Advances in spatial transcriptomics and its application in the musculoskeletal system

Introduction

Since the advent of next-generation sequencing in 2005 (ref. ¹), transcriptomics research has made substantial advances. Bulk RNA sequencing (RNA-seq) provides information on gene expression, RNA structure, and protein translation, and is capable of identifying new genes and deciphering signal networks related to physiological states and pathological processes.^2,3 However, RNA-seq only detects the average gene expression levels of mixed cells, masking phenotypic details of individual cells and the differences in transcriptomes between cells, leading to the potential dilution and oversight of important transcripts in specific cell types.⁴ To overcome this limitation, single-cell RNA sequencing (scRNA-seq) was introduced by Tang et al.⁵ in 2009, achieving the quantification of transcriptomes within individual cells for the first time. It provides a powerful tool for studying cellular heterogeneity, characterizing new cell types and states, and elucidating regulatory networks between cell clusters.⁶ However, scRNA-seq requires the harvest of single live cells from tissues without inducing cell stress or death. During cell separation, the disruption of intercellular connections and changes in the external microenvironment can lead to alterations in the internal transcriptome.⁴ Additionally, the hardness of bone and cartilage tissues and the specific cellular morphology of muscle cells present challenges in preparing single-cell suspensions from these tissues.⁷

Despite the unique data provided by RNA-seq and scRNA-seq for exploring tissue cellular heterogeneity, transcriptomic analysis by either of these techniques results in loss of the spatial context of cells,⁸ which is closely related to biological function.⁹ For example, from the enthesis with higher calcification to the tendon mid-body, and then to the myotendinous junction, the spatial organization of cells within the tendon is crucial for their ability to bear and transmit tensile forces.¹⁰ Similarly, the transcriptional heterogeneity of subpopulations of skeletal stem and progenitor cells (SSPCs) may depend on their diverse spatial positioning and ecological niches within the bone marrow. Even subtle changes in localization or intercellular crosstalk of SSPCs in the bone marrow microenvironment can profoundly impact their functional state or cell fate.¹¹ Furthermore, although researchers can theoretically infer potential mechanisms based on receptor–ligand interactions from RNA-seq and scRNA-seq data, the biological feasibility of such mechanisms still requires further validation, specifically whether the interacting cells are in close spatial...