Book Review: Weiskirchen, R.; Friedman, S.L. Hepatic Stellate Cells: Methods and Protocols, 1st Ed.; Weiskirchen, R., Friedman, S.L., Eds.; Methods in Molecular Biology 2669; Humana Press: New York, NY, USA, 2023; ISBN 978-1-07-163206-2; eISBN: 978-1-0716-3207-9

Hepatic stellate cells (HSCs) are a liver-specific mesenchymal cell type located in the Dissé space between hepatocytes and sinusoidal endothelial cells. They play an important role in liver physiology and pathology. In particular, they are the key cells involved in the initiation, progression and regression of hepatic fibrosis. During chronic liver injury, they produce and respond to a variety of soluble mediators and transdifferentiate from a quiescent to an activated, highly proliferative, myofibroblastic-like, and extracellular matrix producing phenotype [1,2]. Improved understanding of relevant molecular pathways and general features of HSC will be of fundamental importance to uncover potential therapeutic targets. Therefore, many scientists and clinicians are drawn to studying HSC biology and there are many efforts to target HSC as therapeutic targets. In addition, hepatic fibrosis research relies on in vivo mouse models in which damage is experimentally induced. Generation of valuable biological data in the field of HSC biology will require strictly following the standardized protocols and procedures.

In the book, “Hepatic Stellate Cells: Methods and Protocols”, 49 experts from nine countries (Australia, Belgium, Germany, Norway, Republic of Korea, Spain, The Netherlands, the UK, and the USA) have compiled a laboratory manual summarizing up-to-date methods that are presently used in their laboratories in HSC studies.

Each method is written as a readily-reproducible step-by-step protocol in which all necessary materials and reagents required for respective method is provided. A short introduction provides brief background, and each chapter includes detailed notes about potential pitfalls and critical steps in performing these methods. The book covers topics from the isolation of primary HSC from mouse and human, their biological characterization including retinol metabolism, senescence, migration, HSC co-culture systems, immortalized HSC cell lines, HSC-containing spheroid cultures, multiplex immunostaining techniques, HSC secretome analysis, HSC targeting strategies, and guideline to translate experimental findings to preclinical studies and beyond (

Table 1. Content of Hepatic Stellate Cells: Methods and Protocols.

Chapter	Title of Chapter	Authors
1	Isolation, Purification, and Culture of Primary Murine Hepatic Stellate Cells: An Update	Steffen K. Meurer, Sabine Weiskirchen, Carmen G. Tag, and Ralf Weiskirchen
2	Differentiation of Hepatic Stellate Cells from Pluripotent Stem Cells	Raquel A. Martinez Garcia de la Torre and Pau Sancho-Bru
3	Testing Cell Migration, Invasion, Proliferation, and Apoptosis in Hepatic Stellate Cells	Miriam Wankell and Lionel Hebbard
4	Phalloidin Staining for F-Actin in Hepatic Stellate Cells	Sarah K. Schröder, Carmen G. Tag, Sabine Weiskirchen, and Ralf Weiskirchen
5	Retinyl Ester Analysis by Orbitrap Mass Spectrometry	Jeroen W. A. Jansen, Maya W. Haaker, Esther A. Zaal, and J. Bernd Helms
6	Studying Hepatic Stellate Cell Senescence	Sandra A. Serna-Salas, Abel A. Soto-Gámez, Zongmei Wu, Myrthe Klaver, and Han Moshage
7	Isolation of Hepatic Stellate Cells and Lymphocytes for Co-culture Systems	Hee-Hoon Kim, Kyurae Kim, Song Hwa Hong, and Won-Il Jeong
8	Working with Immortalized Hepatic Stellate Cell Lines	Scott L. Friedman and Ralf Weiskirchen
9	Induction of Obstructive Cholestasis in Mice	Ralf Weiskirchen, Sabine Weiskirchen, Carmen G. Tag, and Steffen K. Meurer
10	Mouse Models for Hepatic Stellate Cell Activation and Liver Fibrosis Initiation	Yulia A. Nevzorova, Ralf Weiskirchen, and Christian Liedtke
11	Generation and Culture of Primary Mouse Hepatocyte–Hepatic Stellate Cell Spheroids	Inge Mannaerts, Nathalie Eysackers, and Leo A. van Grunsven
12	Hepatic Stellate Cell Depletion and Genetic Manipulation	Qiuyan Sun and Robert F. Schwabe
13	Human Hepatic Stellate Cells: Isolation and Characterization	Xiao Liu, David A. Brenner, and Tatiana Kisseleva
14	Decellularization of the Human Liver to Generate Native Extracellular Matrix for Use in Automated Functional Assays with Stellate Cells	Emma L. Shepherd, Ellie Northall, Pantelitsa Papakyriacou, Karolina Safranska, Karen K. Sorensen, and Patricia F. Lalor
15	Multiplex Immunostaining to Spatially Resolve the Cellular Landscape in Human and Mouse Livers	Adrien Guillot, Marlene Sophia Kohlhepp, and Frank Tacke
16	Single Cell Secretome Analyses of Hepatic Stellate Cells: Aiming for Single Cell Phenomics	Richell Booijink, Leon Terstappen, and Ruchi Bansal
17	Hepatic Stellate Cell Targeting Using Peptide-Modified Biologicals	Ruchi Bansal and Klaas Poelstra
18	Experimental Workflow for Preclinical Studies of Human Antifibrotic Therapies	Lien Reolizo, Michitaka Matsuda, and Ekihiro Seki

