High level of polarized engraftment of porcine intrahepatic cholangiocyte organoids in decellularized liver scaffolds.

In Europe alone, each year 5500 people require a life-saving liver transplantation, but 18% die before receiving one due to the shortage of donor organs. Whole organ engineering, utilizing decellularized liver scaffolds repopulated with autologous cells, is an attractive alternative to increase the pool of available organs for transplantation. The development of this technology is hampered by a lack of a suitable large-animal model representative of the human physiology and a reliable and continuous cell source. We have generated porcine intrahepatic cholangiocyte organoids from adult stem cells and demonstrate that these cultures remained stable over multiple passages whilst retaining the ability to differentiate into hepatocyte- and cholangiocyte-like cells. Recellularization onto porcine scaffolds was efficient and the organoids homogeneously differentiated, even showing polarization. Our porcine intrahepatic cholangiocyte system, combined with porcine liver scaffold paves the way for developing whole liver engineering in a relevant large-animal model.

Hallmarks of the human intestinal microbiome on liver maturation and function.

As one of the most metabolically complex systems in the body, the liver ensures multi-organ homeostasis and ultimately sustains life. Nevertheless, during early postnatal development, the liver is highly immature and takes about 2 years to acquire and develop almost all of its functions. Different events occurring at the environmental and cellular levels are thought to mediate hepatic maturation and function postnatally. The crosstalk between the liver, the gut and its microbiome has been well appreciated in the context of liver disease, but recent evidence suggests that the latter could also be critical for hepatic function under physiological conditions. The gut-liver crosstalk is thought to be mediated by a rich repertoire of microbial metabolites that can participate in a myriad of biological processes in hepatic sinusoids, from energy metabolism to tissue regeneration. Studies on germ-free animals have revealed the gut microbiome as a critical contributor in early hepatic programming, and this influence extends throughout life, mediating liver function and body homeostasis. In this seminar, we describe the microbial molecules that have a known effect on the liver and discuss how the gut microbiome and the liver evolve throughout life. We also provide insights on current and future strategies to target the gut microbiome in the context of hepatology research.

Large-Scale Production of LGR5-Positive Bipotential Human Liver Stem Cells.

BACKGROUND AND AIMS: The gap between patients on transplant waiting lists and available donor organs is steadily increasing. Human organoids derived from leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5)-positive adult stem cells represent an exciting new cell source for liver regeneration; however, culturing large numbers of organoids with current protocols is tedious and the level of hepatic differentiation is limited. APPROACH AND RESULTS: Here, we established a method for the expansion of large quantities of human liver organoids in spinner flasks. Due to improved oxygenation in the spinner flasks, organoids rapidly proliferated and reached an average 40-fold cell expansion after 2 weeks, compared with 6-fold expansion in static cultures. The organoids repopulated decellularized liver discs and formed liver-like tissue. After differentiation in spinner flasks, mature hepatocyte markers were highly up-regulated compared with static organoid cultures, and cytochrome p450 activity reached levels equivalent to hepatocytes. CONCLUSIONS: We established a highly efficient method for culturing large numbers of LGR5-positive stem cells in the form of organoids, which paves the way for the application of organoids for tissue engineering and liver transplantation.

The role of extracellular matrix on liver stem cell fate: A dynamic relationship in health and disease.

The liver stem cell niche is a specialized and dynamic microenvironment with biomechanical and biochemical characteristics that regulate stem cell behavior. This is feasible due to the coordination of a complex network of secreted factors, small molecules, neural, blood inputs and extracellular matrix (ECM) components involved in the regulation of stem cell fate (self-renewal, survival, and differentiation into more mature phenotypes like hepatocytes and cholangiocytes). In this review, we describe and summarize all the major components that play essential roles in the liver stem cell niche, in particular, growth factor signaling and the biomechanical properties of the ECM.

Biotechnology Challenges to In Vitro Maturation of Hepatic Stem Cells.

