Abstract
Meniscus injuries are common and while surgical strategies have improved, there is a need for alternative therapeutics to improve long-term outcomes and prevent post-traumatic osteoarthritis. Current research efforts in regenerative therapies and tissue engineering are hindered by a lack of understanding of meniscus cell biology and a poorly defined meniscus cell phenotype. This study utilized bulk RNA-sequencing to identify unique and overlapping transcriptomic profiles in cartilage, inner and outer zone meniscus tissue, and passaged inner and outer zone meniscus cells. The greatest transcriptomic differences were identified when comparing meniscus tissue to passaged monolayer cells (> 4,600 differentially expressed genes (DEGs)) and meniscus tissue to cartilage (> 3,100 DEGs). While zonal differences exist within the meniscus tissue (205 DEGs between inner and outer zone meniscus tissue), meniscus resident cells are more similar to each other than to either cartilage or passaged monolayer meniscus cells. Additionally, we identified and validated LUM, PRRX1, and SNTB1 as potential markers for meniscus tissue and ACTA2, TAGLN, SFRP2, and FSTL1 as novel markers for meniscus cell dedifferentiation. Our data contribute significantly to the current characterization of meniscus cells and provide an important foundation for future work in meniscus cell biology, regenerative medicine, and tissue engineering.
Supplementary Information
The online version contains supplementary material available at 10.1038/s41598-024-78580-3.
Keywords: Meniscus, Cartilage, Transcriptomics, Dedifferentiation, Monolayer, Tissue engineering
Subject terms: Biomedical engineering, Cell biology
Introduction
The menisci are fibrocartilaginous tissues located in the knee joint and are essential for proper joint biomechanics. Meniscal injuries are one of the most common knee injuries and remain one of the most common indications for orthopaedic surgery1. However, not all meniscal lesions are amenable to surgical repair, and research is currently aimed towards investigating biomaterials and bioengineered tissues for improved meniscus healing2,3 or replacement of lost and damaged tissue4,5. While significant effort has been made to create meniscus tissue-engineered constructs that can functionally replace damaged tissue6,7, there remain significant gaps in knowledge surrounding meniscus cell biology. Overall, poor characterization of the specific meniscus cell transcriptome and limited understanding of the transcriptomic changes that occur due to monolayer culture expansion remain major hurdles facing the field of meniscus tissue engineering8–12.
It has long been appreciated that the meniscus structure and extracellular matrix (ECM) composition are phenotypically distinct from other musculoskeletal tissues13,14. The meniscus can be divided largely into two zones, the inner and outer zones, which differ in ECM composition, vascularity2, and resident cell morphology7,15,16. Clinically, the inner and outer zones are frequently referred to as white and red zones, respectively, due to the avascularity of the inner zone and the presence of a blood supply in the outer zone. While previous work has characterized the differential expression of ECM and catabolic genes9,10,17 and cell surface markers11,18 of inner and outer zone meniscus cells, a comprehensive evaluation of the transcriptomic profile of these zones is still needed. Therefore, despite the recognition that meniscus cells are a phenotypically distinct cell type, researchers in the field of meniscus cell biology are ultimately left defining meniscus cells in relation to more well-characterized cell types, commonly referring to outer zone cells as “fibroblast-like” and inner zone cells as “chondrocyte-like”7,8,11.
More recently, state-of-the-art genomic technologies have been used to examine the meniscus cell transcriptome, as it relates to embryonic meniscus development19, aging20, mechanotransduction21,22, and osteoarthritis23,24. While these studies have begun to elucidate the genetic signature of meniscus cells, there remains a need to fully characterize the zonal variation in meniscus cells, elucidate the transcriptomic differences between meniscus tissue and articular cartilage, and to further explore the underlying gene expression changes that contribute to dedifferentiation of meniscus cells during monolayer culture. In this study, RNA-sequencing was used to compare 1) cartilage to inner and outer zone meniscus tissue and 2) inner and outer zone meniscus tissue to passaged inner and outer zone monolayer meniscus cells.
Results
Bulk RNA-seq was performed on tissue explants taken from cartilage, inner and outer zone meniscus tissue, and passaged meniscus cells harvested from either the inner or outer zone. Principal component analysis (PCA) plots were used to visualize overarching transcriptomic differences and display a clear separation of meniscus tissue, cartilage, and passaged meniscus cells. When comparing all groups, the first principal component (PC1), accounting for 80% of variance, primarily separated tissue samples from passaged cells, while principal component 2 (PC2, 11% of variance) separated meniscus tissue from cartilage (Fig. 1A). When analyzing only tissue samples, PC1, which accounts for 65% of the variance, separated cartilage from meniscus tissue, while PC2 (20% of variance), separated meniscus tissue samples primarily by zone, albeit with one outer zone sample clustering closer to the inner zone samples (Fig. 1B). When comparing meniscus tissue to passaged meniscus cells, PC1 (88% of variance) separated tissue from monolayer cells, while PC2 (6% of variance) separated meniscus tissue samples, again mostly delineating inner from outer (Fig. 1C). Passaged cells from both inner and outer zones cluster closely together and were not separated by zone in these analyses (Fig. 1A,C).
Fig. 1.
Comparison of Meniscus, Cartilage, and Passage 3 (P3) Meniscus Cells using Principal Component Analysis (PCA) and Differential Gene Expression Analysis. PCA comparing (A) all samples (cartilage, meniscus tissue, and P3 meniscus cells), (B) tissue samples (cartilage and meniscus tissue), or (C) meniscus samples (meniscus tissue and P3 meniscus cells). Shape corresponds to anatomic source (circle: inner zone meniscus, square: outer zone meniscus, triangle: cartilage), color denotes sample type (blue: meniscus tissue, green: P3 meniscus cells, red: cartilage tissue). Total variability accounted for by each principal component is indicated in the axis labels. Volcano plots show genes significantly differentially expressed between (D) inner zone meniscus tissue and cartilage, or (E) outer zone meniscus tissue and cartilage. Each data point is an individual gene. Dashed lines indicate cutoffs for significant genes (FDR adjusted p-value < 0.01 and a |base-2 log fold-change|> 1). Color denotes if the gene was significantly up-regulated (blue) or down-regulated (green) in meniscus tissue relative to cartilage, or not significantly different (black). (F) Number of significantly differentially expressed genes (DEGs) exclusive to or shared between inner and outer zone meniscus tissue comparisons to cartilage.
Pairwise comparisons of DEGs between meniscus tissue and cartilage were performed to better quantify transcriptomic differences. A total of 1,730 genes were significantly differentially expressed (p < 0.01, |LogFC|> 1) between inner zone meniscus tissue and cartilage (Fig. 1D and Supplemental Dataset 1), while 2,731 DEGs were identified between outer zone tissue and cartilage (Fig. 1E and Supplemental Dataset 2). A total of 1,337 of the DEGs were shared between both inner and outer zone comparisons to cartilage. On the other hand, 393 DEGs were exclusive to the inner zone and cartilage comparison, while 1,394 unique DEGs were identified when comparing outer zone tissue to cartilage (Fig. 1F).
To create a cartilage-specific expression profile, genes were sorted by greatest copy number in cartilage and the list of the top 25 expressed genes with logFC > 1 and padj < 0.01 relative to inner (Table 1) and outer zone tissue (Table 2) was generated. When comparing cartilage to inner zone meniscus, differential expression of genes associated with the following were identified: ECM components, including several collagen isoforms (COL9A2, COL16A1, COL9A3, COL3A1), SERPINH1, which is a collagen chaperone25, and several glycoproteins (CHI3L1, BGN, CILP2, and TNC); signal transduction, including GEM, which is a regulator of chondrogenic differentiation26; transcriptional regulation, including AEBP1, which is a transcriptional repressor and is involved in collagen polymerization27; cytoplasmic proteins NUCB1, which is a calcium-binding protein, and SQSTM1, which is involved in bone remodeling; enzymes NT5E, which hydrolyzes 5′-AMP into adenosine and phosphate, and CDO1, which enables cysteine dioxygenase activity; growth factors LTBP1, which is critical for TGF-β activity28, and NDUFA4L2, which is involved in mitochondrial function; and EEF2, which is essential for protein synthesis. Of these top 25 expressed genes, the most highly differentially expressed genes in cartilage relative to inner zone meniscus were ECM genes COL9A2 (logFC = 5.15) and ABI3BP (logFC = 4.64), and transcriptional regulator AEBP1 (logFC = 3.91). Transcriptional differences in cartilage relative to outer zone meniscus tissue identified genes associated predominantly with the ECM, including COL2A1, CILP2, SPARC, ACAN, COL3A1, FMOD, BGN, COMP, and PRELP, which are well-established cartilage matrix components, and protein synthesis, with 10 of the genes encoding ribosomal proteins and two being eukaryotic translation elongation factors. There were also changes in genes associated with signal transduction, such as LCN2, which is a catabolic adipokine29, and transcriptional regulators, including AEBP1. The most highly differentially expressed genes in cartilage compared to outer zone meniscus were ECM genes COL2A1 (logFC = 4.69), CILP2 (logFC = 2.47) and SPARC (logFC = 2.46). Overall, eight of the top 25 highly expressed genes in cartilage were significantly differentially expressed relative to both inner and outer zone meniscus (CILP2, SPARC, BGN, COL3A1, EEF2, LCN2, AEBP1, TPT1).
