BRAFV600E-induced transcriptional changes in cord blood-derived hematopoietic stem and progenitor cells

BRAFV600E is a recurrent mutation in the histiocytoic disorders Langerhans cell histiocytosis (LCH) and Erdheim Chester Disease (ECD). The mutation can be detected in committed monocytes/macrophages, dendritic cells, and hematopoietic stem and progenitor cells (HSPCs). Although the cell of origin that gives rise to these disorders is currently unknown, it was recently suggested that LCH and ECD arise from BRAFV600E-mutant HSPCs. To understand the underlying molecular mechanisms in HSPCs driving this phenotype, we introduced BRAFV600E into healthy human HSPCs using CRISPR/Cas9. Overall design: CD34+ HSPCs isolated from three umbilical cord blood were cultured ex vivo and BRAF wildtype (WT) or BRAFV600E cDNA was introduced via CRISPR/Cas9 and AAV6-mediated knock-in. Cells with correct knock-in and cells without knock-in (RNP) were enriched by FACS based on fluoresecent reporter expression and total RNA was isolated for RNA-seq.