Generation of Human Induced Pluripotent Stem Cell-Derived Bone Marrow Organoids

The human bone marrow niche plays an essential role in supporting and regulating hematopoiesis throughout life. Three dimensional in vitro models to study spatio-temporal control of intrinsic and/or extrinsic factors orchestrating hematopoietic fate decisions in health and disease are needed. Here we present a method to generate a complex bone marrow-like organoid (BMO) from human induced pluripotent stem cells (iPSCs), consisting of hematopoietic, mesenchymal and vascular cells. Our approach expands current co-culture-based models of isolated primary endothelial and stromal cells, or bioengineered microfluidic chips and overcomes some of their intrinsic limitations. We devised a protocol of stepwise mesodermal progenitor differentiation through hemogenic endothelium to generate a complex heterocellular organoid: After embryoid body formation, mesoderm was induced and patterned by a sequential combination of Wnt activation, Nodal inhibition, BMP4, VEGF, bFGF and SCF. Embryoid bodies were embedded into a three-dimensional matrix and stimulated with defined cytokine cocktails in a timely fashion to generate hematopoietic progenitor cells while maintaining endothelial cell generation. At day 10 of differentiation, sprouted embryoid bodies were separated and transferred into a 96-well plate to promote full organoid maturation until day 17. Flow cytometric analysis of dissociated BMOs revealed three main cellular compartments, consisting of hematopoietic (CD45+), mesenchymal (CD45-CD31-CD34-CD271+) and endothelial cells (CD45-CD31+). Analysis of the cellular composition yielded an average content of~40,6% hematopoietic cells, ~39,5% mesenchymal cells, ~6% endothelial cells, ~1,6% HSPCs and ~0,9% MSCs per BMO. Single-cell RNA sequencing of dissociated BMOs confirmed the presence of these distinct cell clusters. Sorted hematopoietic (CD45+CD11b-CD34+-HSPCs) and mesenchymal (CD45-CD31-CD271+CD105+CD90+CD73+-MSCs) stem and progenitor cells showed multilineage differentiation potential in colony-forming-unit (CFU) and trilineage-differentiation assays, respectively. Confocal imaging of whole organoids showed that CD31+ endothelial cells formed a vessel-like network covered by a Collagen IV+ basement membrane and PDGFR-β+ murals cells, resembling pericytes. Ultrastructural analysis by transmission electron microscopy (TEM) revealed capillary-like structures of endothelial cells enclosed by pericytes, which were connected by tight junctions. Importantly, we also detected CXCL12+- CAR-like cells in close association to endothelial cells in our BMOs. Whereas CD45 expressing cells were distributed throughout the organoid, Runx1 expressing cells were found to be localized in distinct clusters. We examined whether the BMO niche promotes maturation of blood progenitors into mature cells of the myeloid lineage. We identified neutrophil progenitor stages from ProNeu to matureNeus by flow cytometry, S100A8/A9 and MPO expressing cells by confocal imaging and cells with characteristic features of neutrophil granulocytes by TEM, showing that granulopoiesis proceeds without the addition of lineage-instructing cytokines as G-CSF or GM-CSF. Next, we tested whether BMOs can be used as a model system to recapitulate the phenotype of monogenic bone marrow failure syndromes. BMOs from iPSCs edited to express a hypomorphic variant of VPS45 showed increased formation of reticulin fibers and a higher abundance of SMA expressing pericytes, reminiscent of increased myelofibrosis in bone marrow biopsies of VPS45 deficient patients. We also started to show that BMOs can be transplanted into immunodeficient NSG-mice with further BMO growth in vivo for up to four months. In ongoing experiments we analyze the contribution of human hematopoietic cells to blood cell chimerism in the host mice. In summary, iPSC-derived BMOs offer a new tool to study hematopoietic development and disease evolution within a complex three-dimensional human environment exhibiting key cellular and structural features of the human bone marrow niche.