Human Induced Pluripotent Stem Cell-Derived Bone Marrow Organoids to Model Hematopoietic Development
Authors: Frenz-Wiessner Stephanie , Fairley Savannah , Buser Maximilian , Goek Isabel , Salewskij Kirill , Jonsson Gustav , zu Putlitz Benedicta , Petersheim Daniel , Illig David , Li Yue , Chen Pin-Hsuan , Kalauz Martina , Conca Raffaele , Sterr Michael , Geuder Johanna , Mizoguchi Yoko , Kotlarz Daniel , Rudelius Martina , Penninger Josef M , Marr Carsten , Klein Christoph 
Published: December 2023

The human bone marrow niche plays an essential role in supporting and regulating hematopoiesis throughout life and inherited bone marrow failure syndromes are often linked to dysfunction of the niche. Recently, methods for generating induced pluripotent stem cell (iPSC)-derived hematopoietic organoids (Motazedian et al., 2020) and bone marrow organoids (Khan et al., 2022) have been developed. We recently presented our approach to generate a model of a complex human bone marrow microenvironment in vitro (ASH annual meeting 2022 ). Here, we characterize our model in greater detail and analyse functionality of bone marrow organoid (BMO)-derived HSPCs.

We devised a feeder- and serum-free protocol to generate a complex human iPSC-derived BMO within three weeks. In comparison to the protocol by Khan et al., we incorporated a sequential combination of Wnt activation and Activin/Nodal inhibition to induce mesoderm formation and patterning. We replicated the method with five different iPSC lines, including fibroblast- and PBMC-derived iPSCs, as well as iPSCs generated from renal epithelial cells isolated from urine samples. Using confocal and two-photon microscopy we visualized the spatial architecture of differentiated BMOs and detected CD45 + cells embedded into a network of CD31 + vascular cells and CD271 + stromal cells. PDGFRβ expressing pericytes were enwrapping the vascular cells. Moreover, stromal cells expressed CXCL12, Leptin receptor and Nestin. Electron microscopy, HE staining of organoid sections and 3D surface rendering showed lumen-forming vascular structures containing hematopoietic cells.

To further elucidate the cell type composition, we performed single-cell RNA sequencing (scRNA-seq) of differentiated BMOs. Clustering of the scRNA-seq data by marker gene expression yielded three main populations: Cells in the endothelial cluster expressed canonical genes of arterial ECs. In the mesenchymal cluster we could distinguish pericytes, vascular smooth muscle cells and osteochondrogenic precursors. In the hematopoietic compartment, we identified cells expressing molecular signatures of granulocytes, monocytes and their progenitors, but also megakaryocytic-like cells and lymphoid progenitors. Remarkably, a cluster of cells expressed genes of fetal HSCs. This suggests that BMOs may recapitulate definitive hematopoiesis. To test T-cell differentiation potential in vitro, we sorted CD34 + HSPCs from dissociated BMOs and differentiated them within an artificial thymic organoid system (ATO). After three weeks we detected CD45 +CD4 +CD8 + cells, some of them expressing CD3 +TCRαβ +. Thus, we postulate that T-cells phenotypically resembling conventional T-cells emerge from BMO-derived HSPCs. To assess functional properties of BMO-derived HSPCs in vivo, we transplanted sorted CD34 + HSPCs into immunodeficient NSG mice and are currently analysing their engraftment potential.

In summary, we provide a novel bone marrow niche model to study hematopoietic development in a complex three-dimensional organoid derived from human iPSCs.