).

The article by Meurer et al. provides an updated method for high-purity isolation of mouse HSCs [3]. The protocol uses a pronase and collagenase digest, followed by density gradient centrifugation using a Nycodenz gradient, and an optional fluorescence-activated cell (FACS) sorting step if extremely high HSC isolates are required. Details are given for presurgical preparation of animals, surgical procedures, liver perfusion, excision of the liver and in vitro digestion, preparation and performance of gradient centrifugation, and subsequent, determination of cell yield, viability, and cell purity, and final culturing and verification of HSC identity. The protocol is provided with many figures and a comprehensive list of complementary notes that should help to easily establish the protocol in a laboratory.

The second chapter by Martinez Garcia de la Torre and Sancho-Bru describes a method by which human pluripotent stem cells (hPSCs) can be differentiated into functional HSCs [4]. The protocol is based on the culturing of hPSCs in the presence of different mixes of growth factors. Following this protocol, it is possible to obtain functionally active HSCs after 12 days that are suitable for many biological applications including liver modeling, drug screening tests and many functional assays.

Wankel and Hebbard provide detailed protocols for in vitro measurement of cell migration, invasion, proliferation and apoptosis in HSC [5]. These protocols are extremely helpful for those interested in studying respective cell features in studies evaluating potential therapeutics. In particular, the methods for measuring migration using scratch-wound assay or in transwell chambers, and assays for measuring HSC invasion, proliferation and apoptosis are outlined in a highly standardized form, which will facilitate conducting studies on HSC biology in a precise and reproducible fashion.

Schröder et al. present an optimized protocol for F-actin staining of HSC using fluorescently labeled phalloidin [6]. The protocol contains all relevant information starting from the pretreatment of glass coverslips, optimal conditions for cell seeding, fixation and permeabilization to final phalloidin stain and nuclear counterstain. The representative images of phalloidin stains provided show that the provided protocol is highly suitable for fast and reliable staining of the F-actin network in HSC.

Jansen and colleagues provide a protocol for the identification and quantification of retinyl ester species of HSC using high-resolution mass spectrometry [7]. In their protocol, the retinyl esters are extracted using the Bligh and Dyer method in which the cells are first homogenized in the dark with a mixture of chloroform and methanol. The organic phase is then separated via high performance liquid chromatography and retinyl esters identified and quantified using mass spectrometry analysis.

The chapter by Serna-Salas and colleagues provides a couple of protocols for studying HSC senescence [8]. The chapter gives a comprehensive background of many current methods and outlines several reliable protocols for detecting cellular senescence. In particular, protocols are stated for determination of senescence-associated β-galactosidase, Lamin B1, and senescence-associated heterochromatic foci formation that are all reliable readouts to detect and quantify cellular senescence. Moreover, the authors review relevant markers to identify cell cycle arrest, altered proliferation, nuclear alterations, and morphological changes associated with HSC senescence.

