To the bones: mapping the skeletal LEPR⁺ pool to component cell types

Abstract

Leptin receptor‐positive skeletal progenitors constitute an essential cell population in the bone, yet their heterogeneity remains incompletely understood. In this issue, Mo et al (2021) report a single‐cell RNA sequencing resource that deconvolutes the pool of LEPR⁺ skeletal cells under homeostatic and various pathologic conditions, uncovering context‐dependent contributions to diverse cell types and functions.

A recent single‐cell sequencing resource uncovers diverse contributions of leptin receptor‐positive bone progenitors to skeletal cell populations.

A major area of progress in bone biology during the recent years has been a shift from considering skeletal cells as primarily defined by morphology or the type of matrix they produce toward resolving specific cell identities based on cell surface or genetic lineage markers. Leptin receptor (LEPR)‐expressing mouse marrow stromal cells are a leading example of this transition. LEPR cells are long‐lived quiescent stromal cells with a progressive contribution to the pool of bone‐forming osteoblasts with age (Zhou et al, 2014). These cells show a high degree of overlap with CXCL12‐abundant reticular cells (CAR cells), another cell definition emerging from studies of marrow cells involved in support of hematopoietic stem cells (Omatsu et al, 2010; Seike et al, 2018; Matsushita et al, 2020). LEPR cells are not present at birth, but instead arise during early adolescence from a population of embryonic skeletal stem cells (Shu et al, 2021). However, many studies on LEPR cells critically rely on cre‐mediated cellular tracing, which specifies lineages of cells and not individual cell types. Moreover, it is largely unclear whether cre‐labeled cells truly constitute a single lineage or multiple lineages that are co‐labeled. Indeed, recent work has identified subsets of peri‐sinusoidal LEPR/CAR cells seemingly poised for adipogenesis, whereas a distinct population of peri‐arteriolar LEPR/CAR cells expressing osteolectin appears to be prone to osteogenesis (Baccin et al, 2020; Shen et al, 2021). Likewise, small numbers of LEPR⁺ cells have been identified in the periosteum (Gao et al, 2019). Thus, LEPR‐cre is likely marking several sets of cells representing distinct lineages with independent functions.

The current work by Mo et al (2021) generates insight into this heterogeneity by applying single‐cell RNA sequencing to LEPR‐cre‐labeled cells to deconvolute this pool into transcriptionally defined cell types. For comparison, similar studies were run on Prrx1‐cre‐labeled cells, which include essentially all skeletal cells in the limb. Additionally, steady‐state conditions in young mice were compared to aging, irradiation, or fracture conditions to determine their respective impact on LEPR lineage cells (Fig 1). Prrx1 lineage cells were shown to divide into nine clusters, including a broad range of mature skeletal cell types. Of these, cells with transcriptional characteristics of bone marrow stromal cells and osteoblast lineage cells were labeled by LEPR‐cre, whereas periosteal cells, chondrocytes, and a discrete set of α‐smooth muscle actin (Acta2)‐expressing cells (Grcevic et al, 2012) were only sparsely represented. Concomitant detection of LEPR lineage‐positive and ‐negative cells in many cell types indicates a model where bone maintains multiple discrete progenitors with both distinct and overlapping functional specializations. Similar to prior reports, different subsets of LEPR/CAR lineage cells express predominantly osteogenic versus adipogenic markers, suggesting that these subsets are skewed toward osteogenesis or adipogenesis (Baccin et al, 2020). Notably, a particular subset of cells expressing adipocyte lineage markers, including adiponectin and lipoprotein lipase, but lacking the mature adipocyte marker Perilipin 1 resembles a recently reported adipogenic progenitor type expressing adiponectin, marrow adipogenic lineage precursors (MALPs; Zhong et al, 2020). If substantiated through transplantation studies, the current findings by Mo et al (2021) would place at least a subset of these MALPs as adipogenic precursors in the LEPR lineage. The finding that LEPR cells are sparse in the periosteal cluster is consistent with a report identifying a LEPR‐negative, Cathepsin K‐lineage periosteal stem cell (Debnath et al, 2018). Small numbers of periosteal LEPR lineage cells were, however, present, especially in imaging studies, and could be expanded by irradiation or the adipogenic stimulus rosiglitazone. This preferential response of LEPR cells to these two stimuli has been consistently observed and suggests a model whereby distinct skeletal progenitors each have distinct functional specializations (Zhou et al, 2014; Matsushita et al, 2020). In such a model, stimulus or pathology‐induced expansion may lead even small populations to have an outsized functional contribution.

