Therapeutic effects of a small molecule agonist of the relaxin receptor ML290 in liver fibrosis.

Fibrosis is an underlying cause of cirrhosis and hepatic failure resulting in end stage liver disease with limited pharmacological options. The beneficial effects of relaxin peptide treatment were demonstrated in clinically relevant animal models of liver fibrosis. However, the use of relaxin is problematic because of a short half-life. The aim of this study was to test the therapeutic effects of recently identified small molecule agonists of the human relaxin receptor, relaxin family peptide receptor 1 (RXFP1). The lead compound of this series, ML290, was selected based on its effects on the expression of fibrosis-related genes in primary human stellate cells. RNA sequencing analysis of TGF-ß1-activated LX-2 cells showed that ML290 treatment primarily affected extracellular matrix remodeling and cytokine signaling, with expression profiles indicating an antifibrotic effect of ML290. ML290 treatment in human liver organoids with LPS-induced fibrotic phenotype resulted in a significant reduction of type I collagen. The pharmacokinetics of ML290 in mice demonstrated its high stability in vivo, as evidenced by the sustained concentrations of compound in the liver. In mice expressing human RXFP1 gene treated with carbon tetrachloride, ML290 significantly reduced collagen content, α-smooth muscle actin expression, and cell proliferation around portal ducts. In conclusion, ML290 demonstrated antifibrotic effects in liver fibrosis.-Kaftanovskaya, E. M., Ng, H. H., Soula, M., Rivas, B., Myhr, C., Ho, B. A., Cervantes, B. A., Shupe, T. D., Devarasetty, M., Hu, X., Xu, X., Patnaik, S., Wilson, K. J., Barnaeva, E., Ferrer, M., Southall, N. T., Marugan, J. J., Bishop, C. E., Agoulnik, I. U., Agoulnik, A. I. Therapeutic effects of a small molecule agonist of the relaxin receptor ML290 in liver fibrosis.

Multisensor-integrated organs-on-chips platform for automated and continual in situ monitoring of organoid behaviors.

Organ-on-a-chip systems are miniaturized microfluidic 3D human tissue and organ models designed to recapitulate the important biological and physiological parameters of their in vivo counterparts. They have recently emerged as a viable platform for personalized medicine and drug screening. These in vitro models, featuring biomimetic compositions, architectures, and functions, are expected to replace the conventional planar, static cell cultures and bridge the gap between the currently used preclinical animal models and the human body. Multiple organoid models may be further connected together through the microfluidics in a similar manner in which they are arranged in vivo, providing the capability to analyze multiorgan interactions. Although a wide variety of human organ-on-a-chip models have been created, there are limited efforts on the integration of multisensor systems. However, in situ continual measuring is critical in precise assessment of the microenvironment parameters and the dynamic responses of the organs to pharmaceutical compounds over extended periods of time. In addition, automated and noninvasive capability is strongly desired for long-term monitoring. Here, we report a fully integrated modular physical, biochemical, and optical sensing platform through a fluidics-routing breadboard, which operates organ-on-a-chip units in a continual, dynamic, and automated manner. We believe that this platform technology has paved a potential avenue to promote the performance of current organ-on-a-chip models in drug screening by integrating a multitude of real-time sensors to achieve automated in situ monitoring of biophysical and biochemical parameters.

Immunogenicity of decellularized porcine liver for bioengineered hepatic tissue.

Liver disease affects millions of patients each year. The field of regenerative medicine promises alternative therapeutic approaches, including the potential to bioengineer replacement hepatic tissue. One approach combines cells with acellular scaffolds derived from animal tissue. The goal of this study was to scale up our rodent liver decellularization method to livers of a clinically relevant size. Porcine livers were cannulated via the hepatic artery, then perfused with PBS, followed by successive Triton X-100 and SDS solutions in saline buffer. After several days of rinsing, decellularized liver samples were histologically analyzed. In addition, biopsy specimens of decellularized scaffolds were seeded with hepatoblastoma cells for cytotoxicity testing or implanted s.c. into rodents to investigate scaffold immunogenicity. Histological staining confirmed cellular clearance from pig livers, with removal of nuclei and cytoskeletal components and widespread preservation of structural extracellular molecules. Scanning electron microscopy confirmed preservation of an intact liver capsule, a porous acellular lattice structure with intact vessels and striated basement membrane. Liver scaffolds supported cells over 21 days, and no increased immune response was seen with either allogeneic (rat-into-rat) or xenogeneic (pig-into-rat) transplants over 28 days, compared with sham-operated on controls. These studies demonstrate that successful decellularization of the porcine liver could be achieved with protocols developed for rat livers, yielding nonimmunogenic scaffolds for future hepatic bioengineering studies.

