Transplantation of patient‐specific bile duct bioengineered with chemically reprogrammed and microtopographically differentiated cells

Abstract Cholangiopathy is a diverse spectrum of chronic progressive bile duct disorders with limited treatment options and dismal outcomes. Scaffold‐ and stem cell‐based tissue engineering technologies hold great promise for reconstructive surgery and tissue repair. Here, we report a combined application of 3D scaffold fabrication and reprogramming of patient‐specific human hepatocytes to produce implantable artificial tissues that imitate the mechanical and biological properties of native bile ducts. The human chemically derived hepatic progenitor cells (hCdHs) were generated using two small molecules A83‐01 and CHIR99021 and seeded inside the tubular scaffold engineered as a synergistic combination of two layers. The inner electrospun fibrous layer was made of nanoscale–macroscale polycaprolactone fibers acting to promote the hCdHs attachment and differentiation, while the outer microporous foam layer served to increase mechanical stability. The two layers of fiber and foam were fused robustly together thus creating coordinated mechanical flexibility to exclude any possible breaking during surgery. The gene expression profiling and histochemical assessment confirmed that hCdHs acquired the biliary epithelial phenotype and filled the entire surface of the fibrous matrix after 2 weeks of growth in the cholangiocyte differentiation medium in vitro. The fabricated construct replaced the macroscopic part of the common bile duct (CBD) and re‐stored the bile flow in a rabbit model of acute CBD injury. Animals that received the acellular scaffolds did not survive after the replacement surgery. Thus, the artificial bile duct constructs populated with patient‐specific hepatic progenitor cells could provide a scalable and compatible platform for treating bile duct diseases.

possible breaking during surgery. The gene expression profiling and histochemical assessment confirmed that hCdHs acquired the biliary epithelial phenotype and filled the entire surface of the fibrous matrix after 2 weeks of growth in the cholangiocyte differentiation medium in vitro. The fabricated construct replaced the macroscopic part of the common bile duct (CBD) and re-stored the bile flow in a rabbit model of acute CBD injury.
Animals that received the acellular scaffolds did not survive after the replacement surgery.
Thus, the artificial bile duct constructs populated with patient-specific hepatic progenitor cells could provide a scalable and compatible platform for treating bile duct diseases. Among the breakthrough applications in the management of the common bile duct (CBD) disorders is the generation of bioengineered bile ducts. [3][4][5][6][7] More recent techniques allow for the production of tridimensional (3D) tubular scaffolds in combination with cells and biologically active molecules to form functional bile duct structures. 2,8 A variety of techniques, biomaterials, and cell types, including primary cholangiocytes 5 and stem cells, 6 have been adopted to create 3D scaffolds promoting cell adhesion and proliferation, and possessing mechanical strength sufficient for transplantation. [9][10][11] However, the tridimensionality in most of the previous works still stayed at the level of simple cylindrical shape. The 3D scaffolds would need a further development toward 3D customization with arbitrary shapes for patient-specific clinical needs.
Here, we introduce a novel combinatorial bioengineering approach to build a new type of artificial bile duct (ABD) for medical applications.
For the 3D customizable shaping, the fabrication method is utilizing our recently published technique for scaffold fabrication using medical image-based 3D printing of a sacrificial template and dip coating of biomaterials. 12 To improve cell-to-matrix topographic cues and increase the mechanical stability, the tubular scaffold was formed as a bilayer construct of a microscale fibrous mat surrounded by a microporous foam layer that reinforced the structure while providing mechanical flexibility. For epithelization of the artificial tubular scaffold, we utilized human chemically derived hepatic progenitor cells (hCdHs) as a compelling cell source of patient-specific stem cells amenable to self-renewal and differentiation toward biliary epithelial lineage. 13 2 | RESULTS AND DISCUSSION

| Fabrication of 3D customized bilayer tubular scaffold
To create a personalized rabbit-size living construct resembling the native CBD, we utilized our recently published approach based on 3D design, 3D printing, postprocessing, and animal experimentation. 12 The fabrication of 3D customized tubular scaffold involved computer-aided design (CAD) based on the magnetic resonance imaging data. From the 2D magnetic resonance cholangiopancreatography images, the target object in biliary tree was three-dimensionally reconstructed and postprocessed as a 3D printable file format (STL) (Figure 1a). According to the 3D model data, a sacrificial template was then fabricated using a water-soluble polymer polyvinylalcohol (PVA) in accordance with a 3D printing method of material extrusion (Figure 1b). To provide a low-stiffness microenvironment supporting cell adhesion and proliferation, a hybrid bilayer film was formed on the surface of the PVA template replacing a simple monolayer used in our earlier publication. 12 To create a fibrous topography of the inner surface of the first layer, electrospinning was performed on the rotating template thereby depositing the as-spun fibrous mat on the circumference of the template (Figure 1c). For the second layer, the fiber-deposited template was dip-coated in salt-suspended polyurethane solution followed by leaching salt particles to form a porous layer around the fiber template ( Figure 1d). The final bilayer tubular scaffold retaining fibrous morphology at the surface of the innermost layer was obtained after removing the core template by dissolving in distilled water.
The correct shape of the extracellular biliary tree was obtained by processing 3D CAD data from medical imaging of rabbit body. The final 3D bilayer tubular scaffold contained a whole anatomical structure composed of CBD and two bifurcated intrahepatic ducts

