BioProject

To investigate molecular differences between HSPCs from young and aged donors, we performed 10x Single Cell Multiome on bone marrow HSPCs from Young, Middle Aged, and Older Aged donors. More...

To investigate molecular differences between HSPCs from young and aged donors, we performed 10x Single Cell Multiome on bone marrow HSPCs from Young, Middle Aged, and Older Aged donors. Overall design: Bone marrow cells from young donors (26-year-old female, and 24-year-old male) were purchased from Lonza, while bone marrow samples from aged donors (70 and 77-year-old females) undergoing hip replacement surgery were collected at the Traumatology and Orthopedics Hospital Lomas Verdes (IMSS), Mexico. These elderly donors were confirmed to have no dysplasia of any hematopoietic lineages by histological and CBC analysis. Ethical approval was obtained from the Institutional Review Board (R-2012-785-092). Patient consent was obtained verbally, and as determined by the Institutional Ethical Board. BM samples were thawed via slow dropwise addition of X-VIVO 10 media (LONZA) with 50% FBS and 100μg/mL DNaseI (Roche). Cells were centrifuged at 400g for 10 min, then dead cell depleted using a commercial kit (EasySep Dead Cell Removal (Annexin V) Kit, STEMCELL) per the manufacturer’s instructions. Cells were resuspended in PBS + 5% FBS and stained for 15 min at RT for fluorescence-activated cell sorting with the following antibodies: anti-CD45RA-FITC (1:50, BD, clone HI100), anti-CD90-PE (1:50, BD, clone 5E10), anti-CD19-BV711 (1:50, BD, clone SJ25C1), anti-CD49f-PE-Cy5 (1:50, BD, clone GoH3), anti-CD271-APC (1:100, BD, ME20.4-1.H4), anti-CD34-APC-Cy7 (1:200, BD, clone 581), anti-CD38-PE-Cy7 (1:200, BD, clone HB7), anti-CD10-AlexaFluor700 (1:50, BD, clone HI10a), anti-CD14-BV605 (1:200, BD, clone M5E2), anti-CD45-V500 (1:50, BD, clone HI30) and anti-CD33-BV421 (1:100, BioLegend, clone WM53). Cells were washed following staining and resuspended in PBS + 2% FBS containing propidium iodide and filtered through 40μm nylon mesh for cell sorting. Lin–CD34+CD38– and Lin–CD34+CD38+ populations were sorted into PBS + 0.04% BSA + EDTA on a BD FACSAria Fusion or BD FACSAria III. Cells were counted and Lin–CD34+CD38– and Lin–CD34+CD38+ cells mixed in the following manner (1:0.33 for 24yM, 1:1 for 26yF, 1:1 for 70yF, and 1:0.5 for 77yF) for downstream 10x Genomics multiome sample preparation by the Princess Margaret Genome Centre. Less...