Disease Modeling in iPSC-Derived Human Bone Marrow Organoids

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. Key features • Stepwise differentiation of human iPSCs into bone marrow organoids containing endothelial, stromal, and hematopoietic compartments. • Efficient engraftment of donor-derived CD34+ HSPCs into mature organoids using a Matrigel-assisted seeding method. • Applicable to imaging, flow cytometry, cytogenetics, and downstream molecular analyses of organoid and donor-derived hematopoietic cells. • Includes renal capsule implantation for in vivo xenograft assessment of organoid-supported hematopoiesis. • Enables disease modeling and ex vivo functional studies of patient-derived hematopoietic cells, including analysis of erythroid and myeloid phenotypes and therapeutic responses.