Population Of Stellate Cells Research Articles

Single-cell transcriptomics stratifies organoid models of metabolic dysfunction-associated steatotic liver disease.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a growing cause of morbidity with limited treatment options. Thus, accurate in vitro systems to test new therapies are indispensable. While recently, human liver organoid models have emerged to assess steatotic liver disease, a systematic evaluation of their translational potential is still missing. Here, we evaluated human liver organoid models of MASLD, comparatively testing disease induction in three conditions: oleic acid, palmitic acid, and TGF-β1. Through single-cell analyses, we find that all three models induce inflammatory signatures, but only TGF-β1 promotes collagen production, fibrosis, and hepatic stellate cell expansion. In striking contrast, oleic acid ameliorates fibrotic signatures and reduces the hepatic stellate cell population. Linking data from each model to gene expression signatures associated with MASLD disease progression further demonstrates that palmitic acid and TGF-β1 more robustly model inflammation and fibrosis. Our findings highlight the importance of stratifying MASLD organoid models by signatures of clinical disease progression, provide a single-cell reference to benchmark future organoid injury models, and allow us to study evolving steatohepatitis, fibrosis, and HSC susceptibility to injury in a dynamic, multi-lineage human in vitro system.

Tipping the balance: New insights into stromal regulation of cancer.

Desmoplastic extracellular matrix (ECM) exerts a seemingly paradoxical role in cancer, restricting or promoting progression. Two recent back-to-back Nature reports shed light on this conundrum, revealing the role of different hepatic stellate cell (HSC) populations and different collagen I cleavage states in directing the progression of cancer.

EP3 enhances adhesion and cytotoxicity of NK cells toward hepatic stellate cells in a murine liver fibrosis model

Natural killer (NK) cells exhibit antifibrotic properties in liver fibrosis (LF) by suppressing activated hepatic stellate cell (HSC) populations. Prostaglandin E2 (PGE2) plays a dual role in innate and adaptive immunity. Here, we found that E-prostanoid 3 receptor (EP3) was markedly downregulated in NK cells from liver fibrosis mice and patients with liver cirrhosis. NK cell-specific deletion of EP3 aggravated hepatic fibrogenesis in mouse models of LF. Loss of EP3 selectively reduced the cytotoxicity of the CD27+CD11b+ double positive (DP) NK subset against activated HSCs. Mechanistically, deletion of EP3 impaired the adhesion and cytotoxicity of DP NK cells toward HSCs through modulation of Itga4-VCAM1 binding. EP3 upregulated Itga4 expression in NK cells through promoting Spic nuclear translocation via PKC-mediated phosphorylation of Spic at T191. Activation of EP3 by sulprostone alleviated CCL4-induced liver fibrosis in mice. Thus, EP3 is required for adhesion and cytotoxicity of NK cells toward HSCs and may serve as a therapeutic target for the management of LF.

UCP1 governs liver extracellular succinate and inflammatory pathogenesis.

Non-alcoholic fatty liver disease (NAFLD), the most prevalent liver pathology worldwide, is intimately linked with obesity and type 2 diabetes. Liver inflammation is a hallmark of NAFLD and is thought to contribute to tissue fibrosis and disease pathogenesis. Uncoupling protein 1 (UCP1) is exclusively expressed in brown and beige adipocytes, and has been extensively studied for its capacity to elevate thermogenesis and reverse obesity. Here we identify an endocrine pathway regulated by UCP1 that antagonizes liver inflammation and pathology, independent of effects on obesity. We show that, without UCP1, brown and beige fat exhibit a diminished capacity to clear succinate from the circulation. Moreover, UCP1KO mice exhibit elevated extracellular succinate in liver tissue that drives inflammation through ligation of its cognate receptor succinate receptor 1 (SUCNR1) in liver-resident stellate cell and macrophage populations. Conversely, increasing brown and beige adipocyte content in mice antagonizes SUCNR1-dependent inflammatory signalling in the liver. We show that this UCP1-succinate-SUCNR1 axis is necessary to regulate liver immune cell infiltration and pathology, and systemic glucose intolerance in an obesogenic environment. As such, the therapeutic use of brown and beige adipocytes and UCP1 extends beyond thermogenesis and may be leveraged to antagonize NAFLD and SUCNR1-dependent liver inflammation.