Cell and organ bioengineering technology as applied to gastrointestinal diseases.

This review illustrates promising regenerative medicine technologies that are being developed for the treatment of gastrointestinal diseases. The main strategies under validation to bioengineer or regenerate liver, pancreas, or parts of the digestive tract are twofold: engineering of progenitor cells and seeding of cells on supporting scaffold material. In the first case, stem cells are initially expanded under standard tissue culture conditions. Thereafter, these cells may either be delivered directly to the tissue or organ of interest, or they may be loaded onto a synthetic or natural three-dimensional scaffold that is capable of enhancing cell viability and function. The new construct harbouring the cells usually undergoes a maturation phase within a bioreactor. Within the bioreactor, cells are conditioned to adopt a phenotype similar to that displayed in the native organ. The specific nature of the scaffold within the bioreactor is critical for the development of this high-function phenotype. Efforts to bioengineer or regenerate gastrointestinal tract, liver and pancreas have yielded promising results and have demonstrated the immense potential of regenerative medicine. However, a myriad of technical hurdles must be overcome before transplantable, engineered organs become a reality.

3D Bioprinted Liver-on-a-Chip for Drug Cytotoxicity Screening.

Tissues on a chip are sophisticated three-dimensional (3D) in vitro microphysiological systems designed to replicate human tissue conditions within dynamic physicochemical environments. However, the current fabrication methods for tissue spheroids on a chip require multiple parts and manual processing steps, including the deposition of spheroids onto prefabricated "chips." These challenges also lead to limitations regarding scalability and reproducibility. To overcome these challenges, we employed 3D printing techniques to automate the fabrication process of tissue spheroids on a chip. This allowed the simultaneous high-throughput printing of human liver spheroids and their surrounding polymeric flow chamber "chips" containing inner channels in a single step. The fabricated liver tissue spheroids on a liver-on-a-chip (LOC) were subsequently subjected to dynamic culturing by a peristaltic pump, enabling assessment of cell viability and metabolic activities. The 3D printed liver spheroids within the printed chips demonstrated high cell viability (>80%), increased spheroid size, and consistent adenosine triphosphate (ATP) activity and albumin production for up to 14 days. Furthermore, we conducted a study on the effects of acetaminophen (APAP), a nonsteroidal anti-inflammatory drug, on the LOC. Comparative analysis revealed a substantial decline in cell viability (<40%), diminished ATP activity, and reduced spheroid size after 7 days of culture within the APAP-treated LOC group, compared to the nontreated groups. These results underscore the potential of 3D bioprinted tissue chips as an advanced in vitro model that holds promise for accurately studying in vivo biological processes, including the assessment of tissue response to administered drugs, in a high-throughput manner.

Bioreactor design and validation for manufacturing strategies in tissue engineering.

The fields of regenerative medicine and tissue engineering offer new therapeutic options to restore, maintain or improve tissue function following disease or injury. To maximize the biological function of a tissue-engineered clinical product, specific conditions must be maintained within a bioreactor to allow the maturation of the product in preparation for implantation. Specifically, the bioreactor should be designed to mimic the mechanical, electrochemical and biochemical environment that the product will be exposed to in vivo. Real-time monitoring of the functional capacity of tissue-engineered products during manufacturing is a critical component of the quality management process. The present review provides a brief overview of bioreactor engineering considerations. In addition, strategies for bioreactor automation, in-line product monitoring and quality assurance are discussed.

The role of the Wnt family of secreted proteins in rat oval "stem" cell-based liver regeneration: Wnt1 drives differentiation.

