Stem Cell

Limb development requires the coordination of multiple cell types, including the limb bud mesoderm and surface ectoderm, by the apical ectodermal ridge (AER), a specialized signaling center secreting numerous morphogens. Characterizing these cell–cell interactions is crucial for understanding limb morphogenesis, but they are challenging to study in vivo. Furthermore, existing in vitro models do not capture the multilineage complexity of the limb. We recently developed a robust 7-day differentiation protocol using mouse embryonic stem cells (mESCs) to generate heterogeneous cultures containing cells with characteristics of the limb bud mesoderm, surface ectoderm, and AER. Dissociating and reaggregating these cultures in low attachment 96-well plates forms budoids, organoids that display certain limb bud–like features. Budoids undergo chondrogenesis-mediated symmetry breaking and elongation within 5 days of culture. Altogether, our protocols have enabled the study of cell–cell interactions in limb development and provide an easily scalable model adaptable for various applications, including drug testing and congenital disorder modeling.

Amyotrophic lateral sclerosis (ALS) is characterized by early and spatially restricted pathology in motor axons, including distal degeneration and accumulation of aggregation-prone proteins such as TDP-43. However, a major limitation in the field has been the lack of approaches that enable robust, quantitative, and compartment-specific analysis of these early axonal events, particularly in human-relevant systems. Here, we describe an integrated experimental and analytical framework that enables quantitative dissection of axonal degeneration and protein aggregation, specifically within distal motor axons. By combining compartmentalized human co-cultures with a dedicated image analysis strategy, this approach enables selective and quantitative analysis of pathological processes specifically within axons, independent of surrounding tissues such as muscle and other cellular compartments. This framework captures both structural degeneration and protein aggregation dynamics at subcellular resolution, enabling spatially resolved quantitative analysis of disease-relevant changes along axons. Importantly, the analytical framework is not limited to TDP-43 but is broadly applicable to diverse aggregation-prone proteins, thereby providing a generalizable platform to study axonal pathology across neurodegenerative diseases. Together, this work provides a scalable approach for investigating axonal pathology as an early and measurable feature of neurodegeneration, with potential applications in mechanistic studies and therapeutic targeting in ALS and related disorders.

The airway epithelium interfaces with the external environment through its apical surface and with the extracellular matrix (ECM) through its basolateral surface. To model this organization in vitro, we developed a decellularized ECM-incorporated apical-out airway organoid (dECM-AoAO) platform in which human bronchial epithelial cells (HBECs) self-assemble around human lung-derived decellularized ECM microparticles (dECM-MPs). This configuration preserves apical-out polarity while enabling direct epithelial–ECM interactions. Here, we describe a protocol for the vacuum filtration and quantification of dECM-MPs, the generation of dECM-AoAOs, and ultimately, whole-mount immunofluorescence staining for organoid characterization.

Embryonic blood formation encompasses the independent generation of different cell types in distinct cellular and anatomical environments, reflecting highly coordinated specific hierarchies of interacting tissues. Despite widespread use of embryonic stem cells (ESC) and induced pluripotent stem cell (iPSC)-based models to attempt to capture blood development in vitro and generate hematopoietic stem cells (HSC), a system that fully captures the spatial and temporal complexity of embryonic hematopoiesis is still lacking. In recent years, gastruloid models have emerged as powerful representations of early development, demonstrating self-organizing behaviors such as symmetry breaking, elongation, multi-axis formation, somitogenesis, and early organogenesis, with striking parallels to embryonic processes. Here, we present a protocol to generate hemogenic gastruloids (haemGx) from mouse ESC (mESC) that closely recapitulates the multi-stage, multi-niche process of blood formation and generates developmentally accurate hematopoietic progenitors. The haemGx model has been proven valuable in understanding embryonic hematopoiesis, as well as an in vitro model of forms of infant leukemia with an embryonic, in utero origin.

Organoids as a drug discovery platform represent an emerging field that continues to refine its tools. NanoBRET (bioluminescence resonance energy transfer) has emerged as a proximity-based and highly sensitive assay to measure protein–protein and protein–ligand interactions. NanoBRET assays were developed and are currently used for 2D cell line experiments. Here, we present the development of the first organoid-compatible Nanoluciferase (Nluc) for 3D model systems. We utilise the Nluc for NanoBRET assays to test drug–target engagement. We describe steps for seeding, transfecting, and replating of mouse colorectal cancer organoids. In addition, we provide detailed procedures for the NanoBRET assay. Various lines of evidence have shown significant difference in drug response between 2D human cell lines and 3D model systems, including patient-derived organoids. Our protocol provides a template for measuring this difference in the context of drug–target engagement.

