Materials and Methods UCB CD34+ and HSC isolations Human CD34+ cells were isolated from umbilical

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Materials and Methods UCB CD34+ and HSC isolations Human CD34+ cells were isolated from umbilical cord blood (UCB) samples collected at the Diakonessenhuis hospital, Leiden after informed consent was given by the mothers. Mononuclear cells were isolated by ficoll (LUMC apothecary) density centrifugation, washed and stored at -80°C until further use. Five to ten UCB samples were selected, thawed and underwent CD34 selection using biotin labelled anti CD34 antibodies and streptavidin coated microbeads (Miltenyi biotech, Bergisch Gladbach, Germany). Isolated Virus production and transduction procedure Lentiviral vectors were contructed in 293T cells by transfection of pEnv VSVG, pMD2 GagPol, pREV and the vector backbone pTGZ-B322…show more content…
Mice were kept on antibiotics (56 mg/l polymixin B (Bupha, Uitgeest), 70 mg/l ciprofloxacin, 80 mg/l amphotheracin B) in autoclaved acidified drinking water until white blood cells counts were recovered at 4 wks post transplant. Blood samples were obtained at 4,8,12,16 and 21 weeks by lateral tail vein incision. At 16 or 21 weeks, mice were killed by O2/CO2 inhalation and thymus, bone marrow and spleen were isolated. Cell suspensions were made by crushing the bones and passing the remaining cells through a 70µm filter. Spleen and thymus were homogenized by passing them through a 70 µm filter. Flowcytometry Erythrocytes in the peripheral blood samples were lysed using an isotonic NH4Cl buffer (LUMC apothecary), after which the cells were stained with the indicated antibodies in PBS/BSA/Sodium azide buffer for 30 minutes on ice. Cells were subsequently sorted using a FACS Aria II (Beckton Dickinson, USA) Spleen and peripheral blood cells were stained using human CD450 v450, CD19 APCCy7, CD3, CD4, CD8, CD13 and CD33. Peripheral blood cells were sorted into lymphocytes and myeloid cells based on their scatter profile and spleen B cell (human CD45+CD19+) and T cell (CD45+CD3+) populations were sorted. Bone marrow cells were stained using human CD45, CD34,CD38, CD90 and CD49f. HSC (human CD45+CD34+CD38-CD90+CD49f+) and CD34+CD38+ populations were sorted. Thymus cells were stained using human CD45, CD1a, CD34, CD3, CD4 and CD8.
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