Pluripotent stem cells (PSCs) are attractive regenerative therapy tools for skeletal tissues. However, a deep understanding of skeletal development is required in order to model this development with PSCs, and for the application of PSCs in clinical settings. Skeletal tissues originate from three types of cell populations: the paraxial mesoderm, lateral plate mesoderm, and neural crest. The paraxial mesoderm gives rise to the sclerotome mainly through somitogenesis. In this process, key developmental processes, including initiation of the segmentation clock, formation of the determination front, and the mesenchymal–epithelial transition, are sequentially coordinated. The sclerotome further forms vertebral columns and contributes to various other tissues, such as tendons, vessels (including the dorsal aorta), and even meninges. To understand the molecular mechanisms underlying these developmental processes, extensive studies have been conducted. These studies have demonstrated that a gradient of activities involving multiple signaling pathways specify the embryonic axis and induce cell-type-specific master transcription factors in a spatiotemporal manner. Moreover, applying the knowledge of mesoderm development, researchers have attempted to recapitulate the in vivo development processes in in vitro settings, using mouse and human PSCs. In this review, we summarize the state-of-the-art understanding of mesoderm development and in vitro modeling of mesoderm development using PSCs. We also discuss future perspectives on the use of PSCs to generate skeletal tissues for basic research and clinical applications.

Osteoarthritis (OA) shows various clinical manifestations depending on the status of its joint components. We aimed to identify the synovial cell subsets responsible for OA pathophysiology by comprehensive analyses of human synovium samples in single-cell resolution. Two distinct OA synovial tissue groups were classified by gene expression profiles in RNA-Seq: inflammatory and fibrotic. The inflammatory group exhibited high expression of inflammatory cytokines, histologically inflammatory infiltrate, and a more severe pain score. The fibrotic group showed higher expression of fibroblast growth factor (FGFs) and bone morphogenetic proteins (BMPs), showed histologically perivascular fibrosis, and showed a lower pain score. In single-cell RNA-Seq (scRNA-Seq) of synovial cells, MERTK ^lo CD206 ^lo macrophages and CD34 ^hi fibroblasts were associated with the inflammatory and fibrotic groups, respectively. Among the 3 fibroblast subsets, CD34 ^lo THY1 ^lo and CD34 ^lo THY1 ^hi fibroblasts were influenced by synovial immune cells, whereas CD34 ^hi fibroblasts were influenced by mural and endothelial cells. Particularly, in CD34 ^hi fibroblast subsets, CD34 ^hi CD70 ^hi fibroblasts promoted proliferation of Tregs, potentially suppressing synovitis and protecting articular cartilage. Elucidation of the mechanisms underlying the regulation of these synovial cell subsets may lead to novel strategies for OA therapeutics.

Although the skeleton is essential for locomotion, endocrine functions, and hematopoiesis, the molecular mechanisms of human skeletal development remain to be elucidated. Here, we introduce an integrative method to model human skeletal development by combining in vitro sclerotome induction from human pluripotent stem cells and in vivo endochondral bone formation by implanting the sclerotome beneath the renal capsules of immunodeficient mice. Histological and scRNA-seq analyses reveal that the induced bones recapitulate endochondral ossification and are composed of human skeletal cells and mouse circulatory cells. The skeletal cell types and their trajectories are similar to those of human embryos. Single-cell multiome analysis reveals dynamic changes in chromatin accessibility associated with multiple transcription factors constituting cell-type-specific gene-regulatory networks (GRNs). We further identify ZEB2, which may regulate the GRNs in human osteogenesis. Collectively, these results identify components of GRNs in human skeletal development and provide a valuable model for its investigation.

Clinically relevant human induced pluripotent stem cell (hiPSC) derivatives require efficient protocols to differentiate hiPSCs into specific lineages. Here we developed a fully defined xeno-free strategy to direct hiPSCs toward osteoblasts within 21 days. The strategy successfully achieved the osteogenic induction of four independently derived hiPSC lines by a sequential use of combinations of small-molecule inducers. The induction first generated mesodermal cells, which subsequently recapitulated the developmental expression pattern of major osteoblast genes and proteins. Importantly, Col2.3-Cherry hiPSCs subjected to this strategy strongly expressed the cherry fluorescence that has been observed in bone-forming osteoblasts in vivo. Moreover, the protocol combined with a three-dimensional (3D) scaffold was suitable for the generation of a xeno-free 3D osteogenic system. Thus, our strategy offers a platform with significant advantages for bone biology studies and it will also contribute to clinical applications of hiPSCs to skeletal regenerative medicine.

Skeletal disorders, such as osteoarthritis and bone fractures, are among the major conditions that can compromise the quality of daily life of elderly individuals. To treat them, regenerative therapies using skeletal cells have been an attractive choice for patients with unmet clinical needs. Currently, there are two major strategies to prepare the cell sources. The first is to use induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs), which can recapitulate the skeletal developmental process and differentiate into various skeletal cells. Skeletal tissues are derived from three distinct origins: the neural crest, paraxial mesoderm, and lateral plate mesoderm. Thus, various protocols have been proposed to recapitulate the sequential process of skeletal development. The second strategy is to extract stem cells from skeletal tissues. In addition to mesenchymal stem/stromal cells (MSCs), multiple cell types have been identified as alternative cell sources. These cells have distinct multipotent properties allowing them to differentiate into skeletal cells and various potential applications for skeletal regeneration. In this review, we summarize state-of-the-art research in stem cell differentiation based on the understanding of embryogenic skeletal development and stem cells existing in skeletal tissues. We then discuss the potential applications of these cell types for regenerative medicine.

Human skeletal elements are formed from distinct origins at distinct positions of the embryo. For example, the neural crest produces the facial bones, the paraxial mesoderm produces the axial skeleton, and the lateral plate mesoderm produces the appendicular skeleton. During skeletal development, different combinations of signaling pathways are coordinated from distinct origins during the sequential developmental stages. Models for human skeletal development have been established using human pluripotent stem cells (hPSCs) and by exploiting our understanding of skeletal development. Stepwise protocols for generating skeletal cells from different origins have been designed to mimic developmental trails. Recently, organoid methods have allowed the multicellular organization of skeletal cell types to recapitulate complicated skeletal development and metabolism. Similarly, several genetic diseases of the skeleton have been modeled using patient-derived induced pluripotent stem cells and genome-editing technologies. Model-based drug screening is a powerful tool for identifying drug candidates. This review briefly summarizes our current understanding of the embryonic development of skeletal tissues and introduces the current state-of-the-art hPSC methods for recapitulating skeletal development, metabolism, and diseases. We also discuss the current limitations and future perspectives for applications of the hPSC-based modeling system in precision medicine in this research field.