Stemline medium

Human brain pericytes (ScienCell Research Laboratories, Carlsbad, CA, USA) were plated and expanded in Stemline medium (Sigma-Aldrich) supplemented with 2% foetal bovine serum (Invitrogen), 1% penicillin/streptomycin (Gibco), 20 ng/ml bFGF (Invitrogen) on gelatin-coated culture flasks (Nunc), and incubated at 37 °C in 5% CO₂ conditions (Heraeus HERAcell 150 CO₂ incubator, Thermo Scientific). Cells grew exponentially with a doubling time of approximately 48 h, reaching ca. 85% confluence after 48–72 h. The cells were seeded in six-well culture plates at 100,000 cells/well for the following experiments. The cells were expanded and used between passages 2–5. As previously described, human brain pericyte–conditioned medium was collected from non-treated pericyte-conditioned medium (CM) and pericytes treated with PDGF-BB (20 ng/ml, RD systems) (CM^PDGFBB) for 72 h (Gaceb et al. 2018 (link)). Briefly, cell medium was centrifuged at 1500g for 5 min, and the supernatant free from cell debris was collected and used for the following experiments. At 72 h, human brain pericyte–conditioned medium shows only a very low contamination with the added PDGF-BB (Gaceb et al. 2018 (link)).

D15 cells differentiated on beads according to cluster B protocols were harvested with 0.25% trypsin/EDTA (Life technologies) and diluted with staining/blocking solution (3% FBS in DPBS). Prior to staining with specific antibody or the corresponding isotype control, cells were incubated with mouse BD Fc block (#553142, BD Biosciences). The antibodies and corresponding isotype controls used were anti-mouse CD11b-APC (clone M1/70) and APC-Rat IgG2a isotype control, both purchased from BD Biosciences. Cells were incubated with antibody for 1 hour on ice, washed twice with 3% FBS in PBS, and then sorted using a FACS Aria flow sorter (BD Bioscience). The live cells were gated using a propidium iodide (PI) stained control and the sorting gates were set using the isotype control stained cells. Both the CD11b positive and the CD11b negative cells were collected. The CD11b positive cells were plated into 2 wells of a 96 well tissue culture plate in Stemline medium (Sigma) containing TPO, IL3 and IL6 (All from R&D) and incubated at 37°C for 1 h to allow the cells to attach. After the incubation period the media were removed and the cells subjected to the pHrodo assay (Life Technologies) as above, then counterstained with calcein green. Cells were subsequently photographed using a Nikon Eclipse 2000 epifluorescence microscope.

As we previously reported [20 (link)], MSC layers at 80% confluence were incubated with 0.3 μg/mL mitomycin C to inhibit cell growth. Ten thousand cells enriched in CD34⁺CD38⁻Lin⁻ cells were seeded on MSC layers in 6-well plates (Corning Inc., Costar, New York, NY, USA) in Stem Line medium (Sigma-Aldrich, St. Louis, MO, USA) with or without the early acting cytokines thrombopoietin (TPO), Flt-3 ligand (FL), stem cell factor (SCF), and interleukin-6 (IL-6) at a concentration of 10 ng/mL (Peprotech, USA). In cultures in which MSC-HPC contact was inhibited, 0.4 μm Transwells (BD) were used. Cultures were taken on day 14, with a medium change on day 7.

We previously established and characterized an adult human brain pericytes line, obtained from brain tissue harvested from individuals undergoing ventriculostomy or surgery for intractable temporal lobe epilepsy as described [23 (link)]. All procedures were performed with informed written consent by the patient for the donation of brain tissue and approved by the ethical committee of the Scania University Hospital, Lund, Sweden. Using flow cytometry, our previous studies show that human brain pericytes express the key pericyte markers including PDGFRb (CD140b), CD146, CD105, and CD13[24 ] [25 ]and are negative for monocyte/macrophage markers CD14, the microglial marker CD11b and the endothelial marker CD31[10 (link),24 ,25 ]. The human brain pericytes were expanded in Stemline medium (Sigma-Aldrich) supplemented with 2% fetal bovine serum (FBS, Invitrogen), 1% Penicillin/Streptomycin (P/S, Gibco), 20 ng/ml basic fibroblast growth factor (bFGF, Invitrogen) on gelatin coated culture flasks (Nunc) and incubated at 37°C in 5% CO₂ conditions (Heraeus HERAcell 150 CO₂ incubator, Thermo Scientific). The cells grew exponentially with a doubling time of approximately 48 hs, reaching approximately 85% confluence after 48-72hs. Commercially available pericytes (ScienCell) were expanded in pericyte medium (ScienCell) according to the manufacturers instructions.