Table 1.
Top 25 expressed genes in cartilage compared to inner zone meniscus tissue.
| Gene symbol | Gene name | LogFC | padj |
|---|---|---|---|
| Cytoplasmic Protein | |||
| NUCB1 | Nucleobindin 1 | 1.26 | 1.83E-06 |
| SQSTM1 | Sequestosome 1 | 1.02 | 1.75E-14 |
| Enzyme | |||
| NT5E | 5'-Nucleotidase Ecto | 1.73 | 4.09E-04 |
| CDO1 | Cysteine Dioxygenase Type 1 | 1.57 | 8.35E-04 |
| Extracellular Matrix | |||
| COL9A2 | Collagen Type IX Alpha 2 Chain | 5.15 | 3.41E-06 |
| ABI3BP | ABI Family Member 3 Binding Protein | 4.64 | 2.85E-28 |
| COL16A1 | Collagen Type XVI Alpha 1 Chain | 3.31 | 3.89E-11 |
| COL9A3 | Collagen Type IX Alpha 3 Chain | 2.88 | 3.20E-03 |
| FBLN7 | Fibulin 7 | 2.65 | 4.96E-06 |
| EFEMP1 | EGF Containing Fibulin Extracellular Matrix Protein 1 | 2.23 | 7.82E-03 |
| TNC | Tenascin C | 1.89 | 1.36E-03 |
| CHI3L1 | Chitinase 3 Like 1 | 1.86 | 6.38E-03 |
| SPARC | Secreted Protein Acidic And Cysteine Rich | 1.64 | 7.29E-04 |
| BGN | Biglycan | 1.54 | 3.91E-06 |
| CILP2 | Cartilage Intermediate Layer Protein 2 | 1.48 | 2.36E-03 |
| SERPINH1 | Serpin Family H Member 1 | 1.48 | 8.89E-10 |
| COL3A1 | Collagen Type III Alpha 1 Chain | 1.20 | 7.34E-03 |
| Growth Factor and Regulator | |||
| LTBP1 | Latent Transforming Growth Factor Beta Binding Protein 1 | 1.20 | 5.98E-05 |
| NDUFA4L2 | NDUFA4 Mitochondrial Complex Associated Like 2 | 1.86 | 1.13E-03 |
| Protein Synthesis | |||
| EEF2 | Eukaryotic Translation Elongation Factor 2 | 1.13 | 1.76E-07 |
| Signal Transduction | |||
| GEM | GTP Binding Protein Overexpressed In Skeletal Muscle | 2.61 | 1.54E-07 |
| LCN2 | Lipocalin 2 | 1.77 | 1.24E-03 |
| Transcriptional Regulation | |||
| AEBP1 | AE Binding Protein 1 | 3.91 | 1.25E-09 |
| TPT1 | Tumor Protein, Translationally-Controlled 1 | 1.11 | 2.18E-04 |
| RPL17-C18orf32 | RPL17-C18orf32 | 1.00 | 1.90E-05 |
Genes with logFC > 1.
Filtered by padj < 0.01.
Genes in bold were selected for qRT-PCR validation.
Table 2.
Top 25 expressed genes in cartilage compared to outer zone meniscus tissue.
| Gene symbol | Gene Name | LogFC | padj |
|---|---|---|---|
| Extracellular Matrix | |||
| COL2A1 | Collagen Type II Alpha 1 Chain | 4.69 | 2.18E-07 |
| CILP2 | Cartilage Intermediate Layer Protein 2 | 2.47 | 5.69E-08 |
| SPARC | Secreted Protein Acidic and Cysteine Rich | 2.46 | 8.75E-08 |
| ACAN | Aggrecan | 2.18 | 4.27E-04 |
| COL3A1 | Collagen Type III Alpha 1 Chain | 1.87 | 7.30E-06 |
| FMOD | Fibromodulin | 1.81 | 2.70E-05 |
| BGN | Biglycan | 1.79 | 3.23E-08 |
| COMP | Cartilage Oligomeric Matrix Protein | 1.61 | 1.32E-03 |
| PRELP | Proline and Arginine Rich End Leucine Rich Repeat Protein | 1.53 | 3.63E-03 |
| Protein synthesis | |||
| EEF2 | Eukaryotic Translation Elongation Factor 2 | 1.57 | 4.87E-14 |
| RPS27A | Ribosomal Protein S27A | 1.27 | 1.27E-06 |
| RPL10 | Ribosomal Protein L10 | 1.23 | 1.98E-05 |
| RPL5 | Ribosomal Protein L5 | 1.22 | 2.03E-06 |
| RPL23 | Ribosomal Protein L23 | 1.20 | 1.24E-06 |
| RPS20 | Ribosomal Protein S20 | 1.19 | 2.01E-05 |
| EEF1G | Eukaryotic Translation Elongation Factor 1 Gamma | 1.14 | 2.12E-05 |
| RPS3 | Ribosomal Protein S3 | 1.12 | 2.13E-05 |
| RPL4 | Ribosomal Protein L4 | 1.06 | 1.20E-06 |
| RPL6 | Ribosomal Protein L6 | 1.01 | 2.02E-06 |
| RPS8 | Ribosomal Protein S8 | 1.01 | 3.52E-04 |
| RPL8 | Ribosomal Protein L8 | 1.01 | 3.75E-05 |
| Signal Transduction | |||
| C2orf40 | ECRG4 Augurin Precursor | 2.36 | 8.51E-03 |
| LCN2 | Lipocalin 2 | 1.72 | 1.22E-03 |
| Transcriptional Regulation | |||
| AEBP1 | AE Binding Protein 1 | 1.84 | 5.81E-03 |
| TPT1 | Tumor Protein, Translationally-Controlled 1 | 1.68 | 3.91E-09 |
Genes with logFC > 1.
Filtered by padj < 0.01.
Genes in bold were selected for qRT-PCR validation.
On the other hand, the top 25 DEGs in inner zone meniscus tissue compared to cartilage showed differences in genes associated with the following: ECM, including glycoprotein LUM, COL1A2, ECM1, which is a matrix protein that regulates chondrogenesis and endochondral ossification30, matrix metalloproteinase (MMP) inhibitor TIMP3, and the mineralization inhibitor MGP; cell membrane proteins, including KCNA6 that encodes a voltage-gated potassium channel; actin cytoskeleton (ACTB, CAPG, and EZR); mitochondrial function (CYP1B1, CYTB); signal transduction, including MT2A, which is a free radical scavenger; transcriptional regulators, including PRRX1, CEBPD, and TRPS1, and HNRNPA2B1, which encodes an RNA binding protein; enzymes, including genes involved in free radical scavenging (SOD3) and metabolism (AKR1B1 and ACSL1); and secreted factor transglutaminase F13A1, which is involved in the coagulation cascade and knockout of this gene protects against collagen-induced arthritis in mice31 (Table 3). Of the 25 most expressed genes, the genes with greatest logFC in inner zone tissue relative to cartilage were ECM genes LUM (logFC = 5.34) and MGP (logFC = 2.81) and secreted protein F13A1 (logFC = 2.85). The top 25 expressed genes in outer zone tissue compared to cartilage largely fell into similar categories as the inner zone, as well as including the growth factor regulator LTBP2 (Table 4). Of the top 25 most expressed genes in outer zone tissue, the genes with greatest logFC relative to cartilage were ECM genes LUM (logFC 6.25) and COL1A2 (logFC 3.11) and the enzyme MMP13 (logFC 3.00). Eleven of the top 25 expressed genes were upregulated in both inner and outer zone meniscus relative to cartilage (CAPG, ACTB, LUM, COL1A2, ECM1, ACSL1, SOD3, CYTB, CYP1B1, PRRX1, HNRNPA2B1).