| Generation of hepatic progenitors and differentiation toward cholangiocytes
To meet high demand of functional biliary epithelial cells for populating ABD constructs for medical applications, we utilized our recently developed methodology designed to obtain a sizable population of hepatic progenitor cells (hCdHs) with high efficiency, purity, and experimental ease-of-use. 14 The hCdHs have documented potential for self-renewal and multi-lineage differentiation and can be rapidly established from the patient-specific human liver biopsies providing an efficient cell-based tool for bioengineered practices.
To generate hCdHs, adult human hepatocytes were isolated from healthy donor liver tissue (Table S1) and directly reprogrammed into bipotent progenitor cells by a combined treatment with two small molecules A83-01 and CHIR99021 in the presence of hepatocyte growth factor (HGF). 14 When hCdHs reached a passage 2, they were subjected to a cholangiocyte differentiation protocol 7 as outlined in a schematic overview ( Figure 3a

| Optimization of fibrous scaffold morphology for enhanced cholangiocyte differentiation
In the field of tissue engineering, the electrospun mats typically act as extracellular-mimicking matrices providing topographic guidance for the seeded cells. 16 in regulating cellular behavior and nanoscale organization of focal adhesion structures, 18 many topographical features of the early scaffolds have been designed using nanoscale configuration. 19,20 However, the slow perfusion mass transport and limited ability to re-create the complexity and hierarchy of the extracellular matrix structures characteristic of native tissues deter the use of nanoscale-engineered F I G U R E 3 Legend on next page. approaches for clinical applications. [21][22][23][24][25] To address these issues, we determined the impact of electrospun sheet scaffold on differentiation properties of hCdH-Chols. For this purpose, hCdHs were resuspended with the same density (10 6 cells/cm 2 ) in reprogramming medium 14 and plated either onto a standard 2D dish or electrospun fiber scaffold.
After 2 days, the medium was replaced to CDM to induce cholangiocyte differentiation for 14 days ( Figure S2a). The results of live/ dead assay showed that the differences in growth conditions did not affect cell viability. At the end point of experiment, hCdH-Chols maintained on the electrospun fiber scaffold exhibited a comparable or slightly higher viability than cells cultured on flat dishes ( Figure S2b).
However, the fiber sheet scaffold provided a more favorable microenvironment for biliary cell differentiation and caused a stronger expression of key marker genes SSRT2, SCR, JAG1, TGR5, CK7, AE2, CFTR, CK19, and AQP1 ( Figure S2c). Consistent with this, the growth rate and stemness features of hCdH-Chols cultured on fibrous scaffold were markedly reduced as judged by RT-qPCR analysis of proliferation (Ki67) and progenitor (CD90, EPCAM) marker gene expression ( Figure S2c).
To extend these data, we then evaluated the effects of fiber sheet architecture on the functional properties of hCdH-Chols. Given that during typical electrospinning process, the concentration and resulting surface tension of polymer solution define the distribution range of fiber diameters, 16

| Epithelization of ABD with hCdH-Chols in vitro
To test whether hCdHs can engraft and survive inside the ABD structures composed of the inner electrospun fibrous layer and surrounded by microporous foam layer for increased stability (Figure 1d

