Liver Stem Cells Research Articles

Human Liver Stem Cell Transplantation Alleviates Liver Fibrosis in a Rat Model of CCl4-Induced Liver Fibrosis.

Background and ObjectivesMesenchymal stem cells (MSCs) elicit therapeutic effects against liver fibrosis in animal models. Human liver stem cells (HLSCs) are cells isolated from human liver tissue that have mesenchymal morphology and express MSC markers. HLSCs also possess intrahepatic stem cell properties. We introduce a rat model of liver fibrosis and trans-portal transplantation of HLSC to demonstrate alleviation of liver fibrosis.Methods and ResultsLiver fibrosis was induced by intraperitoneal injection of Carbon tetrachloride (CCl4). Sprague Dawley rats underwent simultaneous partial hepatectomy of the left hepatic lobe and HLSC transplantation via the portal vein. Gross appearance of the liver observed following CCl4 injection showed cholestasis and surface nodularity. Sirius red staining revealed deposition of collagen fibers in the extracellular matrix (ECM). Following HLSC transplantation, human albumin secreting cells were detected by immunohistochemistry in liver specimens. Quantitative measurements of fibrosis area stained by Sirius red were compared between baseline and post-HLSC transplant (1×107 cells) following 10 weeks of CCl4 treatment liver specimens. Fibrosis area (p<0.05), serum markers of liver inflammation and fibrosis (AST, ALT levels and APRI, p<0.05) significantly decreased from baseline after HLSC transplantation. RNA expression in liver tissues revealed significant decrease in tissue inhibitor of matrix metalloproteinase 1 (TIMP1), TIMP2 expression and increase in hepatocyte growth factor expression following HLSC transplantation (p<0.05).ConclusionsHLSC transplantation effectively reduced the area of liver fibrosis with increased expression of factors promoting ECM degradation. These findings suggest the potential therapeutic role of HLSCs in various liver diseases presenting with liver fibrosis.

Ectopic expansion and vascularization of engineered hepatic tissue based on heparinized acellular liver matrix and mesenchymal stromal cell spheroids

Engineered liver organogenesis is not yet a viable therapeutic option, but ectopic liver histogenesis may be possible. Accumulating evidence has suggested that cell-cell interactions and cell-matrix interactions play an important role in determining the properties of engineered hepatic tissue in vitro and in vivo. In the current study, we utilized heparinized decellularized liver scaffolds and bone marrow mesenchymal stromal cell spheroids to fabricate engineered hepatic tissue, which was subsequently implanted into the omentum of Sprague-Dawley rats with or without liver injury. The survival, liver-specific functions, differentiation level and regenerative potential of the implanted hepatocyte-like cells in this ectopic liver system were evaluated, together with the vascularization status and therapeutic potential of the engineered hepatic tissue. We demonstrated that these hepatic grafts could survive and possess hepatocyte specific function in this ectopic liver system but could also efficiently anastomose with host vascular networks. Furthermore, we found that hepatocyte-like cells within grafts expanded more than 9-fold over the course of 4 weeks in immunocompetent rats with injured livers. Immunostaining revealed that these hepatocyte-like cells could self-organize into cord-like structures in vivo. In addition, these hepatic grafts exhibited therapeutic potential in liver injury induced by CCl4. To our knowledge, this is the first report demonstrating the generation of long-term vascularized hepatic parenchyma at ectopic sites based on decellularized liver scaffolds and stem cells. These results provide an economic and feasible method for engineering hepatic tissue from construction to transplantation. This methodology may be applicable in clinical medicine, especially metabolic liver diseases. Statement of significanceIn this manuscript, we presented an optimized method for the hepatic engineered tissue (HET) from construction to transplantation. The core of this method is utilizing the combination of heparinized decellularized liver scaffolds and stem cell spheroids, which could provide necessary cell-cell and cell-extracellular matrix interactions for HET in vitro and in vivo. We proved that these hepatic grafts could possess hepatocyte specific function and exhibit strong proliferative activity in ectopic liver system, but also able to anastomose with the host vascular networks efficiently and be compatible with the host immune system. This methodology may be possible one day to apply in clinical medicine, especially metabolic liver diseases.

Human Liver Stem Cell Derived Extracellular Vesicles Alleviate Kidney Fibrosis by Interfering with the β-Catenin Pathway through miR29b.

