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. 2008 Feb;56(2):125-38.
doi: 10.1369/jhc.7A7320.2007. Epub 2007 Oct 15.

Chondroitin sulfate sulfation motifs as putative biomarkers for isolation of articular cartilage progenitor cells

Anthony J Hayes  1 , Debbie Tudor, Mari A Nowell, Bruce Caterson, Clare E Hughes
Affiliations

Chondroitin sulfate sulfation motifs as putative biomarkers for isolation of articular cartilage progenitor cells

Anthony J Hayes et al. J Histochem Cytochem. 2008 Feb.

Abstract

Osteoarthritis is a chronic, debilitating joint disease characterized by progressive destruction of articular cartilage. Recently, a number of studies have identified a chondroprogenitor cell population within articular cartilage with significant potential for repair/regeneration. As yet, there are few robust biomarkers of these cells. In this study, we show that monoclonal antibodies recognizing novel chondroitin sulfate sulfation motif epitopes in glycosaminoglycans on proteoglycans can be used to identify metabolically distinct subpopulations of cells specifically within the superficial zone of the tissue and that flow cytometric analysis can recognize these cell subpopulations. Fluorochrome co-localization analysis suggests that the chondroitin sulfate sulphation motifs are associated with a range of cell and extracellular matrix proteoglycans within the stem cell niche that include perlecan and aggrecan but not versican. The unique distributions of these sulphation motifs within the microenvironment of superficial zone chondrocytes, seems to designate early stages of stem/progenitor cell differentiation and is consistent with these molecules playing a functional role in regulating aspects of chondrogenesis. The isolation and further characterization of these cells will lead to an improved understanding of the role novel chondroitin sulfate sulfation plays in articular cartilage development and may contribute significantly to the field of articular cartilage repair.

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Figures

Figure 1
Figure 1
Distribution of novel chondroitin sulfate (CS) sulfation motif (SM) epitopes (green labeling) in immature bovine articular cartilage after immunohistochemical (IHC) labeling with monoclonal antibodies 3B3(−) (A–C), 7D4 (D–F), and 4C3 (G–I). Cell nuclei shown in red. Arrowheads in A,D,G denote pericellular labeling associated with subpopulations of superficial zone cells. Inset in F shows IHC labeling of microvascular pericytes in the calcified zone with monoclonal antibody (MAb) 7D4. Bar = 50 μm.
Figure 2
Figure 2
Distribution of large aggregating proteoglycans (PGs) (hyalectins), aggrecan, and versican (green labeling) in immature bovine articular cartilage. Cell nuclei shown in red. (A–C) Unstained IHC negative control (mouse immunoglobulin). (D–F) Aggrecan is detectable throughout the cartilage matrix, but is particularly prominent pericellularly within the superficial zone (arrowheads) and also around chondrocytes at the mineralization front within the calcified zone. (G–I) IHC labeling of versican occurs specifically within the interterritorial matrix compartment of the superficial zone. Apart from at sites of vascular ingression (asterisk), versican is absent from the underlying cartilage zones. Bar = 50 μm.
Figure 3
Figure 3
Distribution of HSPG2, perlecan, and the small leucine-rich PGs, biglycan, and decorin (green labeling) in immature bovine articular cartilage. Cell nuclei shown in red. (A–C) Perlecan has a strong pericellular presence throughout the tissue but is particularly prominent in the superficial zone (arrowheads). (D–F) Biglycan is detectable throughout the uncalcified cartilage matrix, but within the calcified zone, it has a mainly pericellular distribution. (G–I) Decorin also occurs throughout the uncalcified cartilage matrix, but within the calcified zone, it is present mainly within the territorial matrix compartment. Bar = 50 μm.
Figure 4
Figure 4
Dual labeling of the superficial zone of immature bovine articular cartilage with MAbs toward novel CS SMs [3B3(−), 7D4, and 4C3] and aggrecan (A); versican (B), and perlecan core proteins (C). CS SMs are shown in red (Alexa 594), and PG core proteins are shown in green (Alexa 488). Cell nuclei are depicted in blue (left side only). Regions of red-green co-localization are highlighted by a white overlay mask (right side only). Within the pericellular matrix, all three CS SM epitopes display some degree of overlap with both aggrecan and perlecan, but not versican, core proteins, with the 4C3 CS SM epitope showing the greatest co-localization of the three CS epitopes. Although versican occupies a distinct (interterritorial) matrix compartment, it shares some degree of overlap with the 7D4 CS SM epitope, as a thin band of IHC label at the articular surface (arrowheads). Bar = 25 μm.
Figure 5
Figure 5
Confocal co-localization analysis of the 4C3 CS SM epitope with aggrecan, versican, and perlecan core proteins. (A) Confocal images showing labeling patterns associated with each PG core protein (green) and MAb 4C3 (red). (B) Cytofluorograms showing the frequency distributions of fluorescent intensities from green and red fluorochromes. The yellow diagonal flares denote regions of co-localization. The low-intensity overlapping signal (yellow pixels) evident within the versican/4C3 cytofluorogram represents weak background and electronic noise. Co-localized pixels, as defined within region of interest 1 (area defined by white circle), are represented as a white overlay mask in the underlying cartilage images. Arrowhead demarcates position of arbitrary line segment (yellow dotted line) made through the superficial zone to establish pixel intensity profiles of red and green fluorochromes. (C) Plots comparing the pixel intensity profiles of green and red fluorochromes across line segment (yellow dotted line denoted by arrowhead; above). Note similarity in intensity profiles for both aggrecan and 4C3 and also for perlecan and 4C3. Versican, in contrast, has an almost reciprocal distribution. Bar = 25 μm.
Figure 6
Figure 6
Isolation and flow cytometric analysis of surface-isolated chondrocytes. (A) Suspensions of chondrocytes from the superficial zone IHC labeled with MAbs 3B3(−), 7D4, and 4C3. Bar = 25 μm. (B) Density scatter plot of gated population. (C–E) Histograms showing labeling of cells for each antibody. (F) Box and whiskers plot of immunoreactivity (mean fluorescence intensity) to each antibody (n=4). Box shows the 25% and 75% percentile and median (horizontal line) values. Bars show upper and lower extremes.
Figure 7
Figure 7
A hypothetical model showing the proposed role of differential CS sulfation of matrix and cell-associated PGs in forming a stem cell niche and thereby regulating the proliferation/differentiation state of stem/progenitor cells. (A) Stem cells are protected from the influence of growth factors by a shield of hyaluronic acid and proteoglycans containing non-sulphated or minimally sulphated CS glycosaminoglycans that prevent their binding with cell surface receptors. (B) During stem/progenitor cell division, one of the daughter cells is translocated outside the pericellular shield of non- and minimally sulphated CS proteoglycans, where it becomes susceptible to binding of growth factors. (C) This daughter cell is now completely removed from the stem cell niche, and growth factor–receptor binding leads to cell differentiation/proliferation (green cell). The parent/daughter cell, meanwhile, remains within the low sulphated CS-PG–shielded niche.
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