Single-cell RNA sequencing unravels heterogeneity of skeletal progenitors and cell–cell interactions underlying the bone repair process

Nakayama, Mika; Okada, H.; Seki, Masahide; Suzuki, Yutaka; Chung, Ung‐il; Ohba, Shinsuke; Hojo, Hironori

doi:10.1016/j.reth.2022.05.001

Cited by 9 publications

(8 citation statements)

References 53 publications

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“…To investigate the differentiation process from hiPSCs to osteocytes using the current induction method, scRNA sequencing was performed [43][44][45][46] . One common finding from the scRNA sequencing of primary bone tissues is the presence of stem cell populations.…”

Section: Discussionmentioning

confidence: 99%

3D osteogenic differentiation of human iPSCs reveals the role of TGFβ signal in the transition from progenitors to osteoblasts and osteoblasts to osteocytes

Kawai

Sunaga

Nagata

et al. 2023

Sci Rep

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Although the formation of bone-like nodules is regarded as the differentiation process from stem cells to osteogenic cells, including osteoblasts and osteocytes, the precise biological events during nodule formation are unknown. Here we performed the osteogenic induction of human induced pluripotent stem cells using a three-dimensional (3D) culture system using type I collagen gel and a rapid induction method with retinoic acid. Confocal and time-lapse imaging revealed the osteogenic differentiation was initiated with vigorous focal proliferation followed by aggregation, from which cells invaded the gel. Invading cells changed their morphology and expressed osteocyte marker genes, suggesting the transition from osteoblasts to osteocytes. Single-cell RNA sequencing analysis revealed that 3D culture-induced cells with features of periosteal skeletal stem cells, some of which expressed TGFβ-regulated osteoblast-related molecules. The role of TGFβ signal was further analyzed in the transition from osteoblasts to osteocytes, which revealed that modulation of the TGFβ signal changed the morphology and motility of cells isolated from the 3D culture, suggesting that the TGFβ signal maintains the osteoblastic phenotype and the transition into osteocytes requires down-regulation of the TGFβ signal.

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Section: Discussionmentioning

confidence: 99%

3D osteogenic differentiation of human iPSCs reveals the role of TGFβ signal in the transition from progenitors to osteoblasts and osteoblasts to osteocytes

Kawai

Sunaga

Nagata

et al. 2023

Sci Rep

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“…In addition, the well‐established reference marker genes (Table 2) make it possible to identify known cell clusters and give hints as to some unrecognised rare cell clusters. The scRNA‐seq studies revealed that mesenchymal lineage cells, haematopoietic cells, immune cells, and some other cell types were gathered at the defect site (Nakayama et al ., 2022; Xu et al ., 2022). Immune cells, including macrophages, neutrophils, monocytes, B cells, T cells, etc., occupied the most significant proportion of the collected cells from the bone defect area, followed by the mesenchymal lineage cells or skeletal progenitors as the second‐largest cell cluster, reflecting major contributors and cell population heterogeneity during calvarial bone repair (Nakayama et al ., 2022; Xu et al ., 2022).…”

Section: Unravelling the Cellular And Molecular Bases Of Calvarial In...mentioning

confidence: 99%

“…The scRNA‐seq studies revealed that mesenchymal lineage cells, haematopoietic cells, immune cells, and some other cell types were gathered at the defect site (Nakayama et al ., 2022; Xu et al ., 2022). Immune cells, including macrophages, neutrophils, monocytes, B cells, T cells, etc., occupied the most significant proportion of the collected cells from the bone defect area, followed by the mesenchymal lineage cells or skeletal progenitors as the second‐largest cell cluster, reflecting major contributors and cell population heterogeneity during calvarial bone repair (Nakayama et al ., 2022; Xu et al ., 2022). However, the number of relevant studies is still quite limited, and the clustered cell populations are not understood in sufficiently fine detail, restricting the biological insights into calvarial injury repair gained from scRNA‐seq techniques.…”

Section: Unravelling the Cellular And Molecular Bases Of Calvarial In...mentioning

confidence: 99%

“…The cellular components include MSCs/SSCs/SuSCs, macrophages, and other immune cells (see Section VI). On the molecular level, chemokines and neurotrophins are the two major players, such as chemokine (C–C motif) ligand 5 (CCL5) (Ortinau et al ., 2019), CCL9 (Nakayama et al ., 2022), and nerve growth factor (NGF) (Meyers et al ., 2020; Xu et al ., 2022), etc. Researchers identified long‐term repopulating and functionally distinct postnatal periosteal SSCs, which can be selectively labelled by MX dynamin like GTPase 1 (Mx1) and actin alpha 2, smooth muscle (αSMA) (Ortinau et al ., 2019).…”