| In vivo application
Based on the promising results in vitro, we then explored the feasibility of bioengineered bile ducts to restore a CBD defect using rabbit  During the postimplantation period, most of the serum parameters of liver function remained within the reference range (Table S2 and Figure S4c). A slight increase of biliary enzymes GGT, ALT, and AST observed at 2 weeks post-ABD implantation returned to the basal levels by the 42nd day. The total bilirubin was similarly reduced to the preimplantation level indicating that there was no obvious damage or toxicity in the native CBD.
During autopsy performed at 2 weeks after surgery, the graft retained its place and appeared as an integral part of the CBD (Figure 7a  The subsequent histological evaluation of the cranial and caudal part of the anastomosis sites confirmed that the transplanted ABD construct was well-connected to a native rabbit CBD ( Figure S4a). Double immunofluorescence staining showed that mCherry-tagged hCdH-Chols populating the tubular scaffold expressed lineage-specific marker proteins CK19 and CK7 (Figure 7f; Figure S4b).
To further illustrate a successful integration of ABD with the native bile duct (NBD), a large segment of liver-duodenum-was isolated at 42 days after implantation and sectioned for histological and immunofluorescent evaluation. The H&E staining confirmed that the anastomosis site was free of biliary stricture thereby allowing an open bile passage into intestine (Figure 7e; Movie S5). We then costained the resected specimen with SOX9 and CK19, two classical markers of biliary differentiation. As expected, only the ABD implant displayed a strong immunoreactivity with a human-specific SOX9 antibody ( Figure S4d) clearly demarcating the end-to-end anastomosis from the NBD and revealing the continuity of biliary lining throughout the reconstructed bile duct (Figure 7f). Together, these results establish that our patient-specific bile duct bioengineered with chemically reprogrammed and microtopographically differentiated cells can successfully restore the large-scale defect in the rabbit CBD and thus represent an exciting novel candidate for clinical development.

| CONCLUSIONS
The overall objective of this study was to fabricate and evaluate an implantable bioengineered bile duct construct for medical applications. To test this objective, we (i) optimized our published strategy for scaffold fabrication based on 3D design, 3D printing, and postprocessing; (ii) demonstrated the utility of our hCdHs as a compelling source of patient-specific progenitor cells suitable for epithelization of the ABD structures; (iii) defined the best-fit scaffold architecture for engraftment, proliferation, and differentiation of hCdHs toward biliary lineage; and (iv) assessed the feasibility of fabricated constructs for replacement therapy in animal studies.
To achieve optimal microtopographic characteristics, the tubular scaffolds were engineered as a synergistic combination of two layers.
The inner electrospun fibrous layer was surrounded by outer microporous foam layer that increased the tensile elongation at the break points about 500% reinforcing mechanical durability. Furthermore, we used highly viscous PCL solution to yield fibers with a wide spectrum of diameters ranging from 500 nm to several micrometers. The multiscale architecture of coarse fiber scaffold increased the porosity and permeability thereby improving mass transport. It also offered a more instructive 3D microenvironment that was essential for enhancing The detailed information is included in the previously published paper. 13 For the next step, the electrospinning of polycaprolactone (PCL; MW 80,000; Sigma-Aldrich) solution was performed while rotating the 3D-printed PVA template. The PCL granules were dissolved in a solvent mixture of methylene chloride and dimethyl formaldehyde (DMF) at a ratio of 3:1. The solution concentrations were 10% and 18% (w/v) for generating the fine and coarse electrospun mats, respectively.
In advance of template coating, the two different mats were compared in terms of functionality to induce the cholangiocytes differentiation.
After the comparative experiment, the template coating condition was selected as the concentration of 18% (w/v). For the dip coating step, the fiber-coated template was dipped into a salt-dispersed thermoplastic polyurethane (TPU; DAELIM chemical) solution. The TPU granules were dissolved in DMF at a concentration of 15% (w/v). Thereafter, salt particles sieved with a 45-μm mesh were mixed with the as-prepared TPU solution at a concentration of 400% (w/w). After drying the solvent on the coated part, it was immersed in sonicated water for 2 min to leach out the salt particles. Finally, the core PVA template was removed by immersing in a sonicated water at 50 C for 30 min, thereby obtaining a 3D bilayer tubular construct. To generate entrances for the water penetration, the small parts were cut at the end of the coated template.

| Generation of hCdHs and cholangiocyte differentiation
The study was performed according to protocols approved by the Institutional Review Board of Hanyang University, Seoul, Korea (HYI-16-229-3). Human liver tissues were obtained from three donors operated on in Hanyang University Medical Center (Table S1) with the informed patients' consent. Generation of hCdHs was done following a previously published method. 14 In brief, the primary human hepatocytes were isolated using a two-

| Cholangiocyte differentiation on fiber scaffolds and ABDs
Before cell seeding, scaffolds and bile ducts were cut, washed with ethanol, and sterilized under UV light. After 30 min, the scaffolds and bile duct fragments were coated with 0.1% gelatin (Sigma) and incubated for 30 min at 37 C and 5% CO 2 . Then, excess gelatin was removed and samples were dried out at room temperature for 2 h. 10 6 cells were resuspended in 10 μl of HAC media and seeded inside the ABD constructs. They were then incubated for an hour at 37 C and 5% CO 2 . Half milliliter of reprogramming media was used per well in a four-well plate and replaced with CDM media next day. CDM medium was replaced every 2 days for 14 days.