Table 3.
Top 25 expressed genes in inner zone meniscus tissue compared to cartilage.
| Gene symbol | Gene name | LogFC | Padj |
|---|---|---|---|
| Cell Membrane Protein | |||
| KCNA6 | Potassium Voltage-Gated Channel Subfamily A Member 6 | 1.51 | 9.53E-04 |
| SLA-11 | Major Histocompatibility Complex, Class I, F | 1.17 | 1.59E-07 |
| PLXDC2 | Plexin Domain Containing 2 | 1.06 | 1.82E-03 |
| Cytoskeleton | |||
| EZR | Ezrin | 1.75 | 5.57E-30 |
| CAPG | Capping Actin Protein, Gelsolin Like | 1.14 | 4.08E-05 |
| ACTB | Actin Beta | 1.04 | 1.50E-03 |
| Enzyme | |||
| AKR1B1 | Aldo–Keto Reductase Family 1 Member B | 1.99 | 6.36E-09 |
| RTN4 | Reticulon 4 | 1.28 | 1.98E-07 |
| ACSL1 | Acyl-CoA Synthetase Long Chain Family Member 1 | 1.19 | 2.49E-04 |
| SOD3 | Superoxide Dismutase 3 | 1.10 | 2.46E-03 |
| Extracellular Matrix | |||
| LUM | Lumican | 5.34 | 1.04E-12 |
| MGP | Matrix Gla Protein | 2.81 | 3.67E-03 |
| COL1A2 | Collagen Type I Alpha 2 Chain | 2.20 | 7.65E-07 |
| TIMP3 | TIMP Metallopeptidase Inhibitor 3 | 1.46 | 1.55E-03 |
| ECM1 | Extracellular Matrix Protein 1 | 1.22 | 3.44E-03 |
| Mitochondrial protein | |||
| CYP1B1 | Cytochrome P450 Family 1 Subfamily B Member 1 | 2.55 | 7.97E-16 |
| CYTB | Mitochondrially Encoded Cytochrome B | 1.26 | 2.09E-07 |
| Secreted Protein | |||
| F13A1 | Coagulation Factor XIII A Chai | 2.85 | 4.04E-04 |
| Signal Transduction | |||
| SEMA3D | Semaphorin 3D | 2.62 | 1.88E-06 |
| MT2A | Metallothionein 2A | 1.62 | 7.75E-05 |
| TNFRSF1B | TNF Receptor Superfamily Member 1B | 1.00 | 1.12E-08 |
| Transcriptional Regulation | |||
| PRRX1 | Paired Related Homeobox 1 | 2.16 | 2.55E-26 |
| HNRNPA2B1 | Heterogeneous Nuclear Ribonucleoprotein A2/B1 | 1.29 | 1.01E-17 |
| CEBPD | CCAAT Enhancer Binding Protein Delta | 1.14 | 1.04E-05 |
| TRPS1 | Transcriptional Repressor GATA Binding 1 | 1.03 | 9.87E-05 |
Genes with logFC > 1.
Filtered by padj < 0.01.
Genes in bold were selected for qRT-PCR validation.
Table 4.
Top 25 expressed genes in outer zone meniscus tissue compared to cartilage.
| Gene symbol | Gene name | LogFC | Padj |
|---|---|---|---|
| Cell Membrane Protein | |||
| ANTXR1 | ANTXR Cell Adhesion Molecule 1 | 2.07 | 2.59E-05 |
| Cytoskeleton | |||
| CRYAB | Crystallin Alpha B | 2.71 | 5.04E-06 |
| ACTB | Actin Beta | 1.82 | 3.21E-09 |
| CAPG | Capping Actin Protein, Gelsolin Like | 1.81 | 5.70E-12 |
| FLNA | Filamin A | 1.44 | 3.18E-04 |
| ACTG1 | Actin Gamma 1 | 1.28 | 4.27E-11 |
| CTNNB1 | Catenin Beta 1 | 1.14 | 8.46E-06 |
| CFL1 | CFL1 | 1.00 | 6.97E-08 |
| Enzyme | |||
| MMP13 | Matrix Metallopeptidase 13 | 3.00 | 5.81E-03 |
| MMP3 | Matrix Metallopeptidase 3 | 1.61 | 4.44E-04 |
| ACSL1 | Acyl-CoA Synthetase Long Chain Family Member 1 | 1.16 | 2.59E-04 |
| SOD3 | Superoxide Dismutase 3 | 1.11 | 1.65E-03 |
| Extracellular Matrix | |||
| LUM | Lumican | 6.25 | 1.95E-17 |
| COL1A2 | Collagen Type I Alpha 2 Chain | 3.11 | 3.22E-13 |
| CRISPLD2 | Cysteine Rich Secretory Protein LCCL Domain Containing 2 | 2.34 | 1.57E-04 |
| ECM1 | Extracellular Matrix Protein 1 | 1.26 | 1.65E-03 |
| Growth Factor or Regulator | |||
| LTBP2 | Latent Transforming Growth Factor Beta Binding Protein 2 | 1.80 | 7.73E-04 |
| Mitochondrial Protein | |||
| CYP1B1 | Cytochrome P450 Family 1 Subfamily B Member 1 | 2.31 | 2.27E-13 |
| CYTB | Mitochondrially Encoded Cytochrome B | 1.27 | 8.76E-08 |
| Secreted Protein | |||
| SAA2 | Serum amyloid A protein | 1.43 | 2.57E-03 |
| Signal Transduction | |||
| ANXA2 | Annexin A2 | 1.07 | 1.03E-06 |
| Transcriptional Regulation | |||
| PRRX1 | Paired Related Homeobox 1 | 2.65 | 1.11E-39 |
| CCDC3 | Coiled-Coil Domain Containing 3 | 1.90 | 1.82E-04 |
| HSPA8 | Heat Shock Protein Family A (Hsp70) Member 8 | 1.40 | 2.56E-08 |
| HNRNPA2B1 | Heterogeneous Nuclear Ribonucleoprotein A2/B1 | 1.31 | 2.44E-18 |
Genes with logFC > 1.
Filtered by padj < 0.01.
Genes in bold were selected for qRT-PCR validation.
Several genes were selected for quantitative RT-PCR (qRT-PCR) validation of tissue specific gene expression based on logFC and significant padj. LUM (Fig. 2A, p < 0.001), PRRX1 (Fig. 2B, p < 0.05), and SNTB1 (Fig. 2C, p < 0.0001) were significantly higher in both inner and outer zone meniscus tissue compared to cartilage. Expression of PHLDA1 was significantly higher in inner zone meniscus than cartilage (Fig. 2D, p < 0.05). Interestingly, AEBP1 was decreased in inner zone meniscus compared to both cartilage and outer zone meniscus (Fig. 2E, p < 0.05). There were no detectable differences in the expression of CILP2 between the cartilage and meniscus tissues (Fig. 2F).
Fig. 2.
Quantitative RT-PCR for (A) LUM, (B) PRRX1, (C) SNTB1, (D) PHLDA1, (E) AEBP1, and (F) CILP2 expression in cartilage and inner and outer zone meniscus tissue (N ≥ 10/group). Results are presented as log-twofold change relative to cartilage. The line indicates the mean value for each group. Groups not sharing a letter are significantly different (p < 0.05).
To better understand zonal differences, DEG analysis was performed on inner and outer zone meniscus, identifying a total of 205 DEGs (Supplemental Fig. 1 and Supplemental Dataset 3). The majority of these differentially expressed genes were upregulated in the outer zone tissue (190 genes) compared to the inner zone (15 genes). The most highly expressed genes in the outer zone meniscus included genes associated with the following: signal transduction, including genes related to the vasculature (PLVAP, VWF, PECAM1), the retinoic acid binding protein CRABP1, glycoproteins (GPNMB and THY1), and the glycan-binding protein LGALS1; cytoskeleton, including genes involved in actin regulation (FSCN1, PLEKHO1, MYO1B, and LIMA1); ECM components, such as collagens (COL1A1, COL4A2, and COL18A1) and glycoprotein FBLN2; growth factor CCN2, which is commonly secreted by vascular endothelial cells; and enzymes, including the matrix degrading enzyme MMP2 (Table 5). When analyzing the top DEGs in inner zone meniscus compared to outer zone, genes associated with the following were identified: signal transduction, including genes involved in TGF-β (CHRDL2) and Wnt (RSPO3) signaling; the Ca2+ binding protein CALR3; ECM protein CILP; enzymes, including genes related to matrix degradation (MMP16) and metabolism (ATP6V1B1, LNPEP, and FUT8); and transcriptional regulator OVOL1 (Table 6). Of the most highly expressed genes, the largest DEGs were CD93 (logFC = 10.72), PLVAP (logFC = 9.79), and VWF (logFC = 9.41) in the outer zone and CHRDL2 (logFC = 5.31) and RSPO3 (logFC = 4.55) for the inner zone meniscus.
