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.
Spatial transcriptomics enables genome-wide gene expression profiling while preserving tissue architecture, making it a powerful approach for studying plant developmental transitions. However, preparing small and structurally complex plant tissues for spatial transcriptomics remains technically challenging because samples must be rapidly preserved, precisely oriented, serially sectioned, and accurately positioned within the limited capture area of the Visium slides. Here, we describe an optimized workflow for cryo-embedding, serial cryosectioning, section placement, and data analysis of small plant samples for 10x Genomics Visium spatial transcriptomics. Using maize seedling shoot apices as the target, this protocol includes preparation of custom molds for optimal cutting temperature embedding, rapid fresh sample embedding, serial cryosectioning, section-position marking for Visium HD workflows, and morphological quality assessment of replicate tissue slides before transcript capture. The associated data analysis workflow includes Space Ranger processing, Seurat-based normalization and Harmony integration, anatomical domain annotation, pseudobulk and developmental trend analyses, RNA velocity, pseudotime analysis, transcription factor network analysis, single-cell reference mapping, and 3D transcriptome reconstruction. This computational workflow was developed and tested using maize Visium V1 data, but not Visium HD data. This protocol was used to generate serial spatial transcriptomes of maize shoot apices and developing leaf primordia, enabling reconstruction of gene expression transitions from the shoot apical meristem to sequential leaf developmental stages. The approach is also applicable to other small plant tissues, including Arabidopsis first true leaves and Marchantia thalli.