Single nucleus transcriptomics data integration recapitulates the major cell types in human liver.

The aim of this study was to explore the benefits of data integration from different platforms for single nucleus transcriptomics profiling to characterize cell populations in human liver. We generated single-nucleus RNA sequencing data from Chromium 10X Genomics and Drop-seq for a human liver sample. We utilized state of the art bioinformatics tools to undertake a rigorous quality control and to integrate the data into a common space summarizing the gene expression variation from the respective platforms, while accounting for known and unknown confounding factors. Analysis of single nuclei transcriptomes from both 10X and Drop-seq allowed identification of the major liver cell types, while the integrated set obtained enough statistical power to separate a small population of inactive hepatic stellate cells that was not characterized in either of the platforms. Integration of droplet-based single nucleus transcriptomics data enabled identification of a small cluster of inactive hepatic stellate cells that highlights the potential of our approach. We suggest single-nucleus RNA sequencing integrative approaches could be utilized to design larger and cost-effective studies.

Meningioma-like Tumor of the Skin Revisited: A Distinct CD34+ Dermal Tumor With an Expanded Histologic Spectrum.

The term meningioma-like tumor of the skin (MLTS) was coined in 1993 to designate a particular whorled spindle cell superficial cutaneous tumor. No additional confirmed cases of this entity have been reported to date. Some authors have speculated that these cases might be cellular neurothekeomas. In order to delineate the histologic spectrum and the immunophenotype of this unusual tumor, we studied 5 cases, 2 previously unreported and the 3 original cases. The immunohistochemical findings of case 5, however, were limited to those from the original study. Clinically, the tumor presented as a reddish papule, plaque, or nodule, located in the extremities or trunk. The patient often referred to a recent growth of a longstanding lesion. Histologically, the characteristic whorled spindle and stellate dendritic cell population, commonly in a perivascular arrangement, and variable myxoid component, were consistently found in all cases. A prominent microvasculature was also a constant finding. The presence of large deciduoid cells was conspicuous in one case. A reticular pattern of multivacuolated cells giving a chordoma-like appearance was evident in another case. Tumor cells were diffusely positive for CD34 in all 4 cases studied, and negative for S-100, EMA, NKI-C3, CD68, and smooth muscle markers. No complete loss of retinoblastoma protein was found. No brachyury immunostaining was found in the case with chordoid features. No EWSR1 or NAB2-STAT6 gene fusions were found. From these findings, we demonstrate that MLTS is a distinct CD34 spindle cell benign dermal tumor, unrelated to cellular neurothekeoma, and exhibiting myxoid, deciduoid, or chordoma-like features.

The Wilms' tumor suppressor gene regulates pancreas homeostasis and repair.

The Wilms’ tumor suppressor gene (Wt1) encodes a zinc finger transcription factor that plays an essential role in the development of kidneys, gonads, spleen, adrenals and heart. Recent findings suggest that WT1 could also be playing physiological roles in adults. Systemic deletion of WT1 in mice provokes a severe deterioration of the exocrine pancreas, with mesothelial disruption, E-cadherin downregulation, disorganization of acinar architecture and accumulation of ascitic transudate. Despite this extensive damage, pancreatic stellate cells do not become activated and lose their canonical markers. We observed that pharmacological induction of pancreatitis in normal mice provokes de novo expression of WT1 in pancreatic stellate cells, concomitant with their activation. When pancreatitis was induced in mice after WT1 ablation, pancreatic stellate cells expressed WT1 and became activated, leading to a partial rescue of the acinar structure and the quiescent pancreatic stellate cell population after recovery from pancreatitis. We propose that WT1 modulates through the RALDH2/retinoic acid axis the restabilization of a part of the pancreatic stellate cell population and, indirectly, the repair of the pancreatic architecture, since quiescent pancreatic stellate cells are required for pancreas stability and repair. Thus, we suggest that WT1 plays novel and essential roles for the homeostasis of the adult pancreas and, through its upregulation in pancreatic stellate cells after a damage, for pancreatic regeneration. Due to the growing importance of the pancreatic stellate cells in physiological and pathophysiological conditions, these novel roles can be of translational relevance.

