Fig 1.
Effect of cyclophosphamide in NSG mice 56 days post transplantation.
NSG mice received 20mg/kg or 60mg/kg cyclophosphamide by intraperitoneal injection (day -3, -2) and 200cGy X-ray (day-1) followed by i.v. injection of 1x106 G-hPBMCs or 1x105 CD34+ cells on day 0. Survival (A) and weight loss (B) were monitored until 56 days post transplantation. Each experiment was done in triplicate and every group consisted of at least 7 mice.
Fig 2.
Engraftment of human hematopoietic cells in NSG mice.
Peripheral blood chimerism analysis (% of hCD45+ cells) was performed by flow cytometry 56 days post transplantation. (A; n = 8 donor 1, n = 12 donor 2). In donor 2, % of hCD3/hCD45+ cells was compared on day 28 and day 56 (B; n = 12 mice each). Lung (C) or liver (D) from NSG mice were examined for human CD4, CD8, CD20 and CD68 engraftment by IHC 56 days post transplantation. Three randomly selected sections per slide from at least 3 mice each were placed on the optical photomicroscope and observed under the X20 objective. Data were calculated as: % positive cells = positive nuclei cells ⁄ total cells nuclei x 100. All results are presented as mean ± SEM.
Fig 3.
Histopathology in NSG mouse 56 days post transplantation.
Lung and liver from mice received CD34+ cells (A, B, C, D) or G-hPBMCs (Donor1; E, F, G, H Donor2; I J, K, L) were stained for H&E (lung/liver 10x) and Masson’s trichrome (lung 20x, liver 10x).
Fig 4.
The pathology of lung, liver and spleen from mice with CD34+cells (n = 10) and G-hPBMC (donor 1; n = 8, donor 2; n = 9) were blindly scored 56 days post transplantation (A). A whole slide was scanned and collagen deposition was quantified as a ratio of area of blue staining to total area (B). All results are presented as mean ± SEM.
Fig 5.
Bile duct inflammation in NSG mice 56 days post transplantation.
Liver from mice received G-hPBMCs (donor 2) was stained for hCD4 (A) hCD8 (B) hCD20 (C) hCD68 (D)20x. Histopathology showed ductpenia with lymphocyte infiltration (E; H&E staining 10x), bile duct fibrosis (F; Masson’s trichrome 20x) and macrophage aggregation at bile duct (G, hCD68 positive cells 10x).
Fig 6.
Association between liver pathology and the engraftment level in mice with G-hPBMCs.
Liver pathology score from mice with donor 1 (left; n = 8) was compared with donor 2 (right; more than 18% hCD45+ cells in PB (n = 6), center; less than 18% in PB (n = 3))(A). Correlation between liver pathology score and % hCD68+ cells in liver was analyzed by linear regression (donor 1 and 2; n = 10) (B).
Fig 7.
Healing process beyond 56 days post transplantation.
Lung / liver tissues were taken from mice receiving CD34 (n = 2) and donor 2 G-hPBMCs (n = 7) 56 days post transplantation. The relative expression of mRNA is shown compared to mouse beta actin. (A). Lung from mice receiving CD34+ cells (n = 6) or donor 2 G-hPBMCs (n = 6) were obtained between day 56 and 84 post transplantation and stained for H&E and Masson’s trichrome (20x). The lung pathology from mice with donor 2 G-hPBMCs (white bar, n = 6) was blindly scored and compared with the lung from mice with CD34+cells (black bar, n = 5. One lung was removed due to insufficient size for analysis). All results are presented as mean ± SEM (B). Representative Masson’s trichrome from one of each mouse receiving CD34+cells (C) and G-hPBMCs (D) is shown. The arrow indicates thickened alveolar wall. Total of 15 mice (G-hPBMC n = 9, CD34+ cells n = 6) were examined in two separate transplantations.