Human liver stem-cell-derived extracellular vesicles (HLSC-EVs) exhibit therapeutic properties in various pre-clinical models of kidney injury. We previously reported an overall improvement in kidney function following treatment with HLSC-EVs in a model of aristolochic acid nephropathy (AAN). Here, we provide evidence that HLSC-EVs exert anti-fibrotic effects by interfering with β-catenin signalling. A mouse model of AAN and an in vitro pro-fibrotic model were used. The β-catenin mRNA and protein expression, together with the pro-fibrotic markers α-SMA and collagen 1, were evaluated in vivo and in vitro following treatment with HLSC-EVs. Expression and functional analysis of miR29b was performed in vitro following HLSC-EV treatments through loss-of-function experiments. Results showed that expression of β-catenin was amplified both in vivo and in vitro, and β-catenin gene silencing in fibroblasts prevented AA-induced up-regulation of pro-fibrotic genes, revealing that β-catenin is an important factor in fibroblast activation. Treatment with HLSC-EVs caused increased expression of miR29b, which was significantly inhibited in the presence of α-amanitin. The suppression of the miR29b function with a selective inhibitor abolished the anti-fibrotic effects of HLSC-EVs, resulting in the up-regulation of β-catenin and pro-fibrotic α-Sma and collagen type 1 genes. Together, these data suggest a novel HLSC-EV-dependent regulatory mechanism in which β-catenin is down regulated by HLSC-EVs-induced miR29b expression.

In vitro expansion of fetal liver hematopoietic stem cells

Fetal liver hematopoietic stem and progenitor cells (HSPCs) have been considered appropriate for the management of aplastic anemia owing to their proliferative potential. Bone marrow recovery was possible in some cases; the engraftment potential of these cells, however was unsatisfactory, possibly due to the availability of a smaller number of these cells from a single fetus. The present study explores how we can expand fetal liver hematopoietic stem cells under in vitro conditions. We isolated mononuclear cells from fetal liver and hematopoietic stem cells were identified and analyzed by cell surface marker CD34. CD34+ fetal liver HSPCs cells were separated by magnetic cell sorting positive selection method. HSPCs (CD34+) were cultured by using 5 cytokines, stem cell factor (SCF), granulocyte macrophages-colony stimulating factor (GM-CSF), interleukin-6 (IL-6), Fms-related tyrosine kinase 3 (FLT-3) and erythropoietin (EPO), in 4 different combinations along with supplements, in serum-free culture media for 21 days. Cell viability continued to be greater than 90% throughout 21 days of culture. The cells expanded best in a combination of media, supplements and 5 cytokines, namely SCF, FLT-3, IL6, EPO and GM-CSF to yield a large number of total (CD34+ & CD34-) cells. Even though the total number of nucleated cells increased in culture significantly, levels of CD34 antigen expression declined steadily over this period.

Development of T-cell immunity in a liver and hematopoietic stem cell transplant recipient following coronavirus disease 2019 infection

The outbreak of coronavirus disease 2019 (COVID-19) has disproportionately affected patients with comorbidities, including recipients of solid organ and hematopoietic stem cell transplants (SCT). Upon recovery from COVID-19, the degree of the immunological protection from reinfection remains unclear. Here we describe a 33-year-old patient with erythropoietic protoporphyria (EPP) who had undergone liver transplantation with splenectomy followed by allogeneic SCT in 2013 after an initial failed liver and umbilical cord transplant. The patient developed mild upper respiratory symptoms in the spring of 2020 and was found to have anti-SARS-CoV2 antibodies suggesting past infection. A comprehensive analysis of T cell functionality in peripheral blood from this patient revealed robust in vitro responses against SARS CoV2 antigens Spike (S) 1 and 2, membrane (M) and nucleoprotein (NP), comparable to the reactivity against common antigens from CMV, EBV, Ad and BK viruses, while only low reactivity was seen in healthy donors without documented history of COVID-19. Moreover, the patient displayed a marked recognition of counterpart antigens from related human coronaviruses (hCoVs) 229E, OC43, NL63 and HKU1. Thus, despite lifelong immunosuppression, this survivor of COVID-19 retained a remarkable degree of immunocompetence and showed broad-spectrum T cell memory specific for SARS-CoV2 and related hCoVs including less studied hCoV M and NP antigens. The study highlights the role of cellular immunity after natural COVID-19 infection, suggesting broader use of T cell assays as a tool for risk stratification, measurement of immunocompetence and/or post-infection or post-vaccination protection, and possible T cell-based adoptive immunotherapy strategies in high-risk patients.

