RNAseq analysis of bone marrow mesenchymal stem cells

The phenotype of bone marrow mesenchymal stem cells in the murine Townes model of sickle cell disease is not well characterized. We analyzed differences in the gene signature between SA and SS MSCs. MSCs were defined by CD45-Ter119-CD31-CD51+CD140a+ surface expression. We identified significant differences in SS MSC gene profile and stem cell functionality compared to SA control MSCs. Overall design: Murine bone marrow from SA and SS mice was enzymatically digested, stained with antibodies, and sorted by gating on the live cells, single cells, CD45-, Ter119-, CD31-, CD51+ and CD140a+ population. RNA extraction was performed using the NucleoSpin RNA-XS kit. Library preparation, RNA-seq analysis on the Illumina HiSeq4000 machine and bioinformatics analysis were conducted with the Applied Bioinformatics Laboratory at NYU School of Medicine. SS: Homozygous Townes mice carry a mutation designed with the human hemoglobin a-gene (Hbatm1(HBA)Tow, ha) and a mutation containing the human A?-globin gene and human sickle hemoglobin beta (ßS) that replaced mouse major and minor ß-globin genes (ha/ha::ßS/ßS). SA: Heterozygous Townes mice carry the human hemoglobin a-gene (Hbatm1(HBA)Tow, ha) mutation, one copy of human sickle hemoglobin beta (ßS), and the human wild-type hemoglobin beta (ßA) gene (ha/ha::ßA/ßS).