BMP-eBM and LB-BM show similar cellularity. Bone morphogenetic protein (BMP)-2 (8 μg)/beta-tricalcium phosphate (β-TCP; 40 mg) was implanted into C57BL6, Col1a1(2.3)-GFP; Trap-tdTomato, or Cxcl12-GFP mice. BMP-2–induced ectopic bone marrow (BMP-eBM) was harvested 4 weeks after implantation and compared with native bone marrow in long bones (LB-BM). (A) Representative hematoxylin and eosin (HE) staining images of BMP-eBM and LB-BM. Yellow arrowhead, adipocyte; red arrowhead, red blood cell. (B) Scanning electron microscope images of β-TCP before implantation, BMP-eBM (4 weeks), and LB-BM. Lower panels show high-magnification images of the upper panels. (C) Fluorescence images: upper panel (green, Col1a1(2.3)-GFP; red, Trap-tdTomato); middle panel (red, α-smooth muscle actin [αSMA]); and lower panel (red, periostin in BMP-eBM [4 weeks] and LB-BM). The blue color represents DAPI staining. (D) Quantification analysis of the number of osteoblasts per bone perimeter (N.Ob/B. Pm) and the number of osteoclasts per bone perimeter (N.Oc/B. Pm) in upper image of panel C (n-3). (E-F) Frequency of CXC-chemokine-ligand-12 (CXCL12)-abundant reticular (CAR) cell analyzed by flow cytometric analysis (FCM; n = 4). CAR cell: CD45^–Ter119^–CD31^–Cxcl12-GFP^high. (G) Fluorescence images (green, Cxcl12-GFP and red, Endomucin (Emcn) in BMP-eBM [4 weeks] and LB-BM). The blue color indicates DAPI staining. (H) Frequency of hematopoietic stem cells (HSCs) analyzed by FCM (n = 4). HSC: Lin^–Sca-1⁺c-Kit⁺CD150⁺CD48^–. (I) Cellularity, number of HSCs, and number of CAR cells in BMP-eBM after 2, 4, and 6 weeks of implantation (n = 4). (J) Frequency of common lymphoid progenitor (CLP), common myeloid progenitor (CMP), pre–pro-B, pro-B, pre-B, total B cell, T cell, natural killer (NK) cell, myeloid cell, or erythroid cell (n = 4). CLP, Lin^–Sca-1^lowc-Kit^lowCD127⁺CD135⁺; CMP, Lin^–Sca-1^–c-Kit⁺CD34⁺CD16/32^–; pre–pro-B, B220⁺sIgM^–CD43⁺CD24^–; pro-B, B220⁺sIgM^–CD43⁺CD24⁺; pre-B, B220⁺sIgM^–CD43^–CD24⁺; total B cell, B220⁺; T cell, CD3⁺CD45⁺; NK cell, NK1.1⁺CD3^–; myeloid cell, CD11b⁺Ly6G/C⁺; erythroid cell, Ter119⁺CD45^–. (K) Representative HE staining images of megakaryocytes in BMP-eBM and LB-BM. (L) Cellularity and number of HSCs in BMP-2–implanted or –naive mouse LB-BM (n = 4). (M-P) single-cell RNA sequencing (scRNA-seq) analysis of hematopoietic and CAR cells. (M) Schematic overview. Hematopoietic cells were collected from C57BL6J mice and CAR cells were collected from Cxcl12-GFP mice by collagenase digestion. (N) Uniform manifold approximation and projection (UMAP)-based visualization (14 clusters). Cells were colored by groups (left panel) or clusters (right panel). Biological replicate 1, n = 14 934 cells (BMP-eBM, 5894 cells; LB-BM, 9040 cells). HSPC, hematopoietic stem and progenitor cell; DC, dendritic cell. (O) Frequencies of each hematopoietic cell cluster in panel N. (P) Number of differentially expressed genes (DEGs) (|log2FC| > 1; P < .05) between BMP-eBM and LB-BM for each cell cluster. FC, fold change.