To date the molecular signals regulating activation, proliferation, and differentiation of hepatic oval cells are not fully understood. The Wnt family is essential in hepatic embryogenesis and implicated in hepatic carcinogenesis. This study elucidates novel findings implicating Wnt1 in directing oval cell differentiation during the rat 2-acetylaminofluorene (2AAF) and 2/3 partial hepatectomy (PHx) liver regeneration model. Proteins of Wnt family members were predominantly localized in pericentral hepatocytes during liver injury, oval cell activation, and hepatocyte regeneration. In addition, Wnt message increased coinciding with the rise in oval cell number, whereas protein levels peaked immediately after the height of oval cell proliferation. Immunohistochemical analysis demonstrated nuclear translocation of beta-catenin within oval cells throughout the 2AAF/PHx protocol. Furthermore, RNA interference was used in vivo to confirm the physiological requirement of Wnt1 during the oval cell induction. Ultimately, inhibition of Wnt1 resulted in failure of oval cells to differentiate into hepatocytes and alternatively induced atypical ductular hyperplasia. Taken together, these data indicate that in vivo exposure to Wnt1 shRNA inhibited rat oval cell liver regeneration. In the absence of Wnt1 signaling, oval cells failed to differentiate into hepatocytes and underwent atypical ductular hyperplasia, exhibiting epithelial metaplasia and mucin production. Furthermore, changes in Wnt1 levels are required for the efficient regeneration of the liver by oval cells during massive hepatic injury.

Hepatic stellate cells' involvement in progenitor-mediated liver regeneration.

Earlier studies conducted by our laboratory have shown that suppression of transforming growth factor-beta (TGFbeta)-mediated upregulation of connective tissue growth factor (CTGF) by iloprost resulted in a greatly diminished oval cell response to 2-acetylaminofluorene/partial hepatectomy (2AAF/PH) in rats. We hypothesized that this effect is due to decreased activation of hepatic stellate cells. To test this hypothesis, we maintained rats on a diet supplemented with 2% L-cysteine as a means of inhibiting stellate cell activation during the oval cell response to 2AAF/PH. In vitro experiments show that L-cysteine did, indeed, prevent the activation of stellate cells while exerting no direct effect on oval cells. Desmin immunostaining of liver sections from 2AAF/PH animals indicated that maintenance on the L-cysteine diet resulted in an 11.1-fold decrease in the number of activated stellate cells within the periportal zones. The total number of cells proliferating in the periportal zones of livers from animals treated with L-cysteine was drastically reduced. Further analyses showed a greater than fourfold decrease in the magnitude of the oval cell response in animals maintained on the L-cysteine diet as determined by immunostaining for both OV6 and alpha-fetoprotein (AFP). Global liver expression of AFP as measured by real-time PCR was shown to be decreased 4.7-fold in the L-cysteine-treated animals. These data indicate that the activation of hepatic stellate cells is required for an appropriate oval cell response to 2AAF/PH.

Drug compound screening in single and integrated multi-organoid body-on-a-chip systems.

Current practices in drug development have led to therapeutic compounds being approved for widespread use in humans, only to be later withdrawn due to unanticipated toxicity. These occurrences are largely the result of erroneous data generated by in vivo and in vitro preclinical models that do not accurately recapitulate human physiology. Herein, a human primary cell- and stem cell-derived 3D organoid technology is employed to screen a panel of drugs that were recalled from market by the FDA. The platform is comprised of multiple tissue organoid types that remain viable for at least 28 days, in vitro. For many of these compounds, the 3D organoid system was able to demonstrate toxicity. Furthermore, organoids exposed to non-toxic compounds remained viable at clinically relevant doses. Additional experiments were performed on integrated multi-organoid systems containing liver, cardiac, lung, vascular, testis, colon, and brain. These integrated systems proved to maintain viability and expressed functional biomarkers, long-term. Examples are provided that demonstrate how multi-organoid 'body-on-a-chip' systems may be used to model the interdependent metabolism and downstream effects of drugs across multiple tissues in a single platform. Such 3D in vitro systems represent a more physiologically relevant model for drug screening and will likely reduce the cost and failure rate associated with the approval of new drugs.

