, and wherein each channel contains a volume comprising a culture region; and a lengthwise segment in each of the one or more channels, wherein each lengthwise segment runs longitudinally through the respective channel.Embodiment 101. The scaffold tray system of embodiment 100, wherein the lengthwise segment extends beyond one or more of the open latitudinal ends.Embodiment 102. The scaffold tray system of embodiment 100 or 101, wherein the lengthwise segment comprises a surgical filament.Embodiment 103. The scaffold tray system of any one of embodiments 100-102, wherein each channel has a U-shaped or V-shaped bottom.Embodiment 104. The scaffold tray system of any one of embodiments 100-103, wherein one or more channels are coated with collagen.Embodiment 105. The scaffold tray system of any one of embodiments 100-104, wherein the tray is 3D printed.Embodiment 106. A kit comprising means for preparing a human biliary organoid according to any one of embodiments 42-67, or for performing the method according to any one of embodiments 1-41, 69-87, or 95-99.Embodiment 107. A kit comprising the human biliary organoid according to any one of embodiments 42-67, or the composition of embodiment 68.Embodiment 108. The kit of any of embodiments 106 or 107, wherein one or more of the kit components are provided in separate vials.Embodiment 109. The kit of any of embodiments 106-108, wherein one or more of the kit components are pre-loaded onto one or more assay platform.Embodiment 110. The kit of any of embodiments 106-109, wherein one or more of the kit components are pre-frozen.Embodiment 111. The kit of any of embodiments 106-110, further comprising the scaffold tray of any of embodiments 100-105.Embodiment 112. A biliary implant comprising a lumen-containing tubular structure and extrahepatic cholangiocytes.Embodiment 113. The biliary implant of embodiment 112, further comprising the human biliary organoid (HBO) of any of embodiments 42-67, or the composition of embodiment 68.Embodiment 1 14. The biliary implant of embodiment 1 12 or 113, wherein the HBO and / or extrahepatic cholangiocytes are produced from induced pluripotent stem cells (iPSCs) derived from a subject.Embodiment 115. The biliary implant of embodiment 114, wherein the implant is HLA matched to the subj ect.Embodiment 116. The biliary implant of embodiment 114 or 115, wherein the subject has a cholangiopathic disease or condition.

Claims

CLAIMSWhat is claimed is:

1. An in vitro method of producing a human biliary organoid (HBO), the method comprising:(a) culturing biliary progenitor cells derived from posterior foregut cells in biliary differentiation media (BDM);(b) activating an EGF signaling pathway and a JAK-STAT signaling pathway in the cultured cells of step (a);(c) activating a JAK-STAT, Ras-MAPK, LIFR, and / or OSMR pathway in the treated cells of step (b); and(d) activating an EGF signaling pathway and a JAK-STAT, Ras-MAPK, LIFR, and / or OSMR signaling pathway in the treated cells of step (c); to provide a human biliary organoid comprising extrahepatic cholangiocytes.

2. The method of claim 1, wherein the HBO further comprises intrahepatic cholangiocytes.

3. The method of claim 1 or claim 2, wherein the HBO comprises mature cholangiocyte cells, mesenchymal cells, and / or TROP2-expressing epithelial cells, and / or two or more distinct populations of cholangiocytes.

4. The method of any preceding claim, wherein the cholangiocytes comprise intrahepatic bile duct (IHBD)-like cholangiocytes, gallbladder (GB)-like cholangiocytes, and common bile duct (CBD)-like cholangiocytes.

5. The method of any preceding claim, wherein the HBO comprises at least about 10%-50% mesenchymal cells and at least about 10%-50% cholangiocytes; optionally wherein the HBO comprises at least about 15-35% mesenchymal cells and at least about 15%-35% cholangiocytes.

6. The method of any preceding claim, wherein the HBO further comprises hepatic progenitors, stellate cells, endothelial cells, macrophages, and / or Kupffer cells.

7. The method of any preceding claim, wherein the HBO further comprises about 5%-60% hepatic progenitors, l%-25% stellate cells, l%-25% endothelial cells, l%-25% macrophages, and / or l%-25% Kupffer cells.

8. The method of any preceding claim, wherein the HBO comprises smooth muscle cells, peribiliary gland progenitors, glandular elements, tight junctions, and / or complex structures with cystic motifs communicating via a tubular assembly.