Table 5.
Top 25 expressed genes in outer zone meniscus tissue compared to inner zone meniscus tissue.
| Gene symbol | Gene name | LogFC | padj |
|---|---|---|---|
| Cytoskeleton | |||
| CD93 | CD93 Molecule | 10.72 | 2.76E-05 |
| FSCN1 | Fascin Actin-Bundling Protein 1 | 2.61 | 3.54E-04 |
| PLEKHO1 | Pleckstrin Homology Domain Containing O1 | 2.53 | 2.22E-03 |
| MYO1B | Myosin IB | 2.07 | 7.09E-03 |
| TAGLN2 | Transgelin 2 | 1.87 | 2.61E-04 |
| LIMA1 | LIM domain and actin binding 1 | 1.51 | 1.91E-03 |
| Extracellular Matrix | |||
| COL1A1 | Collagen Type I Alpha 1 Chain | 6.30 | 1.11E-09 |
| COL4A2 | Collagen Type IV Alpha 1 Chain | 4.43 | 8.10E-04 |
| POSTN | Periostin | 3.82 | 5.07E-03 |
| FBLN2 | Fibulin 2 | 2.54 | 4.17E-04 |
| COL18A1 | Collagen Type XVIII Alpha 1 Chain | 2.30 | 4.09E-03 |
| Enzyme | |||
| MMP2 | Matrix Metallopeptidase 2 | 2.89 | 4.24E-04 |
| ANPEP | Alanyl Aminopeptidase | 2.74 | 1.06E-03 |
| UBE2L6 | Ubiquitin Conjugating Enzyme E2 L6 | 1.40 | 8.37E-03 |
| Growth factor | |||
| CNN2 | Calponin 2 | 1.03 | 5.70E-03 |
| Signal transduction | |||
| PLVAP | Plasmalemma Vesicle Associated Protein | 9.79 | 2.20E-05 |
| VWF | Von Willebrand Factor | 9.41 | 4.86E-05 |
| PECAM1 | Platelet And Endothelial Cell Adhesion Molecule 1 | 4.72 | 2.85E-04 |
| CRABP1 | Cellular Retinoic Acid Binding Protein 1 | 4.63 | 3.59E-06 |
| GPNMB | Glycoprotein Nmb | 4.25 | 9.20E-04 |
| LGALS1 | Galectin 1 | 2.44 | 3.80E-03 |
| TMEM119 | Transmembrane Protein 119 | 2.35 | 3.93E-03 |
| THY1 | Thy-1 Cell Surface Antigen | 1.58 | 4.02E-03 |
| Transcriptional regulation | |||
| NFATC4 | Nuclear factor of activated T-cells 4 | 1.26 | 9.12E-03 |
| Unknown | |||
| PALM2 | Paralemmin 2 | 1.19 | 8.54E-03 |
Genes with logFC > 1.
Filtered by padj < 0.01.
Table 6.
Most highly expressed genes in inner zone meniscus tissue compared to outer zone meniscus tissue.
| Gene symbol | Gene name | LogFC | padj |
|---|---|---|---|
| Ca2 + Binding Protein | |||
| CALR3 | Calreticulin 3 | 1.34 | 9.45E-03 |
| Cytoskeleton | |||
| SEMA3E | Semaphorin 3E | 2.67 | 6.52E-04 |
| Extracellular Matrix | |||
| CILP | Cartilage Intermediate Layer Protein 2 | 2.35 | 1.68E-03 |
| Enzyme | |||
| ATP6V1B1 | ATPase H + Transporting V1 Subunit B1 | 2.97 | 8.10E-03 |
| MMP16 | Matrix Metallopeptidase 16 | 1.63 | 1.46E-03 |
| LNPEP | Leucyl And Cystinyl Aminopeptidase | 1.20 | 1.15E-04 |
| FUT8 | Fucosyltransferase 8 | 1.01 | 7.77E-03 |
| Signal transduction | |||
| CHRDL2 | Chordin Like 2 | 5.31 | 9.09E-03 |
| RSPO3 | R-Spondin 3 | 4.55 | 9.30E-03 |
| FIBIN | Fin Bud Initiation Factor Homolog | 1.84 | 6.02E-03 |
| NXPH4 | Neurexophilin 4 | 1.66 | 1.45E-04 |
| STC2 | Stanniocalcin 2 | 1.44 | 6.51E-03 |
| ENKUR | Enkurin | 1.04 | 9.77E-03 |
| Transcriptional regulation | |||
| OVOL1 | Ovo Like Transcriptional Repressor 1 | 2.53 | 3.15E-03 |
| Unknown | |||
| CRISPLD1 | Cysteine Rich Secretory Protein LCCL Domain Containing 1 | 2.11 | 4.09E-03 |
Genes with logFC > 1.
Filtered by padj < 0.01.
The most abundant differences in gene expression were identified when comparing meniscus tissue to passaged meniscus cells. The comparison of inner zone meniscus tissue to passaged inner zone cells yielded 4,266 significant DEGs with 2,122 up-regulated and 2,144 down-regulated in tissue compared to passaged inner zone cells (Fig. 3A and Supplemental Dataset 4). A total of 2,892 significant DEGs were identified between outer zone tissue and passaged outer zone cells (1,573 up-regulated; 1,319 down-regulated) (Fig. 3B and Supplemental Dataset 5). There was substantial overlap in DEGs, with 2,511 of the transcripts identified being shared between both comparisons (Fig. 3C). However, 1,755 genes were unique in the inner zone comparison and 381 genes were unique in the outer zone tissue to passaged cell comparison.
Fig. 3.
Comparison of Tissue vs. Passage 3 (p3) Monolayer Meniscus Cells using Differentially Expressed Gene Analysis and Gene Set Enrichment Analysis (GSEA). Volcano plots show genes significantly differentially expressed between (A) inner zone tissue compared to p3 inner zone meniscus cells and (B) outer zone meniscus tissue versus p3 outer zone meniscus cells. Each data point is an individual gene. Dashed lines indicate cutoffs for significant genes (FDR adjusted p-value < 0.01 and a |base-2 log fold-change|> 1). Color denotes if the gene was significantly up-regulated (blue) or down-regulated (green) in meniscus tissue relative to P3 cells, or not significantly different (black). (C) Number of significantly differentially expressed genes (DEGs) exclusive to inner zone tissue compared to inner zone p3 cells, outer zone tissue compared to outer zone p3 cells, or found in both zones compared to their respective p3 cells. (D) GSEA of inner meniscus tissue versus inner p3 meniscus cells and outer meniscus tissue versus outer p3 meniscus cells. Normalized enrichment scores (NES) of pathways significantly regulated by monolayer culturing in both inner and outer zone samples are shown. A NES > 0 (blue) identifies upregulated pathways and NES < 0 (green) identifies downregulated pathways.
To better identify biological processes associated with gene expression differences between meniscus tissue and passaged cells, Gene Set Enrichment Analysis (GSEA) using the KEGG geneset database was performed. Numerous overlapping KEGG pathways (30 pathways) with an FDR < 0.25 were found between inner and outer zone tissue comparisons to passaged cells from the corresponding zone (Fig. 3D). Nine of these pathways were upregulated in meniscus tissue relative to monolayer cultured cells, while 21 pathways were upregulated in monolayer cells.
When comparing the top 25 genes expressed in passaged inner zone cells compared to inner zone tissue (Table 7) and passaged outer zone cells compared to outer zone tissue (Table 8), genes associated predominantly with the cytoskeleton and ECM were upregulated. In particular, upregulation of genes in the actin, myosin, and tropomyosin families were evident. In the top 25 most expressed genes, the most highly DEGs in passaged inner zone cells relative to tissue were COL1A1 (logFC = 13.40), ACTA2 (logFC = 12.27), and TAGLN (logFC = 6.88). In the passaged outer zone cells relative to tissue, the most highly DEGs were SFRP2 (logFC = 10.38), ACTA2 (logFC = 7.95), and IGFBP2 (logFC = 7.89). Twenty-two of the 25 genes were upregulated in passaged cells from both zones compared to tissue from the corresponding zone.
Table 7.
