Macs cd34 micro bead kit

Cord blood was collected with written informed consent from the mother before delivery at the Japanese Red Cross Cord Blood Bank. Cord blood experiments were approved by the ethical committees of the Japanese Red Cross Cord Blood Bank and National Center for Global Health and Medicine. The sample was processed using Lymphoprep (GE Healthcare) density gradient medium to isolate mononuclear cells. CD34 enrichment was performed by magnetic cell separation using a MACS CD34 Microbead Kit (Miltenyi Biotec). CD34⁺ enriched cells were frozen until use. Frozen cells were thawed in vials in a 37°C water bath and transferred to a 15 mL tube. RPMI-1640 medium containing 10% fetal calf serum (FCS) was slowly added to the suspension while gently swirling to fill the tube. The suspension was centrifuged at 200 × g for 15 min at room temperature. Supernatants were aspirated and the wash step was repeated. An antibody cocktail (50 μL of phosphate-buffered saline (PBS) containing 2% FCS plus 10 μL of anti-CD34-FITC, 2 μL of anti-CD38-PerCP-Cy5.5, 5 μL of anti-CD90-PE-Cy7, and 10 μL of anti-CD45RA-PE) was added to the suspension and kept on ice for 30 min. Cells were washed once with PBS containing 2% FCS and resuspended in PBS containing 2% FCS supplemented with 0.1% propidium iodide (PI). Cells were sorted into StemSpan SFEM-I using a FACSAria IIIu instrument.

Isolation of CD34‐enriched/depleted subpopulations was carried out with the MACs CD34 MicroBead Kit (human cells, Miltenyi Biotec, Bergisch, Germany; #130‐046‐702). Cells were either collected for RNA, put into CFU assays, or for adherent progenitor cells; replated onto matrigel‐coated wells in HSPC medium.

Umbilical cord blood samples (CB) from healthy donors were provided by the Centro de Transfusión de la Comunidad de Madrid. All samples were collected with written consent and agreement from the Centro de Transfusión de la Comunidad de Madrid‘s institutional review board (number PKDEFIN [SAF2017-84248-P]). Mononuclear cells were obtained by fractionation in Ficoll-hypaque according to the manufacturer’s recommendations (GE Healthcare). Purified CB-CD34⁺ cells were obtained using a MACS CD34 Micro-Bead kit (Miltenyi Biotec). Cells were frozen in 10% dimethyl sulfoxide solution and stored in liquid nitrogen until their use.
Mononuclear cells from human BM were obtained by Ficoll- Paque Plus density gradient separation from heparinized BM samples obtained from healthy donors after informed consent. All the procedures were in accordance with the Helsinki Declaration of 1975, and its revision in 2000. Samples were cultured at 1.6×10⁵ cells/cm² in MesenCult medium plus supplements for human cells (Stemcell Technologies). After 24 h, nonadherent cells were discarded. Fresh medium was added and replaced twice a week. At 80% confluence, adherent cells were trypsinized, washed, and seeded at 4×10³ cells/cm². In all the experiments, BM-MSC were used at passages 5 to 8.

Non-mobilized PB was collected from seven healthy donors, and one heterozygous donor, and four patients. The study for the use of samples of human origin was approved by the Ethical Committee of Novara, Italy. MNCs were purified by gradient centrifugation on Ficoll (GE Healthcare) and CD34+ cells were isolated using the MACS CD34+ MicroBeadKit (Miltenyi Biotec) according to the manufacturer's protocol. Isolated cells were cultured for 4 days in HPGM medium (Lonza) supplemented with 1% human serum albumin (Sigma-Aldrich), 50 ng/mL of hSCF, hFlt3-ligand, hTPO, and hIL-3 (Immunotools).