Figure 1. The heterogeneity of LEPR lineage cells in bone marrow and periosteum.

Single‐cell RNA sequencing of LEPR lineage cells reveals distinct cellular populations. Putatively osteogenic lineage cells (LEPR lineage OLCs) and adipogenic lineage cells (LEPR lineage ALCs) are identified based on their expression profile. A distinct slow‐cycling LEPR lineage Notch3⁺ cell population is adjacent to sinusoids and arterioles. In periosteum, LEPR‐cre labels a small subset of periosteal cells (PCs) which can be further separated into LEPR lineage Sca1⁺ PC and LEPR lineage Sca1‐ PC. The differentiation relationships in dotted lines and assignment of osteolectin positivity are speculative and inferred in combination with complimentary data from other recent reports (Baccin et al, 2020; Shen et al, 2021).

Given the heterogeneity in LEPR lineage cells, identifying markers that can subset LEPR cells is of great interest. In this respect, the Notch3‐positive subset of LEPR lineage cells identified by Mo et al (2021) seems notable. Notch3‐expressing LEPR lineage cells express lower levels of osteogenic genes and display a preferential peri‐vascular localization around both sinusoids and arterioles that contrast with the distribution of the Notch3‐negative fraction as distributed throughout the marrow stroma in a reticular pattern. Comparison to recent work discriminating peri‐arteriolar Osteolectin⁺LEPR⁺ cells that are primed for osteogenesis from peri‐sinusoidal Osteolectin‐LEPR⁺ cells that are primed for adipogenesis implies that Notch3 may encompass both of these subsets of LEPR⁺ cells (Shen et al, 2021). Ultimately, use of Notch3 or other similar markers for prospective isolation by FACS of specific LEPR cell subsets for transplantation studies of differentiation and bone formation will represent a key next step for functional annotation of these LEPR lineage cell types.

This work helps to frame the next set of questions regarding LEPR cells: What is the lineage relationship among the several sets of cells marked by LEPR‐cre? Is one cell the source of the others? Or is each population derived from separate progenitors that separately induce LEPR expression? Another interesting feature of LEPR cells is that they only collectively emerge during adolescence, around approximately 5 weeks of age in mice and that the lifespan and proliferative potential appears to differ among LEPR⁺ cell subsets (Shen et al, 2021). Thus, applying approaches similar to those utilized here across both very early stages of LEPR⁺ cell emergence and in aged mice may reveal temporal distinctions in the emergence or later persistence of different LEPR cell types and thereby generate insights into the physiologic specialization of these subsets.

This work also has important limitations, many of which are intrinsic to the current state of single‐cell RNA sequencing, which cannot be directly coupled with prospective isolation of the clusters of interest. Thus, many of the functional properties of the LEPR lineage populations can only be inferred based on gene expression profiles. Ultimately, FACS‐based isolation of specific LEPR⁺ cell types using a combination of a lineage reporter with additional cell surface markers that resolve the specific LEPR cell subsets will be needed. More broadly, there are a number of identified skeletal cell types that are not evident in the current clustering analysis, including non‐stem progenitors in the CTSK lineage. This may in part reflect limitations in the total number of cells sampled for scRNA‐seq studies, but may also reflect that the sparse transcriptional sampling of each cell is insufficient to capture all biologically important cell type distinctions. Thus, these data may still underestimate the number of distinct cell types that comprise the LEPR lineage pool.

In summary, this report by Mo et al (2021) applies single‐cell sequencing to catalogue both LEPR lineage and non‐LEPR lineage skeletal across both normal and a range of pathologic conditions. They reveal a striking degree of heterogeneity in the cell types comprising the pool of LEPR lineage cells and generate data pointing toward a corresponding functional heterogeneity for these cell types. This frames deconvolution of the pool of LEPR lineage cells into its component cell types as a key issue for skeletal cellular biology.

The EMBO Journal (2022) 41: e110343.