Human Liver Stem Cells: A Liver-Derived Mesenchymal Stromal Cell-Like Population With Pro-regenerative Properties.

Human liver stem cells (HLSCs) were described for the first time in 2006 as a new stem cell population derived from healthy human livers. Like mesenchymal stromal cells, HLSCs exhibit multipotent and immunomodulatory properties. HLSCs can differentiate into several lineages under defined in vitro conditions, such as mature hepatocytes, osteocytes, endothelial cells, and islet-like cell organoids. Over the years, HLSCs have been shown to contribute to tissue repair and regeneration in different in vivo models, leading to more than five granted patents and over 15 peer reviewed scientific articles elucidating their potential therapeutic role in various experimental pathologies. In addition, HLSCs have recently completed a Phase 1 study evaluating their safety post intrahepatic injection in infants with inherited neonatal onset hyperammonemia. Even though a lot of progress has been made in understanding HLSCs over the past years, some important questions regarding the mechanisms of action remain to be elucidated. Among the mechanisms of interaction of HLSCs with their environment, a paracrine interface has emerged involving extracellular vesicles (EVs) as vehicles for transferring active biological materials. In our group, the EVs derived from HLSCs have been studied in vitro as well as in vivo. Our attention has mainly been focused on understanding the in vivo ability of HLSC–derived EVs as modulators of tissue regeneration, inflammation, fibrosis, and tumor growth. This review article aims to discuss in detail the role of HLSCs and HLSC-EVs in these processes and their possible future therapeutic applications.

Prenatal Development and Function of Human Mononuclear Phagocytes.

The human mononuclear phagocyte (MP) system, which includes dendritic cells, monocytes, and macrophages, is a critical regulator of innate and adaptive immune responses. During embryonic development, MPs derive sequentially in yolk sac progenitors, fetal liver, and bone marrow haematopoietic stem cells. MPs maintain tissue homeostasis and confer protective immunity in post-natal life. Recent evidence – primarily in animal models – highlight their critical role in coordinating the remodeling, maturation, and repair of target organs during embryonic and fetal development. However, the molecular regulation governing chemotaxis, homeostasis, and functional diversification of resident MP cells in their respective organ systems during development remains elusive. In this review, we summarize the current understanding of the development and functional contribution of tissue MPs during human organ development and morphogenesis and its relevance to regenerative medicine. We outline how single-cell multi-omic approaches and next-generation ex-vivo organ-on-chip models provide new experimental platforms to study the role of human MPs during development and disease.

Brucellosis in transplant recipients: A systematic review.

Brucellosis is a bacterial disease caused by Brucella species. The purpose of this study was to evaluate brucellosis in all types of transplant patients. All the cases of brucellosis in transplant patients with no time and language limitations were searched and retrieved on May 20, 2020, using the following search keywords: (Brucella OR Brucellosis) AND (Transplant OR Transplantation) through the following medical databases: Web of Sciences, Google Scholar, Scopus, PubMed, and regional databases, for example, SID. All clinical features, including the time of transmission (before, during, and after transplantation), treatment protocols and medications, and patients' outcomes were investigated. A total of 14 cases reported in 14 studies (out of 777 studies) were retrieved. Kidney (50%), liver (28.5%), and hematopoietic stem cell transplantation (14.2%) were the most reported types of transplantation. The presentation of brucellosis in 42.8% of the patients occurred in the early post-transplantation period, whereas 57.1% of the cases presented with late onset disease. Brucellosis in transplant recipients seems to be uncommon even in the endemic regions. However, rare cases could be transmitted through bone marrow transplantation and transfusion. Precise screening and meticulous supervision during and after transplantation might lead to a reduction in the frequency of brucellosis.

