Human cd34 microbead kit

Human cord blood CD34⁺ cells (ZenBio Inc., Research Triangle Park, NC, USA) were purified by magnetic activated cell sorting (MACS) using the human CD34 MicroBead Kit (Miltenyi Biotec) according to manufacturer's protocol.

De-identified cord blood samples were purchased from the State University of New York Upstate Medical Center’s Upstate Cord Blood Bank Program. Mononuclear cells were isolated using Ficoll-Paque Plus (Cytiva). CD34 cells were then purified using human CD34 MicroBead kit (Miltenyi Biotec) following the manufacturer’s protocol and cultured in CD34 expansion medium (StemSpan SFEMII (Stemcell Technology), 50 ng/ml SCF, IL-3, IL-6, TPO, FLT3L (Peprotech) and 500 nM UM729 (Stemcell Technology)).

Frozen CD34⁺ HSPCs derived from mobilized peripheral blood or cord blood were purchased from AllCells (Alameda, CA, USA) and thawed according to manufacturer’s instructions. Fresh CD34⁺ HSPCs from cord blood were acquired from donors under informed consent via the Binns Program for Cord Blood Research at Stanford University and used without freezing. Fresh CD34⁺ HSPCs from bone marrow were obtained from Stanford BMT Cell-Therapy Facility after informed consent. CD34⁺ cells were isolated using a human CD34 MicroBead Kit (Miltenyi Biotec, San Diego, CA, USA). Generally, CB-derived HSPCs perform better in HR experiments. CD34⁺ HSPCs were cultured in stem cell retention media consisting of StemSpan SFEM II (Stemcell Technologies, Vancouver, Canada) supplemented with SCF (100 ng/ml), TPO (100 ng/ml), Flt3-Ligand (100 ng/ml), IL-6 (100 ng/ml), UM171 (Stemcell Technologies) (35 nM) and StemRegenin1 (0.75 mM). Mycoplasma contamination testing was not performed. Cells were cultured at 37°C, 5% CO₂, and 5% O₂.

The dorsal aortas were dissected from human embryos CS15-16 (N = 2) then further bisected into ventral and dorsal portions (AoV and AoD respectively). Tissues were dissociated into single cells in 1mg/ml Collagenase-Dispase (Roche) and 0.12 mg/ml of DNase I (Roche) for 35 min in a 37°C rotating water bath and stained with conjugated antibodies anti-human VE-Cadherin-PE (Beckman Coulter, 5μg/ml), anti-human CD45 –v450 (Clone: HI30, BioLegend, RRID:AB_1645574, 6μg/ml) and anti-human CD235A – APC (BD Bioscience, RRID:AB_398499, 0.2μg/ml) for 1 hour at 4°C. Dead cells and erythroid cells were excluded by 7AAD and CD235A staining respectively. VC+CD45-, VC+CD45+ and VC-CD45+ populations were sorted using a FACS Aria-II (BD Bioscience) into Eppendorf tubes containing RLT buffer from the RNeasy Micro kit ready for RNA purification. Data acquisition and analysis was performed using FlowJo software (Tree Star).
For the 10X single cell sequencing, following AoV cell dissociation, CD34+ cells were isolated using the human CD34 MicroBead Kit and LS MACS Columns (both Miltenyi Biotec). This procedure was carried out as per the manufacturer’s guidelines.

Human CB was purchased from the New York Blood Center. The purification of CD34+ cells was described previously^{23 (link)}. Briefly, white blood cells were separated from other types of blood cells by centrifugation. After the wash, CD34+ cells were magnetically labeled with CD34+ MicroBeads and then were separated using MACS column. Human CD34+ MicroBead kit was purchased from Miltenyi Biotec Company.