3127 - Retention of Single Long Term Hematopoietic Stem Cells in Minimal Culture Medium Reveals Essential Components of HSC Function

Abstract

Recent descriptions of single cell gene expression datasets from highly purified long-term hematopoietic stem cells (LT-HSCs) have significantly advanced our understanding of the molecular state of HSCs. Emerging concomitantly, however, is the knowledge that LT-HSCs share a significant amount of their transcriptome with cells that do not possess the functional properties of durable self-renewal and multi-lineage cell output. To discern which genes and pathways drive individual HSC properties and which are bystanders is a major outstanding challenge for the field. We have recently demonstrated that single HSCs (EPCR++CD150+CD48-CD45+Sca-1++, >50% LT-HSCs by single cell transplantation) can be cultured without undergoing division for a period of 710 days with minimal cytokine stimulation (21% single cell survival, 99.2% undivided). Limiting dilution assays of HSCs cultured for 7 days (7dHSCs) estimated the durable multi-lineage HSC frequency at 22%. Single cell transplantation experiments had a similar frequency with both primary (3/10) and secondary (3/3) transplantations demonstrating that single 7dHSCs retained HSC function. Since 7dHSCs share the functional properties of freshly isolated HSCs, they represent a robust comparator population to identify which genes are indispensable for the maintenance of HSC self-renewal and quiescence. We generated RNA-sequencing datasets from freshly isolated HSCs and 7dHSCs and identified 960 genes and a number of biological processes that were significantly down-regulated in 7dHSCs, suggesting that these are dispensable for HSC function. Major down-regulated processes included the response to oxidative stress, ligand-independent apoptosis, and cellular senescence. Interestingly, in all 3 mice successfully transplanted with single 7dHSCs, a lymphoid-deficient (or -HSC) program was observed, suggesting that -HSCs are more resilient to the stress induced by cytokine depletion and represent a potential mechanism of obtaining the molecular program of -HSC. Together, these data highlight the importance of studying HSCs in different contexts to identify common molecular features of their functional properties.