Cd34 isolation kit

Umbilical cord blood (CB) units from normal full-term deliveries were obtained, with the informed consent of the mothers, from the Obstetrics Unit of Saint Louis Hospital, Paris, and collected in placental blood collection bags (Maco Pharma, Tourcoing, France). Blood mononuclear cells were purified by Ficoll density gradient separation (Leucosep, Greiner Bio-one) and Hanks medium (Thermo-Fisher). Low-density cells were recovered and enriched for CD34⁺ cells by automated cell sorting (CD34 isolation kit and autoMACS System, Miltenyi Biotec). CD34⁺ cells were cultured in serum-free expansion medium: Iscove’s Modified Dulbecco’s Medium (IMDM), 15% BIT 9500 (BSA-Insulin-Transferrin, Stem Cell Technologies), 60 ng/mL rh-Stem Cell Factor (SCF), 10 ng/mL rh- interleukin-3 (IL-3), 10 ng/mL rh-IL-6, 2 U/mL rh-Epo, 100 U/mL penicillin and 100 μg/mL streptomycin. After seven days of culture, CD36⁺ cells were isolated with biotin-coupled anti-CD36 antibody and anti-biotin microbeads on an autoMACS System. CD36⁺ EPCs were obtained by lentivirus-mediated transduction with the hTERT and E6/E7 genes from human papillomavirus type 16, as previously described [36 (link)], and were grown in expansion medium to generate a continuous CD36⁺ EPC line.

The CD34⁺ donor hematopoietic stem cells (DHSCs) were prepared using the CD34 isolation kit (Miltenyi Biotech Inc., Auburn, CA). Similarly, T cells were enriched using anti-CD3 coated microbeads with the MACS system via positive selection (Miltenyi Biotech Inc., Auburn, CA). Flow cytometry using appropriate monoclonal antibodies (mAbs) showed that the positively selected populations had 95 - 99% of cells expressing the target epitope. These cells were then used for in vitro assays.

Cord blood was obtained with written informed consent from the Queensland Cord Blood Bank with approval from the Mater Adult Hospital Human Ethics Committee. CD34⁺ hematopoietic progenitor cells were isolated by density gradient enrichment followed by a positive selection using a CD34⁺ isolation kit (Miltenyi Biotec) as previously described (30 (link)). NSG-A2 mice (stock no. 014570) were purchased from Jackson Laboratories. 2–5-day-old NSG-A2 pups received 10 Gy total body irradiation 4 h prior to intrahepatic injection of human CD34⁺ cells. Engraftment of human CD45⁺ cells was confirmed 10–12 weeks later, after which hu mice received 2 s.c. doses of human recombinant huFLT3-L (BioXcell) 4 days apart prior to experimentation. Engrafted mice were injected retro-orbitally with 50 µg poly IC (Invivogen) or 20 µg R848 (Invivogen) alone or in combination and mice were euthanized 2 h later. This study was carried out in accordance with the recommendations of the Australian code for the care and use of animals for scientific purposes (8th Edition). The protocol was approved by the University of Queensland Animal Ethics Committee.

Peripheral venous blood was obtained from healthy adult volunteers, and umbilical cord blood (UCB) was obtained from the Queensland Cord Blood Bank. Written informed consent was obtained for sample acquisition in line with standards established by the Declaration of Helsinki. Study approval was granted by the Mater Human Research Ethics Committee (HREC13/MHS/83 and HREC13/MHS/86). CD34⁺ haematopoietic stem cells (HSCs) were isolated from UCB using a CD34⁺ Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, North Rhine‐Westphalia, Germany) and cryopreserved at −80 °C. Typing for HLA‐A*0201 and HLA‐A*2402 was confirmed by PCR.⁵², ⁷¹

Mononuclear cells (MNCs) were separated from CB and mPB samples (maximum after 1 day from harvesting) by stratification on Lympholyte-H 1.077 g/cm³ gradient (Gibco-Invitrogen, Milan, Italy), followed by red blood cell lysis for 15 min at 4°C. MNCs were then processed on magnetic columns for CD34⁺ cell isolation (mean purity 94 ± 4%) (CD34 Isolation kit; Miltenyi Biotec, Bologna, Italy), as previously described [25 (link)], and treated with our combination of cytokines on the same day. In selected cases, CD34⁺ cells from CB or mPB were cryopreserved in liquid nitrogen and then thawed before testing with the combined inflammatory cytokines. Of note, to minimize the influence of freezing/thawing, only thawed CD34⁺ cells with a survival rate > 80% were used and the thawed CB/mPB cells were studied in the same experiment.