LGR5/R-Spo1/Wnt3a axis promotes stemness and aggressive phenotype in hepatoblast-like hepatocellular carcinoma cell lines

Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a newly defined stem cell marker in endoderm-derived organs such as the small intestine, colon and pancreas. Recently, LGR5 was demonstrated to be an important factor in liver regeneration and stem cell maintenance. Moreover, LGR5 expression is highly up-regulated in various cancers including hepatocellular carcinoma. Herein, we demonstrate that LGR5 expression is specifically observed in certain subset of HCC cell lines with “hepatoblast-like” appearance, characterized by the expression of liver fetal/progenitor markers. Notably, the activation of the canonical Wnt pathway significantly increases the expression of LGR5 in this subset of cell lines, whereas it does not cause any induction of LGR5 expression in mesenchymal like cell lines SNU-475 and SNU-449. Furthermore, we showed that treatment of the hepatoblast-like HCC cell lines HuH-7 and Hep3B with LGR5 ligand R-Spo1 significantly amplifies the induction of LGR5 expression, the phosphorylation of LRP6 and β-catenin resulting in enhanced TCF/LEF activity either alone or in combination with Wnt3a. Consistently, the silencing of the LGR5 gene attenuates the co-stimulatory effect of R-Spo1/Wnt3a on TCF/LEF activity while overexpression of LGR5 enhances it. On the contrary, overexpression of LGR5 does not change TCF/LEF activity induced by R-Spo1/Wnt3a in mesenchymal-like HCC line, SNU-449. Importantly, LGR5-overexpressing cells have increased expression of several Wnt target genes and stemness-related genes including EpCAM and CK19 upon R-Spo1/Wnt3a treatment. LGR5-overexpressing cells also have increased spheroid forming, migration and invasion abilities and stimulation with R-Spo1/Wnt3a augments these abilities of LGR5-overexpressing cells. In addition, ectopic overexpression of LGR5 significantly increases cell proliferation rate independent of R-Spo1/Wnt3a stimulation. Moreover, in vitro tubulogenesis assay demonstrates that treatment with R-Spo1/Wnt3a enhances the sprouting of capillary tubules in only LGR5-overexpressing cells. Finally, R-Spo1/Wnt3a significantly promotes dissemination of LGR5-overexpressing cells in vivo in a zebrafish xenograft model. Our study unravels a tumor-promoting role for LGR5 through activation of canonical Wnt/β-catenin signaling in the hepatoblast-like HCCs. In conclusion, our results suggest that LGR5/R-Spo1/Wnt3a generates an axis that mediates the acquisition of aggressive phenotype specifically in hepatoblast-like subset of HCCs and might represent a valuable target for treatment of HCC tumors with aberrant activation of Wnt/β-catenin pathway.

Evaluation of Ranzoni etal.: Integrative Single-Cell RNA-Seq and ATAC-Seq Analysis of Human Developmental Hematopoiesis.

This article shows an example of the peer review process for "Integrative Single-Cell RNA-Seq and ATAC-Seq Analysis of Human Developmental Hematopoiesis" (Ranzoni etal., 2021).

Population pharmacokinetics of mycophenolic acid in paediatric patients.

Mycophenolic acid (MPA) is widely used in paediatric kidney transplant patients and sometimes prescribed for additional indications. Population pharmacokinetic or pharmacodynamic modelling has been frequently used to characterize the fixed, random and covariate effects of MPA in adult patients. However, MPA population pharmacokinetic data in the paediatric population have not been systematically summarized. The objective of this narrative review was to provide an up-to-date critique of currently available paediatric MPA population pharmacokinetic models, with emphases on modelling techniques, pharmacological findings and clinical relevance. PubMed and EMBASE were searched from inception of database to May 2020, where a total of 11 studies have been identified representing kidney transplant (n = 4), liver transplant (n = 1), haematopoietic stem cell transplant (n = 1), idiopathic nephrotic syndrome (n = 2), systemic lupus erythematosus (n = 2), and a combined population consisted of kidney, liver and haematopoietic stem cell transplant patients (n = 1). Critical analyses were provided in the context of MPA absorption, distribution, metabolism, excretion and bioavailability in this paediatric database. Comparisons to adult patients were also provided. With respect to clinical utility, Bayesian estimation models (n = 6) with acceptable accuracy and precision for MPA exposure determination have also been identified and systematically evaluated. Overall, our analyses have identified unique features of MPA clinical pharmacology in the paediatric population, while recognizing several gaps that still warrant further investigations. This review can be used by pharmacologists and clinicians for improving MPA pharmacokinetic-pharmacodynamic modelling and patient care.