Functional properties of stellate cells in medial entorhinal cortex layer II.

Layer II of the medial entorhinal cortex (MEC) contains two principal cell types: pyramidal cells and stellate cells. Accumulating evidence suggests that these two cell types have distinct molecular profiles, physiological properties, and connectivity. The observations hint at a fundamental functional difference between the two cell populations but conclusions have been mixed. Here, we used a tTA-based transgenic mouse line to drive expression of ArchT, an optogenetic silencer, specifically in stellate cells. We were able to optogenetically identify stellate cells and characterize their firing properties in freely moving mice. The stellate cell population included cells from a range of functional cell classes. Roughly one in four of the tagged cells were grid cells, suggesting that stellate cells contribute not only to path-integration-based representation of self-location but also have other functions. The data support observations suggesting that grid cells are not the sole determinant of place cell firing.

Role of the Wilms' tumor suppressor gene Wt1 in pancreatic development.

The Wilms tumor suppressor gene (Wt1) encodes a transcription factor involved in the development of a number of organs, but the role played by Wt1 in pancreatic development is unknown. The pancreas contains a population of pancreatic stellate cells (PSC) very important for pancreatic physiology. We described elsewhere that hepatic stellate cells originate from the WT1-expressing liver mesothelium. Thus, we checked if the origin of PSCs was similar. WT1 expression is restricted to the pancreatic mesothelium. Between embryonic day (E) 10.5 and E15.5, this mesothelium gives rise to mesenchymal cells that contribute to a major part of the PSC and other cell types including endothelial cells. Most WT1 systemic mutants show abnormal localization of the dorsal pancreas within the mesentery and intestinal malrotation by E14.0. Embryos with conditional deletion of WT1 between E9.5 and E12.5 showed normal dorsal pancreatic bud and intestine, but the number of acini in the ventral bud was reduced approximately 30% by E16.5. Proliferation of acinar cells was reduced in WT1 systemic mutants, but pancreatic differentiation was not impaired. Thus, mesothelial-derived cells constitute an important subpopulation of pancreatic mesodermal cells. WT1 expression is not essential for pancreas development, although it influences intestinal rotation and correct localization of the dorsal pancreas within the mesogastrium. Developmental Dynamics 247:924-933, 2018. © 2018 Wiley Periodicals, Inc.

A comparative study of the neural stem cell niche in the adult hypothalamus of human, mouse, rat and gray mouse lemur (Microcebus murinus)

The adult brain contains niches of neural stem cells that continuously add new neurons to selected circuits throughout life. Two niches have been extensively studied in various mammalian species including humans, the subventricular zone of the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus. Recently, studies conducted mainly in rodents have identified a third neurogenic niche in the adult hypothalamus. In order to evaluate whether a neural stem cell niche also exists in the adult hypothalamus in humans, we performed multiple immunofluorescence labeling to assess the expression of a panel of neural stem/progenitor cell (NPC) markers (Sox2, nestin, vimentin, GLAST, GFAP) in the human hypothalamus and compared them with the mouse, rat and a non-human primate species, the gray mouse lemur (Microcebus murinus). Our results show that the adult human hypothalamus contains four distinct populations of cells that express the five NPC markers: (a) a ribbon of small stellate cells that lines the third ventricular wall behind a hypocellular gap, similar to that found along the lateral ventricles, (b) ependymal cells, (c) tanycytes, which line the floor of the third ventricle in the tuberal region, and (d) a population of small stellate cells in the suprachiasmatic nucleus. In the mouse, rat and mouse lemur hypothalamus, co-expression of NPC markers is primarily restricted to tanycytes, and these species lack a ventricular ribbon. Our work thus identifies four cell populations with the antigenic profile of NPCs in the adult human hypothalamus, of which three appear specific to humans.