9. The method of any preceding claim, wherein the HBO comprises a TROP2-expressing cell population and / or a CK7-expressing cell population.

10. The method of any preceding claim, wherein the HBO cholangiocytes are enriched in one or more genes associated with one or more biological processes selected from bile acid metabolism, bile acid transport, bile salt transport, and bicarbonate transmembrane transporter activity.

11. The method of any preceding claim, wherein the HBO has an elevated expression of one or more mature cholangiocyte marker, and / or reduced expression of one or more hepatoblast markers, relative to a human liver organoid (HLO).

12. The method of claim 11, wherein the one or more cholangiocyte marker comprises one or more of TROP2, cytokeratin 7 (CK7), cytokeratin 19 (CK19), aquaporin 1 (AQP1), multi-drug resistance-associated protein 3 (MRP 3), and bile salt export pump (BSEP),- and / or wherein the one or more hepatoblast marker comprises one or more of alpha fetoprotein (AFP) and albumin (ALB).

13. The method of any preceding claim, wherein the HBO expresses CD68, CD45, CD lib, PECAM1, CDH5, PDGFRA, VIM, ACTA2, HNF4A, DCN, PIN, CALCRL, CK7, CK19, EPCAM, SOX17, ZO1, a-SMA, FLT1, and / or vimentin.

14. The method of any preceding claim, wherein the HBO further comprises a tubular assembly and / or a cystic structure.

15. The method of claim 14, wherein the HBO comprises a tubular assembly.

16. The method of any preceding claim, wherein the HBO transports Rhodamine 123 (Rho 123) and / or bile into a luminal space of the HBO, and wherein the capacity of Rho 123 and / or bile transportation into the luminal space is elevated as compared to a human liver organoid (HLO).

17. The method of any preceding claim, wherein the media in steps (a)-(d) comprises biliary differentiation media.

18. The method of any preceding claim, wherein the biliary differentiation media in one or more of steps (a)-(d) further comprises Vitamin C; optionally wherein the biliary differentiation media in each of steps (a)-(d) further comprises Vitamin C.

19. The method of any preceding claim, wherein the EGF signaling pathway in step (b) and / or step (d) is activated by EGF; wherein the JAK-STAT signaling pathway in step (b) is activated by hGH; and / or wherein the JAK-STAT, Ras-MAPK, LIFR, and / or OSMR signaling pathway in step (c) and / or step (d) is activated by IL-6 and / or OSM; optionally wherein the JAK-STAT, Ras- MAPK, LIFR, and / or OSMR signaling pathway in step (c) and / or step (d) is activated by IL-6.

20. The method of any preceding claim, wherein step (a) occurs for a first time period; step (b) occurs for a second time period; step (c) occurs for a third time period; and step (d) occurs for a fourth time period.

21. The method of claim 20, wherein the first time period is from at least about 0.5-3 days, the second time period is from at least about 1-6 days, the third time period is from at least about 3-9 days; and the fourth time period is from at least about 1-6 days; optionally wherein the first time period is from at least about 1-2 days, the second time period is from at least about 2-4 days, the third time period is from at least about 4-7 days; and the fourth time period is from at least about 2-4 days.

22. The method of any preceding claim, wherein the cells of step (d) self-assemble into the human biliary organoid.

23. The method of any preceding claim, wherein the biliary progenitor cells comprise a SOX17 / PDX1 co-expressing cell population.

24. The method of any preceding claim, wherein the biliary progenitor cells comprise a cell population that is differentiated in vitro from posterior foregut cells to hepatoblasts, and then further differentiated to biliary progenitor cells via exposure to biliary differentiation media (BDM); optionally wherein the BDM further comprises Vitamin C.

25. The method of claim 24, wherein the exposure to biliary differentiation media (BDM) is for at least about 0.5 days to 5 days, or at least about 1 day to 3 days.

26. The method of any preceding claim, further comprising differentiating posterior foregut cells into biliary progenitor cells via in vitro expansion.

27. The method of claim 26, wherein differentiating posterior foregut cells into biliary progenitor cells comprises activating an EGF and an RA signaling pathway in cultured posterior foregut cells prior to differentiation into biliary progenitor cells; optionally wherein the EGF and RA signaling pathway activation is for at least about 1 day to 4 days; optionally wherein the EGF and RA signaling pathway activation is in combination with activation of an FGF signaling pathway and an HGF signaling pathway; optionally wherein the EGF and RA signaling pathway activation is for at least about 1 day to 4 days, and wherein the EGF and RA signaling pathway activation follows at least about 2 days to 6 days of posterior foregut culturing with biliary progenitor differentiation conditions.

