Single-cell RNA-seq analysis identifies meniscus progenitors and reveals the progression of meniscus degeneration

Abstract

Objectives: The heterogeneity of meniscus cells and the mechanism of meniscus degeneration is not well understood. Here, single-cell RNA sequencing (scRNA-seq) was used to identify various meniscus cell subsets and investigate the mechanism of meniscus degeneration.

Methods: scRNA-seq was used to identify cell subsets and their gene signatures in healthy human and degenerated meniscus cells to determine their differentiation relationships and characterise the diversity within specific cell types. Colony-forming, multi-differentiation assays and a mice meniscus injury model were used to identify meniscus progenitor cells. We investigated the role of degenerated meniscus progenitor (DegP) cell clusters during meniscus degeneration using computational analysis and experimental verification.

Results: We identified seven clusters in healthy human meniscus, including five empirically defined populations and two novel populations. Pseudotime analysis showed endothelial cells and fibrochondrocyte progenitors (FCP) existed at the pseudospace trajectory start. Melanoma cell adhesion molecule ((MCAM)/CD146) was highly expressed in two clusters. CD146+ meniscus cells differentiated into osteoblasts and adipocytes and formed colonies. We identified changes in the proportions of degenerated meniscus cell clusters and found a cluster specific to degenerative meniscus with progenitor cell characteristics. The reconstruction of four progenitor cell clusters indicated that FCP differentiation into DegP was an aberrant process. Interleukin 1β stimulation in healthy human meniscus cells increased CD318+ cells, while TGFβ1 attenuated the increase in CD318+ cells in degenerated meniscus cells.

Conclusions: The identification of meniscus progenitor cells provided new insights into cell-based meniscus tissue engineering, demonstrating a novel mechanism of meniscus degeneration, which contributes to the development of a novel therapeutic strategy.

Keywords: arthritis; cytokines; fibroblasts.

Figure 1

A single-cell atlas of healthy human meniscus. (A) Seven healthy human meniscus cell clusters. t-Distributed stochastic neighbour embedding (t-SNE) of 3639 cells (mixed with cell fractions, n=3), annotated post-hoc and coloured by clustering. (B) Heatmap revealing the scaled expression of differentially expressed genes for each cluster. (C) Dot plots showing the expression of the indicated markers for each cell cluster on the t-SNE map. (D) Representative immunohistochemistry staining of MYLK, COL1A1, COL3A1, ZIP8, CD93, BMP2 and CDK1 in white and red zones of healthy human meniscus tissues, and quantification of positive cells displayed by box plot (n=6). Scale bar, 50 µm. **p<0.01. CPC, cartilage progenitor cells; EC, endothelial cells; FC, fibrochondrocytes; FCP, fibrochondrocyte progenitors; PreHTC, prehypertrophic chondrocytes; ProFC, proliferate fibrochondrocytes; RegC, regulatory chondrocytes.

Figure 2

Identification of human meniscus progenitor cells. (A) The 15 most upregulated signal pathways in FCP. (B) Dot plots showing the MCAM expression on t-distributed stochastic neighbour embedding (t-SNE) map and Vin plot. (C) CD146 expression in healthy human meniscus cells determined by flow cytometry (mean±SD; n=3). (D) Alizarin red staining and oil red staining for CD146+ meniscus cells induced to osteogenic differentiation or adipogenic differentiation, respectively (n=5). Scale bar, 50 µm. (E) Colony-forming analysis of CD146+ and CD146− healthy human meniscus cells and quantification. n=5, **p<0.01. (F) IHC staining of MYLK in mice meniscus injury model, and quantification of positive cells. Scale bars, 200 µm (top) and 50 µm (bottom). n≥6, **p<0.01. CFU colony forming unit; CPC, cartilage progenitor cells; EC, endothelial cells; FC, fibrochondrocytes; FCP, fibrochondrocyte progenitors; NOD, nucleotide-binding oligomerisation domain; PreHTC, prehypertrophic chondrocytes; ProFC, proliferate fibrochondrocytes; RegC, regulatory chondrocytes.

Figure 3

Single-cell trajectory branch points demonstrating FCP differentiation. (A, B) Monocle pseudotime trajectory showing the progression of FCP, ProFC, FC, PreHTC and RegC. (C) The expression of the genes in a branch-dependent manner. Each row indicates the standardised kinetic curves of a gene. The centre of the heatmap shows the kinetic curve value at the root of the trajectory. From the centre to the left of the heatmap, the kinetic curve progresses from the root along the trajectory to fate 1. Starting from the right, the curve from the root to fate 2. (D) Pseudotime kinetics of indicated genes from the root of the trajectory to fate 1 (solid line) and the cells up to fate 2 (dashed line). (E) Safranine O/Fast Green staining and immunohistochemistry staining of COL1A1 and MYLK in mice anterior meniscus at 1, 2, 3, 4, 8, 26 and 52 weeks, and quantification of positive cells (n≥3). Scale bar, 100 µm. CPC, cartilage progenitor cells; EC, endothelial cells; FC, fibrochondrocytes; FCP, fibrochondrocyte progenitors; PreHTC, prehypertrophic chondrocytes; ProFC, proliferate fibrochondrocytes; RegC, regulatory chondrocytes.

