Methods
Cells
AML cells were obtained after receipt of informed consent from St Bartholomew’s Hospital. Details of the patient samples are listed in Supplementary Table E1 (online only, available at www.exphem.org). Co-culture experiments were previously described [6]. AML samples were collected at diagnosis, and mononucleated cells were isolated within 24 hours after collection by Ficoll-Paque Plus density gradient (GE Healthcare, France). Cord blood (CB) cells were obtained after receipt of informed consent from the Royal Free Hospital (UK). Both AML and CB sample collections were approved by the East London ethical committee and in accordance with the Declaration of Helsinki. Three to 5 different CB samples were pooled, and mononuclear cells were obtained by density centrifugation. Lineage markers expressing cells were depleted using StemSep columns and human progenitor enrichment cocktail (StemCell Technologies, Vancouver, BC, Canada). CD34+ CD38− cells (hematopoietic stem progenitor cells [HSPCs]) and CD34+ CD38+ cells (hematopoietic progenitor cells [HPCs]) were sorted on a MoFlo cell sorter (DakoCytomation Colorado, Fort Collins, CO, USA) or a BD FACS Aria (BD Biosciences, UK). Gates were set up to exclude nonviable cells and debris. Briefly, lineagedepleted recovered cells were washed twice and stained with antiCD34 Percp, anti-CD38 PE-cy7, AlexaFluor647-conjugated Annexin-V (Invitrogen), and DAPI (4’,6-diamidino-2-phenylindole). The purity of sorted fractions was assessed to ensure the sort quality. The stromal cell line mesenchymal MS-5 and the human osteosarcoma cell line SaOS-2 were obtained from the DSMZ cell bank (Braunschweig Germany) and maintained in Iscove’s modified Dulbecco’s medium (IMDM) containing 10% fetal calf serum (FCS) + 2 mmol/L L-glutamine or in McCoy’s 5a medium containing 15% FCS + 2 mmol/L L-glutamine, respectively.
