Leptin Receptor, a Surface Marker for a Subset of Highly Engrafting Long-term Functional Hematopoietic Stem Cells

The hematopoietic system is sustained by a rare population of hematopoietic stem cells (HSCs), which emerge during early fetal development and then reside in the hypoxic niche of the adult bone marrow microenvironment. Although leptin receptor (Lepr)-expressing stromal cells are well-studied as critical regulators of murine hematopoiesis, the biological implications of Lepr expression on HSCs remain largely unexplored. We hypothesized that Lepr+HSCs are functionally different from other HSCs. Using in vitro and in vivo experimental approaches, we demonstrated that Lepr further differentiates SLAM HSCs into two distinct populations; Lepr+HSCs engrafted significantly higher than Lepr-HSCs and self-renewed more extensively as noted in secondary transplants. To determine how molecular pathways and gene expression profiles affect their functional differences, we performed bulk RNA-sequencing on purified Lepr+HSC/MPP and Lepr-HSC/MPP cells. Our results showed that Lepr+HSCs were characterized by a proinflammatory transcriptomic profile enriched for Type-I Interferon and Interferon-gamma (IFN-?) response pathways, which are known to be critical for the emergence of HSCs in fetal life. Gene set analyses indicated that Lepr+HSCs were more highly enriched for genes associated with long-term HSCs whereas Lepr-HSCs' expression profiles were more closely related to progenitors. We conclude that although Lepr+HSCs represent a minor subset of HSCs, they are highly engrafting cells that possess embryonic-like transcriptomic characteristics, and that Lepr can serve as a useful marker for functional long term HSCs, which may have potential clinical applicability. Overall design: BM cells were collected in ice-cold MACS buffer by crushing spines, pelvis, femurs and tibias from two mice for each replicate (n = 5 replicates). The cells were partially lineage depleted using the direct mouse lineage cell depletion kit (Miltenyi Biotec). Cells were stained for surface markers on ice for 20 min with the following antibodies: APC-mouse lineage cocktail (CD3e, CD11b, CD45R/B220, Ly-76, Ly-6G and Ly-6C; BD Biosciences), PE/Dazzle 594-anti-Ly6A/E (also known as Sca-1; clone D7; Biolegend), APC/Cyanine7-anti-CD117 (also known as c-kit; clone 2B8; Biolegend), BV421 Rat anti-mouse CD150 (clone Q38-480) , FITC-anti-CD48 (clone HM48-1; Biolegend), anti-Lepr-biotin antibody (R&D systems, BAF497, 0.2 ug per one million cells), washed in PBS, then incubated with secondary biotin-conjugated streptavidin-PE/Cy7 for another 20 minutes on ice (Biolegend, 1:500) and washed in PBS. Cells were sorted on the FACSAria or SORP Aria sorters for Lepr+ or Lepr- SLAM-HSC (LSKCD150+CD48-) and SLAM-MPP (LSKCD150-CD48-) directly into 350 uL 2-beta mercaptoethanol containing RLT Plus lysis buffer, and RNA was extract immediately after sorting using Qiagen RNeasy Plus Micro Kit. Non-stranded cDNA libraries were generated by the Indiana University School of Medicine Medical Genomics core and sequenced on a NovaSeq6000 flow cell for paired end sequencing. Data were analyzed for differential expression based on cell type and Lepr status.