28. The method of claim 27, wherein the biliary progenitor differentiation conditions comprise activating an FGF signaling pathway, and a Wnt signaling pathway; optionally wherein the culture media comprises Vitamin C.

29. The method of any one of claims 24-28, wherein activating an EGF signaling pathway comprises culturing with EGF, and wherein activating an RA signaling pathway comprises culturing with RA.

30. The method of any one of claims 24-29, wherein the posterior foregut cells are differentiated by in vitro expansion from definitive endoderm.

31. The method of claim 30, wherein the posterior foregut cells are differentiated from definitive endoderm by activation of a FGF pathway and activation of a Wnt pathway.

32. The method of any one of claims 24-31, wherein the posterior foregut cells are differentiated by in vitro expansion from definitive endoderm derived from pluripotent stem cells; optionally wherein the pluripotent stem cells comprise embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs); optionally wherein the pluripotent stem cells comprise iPSCs.

33. The method of any preceding claim, wherein the biliary progenitor cells are derived in vitro from induced pluripotent stem cells (iPSCs).

34. The method of claim 33, wherein the iPSCs are engineered to lack or have reduced MHC class VII expression.

35. The method of claim 34, wherein the biliary progenitor cells are differentiated from definitive endoderm derived from pluripotent stem cells knocked out for beta-2 microglobulin (B2M) and class II transactivator (CIITA) (B2M- ' CIITA").

36. The method of any preceding claim, wherein the biliary progenitor cells are seeded, cultured, and differentiated into the human biliary organoid in a scaffold tray comprising: a culture plate with a horizontal surface; one or more channels attached to or protruding from the horizontal surface, wherein each channel has a vertical depth, longitudinal sidewalls, and open latitudinal ends, and a volume comprising a culture region; and a lengthwise segment in each of the one or more channels, wherein each lengthwise segment runs longitudinally through the respective channel; and wherein the lengthwise segment is removed from the organoid following culturing and differentiation, and wherein the organoid comprises a lumen-containing tubular system.

37. The method of claim 36, wherein the channels are coated with collagen, to provide a collagen-coated tubular organoid.

38. The method of any one of claims 36-37, wherein the cells are seeded on the scaffold tray at a cell density of at least about 5,000-50,000, 7,500-20,000, or 8,500-15,000 spheroids per channel, and in a seeding volume of at least about 10-500 pL, 50-2500 pL, or 100-150 pL; optionally at a cell density of at least about 10,000 spheroids per channel and in a seeding area of at least about 100-150 pL.

39. The method of any one of claims 36-38, wherein the cells are seeded on the scaffold tray at a spheroid size of at least about 1-1000 pm2, 10-500 pm2, or 100-300 pm2; optionally at a spheroid size of at least about 100-300 pm2.

40. The method of any one of claims 36-39, where the method provides human biliary organoids of consistent size.

41. The method of any one of claims 36-40, wherein the HBO has a tubular diameter of at least about 0.1-20 mm, 0.5-15 mm, 1-10 mm, 5-10 mm, 6-7 mm, or 1-2 mm, and wherein the HBO has a length of at least about 0.1-100 mm, 0.5-50 mm, 50-90 mm, 70-80 mm, 1-30 mm, or 10-20 mm; optionally wherein the HBO has a tubular diameter of at least about 1-2 mm and a length of at least about 10-20 mm.

42. A human biliary organoid (HBO) comprising extrahepatic cholangiocytes.

43. A human biliary organoid (HBO) comprising extrahepatic cholangiocytes, prepared by the method of any of claims 1-41.

44. The human biliary organoid of any one of claims 42 or 43, wherein the HBO is obtained by in vitro differentiation and expansion from definitive endoderm derived from pluripotent stem cells.

45. The human biliary organoid of any one of claims 42-44, further comprising intrahepatic cholangiocytes.

46. The human biliary organoid of any one of claims 42-44, wherein the HBO further comprises mature cholangiocyte cells, mesenchymal cells, and / or TROP2-expressing epithelial cells, and / or two or more distinct populations of cholangiocytes.

