Ggest that each Angptl2 and Angptl3 generally function in vivo to stimulate expansion of fetal liver, and perhaps also adult, HSCs. Our identification of Angptls as development aspects for mouse HSCs suggests that they could also be valuable for ex vivo expansion of human bone marrow or cord blood HSCs. In that case, these components might be valuable in ex vivo expansion of these cells as part of an HSC transplantation or gene therapy protocol.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript METHODSMiceWe purchased C57 BL/6 CD45.two and CD45.1 mice from the Jackson Laboratory or the US National Cancer Deubiquitinase Purity & Documentation Institute and maintained them in the animal facility with the Whitehead Institute for Biomedical Research. All animal experiments were performed together with the approval of your Massachusetts Institute of Technologies Committee on Animal Care. Angiopoietin-like proteins We created Flag uman Angptl2, the coiled-coil domain of human Angptl2, Flag-tagged fibrinogen-like domain of human Angptl2, Flag uman Angptl4, Flag uman Fgl1 and Flaghuman Mfap4 by transient transfection of 293T cells applying Lipofectamine 2000 (Invitrogen). Right after transfection, we cultured cells overnight in Iscove Modified Dulbecco Medium with ten FBS, then washed them with IMDM just before culturing them in serum-free StemSpan medium (BRaf Accession StemCell Technologies) for one more 24 h. We harvested the conditioned medium and applied it in experiments in Figures 1, 2 and 5b. We normally made use of medium from mock-transfected cells as a adverse control. We used serum-free conditioned medium ultured mocktransfected 293T cells for four h ahead of addition of purified Angptl2 or Angptl3 within the experiments in Figure three. To purify Flag-Angptl2 and Flag-Angptl4, we cultured the corresponding plasmidtransfected 293T cells in IMDM with 10 FBS for 48 h or 72 h and collected the conditioned medium for Flag-specific affinity purification.Nat Med. Author manuscript; readily available in PMC 2009 November two.Zhang et al.PagePurified mouse Angptl3 (mAngptl3) expressed by sf21 cells using a baculo-virus expression method was a gift from R D Systems. We bought GST-hAngptl5, a fusion protein of GST and human Angptl5 (hAngptl5) and developed by a cell-free wheat germ in vitro transcriptiontranslation program, from Abnova Corporation. Bacterially expressed hAngptl2 and hAngptl7 were gifts from R D Systems. Production and purification of tagged Angptl2 and Angptl4 We constructed a fusion on the cDNA encoding human Angptl2 (ref. 15) in addition to a Flag peptide sequence (as Flag-hAngptl2) or with Pro100 ys330 of human IgG1 Fc sequence followed by Flag (as human FC lag uman Angptls) at the C terminus. We inserted the DNA in to the pcDNA3.1 ( vector (Invitrogen) downstream on the cytomegalovirus (CMV) promoter. FlagAngptl4 was similarly constructed. We transfected plasmids into 293T cells making use of Lipofectamine 2000 (Invitrogen) and collected serum-containing conditioned medium at 48 h and 72 h after transfection. We added 1 tablet/50 ml from the Complete Protease Inhibitor Cocktail (Roche), 5 g/ml phenylmethylsulfonylfluoride and 100 mM NaCl, and applied the medium to a Flag-specific epitope immunoaffinity column (ANTI-FLAG M2 affinity Gel, Sigma), applying 500 l resin/500 ml conditioned medium. We subsequently washed the column ten instances having a total of one hundred volumes of TBS (50 mM Tris, pH 7.four, 150 mM NaCl) and eluted the FlaghAngptl2 or Flag Angptl4 with 0.1 mg/ml Flag peptide (DYKDDDDK) dissolved in TBS. Cell culture We plated 20 bone marrow SP Sca-1+ CD45+ c.
