Journal: Haematologica

Article Title: In vitro and in vivo evaluation of possible pro-survival activities of PGE2, EGF, TPO and FLT3L on human hematopoiesis

doi: 10.3324/haematol.2018.191569

Figure Lengend Snippet: Prostaglandin E2 (PGE2) has both protective and toxic effects on human CD34 + cells. (A) Cord blood-derived CD34 + cells were subjected to different cytotoxic agents or serum and cytokine withdrawal. Control cells were treated with serum, FLT3L, stem cell factor (SCF), thrombopoietin (TPO) and IL3. PGE2 was added at indicated concentration. After 48 hour (h), cells were stained with AnnexinV/7AAD and specific apoptosis was determined. Bars represent means±Standard Error of Mean (SEM) of 5–6 from 6 independent experiments. P -values were determined using the Mann-Whitney test. (B and C) CD34 + cells were cultured in serum, FLT3L, SCF, TPO and IL3 plus different concentrations of PGE2. Cell count (B) and viability (C) were determined every other day for eight days. Bars represent means±SEM of 4 from 3 independent experiments. P -values were determined using the Mann-Whitney test. (D) After three days of culture in the presence or absence of PGE2 (50 μM), cell cycle status was determined by Ki67 and DAPI staining and flow cytometric analysis. Bars represent means±SEM of 6 from 5 independent experiments. Mann-Whitney test. (E) PGE2-treated CD34 + cells were cultured in the presence of carboxy-fluorescein diacetate succinimidyl ester (CFSE), and CFSE content representing amount of cell divisions was determined by flow cytometry four days later. Bars represent means ± SEM of n=3 independent experiments.

Article Snippet: Thawed cells were cultured in serum-free medium supplemented with 10% ES-FBS (Invitrogen), human TPO (50 ng/mL, Immunotools), FLT3L, SCF, IL3 (100 ng/mL each, Immunotools), human EGF (hEGF) (20 or 200 ng/mL Immunotools), PGE2 (10, 25 or 50 μM, Sigma-Aldrich) and/or cytotoxic drugs (etoposide, taxol, tunicamycin; Sigma-Aldrich).

Techniques: Derivative Assay, Control, Concentration Assay, Staining, MANN-WHITNEY, Cell Culture, Cell Counting, Flow Cytometry