Single-Cell RNA-Sequencing Reveals the Skeletal Cellular Dynamics

Version	Summary	Created by	Modification	Content Size	Created at	Operation
1		Yuki Matsushita	--	2540	2023-06-21 10:55:55	\|
2	references update	Fanny Huang	Meta information modification	2540	2023-06-26 08:21:31	\|

This entry is adapted from the peer-reviewed paper 10.3390/ijms24129814

The bone is an important organ that performs various functions, and the bone marrow inside the skeleton is composed of a complex intermix of hematopoietic, vascular, and skeletal cells. Current single-cell RNA sequencing (scRNA-seq) technology has revealed heterogeneity and sketchy differential hierarchy of skeletal cells.

bone marrow stromal cells (BMSCs) lineage-tracing single-cell RNA-sequencing (scRNA-seq) skeletal stem and progenitor cells (SSPCs)

1. Introduction

Progressive aging has a substantial negative impact on the skeletal system. Diseases of the skeletal system associated with aging, particularly those caused by the loss of bone homeostasis and imbalances in bone metabolism, such as osteoporosis and related bone fractures, significantly shorten a healthy life span ^[1]^[2]^[3]. Cellular senescence is a key contributor to the imbalance in bone homeostasis ^[4]. Senescent cells accumulate in tissues with age, leading to an overall decline in tissue regeneration potential, which is causally linked to degenerative diseases ^[5]^[6]. Elimination of senescent cells prevents age-related bone loss in mice ^[7]. Therefore, dynamic changes in skeletal cells are strongly implicated in skeletal aging.

To address these bone-related negative events, a comprehensive understanding of skeletal cell biology is essential. Skeletal cells are composed of multiple cell types, including skeletal stem cells (SSCs), skeletal progenitor cells, differentiated chondrocytes, osteoblasts, and marrow adipocytes. SSCs are bone tissue-specific stem cells and are located at the pinnacle of the hierarchy of skeletogenesis. Over 50 years have passed since bone marrow SSCs were first established ^[8]. SSCs have been defined as mesenchymal cells, which have the potential of self-renewal and tri-lineage differentiation into chondrocytes, osteoblasts, and adipocytes ^[9]^[10]^[11]. They have been experimentally proven using ex vivo cell culture and/or subsequent in vivo ectopic transplantation. Although ex vivo primary cell culture experiments have been the gold standard for proving the property of SSCs, it is controversial whether these cell culture results alone can define cultured cells as stem cells. The target cells do not always behave as they would in the native biological environment since the culture conditions are quite different from those in the body. In addition, tri-lineage differentiation potential into chondrocytes, osteoblasts, and adipocytes, which is one of the criteria of SSCs, is evaluated under artificial conditions using distinct differentiation induction media. Hence, researchers should carefully define SSCs and their lineage cells by using several ways well-established in the research field of stem cell biology.

It has long been proposed that bone marrow contains multiple types of SSCs, their progenitor cells (SSPCs: skeletal stem and progenitor cells), and their lineage cells; only 0.001% to 0.01% of bone marrow cells contain stem cell populations ^[12]. Among the skeletal cells in bone marrow, bone marrow stromal cells (BMSCs) have been defined as mesenchymal populations, which are located between the outer surfaces of marrow blood vessels and bone surfaces ^[13], suggesting that multiple unclarified skeletal cell types are included in BMSCs. It has been reported that BMSCs are differentiated from SSPCs ^[14]^[15]^[16], while a subpopulation of BMSCs provides downstream skeletal cells as SSPCs ^[17]^[18]. These seemingly opposite roles of BMSCs are complicated. This is because BMSCs themselves have not been completely understood, including their spatial location. Consequently, distinct tissue-specific BMSCs need to be well-defined through basic research. Currently, BMSCs have been elucidated to be heterogeneous populations by using new technology ^[19], and will take us into a new era in skeletal stem cell biology.

Technological breakthrough has provided new insights into skeletal biology. Cell surface marker-based fluorescence-activated cell sorting (FACS) analysis and in vivo lineage-tracing studies are integral methods to investigate cell populations. These approaches enable us to define cellular populations and trace their progeny ^[20]. To adequately characterize a heterogeneous skeletal cell population, single-cell RNA-sequencing (scRNA-seq) analysis is a key technique to complement the previous approaches. scRNA-seq can reveal the molecular signature of individual cells, identify novel cell types, and provide insights into the cellular dynamics in tissues ^[21]. Advances in scRNA-seq technology in recent years have validated its reliability in identifying cell types and analyzing gene expression patterns ^[22].