Top 25 expressed genes in inner zone P3 meniscus cells compared to inner zone tissue.
| Gene symbol | Gene name | LogFC | padj |
|---|---|---|---|
| Extracellular Matrix | |||
| COL1A1 | Collagen Type I Alpha 1 Chain | 13.40 | 4.58E-54 |
| COL1A2 | Collagen Type I Alpha 2 Chain | 5.34 | 2.13E-38 |
| FBN1 | Fibrillin 1 | 4.63 | 8.31E-37 |
| COL5A1 | Collagen Type V Alpha 1 Chain | 3.87 | 5.65E-16 |
| COL5A2 | Collagen Type V Alpha 2 Chain | 3.37 | 7.73E-28 |
| COL3A1 | Collagen Type III Alpha 1 Chain | 3.08 | 7.78E-15 |
| FLNA | Filamin A | 3.04 | 6.53E-16 |
| COL11A1 | Collagen Type XI Alpha 1 Chain | 2.83 | 1.26E-07 |
| THBS1 | Thrombospondin 1 | 2.56 | 1.33E-07 |
| TNC | Tenascin C | 2.11 | 1.30E-04 |
| SPARC | Secreted Protein Acidic and Cysteine Rich | 2.06 | 5.23E-06 |
| COL6A1 | Collagen Type VI Alpha 1 Chain | 1.79 | 4.38E-06 |
| Cytoskeleton | |||
| ACTA2 | Actin Alpha 2, Smooth Muscle | 12.27 | 3.67E-44 |
| TAGLN | Transgelin | 6.88 | 2.34E-21 |
| TPM1 | Tropomyosin 1 | 5.17 | 1.80E-169 |
| CALD1 | Caldesmon 1 | 5.11 | 3.09E-45 |
| MYH9 | Myosin Heavy Chain 9 | 3.26 | 1.04E-11 |
| TPM4 | Tropomyosin 4 | 2.59 | 1.46E-35 |
| ACTB | Actin Beta | 1.79 | 2.16E-09 |
| ACTG1 | Actin Gamma 1 | 1.43 | 3.64E-14 |
| VIM | Vimentin | 2.50 | 1.94E-19 |
| LMNA | Lamin A/C | 1.65 | 5.87E-15 |
| Growth factor | |||
| IGFBP5 | Insulin Like Growth Factor Binding Protein 5 | 1.87 | 2.31E-05 |
| Transcription Factor | |||
| AEBP1 | AE Binding Protein 1 | 3.92 | 2.57E-10 |
| FSTL1 | Follistatin Like 1 | 2.19 | 1.93E-20 |
Genes with logFC > 1.
Filtered by padj < 0.01.
Genes in bold were selected for qRT-PCR validation.
Table 8.
Top 25 expressed genes in outer zone P3 meniscus cells compared to outer zone tissue.
| Gene symbol | Gene name | LogFC | padj |
|---|---|---|---|
| Cytoskeleton | |||
| ACTA2 | Actin Alpha 2, Smooth Muscle | 7.95 | 1.33E-15 |
| TAGLN | Transgelin | 5.93 | 1.00E-12 |
| TPM1 | Tropomyosin 1 | 4.60 | 3.90E-107 |
| CALD1 | Caldesmon 1 | 4.15 | 8.46E-24 |
| MYH9 | Myosin Heavy Chain 9 | 2.51 | 6.75E-06 |
| TPM4 | Tropomyosin 4 | 2.19 | 2.51E-20 |
| ACTB | Actin Beta | 1.31 | 1.97E-04 |
| VIM | Vimentin | 1.86 | 7.22E-09 |
| LMNA | Lamin A/C | 1.33 | 5.05E-08 |
| Extracellular Matrix | |||
| COL1A1 | Collagen Type I Alpha 1 Chain | 6.66 | 4.19E-11 |
| COL1A2 | Collagen Type I Alpha 2 Chain | 4.09 | 4.49E-18 |
| COL4A2 | Collagen Type IV Alpha 2 Chain | 3.70 | 2.10E-03 |
| THBS1 | Thrombospondin 1 | 3.68 | 2.00E-11 |
| FBN1 | Fibrillin 1 | 3.66 | 2.21E-18 |
| COL5A1 | Collagen Type V Alpha 1 Chain | 3.64 | 3.03E-11 |
| COL3A1 | Collagen Type III Alpha 1 Chain | 3.59 | 1.32E-15 |
| COL5A2 | Collagen Type V Alpha 2 Chain | 2.85 | 6.46E-16 |
| FLNA | Filamin A | 2.46 | 1.58E-08 |
| COL11A1 | Collagen Type XI Alpha 1 Chain | 2.44 | 9.41E-05 |
| SPARC | Secreted Protein Acidic and Cysteine Rich | 2.40 | 3.50E-06 |
| COL4A1 | Collagen Type VI Alpha 1 Chain | 1.29 | 5.61E-03 |
| Growth factor | |||
| SFRP2 | Secreted Frizzled Related Protein 2 | 10.38 | 1.08E-15 |
| IGFBP2 | Insulin Like Growth Factor Binding Protein 2 | 7.89 | 2.13E-24 |
| IGFBP5 | Insulin Like Growth Factor Binding Protein 5 | 2.80 | 1.92E-08 |
| Transcription Factor | |||
| AEBP1 | AE Binding Protein 1 | 2.01 | 7.43E-03 |
Genes with logFC > 1.
Filtered by padj < 0.01.
Genes in bold were selected for qRT-PCR validation.
Comparison of the top 25 genes expressed in inner zone meniscus tissue compared to passaged inner zone meniscus cells (Table 9) and outer zone meniscus tissue compared to passaged outer zone meniscus cells (Table 10) identified upregulated genes associated with the following: ECM, including matrix proteins ACAN, COMP, DCN, and FN1; membrane glycoprotein MFGE8; mitochondria, including SOD2 and COX3, which are involved in oxidative phosphorylation; growth factors and regulators, including TPT1, which is a regulator of cell growth and proliferation; iron trafficking (LCN2) and storage (FTL and FTH1); ribosomal proteins, such as RPS2; signal transduction, including SAA3, which enables Toll-like receptor binding, and CLU, which has anti-apoptotic, anti-inflammatory and antioxidant properties in cartilage32; enzymes, including those involved in matrix degradation (CTSD) and metabolism (GAPDH); and secreted protein SAA1, which plays a role in inflammation and tissue injury. Nineteen of these highly expressed genes in meniscus inner and outer zone tissue were shared. The top-most DEGs in inner zone tissue compared to passaged cells were COL2A1 (logFC = 8.63), SAA3 (logFC = 8.47), and SAA1 (logFC = 8.05). In outer zone tissue, SAA3 (logFC = 9.65) and SAA1 (logFC = 9.31) were also among the most DEGs, as well as LCN2 (logFC = 7.79).
Table 9.
Top 25 expressed genes in inner zone tissue compared to inner zone P3 meniscus cells.
| Gene symbol | Gene name | LogFC | padj |
|---|---|---|---|
| Cell Membrane Protein | |||
| MFGE8 | Milk Fat Globule-EGF Factor 8 Protein | 3.32 | 1.58E-16 |
| Extracellular Matrix | |||
| COL2A1 | Collagen 2A1 | 8.63 | 1.49E-23 |
| HAPLN1 | Hyaluronan and Proteoglycan Link Protein 1 | 6.34 | 4.01E-28 |
| MGP | Matrix G1a | 6.03 | 4.43E-12 |
| SPP1 | Secreted Phosphoprotein 1 | 5.35 | 6.51E-38 |
| ACAN | Aggrecan | 4.54 | 6.18E-15 |
| TIMP3 | Tissue inhibitor of metalloproteinase 3 | 4.46 | 3.24E-27 |
| COMP | Cartilage oligomeric matrix protein | 4.35 | 1.19E-20 |
| DCN | Decorin | 2.88 | 1.74E-21 |
| PRELP | Proline and arginine rich end leucine rich repeat protein | 2.24 | 6.85E-06 |
| FN1 | Fibronectin 1 | 1.76 | 7.21E-04 |
| FMOD | Fibromodulin | 1.67 | 7.26E-05 |
| Enzyme | |||
| CTSD | Cathepsin D | 2.61 | 1.97E-15 |
| GAPDH | Glyceraldehyde-3-Phosphate Dehydrogenase | 2.02 | 2.09E-20 |
| Growth Factors and Regulators | |||
| TPT1 | Tumor protein, Translationally-Controlled 1 | 1.84 | 3.01E-11 |
| Iron Homeostasis | |||
| LCN2 | Lipocalin 2 | 6.88 | 4.46E-45 |
| FTL | Ferritin light chain | 2.76 | 4.88E-17 |
| FTH1 | Ferritin heavy polypeptide 1 | 2.73 | 1.29E-27 |
| Mitochondrial Protein | |||
| SOD2 | Superoxide dismutase 2 | 5.59 | 2.83E-61 |
| COX3 | Cytochrome C Oxidase subunit III | 1.47 | 7.80E-17 |
| Ribosomal Protein | |||
| RPS2 | Ribosomal protein S2 | 1.76 | 6.02E-10 |
| RPLP0 | Ribosomal protein lateral stalk subunit P0 | 1.15 | 4.00E-06 |
| Secreted Protein | |||
| SAA1 | Serum amyloid A protein 1 | 8.05 | 4.32E-04 |
| Signal Transduction | |||
| SAA3 | Serum amyloid A protein | 8.47 | 2.65E-86 |
| CLU | Clusterin | 5.33 | 6.74E-45 |
Genes with logFC > 1.
