Transplantable Liver Organoids, Too Many Cell Types to Choose: a Need for Scientific Self-Organization

Purpose of Review: Liver stem cells have been proposed as alternatives or additions for whole liver transplantations to accommodate the donor liver shortage. Various sources of liver stem cells have been described in experimental animal studies. Here we aim to compare the various studies. Recent Findings: Irrespective of the experimental design, the percentage of long-lasting survival and functional recovery of transplanted cells is generally very low. An exception to this are the proliferating hepatocytes transplanted into Fah(-/-) Rag2-/-IL2rg-/- mice; here 4-month post-transplantation around 65% repopulation was observed, and 11/14 mice survived in contrast to zero survival in sham-treated animals. Summary: Taking the different cellular sources for the organoids, the different maturation status of the transplanted cells, and the variable animal models into account, a paper-to-paper comparison is compromised. This lack of objective comparison restricts the translation of these model studies into clinical practice

Companion animal organoid technology to advance veterinary regenerative medicine

First year medical and veterinary students are made very aware that drugs can have very different effects in various species or even in breeds of one specific species. On the other hand, the "One Medicine" concept implies that therapeutic and technical approaches are exchangeable between man and animals. These opposing views on the (dis)similarities between human and veterinary medicine are magnified in regenerative medicine. Regenerative medicine promises to stimulate the body's own regenerative capacity via activation of stem cells and/or the application of instructive biomaterials. Although the potential is enormous, so are the hurdles that need to be overcome before large scale clinical implementation is realistic. It is in the advancement of regenerative medicine that veterinary regenerative medicine can play an instrumental and crucial role. This review describes the discovery of (adult) stem cells in domesticated animals, mainly cats and dogs. The promise of cell-mediated regenerative veterinary medicine is compared to the actual achievements, and this will lead to a set of unanswered questions (controversies, research gaps, potential developments in relation to fundamental, pre-clinical, and clinical research). For veterinary regenerative medicine to have impact, either for human medicine and/or for domesticated animals, answering these questions is pivotal

Morphological characterisation of portal myofibroblasts and hepatic stellate cells in the normal dog liver

BACKGROUND: Hepatic fibrosis is a common outcome of hepatic injury in both man and dog. Activated fibroblasts which develop myofibroblastic characteristics play an essential role in hepatic fibrogenesis, and are comprised of three subpopulations: 1) portal or septal myofibroblasts, 2) interface myofibroblasts and 3) the perisinusoidally located hepatic stellate cells (HSC). The present study was performed to investigate the immunohistochemical characteristics of canine portal myofibroblasts (MF) and HSC in the normal unaffected liver as a basis for further studies on fibrogenesis in canine liver disease. RESULTS: In the formalin-fixed and paraffin embedded normal canine liver vimentin showed staining of hepatic fibroblasts, probably including MF in portal areas and around hepatic veins; however, HSC were in general negative. Desmin proved to react with both portal MF and HSC. A unique feature of these HSC was the positive immunostaining for alpha-smooth muscle actin (α-SMA) and muscle-specific actin clone HHF35 (HHF35), also portal MF stained positive with these antibodies. Synaptophysin and glial fibrillary acidic protein (GFAP) were consistently negative in the normal canine liver. In a frozen chronic hepatitis case (with expected activated hepatic MF and HSC), HSC were negative to synaptophysin, GFAP and NCAM. Transmission electron microscopy (TEM) immunogold labelling for α-SMA and HHF35 recognized the positive cells as HSC situated in the space of Disse. CONCLUSION: In the normal formalin-fixed and paraffin embedded canine liver hepatic portal MF and HSC can be identified by α-SMA, HHF35 and to a lesser extent desmin immunostaining. These antibodies can thus be used in further studies on hepatic fibrosis. Synaptophysin, GFAP and NCAM do not seem suitable for marking of canine HSC. The positivity of HSC for α-SMA and HHF35 in the normal canine liver may eventually reflect a more active regulation of hepatic sinusoidal flow by these HSC compared to other species