A detailed characterization of the adult mouse model of glycogen storage disease Ia.

Glycogen storage disease type Ia (GSDIa) is caused by a genetic defect in the hepatic enzyme glucose-6-phosphatase (G6Pase-alpha), which manifests as life-threatening hypoglycemia with related metabolic complications. A G6Pase-alpha knockout (KO) mouse model was generated to study potential therapies for correcting this disorder. Since then, gene therapy studies have produced promising results, showing long-term improvement in liver histology and glycogen metabolism. Under existing protocols, however, untreated KO pups seldom survived weaning. Here, we present a thorough characterization of the G6Pase-alpha KO mouse, as well as the husbandry protocol for rearing this strain to adulthood. These mice were raised with only palliative care, and characterized from birth through 6 months of age. Once KO mice have survived the very frail weaning period, their size, agility, serum lipids and glycemic control improve dramatically, reaching levels approaching their wild-type littermates. In addition, our data reveal that adult mice lacking G6Pase-alpha are able to mate and produce viable offspring. However, liver histology and glycogen accumulation do not improve with age. Overall, the reliable production of mature KO mice could provide a critical tool for advancing the GSDIa field, as the availability of a robust enzyme-deficient adult offers a new spectrum of treatment avenues that would not be tolerated by the frail pups. Most importantly, our detailed characterization of the adult KO mouse provides a crucial baseline for accurately gauging the efficacy of experimental therapies in this important model.

Establishment of cancer cell lines from rat hepatocholangiocarcinoma and assessment of the role of granulocyte-colony stimulating factor and hepatocyte growth factor in their growth, motility and survival.

BACKGROUND/AIMS: Oval cells (OCs), putative hepatic stem cells, may give rise to liver cancers. We developed a carcinogenesis regimen, based upon induction of OC proliferation prior to carcinogen exposure. In our model, rats subjected to 2-acetylaminofluorene/ partial-hepatectomy followed by aflatoxin injection (APA regimen) developed well-differentiated hepatocholangiocarcinomas. The aim of this study was to establish and characterize cancer cell lines from this animal model. METHODS: Cancer cells were cultured from animals sacrificed eight months after treatment, and single clones were selected. The established cell lines, named LCSCs, were characterized, and their tumorigenicity was assessed in vivo. The roles of granulocyte-colony stimulating factor (G-CSF) and hepatocyte growth factor (HGF) in LCSC growth, survival and motility were also investigated. RESULTS: From primary tumors, six cell lines were developed. LCSCs shared with the primary tumors the expression of various OC-associated markers, including cMet and G-CSF receptor. In vitro, HGF conferred protection from death by serum withdrawal. Stimulation with G-CSF increased LCSC growth and motility, while the blockage of its receptor inhibited LCSC proliferation and migration. CONCLUSIONS: Six cancer cell lines were established from our model of hepatocholangiocarcinoma. HGF modulated LCSC resistance to apoptosis, while G-CSF acted on LCSCs as a proliferative and chemotactic agent.

Isolation and characterization of hepatic stem cells, or "oval cells," from rat livers.

The pace of research on the potential therapeutic uses of liver stem cells or "oval cells" has accelerated significantly in recent years. Concurrent advancements in techniques for the isolation and characterization of these cells have helped fuel this research. Several models now exist for the induction of oval cell proliferation in rodents. Protocols for the isolation and culture of these cells have evolved to the point that they may be set up in any laboratory equipped for cell culture. The advent of magnetic cell sorting has eliminated reliance on expensive flow cytometric sorting equipment to generate highly enriched populations of oval cells. Our laboratory has had much success in using the oval cell surface marker Thy-1 in combination with magnetic sorting to produce material suitable for testing the influence of a myriad of chemical signaling molecules on the oval cell phenotype. This chapter will describe our basic strategy for oval cell induction and isolation. Additionally, two in vitro procedures are described which the reader may find useful in the early stages of developing an oval cell research project.

Stem cell therapy for inherited metabolic disorders of the liver.