Section: Unravelling the Cellular And Molecular Bases Of Calvarial In...mentioning

confidence: 99%

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Dissecting calvarial bones and sutures at single‐cell resolution

Fan

et al. 2023

Biological Reviews

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Cranial bones constitute a protective shield for the vulnerable brain tissue, bound together as a rigid entity by unique immovable joints known as sutures. Cranial sutures serve as major growth centres for calvarial morphogenesis and have been identified as a niche for mesenchymal stem cells (MSCs) and/or skeletal stem cells (SSCs) in the craniofacial skeleton. Despite the established dogma of cranial bone and suture biology, technological advancements now allow us to investigate these tissues and structures at unprecedented resolution and embrace multiple novel biological insights. For instance, a decrease or imbalance of representation of SSCs within sutures might underlie craniosynostosis; dural sinuses enable neuroimmune crosstalk and are newly defined as immune hubs; skull bone marrow acts as a myeloid cell reservoir for the meninges and central nervous system (CNS) parenchyma in mediating immune surveillance, etc. In this review, we revisit a growing body of recent studies that explored cranial bone and suture biology using cutting‐edge techniques and have expanded our current understanding of this research field, especially from the perspective of development, homeostasis, injury repair, resident MSCs/SSCs, immunosurveillance at the brain's border, and beyond.

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“…Single-cell transcriptome sequencing has been used to detect cell heterogeneity during fracture healing in animal models, revealing the potential functions of different types of immune cells, such as B cells and T cells [7]. The above studies were mainly carried out in animal models, and the potential interaction mechanism and functions of immune cells and other cells in different stages of human fracture healing are not well understood.…”

Section: Introductionmentioning

confidence: 99%

Single-cell RNA-seq data reveals a critical role of pro-inflammatory macrophage and fibroblast cells in bone marrow environment after bone fracture

Zhou

Jian

et al. 2023

Preprint

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Single-cell RNA sequencing ("scRNA-Seq") examines the cell population at the single-cell level. The single cell changes in the osteoimmunological microenvironment in fresh and old fractures have not been studied. We used single cell transcriptomics in this study to uncover differences in the molecular composition and cellular signaling in bone tissue from fresh and old fractures.We first searched for and downloaded single-cell omics data from the GEO database, which included both fresh and old fracture samples from patients. After applying UMI detection, reducing the dimensions, and conducting principal component analysis, we visualized the data with tSNE and UMAP and identified the marker genes of the cell subsets. The differences of the differentially expressed genes and the signalling pathways of the cell-cell interaction between the two groups of samples were compared by means of Findmarkers and cellchat.The microenvironment in fracture tissue was analysed using a cell characterisation map, resulting in the identification of 18 distinct cell subsets, comprising of macrophages, fibroblasts, B cells, T cells, neutrophils and plasma cells. In comparison to fresh fractures, there was a significant increase in the number of macrophages in the old fracture samples. The number of fibroblasts was not significantly changed. The results of differential expression gene analysis showed that fibroblasts in old fractures were mainly enriched in immune, inflammatory and neutrophil degranulation reactions. TXNIP expression was significantly upregulated. Macrophages were mainly enriched in inflammatory response, immune response, antigen presentation response and cell migration signalling pathways. Among them, AREG was significantly upregulated in old fractures. In old fractures, the interaction between macrophages and other cells was significantly increased. Macrophages regulate other cells mainly through the ANXA1-FRP1 signalling pathway, thereby influencing the formation of callus and the healing of the fracture. Our findings uncovered that fibroblasts regulate inflammation and immune response via the TXNIP pathway. Macrophages influence fracture healing by changing their population and interacting with other cells via the ANXA1-FRP1 pathway.

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Single-cell RNA sequencing unravels heterogeneity of skeletal progenitors and cell–cell interactions underlying the bone repair process

Cited by 9 publications

References 53 publications

3D osteogenic differentiation of human iPSCs reveals the role of TGFβ signal in the transition from progenitors to osteoblasts and osteoblasts to osteocytes

3D osteogenic differentiation of human iPSCs reveals the role of TGFβ signal in the transition from progenitors to osteoblasts and osteoblasts to osteocytes

Dissecting calvarial bones and sutures at single‐cell resolution

Single-cell RNA-seq data reveals a critical role of pro-inflammatory macrophage and fibroblast cells in bone marrow environment after bone fracture

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