Program: Oral and Poster Abstracts
Session: 501. Hematopoietic Stem and Progenitor Biology: Poster III
Hematology Disease Topics & Pathways:
Biological, HSCs, bone marrow, Therapies, Biological Processes, Cell Lineage, Clinically relevant, hematopoiesis, transplantation, stem cells
Session: 501. Hematopoietic Stem and Progenitor Biology: Poster III
Hematology Disease Topics & Pathways:
Biological, HSCs, bone marrow, Therapies, Biological Processes, Cell Lineage, Clinically relevant, hematopoiesis, transplantation, stem cells
Monday, December 7, 2020, 7:00 AM-3:30 PM
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.
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