Stromal STAT5-mediated trophic activity regulates hematopoietic multipotent progenitor niche factors

Signal transducer and activator of transcription 5 (STAT5a and STAT5b) are intrinsically critical for normal hematopoiesis but are also expressed in stromal cells. However, hematopoiesis-supporting stromal function has not been reported. Here, STAT5ab knockout (KO) was generated with a variety of bone marrow hematopoietic and stromal Cre transgenic mouse strains. Pan-hematopoietic deletion with Vav1-Cre was the positive control for loss of multipotent hematopoietic function but surprisingly dysregulated niche factor mRNA expression and deleted STAT5ab in CD45neg cells. Single cell transcriptome analysis of mouse bone marrow from wild-type or Vav1-Cre KO mice showed major changes in hematopoietic stem cell myeloid commitment genes and upregulated protein translation genes throughout myeloid-primed clusters. Overall design: Bone marrow cells were harvested from the femurs and tibias of 4 Vav1-Cre/+ or Vav1-Cre/+/STAT5abflox/flox mice (8-12 weeks). The cells were lineage depleted using a mouse lineage cell depletion kit (Miltenyi Biotech, Auburn, CA) and stained with FITC-labeled lineage antibodies (Gr-1, Mac-1, B220, Ter119, CD3, CD4, CD8a), anti-c-Kit-APC, anti-Sca-1-PE-Cy7 (Thermo Fisher Scientific, Norcross, GA), and dead cells were excluded by staining with propidium iodide dye. KLS cells were sorted (sorting purity ~6% with 605 CD45neg cells among 10008 total sorted KLS cells) using BD FACSAria III Cell Sorter. For sorting bone marrow stromal cells, cells were isolated as described(79). Briefly, mouse bones (tibia and femur) were isolated and cleaned from 6-8 weeks old mice, then were crushed and cut into tiny pieces with sterile scissors, incubated them in 20 ml of preheated DMEM plus 0.2% (wt/vol; 40 mg) collagenase at 110 rpm at 37°C for 1 hour ). The cell suspension was filtered through a 70-µm cell strainer and bone fragment was further crushed and washed to liberate cells out of the bone into solution. CD45+ cells were depleted with mouse CD45 microbeads from Miltenyi Biotech( Auburn, CA) and stained with anti-Ter119-PE (BD Biosciences, San Jose, CA), anti-CD45-FITC, Anti-CD71-Alexa 700, Anti-Sca-1-PE-Cy7 and Anti-CD140a-APC (Thermo Fisher Scientific, Norcross, GA). CD45negTer119neg/lowCD71negSca-1+CD140a+ MSCs were sorted using BD FACSAria III Cell Sorter. Sorted cells were immediately processed for single cells RNA sequencing.