Bone marrow progenitor cells do not contribute to liver fibrogenic cells

Figure 1 Representative images of histological sections of livers of normal and chimeric animals stained with hematoxylin and eosin or picrosirius. A: hematoxylin and eosin (HE): shows the central vein (CV), portal vein (PV) and regular hepatocyte plates, representing normal architecture of the liver; B: The PV and CV present inflammatory infiltrate (arrows) due to injury of hepatocytes in this region by CCl₄; C: Picrosirius: collagen (red) is present only in the perivascular region of the PV, hepatic artery (HA) (arrows) and lightly present surrounding the VC; D: a high deposition of collagen surrounding the PV and radiating fibers to the CV, indicating initiation of collagen septa (arrows) formation 30 d after injury induction.

Figure 2 Quantification of collagen by morphometric analysis. A significant increase in collagen between the group that received CCl₄ compared to the group that received saline. The groups are represented by box–whisker diagrams in which the values in the boxes represent the medians of collagen content (n = 5) and the bars the 25%-75% range. The Mann-Whitney post-test was used to test for significance. ^aP = 0.0329 vs the Saline 30 d group.

Figure 3 Graphical analysis of subpopulations of peripheral blood of the WT, Saline 30 d and CCl₄ 30 d groups by flow cytometry. Only the animals in CCl₄ 30 d group showed significant differences from wild-type (WT): Total lymphocytes (WT = 55.2% ± 7.4%; Saline 30 d = 56.9% ± 6.6%; CCl₄ 30 d = 24.3% ± 10.2%); Monocytes (WT = 8.1% ± 5.3%; Saline 30 d = 12.7% ± 5.3%; CCl₄ 30 d = 22.7% ± 5.6%); Neutrophils (WT = 10.1% ± 5.0%; Saline 30 d = 18.2% ± 2.1%, CCl₄ 30 d = 43.8% ± 16.3%). One-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparisons was used. ^bP < 0.01, ^cP < 0.001 vs WT.

Figure 4 Flow cytometric analysis of subpopulations of hematopoietic and endothelial progenitor cells in bone marrow of chimeric mice. A: Representative results showing green fluorescent protein (GFP)+ precursor cells in both the Saline 30 d and CCl₄ 30 d groups (side bar = ancestry gating). CD45+/CD34+ (hematopoietic) and CD45+/CD133+ (endothelial) cells were identified; B: Representative results showing GFP-subpopulation precursor cells in the Saline 30 d and CCl₄ 30 d groups (side bar = ancestry gating). No progenitor cells were found in the GFP-subpopulation; C: Graphic of the percentage of GFP+/CD45+/CD34+ cells (hematopoietic progenitor cells); D: GFP+/CD45+/CD133+ (endothelial progenitor cells) in peripheral blood of the Saline 30 d and CCl₄ 30 d groups. No significant difference was detected between the groups by Student’s t test.

Figure 5 Flow cytometric analysis of subpopulations of CD117+ precursor cells in bone marrow of chimeric mice. A: Dot-plot analysis of CD117+ cells found in bone marrow. Side-bar dot-plots represent ancestry gating; B: A significantly greater population of CD117+ cells was observed in liver tissue from the CCl₄ 30 d group compared to the Saline 30 d group (30 d saline = 2.6 ± 1.3%; CCl₄ 30 d = 5.6 ± 1.8%) (^aP = 0.0142). GFP: green fluorescent protein.

Figure 6 Flow cytometric analysis of bone marrow subpopulations in the liver of chimeric mice. A: Dot-plot analysis of cell populations in the liver; B: A significantly greater population of CD45+ cells was observed in liver tissue of animals from the CCl₄ 30 d group compared to the Saline 30 d group (30 d saline = 3.1% ± 2.2%; CCl₄ 30 d = 5.8% ± 1.3%) (^aP = 0.0458). GFP: green fluorescent protein.

Figure 7 Representative analysis of CD38+ hepatic stellate cell subpopulations by flow cytometry. A: Two CD38+ subpopulations were observed: green fluorescent protein (GFP)+/CD38+ and GFP-/CD38+; B: Two subpopulations among GFP-/CD38+ cells were observed: GFP-/CD38+/CD45+ that identifies B lymphocytes, and GFP-/CD38+/CD45 that identifies hepatic stellate cells; C: Analysis of GFP+/CD38+ cells showed that all cells were of hematopoietic origin (CD45+); D: A significant increase of cells GFP/CD38+/CD45- was observed in liver tissue of the CCl₄ 30 d group when compared to the Saline 30 d group (Saline 30 d = 9.3% ± 2.4%; CCl₄ 30 d = 17.5% ± 3.9%, ^bP = 0.004). The statistical test used was Student’s t test.

Figure 8 Representative direct fluorescence images of hepatic sections of chimeric animals after transplantation. Liver samples of chimeric animals after transplantation of green fluorescent protein (GFP)+ bone marrow- mononuclear cell, chimeric animals that received saline (Saline 30 d) and chimeric animals that received CCl₄ (CCl₄ 30 d). In panel A (Salina 30 d), the GFP channel shows the presence of green fluorescence; in the 4, 6-diamino-2-phenylindole panel, nuclei are stained blue in the same field above; the merge panel superposes the two images, where GFP+ cells (arrows) can be seen distributed in major hepatic parenchyma and with fusiform morphology. In panel B (CCl₄ 30 d), GFP+ cells can be seen with greater distribution in the hepatic parenchyma, some fusiform morphology (white arrows) and others with rounded morphology. DAPI: 4,6-diamino-2-phenyl indole.

Figure 9 Immunostaining analysis of α-smooth muscle actin in liver samples from chimeric animals by confocal microscopy. A: α-smooth muscle actin (α-SMA)+ cells in the perivascular region were observed where green fluorescent protein (GFP)+ cells could also be seen, but the two cell types do not co-localize, as can be seen by the orientation of the red and green bars; B: An increase in reactivity against α-SMA around the perivascular region was observed, also with augmentation of GFP+ cells, but again these cell types do not co-localize. In both images the nuclei were stained with TO-PRO3 (blue).