Coincubation as miR-Loading Strategy to Improve the Anti-Tumor Effect of Stem Cell-Derived EVs

Extracellular vesicles are considered a novel therapeutic tool, due to their ability to transfer their cargoes to target cells. Different strategies to directly load extracellular vesicles with RNA species have been proposed. Electroporation has been used for the loading of non-active vesicles; however, the engineering of vesicles already carrying a therapeutically active cargo is still under investigation. Here, we set up a coincubation method to increase the anti-tumor effect of extracellular vesicles isolated from human liver stem cells (HLSC-EVs). Using the coincubation protocol, vesicles were loaded with the anti-tumor miRNA-145, and their effect was evaluated on renal cancer stem cell invasion. Loaded HLSC-EVs maintained their integrity and miR transfer ability. Loaded miR-145, but not miR-145 alone, was protected by RNAse digestion, possibly due to its binding to RNA-binding proteins on HLSC-EV surface, such as Annexin A2. Moreover, miR-145 coincubated HLSC-EVs were more effective in inhibiting the invasive properties of cancer stem cells, in comparison to naïve vesicles. The protocol reported here exploits a well described property of extracellular vesicles to bind nucleic acids on their surface and protect them from degradation, in order to obtain an effective miRNA loading, thus increasing the activity of therapeutically active naïve extracellular vesicles.

Role of prostaglandin E2 in tissue repair and regeneration.

Tissue regeneration following injury from disease or medical treatment still represents a challenge in regeneration medicine. Prostaglandin E2 (PGE2), which involves diverse physiological processes via E-type prostanoid (EP) receptor family, favors the regeneration of various organ systems following injury for its capabilities such as activation of endogenous stem cells, immune regulation, and angiogenesis. Understanding how PGE2 modulates tissue regeneration and then exploring how to elevate the regenerative efficiency of PGE2 will provide key insights into the tissue repair and regeneration processes by PGE2. In this review, we summarized the application of PGE2 to guide the regeneration of different tissues, including skin, heart, liver, kidney, intestine, bone, skeletal muscle, and hematopoietic stem cell regeneration. Moreover, we introduced PGE2-based therapeutic strategies to accelerate the recovery of impaired tissue or organs, including 15-hydroxyprostaglandin dehydrogenase (15-PGDH) inhibitors boosting endogenous PGE2 levels and biomaterial scaffolds to control PGE2 release.

Molecular Modulation of Fetal Liver Hematopoietic Stem Cell Mobilization into Fetal Bone Marrow in Mice.

Development of hematopoietic stem cells is a complex process, which has been extensively investigated. Hematopoietic stem cells (HSCs) in mouse fetal liver are highly expanded to prepare for mobilization of HSCs into the fetal bone marrow. It is not completely known how the fetal liver niche regulates HSC expansion without loss of self-renewal ability. We reviewed current progress about the effects of fetal liver niche, chemokine, cytokine, and signaling pathways on HSC self-renewal, proliferation, and expansion. We discussed the molecular regulations of fetal HSC expansion in mouse and zebrafish. It is also unknown how HSCs from the fetal liver mobilize, circulate, and reside into the fetal bone marrow niche. We reviewed how extrinsic and intrinsic factors regulate mobilization of fetal liver HSCs into the fetal bone marrow, which provides tools to improve HSC engraftment efficiency during HSC transplantation. Understanding the regulation of fetal liver HSC mobilization into the fetal bone marrow will help us to design proper clinical therapeutic protocol for disease treatment like leukemia during pregnancy. We prospect that fetal cells, including hepatocytes and endothelial and hematopoietic cells, might regulate fetal liver HSC expansion. Components from vascular endothelial cells and bones might also modulate the lodging of fetal liver HSCs into the bone marrow. The current review holds great potential to deeply understand the molecular regulations of HSCs in the fetal liver and bone marrow in mammals, which will be helpful to efficiently expand HSCs in vitro.

Cell Sources and Influencing Factors of Liver Regeneration: A Review.

