Cd34 positive selection kit

Human fetal livers and femurs were obtained from aborted fetuses at 15–23 weeks of gestation in accordance with the institutional ethical guidelines of KK Women and Children Hospital, Singapore. All patients gave written informed consent for the donation of their fetal tissues for research. Fetal liver was processed as described previously19 (link). Umbilical cord blood was obtained from the Singapore Cord Blood Bank. CD34⁺ cells from both fetal liver and cord blood were purified with the use of a CD34 positive selection kit (Stem Cell Technologies, Vancouver, BC); the purity of CD34⁺ cells was 90 to 99%. Viable cells were counted by Trypan Blue exclusion of dead cells. All cells were isolated under sterile conditions.

Mice were housed in a barrier facility in individually ventilated plastic cages (Innovive Innorack IVC) with corn cob bedding and a maximum of 5 mice/cage. Mice were provided with environmental enrichment (nesting material), and lighting was timer regulated with 12 h on/12 h off. Humidity was 30–70%, temperature was 68–79 °F, and food and water were provided ad libitum. Mice were observed at the time of dosing every day and were weighed every 2–5 days. Two cohorts of NSG-BLT mice were generated by implanting 8–10-week-old female (mean weight: 26 g) NOD-scid IL-2Rγ^−/− (NSG) mice (stock no. 005557, NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ, Jackson Laboratory) under the kidney capsule with 1-mm³ pieces of human fetal thymus and liver from a single donor for each cohort, as described previously (39 (link), 40 (link)). Three weeks after implantation, mice were injected intravenously with 200,000 viable human CD45⁺ CD34⁺ Lin-1⁻ hematopoietic stem/progenitor cells isolated from the autologous fetal liver with a CD34 positive selection kit (StemCell Technologies) and cryopreserved. The mice were not irradiated prior to cell injection because we have found that this conditioning step is not necessary for robust human leukocyte reconstitution.

Human fetal liver (FL) samples, male and female, 16–23 weeks of age were obtained from Kandang Kerbau Women’s and Children’s Hospital (KKH) with informed and written consent from patients. SingHealth and National Health Care Group Research Ethics Committees Singapore specifically approved this study (CIRB Ref: 2012/064/B), and all experimental procedures were conducted in accordance to the protocol. FLs were processed and digested with collagenase VI (2 mg/ml in Dulbecco’s modified Eagle’s medium (DMEM)) (Thermo Fisher Scientific, USA) for 15 min at 37°C with constant rotation. Digested tissue was passed through a 100 µm mesh to obtain single-cell suspension and isolated for human CD34⁺ cells with a CD34-positive selection kit (STEMCELL Technologies, USA), according to the manufacturer’s instructions. The purity of the CD34⁺ cells was 90–99% as determined by flow cytometry.