Comparative analysis of Cd61 high and Cd61 low populations of HSCs in M.musculus C97/Bl6 mice.

Aging leads to a decline in function of hematopoietic stem cells (HSCs) and increases susceptibility to hematological disease. We found CD61 to be highly expressed in aged HSCs. Here we investigate the role of CD61 in identifying distinct subpopulations of aged HSCs and assess how expression of CD61 affects stem cell function. We show that HSCs with high expression of CD61 are functionality superior and retain self-renewal capacity in serial transplantations. A population of aged HSCs with highest CD61 expression is functionally comparable to young HSCs. These CD61High HSCs display a notably higher quiescence compared to their CD61Low counterparts. We also show that CD61High and CD61Low HSCs are transcriptomically distinct populations within aged HSCs. Collectively, our research identifies CD61 as a key player in maintaining stem cell quiescence during aging, ensuring the preservation of their functional integrity and potential. Moreover, CD61 emerges as a marker to prospectively isolate a superior, highly dormant population of young and aged HSCs, making it a valuable tool both in fundamental and clinical research. 22-24 month Old male mice were used for the isolation of the hematopoietic stem cells (LT-HSCs). The LT-HSCs were sorted for the following markers: Lin-, Sca1+, cKit+, CD48-, CD150+. Each pool of LT-HSC was divided on LT-HSCs CD61High and 3 samples CD61Low as follows: LT-HSC CD61High : top 10% of LT-HSC, LT-HSC CD61Low : bottom 10% of LT-HSC population. In both cases 5,000 LT-HSC cells were sorted into 5uL of RLT Plus buffer. RNA isolation using RNeasy Plus Micro Kit (Quiagen) cDNA conversion was done using SMART-Seq v4 Ultra low input RNA kit for sequencing (TAKARA Bio, 4ng total RNA was used). Kit used for library preparation: Nextera XT DNA Library Prep, preparation was performed according to the protocol. 1ng of amplified cDNA was used and the remaining volume was supplemented with EB buffer. A 0.6x bead cleanup was performed. Samples were eluted in 14μl EB. Tagmentation was used for primer attachment