47. The human biliary organoid of any one of claims 42-46, wherein the cholangiocytes comprise intrahepatic bile duct (IHBD)-like cholangiocytes, gallbladder (GB)-like cholangiocytes, and common bile duct (CBD)-like cholangiocytes.

48. The human biliary organoid of any one of claims 42-47, wherein the HBO comprises 10%- 50% mesenchymal cells and 10%-50% cholangiocytes; optionally 15-35% mesenchymal cells and 15%-35% cholangiocytes.

49. The human biliary organoid of any one of claims 42-48, wherein the HBO further comprises hepatic progenitors, stellate cells, endothelial cells, macrophages, and / or Kupffer cells.

50. The human biliary organoid of claim 42-49, wherein the HBO further comprises 5%-40% hepatic progenitors, l%-25% stellate cells, l%-25% endothelial cells, l%-25% macrophages, and / or l%-25% Kupffer cells.

51. The human biliary organoid of any one of claims 42-50, wherein the HBO comprises smooth muscle cells, peribiliary gland progenitors, glandular elements, tight junctions, and complex structures with cystic motifs communicating via the tubular assembly.

52. The human biliary organoid of any one of claims 42-51, wherein the HBO comprises a TROP2-expressing cell population and / or a CK7-expressing cell population.

53. The human biliary organoid of any one of claims 42-52, wherein the HBO cholangiocytes are enriched in genes associate with one or more biological processes selected from bile acidmetabolism, bile acid transport, bile salt transport, and bicarbonate transmembrane transporter activity.

54. The human biliary organoid of any one of claims 42-53, wherein the HBO has an elevated expression of one or more mature cholangiocyte marker, and / or reduced expression of one or more hepatoblast markers, relative to a human liver organoid (HLO).

55. The human biliary organoid of claim 42-54, wherein the one or more cholangiocyte marker comprise one or more of TROP2, cytokeratin 7 (CK7), cytokeratin 19 (CK19), aquaporin 1 (AQP1), multi-drug resistance-associated protein 3 (MRP3), and bile salt export pump (BSEP), and / or wherein the one or more hepatoblast marker comprises one or more of alpha fetoprotein (AFP) and albumin (ALB).

56. The human biliary organoid of any one of claims 42-55, wherein elevated expression of one or more mature cholangiocyte marker, and / or reduced expression of one or more hepatoblast markers, comprises a 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, or greater expression increase or decrease, respectively, relative to a human liver organoid (HLO).

57. The human biliary organoid of any one of claims 42-56, wherein the HBO expresses CD68, CD45, GDI lb, PECAM1, CDH5, PDGFRA, VIM, ACTA2, HNF4A, DCN, PTN, CALCRL, CK7, CK19, EPCAM, S0X17, ZO1, a-SMA, FLT1, and / or vimentin.

58. The human biliary organoid of any one of claims 42-57, wherein the HBO comprises intrahepatic bile duct (IHBD)-like cholangiocytes expressing JAG1, HES1, S0X4 and / or BICC1, gallbladder (GB)-like cholangiocytes expressing CA4, S0X17, KRT7, ABCC3,' and common bile duct (CBD)-like cholangiocytes expressing TFF2, LYZ, and / or SLC28A3, and having lower expression of CA4 and S0X4 in relation to the IHBD-like cholangiocytes and GB-like cholangiocytes.

59. The human biliary organoid of any one of claims 42-58, wherein the HBO further comprises a tubular assembly and / or a cystic structure.

60. The human biliary organoid of claim 59, wherein the HBO comprises a tubular assembly.

61. The human biliary organoid of any one of claims 42-60, wherein the HBO transports Rhodamine 123 (Rho 123) and / or bile into a luminal space of the HBO, and wherein the capacity of Rho 123 and / or bile transportation into the luminal space is elevated as compared to a human liver organoid (HLO).

62. The human biliary organoid of any one of claims 42-61, wherein the HBO is obtained by in vitro differentiation and expansion of biliary progenitor cells.

63. The human biliary organoid of any one of claims 42-62, wherein the biliary progenitor cells comprise a SOX17 / PDX1 co-expressing cell population.

64. The human biliary organoid of any one of claims 42-63, wherein the HBO is hypoimmunogenic.

65. The human biliary organoid of claim 64, wherein the hypoimmunogenic HBO is engineered to lack or have reduced MHC class I / II expression.

