Evolution of organoid technology: Lessons learnt in Co-Culture systems from developmental biology.

In recent years, the development of 3D organoids has opened new avenues of investigation into development, physiology, and regenerative medicine. Organoid formation and the process of organogenesis share common developmental pathways; thus, our knowledge of developmental biology can help model the complexity of different organs to refine organoids into a more sophisticated platform. The developmental process is strongly dependent on complex networks and communication of cell-cell and cell-matrix interactions among different cell populations and their microenvironment, during embryogenesis. These interactions affect cell behaviors such as proliferation, survival, migration, and differentiation. Co-culture systems within the organoid technology were recently developed and provided the highly physiologically relevant systems. Supportive cells including various types of endothelial and stromal cells provide the proper microenvironment, facilitate organoid assembly, and improve vascularization and maturation of organoids. This review discusses the role of the co-culture systems in organoid generation, with a focus on how knowledge of developmental biology has directed and continues to shape the development of more evolved 3D co-culture system-derived organoids.

In vitro modeling of liver fibrosis in 3D microtissues using scalable micropatterning system.

Liver fibrosis is the late consequence of chronic liver inflammation which could eventually lead to cirrhosis, and liver failure. Among various etiological factors, activated hepatic stellate cells (aHSCs) are the major players in liver fibrosis. To date, various in vitro liver fibrosis models have been introduced to address biological and medical questions. Availability of traditional in vitro models could not fully recapitulate complicated pathology of liver fibrosis. The purpose of this study was to develop a simple and robust model to investigate the role of aHSCs on the progression of epithelial to mesenchymal transition (EMT) in hepatocytes during liver fibrogenesis. Therefore, we applied a micropatterning approach to generate 3D co-culture microtissues consisted of HepaRG and human umbilical cord endothelial cells (HUVEC) which co-cultured with inactivated LX-2 cells or activated LX-2 cells, respectively, as normal or fibrotic liver models in vitro. The result indicated that the activated LX-2 cells could induce EMT in HepaRG cells through activation of TGF-ß/SMAD signaling pathway. Besides, in the fibrotic microtissue, physiologic function of HepaRG cells attenuated compared to the control group, e.g., metabolic activity and albumin secretion. Moreover, our results showed that after treatment with Galunisertib, the fibrogenic properties decreased, in the term of gene and protein expression. In conclusion, it is proposed that aHSCs could lead to EMT in hepatocytes during liver fibrogenesis. Furthermore, the scalable micropatterning approach could provide enough required liver microtissues to prosper our understanding of the mechanisms involved in the progression of liver fibrosis as well as high throughput (HT) drug screening.

Prenatal liver stromal cells: Favorable feeder cells for long-term culture of hepatic progenitor cells.

Clinical and pharmaceutical applications of primary hepatocytes (PHs) are limited due to inadequate number of donated livers and potential challenges in successful maintenance of PHs in culture. Freshly isolated hepatocytes lose their specific features and rapidly de-differentiate in culture. Bipotent hepatoblasts, as liver precursor cells that can differentiate into both hepatocytes and cholangiocytes (Alb- and Ck19-positive cells, respectively), could be used as an alternative and reliable cell source to produce enough PHs for drug discovery or possible clinical applications. In this study, growth factor-free coculture systems of prenatal or postnatal murine liver stromal cells (pre-LSCs or post-LSCs, respectively) were used as feeder cells to support freshly isolated mice hepatoblasts. DLK1-positive hepatoblasts were isolated from mouse fetuses (E14.5) and cocultured with feeder cells under adherent conditions. The hepatoblasts' bipotent features, proliferation rate, and colony formation capacity were assessed on day 5 and 7 post-seeding. Immunofluorescence staining showed that the hepatoblasts remained double positive for Alb and Ck19 on both Pre- and Post-LSCs, after 5 and 7 days of coculture. Moreover, application of pre-LSCs as feeder cells significantly increased the number of DLK1-positive cells and their proliferation rate (ie, increased the number of Ki-67 positive cells) on day 7, compared to Post-LSCs group. Finally, to address our ultimate goal, which was an extension of hepatoblasts ex vivo maintenance, 3D spheres of isolated hepatoblasts were, cultured in conditioned medium (CM) derived from pre-LSCs until day 30. It was observed that the CM derived from Pre-LSCs could successfully prolong the maintenance of hepatic progenitor cells (HPCs) in 3D suspension culture.