Comparison of different methods to obtain and store liver biopsies for molecular and histological research

BACKGROUND: To minimize the necessary number of biopsies for molecular and histological research we evaluated different sampling techniques, fixation methods, and storage procedures for canine liver tissue. For addressing the aim, three biopsy techniques (wedge biopsy, Menghini, True-cut), four storage methods for retrieval of RNA (snap freezing, RNAlater, Boonfix, RLT-buffer), two RNA isolation procedures (Trizol and RNAeasy), and three different fixation protocols for histological studies (10% buffered formalin, RNAlater, Boonfix) were compared. Histological evaluation was based on hematoxylin-eosin (HE) and reticulin (fibrogenesis) staining, and rubeanic acid and rhodanine stains for copper. Immunohistochemical evaluation was performed for cytokeratin-7 (K-7), multidrug resistance binding protein-2 (MRP-2) and Hepar-1. RESULTS: RNA quality was best guaranteed by the combination of a Menghini biopsy with NaCl, followed by RNAlater preservation and RNAeasy mini kit extraction. These results were confirmed by quantitative RT-PCR testing. Reliable histological assessment for copper proved only possible in formalin fixed liver tissue. Short formalin fixation (1-4 hrs) improved immunohistochemical reactivity and preservation of good morphology in small liver biopsies. CONCLUSION: At least two biopsies (RNAlater and formalin) are needed. Since human and canine liver diseases are highly comparable, it is conceivable that the protocols described here can be easily translated into the human biomedical field

Regenerative and fibrotic pathways in canine hepatic portosystemic shunt and portal vein hypoplasia, new models for clinical hepatocyte growth factor treatment

BACKGROUND: We analyzed two spontaneous dog diseases characterized by subnormal portal perfusion and reduced liver growth: (i) congenital portosystemic shunts (CPSS) without fibrosis and (ii) primary portal vein hypoplasia (PPVH), a disease associated with fibrosis. These pathologies, that lack inflammation or cholestasis, may represent simplified models to study liver growth and fibrosis. To investigate the possible use of those models for hepatocyte growth factor (HGF) treatment, we studied the functionality of HGF signaling in CPSS and PPVH dogs and compared this to aged-matched healthy controls. RESULTS: We used quantitative real-time polymerase chain reaction (Q-PCR) to analyze the mRNA expression of HGF, transforming growth factor β1 (TGF-β1), and relevant mediators in liver biopsies from cases with CPSS or PPVH, in comparison with healthy control dogs. CPSS and PPVH were associated with a decrease in mRNA expression of HGF and of MET proto-oncogene (c-MET). Western blot analysis confirmed the Q-PCR results and showed that intracellular signaling components (protein kinase B/Akt, ERK1/2, and STAT3) were functional. The TGF-β1 mRNA levels were unchanged in CPSS whereas there was a 2-fold increase in PPVH indicating an active TGF-β1 pathway, consistent with the observation of fibrosis seen in PPVH. Western blots on TGF-β1 and phosphorylated Smad2 confirmed an activated pro-fibrotic pathway in PPVH. Furthermore, Q-PCR showed an increase in the amount of collagen I present in PPVH compared to CPSS and control, which was confirmed by Western blot analysis. CONCLUSION: The pathophysiological differences between CPSS and PPVH can adequately be explained by the Q-PCR measurements and Western blots. Although c-MET levels were reduced, downstream signaling seemed to be functional and provides a rational for HGF-supplementation in controlled studies with CPSS and PPVH. Furthermore both diseases may serve as simplified models for comparison with more complex chronic inflammatory diseases and cirrhosis

The establishment and characterization of the first canine hepatocellular carcinoma cell line, which resembles human oncogenic expression patterns

