Cholangiocyte organoids can repair bile ducts after transplantation in the human liver.

Organoid technology holds great promise for regenerative medicine but has not yet been applied to humans. We address this challenge using cholangiocyte organoids in the context of cholangiopathies, which represent a key reason for liver transplantation. Using single-cell RNA sequencing, we show that primary human cholangiocytes display transcriptional diversity that is lost in organoid culture. However, cholangiocyte organoids remain plastic and resume their in vivo signatures when transplanted back in the biliary tree. We then utilize a model of cell engraftment in human livers undergoing ex vivo normothermic perfusion to demonstrate that this property allows extrahepatic organoids to repair human intrahepatic ducts after transplantation. Our results provide proof of principle that cholangiocyte organoids can be used to repair human biliary epithelium

Reconstruction of the mouse extrahepatic biliary tree using primary human extrahepatic cholangiocyte organoids

Treatment of common bile duct disorders such as biliary atresia or ischaemic strictures is limited to liver transplantation or hepatojejunostomy due to the lack of suitable tissue for surgical reconstruction. Here, we report a novel method for the isolation and propagation of human cholangiocytes from the extrahepatic biliary tree and we explore the potential of bioengineered biliary tissue consisting of these extrahepatic cholangiocyte organoids (ECOs) and biodegradable scaffolds for transplantation and biliary reconstruction in vivo. ECOs closely correlate with primary cholangiocytes in terms of transcriptomic profile and functional properties (ALP, GGT). Following transplantation in immunocompromised mice ECOs self-organize into tubular structures expressing biliary markers (CK7). When seeded on biodegradable scaffolds, ECOs form tissue-like structures retaining biliary marker expression (CK7) and function (ALP, GGT). This bioengineered tissue can reconstruct the wall of the biliary tree (gallbladder) and rescue and extrahepatic biliary injury mouse model following transplantation. Furthermore, it can be fashioned into bioengineered ducts and replace the native common bile duct of immunocompromised mice, with no evidence of cholestasis or lumen occlusion up to one month after reconstruction. In conclusion, ECOs can successfully reconstruct the biliary tree following transplantation, providing proof-of-principle for organ regeneration using human primary cells expanded in vitro

Interleukin-13 Activates Distinct Cellular Pathways Leading to Ductular Reaction, Steatosis, and Fibrosis

Fibroproliferative diseases are driven by dysregulated tissue repair responses and are major cause of morbidity and mortality as they affect nearly every organ system. Type-2 cytokine responses are critically involved in tissue repair; however, the mechanisms that regulate beneficial regeneration versus pathological fibrosis are not well understood. Here, we have shown that the type-2 effector cytokine interleukin-13 simultaneously, yet independently, directed hepatic fibrosis and the compensatory proliferation of hepatocytes and biliary cells in progressive models of liver disease induced by interleukin-13 over-expression or following infection with Schistosoma mansoni. Using transgenic mice with interleukin-13 signaling genetically disrupted in hepatocytes, cholangiocytes, or resident tissue fibroblasts, we have revealed direct and distinct roles for interleukin-13 in fibrosis, steatosis, cholestasis, and ductular reaction. Together, these studies show that these mechanisms are simultaneously controlled but distinctly regulated by interleukin-13 signaling. Thus, it may be possible to promote interleukin-13-dependent hepatobiliary expansion without generating pathological fibrosis

Disease modeling using human induced pluripotent stem cells: lessons from the liver

Human pluripotent stem cells (hPSCs) have the capacity to differentiate into any of the hundreds of distinct cell types that comprise the human body. This unique characteristic has resulted in considerable interest in the field of regenerative medicine, given the potential for these cells to be used to protect, repair, or replace diseased, injured, and aged cells within the human body. In addition to their potential in therapeutics, hPSCs can be used to study the earliest stages of human development and to provide a platform for both drug screening and disease modeling using human cells. Recently, the description of human induced pluripotent stem cells (hIPSCs) has allowed the field of disease modeling to become far more accessible and physiologically relevant, as pluripotent cells can be generated from patients of any genetic background. Disease models derived from hIPSCs that manifest cellular disease phenotypes have been established to study several monogenic diseases; furthermore, hIPSCs can be used for phenotype-based drug screens to investigate complex diseases for which the underlying genetic mechanism is unknown. As a result, the use of stem cells as research tools has seen an unprecedented growth within the last decade as researchers look for in vitro disease models which closely mimic in vivo responses in humans. Here, we discuss the beginnings of hPSCs, starting with isolation of human embryonic stem cells, moving into the development and optimization of hIPSC technology, and ending with the application of hIPSCs towards disease modeling and drug screening applications, with specific examples highlighting the modeling of inherited metabolic disorders of the liver. This article is part of a Special Issue entitled Linking transcription to physiology in lipodomics