Evaluation of triptolide pro-drug (Minnelide) as an anti-stromal and anti-tumoral therapeutic option for pancreatic cancer.

262 Background: In pancreatic cancer, the stromal microenvironment is considered to be the major reason behind the failure of conventional and targeted therapy. The desmoplastic stroma is responsible for compression of vasculature in the tumor resulting in impaired drug delivery. Pancreatic stellate cells contribute extensively to this desmoplastic reaction. Our group has recently evaluated a water-soluble pro-drug of triptolide (Minnelide) in pre-clinical studies for pancreatic cancer with very promising results. The current study evaluates the ability of Minnelide to deplete the stromal architecture and the stellate cell population in pancreatic cancer resulting in increased drug delivery. Methods: Patient tumor xenografts in SCID mice (PDX) and KRASG12DTP53-PDX-Cre spontaneous pancreatic tumor mice (KPC) were treated with 0.42mg/kg Minnelide. Vascular patency was studied by terminal perfusion of mice with 4% PFA following injection with tomato lectin. Drug delivery was determined by quantitating accumulated doxorubicin in the tumor tissue. Cell viability was studied by MTT assay and the invasion of cells in vitro was studied using Boyden Chamber assay. Results: Minnelide treatment decreased Hyaluronic acid (HA) in PDX (48% of control) as well as in KPC model (52% of control). Expression of HA synthase (HAS) genes were decreased in both tumor models in response to Minnelide (22% of control in PDX and 35% of control in KPC tumors). HAS Enzyme activity also consistently decreased in both the models after treatment with Minnelide (28% of control in PDX and 31% of control in KPC). In vitro, triptolide decreased the invasion of the tumor cells towards the stellate cells indicating an inhibition of crosstalk. Treatment with Minnelide resulted in “open” and functional vessels in the treated animals resulting in increased drug delivery. Median survival of animals increased by 38 days after treatment with Minnelide. Conclusions: Our study showed that Minnelide depleted the stromal architecture leading to increased functional vasculature and enhanced drug delivery in the tumor. This is an extremely promising observation for Minnelide that is currently under clinical trial.

Spontaneous multicentric myxoma of the dermal nerve sheaths in farmed European eels Anguilla anguilla.

This report describes a peripheral nerve sheath tumour in 8 European eels Anguilla anguilla L. from a fish farm located in Croatia. The newborn tissue appeared as smooth and soft skin nodules without pronounced colour change. Nodules were dome-shaped with a pale crater and were present on different body areas. In general, nodules were located as series of differently sized protrusions extending along the lateral line on both sides of the fish, as well as sensory canals on the head. Cut sections showed a homogeneous, pale white-grey texture. Histologically, the pathological tissue was located in the dermis, occasionally intruding into the hypodermis, and pushing as a space-occupying mass against the underlying muscle tissue without any evident boundaries. The pressure also caused changes in the overlying epidermis, such as atrophy, spongiosis and erosion. In some areas, the epidermis was 1 cell thick and club and goblet cells had completely disappeared. Ultimately, these changes resulted in shallow ulceration. Tumour tissue was characterized by a scant population of spindle or stellate cells, with oval, hyperchromatic nuclei and pale cytoplasm embedded in a copious myxoid matrix. Cells were arranged in fascicles and whorls, extending in a poorly defined manner among the dermal collagen bundles. Occasionally, adipose cells were also detected, mainly in the central portion of the bulges. Myxoid areas appeared rich in metachromatic and alcianophilic mucous ground substance. Reticular fibres and collagenous connective tissue were scarce. Immunohistochemistry (IHC) using antibodies against S-100 and glial fibrillary acidic protein caused a slight positive reaction in neoplastic dendritic cells. High magnification showed the immunostaining to be cytoplasmic in all tumour cells. IHC with anti-calretinin antibody gave only negative results. Macroscopic, histological, histochemical and immunohistochemical findings were consistent with a diagnosis of multicentric myxoma of the dermal nerve sheaths, a tumour not yet reported in fish.