The incidence of liver disease is increasing globally. The only curative therapy for severe end-stage liver disease, liver transplantation, is limited by the shortage of organ donors. In vitro models of liver physiology have been developed and new technologies and approaches are progressing rapidly. Stem cells might be used as a source of liver tissue for development of models, therapies, and tissue-engineering applications. However, we have been unable to generate and maintain stable and mature adult liver cells ex vivo. We review factors that promote hepatocyte differentiation and maturation, including growth factors, transcription factors, microRNAs, small molecules, and the microenvironment. We discuss how the hepatic circulation, microbiome, and nutrition affect liver function, and the criteria for considering cells derived from stem cells to be fully mature hepatocytes. We explain the challenges to cell transplantation and consider future technologies for use in hepatic stem cell maturation, including 3-dimensional biofabrication and genome modification.

Self-assembled liver organoids recapitulate hepatobiliary organogenesis in vitro.

Several three-dimensional cell culture systems are currently available to create liver organoids. In gneral, these systems display better physiologic and metabolic aspects of intact liver tissue compared with two-dimensional culture systems. However, none reliably mimic human liver development, including parallel formation of hepatocyte and cholangiocyte anatomical structures. Here, we show that human fetal liver progenitor cells self-assembled inside acellular liver extracellular matrix scaffolds to form three-dimensional liver organoids that recapitulated several aspects of hepatobiliary organogenesis and resulted in concomitant formation of progressively more differentiated hepatocytes and bile duct structures. The duct morphogenesis process was interrupted by inhibiting Notch signaling, in an attempt to create a liver developmental disease model with a similar phenotype to Alagille syndrome. Conclusion: In the current study, we created an in vitro model of human liver development and disease, physiology, and metabolism, supported by liver extracellular matrix substrata; we envision that it will be used in the future to study mechanisms of hepatic and biliary development and for disease modeling and drug screening. (Hepatology 2018;67:750-761).

Hepatocyte-like cells generated by direct reprogramming from murine somatic cells can repopulate decellularized livers.

Direct reprogramming represents an easy technique to generate induced hepatocytes (iHeps) from somatic cells. However, current protocols are accompanied by several drawbacks as iHeps are heterogenous and lack fully mature phenotypes of primary hepatocytes. Here, we established a polycistronic expression system to induce the direct reprogramming of mouse embryonic fibroblasts towards hepatocytes. The resulting iHeps are homogenous and display key properties of primary hepatocytes, such as expression of hepatocyte markers, albumin secretion, and presence of liver transaminases. iHeps also possess the capacity to repopulate decellularized liver tissue and exhibit enhanced hepatic maturation. As such, we present a novel strategy to generate homogenous and functional iHeps for applications in tissue engineering and cell therapy.

Naturally-Derived Biomaterials for Tissue Engineering Applications.

Naturally-derived biomaterials have been used for decades in multiple regenerative medicine applications. From the simplest cell microcarriers made of collagen or alginate, to highly complex decellularized whole-organ scaffolds, these biomaterials represent a class of substances that is usually first in choice at the time of electing a functional and useful biomaterial. Hence, in this chapter we describe the several naturally-derived biomaterials used in tissue engineering applications and their classification, based on composition. We will also describe some of the present uses of the generated tissues like drug discovery, developmental biology, bioprinting and transplantation.

De- and recellularization of the pig uterus: a bioengineering pilot study.

Absolute uterine factor infertility, or the absence of a functional uterus, has a prevalence of 3%-5% in the general population. Despite the great strides being made in reproductive medicine, patients diagnosed with absolute uterine factor infertility remain untreatable. The only available solution has been gestational surrogacy, but recently the Brannström group presented a viable alternative by reporting the first successful live birth after uterus transplantation. Similar to other transplantations, this approach has inherent limitations such as the paucity of donor organs and the need for long-term immunosuppression. Whole organ de- and recellularization, a novel tissue engineering approach within the field of regenerative medicine, could eventually provide another solution. Several groups have described animal models in which they have performed decellularization of whole uteri, while maintaining the extracellular matrix to enable recellularization attempts. Our work offers a new perspective; in decellularizing the porcine uterus, this constitutes the first pilot study using large whole reproductive organs. We demonstrated the preservation of a reusable/functional extracellular matrix while maintaining its vascular network. Furthermore, we report the first use of human side population stem cells in the successful recellularization of small acellular disk scaffolds procured from the decellularized organs. To conclude, this research opens new avenues in whole uterus bioengineering, opening the way towards the transplantation of functional bioengineered uteri into humans.

