Hbfgf

Human muscle biopsies from the M. rectus abdominis were collected upon ethical approval and with informed consent of 6 hospitalized patients undergoing abdominal surgery under general anesthesia. The patients were selected according to strict inclusion and exclusion criteria, assuring the optimal quality of the muscle biopsy (e.g., no muscular dystrophy, hormonal therapy, and chronic infectious diseases). The samples were processed according to established protocols [29 (link)]. Briefly, each muscle biopsy was first minced and digested with collagenase type I 0.2% (w/v) (Sigma) and dispase 0.4% (w/v) (Gibco). The enzymatic reaction was terminated with medium containing 10% FBS. Individual fibers were then liberated by rigorous pipetting and filtered through a strainer with a pore size of 100 μm. After centrifugation, the pellet was resuspended in culture medium and the muscle fibers transferred into 35 mm dishes coated with collagen type I (1 mg/ml) (BD). The culture medium consisted of DMEM/F12, 1% penicillin/streptomycin, 18% FBS, 10 ng/ml hEGF (Sigma), 1 ng/ml hbFGF (Sigma), 10 μg/ml human insulin (Sigma), and 0.4 μg/ml dexamethasone (Sigma) [29 (link)]. After 24 h, a fibroblast reduction step was performed by replating the cells. The cultured hMPCs were characterized as published before [14 (link), 29 (link)].

Microvessels were fabricated in collagen and hylauronan (HA) composite hydrogels polymerized inside polydimethylsiloxane (PDMS)-based microfluidic devices fabricated using soft lithography^{43 (link)}. p20–p23 human cerebral microvascular endothelial cells (hCMEC/D3) and p7–p15 GFP-labeled human brain vascular pericytes (hBVP) (Neuromics) were seeded in the hydrogels at densities of 2 M/mL and 0.4 M/mL, respectively. These ratios were obtained from a previous study that used co-cultures of human blood outgrowth endothelial cells and human pericytes to form microvasculature networks^{13 (link)}. hCMEC/D3 were cultured in Endothelial Cell Basal Medium (PromoCell) supplemented with 5 µg/mL ascorbic acid (Sigma), 1 ng/mL hBFGF (Sigma), 1/100 chemically defined lipid concentrate (Thermo Fisher), 5% fetal bovine serum (VWR Life Science), 10 mM HEPES (Quality Biological), 1.4 µM hydrocortisone (Sigma), and 1% penicillin-streptomycin (Corning). hBVP were cultured in DMEM (Corning) supplemented with 10% FBS, 1% penicillin-steptomycin, and 1X MEM Amino Acid Solution (Thermo Fisher). Final hydrogel formulation concentrations consisted of 3 mg/mL HA (Sigma), 5 mg/mL collagen type I (MP Biomedical), and 0.85–1 mg/mL Matrigel (Corning). These reagents were combined with 0.1 M sodium hydroxide (NaOH) and 10x phosphate buffer solution (PBS) to facilitate polymerization and maintain physiological pH.

Human induced pluripotent stem cells (SBAD-02-01) were kindly supplied Dr Zameel Cader, University of Oxford. Expansion and differentiation of hiPSCs was done on Matrigel (BD Biosciences) coated 6-well plates (Nunc) in mTeSR1 medium (STEMCELL Technologies). Differentiation of SBAD-02-01 was performed as described [15 (link)]. Briefly, cells were thawed from the frozen vial and were expanded until 70% confluent on a Matrigel coated 6 well plate. After expansion, cells were passaged by use of accutase solution (Sigma-Aldrich) and seeded at 7.5x10³ hiPSC/cm² on Matrigel coated 6-well plates. Cells were further expanded in mTeSR1 medium. After achieving an optimal cell density of 2.5 x10⁴–3.5 x10⁴ hiPSC/cm², the cells were shifted to unconditioned media (UC) for 6 days. UC medium consisted of 78.5% DMEM/F-12 + 20% KnockOutTM Serum Replacement (Life Technologies) + 1% MEM NEAA (Sigma-Aldrich) + 1mM L-glutamine (Sigma-Aldrich) + 0.1 mM β-mercaptoethanol (Sigma-Aldrich). The medium was changed completely every day. Cells were then cultured for 2 days in human Endothelial-SFM (Life Technologies), containing 0.5% B27^TM (50x) (Thermo Fisher Scientific) supplemented with 20 ng/mL hbFGF (PeproTech) and 10 μM all-trans Retinoic Acid (Sigma-Aldrich), called EC medium + hbFGF + RA.