EBF1-mutant bone marrow stroma confers long-term changes in hematopoietic stem cell potential

Crosstalk between mesenchymal stromal cells (MSCs) and hematopoietic stem and cells (HSCs) is essential for hematopoietic homeostasis and lineage output. Ebf1-deficient MSCs have reduced mesenchymal lineage potential. Ebf1 deletion in Cxcl12-abundant reticular (CAR) cells and PDGFRα+Sca1+CD45-CD31-Lin- (PαS) cells in the bone marrow decreased the numbers of HSPCs and myeloid cells. EBF1 in the bone marrow niche regulates a transcriptional program that determines the functional interactions with HSCs and the long-term balance between the myeloid and lymphoid cell fates. Intact bones (femora and tibia) were chopped into small fragments and crushed in FACS buffer (PBS-2% FCS) using pestle and mortar. For the MSC cells isolation, the bone chips were digested with 0.3% collagenase type II and 0.02% DNase I at 37°C for 45 min with gentle shaking. After enzymatic digestion, the cells were filtered, washed with FACS buffer, and stained accordingly. CAR cells were identified as CD45-CD31-Lin-PDGFRα+Sca1-, PαS cells were identified as CD45-CD31-Lin-PDGFRα+ Sca1+. BD FACSAria Fusion was used for fluorescence-activated cell sorting.