Immune responses to bioengineered organs.

PURPOSE OF REVIEW: Organ donation in the United States registered 9079 deceased organ donors in 2015. This high percentage of donations allowed organ transplantation in 29 851 recipients. Despite increasing numbers of transplants performed in comparison with previous years, the numbers of patients that are in need for a transplant increase every year at a higher rate. This reveals that the discrepancy between the demand and availability of organs remains fundamental problem in organ transplantation. RECENT FINDINGS: Development of bioengineered organs represents a promising approach to increase the pool of organs for transplantation. The technology involves obtaining complex three-dimensional scaffolds that support cellular activity and functional remodeling though tissue recellularization protocols using progenitor cells. This innovative approach integrates cross-thematic approaches from specific areas of transplant immunology, tissue engineering and stem cell biology, to potentially manufacture an unlimited source of donor organs for transplantation. SUMMARY: Although bioengineered organs are thought to escape immune recognition, the potential immune reactivity toward each of its components has not been studied in detail. Here, we summarize the host immune response toward different progenitor cells and discuss the potential implications of using nonself biological scaffolds to develop bioengineered organs.

Cardiovascular risk assessment in inflammatory bowel disease with metabolic dysfunction-associated steatotic liver disease.

BACKGROUND AND AIMS: Inflammatory bowel disease (IBD) has been reported to increase the risk of early atherosclerosis even in young patients. Moreover, metabolic dysfunction-associated steatotic liver disease (MASLD), which has been linked to IBD, is a well-recognized but underdiagnosis entity related to cardiovascular risk. We analyze the impact of MASLD in IBD patients' cardiovascular risk through both advanced lipoprotein profile sorted by nuclear magnetic resonance spectroscopy, and carotid artery intima-media thickness (CIMT). METHODS: Cross-sectional cohort study which involves 941 IBD adult outpatients. Of them, 50 patients with IBD who met criteria for MASLD and 50 with IBD without MASLD, matched by sex and age were included. Alterations in CIMT were evaluated considering abnormal measures above the 75th percentile adjusted for sex and age. Specific advanced lipoprotein profile was also carried out. RESULTS: Most of the patients had an abnormal CIMT (58%). MASLD (OR=5.05, CI 95%=1.71-14.92) and female sex (OR=3.32, CI 95%=1.03-10) were significantly associated with CIMT alterations. Dense LDL particles (with high cholesterol composition in general cohort (OR=3.62, 95% CI=1.07-12.19) and high triglycerides density in young subgroup (OR=6.25, 95% CI=1.04-50) but not total LDL cholesterol were associated with CIMT alterations. CONCLUSIONS: MASLD and female sex are associated with early atherosclerosis in IBD patients. Dense LDL particle in combination with vascular imaging findings should be evaluated as non-invasive tools in the management of cardiovascular risk in IBD patients.

Scheimpflug-Based Corneal Biomechanical Analysis As A Predictor of Glaucoma in Eyes With High Myopia.