2. Spatiotemporal-Specific Skeletal Stem Cells

SSCs play important roles in the development, maintenance, and regeneration of bone tissues. SSCs have the potential of self-renewal and differentiation into osteoblasts, chondrocytes, adipocytes, and stromal cells, which are necessary for bone growth and repair ^[9]^[10]^[11]. The concept of SSCs originated from studies in which autologous fragments of bone marrow or its cell suspensions were found to generate skeletal tissue after heterotopic transplantation ^[8]^[23]. Currently, SSCs in mouse long bones are identified by two distinct approaches: FACS-based isolation using appropriate cell surface markers, and an in vivo lineage-tracing approach. These approaches have the benefit of excluding hematopoietic and endothelial cells and can select target cells. In the former, mouse SSCs (mSSCs) are defined as those having several markers, including CD51⁺CD90⁻CD105⁻CD200⁺ ^[24], PDGFRα⁺Sca1⁺CD45⁻TER119⁻ ^[25], and CD73⁺CD31⁻ ^[26]. The latter, in vivo lineage-tracing approach using mouse genetic models, has revealed several types of SSCs at distinct locations in long bones, such as parathyroid hormone-related protein (PTHrP)⁺ growth plate stem cells residing within the resting zone of the growth plate in the early postnatal stage ^[27], cathepsin K⁺ periosteal stem cells in the early developmental stage ^[28], CXCL12⁺LepR⁺ reticular stromal cells in adult to aged bone marrows ^[17]^[18], and fibroblast growth factor receptor 3 (FGFR3)⁺ endosteal stromal cells in young bone marrows ^[29]. Notably, these populations targeted by distinct cre or creER lines are supposed to include the abovementioned SSC population. These cre driver lines not only mark SSCs but also other cell types. These lineage-tracing-based SSC identification processes are often performed together with the FACS-based cell surface marker isolation approach. This combined approach can identify bona fide SSCs along with their different spatial allocations and cellular dynamics ^[27]^[28]^[29]. These small populations of highly clonogenic SSCs present in each bone compartment play important roles in bone maintenance and regeneration.

3. Heterogeneity of BMSCs by scRNA-Seq

BMSCs are a versatile mesenchymal cell population supporting key skeletal functions. A part of the mesenchymal cells, residing in the perivascular regions surrounding sinusoids or distal arteries, are believed to contain SSPC populations ^[18]^[30]^[31]. In fact, human skeletal progenitor cells in the bone marrow have been reported to be located in the perisinusoidal area ^[9]. The majority of BMSCs are reticular in shape and adjacent to sinusoidal vessels. These reticular cells express C-X-C motif chemokine ligand 12 (CXCL12, also known as stromal cell-derived factor 1, SDF1) ^[32]^[33]^[34], leptin receptor (LepR) ^[17]^[34], stem cell factor (SCF, also known as KIT ligand) ^[35], and early B-cell factor 3 (Ebf3) ^[18]. Among all skeletal cells, these reticular stromal cells are one of the major populations in the bone marrow and are considered to possess various properties. Reticular stromal cells represent 0.3% of all bone marrow cells, including hematopoietic, vascular, and skeletal cells. CXCL12- and LepR-expressing reticular stromal cells predominantly occupy the marrow cavity among all skeletal cells ^[17]^[34]. These CXCL12⁺LepR⁺ reticular cells have been defined as total BMSCs. LepR⁺ cells in adult bone marrow almost entirely overlap (approximately 90%) with CXCL12⁺ cells ^[34]^[36]. They maintain a hematopoietic microenvironment by expressing cytokines and are in physical contact with each other; in addition, they are critical sources of hematopoietic stem cell niche factors ^[37]^[38]^[39]. These cells are required for the proliferation of hematopoietic stem cells, and lymphoid and erythroid progenitors ^[37]. In addition, they are a major source of bone cells and adipocytes in adult bone marrow and functionally regulate osteogenesis and adipogenesis ^[38]^[40]^[41]^[42]^[43]. In recent years, it has become clear that BMSCs are heterogeneous, thereby making it difficult to accurately describe them by the umbrella term, BMSCs.

scRNA-seq is a progressive technology used to characterize heterogeneous cell populations, allowing rapid mapping of the BMSC landscape. scRNA-seq studies have identified several types of BMSCs. Baryawno et al. identified mesenchymal stromal cells (showing high expression of LepR), stroma-descendent osteolineage cells, bone marrow-derived endothelial cells, and other stromal cell types ^[44]. Tikhonova et al. performed scRNA-seq analyses of the bone marrow microenvironment at a steady state and identified two vascular endothelial-cadherin (VE-Cad)⁺ endothelial cells, four LepR⁺ perivascular, and three 2.3 kb type 1 collagen (COL2.3)⁺ osteolineage clusters, and a subpopulation of proliferative cells ^[19]. Analyses of the formation of fibroblastic colony-forming units (CFU-F) and the trajectory of differentiation revealed that the LepR⁺ compartment possesses multilineage differentiation potential and SSC activity.