Modern medicine has conquered an enormous spectrum of health concerns, from the neonatal to the geriatric, the chronically ill to the acutely injured. Among the unmet challenges remaining in modern medicine are inborn disorders of metabolism within the liver. Such inherited metabolic disorders (IMDs) often leave an otherwise healthy individual with a crippling imbalance. As the principal regulator of the body's many metabolic pathways, malencoded hepatic enzymes can drastically disrupt homeostasis throughout the entire body. Severe phenotypes are usually detected within the first few days of life, and treatments range from palliative lifestyle modifications to aggressive surgical procedures. While orthotopic liver transplantation is the single last resort "cure" for these conditions, research during the past few years has brought new therapeutic technologies ever closer to the clinic. Stem cells, therapeutic viral vectors, or a combination thereof, are projected to be the next, best, and final cure for IMDs, which is well-reflected by this generation's research initiatives.

An Industry-Driven Roadmap for Manufacturing in Regenerative Medicine.

Regenerative medicine is poised to become a significant industry within the medical field. As such, the development of strategies and technologies for standardized and automated regenerative medicine clinical manufacturing has become a priority. An industry-driven roadmap toward industrial scale clinical manufacturing was developed over a 3-year period by a consortium of companies with significant investment in the field of regenerative medicine. Additionally, this same group identified critical roadblocks that stand in the way of advanced, large-scale regenerative medicine clinical manufacturing. This perspective article details efforts to reach a consensus among industry stakeholders on the shortest pathway for providing access to regenerative medicine therapies for those in need, both within the United States and around the world. Stem Cells Translational Medicine 2018;7:564-568.

Environmental Toxin Screening Using Human-Derived 3D Bioengineered Liver and Cardiac Organoids.

INTRODUCTION: Environmental toxins, such as lead and other heavy metals, pesticides, and other compounds, represent a significant health concern within the USA and around the world. Even in the twenty-first century, a plethora of cities and towns in the U.S. have suffered from exposures to lead in drinking water or other heavy metals in food or the earth, while there is a high possibility of further places to suffer such exposures in the near future. METHODS: We employed bioengineered 3D human liver and cardiac organoids to screen a panel of environmental toxins (lead, mercury, thallium, and glyphosate), and charted the response of the organoids to these compounds. Liver and cardiac organoids were exposed to lead (10 µM-10 mM), mercury (200 nM-200 µM), thallium (10 nM-10 µM), or glyphosate (25 µM-25 mM) for a duration of 48 h. The impacts of toxin exposure were then assessed by LIVE/DEAD viability and cytotoxicity staining, measuring ATP activity and determining IC50 values, and determining changes in cardiac organoid beating activity. RESULTS: As expected, all of the toxins induced toxicity in the organoids. Both ATP and LIVE/DEAD assays showed toxicity in both liver and cardiac organoids. In particular, thallium was the most toxic, with IC50 values of 13.5 and 1.35 µM in liver and cardiac organoids, respectively. Conversely, glyphosate was the least toxic of the four compounds, with IC50 values of 10.53 and 10.85 mM in liver and cardiac organoids, respectively. Additionally, toxins had a negative influence on cardiac organoid beating activity as well. Thallium resulting in the most significant decreases in beating rate, followed by mercury, then glyphosate, and finally, lead. These results suggest that the 3D organoids have significant utility to be deployed in additional toxicity screening applications, and future development of treatments to mitigate exposures. CONCLUSION: 3D organoids have significant utility to be deployed in additional toxicity screening applications, such as future development of treatments to mitigate exposures, drug screening, and environmental toxin detection.

Urothelium with barrier function differentiated from human urine-derived stem cells for potential use in urinary tract reconstruction.