66. The human biliary organoid of claim 65, wherein the HBO is derived from iPSCs which are knocked out for beta-2 microglobulin (B2M) and class II transactivator (CIITA) (B2M- ' CIITA" ).

67. The human biliary organoid of claim 66, wherein the HBO expresses CD34 and / or CD68.

68. An in vitro composition comprising the HBO of any of claims 42-67.

69. A method of treating a cholangiopathic condition, the method comprising: transplanting, into a subject having a cholangiopathic condition, a human biliary organoid (HBO) according to any one of claims 42-67, or the composition of claim 68, to provide in vivo engraftment of the human biliary organoid.

70. A method of treating a cholangiopathic condition, the method comprising: surgically performing a biliary bypass in a subject having a cholangiopathic condition, by engrafting a human biliary organoid (HBO) according to any one of claims 42-67, or the composition of claim 68, between the gallbladder and duodenum of the subject, wherein the bypass reconstructs continuity of an extrahepatic bile duct pathway, restores bile flow, and / or allows for bile drainage.

71. The method of claim 69, wherein the transplanting comprises: transplanting the HBO at a base of a liver in the subject; and allowing the HBO to engraft.

72. The method of claim 69 or 70, wherein the method comprises ligating a bile duct, or performing a bile duct graft, in a subject.

73. The method of any one of claims 69-72, wherein the HBO comprises extrahepatic ductal features; optionally wherein the extrahepatic ductal features comprise one or more of hepatic ducts, gallbladder epithelium, and / or intrahepatic ducts.

74. The method of any one of claims 69-73, wherein the HBO, following engraftment, is innervated.

75. The method of any one of claims 69-74, wherein the HBO, following engraftment, comprises a reconstructed bile duct comprising mature reconstructed biliary components and a mesenchymal population, and / or expresses c-Kit, S100, calretinin vimentin, CK7, and / or CK19.

76. The method of claim 75, wherein the mesenchymal population is in close proximity to luminal CK7 and CK19 positive cells.

77. The method of any one of claims 75-76, wherein the HBO, following engraftment, further comprises interstitial cells of Cajal -like cells within the mesenchyme of the reconstructed bile duct.

78. The method of any one of claims 69-77, wherein the engraftment reconstructs one or more extrahepatic bile duct pathway, and / or wherein the engrafted HBO resists ischemic damage and maintains ductal integrity.

79. The method of any one of claims 69-78, wherein the HBO comprises a hypoimmunogenic HBO; optionally wherein the HBO is engineered to lack or have reduced MHC class I / II expression and wherein the subject has hypoxia.

80. The method of any one of claims 69-79, wherein the disease is a cholangiopathic disease or condition.

81. The method of any one of claims 69-80, wherein the cholangiopathic disease or condition comprises biliary stricture, hypoxia, jaundice, inflammation, bile obstruction, congenital anomaly, ischemic attack, immune-related pathology, transplantation-associated complication, surgical- associated complication, biliary injury and / or trauma, and / or iatrogenic condition; optionally wherein the bile obstruction is due to gallstone; and / or wherein the biliary injury and / or trauma is from intraoperative bile duct injury; optionally wherein the intraoperative bile duct injury occurs during cholecystectomy, hepatic resection, or surgery requiring mobilization or dissection near a biliary tree or bile duct; and / or wherein the iatrogenic condition is drug induced and / or endoscopy induced.

82. The method of any one of claims 69-81, wherein the cholangiopathic disease comprises a drug-induced cholangiopathy.

83. The method of any one of claims 69-82, wherein the subject, after engraftment, has resistance to hypoxia and / or reperfusion injury.

84. The method of claim 83, wherein the subject is a mammal; optionally wherein the subject is a mouse or a human; optionally wherein the subject is a pediatric human subject.

85. The method of any one of claims 69-84, wherein the HBO is prepared from subject-derived cells; optionally from subject-derived induced pluripotent stem cells (iPSCs).

86. A method for screening a compound or composition, wherein the compound or composition to be screened comprises one or more exogenous agent, the method comprising: contacting the HBO of any of claims 42-67 with the compound or composition; culturing the HBO with the compound or composition for a period of time; and assessing one or more effects of the compound or composition on the HBO, thereby screening the compound or composition.