Purpose: To address if corneal biomechanical behavior has a predictive value for the presence of glaucomatous optical neuropathy in eyes with high myopia. Patients and Methods: This observational cross-sectional study included 209 eyes from 108 consecutive patients, divided into four groups: high myopia and primary open-angle glaucoma (POAG) - HMG, n = 53; high myopia without POAG - HMNG, n = 53; non-myopic with POAG - POAG, n = 50; non-myopic and non-POAG- NMNG, n = 53. Biomechanical assessment was made through a Scheimpflug-camera-based technology. Receiver operating characteristic curves were made for the discrimination between groups. Multivariable logistic regression models were performed to address the predictive value of corneal biomechanics for the presence of glaucoma. Results: Areas Under the Receiver Operating Characteristic (AUROCs) above 0.6 were found in 6 parameters applied to discriminate between HMG and HMNG and six parameters to discriminate between POAG and NMNG. The biomechanical models with the highest power of prediction for the presence of glaucoma included 5 parameters with an AUROC of 0.947 for eyes with high myopia and 6 parameters with an AUROC of 0.857 for non-myopic eyes. In the final model, including all eyes, and adjusted for the presence of high myopia, the highest power of prediction for the presence of glaucoma was achieved including eight biomechanical parameters, with an AUROC of 0.917. Conclusion: Corneal biomechanics demonstrated differences in eyes with glaucoma and mainly in myopic eyes. A biomechanical model based on multivariable logistic regression analysis and adjusted for high myopia was built, with an overall probability of 91.7% for the correct prediction of glaucomatous damage.

Crohn´s disease is an independent risk factor for liver fibrosis in patients with inflammatory bowel disease and non-alcoholic fatty liver disease.

BACKGROUND AND AIMS: Controversial data have been reported regarding the prevalence of Non-Alcoholic Fatty Liver Disease (NAFLD) in Inflammatory Bowel Disease (IBD) population and IBD-related risk factors. The aim of the study was to assess the prevalence and risk factors associated with NAFLD and liver fibrosis in IBD participants compared with non-IBD controls. METHODS: Cross-sectional, case-control study including 741 IBD cases and 170 non-IBD controls, matched by sex and age. All participants underwent liver ultrasound, transient elastography and laboratory tests. A logistic regression multivariable analysis was performed adjusting for classic metabolic risk factors and history of systemic steroid use. RESULTS: The prevalence of NAFLD and significant liver fibrosis was 45 % and 10 % in IBD group, and 40 % and 2.9 % in non-IBD group (p = 0.255 and 0.062, respectively). Longer IBD duration (aOR 1.02 95% CI (1.001-1.04)) and older age at IBD diagnosis (aOR 1.02 95 % CI (1.001-1.04)) were independent risk factors for NAFLD in IBD group. Crohn´s Disease was an independent risk factor for significant liver fibrosis in participants with IBD and NAFLD (aOR 3.97 95 % CI (1.78-8.96)). NAFLD occurred at lower BMI levels in IBD group with NAFLD compared to non-IBD group with NAFLD (aOR 0.92 95 % CI (0.87-0.98)). CONCLUSIONS: Although we found no differences in the prevalence of NAFLD and liver fibrosis between IBD group and non-IBD group, our findings suggest that liver fibrosis progression should be closely monitored in patients with concomitant CD and NAFLD, more in particular in those with long standing active disease.

Cell transplantation-based regenerative medicine in liver diseases.

This review aims to evaluate the current preclinical state of liver bioengineering, the clinical context for liver cell therapies, the cell sources, the delivery routes, and the results of clinical trials for end-stage liver disease. Different clinical settings, such as inborn errors of metabolism, acute liver failure, chronic liver disease, liver cirrhosis, and acute-on-chronic liver failure, as well as multiple cellular sources were analyzed; namely, hepatocytes, hepatic progenitor cells, biliary tree stem/progenitor cells, mesenchymal stromal cells, and macrophages. The highly heterogeneous clinical scenario of liver disease and the availability of multiple cellular sources endowed with different biological properties make this a multidisciplinary translational research challenge. Data on each individual liver disease and more accurate endpoints are urgently needed, together with a characterization of the regenerative pathways leading to potential therapeutic benefit. Here, we critically review these topics and identify related research needs and perspectives in preclinical and clinical settings.

Unravelling the cellular response to the SARS-COV-2 vaccine in inflammatory bowel disease patients on biologic drugs.

