Fig. 3

Mature and actively dividing cells in BM are affected by 5-FU whereas primitive and relatively quiescent stem cells are spared. a The total BM nucleated cells isolated from two femurs and two tibias of the mice were counted on D2, D4, and D10 after 5-FU treatment and compared with control. Similar to histological data (Fig. 2); the cell count decreased up to D4 (7-fold) and is increased on D10 (n = 10, ***p ≤ 0.001). b The percentage of various bone marrow subtypes based on Lin and CD45 expression were evaluated on D4 and D10 after 5-FU treatment. Note that while the percentage of Lin⁺/CD45⁺ subtypes decreased on D4, its percentage increased with hematopoietic recovery. Percentage of Lin^–/CD45^– (enriched in VSELs) increased on D4 and slightly reduced by D10. The Lin^–/CD45⁺ (enriched in HSCs) percentage also increased by D4, and remained high on D10 (n = 10, ***p ≤ 0.001). c Upregulation in the pluripotent, primordial germ cell and proliferation specific transcripts is observed in BM cells that survive 5-FU on D4. The upregulation of these markers confirms the presence and involvement of primitive stem cells in regeneration. The transcript levels on D4 are shown compared with control (n = 6, *p ≤ 0.05; **p ≤ 0.01). Bars in a, b represent average ± SD and in c average ± SEM. d–h Characterization of cells on D4 that survived 5-FU treatment. d H & E staining shows VSELs which are spherical in shape and small in size with high nucleo-cytoplasmic ratio located near the endosteal region. Inset (e) shows cells at higher magnification. f Nuclear OCT-4⁺ VSELs are clearly visualized in bone marrow sections cells in clusters (*) or singly (arrow) along the endosteal region; these cells also express (g) cell-surface SSEA-1and (h) nuclear SCA-1. Presence of nuclear OCT-4, SSEA-1, and SCA-1 confirms that VSELs survive 5-FU treatment. Nuclei are counterstained with DAPI. Scale bar = 20 μm for H & E and immunohistochemistry images, scale bar = 10 μm for immunofluorescence images. Day 2-FU, Day 4-FU, Day 10-FU days post 5-FU treatment, BM bone marrow, 5-FU 5-fluorouracil, H & E Hematoxylin and Eosin, OCT-4 octamer binding transforming factor-4, SSEA-1 stage-specific embryonic antigen-1, SCA-1, stem cell antigen-1, DAPI 4′,6-diamidino-2-phenylindole