Cd34 pe cy7

All the samples used for microarray analysis were FACS-sorted. Haemogenic endothelium panel: CD34 PE-Cy7 (8G12; BD), FLK1 AF647 (89106; BD), CD235a/glycophorin (GLY)-A FITC (11E4B-7-6; Coulter), and CD43 PE (1G10; BD). Fetal-liver HSCs were purchased from StemCell Technologies and stained with HSC panel: CD38 PE-Cy5 (LS198-4-3; Clontech), CD34 PE-Cy7 (8G12; BD), and CD45 PE (HI30; BD). Between 10,000 and 50,000 cells were sorted for each cell type with two or three biological replicates. An RNAeasy Microkit (Qiagen) was used to collect and prepare total RNA for microarray analysis. The Ovation Picokit (Nugen) was used for preamplification, where required. Gene expression profiling was performed on Affymetrix 430 2.0 gene chips according to standard protocol. Microarray data were analysed according to standard protocol using R/Bioconductor.

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).

For ASC phenotype analysis, cells were detached with non-enzymatic cell dissociation solution (ATCC Manassas, VA, USA). In the next step, ASCs were washed with FACS buffer (phosphate-buffered saline, 0.1% NaN₃, 1% FCS). Then, 5 × 10⁴ cells were suspended in 50 μL of FACS buffer and stained with antibodies against the following surface markers: CD90-FITC, CD105-PE, CD73-APC (eBioscience, San Diego, CA, USA), CD34-PE-Cy7, CD45-PE, CD19-PE, and CD14-APC (BD Pharmingen, San Diego, CA, USA). After the washing step, cells were acquired and analysed using a FACSCanto cell sorter/cytometer and Diva software. The gating strategy is shown in Figure S1 in the Supplementary Materials.

The following fluorochrome conjugated antibodies were used to label cell surface antigens: CD34 PE-Cy7, CD31 APC, CD73 PE, CD146 PE, CD45 PE, HLA DR FITC, and glycophorin A PE (BD Pharmingen). The relevant mouse isotypes (BD Pharmingen) were used as control. Cells were stained with labelled antibodies in the dark at 4°C for 45 min. A minimum of 30 000 events were acquired on a BD LSR II flow cytometer and the results were analyzed using BD FACSDiva software.

Immunostaining and flow cytometry analyzes (FACS) were performed to detect and confirm the presence of specific cell surface antigens characteristic for hASCs. All mouse antibodies used [CD 29-PE (BD 562801), CD 34-PE-Cy7 (BD 560710), CD 44-APC (BD 559942), CD 45-PerCP (BD 557235), CD 73b-FITC (BD), CD 90-APC-Cy7 (BD 561401), CD 105- Percp-Cy5.5 (BD 560819) and streptavidin (BD 554066)] were purchased from BD Biosciences (USA). Fluorochrome-conjugated mouse immunoglobulin was used as isotype control. Single cell suspensions of hASC were subsequently analyzed on a Becton–Dickinson FACSCalibur flow cytometer to obtain at least ten thousand cells. Samples were analyzed by FlowJo software (TreeStar, USA).