Liver regeneration (LR) is a set of complicated mechanisms between cells and molecules in which the processes of initiation, maintenance, and termination of liver repair are regulated. Although LR has been studied extensively, there are still numerous challenges in gaining its full understanding. Cells for LR have a wide range of sources and the feature of plasticity, and regeneration patterns are not the same under different conditions. Many patients undergoing partial hepatectomy develop cirrhosis or steatosis. The changes of LR in these cases are not clear. Many types of cells participate in LR. Hepatocytes, biliary epithelial cells, hepatic progenitor cells, and human liver stem cells can serve as the cell sources for LR. However, different types and degrees of damage trigger the response from the most suitable cells. Exploring the cell sources of LR is of great significance for accelerating recovery of liver function under different pathological patterns and developing a cell therapy strategy to cope with the shortage of donors for liver transplantation. In clinical practice, the background of the liver influences regeneration. Fibrosis and steatosis create different LR microenvironments and signal molecule interaction patterns. In addition, factors such as partial hepatectomy, aging, platelets, nerves, hormones, bile acids, and gut microbiota are widely involved in this process. Understanding the influencing factors of LR has practical value for individualized treatment of patients with liver diseases. In this review, we have summarized recent studies and proposed our views. We discuss cell sources and the influential factors on LR to help in solving clinical problems.

Screening and identification of key genes between liver hepatocellular carcinoma (LIHC) and cholangiocarcinoma (CHOL) by bioinformatic analysis.

Background:Liver hepatocellular carcinoma (LIHC) and cholangiocarcinoma (CHOL) are common primary liver cancers worldwide. Liver stem cells have biopotential to differentiate into either hepatocytes and cholangiocytes, the phenotypic overlap between LIHC and CHOL has been acceptable as a continuous liver cancer spectrum. However, few studies directly investigated the underlying molecular mechanisms between LIHC and CHOL.Method:To identify the candidate genes between LIHC and CHOL, three data series including GSE31370, GSE15765 and GSE40367 were downloaded from Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) were identified, and function enrichment analyses were performed. The protein-protein interaction network (PPI) was constructed and the module analysis was performed using STRING and Cytoscape.Results:A total of 171 DEGs were identified, consisting of 49 downregulated genes and 122 upregulated genes. Compared with CHOL, the enriched functions of the DEGs mainly included steroid metabolic process, acute inflammatory response, coagulation. Meanwhile, the pathway of KEGG enrichment analyses showed that the upregulated gene(s) were mainly enriched complement and coagulation cascades, cholesterol metabolism and PPAR signaling pathway, while the downregulated gene(s) were mainly enriched in ECM-receptor interaction, focal adhesion, bile secretion. Similarly, the most significant module was identified and biological process analysis revealed that these genes were mainly enriched in regulation of blood coagulation, acute inflammatory response, complement and coagulation cascades. Finally, two (ITIH2 and APOA2) of 10 hub genes had been screened out to help differential diagnosis.Conclusion:171 DEGs and two (ITIH2 and APOA2) of 10 hub genes identified in the present study help us understand the different molecular mechanisms between LIHC and CHOL, and provide candidate targets for differential diagnosis.

Tanshinone IIA exerts therapeutic effects by acting on endogenous stem cells in rats with liver cirrhosis

Background and ObjectiveLiver cirrhosis (LC), the major pathway for the progression and development of chronic liver disease, is an advanced stage of liver disease. It is the third most common chronic noncommunicable disease after cardiovascular diseases and malignant tumors. Tanshinone IIA (Tan), an extract of Salvia miltiorrhiza (S. miltiorrhiza), has been proven to promote the proliferation and differentiation of stem cells. Moreover, its protective effect in liver injury has received widespread attention. The present study investigated whether Tan plays a therapeutic role in LC by promoting endogenous stem cell proliferation and differentiation. Materials and MethodsLC models were established by intraperitoneal injection of an olive oil solution containing 50 % carbon tetrachloride (CCL4) combined with 10 % alcohol in the drinking water. After successful model establishment, the animals were randomly divided into four groups and injected with physiological saline or low-, medium-, or high-dose (10, 20, or 40 mg/kg) Tan for seven consecutive days. The protective effect of Tan on LC was observed by western blotting, serological examination and histopathological staining. Furthermore, immunofluorescence double-labeling of 5-bromo-2-deoxyuridine (BrdU) and the liver cell markers albumin and CK-18 or the liver stem cell markers EPCAM and OV-6 was used to evaluate the proliferation and differentiation of endogenous liver stem cells. ResultsWe confirmed successful establishment of the LC model by observing transaminase levels and hematoxylin-eosin (HE) and Masson staining of liver sections in CCL4-treated and healthy rats. After Tan treatment, HE and Masson staining of paraffin sections of liver tissue showed that Tan treatment significantly improved histological injury to the liver. Serological tests showed that albumin-bilirubin (ALBI) scores and models for end-stage liver disease (MELD) were lower. Immunofluorescence and immunohistochemical staining showed that the newly proliferated cells were colocalized with ALB, OV-6, EPCAM, and CK-18, indicating that new expression of these markers occurred after Tan injection. All results were most significant in the medium-dose treatment group. ConclusionTan can alleviate liver injury induced by CCL4 combined with alcohol in rats and plays a therapeutic role in LC by promoting the proliferation and differentiation of endogenous liver stem cells.

