Cell Biology

A rigorous determination of effector contributions of tumor-infiltrating immune cells is critical for identifying targetable molecular mechanisms for the development of novel cancer immunotherapies. A tumor/immune cell–admixture model is an advantageous strategy to study tumor immunology as the fundamental methodology is relatively straightforward, while also being adaptable to scale to address increasingly complex research queries. Ultimately, this method can provide robust experimental information to complement more traditional murine models of tumor immunology. Here, we describe a tumor/macrophage-admixture model using bone marrow–derived macrophages to investigate macrophage-dependent tumorigenesis. Additionally, we provide commentary on potential branch points for optimization with other immune cells, experimental techniques, and cancer types.

Human neuromuscular diseases represent a diverse group of disorders with unmet clinical need, ranging from muscular dystrophies, such as Duchenne muscular dystrophy (DMD), to neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS). In many of these conditions, axonal and neuromuscular synapse dysfunction have been implicated as crucial pathological events, highlighting the need for in vitro disease models that accurately recapitulate these aspects of human neuromuscular physiology. The protocol reported here describes the co-culture of neural spheroids composed of human pluripotent stem cell (PSC)–derived motor neurons and astrocytes, and human PSC-derived myofibers in 3D compartmentalised microdevices to generate functional human neuromuscular circuits in vitro. In this microphysiological model, motor axons project from a central nervous system (CNS)–like compartment along microchannels to innervate skeletal myofibers plated in a separate muscle compartment. This mimics the spatial organization of neuromuscular circuits in vivo. Optogenetics, particle image velocimetry (PIV) analysis, and immunocytochemistry are used to control, record, and quantify functional neuromuscular transmission, axonal outgrowth, and neuromuscular synapse number and morphology. This approach has been applied to study disease-specific phenotypes for DMD and ALS by incorporating patient-derived and CRISPR-corrected human PSC-derived motor neurons and skeletal myogenic progenitors into the model, as well as testing candidate drugs for rescuing pathological phenotypes. The main advantages of this approach are: i) its simple design; ii) the in vivo–like anatomical separation between CNS and peripheral muscle; and iii) the amenability of the approach to high power imaging. This opens up the possibility for carrying out live axonal transport and synaptic imaging assays in future studies, in addition to the applications reported in this study.

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Graphical abstract abbreviations: Channelrhodopsin-2 (CHR2+), pluripotent stem cell (PSC), motor neurons (MNs), myofibers (MFs), neuromuscular junction (NMJ).

In the peripheral nervous system, Schwann cells are the primary type of glia; their in vitro differentiation and dedifferentiation system has not been described in detail in the literature. Thus, an in vitro differentiation and dedifferentiation system of rat Schwann cells is described in this protocol. These cultures and systems may be used to investigate the morphological and biochemical effects of drug interventions or lentivirus-mediated gene transfer on Schwann cells during differentiation or dedifferentiation.

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Cardiac fibroblasts are one of the major constituents of a healthy heart. Cultured cardiac fibroblasts are a crucial resource for conducting studies on cardiac fibrosis. The existing methods for culturing cardiac fibroblasts involve complicated steps and require special reagents and instruments. The major problems faced with primary cardiac fibroblast culture are the low yield and viability of the cultured cells and contamination with other heart cell types, including cardiomyocytes, endothelial cells, and immune cells. Numerous parameters, including the quality of the reagents used for the culture, conditions maintained during digestion of the cardiac tissue, composition of the digestion mixture used, and age of the pups used for culture determine the yield and purity of the cultured cardiac fibroblasts. The present study describes a detailed and simplified protocol to isolate and culture primary cardiac fibroblasts from neonatal murine pups. We demonstrate the transdifferentiation of fibroblasts into myofibroblasts through transforming growth factor (TGF)-β1 treatment, representing the changes in fibroblasts during cardiac fibrosis. These cells can be used to study the various aspects of cardiac fibrosis, inflammation, fibroblast proliferation, and growth.

Primary hematopoietic stem and progenitor cell (HSPC)-derived megakaryocytes are a valuable tool for translational research interrogating disease pathogenesis and developing new therapeutic avenues for patients with hematologic disorders including myeloproliferative neoplasms (MPNs). Thrombopoietin (TPO)-independent proliferation and megakaryocyte differentiation play a central role in the pathogenesis of essential thrombocythemia and myelofibrosis, two MPN subtypes that are characterized by increased numbers of bone marrow megakaryocytes and somatic mutations in either JAK2, CALR, or MPL. However, current culture strategies generally use healthy HSPCs for megakaryocyte production and are not optimized for the investigation of TPO-independent or TPO-hypersensitive growth and megakaryocyte-directed differentiation of primary patient–derived HSPCs. Here, we describe a detailed protocol covering all necessary steps for the isolation of CD34⁺ HSPCs from the peripheral blood of MPN patients and the subsequent TPO-independent differentiation into CD41⁺ megakaryocytes using both a collagen-based colony assay and a liquid culture assay. This protocol provides a novel, reproducible, and cost-effective approach for investigating megakaryocyte growth and differentiation properties from primary MPN patient cells that can be easily adapted for research on other megakaryocyte-related disorders.