Filtered by padj < 0.01.
Table 10.
Top 25 expressed genes in outer zone tissue compared to outer zone P3 meniscus cells.
| Gene symbol | Gene name | LogFC | padj |
|---|---|---|---|
| Cell Membrane Protein | |||
| MFGE8 | Milk Fat Globule-EGF Factor 8 Protein | 2.44 | 1.91E-07 |
| Extracellular Matrix | |||
| SPP1 | Secreted Phosphoprotein 1 | 6.46 | 1.23E-43 |
| MGP | Matrix G1a | 6.16 | 6.58E-10 |
| ACAN | Aggrecan | 4.69 | 1.61E-12 |
| COMP | Cartilage oligomeric matrix protein | 2.86 | 1.57E-07 |
| DCN | Decorin | 2.64 | 2.22E-14 |
| CCDC80 | Coiled-coil domain containing 80 | 2.02 | 1.11E-03 |
| FN1 | Fibronectin 1 | 1.97 | 1.10E-03 |
| Enzyme | |||
| CTSD | Cathepsin D | 2.29 | 1.41E-09 |
| HTRA1 | Serine protease HTRA1 | 1.95 | 4.67E-06 |
| GAPDH | Glyceraldehyde-3-Phosphate Dehydrogenase | 1.86 | 7.47E-14 |
| CYP1B1 | CYP450 Family 1 Subfamily B Member 1 | 1.16 | 1.80E-03 |
| Growth Factors and Regulators | |||
| NBL1 | DAN Family BMP antagonist | 3.96 | 7.91E-34 |
| TPT1 | Tumor protein, Translationally-Controlled 1 | 1.12 | 6.62E-04 |
| Iron Homeostasis | |||
| LCN2 | Lipocalin 2 | 7.79 | 1.24E-45 |
| FTH1 | Ferritin heavy polypeptide 1 | 2.48 | 4.07E-18 |
| FTL | Ferritin light chain | 1.78 | 3.56E-06 |
| Mitochondrial Protein | |||
| SOD2 | Superoxide dismutase 2 | 5.19 | 5.06E-42 |
| SOD3 | Superoxide dismutase 3 | 3.05 | 2.17E-16 |
| ATP6 | ATP synthase 6 | 2.19 | 4.07E-13 |
| COX3 | Cytochrome C oxidase subunit III | 1.24 | 1.17E-09 |
| Ribosomal Protein | |||
| RPS2 | Ribosomal protein S2 | 1.23 | 2.46E-04 |
| Secreted Protein | |||
| SAA1 | Serum amyloid A protein 1 | 9.31 | 4.24E-04 |
| Signal Transduction | |||
| SAA3 | Serum amyloid A protein | 9.65 | 3.07E-89 |
| CLU | Clusterin | 4.90 | 4.42E-30 |
Genes with logFC > 1.
Filtered by padj < 0.01.
Quantitative RT-PCR was used to validate expression of ACTA2 (Fig. 4A), TAGLN (Fig. 4B), SFRP2 (Fig. 4C), and FSTL1 (Fig. 4D) in passaged cells and meniscus tissue obtained from both the inner and outer zones. Expression of each of these genes was upregulated in both the inner and outer zone passaged cells compared to inner and outer zone tissue (p < 0.0001). There were no detectable differences in expression between passaged cells obtained from the inner and outer zone or tissue obtained from either zone except for SFRP2, which was downregulated in inner zone meniscus tissue compared to outer zone meniscus tissue (p < 0.001).
Fig. 4.
qRT-PCR for (A) ACTA2, (B) TAGLN, (C) SFRP2, and (D) FSTL1 expression in passage 3 (P3) inner and outer zone meniscus cells and inner and outer zone meniscus tissue (n = 4/group). Results are presented as log-twofold change relative to the meniscus outer zone tissue. The line indicates the mean value for each group. Groups not sharing a letter are significantly different (p < 0.001).
Discussion
Despite the common comparison of inner zone meniscus cells to chondrocytes and outer zone meniscus cells to fibroblasts7,8,11, our findings show that inner and outer zone meniscus cells have a distinct transcriptomic profile and are more similar to each other than to either articular cartilage or passaged meniscus cells. When comparing by PCA, it is noted that inner meniscus tissue clusters slightly closer to cartilage and outer zone meniscus tissue clusters closer to monolayer cells. However, differences between meniscus tissue and articular cartilage or monolayer cells are much greater than differences between the zones of the meniscus. Similarly, DEG analysis shows that inner and outer zone meniscus tissues are more closely related to each other than to articular cartilage or monolayer cultured cells. More than 3,100 DEGs were identified between meniscus tissue and cartilage compared to 205 between inner and outer zone meniscus tissue. Of these DEGs between meniscus and cartilage, 1337 were shared between inner and outer meniscus, suggesting that the transcriptome of inner and outer meniscus cells differ from cartilage in a similar way. Interestingly, prior work using single cell RNA-Seq revealed new chondrocyte subtypes that appear in degenerated human meniscus tissue, suggesting that these chondrocyte populations in the meniscus tissue may be therapeutic targets33. When comparing meniscus tissue and passaged cells, more than 4,600 DEGs were identified. These data show that in situ meniscus cells are a distinct cell type, substantially different from chondrocytes in articular cartilage, and these cells undergo significant transcriptomic changes when passaged.
When looking at the top 25 abundantly expressed genes in cartilage compared to meniscus tissue, there were major differences in ECM-associated genes, as well as genes associated with signal transduction and transcriptional regulation. When comparing cartilage to outer zone meniscus tissue, several expected genes, including COL2A1 and ACAN were identified. Cartilage experiences more compressive loads than the outer zone of the meniscus. Therefore, in cartilage, the relatively increased expression of genes that encode negatively-charged proteoglycans34 that can resist compressive loads, such as ACAN, FMOD, and BGN, is expected. Interestingly, this analysis also identified the upregulation of numerous ribosomal proteins in cartilage compared to outer zone tissue. While ribosomal proteins play an essential role in protein translation, their extra-ribosomal functions, including roles in tumorigenesis, immune signaling, and development35 may prove to be interesting targets for future studies. On the other hand, the top 25 expressed genes in meniscus compared to cartilage include previously studied genes such as COL1A2, as well as several other genes associated with ECM, mitochondrial proteins, signal transduction, and proteins related to transcriptional regulation36,37. Increased expression of genes encoding mitochondrial proteins in meniscus tissue compared to cartilage, such as CYP1B1 and CTYB, may point to differences in metabolism between the tissues, while increased expression of actin family genes CAPG, ACTB, and ACTG1 may indicate differences in cell mechanobiology, migration, and/or motility38.