Satellite cells are adult skeletal muscle stem cells that play essential roles in muscle regeneration. Understanding their behavior is critical for elucidating the mechanisms of muscle repair and advancing muscle regenerative therapies. This requires efficient methods for genetic manipulation in these cells. Retroviral-mediated gene delivery is commonly used for stable transgene expression in immortalized cell lines. However, existing approaches are not optimized for primary satellite cells, often resulting in variable efficiency and inconsistent outcomes. Here, we describe an optimized protocol for satellite cell isolation and culture, as well as retroviral production and infection of primary satellite cells that achieves high transduction efficiency. The satellite cell isolation procedure enriches for myofiber fragments prior to satellite cell release, thereby reducing contamination by non-myogenic cells and improving cell purity. Another key feature of this protocol is the concentration of retroviral particles and their resuspension in satellite cell growth medium prior to infection, which minimizes satellite cell exposure to packaging cell-conditioned medium. Compared to standard approaches, this protocol improves both infection efficiency and reproducibility. It is readily adaptable to a wide range of downstream applications, including microscopies, biochemical assays, and molecular biology analyses.

Organoids are three-dimensional cell structures derived from stem cells that recapitulate the architecture and function of native tissues. Histological analysis of organoids is essential for assessing their structure, cellular composition, and responses to experimental conditions. However, their small size and fragility make standard paraffin embedding workflows difficult. Here, we describe a robust and reproducible protocol for the fixation, paraffin embedding, and sectioning of human organoids, enabling high-quality histological and immunostaining analysis. The method involves direct fixation within the culture matrix and inclusion in HistoGel to prevent organoid loss during processing. The protocol is compatible with hematoxylin–eosin (H&E) staining and multiplex immunofluorescence. Critical steps, troubleshooting, and adaptations for intestinal and cardiac organoids are discussed. This cost-effective and accessible method supports long-term preservation and detailed structural analysis of organoid models.

In vitro vascular models are most informative when they recapitulate endothelial assembly within a 3D microenvironment. Blood vessel organoids (BVOs) enable the study of vascular heterogeneity, function, and organ-instructive cues in development, homeostasis, and disease. Here, we present a robust stepwise method to generate murine blood vessel organoids (mBVOs) from feeder-dependent mouse embryonic stem cells (mESCs) of common genetic backgrounds. Embryoid bodies (EBs) are formed using strain-specific seeding densities (day 0–3), followed by mesoderm induction (day 3–6) and vascular induction (day 6–8). Induced EBs are embedded in collagen I with Geltrex to drive sprouting and network formation (day 8–13). Vascular networks are microdissected and grown in suspension to yield mature mBVOs (day 21–30). The inclusion of a Cre-inducible VE-cadherin-GFP reporter line enables a quantitative quality control, reducing variability by excluding poorly differentiated organoids. The protocol reliably produces ~100 mBVOs per differentiation and is compatible with engineered mouse strains for gain- and loss-of-function studies, functional assays of vascular plasticity, and syngeneic grafting to assess perfusion. Thus, mBVOs provide a scalable and traceable 3D platform that bridges endothelial assays, mouse models, and human organoid systems.

The thymus is critical for the establishment of a functional and self-tolerant adaptive immune system, but it involutes with age, resulting in reduced naive T-cell output. Generation of a functional human thymus from human pluripotent stem cells (hPSCs) is an attractive regenerative medicine strategy. Direct differentiation of thymic epithelial progenitors (TEPs) from hPSCs has been demonstrated in vitro, but functional thymic epithelial cells (TECs) develop only after transplantation of TEPs in vivo. Functional human reaggregated thymic organoid cultures (RTOCs) and artificial thymic organoids (ATOs) cultured at the air–liquid interface support T-cell development in vitro and in vivo and permit the interrogation of human thymic function and T-cell development. However, these approaches require access to primary human tissues or murine bone marrow stromal cells, are allogeneic, and do not support negative selection. Recently, we reported the directed differentiation of induced PSCs (iPSCs) to functional thymic epithelial progenitors (TEPs) that support murine T-cell development after transplantation in nude mice. Here, we combined hPSC-derived TEPs, hematopoietic progenitor cells (HPCs), and mesenchymal cells, differentiated from the same hPSC line, and generated functional isogenic stem cell–derived thymic organoids (sTOs). Our revised protocol improves our TEP differentiation process and allows the generation of functional isogenic, patient-specific thymic organoids in vitro.

Bovine muscle satellite cells (MuSC) and fibro-adipogenic progenitor cells (FAP) are muscle resident stem cells that are responsible for postnatal muscle growth, intramuscular fat deposition, and extracellular matrix generation. These cells are of increasing interest for the cultivated meat community due to their ability to generate all the major components of meat; additionally, these cells are of interest to conventional animal science research to elucidate mechanisms to improve meat quality. To use these cells for these goals, efficient and accurate cell isolation, culture, and differentiation are essential to evaluate their cell fate decisions and behaviors. In this protocol, we detail a simultaneous isolation of both MuSCs and FAPs with multiple intermediate stopping points, allowing for flexibility for day-of time constraints. We also detail improved growth conditions to maximize cell expansion and procedures to assess cell differentiation. This protocol provides a flexible isolation procedure that is compatible with sampling in modern slaughterhouses or from biopsies. Additionally, the differentiation procedures provide improved differentiation but still allow in vitro treatment and assessment.