Review on Kidney-Liver Crosstalk: Pathophysiology of Their Disorders.

Kidney-liver crosstalk plays a crucial role in normal and certain pathological conditions. In pathologic states, both renal-induced liver damage and liver-induced kidney diseases may happen through these kidney-liver interactions. This bidirectional crosstalk takes place through the systemic conditions that mutually influence both the liver and kidneys. Ischemia and reperfusion, cytokine release and pro-inflammatory signaling pathways, metabolic acidosis, oxidative stress, and altered enzyme activity and metabolic pathways establish the base of this interaction between the kidneys and liver. In these concomitant kidney-liver diseases, the survival rates strongly correlate with early intervention and treatment of organ dysfunction. Proper care of a nephrologist and hepatologist and the identification of pathological conditions using biomarkers at early stages are necessary to prevent the complications induced by this complex and potentially vicious cycle. Therefore, understanding the characteristics of this crosstalk is essential for better management. In this review, we discussed the available literature concerning the detrimental effects of kidney failure on liver functions and liver-induced kidney diseases.

Cell and cell-derivative-based therapy for liver diseases: current approaches and future promises.

INTRODUCTION: According to the recent updates from World Health Organization, liver diseases are the 12th most common cause of mortality. Currently, orthotopic liver transplantation (OLT) is the most effective and the only treatment for end-stage liver diseases. Owing to several shortcomings like finite numbers of healthy organ donors, lifelong immunosuppression, and complexity of the procedure, cell and cell-derivatives therapies have emerged as a potential therapeutic alternative for liver diseases. Various cell types and therapies have been proposed and their therapeutic effects evaluated in preclinical or clinical studies, including hepatocytes, hepatocyte-like cells (HLCs) derived from stem cells, human liver stem cells (HLSCs), combination therapies with various types of cells, organoids, and implantable cell-biomaterial constructs with synthetic and natural polymers or even decellularized extracellular matrix (ECM). AREAS COVERED: In this review, we highlighted the current status of cell and cell-derivative-based therapies for liver diseases. Furthermore, we discussed future prospects of using HLCs, liver organoids, and their combination therapies. EXPERT OPINION: Promising application of stem cell-based techniques including iPSC technology has been integrated into novel techniques such as gene editing, directed differentiation, and organoid technology. iPSCs offer promising prospects to represent novel therapeutic strategies and modeling liver diseases.

Conjugated Linoleic Acid Treatment Attenuates Cancerous features in Hepatocellular Carcinoma Cells.

Background: A growing number of hepatocellular carcinoma (HCC), and recurrence frequency recently have drawn researchers' attention to alternative approaches. The concept of differentiation therapies (DT) relies on inducing differentiation in HCC cells in order to inhibit recurrence and metastasis. Hepatocyte nuclear factor 4 alpha (HNF4α) is the key hepatogenesis transcription factor and its upregulation may decrease the invasiveness of cancerous cells by suppressing epithelial-mesenchymal transition (EMT). This study aimed to evaluate the effect of conjugated linoleic acid (CLA) treatment, natural ligand of HNF4α, on the proliferation, migration, and invasion capacities of HCC cells in vitro. Materials and Method. Sk-Hep-1 and Hep-3B cells were treated with different doses of CLA or BIM5078 [1-(2'-chloro-5'-nitrobenzenesulfonyl)-2-methylbenzimidazole], an HNF4α antagonist. The expression levels of HNF4a and EMT related genes were evaluated and associated to hepatocytic functionalities, migration, and colony formation capacities, as well as to viability and proliferation rate of HCC cells. Results: In both HCC lines, CLA treatment induced HNF4α expression in parallel to significantly decreased EMT marker levels, migration, colony formation capacity, and proliferation rate, whereas BIM5078 treatment resulted in the opposite effects. Moreover, CLA supplementation also upregulated ALB, ZO1, and HNF4α proteins as well as glycogen storage capacity in the treated HCC cells. Conclusion: CLA treatment can induce a remarkable hepatocytic differentiation in HCC cells and attenuates cancerous features. This could be as a result of HNF4a induction and EMT inhibition.

Promoting Maturation of Human Pluripotent Stem Cell-Derived Renal Microtissue by Incorporation of Endothelial and Mesenchymal Cells.

