Isolation and Culture of CD34+ Cells from CML Patients

Bone marrow samples were collected from 11 cases of newly diagnosed CML patients at the Department of Hematology of the Second Hospital of Dalian Medical University. Patients were diagnosed according to French–American–British classification. Informed consent was obtained from all patients in accordance with the Declaration of Helsinki, and all manipulations were approved by the Medical Science Ethic Committee of Dalian Medical University. Mononuclear cells were isolated by density gradient centrifugation using Lymphoprep, and cryopreserved. In addition, 5 potential donors for allogeneic bone marrow transplantation were used to purify healthy hematopoietic cells. Human CD34⁺ cells were enriched from bone marrow mononuclear cells using MiniMACS (Miltenyi Biotech, Bergisch Gladbach, Germany) following the manufacturer’s instructions [27 (link)]. Confirmation of CD34⁺ cells phenotype and purity was assessed by flow cytometry analysis using CD34-PE-Cy7 (BD Biosciences, San Diego, CA). Purified CD34⁺ cells were grown in serum-free hematopoietic growth medium (HPGM; Lonza) supplemented with 10 ng/mL recombinant human interleukin 3 (rhIL-3), 10 ng/mL rhIL-6, and 50 ng/mL recombinant human stem cell factor (PeproTech) in a humidified incubator at 37 °C and 5% CO₂/95% air (v/v).