Suboptimal vaccine response is a significant concern in patients with Inflammatory Bowel Disease (IBD) receiving biologic drugs. This single-center observational study involved 754 patients with IBD. In Phase I (October 2020-April 2021), 754 IBD participants who had not previously received the SARS-CoV-2 vaccine, underwent blood extraction to assess the seroprevalence of SARS-CoV-2 infection and IBD-related factors. Phase II (May 2021-October 2021) included a subgroup of 52 IBD participants with confirmed previous SARS-CoV-2 infection, who were studied for humoral and cellular response to the SARS-CoV-2 vaccine. In Phase I, treatment with anti-TNF was associated with lower rates of seroconversion (aOR 0.25 95% CI [0.10-0.61]). In Phase II, a significant increase in post-vaccination IgG levels was observed regardless of biologic treatment. However, patients treated with anti-TNF exhibited significantly lower IgG levels compared to those without IBD therapy (5.32 ± 2.47 vs. 7.99 ± 2.59 U/ml, p = 0.042). Following vaccination, a lymphocyte, monocyte, and NK cell activation pattern was observed, with no significant differences between patients receiving biologic drugs and those without IBD treatment. Despite lower seroprevalence and humoral response to the SARS-CoV-2 vaccine in patients treated with anti-TNF, the cellular response to the vaccine did not differ significantly from that patients without IBD therapy.

Regenerative medicine as applied to general surgery.

The present review illustrates the state of the art of regenerative medicine (RM) as applied to surgical diseases and demonstrates that this field has the potential to address some of the unmet needs in surgery. RM is a multidisciplinary field whose purpose is to regenerate in vivo or ex vivo human cells, tissues, or organs to restore or establish normal function through exploitation of the potential to regenerate, which is intrinsic to human cells, tissues, and organs. RM uses cells and/or specially designed biomaterials to reach its goals and RM-based therapies are already in use in several clinical trials in most fields of surgery. The main challenges for investigators are threefold: Creation of an appropriate microenvironment ex vivo that is able to sustain cell physiology and function in order to generate the desired cells or body parts; identification and appropriate manipulation of cells that have the potential to generate parenchymal, stromal and vascular components on demand, both in vivo and ex vivo; and production of smart materials that are able to drive cell fate.

The use of whole organ decellularization for the generation of a vascularized liver organoid.

UNLABELLED: A major roadblock to successful organ bioengineering is the need for a functional vascular network within the engineered tissue. Here, we describe the fabrication of three-dimensional, naturally derived scaffolds with an intact vascular tree. Livers from different species were perfused with detergent to selectively remove the cellular components of the tissue while preserving the extracellular matrix components and the intact vascular network. The decellularized vascular network was able to withstand fluid flow that entered through a central inlet vessel, branched into an extensive capillary bed, and coalesced into a single outlet vessel. The vascular network was used to reseed the scaffolds with human fetal liver and endothelial cells. These cells engrafted in their putative native locations within the decellularized organ and displayed typical endothelial, hepatic, and biliary epithelial markers, thus creating a liver-like tissue in vitro. CONCLUSION: These results represent a significant advancement in the bioengineering of whole organs. This technology may provide the necessary tools to produce the first fully functional bioengineered livers for organ transplantation and drug discovery.

Cellular therapy and bioartificial approaches to liver replacement.

PURPOSE OF REVIEW: The success of liver transplantation has increased over the past 20 years due to improved immunosuppressive medications, surgical technique and donor-recipient selection. To date, the number of patients waiting for a liver transplant exceeds the number of transplants performed yearly by over a 2 : 1 ratio. Despite efforts to expand the donor pool, mortality of patients waiting for a liver remains high due to the shortage of donor organs. Herein, we discuss options for liver replacement that are currently under development. RECENT FINDINGS: Extracorporeal bioactive liver perfusion devices were investigated in the late 1990s and preliminarily demonstrated safety but failed to show clinical efficacy. Current research is ongoing, but the focus has shifted to xenotransplantation of whole organs, organ engineering and cell transplantation. These new modalities are limited to small and large animal studies and each present unique advantages and limitations. SUMMARY: Discovery of new sources of organs or cells to replace a damaged liver may be the only long-term solution to provide definitive therapy to all patients who require transplantation. The past 2 years have seen notable achievements in xenotransplantation, tissue engineering and cell transplantation. Though challenges remain, now identified, they may be readily solved.

