Proliferation and survival analyses of engrafted CD34⁺ HSPCs in human bone marrow organoids. (A) The CellTrace proliferation assay of engrafted CD34⁺ HSPCs on days 2, 5, 10, and 15. CD34⁺ HSPCs (1 × 10⁶) were pooled from 3 independent MDS samples and stained with CellTrace Far Red dye. CD34⁺ stained HSPCs (5 × 10³) were engrafted into each organoid and at least 10 organoids were pooled for each flow cytometry assay. The CellTrace intensity of engrafted CD34⁺ cells was tested on days 2, 5, 10, and 15 after engraftment. (B-C) Cell death of the engrafted CD34⁺ cells was assessed using DAPI (B) and quantified in panel C. (D) The total output of hematopoietic cells per 1000 engrafted CD34⁺ HSCPs was assessed using cell-counting beads. (E) Secondary engraftment of CD34⁺ HSPCs. CellTrace+ HSPCs were sorted using FACS on day 3 after primary engraftment. Live sorted HSPCs (5 × 10³) were engrafted into each human bone marrow organoid. The secondary engraftment was assessed with whole-mount 3D imaging. (F) Flow cytometry analysis of the indicated cell lineages in the CellTrace+ engrafted donor cells in the secondary engrafted organoids. The percentages of the indicated populations are presented. APC, antigen-presenting cell; DAPI, 4′,6-diamidino-2-phenylindole.