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Human Migratory Meniscus Progenitor Cells Are Controlled via the TGF-β…

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 타이틀

- Human Migratory Meniscus Progenitor Cells Are Controlled via the TGF-β Pathway

 저자

- Hayat Muhammad et al

 게시일

- 11 November 2014,

 게시장소

- Volume 3, Issue 5, Pages 789–803



 

Summary

Degeneration of the knee joint during osteoarthritis often begins with meniscal lesions. Meniscectomy, previously performed extensively after meniscal injury, is now obsolete because of the inevitable osteoarthritis that occurs following this procedure. Clinically, meniscus self-renewal is well documented as long as the outer, vascularized meniscal ring remains intact. In contrast, regeneration of the inner, avascular meniscus does not occur. Here, we show that cartilage tissue harvested from the avascular inner human meniscus during the late stages of osteoarthritis harbors a unique progenitor cell population. These meniscus progenitor cells (MPCs) are clonogenic and multipotent and exhibit migratory activity. We also determined that MPCs are likely to be controlled by canonical transforming growth factor β (TGF-β) signaling that leads to an increase in SOX9 and a decrease in RUNX2, thereby enhancing the chondrogenic potential of MPC. Therefore, our work is relevant for the development of novel cell biological, regenerative therapies for meniscus repair.

Graphical Abstract

 

Image for unlabelled figure

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Introduction

In the elderly, osteoarthritis (OA) is the most common musculoskeletal disease (Reginster, 2002) and will be the fourth leading cause of disability by the year 2020 (Woolf and Pfleger, 2003). The knee is particularly prone to meniscal lesions that lead to OA (Englund et al., 2012), and a high interdependency of OA and meniscus lesions has been described (Brophy et al., 2012). In fact, meniscal injuries are the most common knee injury and account for more than 50% of the 1.5 million knee arthroscopies performed annually (Englund et al., 2008 and Lohmander et al., 2007). The prevalence of meniscal tears increases with age (Loeser, 2013) and may be as high as 56% in men aged 70–90 years old (Englund et al., 2008). Lack of robust meniscal repair in adults with or without surgical intervention has led to the development of allografts or bioengineered meniscal substitutes (Haddad et al., 2013 and Steinert et al., 2007), and, whereas these fill the space void created following removal of the meniscus, clinical, radiological, and MRI evaluations show no protection against the development of OA (Hommen et al., 2007). The specific reasons for this lack of effect are unknown; however, a failure to successfully remodel the allograft into living tissue is one likely factor (Steadman and Rodkey, 2005). Almost all patients eventually require joint replacement (Lohmander and Roos, 2007).

The meniscus is best described as a fibrocartilage (Benjamin and Evans, 1990) comprising an outer, “red vascularized” part and an inner “white, unvascularized” part harboring round fibrochondrocytes (Hellio Le Graverand et al., 2001). Additionally, the outer surface of the meniscus is enclosed by a superficial layer with flattened, elongated fibroblast-like cells that predominantly synthesize collagen type I (McDevitt and Webber, 1990), whereas the round fibrochondrocytes from the inner part also produce collagen type II (Chevrier et al., 2009).

It has long been known that the outer, vascularized part of the meniscus is often able to successfully repair itself after injury allowing for normal meniscus function, whereas the inner meniscus produces a repair tissue in response to injury but cannot functionally regenerate. Experimental animal studies support the idea that the meniscus possesses regeneration activity. For example, fibrochondrocytes have been isolated from bovine meniscus tissue (Mauck et al., 2007) and migrating cells are found in healthy adult rabbit meniscus (Webber et al., 1989). Regeneration of the vascular, outer part of the meniscus might be due to the presence of CD34-positive cells (Osawa et al., 2013) or to mesenchymal cells that can be released by collagenase digestion of human meniscus (Segawa et al., 2009). Because application of growth factors and fibrin clots have elicited responses by cells resident in the inner meniscus, several authors have speculated about the importance of activation of meniscal repair cells (Petersen et al., 2005). Up until today, the question remains, as to whether the inner, avascular part of the human meniscus harbors multipotent cells capable of regeneration.

Here, we describe isolation of human meniscus progenitor cells (MPCs) from the inner meniscus harvested from late-stage OA patients prior to knee replacement and show that the regenerative potential of these cells is governed by transforming growth factor β (TGF-β) signaling.

Results

Meniscus Tissue Histology and Molecular Composition

By combining and modifying available classification systems for OA specimens (Pauli et al., 2011 and Zhang et al., 2011), we developed a means to discriminate healthier human menisci from their diseased counterparts. Intact meniscus tissue is composed of a superficial zone with flattened cells that primarily synthesize collagen type I (McDevitt and Webber, 1990). This architecture remains in healthier human menisci obtained from patients suffering from OA but is always absent in more disease samples (Figures 1A and 1B). The inner zone, containing more rounded cells that secrete collagen type I and type II, is present regardless of disease severity (Figures 1C and 1F). In diseased human menisci (score greater 4), 23% of the samples exhibit abnormal calcifications (Figure 1D) and 53% display anomalous clusters of cells (Figure 1E).


Characterization of Human Healthy and Diseased Meniscus Tissue(A) ...
Figure 1. 

Characterization of Human Healthy and Diseased Meniscus Tissue

(A) Immunohistochemistry of collagen type I in healthier meniscus (arrow); inset: low magnification of a human healthier meniscus stained for collagen type I; the black line indicates the border between the vascular part on the right side and the avascular part on the left side.

