American Society of Hematology

Figure 6

$Figure 6. SPARC deficiency in the BM stroma associates with an enhanced myelopoietic response to TPO. (A) Histomorphologic analysis performed on hematoxylin and eosin-stained BM sections from TPO-treated mouse chimeras shows that mice with Sparc−/− stroma (WT > Sparc−/−, Sparc−/− > Sparc−/−) have significantly higher numbers of immature granulocyte precursors (green arrows) and highly atypical/dysplastic MKs (red arrows) compared with chimeras having WT stroma (WT > WT, Sparc−/− > WT). Four representative sections (1 per group) of the 20 evaluated are shown. Original magnifications ×400. Scale bars represent 50 μm. (B) Differential hematopoietic cell counts (mean ± SD) performed on BM sections of TPO-treated chimeric mice showing the different expansion of myeloid cell fractions of mice with a Sparc−/− or WT BM stroma. In each BM sample, the number of neutrophils, eosinophils, morphologically immature myeloid cells, erythroid cells, MKs, and lymphoid cells was counted of 10 HPFs. *P < .05. **P < .01. Data are relative to counts performed on 2 different BM sections, per mice, per group. (C-D) Hematopoiesis in chimeric mice was analyzed using a clonogenic colony culture assay. BM cells from chimeric mice were seeded in Methocult M3434 for 10 days, and the colonies that formed were scored and photographed under an inverted microscope. (C) Representative images highlighting the enrichment of myeloid colonies in chimeric mice with Sparc−/− stroma. (D) The relative number of BM CFU-GM myeloid colonies (mean ± SD) is significantly increased in Sparc−/− recipient BM chimeras compared with WT recipient chimeras. *P < .05. **P < .01. One representative experiment of the 3 performed with 5 mice per group is shown. (E) Hematopoietic progenitor cell numbers expressed as a percentage (mean ± SD) of total BM, showing the significant increase of GMP precursors in TPO-treated mice with Sparc−/− stroma compared with the WT counterparts. *P < .05. The data represent 1 experiment of the 3 performed with 5 mice per group. (F) Flow cytometric analysis of Gr1+CD11b+ circulating myeloid cells performed on the PB mononuclear cells of mouse chimeras. *P < .05. **P < .01. The data represent 1 experiment of the 3 performed with 5 mice per group. (G) Morphologic analysis of hematoxylin and eosin-stained PB smears from BM chimeras show that circulating granulocytes from chimeric mice with Sparc−/− recipient stroma are enriched in immature forms, including pseudo-Pelger-Hüet and band-form nuclei, compared with circulating granulocytes from chimeras with WT stroma. Representative examples of circulating granulocytes from 2 smears per condition (of the 5 evaluated per experiment) are shown. Original magnifications ×1000.$

SPARC deficiency in the BM stroma associates with an enhanced myelopoietic response to TPO. (A) Histomorphologic analysis performed on hematoxylin and eosin-stained BM sections from TPO-treated mouse chimeras shows that mice with Sparc^−/− stroma (WT > Sparc^−/−, Sparc^−/− > Sparc^−/−) have significantly higher numbers of immature granulocyte precursors (green arrows) and highly atypical/dysplastic MKs (red arrows) compared with chimeras having WT stroma (WT > WT, Sparc^−/− > WT). Four representative sections (1 per group) of the 20 evaluated are shown. Original magnifications ×400. Scale bars represent 50 μm. (B) Differential hematopoietic cell counts (mean ± SD) performed on BM sections of TPO-treated chimeric mice showing the different expansion of myeloid cell fractions of mice with a Sparc^−/− or WT BM stroma. In each BM sample, the number of neutrophils, eosinophils, morphologically immature myeloid cells, erythroid cells, MKs, and lymphoid cells was counted of 10 HPFs. *P < .05. **P < .01. Data are relative to counts performed on 2 different BM sections, per mice, per group. (C-D) Hematopoiesis in chimeric mice was analyzed using a clonogenic colony culture assay. BM cells from chimeric mice were seeded in Methocult M3434 for 10 days, and the colonies that formed were scored and photographed under an inverted microscope. (C) Representative images highlighting the enrichment of myeloid colonies in chimeric mice with Sparc^−/− stroma. (D) The relative number of BM CFU-GM myeloid colonies (mean ± SD) is significantly increased in Sparc^−/− recipient BM chimeras compared with WT recipient chimeras. *P < .05. **P < .01. One representative experiment of the 3 performed with 5 mice per group is shown. (E) Hematopoietic progenitor cell numbers expressed as a percentage (mean ± SD) of total BM, showing the significant increase of GMP precursors in TPO-treated mice with Sparc^−/− stroma compared with the WT counterparts. *P < .05. The data represent 1 experiment of the 3 performed with 5 mice per group. (F) Flow cytometric analysis of Gr1⁺CD11b⁺ circulating myeloid cells performed on the PB mononuclear cells of mouse chimeras. *P < .05. **P < .01. The data represent 1 experiment of the 3 performed with 5 mice per group. (G) Morphologic analysis of hematoxylin and eosin-stained PB smears from BM chimeras show that circulating granulocytes from chimeric mice with Sparc^−/− recipient stroma are enriched in immature forms, including pseudo-Pelger-Hüet and band-form nuclei, compared with circulating granulocytes from chimeras with WT stroma. Representative examples of circulating granulocytes from 2 smears per condition (of the 5 evaluated per experiment) are shown. Original magnifications ×1000.

This Feature Is Available To Subscribers Only