How Donor and Surgical Factors Affect the Viability and Functionality of Human Hepatocytes Isolated From Liver Resections.

Liver resections are a significant source of primary human hepatocytes used mainly in artificial liver devices and pharmacological and biomedical studies. However, it is not well known how patient-donor and surgery-dependent factors influence isolated hepatocytes' yield, viability, and function. Hence, we aimed to analyze the impact of all these elements on the outcome of human hepatocyte isolation. Patients and methods: Hepatocytes were isolated from liver tissue from patients undergoing partial hepatectomy using a two-step collagenase method. Hepatocyte viability, cell yield, adhesion, and functionality were measured. In addition, clinical and analytical patient variables were collected and the use or absence of vascular clamping and its type (continuous or intermittent) plus the ischemia times during surgery. Results: Malignant disease, previous chemotherapy, and male gender were associated with lower hepatocyte viability and isolation cell yields. The previous increase in transaminases was also associated with lower yields on isolation and lower albumin production. Furthermore, ischemia secondary to vascular clamping during surgery was inversely correlated with the isolated hepatocyte viability. An ischemia time higher than 15 min was related to adverse effects on viability. Conclusion: Several factors correlated with the patient and the surgery directly influence the success of human hepatocyte isolation from patients undergoing liver resection.

Safety and efficacy of BCG re-vaccination in relation to COVID-19 morbidity in healthcare workers: A double-blind, randomised, controlled, phase 3 trial.

Background: BCG vaccination prevents severe childhood tuberculosis (TB) and was introduced in South Africa in the 1950s. It is hypothesised that BCG trains the innate immune system by inducing epigenetic and functional reprogramming, thus providing non-specific protection from respiratory tract infections. We evaluated BCG for reduction of morbidity and mortality due to COVID-19 in healthcare workers in South Africa. Methods: This randomised, double-blind, placebo-controlled trial recruited healthcare workers at three facilities in the Western Cape, South Africa, unless unwell, pregnant, breastfeeding, immunocompromised, hypersensitivity to BCG, or undergoing experimental COVID-19 treatment. Participants received BCG or saline intradermally (1:1) and were contacted once every 4 weeks for 1 year. COVID-19 testing was guided by symptoms. Hospitalisation, COVID-19, and respiratory tract infections were assessed with Cox proportional hazard modelling and time-to-event analyses, and event severity with post hoc Markovian analysis. This study is registered with ClinicalTrials.gov, NCT04379336. Findings: Between May 4 and Oct 23, 2020, we enrolled 1000 healthcare workers with a median age of 39 years (IQR 30-49), 70·4% were female, 16·5% nurses, 14·4% medical doctors, 48·5% had latent TB, and 15·3% had evidence of prior SARS-CoV-2 exposure. Hospitalisation due to COVID-19 occurred in 15 participants (1·5%); ten (66·7%) in the BCG group and five (33·3%) in the placebo group, hazard ratio (HR) 2·0 (95% CI 0·69-5·9, p = 0·20), indicating no statistically significant protection. Similarly, BCG had no statistically significant effect on COVID-19 (p = 0·63, HR = 1·08, 95% CI 0·82-1·42). Two participants (0·2%) died from COVID-19 and two (0·2%) from other reasons, all in the placebo group. Interpretation: BCG did not protect healthcare workers from SARS-CoV-2 infection or related severe COVID-19 disease and hospitalisation. Funding: Funding provided by EDCTP, grant number RIA2020EF-2968. Additional funding provided by private donors including: Mediclinic, Calavera Capital (Pty) Ltd, Thys Du Toit, Louis Stassen, The Ryan Foundation, and Dream World Investments 401 (Pty) Ltd. The computations were enabled by resources in project SNIC 2020-5-524 provided by the Swedish National Infrastructure for Computing (SNIC) at UPPMAX, partially funded by the Swedish Research Council through grant agreement No. 2018-05,973.