Hydrogel Differentiation Patterns of HSPCs and MSPCs

HSPCs and MSPCs were cultured at increasing ratios of 1:0, 1:1, 1:10, 0:1 and 0:10 (HSPCs–MSPCs) in 4, 5 and 7.5 wt% methacrylamide-functionalized gelatin (GelMA) [49 (link), 50 (link)] at constant crosslinking conditions (85% functionalization, 0.1% lithium acylphosphinate photoinitiator (PI) and 7.14 mW/cm² UV light for 30 seconds) for a total of 15 conditions (Fig. 1A) [33 (link), 46 (link), 51 ]. HSPC seeding density was kept constant at 1×10⁵ HSPCs/mL, and cells were encapsulated in 5 mm diameter hydrogels (20 μL) and cultured for 7 days in 300 μL SFEM media (#09650, Stemcell Technologies) supplemented with 100 ng/mL SCF (#250–03, Peprotech) and 0.1% PenStrep, changed every 2 days. Media was collected from each sample at day 2, 4 and 6 and stored at −80°C for use in secretome analysis. Hematopoietic differentiation patterns in response to hydrogel and seeding condition were previously reported by Gilchrist et al. and are publicly available [46 (link)]. Importantly, the collected media and hematopoietic lineage patterns were from the same samples, allowing for direct mapping of sample conditioned media to HSC phenotype.