Inhibition Of Lysine-Specific Demethylase 1a (Lsd1) Supports Human Hscs In Culture By Preserving Their Immature State

It has been a long-standing goal to gain a better understanding of the genes that regulate the self-renewal of HSCs in order to develop optimal culture conditions in which HSCs can be expanded for clinical benefit. In this study, we asked whether inhibition of Lysine-specific histone demethylase 1A (LSD1) would improve expansion of cultured human HSCs derived from umbilical cord blood (UCB). LSD1 modulates gene expression by eliminating di- and mono-methyl groups on H3 lysine K4 and K9 residues and has been shown to negatively impact self-renewal of mouse HSCs. To evaluate the effect of LSD1 inhibition we treated UCB CD34+ cells with three different LSD1 inhibitors (2-PCPA, GSK-LSD1 and RN1) and expanded the cultures for 6 days. Remarkably, treatment with either 2-PCPA or GSK-LSD1 resulted in a more than 10-fold increase of the HSC enriched CD34+EPCR+ population compared to controls. Using CFSE labeling, we found that 2-PCPA did not significantly alter the cell division rate of the cultured CD34+ cells and that the expansion of CD34+EPCR+ cells is not due to increased proliferation, but rather reduced differentiation of the immature HSPCs. Further, we mapped the early transcriptional changes triggered by 2-PCPA in HSCs and found that gene sets corresponding to UCB and fetal liver HSCs were significantly enriched upon 2-PCPA treatment. This suggests that 2-PCPA indeed restricts differentiation and preserves the HSC state upon ex vivo culture. Finally, the frequency of functional HSCs in DMSO and 2-PCPA treated cultures were measured using limiting dilution analysis (LDA). Cultures treated with 2-PCPA showed a 5-fold higher content of long-term repopulating cells compared to DMSO treated controls (1 in 615 vs 1 in 3041, p=0.03). Altogether our data suggest that LSD1 inhibition supports both phenotypic and functional HSCs in culture by preserving the immature state.