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

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.

Association of Microbial Dysbiosis with Gallbladder Diseases Identified by Bile Microbiome Profiling.

BACKGROUND: Cholecystitis is an important risk factor for gallbladder cancer, but the bile microbiome and its association with gallbladder disease has not been investigated fully. We aimed to analyze the bile microbiome in normal conditions, chronic cholecystitis, and gallbladder cancer, and to identify candidate bacteria that play an important role in gallbladder carcinogenesis. METHODS: We performed metagenome sequencing on bile samples of 10 healthy individuals, 10 patients with chronic cholecystitis, and 5 patients with gallbladder cancer, and compared the clinical, radiological, and pathological characteristics of the participants. RESULTS: No significant bacterial signal was identified in the normal bile. The predominant dysbiotic bacteria in both chronic cholecystitis and gallbladder cancer were those belonging to the Enterobacteriaceae family. Klebsiella increased significantly in the order of normal, chronic cholecystitis, and gallbladder cancer. Patients with chronic cholecystitis and dysbiotic microbiome patterns had larger gallstones and showed marked epithelial atypia, which are considered as precancerous conditions. CONCLUSION: We investigated the bile microbiome in normal, chronic cholecystitis, and gallbladder cancer. We suggest possible roles of Enterobacteriaceae, including Klebsiella, in gallbladder carcinogenesis. Our findings reveal a possible link between a dysbiotic bile microbiome and the development of chronic calculous cholecystitis and gallbladder cancer.

Hepatic patch by stacking patient-specific liver progenitor cell sheets formed on multiscale electrospun fibers promotes regenerative therapy for liver injury.

Recently, use of cell sheets with bio-applicable fabrication materials for transplantation has been an attractive approach for the treatment of patients with liver failure. However, renewable and scalable cell sources for engineered tissue patches remain limited. We previously reported a new type of proliferating bipotent human chemically derived hepatic progenitor cells (hCdHs) developed by small molecule-mediated reprogramming. Here, we developed a patient-specific hepatic cell sheet constructed from liver biopsy-derived hCdHs on a multiscale fibrous scaffold by combining electrospinning and three-dimensional printing. Analysis of biomaterial composition revealed that the high-density electrospun sheet was superior in increasing the functional properties of hCdHs. Furthermore, the hepatic patch assembled by multilayer stacking with alternate cell sheets of hCdHs and human umbilical vein endothelial cells (HUVECs) recapitulated a liver tissue-like structure, with histological and morphological shape and size similar to those of primary human hepatocytes, and exhibited a significant increase in hepatic functions such as albumin secretion and activity of cytochrome P450 during in vitro hepatic differentiation compared with that in hCdH cells cultured in a two-dimensional monolayer. Interestingly, in the hepatic patch, the induction of functional hepatocytes was associated with both the electrospun fibrous-facilitated oncostatin M signaling and selective activation of AKT signaling by HUVECs. Notably, upon transplantation into a mouse model of therapeutic liver repopulation, the hepatic patch effectively repopulated the damaged parenchyma and induced the restoration of liver function with healthy morphology in the lobe and an improved survival rate (>70%) in mice. Overall, these results suggested that liver biopsy-derived hCdHs can be an efficient alternative source for developing hepatic cell sheets and patches with potential clinical applications in tissue engineering to advance liver regeneration.

Small molecule-mediated reprogramming of human hepatocytes into bipotent progenitor cells.

BACKGROUND & AIMS: Currently, much effort is directed towards the development of new cell sources for clinical therapy using cell fate conversion by small molecules. Direct lineage reprogramming to a progenitor state has been reported in terminally differentiated rodent hepatocytes, yet remains a challenge in human hepatocytes. METHODS: Human hepatocytes were isolated from healthy and diseased donor livers and reprogrammed into progenitor cells by 2 small molecules, A83-01 and CHIR99021 (AC), in the presence of EGF and HGF. The stemness properties of human chemically derived hepatic progenitors (hCdHs) were tested by standard in vitro and in vivo assays and transcriptome profiling. RESULTS: We developed a robust culture system for generating hCdHs with therapeutic potential. The use of HGF proved to be an essential determinant of the fate conversion process. Based on functional evidence, activation of the HGF/MET signal transduction system collaborated with A83-01 and CHIR99021 to allow a rapid expansion of progenitor cells through the activation of the ERK pathway. hCdHs expressed hepatic progenitor markers and could self-renew for at least 10 passages while retaining a normal karyotype and potential to differentiate into functional hepatocytes and biliary epithelial cells in vitro. Gene expression profiling using RNAseq confirmed the transcriptional reprogramming of hCdHs towards a progenitor state and the suppression of mature hepatocyte transcripts. Upon intrasplenic transplantation in several models of therapeutic liver repopulation, hCdHs effectively repopulated the damaged parenchyma. CONCLUSION: Our study is the first report of successful reprogramming of human hepatocytes to a population of proliferating bipotent cells with regenerative potential. hCdHs may provide a novel tool that permits expansion and genetic manipulation of patient-specific progenitors to study regeneration and the repair of diseased livers. LAY SUMMARY: Human primary hepatocytes were reprogrammed towards hepatic progenitor cells by a combined treatment with 2 small molecules, A83-01 and CHIR99021, and HGF. Chemically derived hepatic progenitors exhibited a high proliferation potential and the ability to differentiate into hepatocytes and biliary epithelial cells both in vitro and in vivo. This approach enables the generation of patient-specific hepatic progenitors and provides a platform for personal and stem cell-based regenerative medicine.

