3149 – COMPARISON OF PRE-ENRICHMENT STRATEGIES FOR SORTING OF MOUSE LIN-CKIT+SCA1+ CELLS

The short life span of blood cells requires hematopoiesis to be a continuous ongoing process. In adult mammals, blood production takes place in the bone marrow. This tissue is home to hematopoietic stem cells (HSCs) that possess the ability to both self-renew and differentiate into mature blood cells. HSCs are widely studied in cancer research, as they are the source of various hematological malignancies. Such cancers develop in response to a progressive accumulation of mutations that confer a selective growth advantage to HSCs. In the case of leukemia, the mutations will lead to a differentiation blockade, causing dysfunctional blood cells to accumulate. Mouse models are the system of choice to study blood cancers given the complexity of the hematopoietic system. Thus, in order to study HSCs, we need to be able to isolate them from mice. Several HSC subpopulations have been defined, however, the most widely studied are the Lin-cKit+Sca1+ (LKS) cells. The best way to isolate LKS cells is by FACS sorting. Yet, the frequency of LKS cells in the bone marrow being very low (< 0.1%), a pre-enrichment step is necessary to decrease the sorting time. With the aim of identifying the most suitable pre-enrichment strategy, we tested several magnetic kits and compared them based on enrichment efficiency and final LKS yield. Four approaches were tested: lineage depletion (Lin-), cKit-enrichment (cKit+), Sca1-enrichment (Sca1+) and a combination of lineage depletion followed by Sca1-enrichment (Lin- Sca1+). Our results show that the best strategy are lineage depletion and Sca1-enrichment. Combining these two methods highly increased the LKS enrichment, however, the final LKS yield was low due to increased cell loss from performing a second enrichment step. The short life span of blood cells requires hematopoiesis to be a continuous ongoing process. In adult mammals, blood production takes place in the bone marrow. This tissue is home to hematopoietic stem cells (HSCs) that possess the ability to both self-renew and differentiate into mature blood cells. HSCs are widely studied in cancer research, as they are the source of various hematological malignancies. Such cancers develop in response to a progressive accumulation of mutations that confer a selective growth advantage to HSCs. In the case of leukemia, the mutations will lead to a differentiation blockade, causing dysfunctional blood cells to accumulate. Mouse models are the system of choice to study blood cancers given the complexity of the hematopoietic system. Thus, in order to study HSCs, we need to be able to isolate them from mice. Several HSC subpopulations have been defined, however, the most widely studied are the Lin-cKit+Sca1+ (LKS) cells. The best way to isolate LKS cells is by FACS sorting. Yet, the frequency of LKS cells in the bone marrow being very low (< 0.1%), a pre-enrichment step is necessary to decrease the sorting time. With the aim of identifying the most suitable pre-enrichment strategy, we tested several magnetic kits and compared them based on enrichment efficiency and final LKS yield. Four approaches were tested: lineage depletion (Lin-), cKit-enrichment (cKit+), Sca1-enrichment (Sca1+) and a combination of lineage depletion followed by Sca1-enrichment (Lin- Sca1+). Our results show that the best strategy are lineage depletion and Sca1-enrichment. Combining these two methods highly increased the LKS enrichment, however, the final LKS yield was low due to increased cell loss from performing a second enrichment step.