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most worldwide frequent primary carcinomas resulting in the death of many cirrhotic patients. Unfortunately, the molecular mechanisms of this cancer are not well understood; therefore, we need a good model system to study HCC. The dog is recognized as a promising model for human medical research, namely compared with rodents. The objective of this study was to establish and characterize a spontaneous canine tumor cell line as a potential model for studies on HCC. RESULTS: Histomorphological, biochemical, molecular biological and quantitative assays were performed to characterize the canine HCC cell line that originated from a dog with a spontaneous liver tumor. Morphological investigations provided strong evidence for the hepatocytic and neoplastic nature of the cell line, while biochemical assays showed that they produced liver-specific enzymes. PCR analysis confirmed expression of ceruloplasmin, alpha-fetoprotein and serum albumin. Quantitative RT-PCR showed that the canine HCC cell line resembles human HCC based on the measurements of expression profiles of genes involved in cell proliferation and apoptosis. CONCLUSIONS: We have developed a novel, spontaneous tumor liver cell line of canine origin that has many characteristics of human HCC. Therefore, the canine HCC cell line might be an excellent model for comparative studies on the molecular pathogenesis of HCC

Fibroblast growth factor-21 (FGF21) analogs as possible treatment options for diabetes mellitus in veterinary patients

Fibroblast growth factors (FGFs) are involved in numerous metabolic processes. The endocrine subfamily of FGFs, consisting of FGF19, FGF21, and FGF23, might have beneficial effects in the treatment of diabetes mellitus (DM) and/or obesity. The analog with the greatest potential, FGF21, lowers blood glucose levels, improves insulin sensitivity, and induces weight loss in several animal models. In this review we summarize recent (pre)clinical findings with FGF21 analogs in animal models and men. Furthermore, possible applications of FGF21 analogs for pets with DM will be discussed. As currently, information about the use of FGF21 analogs in pet animals is scarce

MIQE précis: Practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments

The conclusions of thousands of peer-reviewed publications rely on data obtained using fluorescence-based quantitative real-time PCR technology. However, the inadequate reporting of experimental detail, combined with the frequent use of flawed protocols is leading to the publication of papers that may not be technically appropriate. We take the view that this problem requires the delineation of a more transparent and comprehensive reporting policy from scientific journals. This editorial aims to provide practical guidance for the incorporation of absolute minimum standards encompassing the key assay parameters for accurate design, documentation and reporting of qPCR experiments (MIQE précis) and guidance on the publication of pure 'reference gene' articles

Human Cholangiocytes Form a Polarized and Functional Bile Duct on Hollow Fiber Membranes

Liver diseases affect hundreds of millions of people worldwide; most often the hepatocytes or cholangiocytes are damaged. Diseases of the biliary tract cause severe patient burden, and cholangiocytes, the cells lining the biliary tract, are sensitive to numerous drugs. Therefore, investigations into proper cholangiocyte functions are of utmost importance, which is restricted, in vitro, by the lack of primary human cholangiocytes allowing such screening. To investigate biliary function, including transepithelial transport, cholangiocytes must be cultured as three-dimensional (3D) ductular structures. We previously established murine intrahepatic cholangiocyte organoid-derived cholangiocyte-like cells (CLCs) and cultured them onto polyethersulfone hollow fiber membranes (HFMs) to generate 3D duct structures that resemble native bile ducts at the structural and functional level. Here, we established an efficient, stepwise method for directed differentiation of human intrahepatic cholangiocyte organoids (ICOs) into CLCs. Human ICO-derived CLCs showed key characteristics of cholangiocytes, such as the expression of structural and functional markers, formation of primary cilia, and P-glycoprotein-mediated transport in a polarized fashion. The organoid cultures exhibit farnesoid X receptor (FXR)-dependent functions that are vital to liver bile acid homeostasis in vivo. Furthermore, human ICO-derived CLCs cultured on HFMs in a differentiation medium form tubular architecture with some tight, confluent, and polarized monolayers that better mimic native bile duct characteristics than differentiated cultures in standard 2D or Matrigel-based 3D culture plates. Together, our optimized differentiation protocol to obtain CLC organoids, when applied on HFMs to form bioengineered bile ducts, will facilitate studying cholangiopathies and allow developing therapeutic strategies