87. The method of claim 86, wherein the assessed effect comprises therapeutic efficacy and / or toxicity of the compound or composition.

88. The human biliary organoid of any one of claims 42-67, or the composition of claim 68, for use in a disease model.

89. Use of the human biliary organoid of any one of claims 42-67, or the composition of claim 68, in a disease model.

90. The use of claim 89, wherein the disease is a cholangiopathic disease or condition.

91. The use of claim 90, wherein the cholangiopathic disease or condition comprises biliary stricture, hypoxiajaundice, inflammation, bile obstruction, congenital anomaly, ischemic attack, immune-related pathology, transplantation-associated complication, surgical-associated complication, biliary injury and / or trauma, and / or iatrogenic condition.

92. The use of claim 90, wherein the cholangiopathic disease comprises a drug-induced cholangiopathy.

93. The use of claim 90, wherein the disease model is a hypoxia-induced inflammation disease model.

94. The use of claim 93, wherein the HBO is hypoimmunogenic, and wherein the hypoxia- induced inflammation is reduced.

95. A method of forming an organoid comprising a lumen-containing tubular system, the method comprising: seeding progenitor cells in a scaffold tray comprising: a culture plate with a horizontal surface; one or more channels attached to or protruding from the horizontal surface, wherein each channel has a vertical depth, longitudinal sidewalls, and open latitudinal ends, and a volume comprising a culture region; and a lengthwise segment in each of the one or more channels, wherein each lengthwise segment runs longitudinally through the respective channel; culturing and differentiating the seeded progenitor cells in the culture region for a period of time sufficient to develop a tubular system around the lengthwise segment; and removing the lengthwise segment from the organoid following culturing and differentiation, thus providing an organoid comprising a lumen-containing tubular system.

96. The method of claim 95, wherein the lengthwise segment extends beyond one or more of the open latitudinal ends.

97. The method of claim 95 or 96, wherein the lengthwise segment comprises a surgical filament.

98. The method of any one of claims 95-97, wherein each channel has a U-shaped or V-shaped bottom.

99. The method of claim 95-98, wherein one or more channels are coated with collagen, to provide a collagen-coated tubular organoid.

100. A scaffold tray system for producing a tubular organoid, the system comprising: a culture plate with a horizontal surface; one or more channels attached to or protruding from the horizontal surface, wherein each channel has a vertical depth, longitudinal sidewalls, and open latitudinal ends, and wherein each channel contains a volume comprising a culture region; and- I l l -a lengthwise segment in each of the one or more channels, wherein each lengthwise segment runs longitudinally through the respective channel.

101. The scaffold tray system of claim 100, wherein the lengthwise segment extends beyond one or more of the open latitudinal ends.

102. The scaffold tray system of claim 100 or 101, wherein the lengthwise segment comprises a surgical filament.

103. The scaffold tray system of any one of claims 100-102, wherein each channel has a U- shaped or V-shaped bottom.

104. The scaffold tray system of any one of claims 100-103, wherein one or more channels are coated with collagen.

105. The scaffold tray system of any one of claims 100-104, wherein the tray is 3D printed.

106. A kit comprising means for preparing a human biliary organoid according to any one of claims 42-67, or for performing the method according to any one of claims 1-41, 69-87, or 95-99.

107. A kit comprising the human biliary organoid according to any one of claims 42-67, or the composition of claim 68.

108. The kit of any of claims 106 or 107, wherein one or more of the kit components are provided in separate vials.

109. The kit of any of claims 106-108, wherein one or more of the kit components are pre- loaded onto one or more assay platform.

110. The kit of any of claims 106-109, wherein one or more of the kit components are prefrozen.

111. The kit of any of claims 106-110, further comprising the scaffold tray of any of claims 100- 105.

112. A biliary implant comprising a lumen-containing tubular structure and extrahepatic cholangiocytes.

113. The biliary implant of claim 112, further comprising the human biliary organoid (HBO) of any of claims 42-67, or the composition of claim 68.

114. The biliary implant of claim 112 or 113, wherein the HBO and / or extrahepatic cholangiocytes are produced from induced pluripotent stem cells (iPSCs) derived from a subject.

115. The biliary implant of claim 114, wherein the implant is HLA matched to the subject.

116. The biliary implant of claim 114 or 115, wherein the subject has a cholangiopathic disease or condition.

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