Transcriptional remodeling of human hematopoeitic stem cells in vitro by Interleukin-1β

Trained immunity is a phenomenon where innate immune cells develop and long-lasting pro-inflammatory reprogramming mediated by epigenetic and metabolic reprogramming. Trained immunity can be peropheral (circulating monocytes) or central (bone marrow) and it can be caused by exposure to β-glucan, BCG and Western Type Diet vial IL-1β signaling. HPCs were isolated from bone marrow mononuclear cells by CD34+ mangenitc separation. CD34+ cells were then exposed for 4 hours to 10 ng/ml IL1b. After incubation, cells were washed and seeded in 24-well plates at a density of 50,000 cells per well for expansion (10 days) and differentiation into monocytes for 7 days (see details in Growth protocol). At day 7 of differentiation, pan monocytes were magenetically separated. 500.000 cells per condition were then incubated for 4 hours with medium only or LPS 10 ng/ml; after the incubation time, the cells were washed and stored in RNAlater until further RNA extraction. RNA isolation was done using the QIAGEN AllPrep DNA/RNA micro-kit according to manufacturer’s instructions.