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. Author manuscript; available in PMC: 2019 Nov 1.
Published in final edited form as: Br J Haematol. 2018 Aug 14;183(3):445–456. doi: 10.1111/bjh.15548

Fig 3.

Fig 3.

The AML-MSC COX2-PG secretome promotes HSPC/myeloid expansion in humanized recipients. (A) Schematic representation of the experimental design. Reduction of prostaglandin (PG) biosynthesis by COX2 inhibitor (COX2i) in mesenchymal stromal cells (MSCs) from Fanconi anaemia (FA) patients with acute myeloid leukaemia (FA-AML). FA-AML MSCs were pre-treated with COX2i (celecoxib, 10 μmol/l) for 2 h followed by co-culture with normal bone marrow (BM) CD34+ cells for two weeks. The progenies were then transplanted into sublethally-irradiated NSGS recipients. In another set of experiments, normal BM CD34+ cells were co-cultured on healthy donor (HD) MSCs for 2 weeks followed by BM transplantation into NSGS mice. The recipients were then injected with 16–16 dimethyl-PGE2 (dmPGE2; 10 μg/kg body weight) twice per day for 7 days starting at 24 h after transplantation. (B) COX2i celecoxib reduces PG production in FA-AML MSCs. FA-AML MSCs were pre-treated with COX2i (COX2i; celecoxib, 10 μmol/l) or vehicle (5% dimethyl sulphoxide) for 2 h. The levels of the indicated PGs in MSCs were measured by enzyme-linked immunosorbent assay. Results are means ± SD of three independent experiments (n = 9 for each group). (C) COX2i celecoxib prevents haematopoietic stem and progenitor cell (HSPC)/myeloid expansion of FA-AML MSC-co-cultured cells. Co-cultured cells described in (B) were transplant into sublethally-irradiated NSGS mice (5 × 105 cells/mouse; n = 6). Total human engraftment (hCD45+), HSPCs (CD34+) and myeloid (CD33+)/lymphoid (CD19+) cells were analysed by flow cytometry eight weeks post-transplant. Results are means ± SD of three independent experiments (n = 9–12 for each group). (D) dmPGE2 treatment induces HSPC/myeloid expansion in NSGS recipients. The NSGS recipient mice transplanted with HD MSC co-cultured cells were subjected to daily i.p. injection of dmPGE2 (10 μg/kg body weight) or vehicle (Neat oil) twice per day for 7 days starting at 24 h post-transplant. Total human engraftment (hCD45+), HSPCs (CD34+) and myeloid (CD33+)/lymphoid (CD19+) cells of the recipients were analysed by flow cytometry. Results are means ± SD of three independent experiments (n = 9–12 for each group). *P < 0·05, **P <0·01. [Colour figure can be viewed at wileyonlinelibrary.com]