To accurately map BMSCs, Zhong et al. performed scRNA-seq analyses using Col2a1-cre mouse long bones at 1, 3, and 16 months. They found subpopulations of BMSCs, including clusters of early, intermediate, and late mesenchymal progenitors and lineage-committed progenitors ^[40]. Interestingly, the pseudotime trajectory represented early mesenchymal progenitors expressing SSC marker genes at one end, whereas osteoblasts and adipocytes are at the opposite and distinct ends, thereby implying that a subset of BMSCs has the potential to bi-differentiate into osteogenic and adipogenic lineages in physiological conditions. In addition, LepR⁺ cells in the adult bone marrow consist of mesenchymal progenitors and a unique cell type that expresses adipocyte markers, including Adipoq but does not contain lipid droplets. The adipogenic cell type is termed the bone marrow adipogenic lineage precursor (MALP). Wolock et al. performed scRNA-seq to analyze non-hematopoietic (CD45⁻/Ter119⁻) and non-epithelial/non-endothelial cells (CD31⁻) and identified that CXCL12-abundant reticular (CAR) cells are highly enriched in multipotent stromal cells, adipocyte-progenitor cells, and osteoblast progenitors ^[45]. scRNA-seq analysis of CXCL12⁺ BMSCs using young Cxcl12-GFP mice identified two major populations, including preadipocyte-like reticular cells: Adipo-CAR cells and preosteoblast-like cells named Osteo-CAR cells ^[34]. Interestingly, these two populations have been predicted to be quite distinct ^[38]. Lepr-cre labels most BMSCs and osteogenic lineage cells in adult long bones, and these BMSCs include adipogenic populations, such as Adipo-CAR cells or MALPs as per scRNA-seq analysis of young adult and aging Lepr-cre mice ^[46].

Paired-related homeobox protein 1 (Prrx1) is a transcription factor that is prominently expressed in the mesenchyme during the crucial stages of craniofacial and limb development ^[47]. Thus, Prrx1-cre labels all limb skeletal cells in the appendicular skeleton ^[48]. Currently, scRNA-seq for Prrx1-cre lines at young and aged stages revealed the transitional stromal populations between typical skeletal cell types. Through the process of single-cell data downscaling and visualization analysis by using UMAP plots, osteoblast–chondrocyte transitional (OCT) cells and osteoblast–reticular transitional (ORT) cells, which have intermediate properties of typical gene expressions of both, are identified as a type of BMSCs. Among them, Fgfr3 is highly expressed in the OCT cluster. ORT cells marked by growth arrest-specific 1 (Gas1), regarded as a cell cycle inhibitor ^[49], are located between osteoblast and Cxcl12 highly expressed reticular populations. Bioinformatics investigation using RNA velocity analysis revealed that the OCT population provides osteoblasts, ORT cells, and pre-adipocyte-like reticular stromal cells at the young stage. In contrast, these once differentiated reticular stromal cells behave as the origin of skeletal cells at an aged stage ^[29], suggesting that cellular origins’ shift of BMSCs may occur with the advance of age. This idea could be the key to uncovering the biology of BMSCs.

Taken together, these scRNA-seq approaches elucidate the heterogeneity of BMSCs. They are grouped into several specific types, such as osteogenic and chondrogenic bi-potential mesenchymal progenitors, osteogenic and adipogenic progenitors, and osteo- or adipose-lineage-determined progenitors. Importantly, spatiotemporal multi-potential SSPCs are included in a subset of BMSCs. However, it is worth noting that scRNA-seq analysis can only provide an approximate representation of the diverse cell populations within the BMSCs. It cannot strictly show the interactions with peripheral vascular and hematopoietic cells. In addition, currently published scRNA-seq analyses have lost spatial information. Therefore, it is imperative to develop a strategy for high-dimensional integrated analyses and spatial transcriptomic analyses, which approach will enable a more comprehensive understanding of the complex cellular interactions and dynamics within the bone marrow stromal microenvironment. Furthermore, researchers strongly believe that enhancing the accuracy of single-cell sequencing analysis can be achieved by effectively integrating the biological information acquired from in vivo cell profiling with single-cell sequencing analysis. This synergy holds great potential for improving researchers understanding of the complex cellular landscape and enhancing the reliability of single-cell sequencing data.