BACKGROUND: Autologous urothelial cells are often obtained via bladder biopsy to generate the bio-engineered urethra or bladder, while urine-derived stem cells (USC) can be obtained by a non-invasive approach. The objective of this study is to develop an optimal strategy for urothelium with permeability barrier properties using human USC which could be used for tissue repair in the urinary tract system. METHODS: USC were harvested from six healthy adult individuals. To optimize urothelial differentiation, five different differentiation methods were studied. The induced cells were assessed for gene and protein expression markers of urothelial cells via RT-PCR, Western blotting, and immunofluorescent staining. Barrier function and ultrastructure of the tight junction were assessed with permeability assays and transmission electron microscopy (TEM). Induced cells were both cultured on trans-well membranes and small intestinal submucosa, then investigated under histology analysis. RESULTS: Differentiated USC expressed significantly higher levels of urothelial-specific transcripts and proteins (Uroplakin III and Ia), epithelial cell markers (CK20 and AE1/AE3), and tight junction markers (ZO-1, ZO-2, E-cadherin, and Cingulin) in a time-dependent manner, compared to non-induced USC. In vitro assays using fluorescent dye demonstrated a significant reduction in permeability of differentiated USC. In addition, transmission electron microscopy confirmed appropriate ultrastructure of urothelium differentiated from USC, including tight junction formation between neighboring cells, which was similar to positive controls. Furthermore, multilayered urothelial tissues formed 2 weeks after USC were differentiated on intestine submucosal matrix. CONCLUSION: The present study illustrates an optimal strategy for the generation of differentiated urothelium from stem cells isolated from the urine. The induced urothelium is phenotypically and functionally like native urothelium and has proposed uses in in vivo urological tissue repair or in vitro urethra or bladder modeling.

Optical Tracking and Digital Quantification of Beating Behavior in Bioengineered Human Cardiac Organoids.

Organoid and organ-on-a-chip technologies are rapidly advancing towards deployment for drug and toxicology screening applications. Liver and cardiac toxicities account for the majority of drug candidate failures in human trials. Liver toxicity generally produces liver cell death, while cardiac toxicity causes adverse changes in heart beat kinetics. In traditional 2D cultures, beating kinetics can be measured by electrode arrays, but in some 3D constructs, quantifying beating kinetics can be more challenging. For example, real time measurements of calcium flux or contractile forces are possible, yet rather complex. In this communication article, we demonstrate a simple sensing system based on software code that optically analyzes video capture files of beating cardiac organoids, translates these files in representations of moving pixels, and quantifies pixel movement activity over time to generate beat kinetic plots. We demonstrate this system using bioengineered cardiac organoids under baseline and drug conditions. This technology offers a non-invasive, low-cost, and incredibly simple method for tracking and quantifying beating behavior in cardiac organoids and organ-on-a-chip systems for drug and toxicology screening.

Multi-tissue interactions in an integrated three-tissue organ-on-a-chip platform.

Many drugs have progressed through preclinical and clinical trials and have been available - for years in some cases - before being recalled by the FDA for unanticipated toxicity in humans. One reason for such poor translation from drug candidate to successful use is a lack of model systems that accurately recapitulate normal tissue function of human organs and their response to drug compounds. Moreover, tissues in the body do not exist in isolation, but reside in a highly integrated and dynamically interactive environment, in which actions in one tissue can affect other downstream tissues. Few engineered model systems, including the growing variety of organoid and organ-on-a-chip platforms, have so far reflected the interactive nature of the human body. To address this challenge, we have developed an assortment of bioengineered tissue organoids and tissue constructs that are integrated in a closed circulatory perfusion system, facilitating inter-organ responses. We describe a three-tissue organ-on-a-chip system, comprised of liver, heart, and lung, and highlight examples of inter-organ responses to drug administration. We observe drug responses that depend on inter-tissue interaction, illustrating the value of multiple tissue integration for in vitro study of both the efficacy of and side effects associated with candidate drugs.

Organoid-on-a-chip and body-on-a-chip systems for drug screening and disease modeling.

In recent years, advances in tissue engineering and microfabrication technologies have enabled rapid growth in the areas of in vitro organoid development as well as organoid-on-a-chip platforms. These 3D model systems often are able to mimic human physiology more accurately than traditional 2D cultures and animal models. In this review, we describe the progress that has been made to generate organ-on-a-chip platforms and, more recently, more complex multi-organoid body-on-a-chip platforms and their applications. Importantly, these systems have the potential to dramatically impact biomedical applications in the areas of drug development, drug and toxicology screening, disease modeling, and the emerging area of personalized precision medicine.