A computational model of the stellate cell microcircuit in the auditory brainstem

The cochlear nucleus (CN) is the first processing center of auditory signals from the cochlea. Several distinct neuronal circuits have been identified that form different parallel representations of auditory information from auditory nerve fibers (ANFs). One cell circuit of interest is the stellate cell microcircuit of the ventral cochlear nucleus (VCN), which is centered on two populations of stellate cells, known as T and D stellate cells [1]. The output of the T stellate (TS) cell population has been shown experimentally to accurately encode important frequency components over a wide range of input sound pressure levels (SPL) and in the presence of significant signal noise [2]. It is thought that one purpose of the stellate cell circuit is to increase the dynamic range and the signal-to-noise ratio (SNR) of the incoming signal at all levels in order to more accurately highlight the most significant frequencies. A clear application of this in speech processing is the identification of vowels, which requires detection of the first two or three formant frequencies. A neural network model of the stellate microcircuit was created using leaky integrate-and-fire neurons for each cell in the network. The key mechanisms incorporated to achieve SNR and dynamic range improvement were wide-band lateral inhibition and selective processing of excitatory and inhibitory inputs [3]. The number and type of cell inputs, the network connectivity, and the time constants associated with cellular dynamics were made, where possible, to agree with experimental observations. In addition, the performance of the model in response to a variety of stimuli was adjusted to achieve the desired signal processing functions regarding noise reduction, peak identification and dynamic range. To quantify the enhancement achieved by the network, two signal-to-noise metrics were used. SNR metric 1 measures the level of noise reduction and is calculated as the ratio of the average output rate of cells within 1.5 critical bands of the known stimulus frequency to the average background output rate. SNR metric 2 measures the level of peak enhancement and is calculated as the ratio of the maximum output rate of the cells within 1.5 critical bands of the known stimulus frequency to the maximum background output rate. The metrics were calculated for the input signal, the input ANF population, and the TS cell population. Each metric was calculated for the first three formants of the vowel /o:/ and for a range of input SNR values. The SNR metric 1 was significantly higher than for the ANF population for the majority of input SNR levels. Similarly, SNR metric 2 was also higher for the TS cell population than for the ANF population. This demonstrates that the TS cells improved the ability to decode the spectral information in noise and the model illustrates the mechanisms by which this is achieved.

Isolation and characterization of islet stellate cells in rat

The central role of PSCs in pancreatic fibrogenesis is well established. However, the mechanism responsible for the islet fibrosis presenting in the late stage of T2DM has not been fully elucidated. This study was designed to determine whether the endocrine pancreatic islets contain cells resembling PSCs. PSCs were isolated from pancreas using standard explants techniques. A similar method was used to acquire ISCs. Adherent ISCs with a stellate, angular morphology migrated from the edge of cultured islets within 48 h of primary culture. ISCs contained fewer lipid droplets than equivalent PSCs, and their rapid disappearance accompanied by the increased expression of α-SMA suggested that ISCs were more rapidly activated than PSCs in vitro. They expressed α-SMA, vimentin, GFAP and were positive for ECM components col-I, col-III and FN, all of which are characteristics of classical PSCs. However, ISCs differed from PSCs by having reduced rates of proliferation and migration in vitro. Our in vitro study shows that isolated islets contain a population of stellate cells which are phenotypically similar but not identical to PSCs. In view of the established role of PSCs in pancreatic fibrosis, we suggest that these may contribute to islet fibrosis in T2DM.

Characterization of the hepatic stellate cell populations in carbon tetrachloride liver rat through Golgi‐Cox Method.

