H flt3 ligand

Mononuclear cells were isolated from umbilical cord blood from anonymous healthy donors by density centrifugation using Ficoll (Biocoll, Merck Millipore). Human CD34+ cells were then enriched in the sample by immunomagnetic beads using an AutoMACSpro (Miltenyi Biotec). After collection, enriched CD34+ cells were frozen in a cryopreservation medium containing 90% of fetal bovine serum (Eurobio) and 10% of dimethylsulfoxide (Sigma) and stored in liquid nitrogen.
After thawing, the CD34+ cells were cultured in a 96-well plate in a humidified 5% CO2 incubator at 37°C. Cells were cultured in prestimulation medium made of XVivo (Lonza) supplemented with penicillin/streptomycin (respectively, 100 U/mL and 100 μg/mL; Gibco, Thermo Fisher Scientific), 50 ng/ml h-FLT3-ligand, 25 ng/ml h-SCF, 25 ng/ml h-TPO, and 10 ng/ml h-IL3 (Miltenyi) final concentration.

Peripheral blood or bone marrow samples derived from AML patients between 2018 and 2020 were obtained from the UCT Biobank of the University Hospital Frankfurt. The use of peripheral blood and bone marrow aspirates was approved by the Ethics Committee of Frankfurt University Hospital (approval no. SHN-03-2017). All patients gave informed consent to the collection of samples and to the scientific analysis of their data and of biomaterial obtained for diagnostic purposes according to the Declaration of Helsinki.
Mononuclear cell (MNC) fractions were purified by gradient centrifugation with Biocoll cell separation solution (Merck Millipore, Darmstadt, Germany). Leukemic cells were enriched by negative selection with a combination of CD3-, CD19- and CD235a-microbeads (all obtained from Miltenyi Biotec, Bergisch Gladbach, Germany, 130–050-301, 130–050-101, 130–050-501) according to the manufacturer’s instructions and separated by the autoMACS™ Pro Separator (Miltenyi Biotec). FACS staining and treatment for viability assays of AML blasts was executed immediately after isolation. Culture medium for AML blasts consisted of IMDM (Biochrom) supplemented with 10% FBS, 4 mM L-glutamine, 25 ng/ml hTPO, 50 ng/ml hSCF, 50 ng/ml hFlt3-Ligand and 20 ng/ml hIL-3 (all obtained from Miltenyi Biotec, 130–094-013, 130–096-695, 130–096-479, 130–095-069).

Primary human AML samples were obtained with informed consent and used in agreement with the Declaration of Helsinki upon the approval of the local ethic committees of the Goethe University Frankfurt (approval number 329–10). Samples were maintained in X-Vivo10 medium (Lonza) supplemented with 10% FCS (Hyclone/Perbio Science), with the addition of 20 ng/ml hIL-3, 50 ng/ml hSCF, 25 ng/ml hTPO and 50 ng/ml hFLT3-ligand (Miltenyi, Bergisch-Gladbach, Germany).