Directed differentiation of human pluripotent stem cells (hPSCs) uses a growing number of small molecules and growth factors required for in vitro generation of renal lineage cells. Although current protocols are relatively inefficient or expensive. The first objective of the present work was to establish a new differentiation protocol for generating renal precursors. We sought to determine if inducer of definitive endoderm 1 (IDE1), a cost-effective small molecule, can be used to replace activin A. Gene expression data showed significantly increased expressions of nephrogenic markers in cells differentiated with 20 nM IDE1 compared with cells differentiated with activin A. Thus, renal lineage cells could be generated by this alternative approach. Afterward, we determined whether coculture of endothelial and mesenchymal cells could increase the maturation of three-dimensional (3D) renal structures. For this purpose, we employed a 3D coculture system in which hPSC-derived kidney precursors were cocultured with endothelial cells (ECs) and mesenchymal stem cells (MSCs), hereafter named RMEM (renal microtissue derived from coculture of renal precursors with endothelial and mesenchymal stem cells). hPSC-derived kidney precursors were cultured either alone [renal microtissue (RM)] or in coculture with human umbilical vein endothelial cells and human bone marrow-derived mesenchymal stem cells at an approximate ratio of 10:7:2, respectively. Immunofluorescent staining showed expressions of kidney-specific markers synaptopodin, LTL, and E-cadherin, as well as CD31+ ECs that were distributed throughout the RMEMs. Quantitative real-time polymerase chain reaction analysis confirmed a significant increase in gene expressions of the renal-specific markers in RMEMs compared with RMs. These findings demonstrated that renal precursors cocultured with endothelial and MSCs showed greater maturity compared with RMs. Moreover, ex ovo transplantation induced further maturation in the RMEM constructs. Our novel approach enabled the generation of RMEM that could potentially be used in high-throughput drug screening and nephrotoxicology studies.

Tissue-Specific Microparticles Improve Organoid Microenvironment for Efficient Maturation of Pluripotent Stem-Cell-Derived Hepatocytes.

Liver organoids (LOs) are receiving considerable attention for their potential use in drug screening, disease modeling, and transplantable constructs. Hepatocytes, as the key component of LOs, are isolated from the liver or differentiated from pluripotent stem cells (PSCs). PSC-derived hepatocytes are preferable because of their availability and scalability. However, efficient maturation of the PSC-derived hepatocytes towards functional units in LOs remains a challenging subject. The incorporation of cell-sized microparticles (MPs) derived from liver extracellular matrix (ECM), could provide an enriched tissue-specific microenvironment for further maturation of hepatocytes inside the LOs. In the present study, the MPs were fabricated by chemical cross-linking of a water-in-oil dispersion of digested decellularized sheep liver. These MPs were mixed with human PSC-derived hepatic endoderm, human umbilical vein endothelial cells, and mesenchymal stromal cells to produce homogenous bioengineered LOs (BLOs). This approach led to the improvement of hepatocyte-like cells in terms of gene expression and function, CYP activities, albumin secretion, and metabolism of xenobiotics. The intraperitoneal transplantation of BLOs in an acute liver injury mouse model led to an enhancement in survival rate. Furthermore, efficient hepatic maturation was demonstrated after ex ovo transplantation. In conclusion, the incorporation of cell-sized tissue-specific MPs in BLOs improved the maturation of human PSC-derived hepatocyte-like cells compared to LOs. This approach provides a versatile strategy to produce functional organoids from different tissues and offers a novel tool for biomedical applications.

Modeling Hepatotropic Viral Infections: Cells vs. Animals.

The lack of an appropriate platform for a better understanding of the molecular basis of hepatitis viruses and the absence of reliable models to identify novel therapeutic agents for a targeted treatment are the two major obstacles for launching efficient clinical protocols in different types of viral hepatitis. Viruses are obligate intracellular parasites, and the development of model systems for efficient viral replication is necessary for basic and applied studies. Viral hepatitis is a major health issue and a leading cause of morbidity and mortality. Despite the extensive efforts that have been made on fundamental and translational research, traditional models are not effective in representing this viral infection in a laboratory. In this review, we discuss in vitro cell-based models and in vivo animal models, with their strengths and weaknesses. In addition, the most important findings that have been retrieved from each model are described.

Engineering a Model to Study Viral Infections: Bioprinting, Microfluidics, and Organoids to Defeat Coronavirus Disease 2019 (COVID-19).

