Olympus helped draw the world's first single-cell transcriptome map of intervertebral discs

The author of this article: Christine Zheng, Army Characteristic Medical Center, Army Military Medical University The author of this article: Christine Zheng, The Army Characteristic Medical Center of Army Military Medical University.

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Degenerative disc disease (DDD) is the main cause of pain in the waist and legs, which not only affects the quality of life of patients, but also brings a heavy burden to the medical and economic resources of the society
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At present, the treatment of DDD mainly relies on methods such as bed rest, rehabilitation, drugs, intervention and surgery, but these methods can only alleviate the symptoms of patients, and it is difficult to reconstruct the steady-state environment of the intervertebral disc (IVD)
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Therefore, it is urgent to explore the physiology and pathological mechanism of intervertebral disc in depth, which has great guiding significance for the treatment of degenerative disc disease
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Due to the high heterogeneity of the intervertebral disc cells and the complex cellular microenvironment, it is difficult for previous studies to analyze molecular events in the intervertebral disc through bulk RNA sequencing to meet the needs of in-depth research.
The physiological and pathological mechanisms of the intervertebral disc are still far from being elucidated
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Recently, Liu Peng's research group from the Department of Orthopedics, Army Special Medical Center (Chongqing Daping Hospital) of the Army Military Medical University and Liu Bing's research group from the Fifth Medical Center of the Chinese People's Liberation Army With the support of the research group, the first human intervertebral disc cell map was drawn, and a cover research paper entitled Spatially defined single-cell transcriptional profiling characterizes diverse chondrocyte subtypes and nucleus pulposus progenitors in human intervertebral discs was published in Bone Research.

