Cd34 conjugated microbeads

In order to exclude the contamination of human iPSCs, the generated EPCs were purified by using MACS according to the manufacture’s instruction. In brief, the differentiated cells (10⁷ cells) were incubated with 10 μl CD34-conjugated microbeads (Miltenyi Biotec Inc) in the refrigerator for 20 min, followed by wash in PBS for twice. The beads-binding cells were separated using an autoMACS separator (Miltenyi Biotec Inc). All CD34⁺ cells were resuspended with EPC culture medium and cultured in a regular humidified incubator.
For flow cytometry analysis, the purified EPCs were incubated with FITC-conjugated CD34, FITC-conjugated KDR, FITC-conjugated Oct3/4 or FITC-conjugated IgG for 30 min (5 μl, eBioscience) in the dark. All antibodies were purchased from eBioscience. After incubation, all samples were analyzed under flow cytometry (Accuri C6 flow cytometer). 10,000 events were collected for data analysis.

Bone marrow samples were stratified on Ficoll Histopaque 1077 (Sigma-Aldrich, St. Louis, MO, USA, catalogue number 10771) and centrifuged at 300 g for 30 min at 25°C. Mononuclear cells sedimented at the interphase were then collected, washed twice with PBS and assessed for viability by trypan blue staining (ThermoFisher, catalogue number 15250061). An average of 1 × 10⁸ bone marrow mononuclear cells (BM-MNCs) was labelled with CD34-conjugated microbeads (Miltenyi, Woking, UK) and immunomagnetically sorted. CD34-depleted cells were labelled with CD45-conjugated microbeads (Miltenyi) and further sorted. The CD34–CD45 double-negative population was labelled with CD146-conjugated microbeads (Miltenyi) and enriched through immunomagnetic sorting. The purity of the selected cell population was assessed by flow cytometry (see below). Samples with a purity below 90% were excluded from the study.
The CD34⁻CD45⁻CD146⁺ cell fraction was then seeded onto 24-well plates at a density of 1 × 10³ to 5 × 10³ cells per cm² and expanded in an α-MEM basal media (ThermoFisher Scientific, catalogue number 32561-029) supplemented with 20% FBS (ThermoFisher Scientific, catalogue number 16000044). Four to six cell lines per group were studied between passage three and seven in the subsequent experiments.

Human BM pericytes (BM-PCs) and stromal cells (BM-SCs) were obtained as previously reported [24 (link), 25 (link)]. Once approximately 90% confluent, BM-MSCs were trypsinised and sorted for isolation of PCs. Around 1 × 10⁷ BM cells were resuspended in pre-cooled column buffer containing 0.5% (w/v) BSA and 2 mM EDTA in DPBS, labelled with CD45-conjugated microbeads (Miltenyi) for 15 min at 4℃ before subsequently being sorted by magnetic separation. The CD45-negative cells were then labelled with CD34-conjugated microbeads (Miltenyi), followed by magnet sorting. CD45^neg CD34^neg cells were labelled with CD146-conjugated microbeads (Miltenyi). The CD34^neg CD45^neg CD146^pos population, collected through immunomagnetic sorting, was considered BM-PCs. The remaining cells (CD45^negCD34^pos and CD45^negCD34^negCD146^neg populations) were pooled and considered BM-SCs. The purity of BM-PCs was assessed by immunocytochemistry staining (data not shown). Cell lines between passage 3 to passage 6 were used for subsequent experiments.