Homing and maintenance of murine hematopoiesis in ectopic niches. (A) Left: representative macroscopic photograph of an ossicle generated by 2 × 106 BM-MSCs at 10 weeks posttransplant (millimeter scale is shown). Abundant hematopoietic tissue within the transplant is reflected by purple coloring. Middle: flow cytometric assessment of the hematopoietic tissue after crushing the ossicle confirmed the presence of mouse CD45+ cells. Right: immunohistochemical staining for TER119 marks erythroid cells within the invaded marrow space. (B) Upper left, upper right, and lower left: hematoxylin and eosin staining illustrating invasion of all three major blood cell lineages, including leukocytes and myeloid precursors (black arrowheads), erythrocytes with foci of immature red cells (black oval), as well as multinucleated megakaryocytes (Me) adjacent to bone (b) and/or BM sinusoids (asterisks). Lower right: immunohistochemistry for murine CD45 (mCD45) showing murine and hematopoietic origin of migrated cells within the ectopic marrow niche. Megakaryocytes (Me) and BM sinusoids (asterisks) are shown. (C) BM-MSC-derived marrow niches are colonized by immature murine HSPCs. Representative flow cytometric contour plots showing analysis of c-kit and Sca-1 surface expression on lineage− cells (left plots) within BM-MSC-derived ossicles (upper row) and the respective mouse BMs (lower row). Lineage−, c-kit+, Sca-1+ cells (rectangular gate) were used for further analysis of murine long-term HSCs. Representative contour plots depict gating on CD150+ LT-HSCs within LSK/CD34−/CD135− cells (right plots). Bar graph summarizes percentage (mean ± SD) of c-kit+/Sca-1+ cells within the lineage− (lin−) population (n = 4; Student t test P value not significant).
