LSD1/KDM1A and GFI1B orchestrate hematopoietic emergence from induced pluripotent stem cells
Differentiation of induced pluripotent stem cells to hematopoietic lineages holds great promise to produce novel, or replace existing cellular advanced therapeutic and medicinal products. During embryogenesis, hemogenic endothelial cells give rise to hematopoietic stem and progenitor cells through a process called endothelial to hematopoietic transition. Understanding the process of endothelial to hematopoietic transition in iPSC is crucial to establish protocols to generate bone marrow repopulating hematopoietic stem cells for therapeutic use. The transcription factor Gfi1b and its cofactor, the chromatin demethylase Lsd1 (KDM1A), are essential to produce functional hematopoietic stem and progenitor cells during endothelial to hematopoietic transition in mice.
To study the role of GFI1B and LSD1 in this process, we used iPSC lines derived from patients with a bleeding disorder that express a dominant negative dysfunctional GFI1BQ287*, and we treated wild type iPSC with an irreversible LSD1 inhibitor. Single cell RNA-sequencing was used to uncover the effects and differentiation potential of CD144+/CD309+ progenitor cells with hemogenic endothelial capacity.
GFI1BQ287* patient lines nor inhibition of LSD1 affected the formation of CD144+/CD309+ progenitor cells with hemogenic endothelial capacity during iPSC differentiation. However, the hematopoietic committed population as well as the yield of hematopoietic progenitors was severely reduced in GFI1BQ287* iPSC and completely absent upon inhibiting LSD1. To uncover the molecular mechanisms underlying the affected hematopoietic commitment via hemogenic endothelial in these conditions, we performed single cell RNA sequencing (scRNAseq) on the heterogeneous hemogenic endothelial population harvested from iPSC that were differentiated in absence or presence of a LSD1 inhibitor. scRNAseq revealed a dynamic expression of genes associated with endothelial to hematopoietic transition in vivo, and a complete absence of cells with a hematopoietic gene expression profile in LSD1 treated conditions. Furthermore, within the endothelial cell population, we identified gene modules that are specifically associated with LSD1 inhibition.
Based on the results we suggest that the endothelial program during endothelial to hematopoietic transition is partly controlled by LSD1/GFI1B and not efficiently down-regulated upon functional impairment of LSD1, or upon expression of the dominant negative GFI1BQ287*. In conclusion we showed a crucial role of the LSD1/GFI1B axis in emergence of hematopoiesis from iPSC and expect that the scRNAseq dataset will help to unravel the molecular events that drive hematopoiesis from iPSC.