In the contribution of Kim and coworkers, efficient methods are summarized to isolate and purify mouse HSCs and hepatic lymphocytes using density gradient centrifugation and flow cytometry [9]. The presented protocols are suitable to purify cells in high purity and yield, which allows using these cells extensively in direct and indirect co-culturing experiments. It is obvious that the presented protocols will help many researchers to gain a deeper understanding of the immunoregulatory roles of HSCs in both healthy and diseased liver. Moreover, this co-culture system is a significant asset in testing drugs in vitro due to their capacity to interfere with interactions between HSCs and lymphocytes in the pathogenesis of liver disease.

Friedman and Weiskirchen have summarized issues that need to be considered when working with established HSC lines, including experimental recommendations and procedures for media preparation, culture conditions, storage, cryopreservation, banking, and parceling [10]. In addition, information is outlined about safety guidelines, biosafety risk assessment, cell line authentication, and contamination testing. This article further lists detailed features and characteristics for relevant continuous mouse, rat and human HSC lines and provides information on how individual cell lines can be identified based on their morphological, genetic, or biochemical characteristics. As such, this chapter is a comprehensive reference work for those who are attempting to work with immortalized HSC lines.

Weiskirchen et al. provide a detailed protocol for the induction of obstructive cholestasis in mice [11]. The protocol provided for experimental bile duct ligation (BDL) surgery is technically simple and quick to perform. Individual steps of the protocol starting from preparing the workplace, to presurgical animal preparation, and necessary surgical procedures are extensively illustrated. Furthermore, information is provided about postoperative treatment of animals subjected to BDL, essential controls, and suitable readouts that allow monitoring of disease progression.

Information and protocols for two other mouse liver fibrosis models are summarized by Nevzorova et al. [12]. In their chapter, the authors describe the individual steps necessary to induce hepatic fibrosis by repeated administration of carbon tetrachloride or by administering a combination of alcohol plus Western diet, which is known as the DUAL model. In addition, the authors provide a brief overview of many other mouse models associated with HSC and fibrosis that serve as useful experimental tools in hepatic fibrosis research.

Mannerts and colleagues provide a protocol for the simultaneous isolation of mouse hepatocytes and HSC from the same mouse using a Percoll gradient-based isolation protocol for hepatocytes and a flow cytometry-based isolation protocol for HSC [13]. Importantly, the purified cells isolated can be used for many applications including mono-layer cultures, co-culture or even the preparation of free-floating spheroid cultures, which facilitates the study of 3D interactions between these hepatic subpopulations in culture. The figures included in this contribution are very helpful to allow rapid adaptation of respective protocols in any laboratory.

Sun and Schwabe have reviewed and compared different Cre-dependent and Cre-independent methods for genetic labeling, gene knockout, HSC tracing and depletion, as well as their application to different disease models [14]. The sophisticated protocols detailing the conditional deletion of genes in HSC, in vivo labeling and tracing of HSC, and in vivo depletion of HSC provide a highly informative reference manual for respective mouse studies.

Liu et al. describe a perfusion/gradient centrifugation-based method for the isolation, culturing and cryopreservation of highly purified and viable human HSC from normal and diseased livers [15]. Notably, the protocol enables the isolation of human HSC that range from normal to moderately fibrotic from the left liver lobe of donor livers prepared for transplantation, but declined for various reasons. Because the HSC are freshly prepared from completely intact, generally healthy liver, the yield and viability of cells is much higher than human HSC isolated from resected/explanted livers.

Shepherd and colleagues describe strategies for decellularizing cirrhotic and healthy human liver specimens to generate native extracellular matrix for use in automated functional assays with HSC [16]. The workflow and precise protocols for validation of successful decellularization methods are presented in a very understandable way. The cell-free material produced using this method, is useful for many studies including in vitro studies investigating extracellular matrix effects or for studies aiming to use these materials as a scaffold for repopulation studies. The materials generated by this protocol will further provide novel opportunities to study matrix remodeling, HSC-matrix interactions, and the impact of extracellular matrix on HSC senescence, apoptosis, and cell functions.