A humidity-sensitive hydrogel-Bacillus spore composite for micropatterning of biomolecular gradients

A composite material consisting of Bacillus subtilis spores suspended in a humidity sensitive hydrogel can be used to pattern biomolecules in different concentrations directly onto glass surfaces using a mechanical micromanipulator. By altering the relative humidity surrounding the composite gel during deposition, surface concentration of patterned biomolecules can be controlled and varied to create user-defined, biomolecular surface concentrations. (C) 2013 AIP Publishing LLC

Maturation of induced pluripotent stem cell derived hepatocytes by 3D-culture

Induced pluripotent stem cell derived hepatocytes (IPSC-Heps) have the potential to reduce the demand for a dwindling number of primary cells used in applications ranging from therapeutic cell infusions to in vitro toxicology studies. However, current differentiation protocols and culture methods produce cells with reduced functionality and fetal-like properties compared to adult hepatocytes. We report a culture method for the maturation of IPSC-Heps using 3-Dimensional (3D) collagen matrices compatible with high throughput screening. This culture method significantly increases functional maturation of IPSC-Heps towards an adult phenotype when compared to conventional 2D systems. Additionally, this approach spontaneously results in the presence of polarized structures necessary for drug metabolism and improves functional longevity to over 75 days. Overall, this research reveals a method to shift the phenotype of existing IPSC-Heps towards primary adult hepatocytes allowing such cells to be a more relevant replacement for the current primary standard

Selective Weighing of Individual Microparticles Using a Hybrid Micromanipulator-Nanomechanical Resonator System

We report a system based on a combination of a micromanipulator and a cantilever-based differential resonator for selecting and weighing individual micro-scale particles. Instead of relying on probabilistic attachment of particles on sensor surfaces, the system can specifically select and weigh individual micro-entities. The micromanipulator is able to move particles from a native media to the surface of a resonator that can weigh particles with pg-level resolution. The system allows individually manipulating and weighing a wide variety of entities that can be visualized under a microscope, ranging from cell spheres to spore clusters and single diatom algae

Counting the Number of Magnesium Ions Bound to the Surface-Immobilized Thymine Oligonucleotides That Comprise Spherical Nucleic Acids

Label-free studies carried out under aqueous phase conditions quantify the number of Mg²⁺ ions binding to surface-immobilized T₄₀ sequences, the subsequent reordering of DNA on the surface, and the consequences of Mg²⁺ binding for DNA–DNA interactions. Second harmonic generation measurements indicate that, within error, 18–20 Mg²⁺ ions are bound to the T₄₀ strand at saturation and that the metal–DNA interaction is associated with a near 30% length contraction of the strand. Structural reordering, evaluated using vibrational sum frequency generation, atomic force microscopy, and dynamic light scattering, is attributed to increased charge screening as the Mg²⁺ ions bind to the negatively charged DNA, reducing repulsive Coulomb forces between nucleotides and allowing the DNA single strands to collapse or coil upon themselves. The impact of Mg²⁺ binding on DNA hybridization and duplex stability is assessed with spherical nucleic acid (SNA) gold nanoparticle conjugates in order to determine an optimal working range of Mg²⁺ concentrations for DNA–DNA interactions in the absence of NaCl. The findings are consistent with a charge titration effect in which, in the absence of NaCl, (1) hybridization does not occur at room temperature if an average of 17.5 or less Mg²⁺ ions are bound per T₄₀ strand, which is not reached until the bulk Mg²⁺ concentration approaches 0.5 mM; (2) hybridization proceeds, albeit with low duplex stability having an average <i>T</i>_m of 31(3)°C, if an average of 17.5–18.0 Mg²⁺ ions are bound; and (3) highly stable duplexes having a <i>T</i>_m of 64(2)°C form if 18.5–19.0 Mg²⁺ ions are bound, corresponding to saturation of the T₄₀ strand