Meniscus plays an important role in joint stability, shock absorption, contact force distribution and joint lubrication. It contains different cell types, including chondrocyte morphology cells and fibrocyte morphology cells. However, The cell type, distribution and biomarkers of meniscus, and the biological targets for meniscal degeneration are still unclear. 。
In December 7, 2019, Professor Liao Weiming, the First Affiliated Hospital of Zhongshan University, and Professor Zhang Zhiqi's research group described the cell composition of meniscus in detail by ScRNA-Seq. A progenitor cell with potential application in meniscal tissue engineering was identified, and the mechanism of meniscus degeneration was demonstrated. And pass TGF beta 1 To treat meniscus degeneration effectively, a potential therapeutic strategy for meniscus degeneration is proposed. 。 The single cell RNA sequencing experiments and data analysis were carried out in this study. Strong Ice Technology Participate in support. The research results are published in the title "Single- cell RNA- seq
analysis identifies meniscus progenitors and reveals the progression progression". Annals of the Rheumatic
Diseases (IF=14.299) Journal.
Sequencing sample information:
Healthy meniscus samples and degenerative meniscus samples
BD Rhapsody system
1. ScRNA-Seq Revealing the cell types of healthy human meniscus
The researchers first performed ScRNA-Seq on the meniscus tissue of healthy people, and t-SNE analysis of 3639 cells. Separate out Seven A subpopulation of two newly discovered cell subsets. :
(1) endothelial cells (EC); (2) chondrocyte progenitor cells (CPC); (3) regulatory chondrocytes (RegC); (4) fibrochondrocytes (FC); (5) prehypertrophic chondrocytes (PreHTC); Fibrochondrocyte progenitor cells (FCP); proliferating fibrochondrocytes (ProFC). The distribution of cell subsets was further determined by immunohistochemistry. FCP is mainly distributed on the surface of the meniscus, RegC is located in the middle of the meniscus, EC exists around the vessels of the red area, and PreHTC is distributed in the white area.
Fig. 1 single cell map of healthy human meniscus
Two Analysis of the differentiation locus of meniscus cells in healthy volunteers
The researchers found that FCP and EC existed at the beginning of the differentiation locus. In order to further explore whether FCP has the properties of progenitor cells, CD146+ meniscal cells were selected according to the high expression genes and their ability to differentiate and clone was verified. Through colony formation and differentiation potential analysis, CD146+ cells can differentiate into osteoblasts and adipocytes and form colonies. It is suggested that in meniscal cells of healthy people, FCP It has the characteristics of progenitor cells.
Fig. 2 Identification of human meniscus progenitor cells
In order to study the differentiation pathway of FCP, the researchers reconstructed a new differentiation locus (map 3A) by FCP, ProFC, FC, PreHTC and RegC. The locus was initially composed of FCP and ProFC, followed by PreHTC, while terminal Fate 1 was FC and terminal Fate 2 was composed of RegC (Fig. 3B). We further analyzed the gene expression patterns of different subsets in FCP differentiation to Fate1 and Fate2 (3C, 3D).
In order to determine the cell differentiation locus, the researchers analyzed the differentially expressed genes and expression patterns of various subgroups, and studied the development of menisci in vivo. The expression of marker gene in meniscus of mice at 1, 2, 3, 4, 8, 26 and 52 weeks was observed (Fig. 3E): the expression of COL1A1 increased gradually after birth, reached its peak at 4 weeks, then decreased gradually, and MYLK was 3 weeks later. The amount of expression decreased significantly. The trend of gene expression in vivo is consistent with the trend of sequencing analysis. Further verified FCP The differentiation path.
Fig. 3 bifurcation analysis of single cell locus in FCP differentiation
Three Single cell map of meniscus and degenerative meniscus in healthy population comparative analysis
The researchers first observed the changes in the structure of the tissues and found that the collagen fibers of the degenerative meniscus were in disorder (Fig. 4A). Then ScRNA-Seq analysis of healthy and degenerative meniscus (Fig. 4B-E) was carried out. The proportion of cell subsets in degenerative meniscus changed significantly, and three new subgroups were found. (1) monocyte derived dendritic cells (MoDC); (2) hypertrophic chondrocytes (HTC); (3) degeneration of meniscus progenitor cells (DegP).
Fig. 4 Comparison of single cell profiles between meniscus and degenerative meniscus in healthy volunteers
Four Single cell differentiation locus hints DegP It is the key factor of meniscus degeneration.
The researchers found that DegP (CD318+ cells) can form colonies and differentiate into multiple cells through colony forming and multipotential differentiation analysis. It is proved that DegP has progenitor cell characteristics in degenerative meniscus. Therefore, the researchers re selected four subgroups of FCP, ProFC, CPC and DegP to construct the differentiation locus. The origin of the locus was mainly composed of FCP and ProFC, and Fate 1 and Fate 2 were composed of DegP and CPC respectively (Fig. 5A, DegP).
By comparing the branch gene expression of healthy and degenerative meniscus, it was found that the differentiation of FCP to DegP was abnormal in the process of meniscus degeneration (5C, 5D). The researchers used immunohistochemical staining to confirm that the DegP marker gene (GAS1, DNER) was highly expressed in the area of meniscal lesions and cell proliferation (5E).
In addition, the researchers also used IL-1 beta to stimulate healthy and degenerative meniscal cells, with similar results: CD146+ cells decreased and CD318+ cells increased. Prompt proinflammatory mediators IL-1 beta Induce DegP Activation is the mechanism of meniscus degeneration.
Fig. 5 single cell differentiation locus suggests that DegP is a key factor in meniscus degeneration.
Five TGF beta Activation of signal pathway can inhibit degeneration of meniscus. CD318+ Cell growth
Activation of TGF beta signaling pathway enhances the differentiation ability of meniscal stem cells. The TGF beta signaling pathway was upregulated in healthy meniscus cells, and its ligand TGF beta 1 was highly expressed (Fig. 6A-D). Therefore, the researchers used TGF beta 1 to stimulate the degeneration of meniscus cells in vitro, and found that the number of CD318+ cells decreased significantly and the expression of COL1A1 (FCP marker gene) increased (6E, 6F). TGF beta There may be a delay in meniscus withdrawal. Variable function 。
Figure 6. Activation of the TGF beta pathway can inhibit the increase of Cd318+ cells in the degenerative meniscus.
In summary, the researchers used ScRNA-Seq to describe the map of healthy and degenerative meniscal cells, and identified important CD146+ progenitor cells in meniscal tissue engineering. Secondly, the researchers also confirmed that DegP activation mediated by proinflammatory mediators is a possible mechanism of meniscus degeneration. TGF beta can reduce the proportion of DegP, which may become a therapeutic strategy for delaying meniscal degeneration.