The team of Guillot describes a sequential immunostaining protocol consisting of repetitive cycles of immunostaining and chemically induced antibody stripping that can be readily applied to various formalin-fixed tissues [17]. The outlined multiple staining immunostaining protocol is straightforward and does not require specific equipment or reagent kits. Tissue section preparation and the individual cycles consisting of staining and stripping are rather simple. In addition, counterstaining, mounting, image processing, and final data generation can be performed using common laboratory equipment. The representative images depicted demonstrate that the methodology is highly suitable to establish complex cellular expression landscapes composed of multiple marker proteins.

Booijink et al. describe a modified enzyme-linked immunosorbent spot (ELISpot) assay suitable to visualize single HSC collagen type I secretion [18]. As such, this methodology is ideally suited to analyze the effect of different stimuli, inhibitors, conditioned medium on extracellular matrix production at single HSC level. Although this methodology is quite sophisticated and still being developed, the possibilities it offer will facilitate future establishment of novel high-throughput technologies for in-depth analysis of single HSC at all different levels (phenotype, secretome, transciptome, and genome).

The chapter by Bansal and Poelstra provides background and protocols for the generation of PDGF-β-receptor targeted (mimetic) IFNγ constructs to activated HSCs [19]. In particular, the authors focus on materials and methods for the construction of PEGylated interfereon-γ (IFNγ)-conjugated PDGF receptor type β (PDGFRβ)-recognizing cyclic peptide (IFNγ-PEG-PPB), human serum albumin modified with a PDGFRβ-recognizing cyclic peptide that is coupled to PEG-IFNγ (PPB-HSA-PEG-IFNγ), and a chimeric molecule containing peptidomimetic IFNγ signaling peptide coupled via heterobifunctional PEG linker to a bicyclic PDGFRβ-binding peptide (IFNγ-PEG-BiPPB). The synthesis methods are widely applicable and adaptable to conjugate other receptor-recognizing peptides, proteins or drug carriers to specifically deliver biological and small molecule therapeutics or diagnostics to HSCs or other target cells.

In the last chapter, Reolizo and colleagues summarize current experimental approaches for translating experimental findings to humans from in vitro cell culture models, in vivo animal models, and new experimental tools [20]. The chapter demonstrates that the drug discovery process is highly complex and that the process from target selection, target validation, performance of carefully designed clinical trials, and final development and registration of a new drugs, highlighting the obstacles that might delay or even prevent translation of promising antifibrotic drugs from animal models into the clinics.

The images on the front cover of this book were kindly provided by Shuang Wang from the Division of Liver Diseases, Icahn School of Medicine at Mount Sinai, New York, NY (Figure 1). They show high-resolution 3D reconstructed stains of HSC marker Desmin with (left) and without (middle) additional nuclear stain and the surface and spot segmentation of Desmin (right) from tissue-cleared murine nonalcoholic steatohepatitis (NASH) livers, as assessed using the Imaris microscopy imaging analysis software (Imaris). These impressive images adequately demonstrate the dense autocrine contacts of HSCs in the diseased liver that are linked to progressive disease severity.

In summary, the book “Hepatic Stellate Cells: Methods and Protocols” includes 18 chapters devoted to exciting research protocols that are widely used in HSC biology research. It features standardized methods to isolate, evaluate, and manipulate HSC and further provides strategies to target this cell type. All protocols are represented in an easy step-to-step format, necessary materials and reagents are listed, and tips to avoid potential pitfalls are listed. As such, this book is ideally suited to readily using respective protocols for own work. The cost of the hardcover book version (ISBN: 978-1-0716-3206-2) is about EUR 215, while the softcover (ISBN: 978-1-0716-3209-3) and eBook version (ISBN: 978-1-0716-3207-9) are cheaper.

As editors, we believe that this book is suitable for both beginners and experts working in the field of HSC biology in health and disease. We hope that the protocols will expedite the implementation of the state-of-the-art standardized methods for interested investigators or trainees.

Finally, we sincerely thank the outstanding expert authors and their teams for their excellent contributions to this book.