PCR validation of several genes showed trends that largely aligned with our RNA-seq results. In particular, meniscus tissue showed significantly higher expression of LUM, PRRX1, and SNTB1 compared to cartilage. Previous studies in mice homozygous for a null mutation of the small leucine-rich proteoglycan lumican showed reduced tensile strength, as well as thicker collagen fibrils and non-uniform interfibrillar spacing in other connective tissues39. Therefore, increased expression of LUM in meniscus may indicate an important role of lumican in collagen structure, function, and matrix organization. Prior work has shown upregulation of LUM in meniscus but not cartilage following anterior cruciate ligament transection in rats40 but an increase in lumican fragmentation in degenerated human cartilage and menisci41. PRRX1, a transcription factor that binds to the promoter region of TGF-β1, is associated with early limb development, joint morphogenesis42,43, regulation of proliferation and differentiation44, and can promote meniscus repair45. While not one of the most abundantly expressed genes in the inner or outer zone meniscus tissue, SNTB1 was highly upregulated in both the inner and outer zone tissue compared to cartilage. While the specific role of SNTB1 has not been studied in meniscus, its role in Wnt/β-catenin signaling has implications in OA development46–48. Interestingly, expression of PHLDA1 and AEBP1 was specific to meniscus tissue zone with PHLDA1 expression increased in inner zone meniscus tissue compared to cartilage and AEBP1 expression decreased in inner zone meniscus tissue compared to cartilage and outer zone meniscus. PHLDA1 encodes a protein involved in apoptosis and upregulation of this gene is associated with reduced cell growth and cell death49,50, while AEBP1 encodes for secreted aortic carboxypeptidase-like protein (ACLP), which plays a role in collagen polymerization27. Variants in AEBP1 cause ultrastructural changes in collagen organization and have been identified in patients with Ehlers-Danlos27, which clinically results in poor wound healing, joint hypermobility, and osteoporosis51. The differential expression of these two genes may have interesting implications in the collagen structure and differential healing abilities between the inner and outer zones. Overall, these robustly and differentially expressed genes provide interesting targets for further research in distinguishing between cartilage and meniscus tissue.
Our findings illustrate the global transcriptomic differences between inner and outer zone meniscus tissue. The larger number of upregulated genes in the outer zone is likely due to the presence of vasculature and vascular-associated cells in the outer zone, which are not found in the inner avascular zone52. Furthermore, differences in gene expression profiles between the zones may also be driven by the structural adaptations of the tissue, which is built to withstand the different magnitudes of compressive and tensile strains53 in vivo. The expression and arrangement of collagen fibers in the meniscus tissue is critically important for resisting the circumferential hoop stresses experienced by the tissue. The outer zone expressed higher levels of several collagens than the inner zone, including COL1A1, COL4A2, and COL18A1. CILP was the only ECM component that was upregulated in the inner zone compared to outer zone but the function of CILP in the meniscus tissue is currently unclear. Interestingly, CILP protein levels are decreased in end-stage OA menisci54; however, this study did not compare inner and outer zone protein concentrations. While PCA largely showed separation of inner and outer zone meniscus tissue, there was one outer zone sample that clustered with the inner zone tissue samples. There is no physical delineation between the inner and outer zones of the meniscus. Indeed, some studies separate the outer third from the inner two-thirds10,17,18, while others separate at the midline of the tissue55 or into thirds9,11. Explants for RNA-seq were harvested from the inner two-thirds and outer one-third of the meniscus. Interestingly, the two meniscus tissue samples that overlapped in the PCA were taken closer to the anterior and posterior horn, while the other samples were taken closer to the mid-body of the meniscus. It may be that the inner/outer delineation becomes less relevant approaching the horn region and/or that the cells of the horn region are a third distinct phenotype56. Indeed, it is likely that meniscus cells vary in all three dimensions, as differences between cells of the superficial and deep regions have also been reported11,57. To diminish the confounding effects of potential tissue overlap, samples obtained for qRT-PCR validation were harvested from only the inner one-third and outer one-third of the meniscus tissue, which may explain some of the differences detected between the RNA-seq findings and qRT-PCR. This further separation allowed the detection of significantly lower expression of AEBP1 and SFRP2 in the inner zone tissue as compared to the outer zone tissue. SFRP2 is a marker for bone marrow-derived stem cells and skeletal stem cell multipotency58. Given the vascular distribution in meniscus tissue, it is possible that multipotent stem cells may be localized more to the outer zone than the inner zone. While our study has identified overall transcriptomic differences between zones, further work must be performed to identify distinct cell populations between zones of the meniscus. Single-cell sequencing techniques have been utilized to elucidate the cellular landscape of human menisci and how these change with tissue degeneration33,59. Leveraging single-cell sequencing analysis may help to determine whether these zonal transcriptomic differences represent phenotypic changes of a single predominant meniscal cell type or changing proportions of multiple cell types.
In this study, we also revealed profound global transcriptomic changes that occur due to passaging of meniscus cells. There are more DEGs and larger fold-change differences between meniscus tissue and passaged cells than any other pairwise comparison. The passaged cells were not donor-matched to the tissue samples, which may have decreased the specificity of RNA-seq to identify changes specific to monolayer passaging. However, the qPCR validation of select genes was performed in a separate cohort of donor samples for both tissue and isolated cells, supporting the generalizability of the measured DEGs. The majority of DEGs and KEGG pathways were shared between inner and outer zone comparisons, suggesting that passaging results in cells that are transcriptionally similar regardless of zone of origin and that the process of dedifferentiation may be similar as well. This is also evidenced by the tight clustering of inner and outer zone cells on the PCA plots. When analyzing GSEA data, metabolic pathways were well-represented in the comparison of meniscus tissue to passaged cells. Oxidative phosphorylation, pentose phosphate pathway, and steroid biosynthesis were all more highly expressed in tissue than passaged cells, while citrate cycle (TCA), nitrogen metabolism, and inositol phosphate metabolism were more highly expressed in passaged cells, suggesting a major shift in energy metabolism that occurs with cellular dedifferentiation. Passaged cells also exhibited increased expression of pathways related to cell–cell and cell–matrix interactions, including adherens junction, focal adhesion, tight junction, and ECM-receptor interaction, indicating transcriptional changes related to their physical environment. Identification of some unexpected pathways, such as arrhythmogenic right ventricular cardiomyopathy (ARVC) and viral myocarditis, were likely due to cytoskeletal changes as many of the top genes driving these associations are members of the integrin, myosin, and actin families. Given that the tissue explants were maintained in the same culture medium as the monolayer cells for three days before RNA extraction, these transcriptomic findings are not attributable to in vitro culture. However, alterations in the physical microenvironment likely predominantly drive these changes and our findings reflect the effect of removing the three-dimensional ECM-cell interactions and placing cells in a two-dimensional culture dish. While it is known that primary meniscus cells dedifferentiate in culture8–12, prior studies have largely focused on just a few genes based on changes observed during chondrocyte dedifferentiation. Therefore, our data provides an unbiased comprehensive picture of the global gene expression changes in both the inner and outer zone cells due to passaging and provides a broader set of meniscus dedifferentiation markers that can be utilized in meniscus biology and tissue engineering studies to recapitulate the native meniscus phenotype.
When assessing the top 25 expressed genes in monolayer passaged meniscus cells, numerous cytoskeleton-associated transcripts, particularly in the actin and tropomyosin families, were upregulated compared to meniscus tissue. Changes in expression of ACTA2, smooth muscle alpha-2 actin, and TGLN, an early marker of smooth muscle differentiation, together with alterations in actin polymerization and cell morphology have previously been implicated in chondrocyte dedifferentiation60. These changes in gene expression indicate an acquisition of contractile properties, which may have major implications for tissue engineering applications. ACTA2 expression has been identified as an important marker in meniscus tissue healing61 and smooth muscle actin immunostaining is increased in the superficial zone of torn menisci but is absent in normal and OA menisci57. However, studies in chondrocytes have shown that excessive tissue contraction and shrinkage secondary to expression of contractile proteins may prevent integration of implanted tissues60. Therefore, further work is needed to understand the role of ACTA2 in meniscus tissue healing. FSTL-1, which was also upregulated by meniscus cell passaging, is associated with activation of NF-κB signaling pathways, an important pathway for inflammatory signaling and OA development62,63, and thus its upregulation in passaged cells may have implications for in vitro OA studies. Finally, expression of both SFRP2 and TAGLN were upregulated in passaged cells compared to meniscus tissue. SFRP2, a modulator of canonical Wnt signaling, is expressed in the inner and middle regions of the embryonic mouse meniscus64. While the roles of these genes have not been studied in meniscus, their increased expression in passaged meniscus cells suggests these are markers of the dedifferentiated passaged meniscal cells. Transcriptomic data from this study serves as an important foundation for exploring the factors and pathways that drive dedifferentiation and provides benchmarks for restoring the native meniscus phenotype.
Future studies are necessary to address some of the limitations of this work. In this study, due to tissue availability from our abattoir, we were only able to assess female tissues. Analyzing the effect of sex on these transcriptomic findings is an important next step. Furthermore, in this study, we used a 3-day preculture period for the tissue samples prior to RNA extraction. This step was intended to standardize culture conditions experienced by the tissues and cells to allow direct comparison of the expression profiles of the cells existing in the native 3D ECM versus monolayer culture on tissue culture plastic, including nutrient and gas composition of the media and incubation temperature. The preculture period most likely resulted in transcriptomic changes relative to the in vivo state12. However, in this study, the preculture period allowed us to compare tissues and cells that had experienced similar culture conditions to isolate the effects of the 3D ECM and cell passaging. In addition to comparisons of freshly isolated porcine tissues, it would also be interesting to investigate differences in human meniscus and cartilage samples.
