Leptin Receptor As a Marker for a Subset of Long-Term Functional Hematopoietic Stem Cells
Authors: Trinh Thao , Ropa James , Aljoufi Arafat , Cooper Scott , Srour Edward F. , Sinn Anthony , Broxmeyer Hal E. 
Published: February 2021

The hematopoietic system is maintained by the hematopoetic stem and progenitor cells (HSCs/HPCs), a group of rare cells that reside in a hypoxic bone marrow (BM) microenvironment. Leptin (Lep) is well-known for its neuroendocrine and immunological functions, and its receptor (Lepr) has been studied extensively in the BM niche cells. Yet, its biological implications in HSC/HPC biology remained largely unknown. In this study, we hypothesized that Lepr-expressing HSCs/HPCs are functionally and transcriptomically distinct from their negative counterparts. To test our hypothesis, we utilized both in vitro and in vivo approaches. We first employed Fluorescence-activated cell sorting (FACS) analysis to confirm expression of Lepr on HSCs/HPCs in adult mouse BM. We then isolated equal numbers of Lepr+Lineage-Sca1+cKit+ (LSK cells - a heterogenous population of long-term, short-term HSCs and multipotent HPCs) and Lepr-LSK cells from C57BL/6 (CD45.2+) mouse BM to perform colony-forming unit (CFU) assay and competitive transplantation assay, which also included using competitor cells from BoyJ (CD45.1+) unseparated BM and lethally-irradiated F1 (CD45.1+CD45.2+) as hosts. To determine whether Lepr can further hierarchize HSCs into two distinct populations, we repeated the competitive transplants using freshly isolated C57BL/6 Lepr+HSCs or Lepr-HSCs cells instead. At the end of primary transplants, whole BM were analyzed for donor chimerisms in the peripheral blood (PB) and BM as well as transplanted in a non-competitive fashion into lethally-irradiated secondary recipients. To gain mechanistic insights, we assessed homing potential as homing plays a role in increased engraftment. We also performed bulk RNA-seq using freshly sorted BM Lepr+HSCs or Lepr-HSCs to elucidate potential molecular pathways that are responsible for the differences in their functional capacity. By phenotypic studies, our FACS analyses showed that Lepr+ cells represented a smaller population within the hematopoietic compartment in the BM. However, HSCs contained a higher percentage of Lepr+ cells than other HPC populations. By functional assessments, Lepr+LSK cells were more highly enriched for colony-forming progenitor cells in CFU assay as compared to Lepr-LSK cells. Interestingly, Lepr+LSK cells exhibited more robust engraftment capability in primary transplants and substantial self-renewal capacity in secondary transplants throughout different time points in both PB and BM. In addition, Lepr+HSCs showed significantly higher donor chimerisms in PB month 1, 2, 4 and BM month 4 with similar lineage output compared to Lepr-HSCs. Higher engraftment could be due to increased homing of HSCs to the BM; however, Lepr+HSCs and Lepr-HSCs showed similar homing capacity as well as levels of surface CXCR4 expression. Molecularly, Fast Preranked Gene Set Enrichment Analysis (FGSEA) showed that Lepr+HSCs were enriched for Type-I Interferon and Interferon-gamma response pathways with Normalized Enrichment Scores of 2 or higher. Lepr+HSC transcriptomic study also revealed that these cells as compared to Lepr-HSCs expressed significantly higher levels of genes involved in megakaryopoiesis and proinflammatory immune responses including the NF-κB subunits (Rel and Relb). Interestingly, both IFN-γ and NF-κB signalings have been demonstrated to be critical for the emergence of HSCs from the hemogentic endothelium during embryonic development. In summary, although Lepr+LSK cells occupied a minor fraction compared to their negative counterparts in the BM, they possessed higher colony-forming capacity and were more highly enriched for long-term functional HSCs. In line with this, Lepr+HSCs engrafted significantly higher and self-renewed more extensively than Lepr-HSCs, suggesting that Lepr not only can be used as a marker for functional HSCs but also further differentiate HSCs into two functionally distinguishable populations. Intriguingly, Lepr+HSCs were characterized with a proinflammatory transcriptomic profile that was previously suggested to be critical for the development of HSCs in the embryo. All together, our work demonstrated that Lepr+HSCs represent a subset of highly engrafting adult BM HSCs with an embryonic-like transcriptomic signature. This can have potential therapeutic implications in the field of hematopoietic transplantation as Lepr is highly conserved between mice and human.

Disclosures
No relevant conflicts of interest to declare.