Golgi‐Cox method was used to characterize the hepatic stellate cell (HSC) populations in CCl4 livers. In wistar rats, damage liver was induced by CCl4 administration during two months. After, rats were sacrifiezed and liver tissues were impregnated with the Golgi‐Cox stain, processed and analyzed by Sholl method under light microscopy adapted to a camera lucida. Location, soma cell size, and cytoplasmic processes from HSCs were analyzed. Damage tissue was evaluated with METAVIR score. HSCs were identified for their classical stellate form, and their cytoplasmic processes, making and extense network of reticular fibers. In accord with their soma cell size and location into the hepatic lobule, we identified periportal, perivenular and perisinusoidal hepatic stellate cells. Periportal and perivenular had the larger soma cells, whereas perisinusoidal had the smaller. The morphological changes of the HSCs in CCl4 livers consisted in an increase in the soma cell size, cut and frayly appearance of emerging cytoplasmic processes, and a significant decrease in the number of HSCs. All changes were less pronunciated in tissues with METAVIR score 1 and 2, particularly in the perisinusoidal hepatic stelate cells. HSC populations suffer morphological changes in CCl4 livers, however perisinusoidall cells are the last affected, suggesting that their preservation could be related to the regeneration liver.

Mechanistic Insights into Self-Reinforcing Processes Driving Abnormal Histogenesis During the Development of Pancreatic Cancer

Pancreatic ductal adenocarcinoma, one of the most feared lethal and painful diseases, is increasing in incidence. The poor prognosis of pancreatic ductal adenocarcinoma-affected patients primarily is owing to our inability to develop effective therapies. Mechanistic studies of genetic, epigenetic, and cell-to-cell signaling events are providing clues to molecular pathways that can be targeted in an attempt to cure this disease. The current review article seeks to draw inferences from available mechanistic knowledge to build a theoretical framework that can facilitate these approaches. This conceptual model considers pancreatic cancer as a tissue disease rather than an isolated epithelial cell problem, which develops and progresses in large part as a result of three positive feedback loops: i) genetic and epigenetic changes in epithelial cells modulate their interaction with mesenchymal cells to generate a dynamically changing process of abnormal histogenesis, which drives more changes; ii) the faulty tissue architecture of neoplastic lesions results in unsynchronized secretion of signaling molecules by cells, which generates an environment that is poor in oxygen and nutrients; and iii) the increased metabolic needs of rapidly dividing cells serve as an evolutionary pressure for them to adapt to this adverse microenvironment, leading to the emergence of resistant clones. We discuss how these concepts can guide mechanistic studies, as well as aid in the design of novel experimental therapeutics.

Recruitment and Activation of Pancreatic Stellate Cells from the Bone Marrow in Pancreatic Cancer: A Model of Tumor-Host Interaction

Background and AimsChronic pancreatitis and pancreatic cancer are characterised by extensive stellate cell mediated fibrosis, and current therapeutic development includes targeting pancreatic cancer stroma and tumor-host interactions. Recent evidence has suggested that circulating bone marrow derived stem cells (BMDC) contribute to solid organs. We aimed to define the role of circulating haematopoietic cells in the normal and diseased pancreas.MethodsWhole bone marrow was harvested from male β-actin-EGFP donor mice and transplanted into irradiated female recipient C57/BL6 mice. Chronic pancreatitis was induced with repeat injections of caerulein, while carcinogenesis was induced with an intrapancreatic injection of dimethylbenzanthracene (DMBA). Phenotype of engrafted donor-derived cells within the pancreas was assessed by immunohistochemistry, immunofluorescence and in situ hybridisation.ResultsGFP positive cells were visible in the exocrine pancreatic epithelia from 3 months post transplantation. These exhibited acinar morphology and were positive for amylase and peanut agglutinin. Mice administered caerulein developed chronic pancreatitis while DMBA mice exhibited precursor lesions and pancreatic cancer. No acinar cells were identified to be donor-derived upon cessation of cerulein treatment, however rare occurrences of bone marrow-derived acinar cells were observed during pancreatic regeneration. Increased recruitment of BMDC was observed within the desmoplastic stroma, contributing to the activated pancreatic stellate cell (PaSC) population in both diseases. Expression of stellate cell markers CELSR3, PBX1 and GFAP was observed in BMD cancer-associated PaSCs, however cancer-associated, but not pancreatitis-associated BMD PaSCs, expressed the cancer PaSC specific marker CELSR3.ConclusionsThis study demonstrates that BMDC can incorporate into the pancreas and adopt the differentiated state of the exocrine compartment. BMDC that contribute to the activated PaSC population in chronic pancreatitis and pancreatic cancer have different phenotypes, and may play important roles in these diseases. Further, bone marrow transplantation may provide a useful model for the study of tumor-host interactions in cancer and pancreatitis.

