Leptin Receptor As a Functional Marker for Long-Term Repopulating Hematopoietic Stem Cells

Hematopoietic cell transplantation is an invaluable life-saving regimen for patients affected by malignant and non-malignant hematological disorders. However, successful clinical outcomes depend on the abilities of hematopoietic stem (HSCs) and progenitor cells (HPCs) to home to the bone marrow (BM) and then reconstitute a healthy new blood system. Leptin (Lep), a metabolic hormone well-characterized for its regulations of appetite and body weight by acting on the hypothalamus neurons, has a WSXWS motif of the type I cytokine receptor family and has reported hematopoietic effects (Cioffi et al., Nat Med 1996, Bennett et al., Curr Biol 1996, Umemoto et al., Blood 1997, Gainsford et al. Proc Natl Acad Sci USA 1996, Claycombe et al., Proc Natl Acad Sci USA 2008). These studies were however mostly limited to in vitro assays. Recent work demonstrated that Lep receptor(r)⁺ stromal cells were indispensable for maintenance of HSC/HPC (Comazzetto et al., Cell Stem Cell 2019, Himburg et al., Cell Stem Cell 2018, Zhou et al., Nat Cell Biol 2017). Yet, whether Lepr expression on HSC/HPC has effects on their in vivo functions remain largely unknown. We hypothesized that environmental factors that affect metabolism of HSCs and HPCs, such as those modulated by Lep/Lepr interactions, may be involved in HSC/HPC regulation and the engraftment of these cells.

Using flow cytometry analysis, we first assessed expression levels of Lepr on HSCs and HPCs. While only a low percentage of mouse BM HSC/HPC expressed Lepr, both the percentages of Lepr⁺HSCs (28.5% Lepr⁺LT-HSC and 17.2% Lepr⁺ST-HSC) and mean fluorescence intensity (MFI) of surface Lepr on these cells are significantly higher than that of Lepr⁺HPCs such as CMP, GMP and CLP (3.8%, 1.5%, 0.7% Lepr⁺ respectively). Despite the fact that HPCs express a lower level of Lepr, intact Lep/Lepr signaling was critical for their functions. This was illustrated by in vitro colony assay of cells taken from Lepr knockout (^-/^-) mouse BM in which significantly fewer absolute numbers per femur of HPC-derived colonies (CFU-GM, CFU-GEMM, BFU-E) formed compared to WT controls, and these progenitors were in a slow or non-cycling state. To evaluate how Lepr expression affects in vivo HSC/HPC functions, equal numbers of BM C57BL/6 (WT; CD45.2⁺) Lepr ^- Lineage^-Sca1⁺cKit⁺ (LSK) vs. Lepr⁺LSK cells were sorted and each transplanted with competitive BoyJ (CD45.1⁺) cells into lethally irradiated CD45.2⁺/CD45.1⁺ F1 recipients. A consistently higher engraftment capacity of Lepr⁺LSK cells was manifested in comparison to Lepr ^- LSK cells as noted in peripheral blood (PB) at months 1-6 chimerism post-transplant (91% vs 1.1% at month 6). Lepr⁺HSCs and Lepr⁺MPPs expressed similar levels of surface CXCR4 in comparison to corresponding Lepr ^- populations, suggesting that homing differences may not explain increased engraftment of Lepr⁺ LSK. At month 6, Lepr⁺LSK, but not Lepr ^- cells, demonstrated a significant myeloid-biased engraftment (0.24 vs 0.03 respectively for myeloid/lymphoid ratios). This is consistent with the phenotypic finding that compared to Lepr ^-LSK cells, Lepr⁺LSK cells contained a significantly lowered percentage of MPP4 progenitor cells (3.6% vs 36%), which have been demonstrated as a lymphoid-biased subset of MPPs (Pietras et al., Cell Stem Cell 2015). In addition, Lepr⁺LSK cells contained three-fold fewer progenitors as determined by in vitro colony assays. These findings demonstrated that Lepr⁺LSK cells were enriched for long-term hematopoietic repopulating HSCs, while its counterpart Lepr ^-LSK cells contained mostly HPCs. The data also suggested that absence of Lepr expression may play a role in fate-decision skewing HSCs towards MPP4 production.

For beginning efforts at mechanistic insight, we hypothesized that Lepr⁺ HSCs and Lepr⁺MPP may be different than Lepr ^- cells in mitochondrial activity. Compared to Lepr ^- cells, Lepr⁺HSC and Lepr⁺MPP cells interestingly possessed more robust mitochondrial metabolism, as demonstrated by their mitochondria having significantly higher membrane potential (measured by JC-1 assay).

In summary, Lep/Lepr signaling appears to be a functional ligand-receptor axis for maintaining HSC/HPC homeostasis and differentiation cell bias. Moreover, Lepr expression may serve as a functional marker for long-term repopulating HSCs, which has potential translational possibilities, as Lepr is highly conserved between mice and humans.