Stem Cell

Human bone marrow organoids provide a tractable three-dimensional platform for modeling hematopoiesis and hematologic disease in a human niche–like context. Here, we describe a stepwise protocol for utilizing human induced pluripotent stem cell (iPSC)-derived bone marrow organoids that support autonomous hematopoiesis for hematopoietic disease modeling, mouse xenograft hematopoiesis, and drug sensitivity testing. The workflow combines embryoid body formation, early mesoderm/angiogenic induction under hypoxia, hemogenic endothelial commitment, maturation within a collagen-containing hydrogel, and subsequent suspension culture as individual organoids. The resulting organoids contain endothelial, stromal, and hematopoietic components and reproduce key structural and cellular features of human marrow. We further describe procedures for engraftment of normal donor- or patient-derived CD34+ cells and implantation of mature organoids under the renal capsule of immunodeficient mice to assess in vivo hematopoietic maintenance. In prior applications of this platform, donor-derived CD34+ cells were shown to engraft within the organoid niche and undergo multilineage differentiation, enabling detection of selective erythroid defects caused by DDX41 deficiency and assessment of therapeutic suppression of JAK2V617F-mutant patient-derived hematopoietic cells in a human marrow–like microenvironment. This protocol, therefore, enables disease modeling, in vivo xenograft assessment, and ex vivo functional analysis of patient-derived hematopoietic cells using relatively small input samples.

Whole-mount 3D imaging of multilayered biological tissues enables quantitative analysis of cell states and organization in their spatial context. However, extracting unbiased and meaningful quantitative information from dense, multilayered samples remains challenging due to imaging artifacts, increased density, and limited signal-to-noise ratio. Open source bioimage analysis workflows tailored to this type of analysis are scarce, and analysis bottlenecks like image curation or cell segmentation are seldom available without coding expertise. Here, we present a step-by-step computational protocol for the analysis of dense 3D organoid datasets using the Tapenade (Thorough Analysis PipEliNe for Advanced DEep imaging) workflow. Starting from multichannel image stacks, the protocol guides users through software installation, registration and fusion of multi-view datasets, preprocessing, and nuclei segmentation. It further details the generation of quantitative outputs, including morphometric measurements, deformation fields, and spatial correlation analyses. The workflow can be executed through open-source Python scripts or user-friendly Napari interfaces, allowing interactive parameter tuning and 3D visualization at each stage. This pipeline provides an accessible and modular framework for nonspecialist users to perform reproducible, multiscale quantitative analysis of 3D organoid images, while retaining flexibility for advanced users to customize individual steps.

Human induced pluripotent stem cell (hiPSC)-derived liver organoids have emerged as valuable models for studying human liver development. However, existing organoid systems often lack developmentally matched cell populations, particularly fetal hepatic stellate cells (HSCs), limiting their ability to recapitulate key developmental processes. Current approaches for generating HSCs rely on primary cells, immortalized cell lines, or hiPSC differentiation methods that frequently produce activated HSC-like cells and often require cell sorting. Here, we describe an efficient protocol for generating expandable fetal-like HSCs from hiPSCs through a stepwise differentiation strategy that mimics embryonic HSC development. The resulting cells can be robustly expanded while maintaining characteristic molecular and functional features of fetal HSCs. This protocol provides a reproducible and scalable source of fetal-like HSCs without cell sorting and supports the generation of multicellular liver organoids containing developmentally relevant stromal components. Beyond the validation of the protocol in studies of liver maturation and vascularization, it can be applied to investigations of HSC biology and congenital liver diseases.

The mucus layer lining the human stomach is a critical barrier that protects the underlying epithelium from gastric acid and harmful pathogens such as Helicobacter pylori. The efficacy of this barrier relies on the structural integrity of the mucus, which is determined by various biochemical and biophysical features. Human gastric organoids—3D cellular models that resemble the stomach—contain mucus and have been used to investigate gastric disease. The luminal compartment of three-dimensional epithelial organoids represents a physiologically relevant but experimentally inaccessible microenvironment. In gastric organoids, luminal accumulation of mucus creates a confined viscoelastic hydrogel that mimics native gastric mucus. However, the small volume and topological confinement of organoids preclude conventional bulk rheometry. Here, we describe a particle tracking microrheology (PTM) protocol to measure the viscoelastic properties of the mucus within intact organoid lumina following microinjection of fluorescent microspheres. High-speed fluorescence imaging and particle trajectory analysis enable the quantification of viscous and elastic properties of the mucus through calculation of mean squared displacement (MSD), diffusive scaling exponent (alpha), and frequency-dependent storage (G’) and loss (G’’) moduli. This method enables rheological measurements in nanoliter-scale compartments without disrupting organoid architecture. We further discuss the impact of mucus heterogeneity and microstructure on scale-dependent mechanical behavior. This protocol is broadly applicable to other organoid systems and can be adapted to Transwell or organ-on-chip platforms for in situ luminal measurements.

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.

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 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.

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.

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.