Single-cell profiling unravels bone marrow stromal remodeling driven by leptin receptor-expressing mesenchymal precursors in myelofibrosis

In order to comprehensively characterize the cellular constituents of the bone marrow microenvironment in primary myelofibrosis, single-cell RNA sequencing was performed on bone marrow stromal cells isolated from mice transplanted with MPLW515L-transduced hematopoietic cells. We profiled non-hematopoietic bone marrow cells from Lepr-Cre; tdTomato recipient mice transplanted with hematopoietic cells transduced with MPLW515L or empty vector retrovirus. Following onset of fulminant disease at 30-35 days after transplantation, bone marrow stromal cells were enriched by FACS using negative markers (CD45, Ter119, MPLW515L-GFP, SYTOX Green) to exclude hematopoietic/dead cells and a positive live cell marker (Calcein Violet-AM) to exclude tissue debris. Single cells isolated by FACS were profiled using droplet-based scRNA-seq (10x Genomics). We analyzed 4 MPLW515L mice (2 were pooled together for a total of 3 samples) and 2 empty vector control mice (pooled together into 1 sample). After data filtering and exclusion of contaminating hematopoietic cells (mostly erythroid progenitors), we ended up with 1,765 control cells and 2,184 MPLW515L cells, which were used for our downstream analyses.