American Society of Hematology

Figure 7.

$The properties of MF LCs and SVECs. (A, upper) Representative photomicrograph of hematoxylin and eosin staining of the red pulp of normal and MF spleens. Scale bar, 50 μm. (Middle) Matching samples stained with both anti-CD8α (brown) and anti-CD34 (magenta) antibodies. LCs are stained brown, SVECs are stained magenta. Scale bar, 20 μm. (Bottom) Reticulin staining of the matching samples as earlier. Scale bar, 50 μm. Pictures of all 3 panels were taken at 400× original magnification. (B) Representative FACS profiles of normal or MF nonhematopoietic cell fraction from collagenase digested nonsuspensive part of spleen tissues, which are CD45−CD3− and RBC-free. The percentages of LCs and SVECs are indicated in the profiles. (C) Percentage of normal or MF LCs and SVECs relative to total viable CD45−CD3−, RBC-free, nonhematopoietic splenic cells as in (B). Data are shown as mean ± SD, n > 3. ******P = 3.06E-06 by 2-sample Student t test. The result represents an average of 4 independent experiments with spleens from 4 patients with MF and 6 normal individuals. (D) The heat map of relative gene expression intensity for MF SVEC or LC gene signature. (Upper) MF SVECs; (lower) MF LCs. (E) DAVID Functional Annotation Clustering analysis of MF LC gene signature for downregulated genes in MF compared with normal LCs. (F) A network of energy production, nucleic acid metabolism, and small molecule biochemistry by IPA analysis of genes differentially expressed in MF compared with normal LCs. Network score, 27; focus molecules, 34; green, gene expression downregulated in MF LCs; red: gene expression upregulated in MF LCs; dotted arrow lines, direction of regulation. (G) z-scores of apoptosis, cell death, and necrosis by IPA disease or function analysis for MF LCs and SVECs compared with their normal counterparts. Activation z-score ≥2 predicts the increase of a function; ≤−2 predicts the decrease of a function. P value indicates the significance of an association with the change of a related function. (H) z-scores of cell movement, migration, and outgrowth of cells by IPA disease or function analysis for MF LCs and SVECs compared with their normal counterparts. MF LCs did not have any significant change with a qualified P value in these annotations. (I) DAVID Functional Annotation Clustering analysis of upregulated genes in MF SVECs compared with normal SVECs. (J) The heat map of relative average expression intensity for selected genes by normal or MF SVECs and LCs. (K) The number of colony forming units (CFUs) formed in the colony-forming assay after the cocultures of 3000 normal or MF splenic CD34+ cells with normal SVEC cell layers for 4 days (left graph) or 6 days (right graph). CD34+ cells of 1 MF and 2 normal spleens obtained from different individual donors were cocultured in parallel with each of the 2 lines of normal SVECs (#1 and #2). Each CD34+ cell coculture was plotted individually (blue, normal; red, MF). (L) The number of assayable CFUs after coculture of 3000 normal splenic CD34+ cells from 3 different donors each with 2 lines of normal or 1 line of MF SVECs for 4 to 6 days. IL1R1, interleukin 1 receptor type 1; NADH, nicotinamide adenine dinucleotide hydride.$

The properties of MF LCs and SVECs. (A, upper) Representative photomicrograph of hematoxylin and eosin staining of the red pulp of normal and MF spleens. Scale bar, 50 μm. (Middle) Matching samples stained with both anti-CD8α (brown) and anti-CD34 (magenta) antibodies. LCs are stained brown, SVECs are stained magenta. Scale bar, 20 μm. (Bottom) Reticulin staining of the matching samples as earlier. Scale bar, 50 μm. Pictures of all 3 panels were taken at 400× original magnification. (B) Representative FACS profiles of normal or MF nonhematopoietic cell fraction from collagenase digested nonsuspensive part of spleen tissues, which are CD45⁻CD3⁻ and RBC-free. The percentages of LCs and SVECs are indicated in the profiles. (C) Percentage of normal or MF LCs and SVECs relative to total viable CD45⁻CD3⁻, RBC-free, nonhematopoietic splenic cells as in (B). Data are shown as mean ± SD, n > 3. ******P = 3.06E-06 by 2-sample Student t test. The result represents an average of 4 independent experiments with spleens from 4 patients with MF and 6 normal individuals. (D) The heat map of relative gene expression intensity for MF SVEC or LC gene signature. (Upper) MF SVECs; (lower) MF LCs. (E) DAVID Functional Annotation Clustering analysis of MF LC gene signature for downregulated genes in MF compared with normal LCs. (F) A network of energy production, nucleic acid metabolism, and small molecule biochemistry by IPA analysis of genes differentially expressed in MF compared with normal LCs. Network score, 27; focus molecules, 34; green, gene expression downregulated in MF LCs; red: gene expression upregulated in MF LCs; dotted arrow lines, direction of regulation. (G) z-scores of apoptosis, cell death, and necrosis by IPA disease or function analysis for MF LCs and SVECs compared with their normal counterparts. Activation z-score ≥2 predicts the increase of a function; ≤−2 predicts the decrease of a function. P value indicates the significance of an association with the change of a related function. (H) z-scores of cell movement, migration, and outgrowth of cells by IPA disease or function analysis for MF LCs and SVECs compared with their normal counterparts. MF LCs did not have any significant change with a qualified P value in these annotations. (I) DAVID Functional Annotation Clustering analysis of upregulated genes in MF SVECs compared with normal SVECs. (J) The heat map of relative average expression intensity for selected genes by normal or MF SVECs and LCs. (K) The number of colony forming units (CFUs) formed in the colony-forming assay after the cocultures of 3000 normal or MF splenic CD34⁺ cells with normal SVEC cell layers for 4 days (left graph) or 6 days (right graph). CD34⁺ cells of 1 MF and 2 normal spleens obtained from different individual donors were cocultured in parallel with each of the 2 lines of normal SVECs (#1 and #2). Each CD34⁺ cell coculture was plotted individually (blue, normal; red, MF). (L) The number of assayable CFUs after coculture of 3000 normal splenic CD34⁺ cells from 3 different donors each with 2 lines of normal or 1 line of MF SVECs for 4 to 6 days. IL1R1, interleukin 1 receptor type 1; NADH, nicotinamide adenine dinucleotide hydride.

This Feature Is Available To Subscribers Only