While the number of studies related to severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is constantly growing, it is essential to provide a framework of modeling viral infections. Therefore, this review aims to describe the background presented by earlier used models for viral studies and an approach to design an "ideal" tissue model for SARS-CoV-2 infection. Due to the previous successful achievements in antiviral research and tissue engineering, combining the emerging techniques such as bioprinting, microfluidics, and organoid formation are considered to be one of the best approaches to form in vitro tissue models. The fabrication of an integrated multi-tissue bioprinted platform tailored for SARS-CoV-2 infection can be a great breakthrough that can help defeat coronavirus disease in 2019.

Protective Effects of Royal Jelly on Oxymetholone-Induced Liver Injury in Mice.

BACKGROUND: The present study was carried out to investigate the possible protective effects of royal jelly (RJ) on oxymetholone (OXM)-induced oxidative liver injuries in mice. METHODS: In total, 32 adult male NMRI mice were divided into four groups of eight mice each. Mice in groups 1 and 2 were orally administered 5 mg/kg/day OXM for 30 days. At the same time, mice in group 3 received RJ at a dose of 100 mg/kg/day. Saline control and RJ control groups were also included in this study. RESULTS: Administration of 5 mg/kg OXM resulted in a significant decrease in total antioxidant capacity and catalase activity, as well as a significant increase in malondialdehyde (P<0.05). In addition, OXM-administrated mice showed a slight increase in liver enzymes, including alanine amino transferase, aspartate amino transferase, and alkaline phosphatase. Although OXM caused histopathological changes in the liver, RJ could significantly improve all of the above-mentioned parameters at a dose of 100 mg/kg. CONCLUSION: The results of the present study indicated that RJ has a partially protective effect on OXM-induced liver toxicity in mice.

Protective Effect of Royal Jelly on In Vitro Fertilization (IVF) in Male Mice Treated with Oxymetholone.

OBJECTIVE: This study aimed to investigate the effects of royal jelly (RJ) on catalase, total antioxidant capacity and embryo development in adult mice treated with oxymetholone (OXM). MATERIALS AND METHODS: In this exprimental study, 32 male and 96 female adult Naval Medical Research Institute (NMRI) mice (7-9 weeks of age) with a ratio of 1:3 for fertili- zation purposes were randomly divided into 4 groups as follows: i. Control group (n=8) receiving 0.1 ml/mice saline daily by gavage for 30 day, ii. RJ group (n=8) treated with RJ at a dose of 100 mg/kg daily by gavage for 30 days, iii. OXM group (n=8) receiving OXM at the dose of 5 mg/kg daily by gavage for 30 days and iv. RJ+OXM group (n=8) receiving RJ at the dose of 100 mg/kg daily by gavage concomitant with 100 mg/kg OXM adminis- tration for 30 days. RESULTS: Analysis revealed a significant reduction in catalase, total antioxidant, as well as embryo development in OXM group (P<0.05). However, RJ group showed a salient recovery in the all of the above mentioned parameters and embryo toxicity. CONCLUSION: The results of this study indicated a partially protective effect of RJ against OXM-induced embryo toxicity.

Protective effect of royal jelly on the sperm parameters and testosterone level and lipid peroxidation in adult mice treated with oxymetholone.

UNLABELLED: Objectives : The aim of the present study was to evaluate protective effect of royal jelly on sperm parameters, testosterone level, and malondialdehyde (MDA) production in mice. MATERIALS AND METHODS: Thirty-two adult male NMRI mice weighing 30±2 g were used. All the animals were divided into 4 groups. CONTROL GROUP: received saline 0.1 ml/mouse/day orally for 30 days. Royal jelly group (RJ): received royal jelly at dose of 100 mg/kg daily for 30 days orally. Oxymetholone group: the received Oxymetholone (OX) at dose of 5 mg/kg daily for 30 days orally. Royal jelly+Oxymetholone group: received royal jelly at dose of 100 mg/kg/day orally concomitant with OX administration. Sperm count, sperm motility, viability, maturity, and DNA integrity were analyzed. Furthermore, serum testosterone and MDA concentrations were determined. RESULTS: In Oxymetholone group, sperm count, motility as well as testosterone concentration reduced significantly (p<0.05), while significant (p<0.05) increases in immature sperm, sperm with DNA damaged, and MDA concentration were announced in Oxymetholone group in comparison with control group and Royal jelly+Oxymetholone group. RJ caused partially amelioration in all of the above- mentioned parameters in Royal Jelly+Oxymetholone group. CONCLUSION: In conclusion, RJ may be used in combination with OX to improve OX-induced oxidative stress and male infertility.