Nonintegrating Direct Conversion Using mRNA into Hepatocyte-Like Cells.

Recently, several researchers have reported that direct reprogramming techniques can be used to differentiate fibroblasts into hepatocyte-like cells without a pluripotent intermediate step. However, the use of viral vectors for conversion continues to pose important challenges in terms of genome integration. Herein, we propose a new method of direct conversion without genome integration with potential clinical applications. To generate hepatocyte-like cells, mRNA coding for the hepatic transcription factors Foxa3 and HNF4α was transfected into mouse embryonic fibroblasts. After 10-12 days, the fibroblasts converted to an epithelial morphology and generated colonies of hepatocyte-like cells (R-iHeps). The generated R-iHeps expressed hepatocyte-specific marker genes and proteins, including albumin, alpha-fetoprotein, HNF4α, CK18, and CYP1A2. To evaluate hepatic function, indocyanine green uptake, periodic acid-Schiff staining, and albumin secretion were assessed. Furthermore, mCherry-positive R-iHeps were engrafted in the liver of Alb-TRECK/SCID mice, and we confirmed FAH enzyme expression in Fah1RTyrc/RJ models. In conclusion, our data suggest that the nonintegrating method using mRNA has potential for cell therapy.

Prolongation of liver-specific function for primary hepatocytes maintenance in 3D printed architectures.

Isolated primary hepatocytes from the liver are very similar to in vivo native liver hepatocytes, but they have the disadvantage of a limited lifespan in 2D culture. Although a sandwich culture and 3D organoids with mesenchymal stem cells (MSCs) as an attractive assistant cell source to extend lifespan can be used, it cannot fully reproduce the in vivo architecture. Moreover, long-term 3D culture leads to cell death because of hypoxic stress. Therefore, to overcome the drawback of 2D and 3D organoids, we try to use a 3D printing technique using alginate hydrogels with primary hepatocytes and MSCs. The viability of isolated hepatocytes was more than 90%, and the cells remained alive for 7 days without morphological changes in the 3D hepatic architecture with MSCs. Compared to a 2D system, the expression level of functional hepatic genes and proteins was higher for up to 7 days in the 3D hepatic architecture. These results suggest that both the 3D bio-printing technique and paracrine molecules secreted by MSCs supported long-term culture of hepatocytes without morphological changes. Thus, this technique allows for widespread expansion of cells while forming multicellular aggregates, may be applied to drug screening and could be an efficient method for developing an artificial liver.

Three-Dimensional Bioprinting of Hepatic Structures with Directly Converted Hepatocyte-Like Cells.

Three-dimensional (3D) bioprinting technology is a promising new technology in the field of bioartificial organ generation with regard to overcoming the limitations of organ supply. The cell source for bioprinting is very important. Here, we generated 3D hepatic scaffold with mouse-induced hepatocyte-like cells (miHeps), and investigated whether their function was improved after transplantation in vivo. To generate miHeps, mouse embryonic fibroblasts (MEFs) were transformed with pMX retroviruses individually expressing hepatic transcription factors Hnf4a and Foxa3. After 8-10 days, MEFs formed rapidly growing hepatocyte-like colonies. For 3D bioprinting, miHeps were mixed with a 3% alginate hydrogel and extruded by nozzle pressure. After 7 days, they were transplanted into the omentum of Jo2-treated NOD Scid gamma (NSG) mice as a liver damage model. Real-time polymerase chain reaction and immunofluorescence analyses were conducted to evaluate hepatic function. The 3D bioprinted hepatic scaffold (25 × 25 mm) expressed Albumin, and ASGR1 and HNF4a expression gradually increased for 28 days in vitro. When transplanted in vivo, the cells in the hepatic scaffold grew more and exhibited higher Albumin expression than in vitro scaffold. Therefore, combining 3D bioprinting with direct conversion technology appears to be an effective option for liver therapy.

Design and Fabrication of a Thin-Walled Free-Form Scaffold on the Basis of Medical Image Data and a 3D Printed Template: Its Potential Use in Bile Duct Regeneration.