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes(1,2). Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A(1, 2). Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis(3). Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension(4). Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes(5). Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Constitutive release of powerful antioxidant-scavenging activity by hepatic stellate cells: Protection of hepatocytes from ischemia/reperfusion injury

Within the liver, reactive oxygen species produced by infiltrating blood cells and Kupffer cells (resident macrophages) can injure hepatocytes. We hypothesized that hepatocyte survival is influenced by the relatively small juxtaposed population of hepatic stellate cells (HSCs). We used cultures of primary rat hepatocytes as targets for superoxide-induced damage, which was determined by crystal violet assay and lactate dehydrogenase release. An HSC-conditioned medium prevented the superoxide-induced death of hepatocytes, and the protective factor released by HSCs was a protein or proteins (apparent molecular weight > 100 kDa) resistant to heat (70°C) and pH (4.5-8.5). The protein or proteins were partially purified on DE52 cellulose, and the active fraction contained no detectable levels of superoxide dismutase: after separation by Sephadex G-100 gel filtration, the antioxidant activity could be reconstituted by the combination of 2 protein peaks, and this reconstituted activity was protective both in vitro and against liver ischemia/reperfusion injury in intact rats. Mass spectrometry proteomic studies confirmed that this activity could not be attributed to any previously identified antioxidant protein. Thus, HSCs protect hepatocytes against oxidative damage through the production of a novel protein, the further purification of which may lead to the isolation of a powerful oxygen radical scavenger with clinical applications.

Relationship between plasma cells and hepatic stellate cells in autoimmune hepatitis

Autoimmune hepatitis is an inflammatory chronic disease of the liver, which frequently results in cirrhosis. The present study aimed to verify the relationship between plasma cells and stellate cells in autoimmune hepatitis. Thirty-three pre-treatment, 11 post-treatment, and 10 normal liver biopsies were reviewed. Sirius Red staining (for semi-quantitative analysis of hepatic fibrosis) and immunohistochemistry were carried out: double staining for smooth muscle α-actin and plasma cell marker (for detection and localization of activated hepatic stellate cells and plasma cells, respectively); and single staining for glial fibrillary acid protein (for detection of hepatic stellate cells). We found an increase in the stellate cell population, mainly with an activated phenotype in autoimmune hepatitis, compared to the control group (liver specimens with no histological evidence of liver disease, obtained from patients undergoing hepatic resection for benign liver mass). A positive significant correlation was observed between stellate cells and scores of fibrosis (measured by Sirius Red) and the number of plasma cells. Additionally, there was a co-localization of plasma cells and activated stellate cells. We also observed a reduction in the number of plasma cells, hepatic stellate cells, and fibrosis in patients who had successfully been treated and had a second liver biopsy post-treatment. Our findings support that the number of plasma cells can be a surrogate marker for the severity of liver disease, reflecting the number of hepatic stellate cells and the amount of fibrosis. It remains to be seen if this is a result of a direct interaction between the plasma cells and hepatic stellate cells or the response to the same stimulus that affects both cellular types.