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Schematic representation of the isolation of CD34⁺ progenitor cells and subsequent TPO-independent megakaryocyte differentiation

Conjunctival disorders are multivariate degenerative ocular surface diseases that can jeopardize ocular function and impair visual capacity in severe cases. The recent development of stem cell technologies has shed a new light on the treatment of conjunctival disorders as the regenerative medicine using endogenous stem cells becomes a potential therapeutic strategy. However, the efficient in vitro expansion of the endogenous stem cells dominating the conjunctival regeneration, the conjunctival stem cells (CjSCs), remains challenging. Existing protocols largely adopted primary culture using feeder layers, which has limited efficiency and risk of contamination. Here, we report a protocol for the isolation and expansion of primary CjSCs derived from human or animal tissues. This protocol adopts collagenase-based enzymatic digestion to release the primary cells from conjunctival tissues and utilizes a feeder-free culture strategy based on a small molecule inhibitor cocktail that stimulates the expansion of CjSCs. The CjSCs generated with this method were rapidly dividing and highly homogeneous. They also expressed characteristic stem cell markers and exhibited differentiation potency. These findings marked an important step forward in building stable CjSCs in vitro expansion, which will help researchers better understand the biology of ocular surface stem cells and develop innovative regenerative medicine approaches for ocular surface diseases.

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Macrophages are a heterogeneous class of innate immune cells that offer a primary line of defense to the body by phagocytizing pathogens, digesting them, and presenting the antigens to T and B cells to initiate adaptive immunity. Through specialized pro-inflammatory or anti-inflammatory activities, macrophages also directly contribute to the clearance of infections and the repair of tissue injury. Macrophages are distributed throughout the body and largely carry out tissue-specific functions. In skeletal muscle, macrophages regulate tissue repair and regeneration; however, the characteristics of these macrophages are not yet fully understood, and their involvement in skeletal muscle aging remains to be elucidated. To investigate these functions, it is critical to efficiently isolate macrophages from skeletal muscle with sufficient purity and yield for various downstream analyses. However, methods to prepare enriched skeletal muscle macrophages are scarce. Here, we describe in detail an optimized method to isolate skeletal muscle macrophages from mice. This method has allowed the isolation of CD45⁺/CD11b⁺ macrophage-enriched cells from young and old mice, which can be further used for flow cytometric analysis, fluorescence-activated cell sorting (FACS), and single-cell RNA sequencing.

Entosis is a process where a living cell launches an invasion into another living cell’s cytoplasm. These inner cells can survive inside outer cells for a long period of time, can undergo cell division, or can be released. However, the fate of most inner cells is lysosomal degradation by entotic cell death. Entosis can be detected by imaging a combination of membrane, cytoplasmic, nuclear, and lysosomal staining in the cells. Here, we provide a protocol for detecting entosis events and measuring the kinetics of entotic cell death by time-lapse imaging using tetramethylrhodamine methyl ester (TMRM) staining.

Babesiosis is a tick-borne disease caused by pathogens belonging to the genus Babesia. In humans, the disease presents as a malaria-like illness and can be fatal in immunocompromised and elderly people. In the past few years, human babesiosis has been a rising concern worldwide. The disease is transmitted through tick bite, blood transfusion, and transplacentally in rare cases, with several species of Babesia causing human infection. Babesia microti, Babesia duncani, and Babesia divergens are of particular interest because of their important health impact and amenability to research inquiries. B. microti, the most commonly reported Babesia pathogen infecting humans, can be propagated in immunocompetent and immunocompromised mice but so far has not been successfully continuously propagated in vitro in human red blood cells (hRBCs). Conversely, B. divergens can be propagated in vitro in hRBCs but lacks a mouse model to study its virulence. Recent studies have highlighted the uniqueness of B. duncani as an ideal model organism to study intraerythrocytic parasitism in vitro and in vivo. An optimized B. duncani in culture and in mouse (ICIM) model has recently been described, combining long-term continuous in vitro culture of the parasite in human red blood cells with an animal model of parasitemia (P) and lethal infection in C3H/HeJ mice. Here, we provide a detailed protocol for the use of the B. duncani ICIM model in research. This model provides a unique and sound foundation to gain further insights into the biology, pathogenesis, and virulence of Babesia and other intraerythrocytic parasites, and has been validated as an efficient system to evaluate novel strategies for the treatment of human babesiosis and possibly other parasitic diseases.

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ICIM model [Adapted and modified from Pal et al. (2022)]

Skeletal muscle stem cells differentiated from human-induced pluripotent stem cells (hiPSCs) serve as a uniquely promising model system for investigating human myogenesis and disease pathogenesis, and for the development of gene editing and regenerative stem cell therapies. Here, we present an effective and reproducible transgene-free protocol for derivation of human skeletal muscle stem cells, iMyoblasts, from hiPSCs. Our two-step protocol consists of 1) small molecule-based differentiation of hiPSCs into myocytes, and 2) stimulation of differentiated myocytes with growth factor-rich medium to activate the proliferation of undifferentiated reserve cells, for expansion and cell line establishment. iMyoblasts are PAX3⁺/MyoD1⁺ myogenic stem cells with dual potential to undergo muscle differentiation and to self-renew as a regenerative cell population for muscle regeneration both ex vivo and in vivo. The simplicity and robustness of iMyoblast generation and expansion have enabled their application to model the molecular pathogenesis of Facioscapulohumeral Muscular Dystrophy and Limb-Girdle Muscular Dystrophies, to both ex vivo and in vivo muscle xenografts, and to respond efficiently to gene editing, enabling the co-development of gene correction and stem cell regenerative therapeutic technologies for the treatment of muscular dystrophies and muscle injury.

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