4. Cellular Dynamics of BMSCs

Bone is a dynamic and non-stop tissue that undergoes constant remodeling throughout our lives ^[50]. Multiple types of cells play a crucial role in this process by regulating bone remodeling similar to osteocytes ^[51]. Consistent with these scRNA-seq results, biological data using an in vivo lineage-tracing study with tamoxifen-inducible creER or constitutively active cre lines show the heterogeneity and the dynamics of BMSCs. Multiple studies address the dynamics of a major BMSCs population, which expresses CXCL12 and LepR ^[17]^[44]. Lepr-cre-marked reticular stromal cells are the major source of osteoblasts and adipocytes in adult bone marrow. These cells are in the bone marrow and are hardly detected in bones at 2 months of age. However, Lepr-cre⁺ osteoblasts gradually increase from 6 to 14 months of age ^[17]. A lineage-tracing study using a tamoxifen-inducible Lepr-creER line revealed that Lepr-creER⁺ marrow stromal cells at the perinatal stage decrease and do not differentiate into osteoblasts with the advance of age. In contrast, adult Lepr-creER⁺ cells become the main source of osteoblasts ^[43]. A transcription factor Ebf3 is expressed in CXCL12⁺LepR⁺ BMSCs and Ebf3⁺ cells marked by Ebf3-creER behaving as SSPCs in adult bone marrow ^[18]. Hence, CXCL12⁺LepR⁺ reticular stromal cells play a role as SSPCs in the adult stage. The Cxcl12-creER line has revealed the unique cell fate of an adipogenic subset of CXCL12⁺LepR⁺ reticular stromal cells called Adipo-CAR cells. Cxcl12-creER precisely marks a relatively quiescent subset of CXCL12⁺LepR⁺ cells in the central marrow space after a tamoxifen injection ^[34]. Adipo-CAR cells spontaneously differentiate into Perillipin⁺ marrow adipocytes. Other adipogenic reticular stromal cells, called MALPs, also contribute to marrow adipocytes. Short-term ablation of CXCL12⁺LepR⁺ cells in vivo using diphtheria toxin significantly reduces the number of hematopoietic stem and progenitor cells ^[37]. BMSC ablation using Adipoq-cre; DTR mouse with diphtheria toxin injection showed de novo trabecular formation ^[40]. Interestingly, LepR deletion in BMSCs using Prrx1-cre increases osteogenesis and decreases adipogenesis ^[41], whereas CXCL12 deletion using Prrx1-cre and Osx-cre decreases osteogenesis and increases adipogenesis ^[42]. A study of Adipoq-creER mice revealed that Perillipin⁻ MALPs differentiate into Perillipin⁺ adipocytes in the marrow space ^[40]. These Cxcl12-creER⁺ cells are dormant and remain in primitive regions of the bone marrow space, suggesting that adipogenic Cxcl12-creER⁺ cells may be derived from other mesenchymal precursor cells, such as type II collagen⁺ cells adjacent to the growth plate or PTHrP⁺ resting chondrocytes ^[27]^[52] as scRNA-seq data mentions the existence of their upstream cells ^[40]. In fact, CXCL12⁺LepR⁺ BMSCs have been proposed to be differentiated cells, which are located downstream of SSPCs during development ^[14]^[15]^[27]^[52]^[53]^[54]. Several studies have elucidated the origins of these reticular cells. Early postnatal mesenchymal cells in the metaphyseal area marked by Gli1-creER, Pdgfrb-creER, and Lepr-creER contribute to reticular cells ^[15]^[55]. Early postnatal Osx⁺ cells also transform into whole reticular cells ^[14]. Moreover, in the embryonic developmental stage, chondrogenic cells inside the cartilage template marked by Fgfr3-creER become postnatal metaphyseal reticular cells ^[16]. In contrast, the outer layer of perichondrial cells marked by Dlx5-creER surrounding the cartilage template contributes to diaphyseal reticular cells, although the Osx⁺ inner layer of perichondrial cells contribute to skeletal cells transiently ^[16]. Recently, endosteal stromal populations were spatiotemporally identified. Juvenile Fgfr3-creER⁺ cells located in the endosteal surface robustly contribute to CXCL12⁺LepR⁺ reticular stromal cells and osteoblasts.

In summary, BMSC heterogeneity has been elucidated. The central parts of BMSCs are marked by CXCL12 and LepR. They include multipotent progenitors, pre-osteogenic BMSCs (Osteo-CAR cells), and pre-adipogenic BMSCs (Adipo-CAR cells and MALPs). These BMSCs behave as SSPCs in adult and aged stages. In contrast, Fgfr3⁺ endosteal BMSCs contribute to skeletogenesis mainly at the young stage.

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Subjects: Cell Biology

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Sixun Wu

Shinsuke Ohba

Yuki Matsushita

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Update Date: 26 Jun 2023

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