Three-dimensional (3D) printing, combined with medical imaging technologies, such as computed tomography and magnetic resonance imaging (MRI), has shown a great potential in patient-specific tissue regeneration. Here, we successfully fabricated an ultrathin tubular free-form structure with a wall thickness of several tens of micrometers that is capable of providing sufficient mechanical flexibility. Such a thin geometry cannot easily be achieved by 3D printing alone; therefore, it was realized through a serial combination of processes, including the 3D printing of a sacrificial template, the dip coating of the biomaterial, and the removal of the inner template. We demonstrated the feasibility of this novel tissue engineering construct by conducting bile duct surgery on rabbits. Moving from a rational design based on MRI data to a successful surgical procedure for reconstruction, we confirmed that the presented method of fabricating scaffolds has the potential for use in customized bile duct regeneration. In addition to the specific application presented here, the developed process and scaffold are expected to have universal applicability in other soft-tissue engineering fields, particularly those involving vascular, airway, and abdominal tubular tissues.

Three-dimensional (3D) printing of mouse primary hepatocytes to generate 3D hepatic structure.

PURPOSE: The major problem in producing artificial livers is that primary hepatocytes cannot be cultured for many days. Recently, 3-dimensional (3D) printing technology draws attention and this technology regarded as a useful tool for current cell biology. By using the 3D bio-printing, these problems can be resolved. METHODS: To generate 3D bio-printed structures (25 mm × 25 mm), cells-alginate constructs were fabricated by 3D bio-printing system. Mouse primary hepatocytes were isolated from the livers of 6-8 weeks old mice by a 2-step collagenase method. Samples of 4 × 107 hepatocytes with 80%-90% viability were printed with 3% alginate solution, and cultured with well-defined culture medium for primary hepatocytes. To confirm functional ability of hepatocytes cultured on 3D alginate scaffold, we conducted quantitative real-time polymerase chain reaction and immunofluorescence with hepatic marker genes. RESULTS: Isolated primary hepatocytes were printed with alginate. The 3D printed hepatocytes remained alive for 14 days. Gene expression levels of Albumin, HNF-4α and Foxa3 were gradually increased in the 3D structures. Immunofluorescence analysis showed that the primary hepatocytes produced hepatic-specific proteins over the same period of time. CONCLUSION: Our research indicates that 3D bio-printing technique can be used for long-term culture of primary hepatocytes. It can therefore be used for drug screening and as a potential method of producing artificial livers.

Simple Maturation of Direct-Converted Hepatocytes Derived from Fibroblasts.

Target cells differentiation techniques from stem cells are developed rapidly. Recently, direct conversion techniques are introduced in various categories. Unlike pluripotent stem cells, this technique enables direct differentiation into the other cell types such as neurons, cardiomyocytes, insulin-producing cells, and hepatocytes without going through the pluripotent stage. However, the function of these converted cells reserve an immature phenotype. Therefore, we modified the culture conditions of mouse direct converted hepatocytes (miHeps) to mature fetal characteristics, such as higher AFP and lower albumin (ALB) expression than primary hepatocytes. First, we generate miHeps from mouse embryonic fibroblasts (MEFs) with two transcription factors HNF4α and Foxa3. These cells indicate typical epithelial morphology and express hepatic proteins. To mature hepatic function, DMSO is treated during culture time for more than 7 days. After maturation, miHeps showed features of maturation such as exhibiting typical hepatocyte-like morphology, increased up-regulated ALB and CYP enzyme gene expression, down-regulated AFP expressions, and acquired hepatic function over time. Thus, our data provides a simple method to mature direct converted hepatocytes functionally and these cells enable them to move closer to generating functional hepatocytes.

Generation of Multilayered 3D Structures of HepG2 Cells Using a Bio-printing Technique.

BACKGROUND/AIMS: Chronic liver disease is a major widespread cause of death, and whole liver transplantation is the only definitive treatment for patients with end-stage liver diseases. However, many problems, including donor shortage, surgical complications and cost, hinder their usage. Recently, tissue-engineering technology provided a potential breakthrough for solving these problems. Three-dimensional (3D) printing technology has been used to mimic tissues and organs suitable for transplantation, but applications for the liver have been rare. METHODS: A 3D bioprinting system was used to construct 3D printed hepatic structures using alginate. HepG2 cells were cultured on these 3D structures for 3 weeks and examined by fluorescence microscopy, histology and immunohistochemistry. The expression of liverspecific markers was quantified on days 1, 7, 14, and 21. RESULTS: The cells grew well on the alginate scaffold, and liver-specific gene expression increased. The cells grew more extensively in 3D culture than two-dimensional culture and exhibited better structural aspects of the liver, indicating that the 3D bioprinting method recapitulates the liver architecture. CONCLUSIONS: The 3D bioprinting of hepatic structures appears feasible. This technology may become a major tool and provide a bridge between basic science and the clinical challenges for regenerative medicine of the liver.