Fig 1.
Transplantation model of MPLW515L MPN.
C57BL/6j bone marrow transplantation recipients received MPLW515L, MPLWT, and empty vector control (EV) infected bone marrow (BM) for 17 days. (A) spleen size (B) spleen weight (g), (C) liver weight (g), (D) H&E and reticulin stained spleens (E) H&E, reticulin, and trichrome stained bone marrow sections, (F) WBC counts (G) platelet counts (H) RBC counts and (I) hematocrit (Hct) levels were determined on day 17. Data using four mice in one representative experiment out of three replicates with statistical analysis using two-tailed t test. Significant p-values <0.05 are shown.
Fig 2.
(A) Provides a diagram outlining the procedures in this experiment. BM from B6.SJL-Ptprca (CD45.1+) mice was infected with MPLW515L-GFP virus and mixed 1:0 (100%, n = 4), 1:1 (50%, n = 3 with one death on day 17), 1:4 (20%, n = 3) and 0:1 (0%, n = 3) with BM from B6.SJL-Ptprca (CD45.1+) mice infected with MPLWT virus. Populations were injected into lethally irradiated C57BL/6j-Ptprcb (CD45.2+) recipient mice and then (B, E) platelet count, (C, F) WBC count (D, G) RBC count were determined on day 17 (B, C, D) or day 34 (E, F, G) post-transplantation. On day 34, all mice were euthanized and (H) total BM cell number from two femurs and two tibia (I) spleen weight, and (J) pictures of spleens were determined. This is characteristic of two additional experiments. Data is shown for one representative experiment out of three with statistical analysis conducted using ANOVA. Significant p-values <0.05 are shown.
Fig 3.
(A) Diagram of study methods for isolation of MSC populations. MPLWT was used as a comparator to account for any possible influence of viral infection on the recovery or activity of the isolated MSCs. (B) mRNA of collagen I (Col1A1), (C) mRNA for collagen III (Col3A1), (D, E) qRT-PCR for EGFP and CD45, respectively using hematopoietic cells expressing MPLW515L (BM mu), BaF3 B-cell leukemia cell line overexpressing MPLW515L (Baf3MPLW515L) as a positive control, MSCs from mice expressing MPLWT (MSC wt) and MSCs derived from bone marrow of two mice expressing MPLW515L (MSC mu1 and mu2). Cells exposed to no differentiation-inducing media and differentiation-inducing media specific for adipocytes and chondrocytes. Stain with Oil-red-O (F) or alcian blue (G) to detect adipogenic and chondrogenic populations. (H) Collagen I, III, and IV complexes stained after 72 hours of culture on glass slides. DAPI stained nuclei are shown in gray. Merged images shown are representative of 3–4 slides per test condition.
Fig 4.
Production of TGFβ and anti-TGFβ in MPLWT and MPLW515L-transduced bone marrow-derived MSC.
(A) Diagram showing conditions used for this experiment. mRNA expression (B) and secreted levels (C) of TGFβ from MSCs derived from mice transplanted with bone marrow expressing MPLWT or MPLW515L retroviruses. Collagen I (Col1A1) (D) and III (Col3A1) (E) mRNA expressed in these cells in the presence of anti-TGFβ (1 and 5 μg/ml) monoclonal antibody during a three day culture period. (F) Collagen I (Col1A1) mRNA in MSCs derived from MPLWT mice under treatment with TGFβ and anti-TGFβ antibody. Relative expression to TATA binding protein (TBP) control is shown. Statistical analysis was performed using 2-sided t test with significance <0.05. Experiments are shown for three biological replicates.
Fig 5.
Elevated signaling pathways induced stably in MSCs from animals with MPN.
(A) Western blot analysis with (B) quantification of the bands for pSmad3, Smad3, pSTAT3, STAT3, and β-actin. Shown are cell extracts from WT1 and WT2 cell lines (MSCs derived from bone marrow expressing MPLWT) and MU1 and MU2 (MSCs derived from bone marrow expressing MPLW515L). The experimental results are representative of three. Statistical analysis was performed using 2-sided t test with significance <0.05.
Fig 6.
FGFb reverses the fibrotic phenotype of MPLW515L- MSCs.
FGFb and LIF maintain MSCs in a pluripotent state, but stimulate proliferation and expansion. (A, B) MSCs derived from human bone marrow were exposed to increasing concentrations (0.5, 1, 5 ng/ml) of TGFβ to enhance collagen I (green) and collagen III (red) in three-dimensional culture conditions in the absence (A) and presence of 20 ng/ml FGFb (B). (C) MSCs from three different mice expressing MPLWT or MPLW515L which has increased expression of collagen I, III, and IV. Presence of 20 ng/ml FGFb reduces collagen deposition in the culture after 72 hours. Cell numbers were increased after continuous culture in this growth factor (data not shown). Quantification of collagen I (D) collagen III, (E) collagen IV (F) fibers under each condition shown with p-values indicated based on analysis using a 2-sided t-test.