Figure 4

Comparison of the single cell landscape between healthy human meniscus and degenerated meniscus. (A) Representative polarised light microscopy images of healthy human and degenerated meniscus. The white and red colours in the angle images are 90° apart in orientation. Dashed lines indicate the surface of the meniscus. Scale bar, 100 µm. (B) Merged t-distributed stochastic neighbour embedding (t-SNE) of single-cell RNA sequencing of healthy meniscus cells and degenerated meniscus cells. (C) Twelve healthy human and degenerated meniscus cell clusters at t-SNE. (D) Proportion of each cluster to the total cells. (E) Proportion of healthy and degenerated meniscus cells in each cluster. (F) Expression of representative marker genes in Vin plot. (G) Heatmap revealing the scaled expression of differentially expressed genes for each cluster. (H) CD146 and CD318 expression in healthy human meniscus cells and degenerated meniscus cells determined by flow cytometry. n≥5, **p<0.01. (I) Representative IHC staining of COL1A1 and COL2A1 healthy human meniscus and degenerated meniscus, and quantification of positive cells. Scale bar, 50 µm. n≥5, **p<0.01. (J) The expression of indicated marker genes in human healthy meniscus cells and degenerated meniscus cells were detected by qRT-PCR. *p<0.05, **p<0.01, otherwise, not significant. n=3, *p<0.05, **p<0.01. CPC, cartilage progenitor cells; DegP, degenerated meniscus progenitor cells; EC, endothelial cells; FC, fibrochondrocytes; FCP, fibrochondrocyte progenitors; HTC, hypertrophic chondrocytes; MoDC, monocyte-derived dendritic cells; PreHTC, prehypertrophic chondrocytes; ProFC, proliferate fibrochondrocytes; RegC, regulatory chondrocytes.

Figure 5

Identification of degenerated meniscus progenitor cells (DegP) as a key element for meniscus degeneration. (A, B) Monocle pseudotime trajectory showing the progression of FCP, ProFC, CPC and DegP. (C) From the centre to the left of the heatmap, the kinetic curve from the root along the trajectory to fate 1. Starting from the right, the curve from the root to fate 2. FCP markers MYLK and MCAM, DegP markers GAS1, Rab3B and CDCP1 and CPC markers CDK1 and BIRC5 expressed from the root to each branch. (D) Pseudotime kinetics of indicated genes from the root of the trajectory to fate 1 (solid line) and the cells up to fate 2 (dashed line). (E) Representative IHC staining of MYLK, GAS1 and DNER in healthy human meniscus and degenerated meniscus, and quantification of positive cells. Scale bar, 50 µm. n=6, **p<0.01. (F) Healthy human meniscus cells were treated with 5 ng/mL IL-1β for 48 hours or 96 hours. Phosphate buffer saline (PBS) was used as a negative control. CD146 and CD318 expression was determined by flow cytometry. n≥5, * versus control, p<0.05; & versus IL-1β (48 hours), p<0.05. CPC, cartilage progenitor cells; DeP, degenerated meniscus progenitor cell; FCP, fibrochondrocyte progenitors; ProFC, proliferate fibrochondrocytes.

Figure 6

Activation of TGFβ signalling pathway attenuates the increase in CD318+ cells in degenerated meniscus. (A) The expression of TGFβ1 on merged and split t-distributed stochastic neighbourembedding map. (B) IHC staining of TGFβ1 on human healthy meniscus and degenerated meniscus. n=6, **p<0.01. (C) Volcano plot comparing the gene expression between FC-1 and FC-2. Each plot represents one gene. (D) Volcano plot comparing the gene expression between PreHTC-1 and PreHTC-2. Each plot represents one gene. (E) Human degenerated meniscus cells were treated with 5 ng/mL TGFβ1 for 48 hours or 96 hours. PBS was used as a negative control. CD318 expression was determined by flow cytometry (n≥5). * vs control, p<0.05; & vs TGFβ1 (48 hours), p<0.05. (F) Human degenerated meniscus cells were treated with 5 ng/mL TGFβ1 or PBS as negative control. The expression of indicated marker genes were detected by qRT-PCR. n=3, **p<0.01.