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The paper uses single-cell RNA sequencing (scRNA-seq), immunohistochemistry, immunofluorescence, flow sorting and other technologies to analyze the cells in the nucleus pulposus, annulus fibrosus and cartilage endplates of human intervertebral discs, and systematically map the intervertebral discs The single-cell transcription map accurately reveals the heterogeneity between cells and provides new ideas for clinical treatment strategies for degenerative disc diseases
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The researchers collected 11 intervertebral discs and separated the nucleus pulposus, annulus fibrosus, and cartilage endplate tissues, separated and extracted cells from the above tissues, and performed single-cell transcriptome sequencing on the cells
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After quality control and multi-joint elimination, the author sequenced 108,108 single cells, analyzed 2,651 differentially expressed genes (DEGs), and visualized the sequencing data with tSNE, and finally obtained 9 subgroups
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These subpopulations include 3 SOX9+ chondrocyte subpopulations, chordal cells, stromal cells, pericytes, endothelial cells, nucleus pulposus progenitor cells, and blood cells
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Subsequently, the author performed immunohistochemical staining on the samples to verify the spatial positioning of several major cell subgroups in the intervertebral disc (Figure 1)
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In addition, in order to verify that the heterogeneity of intervertebral disc cells is highly conserved among species, the authors compared the single-cell transcriptome sequencing data of human and rat intervertebral disc cells and found that most of the cell subpopulations in human intervertebral discs are large.
The same exists in rat intervertebral discs, including nucleus pulposus progenitor cells, endothelial cells, and pericytes
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This paper uses single-cell RNA sequencing (scRNA-seq), immunohistochemistry, immunofluorescence, flow sorting and other technologies to analyze the cells in the nucleus pulposus, annulus fibrosus, and cartilage endplates of human intervertebral discs, and systematically map the intervertebral discs.
Single-cell transcription atlas accurately reveals the heterogeneity between cells, providing new ideas for clinical treatment strategies for degenerative disc diseases
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Figure 1: AF (annulus fibrous) NP (nucleus pulposus) CEP (cartilage endplate) immunohistochemical staining in healthy human intervertebral disc The author of immunohistochemical staining of) through further research, according to the difference in function, divided the intervertebral disc chondrocytes into three types: three types 1.
Regulated chondrocytes are regulated by active growth factor expression and cartilage signaling pathways, which are in the cartilage ECM Play an important role in steady state
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Highly differentiated steady-state chondrocytes are in a resting state and are mainly responsible for the deposition of ECM
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The effector chondrocytes are in a state of high metabolic activity, which is inseparable from maintaining the synthesis of ECM in the intervertebral disc, and has the characteristics of high expression of PRG4, which can reduce shear stress and infection damage, and has a protective effect on joints
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In addition, the author's team found through immunostaining that there is a subpopulation of PDGFRA+PROCR+ cartilage progenitor cells in the nucleus pulposus area.
This group of cells has potential stemness and specifically expresses PRRX1 (Figure 2)
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Regulating chondrocytes are regulated by active growth factor expression and cartilage signal pathways, and play an important role in cartilage ECM homeostasis
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Highly differentiated steady-state chondrocytes are in a resting state and are mainly responsible for the deposition of ECM
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The effector chondrocytes are in a state of high metabolic activity, which is inseparable from maintaining the synthesis of ECM in the intervertebral disc, and has the characteristics of high expression of PRG4, which can reduce shear stress and infection damage, and has a protective effect on joints
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Figure 2: Immunofluorescence staining showing the co-expression of PDGFRA/PROCR/PRRX1 in human intervertebral disc cells Optionally, different cell subgroups were purified and enriched
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Through in vitro experiments, researchers found that PROCR+ subpopulations have strong in vitro cloning ability compared with PROCR- cell subpopulations
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The results of immunofluorescence staining indicated that the transcription factor p-SMAD3, a transcription factor related to chondrogenesis, was highly activated in the PROCR+ cell subset (Figure 3)
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The final in vitro differentiation experiment confirmed that PROCR+ progenitor cells have the three-lineage differentiation ability of osteogenic, chondrogenic and adipogenic (Figure 4)
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Figure 3: Immunofluorescence staining reveals the difference in the expression of p-SMAD3 in the PROCR+ and PROCR- subgroups Figure 3: Immunofluorescence staining reveals the difference in the expression of p-SMAD3 in the PROCR+ and PROCR- subgroups Figure 4: The PROCR+ subgroup undergoes osteogenesis, Alizarin Red, Oil Red O, Alcian Blue, Safranin O-Fast Green Staining after Adipogenesis and Cartilage Induction Figure 4: Alizarin Red and Oil after Osteogenesis, Adipogenesis, and Cartilage Induction of PROCR+ Subgroups Red O, Alcian Blue, and Safranin O-Fast Green Staining To sum up, this study used single-cell expression profile sequencing to draw a single-cell map of human intervertebral discs for the first time, defining a subpopulation of chondrocytes at the single-cell level.
Let us He has a new understanding of the heterogeneity of intervertebral disc cells; and the author analyzes and locates these different subgroups spatially, and reveals their regulatory characteristics through genetic analysis
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In addition, the author's team identified a group of progenitor cells with stem potential in the nucleus pulposus, providing a new theoretical basis for the treatment of human intervertebral disc degenerative diseases
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The images of tissue slices in this article are mainly obtained using the Olympus VS200 research-grade full slide scanning system.
The system supports five common observation methods such as bright field and fluorescence.
The X Line high-performance objective lens can provide users with flat Images with better field characteristics; automatic focus and automatic sample detection functions can greatly improve the shooting efficiency of the system without losing accuracy; the powerful function of TruAI deep learning technology can accurately identify the morphological characteristics of the sample and accurately segment the target Area, on the premise of being simple and easy to learn, it helps users save the time of analyzing images
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Olympus VS200 research-grade full slide scanning system X Line TruAI deep learning technology.
In addition, the immunofluorescence staining image of cells is mainly obtained by Olympus' new generation of ultra-high-resolution turntable confocal system SpinSR10, which has Olympus The ultra-high-resolution module available in Sturdish does not require special fluorescent labeling dyes, and the XY resolution can reach 110 nm, which can easily analyze fine structures; the confocal scanning unit based on the turntable can achieve high frame rate image acquisition without missing every moment; this The extremely low phototoxicity of the system does little damage to the specimens, and is especially suitable for the observation of living cells
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The ultra-high-resolution turntable confocal system SpinSR10 XY resolution can reach 110 nm.
Professor Liu Peng from the Department of Orthopedics, Army Special Medical Center (Daping Hospital), Army Military Medical University, and Researcher Liu Bing from the Fifth Medical Center of the PLA General Hospital are the co-corresponding authors of the paper.
Gan Postdoctoral Yibo and Dr.
He Jian are the co-first authors of the paper, and the Army Special Medical Center is the first completion unit
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This work was also participated and supported by the research group of Professor Chen Lin from the Army Specialized Medical Center
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Professor Liu Peng from the Department of Orthopaedics, Army Special Medical Center (Daping Hospital) of the Army Military Medical University and researcher Liu Bing from the Fifth Medical Center of the PLA General Hospital are the co-corresponding authors of the paper.
Postdoctoral fellow Gan Yibo and Dr.
He Jian are the co-first authors of the paper.
The Army Characteristic Medical Center is the first completed unit
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This work was also participated and supported by the research group of Professor Chen Lin from the Army Specialized Medical Center
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