Overall, we have identified and characterized the global transcriptomic differences between cartilage, inner and outer zone meniscus tissue, and passaged meniscus cells, allowing us to reveal differences between these connective tissues and changes in phenotype due to monolayer culturing. Comparing the expression profiles of cultured meniscus cells to meniscus tissue resident cells has revealed the radical transcriptomic changes that occur during in vitro cell passaging. These results provide comprehensive, unbiased transcriptomic changes and numerous novel targets for characterizing inner and outer zone meniscus cells and tissue constructs for regenerative medicine approaches. We have validated LUM, PRRX1, and SNTB1 as markers for meniscus tissue and ACTA2, TAGLN, SFRP2, and FSTL1 as novel markers for meniscus cell dedifferentiation. Finally, this work provides comprehensive meniscus gene expression profiles and can serve as a resource for future studies of meniscus cell biology, regenerative medicine, and tissue engineering.
Materials and methods
RNA sequencing experiments
Tissue sample collection
Porcine stifle joints from skeletally mature 2–3 year old female pigs were obtained from a local abattoir. Explants from cartilage (N = 3) were harvested using a scalpel to remove full thickness sections of articular cartilage from the femoral surface, which were approximately 5 mm x 5 mm. Meniscus tissue explants were obtained using a 5 mm biopsy punch inserted perpendicular to the femoral surface of the meniscus. Explants were cut to approximately 2 mm in depth, preserving the femoral surface for culture. Explants were taken from the inner two-thirds and outer one-third of the medial menisci (N = 3/zone; Supplemental Fig. 2) and placed in DMEM-HG (DMEM-HG; Catalog # 11,995,073; Gibco, Carlsbad, CA) with 10X antibiotic/antimycotic (Catalog # 15,240,062; Gibco) for one hour. Explants were subsequently maintained in culture media composed of DMEM-HG, 10% fetal bovine serum (Catalog # SH30396.03; HyClone, Logan, UT), 1X non-essential amino acids (Catalog # 11,140,050; Gibco), 1X antibiotic/antimycotic, 10 mM HEPES (Cat # 15,630,080; Gibco), 40 μg/mL L-proline (SKU # H54409; Sigma-Aldrich, St. Louis, MO), and 50 μg/mL ascorbate (SKU # A8960; Sigma-Aldrich). Media was changed the day after explants were harvested and explants were cultured for an additional two days at 37 °C, 5% CO2 for a total of three days. Samples were rinsed in phosphate buffered saline (PBS; catalog # 10,010–023; Gibco), flash frozen in liquid nitrogen, and stored at -80 °C until RNA extraction.
Tissue RNA extraction
Explants were pulverized in TRIzol (Catalog # 15,596,026; Life Technologies, Carlsbad, CA) using a freezer mill (Model 6875; SPEX SamplePrep, Metuchen, NJ) under liquid nitrogen12,21. Samples underwent three, two-minute cycles at maximum frequency with two minutes precooling between cycles. RNA was extracted by TRIzol/chloroform separation, resuspended in lysis buffer, and column purified according to the Norgen Total RNA Purification Plus extraction kit protocol (Catalog # 48,300; Norgen Biotek Corp, Ontario, Canada).
Cell isolation and monolayer culture
Meniscus cells were isolated from the outer one-third or inner two-thirds of the porcine medial meniscus (N = 3 inner zone, N = 2 outer zone). Tissue was minced and processed by sequential digestion with 0.5% (w/v) pronase (SKU 53,702; EMD Millipore Corp., Temecula, CA) for one hour and 0.2% (w/v) collagenase type I (Catalog # LS004197; Worthington Biochemical Corp., Lakewood, NJ) for 16 h in complete culture media12,21. Cells were seeded at 5 million cells per 10 cm tissue culture plate and maintained in complete culture media. Media was replaced every 2–3 days and cells were passaged at 90% confluence (5–6 days for passage 1 and then 2–3 days for subsequent passages). Cell pellets were collected at the third passage, flash frozen in liquid nitrogen, and stored at -80 °C until RNA extraction was performed using the Norgen Total RNA Purification Plus extraction kit.
RNA sequencing
RNA-seq was performed by the Duke Center for Genomic and Computational Biology (Durham, NC). Reads that were 20nt or longer after trimming were mapped to the Sscrofa11.1v91 version of the pig genome and transcriptome65, using the STAR RNA-seq alignment tool66. Subsequent analysis was performed only for genes that mapped to a single genomic location. Using the HTSeq tool, gene counts were compiled, and subsequent analysis performed on genes with at least 10 reads. Normalization and differential expression analyses were performed using the DESeq2 Bioconductor67 package within the R statistical programming environment. Principal component analysis (PCA) was performed using the variance stabilizing transformation of DESeq normalized gene counts. False discovery rate (FDR) was calculated to control for multiple hypothesis testing of differential expression for pairwise comparisons. Genes expression changes with a base-2 log fold-change (LogFC) magnitude greater than one (|LogFC|> 1) and FDR adjusted p-value < 0.01 (padj < 0.01) were considered significant. The “Top 25 Expressed Genes” tables were created by identifying the most highly expressed genes by transcript count and filtering based on logFC > 1 and padj < 0.01 when compared to the second group. Gene set enrichment analysis (GSEA)68,69 was performed using the KEGG pathway geneset database70 comparing inner and outer zone meniscus tissue to passaged cells from corresponding zones. Genesets with FDR q-value < 0.25 that were commonly up- and down-regulated in both inner tissue versus passaged inner cells and outer zone tissue versus passaged outer cells were identified.
Gene expression validation experiments
Sample collection
Cartilage (N = 13), meniscus tissue (N = 13/zone), and passaged meniscus cells (N = 3/zone) were isolated from 2–3 year old skeletally mature female pigs to validate the RNA- seq results. Meniscus tissue was harvested from the outer one-third and inner one-third of the medial menisci to definitively separate the inner and outer meniscal zones (Supplemental Fig. 2). All other harvest, culture, and RNA extraction procedures were performed as described above for both explants and cells.
Reverse transcription-quantitative polymerase chain reaction
RNA samples were reverse transcribed using the SuperScript VILO complementary DNA Synthesis Kit (Catalog # 11,754,050; Life Technologies). Quantitative polymerase chain reaction (qPCR) was performed using PowerUP SYBR Green Master Mix (Catalog # A25776; Thermo Fisher Scientific Baltics UAB) and the StepOne Plus Real-time PCR System (Model 4,376,374; Applied Biosystems). Supplemental Table 1 lists the porcine specific PCR primers for each target. Relative fold-change was determined by the 2-∆∆Ct method71 using 18S as a reference gene and a single cartilage (Fig. 2) or outer zone meniscus tissue (Fig. 4) sample as the calibrator. There was no expression detected for SNTB1 in 4 cartilage samples so a Ct of 40 (equivalent to the maximum number of PCR cycles) was assigned. Results are presented as log2 fold-change relative to the geomean of the indicated group. A Grubbs’ test was performed to identify outliers, which were subsequently removed. For data that was normally distributed, one-way ANOVAs with Tukey’s post-hoc analyses were performed. LUM expression was not normally distributed and thus a Kruskal–Wallis test with Dunn’s multiple comparison was performed. Groups were considered statistically significant at p < 0.05.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Acknowledgements
We thank Dr. David Corcoran from the Duke Genomic Analysis and Bioinformatics Core for assistance with the RNASeq analysis.
Author contributions
The study was conceived and designed by BDA and ALM. KF, BDA, AMK, and DADC were responsible for data collection and analysis. KF, BDA, AMK, and ALM participated in statistical analysis and interpretation of the data. KF and BDA drafted the manuscript, and all authors participated in the revision process. All authors have critically evaluated the manuscript, approved the final version, and are responsible for the integrity of the manuscript.
Funding
This work was supported in part by NIH grants AR073221, AR079184, AR078245, and T32GM144291.
Data availability
The datasets presented in this study can be found in the NCBI Gene Expression Omnibus (GEO) under accession numbers GSE275100.
Declarations
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
These authors have contributed equally to this work and share first authorship.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
The datasets presented in this study can be found in the NCBI Gene Expression Omnibus (GEO) under accession numbers GSE275100.