Journal: Cell stem cell

Article Title: Gli1 + mesenchymal stromal cells are a key driver of bone marrow fibrosis and an important cellular therapeutic target

doi: 10.1016/j.stem.2017.03.008

Figure Lengend Snippet: (A) Bigenic Gli1CreERt2;tdTomato mice received tamoxifen (3x10mg p.o.) and were lethally irradiated 10 days after the last tamoxifen dose and transplanted with 2x105 c-kit purified bone marrow (BM) cells from wild type littermates that had been transduced with either control (n=10) or Jak2(V617F) (n=10) cDNA (both MCSV-IRES-GFP retroviral backbone vector). Mice were injected with GANT61 (50mg/kg body weight) or vehicle (corn oil / ethanol) every other day between 8 and 17 weeks after transplantation (control + vehicle n=3, 2 males; control+GANT61 n=5, 3 males; Jak2(V617F) + vehicle n=4, 3 males; Jak2(V617F) + GANT61 n=4, 3 males). (B) Grading of reticulin fibrosis. ***p<0.001 versus all other groups by one way ANOVA with posthoc Tukey; mean±SEM. (C) Spleen weight was determined as a percentage of the body weight. *p<0.05, **p<0.001 by one way ANOVA with posthoc Tukey; mean±SEM. (D) Representative images of sternal bones. Scale bars 50μm, inserts 25μm. (E) Quantification of all alpha smooth muscle actin expressing cells (α-SMA+), Gli1 cells (tdTomato+) and Gli1-derived myofibroblasts (α-SMA+/tdTomato+) in the BM of mice transplanted with Jak2(V617F) (Jak2) or control that have been treated with GANT61 or vehicle. ***p<0.001 by one way ANOVA with posthoc Tukey; mean±SEM. (F) Representative flow cytometric plots and quantification of Gli1+ cells and within the lineage depleted BM. *p<0.05 by one way ANOVA with posthoc Tukey; mean±SEM. (G) Analysis of long-term (LT; linlowSca1+ckit+CD48-CD150+) and multipotent progenitor cells (MPP, linlowSca1+ckit+CD48+CD150-) as well as erythroid cells (Gr1-CD11b-CD3-CD19-Ter119+) by flow cytometry. *p<0.05, **p<0.01 by one way ANOVA with posthoc Tukey; mean±SEM. (H) c-kit+ purified HSPCs from wild type mice were transduced with either control or Jak2(V617F) cDNA (both MCSV-IRES-GFP retroviral backbone vector). 48 hours after transduction, cells were treated with GANT61 or vehicle. The gene marking (GFP+) was quantified by flow cytometry 48 hours after treatment. ***p<0.001 versus all other groups by one way ANOVA with posthoc Tukey; mean±SEM. (I) Quantification of the mean fluorescence intensity (MFI) and representative flow cytometric analysis of levels of phospho-STAT5 (p-STAT5) in c-kit+ cells transduced with Jak2(V617F) or control (GFP+) 48 hours after treatment with GANT61 or vehicle. GFP+ cells are shown in the histogram. ***p<0.001 versus all other groups by one way ANOVA with posthoc Tukey; mean±SEM. (J) c-kit+ purified HSPCs were transduced with Jak2(V617F) or control cDNA, were sort-purified by GFP-expression 48 hours after transduction and treated with GANT61 or vehicle 24 hours later. 24 hours after treatment, cells were harvested for RNA isolation and qt-RT-PCRs. Relative mRNA expression for Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), Akt and Gli1 is shown. *p<0.05 ns=non-signifcant by one way ANOVA with posthoc Tukey; mean±SEM. See also Figure S4.

Article Snippet: The murine Jak2 cDNA was cloned into the retroviral vector MSCV-IRES EGFP (a gift from Tannishtha Reya Addgene #20672).

Techniques: Irradiation, Purification, Transduction, Control, Retroviral, Plasmid Preparation, Injection, Transplantation Assay, Expressing, Derivative Assay, Flow Cytometry, Fluorescence, Isolation

Journal: Cell stem cell

Article Title: Gli1 + mesenchymal stromal cells are a key driver of bone marrow fibrosis and an important cellular therapeutic target

doi: 10.1016/j.stem.2017.03.008

Figure Lengend Snippet: Bigenic Gli1CreER;tdTomato mice were injected with tamoxifen (3x10mg p.o.) at 8 weeks of age, lethally irradiated at 10 days after the last tamoxifen dose and received c-kit enriched hematopoietic stem cells from wildtype littermates expressing either Thrombopoietin-cDNA (ThPO, n=5, 3 males) or control-cDNA (control, n=4, 3 males; both lentiviral SFFV-iGFP vector backbone). Mice were sacrificed at 70 days after transplantation. Gli1+ cells were sort-purified as lin-GFP-dtTomato+ and subjected to RNA sequencing. (A) Principal component analysis (PCA). (B) Heatmap representation with hierarchical clustering (C) Significantly directional enriched (median FDR >0.05) pathways. (D) Fragments per kilobase of exon per million fragments mapped (FPKM) values of CXCL4 in control and ThPO group; mean±SEM. (E) Cytokine analysis in serum and cytokine-free medium supernatant of ckit+ cells expressing ThPO or control (lentiviral SFFV-iGFP vector backbone) or Jak2(V617F) or control (both MCSV-IRES-GFP retroviral backbone). n=2. (F) FPKM value of stromal derived factor 1 (SDF 1/ CXCL12) in control and ThPO group; mean±SEM. (G) Representative images of bone marrow from Gli1CreERt2;tdTomato mice transplanted with bone marrow from wildtype littermates expressing either ThPO or control-cDNA stained with the neutral lipid staining LipidTOX. Scale bars 50μm. Arrow indicating LipidTOX positive Gli1+ cells. (H) Model for stroma-hematopoiesis interactions in bone marrow fibrosis. See also Figure S5.

Article Snippet: The murine Jak2 cDNA was cloned into the retroviral vector MSCV-IRES EGFP (a gift from Tannishtha Reya Addgene #20672).

Techniques: Injection, Irradiation, Expressing, Control, Plasmid Preparation, Transplantation Assay, Purification, RNA Sequencing, Retroviral, Derivative Assay, Staining