Homing and maintenance of multilineage human hematopoiesis and functional HSPCs in ectopic niches. (A) Schematic illustration of the experimental procedure for studying homing and engraftment of human hematopoiesis. Hematopoietic chimerism (human/mouse) was induced by transplanting UCB-derived CD34⁺ HSPCs in advance (8-10 weeks) of BM-MSC transplants (1st Tx). After formation of ectopic niches by BM-MSC (indicated by purple color), hematopoietic cells were recovered and either subjected to flow cytometric engraftment analysis or transplanted into irradiated secondary recipients (2nd Tx). (B) Bar graphs show percentage of human chimerism and corresponding leukocyte subsets, including B cells, T cells, and myeloid cells within human CD45-positive cells within ossicles (n = 14; 5 different UCB transplants) of primary recipients (left graph) and within BM (n = 3; 3 different primary UCB transplants) of secondary recipients (right graph). (C) Representative flow-cytometric contour plots depicting (i) representative gating of human B cells (CD19) and T-cells (CD3) within human CD45-positive cells; (ii) T-cell subpopulation analysis revealing CD4⁺ T-helper cells, CD8⁺ cytotoxic T-cells, and a minor proportion of double-positive T cells; and (iii) CD33-positive myeloid cells comprising substantial amounts of CD14⁺ monocytes and CD15⁺ granulocytes. (D) Human cells within ossicles were analyzed for CD34, CD38, CD90, and CD45RA by flow cytometry. The left plot is gated on lineage negative live cells, whereas the right plots are gated on lineage-negative CD34⁺CD38⁻ cells. The latter cells contain human HSCs (CD90⁺/CD45RA⁻), multipotent progenitor cells (MPPs, CD90⁻/CD45RA⁻), and lymphoid primed multipotent progenitor cells (LMPPs, CD90⁻/CD45RA⁺). Data shown are representative of multiple samples. Numbers within the plots indicate percentages of the respective parent population. (E) Immunohistochemistry of explanted ossicles confirms flow cytometric results. Representative photographs of immunohistochemical stainings using antibodies specifically binding human vimentin (huVim), huCD45, huCD19, huCD3, huCD34, human glycophorin A (huGpA), huCD15, huCD14, and huFoxP3. MNCs, mononuclear cells.