The Conundrum of the Pericentral Hepatic Niche: WNT/-Catenin Signaling, Metabolic Zonation, and Many Open Questions.

WNT/-catenin signaling promotes stemness, proliferation, and cell fate decisions in various tissue stem cell compartments, which maintain organs with a high turnover of cells (e.g., skin, stomach, and gut). Thus, the -catenin target genes AXIN2 and LGR5 are widely considered as tissue stem cell markers. In contrast, AXIN2 and LGR5 are expressed in pericentral hepatocytes, which do not show overt proliferation during liver homeostasis. Given the low hepatocyte turnover, the liver does not require constant high rates of proliferation, whereas WNT/-catenin signaling is critical for metabolic zonation. Yet, WNT/-catenin pathway upregulation, including AXIN2 and LGR5 induction in hepatocytes throughout the liver, enables hepatocyte regeneration in response to various injuries. In this brief review, I discuss the role of WNT/-catenin signaling in controlling metabolic zonation and the conundrum around pericentral hepatocytes that have been proposed as liver stem cells.

Ready to go 3D? A semi-automated protocol for microwell spheroid arrays to increase scalability and throughput of 3D cell culture testing

3-dimensional (3D) cell cultures are being increasingly recognized as physiologically more relevant in vitro models than traditional monolayer cultures, because they better mimic in vivo-like microenvironment, cell-cell and cell-extracellular matrix interactions. Nevertheless, the broader use of 3D models might be limited by requirements for special consumables, equipment, or skills for 3D cell cultures, and by their limited throughput and scalability. In this study, we optimized and adapted a commercially available agarose-micromolding technique to produce scaffold-free spheroid cultures. Brightfield microscopy was used for routine nondestructive and noninvasive evaluation of spheroid formation and growth. The workflow is compatible with manual, as well as high speed automated microscopic image acquisition, and it is supplemented with an in-house developed macro ‘Spheroid_Finder’ for open source software Fiji to facilitate rapid automated image analysis. This protocol was used to characterize and quantify spheroid formation and growth of two different hepatic cell lines, hTERT immortalized, but non-cancerous, adult human liver stem cell line HL1-hT1, and human hepatocellular carcinoma cell line HepG2, as well as their responses to a model antiproliferative and cytotoxic agent, 5-fluorouracil. The complete protocol provides a simple and ready-to-use solution to initiate scaffold-free spheroid cultures in any laboratory with standard equipment for mammalian in vitro cell culture work. Thus, it allows to increase throughput and scale of spheroid culture experiments, which can be greatly utilized in different areas of biomedical, pharmaceutical and toxicological research.

The mutational impact of culturing human pluripotent and adult stem cells

Genetic changes acquired during in vitro culture pose a risk for the successful application of stem cells in regenerative medicine. To assess the genetic risks induced by culturing, we determined all mutations in individual human stem cells by whole genome sequencing. Individual pluripotent, intestinal, and liver stem cells accumulate 3.5 ± 0.5, 7.2 ± 1.1 and 8.3 ± 3.6 base substitutions per population doubling, respectively. The annual in vitro mutation accumulation rate of adult stem cells is nearly 40-fold higher than the in vivo mutation accumulation rate. Mutational signature analysis reveals that in vitro induced mutations are caused by oxidative stress. Reducing oxygen tension in culture lowers the mutational load. We use the mutation rates, spectra, and genomic distribution to model the accumulation of oncogenic mutations during typical in vitro expansion, manipulation or screening experiments using human stem cells. Our study provides empirically defined parameters to assess the mutational risk of stem cell based therapies.