(B) Healthier human meniscus with an intact superficial zone and flattened cells (arrow).

(C) Diseased meniscus with a completely degenerated superficial zone where only the round cells of the inner zone remain (arrow).

(D and E) Calcifications (D) and cell clusters (E) are signs of OA.

(F) Immunohistochemistry of collagen type II in diseased meniscus (arrows); inset: low magnification of a human diseased meniscus stained for collagen type II. Magnification in (A)–(F), scale bar, 150 μm; inset bar, 1.7 cm.

(G) Heatmap of the top 100 differentially expressed genes with p values < 0.001. The red color indicates upregulated genes, and the blue color indicates downregulated genes. The upregulated genes in diseased meniscus compared with healthier tissue are ACTR2, KRT, HSPD1, CALU, HSPA9, BAX, CCDC80, C17orf59, HSP90AB1, CDC42, HNRNPH1, FKBP9, MT1DP, G3BP1, CTTN, COL6A1, TAF15, ELAVL1, MCL1, AL, HSPA4, CXorf40A CXorf40B, TCP1, RP5, VHL, PRPF4, BAG5, RIOK3, FCF1, SPTLC1, APH1A, RNF170, RCN3, DENR, CAPZB, MBNL1, CASC4, ASB1, MRPS10, RAB23, PAAF1, FARSB, NASP, NUDC, ZNF346, RIOK1, and GAR1. The downregulated genes in diseased meniscus compared with healthier tissue are RNF103, SLC41A3, SNX19, GTFIP1, ERF, NBPF10 NBPF3, OSBPL10, ADAM15, KIAA0930, ZMAT3, AL845464.3, ANTXR1, ENDOD1, ZNFX1, GLT8D2, RP5-1022P6.4, ENG, POMZP3, C11orf95, LPCAT1, EPN2, POMZP3, RAB11FIP3, TBC1D2B, PCDHGC3, MMP14, TRPM4, HDAC5, ABL1, SOX9, Antxr1, BCAN, ZP3, ABR, SNX33, RPS6KA4, CXCR7, NCOR2, PLEC, GPC1, SEMA3C, PDGFRB, LUM, ACAN, CSPG4, OAS1, TIMP2, VCAM1, LAM5, PRSS54, PCDHB1, and FMOD.

(H) Selected microarray data of genes that are known from the literature to be highly expressed in healthy hyaline cartilage tissue (SOX9, PDFGFRB, ACAN, TIMP2, and FMOD). These genes were significantly downregulated in cells derived from diseased tissue compared with cells from healthier meniscus tissue. Only COL6, as indicator gene of an active pericellular matrix of chondrocytes (Poole et al., 1992), is upregulated.

(I) Peptide abundance of selected genes shown as the mean of four experiments that analyzed cells that migrated out from meniscus from healthier and diseased samples in vitro (SD).

Figure options

Using microarrays, we then analyzed cells obtained by culturing tissue explants from the inner zone of diseased and healthier human menisci. We focused on the top 100 most abundantly expressed genes, and among these, we identified 48 as being upregulated, i.e., exhibiting a positive fold change, and 52 as being downregulated, i.e., exhibiting a negative fold change (p value <0.001, as listed in the figure legend). Among these top 100 genes, we found only four of the 15 arbitrarily defined potential marker genes: TIMP2, SOX9, ACAN, and MMP14, stressing the importance of these particular genes for discriminating healthier meniscal samples from diseased ones. When we analyzed eight human meniscus tissue samples, the 100 top genes clustered into two groups, healthier and diseased, as shown in the heatmap (Figure 1G). A selection is listed (Figure 1H), and the complete results can be found under GEO (accession number GSE52042).

To complement our microarray analyses, we performed proteomics analyses on cells grown from the healthier and diseased meniscus samples and could identify approximately 4,000 proteins as expressed by meniscal explant cells. However, only a small number of proteins produced by meniscal cells are known to be relevant for OA and meniscus pathology (as listed in Figure 1I), suggesting that for the meniscus, like other tissues studied so far, the correlation between transcriptome and proteome data is weak (Haider and Pal, 2013). Upon close examination, several signaling pathway mediators stood out as being differentially expressed between cells from healthier and diseased meniscal samples. In particular, SMAD2, a mediator of the canonical TGF-β/activin signaling pathway was more abundant in healthier meniscus cells (Figure 1I; for a full listing, visit http://www.miosge.med.uni-goettingen.de/de/?id=17). To follow up on this finding, we performed immunohistochemical (IHC) staining and found that meniscus samples that received a high disease score exhibited a reduced IHC staining for TGF-β and SMAD2. Consistent with these findings, proteome analysis and western blotting of diseased specimens also showed a reduction in SMAD2 protein and an upregulation of RUNX2 compared to healthier specimens. These results, together with existing literature, indicated that the TGF-β/BMP pathway, with its dual osteogenic and chondrogenic actions (Massagué, 2012), was a good candidate to investigate in greater detail.

TGF-β/BMP Signaling in Human Osteoarthritic Meniscus Tissue

Because the deregulation of TGF-β family proteins has been described to be important for meniscus pathology (de Mulder et al., 2013), we examined the TGF-β status of our samples. We observed greater staining for TGF-β3 in healthier (Figure 2A) tissue compared with diseased (


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