Mesenchymal Stem Cell-Like Properties of Orbital Fibroblasts in Graves' Orbitopathy

Abstract

Purpose: Graves' orbitopathy (GO) is a sight-threatening autoimmune disorder causing extraocular muscle fibrosis, upper lid retraction and eye bulging due to orbital fat expansion. These clinical features are mediated by aspects of orbital fibroblasts differentiation, including adipogenesis and fibrosis. Our previous work suggested that this dual phenotype might be a manifestation of mixed cell populations, partially linked to the expression of mesenchymal stem cell (MSC) marker CD90. Thus, we set out to determine whether GO orbital fibroblasts displayed MSC properties.

Methods: Control and GO orbital fibroblasts previously characterized for CD90 and CD45 expression were analyzed by flow cytometry for classical MSC positive (CD73, CD105) and negative (CD14, CD19, HLA-DR, and CD34) markers. Graves' orbitopathy fibroblasts were tested further for their ability to undergo lineage specific differentiation following standard protocols.

Results: Control and GO fibroblasts strongly expressed CD73 and CD105, with a higher percentage of positive cells and stronger expression levels in GO. Neither cell type expresses CD14, CD19, and HLA-DR. Protein CD34 was expressed at low levels by 45% to 70% of the cells, with its expression significantly lower in GO cells. Graves' orbitopathy fibroblasts displayed features of osteogenesis (calcium deposits, and osteocalcin [BGLAP] and osteonectin [SPARC] expression), chondrogenesis (glycosaminoglycan production; SOX9 and aggrecan [ACAN] expression), myogenesis (α-smooth muscle actin expression), and neurogenesis (β-III tubulin expression) upon differentiation.

Conclusions: Our findings suggest that orbital fibroblasts contain a population of cells that fulfil the criteria defining MSC. This subpopulation may be increased in GO, possibly underlying the complex differentiation phenotype of the disease.

Figure 1

Graves' orbitopathy and control orbital fibroblasts express MSC markers. The expression of MSC markers CD105, CD73, CD14, CD19, HLA-DR, and CD34, as well as CD221 (IGF-1R) was analyzed in GO and control fibroblasts using flow cytometry. (A) Representative flow charts for individual markers in one GO (line HO1) and one control (line CO2) fibroblast line. Gray areas represent specific marker expression profile, with the percentage of positive cells as indicated. White areas show the distribution of the fluorescence using nonspecific matching IgG isoform control. (B, C) Percentage of cells expressing the indicated marker (B) and geometric mean fluorescence intensity (gMFI) for each marker (C). Shown is the mean ± SEM for 3 GO and 3 control fibroblast lines, with n = 3 for each marker in each cell line. *Statistically significant difference between control and GO cells (P < 0.05).

Figure 2

Mesenchymal stem cell marker expression is correlated with disease profile. Pearson product-moment correlation analysis was performed between (A) expression levels (mean gMFI) of CD34 versus CD73 (circles) and CD105 (triangles); (B) percentage of cells expressing CD221 versus CD73 and CD105; and (C) expression levels of CD221 versus CD73 and CD105. Each point represents averaged data (n = 3) for each control (filled symbols) and GO (empty symbols) cell line. Statistically significant correlations: *P < 0.05, **P < 0.01.

Figure 3

Graves' orbitopathy fibroblasts demonstrate osteogenic and chondrogenic lineage differentiation. Graves' orbitopathy fibroblasts (lines HO1-3) were induced toward osteogenic (A–C) and chondrogenic (G/G'–I/ I') differentiation using specific media or kept in control medium under the same conditions (“undifferentiated”; [D–F] and [J/J'–L/L'], respectively). (A–F) Cells were stained with alizarin red to evaluate calcium deposits (brown areas). (G–L') Cell pellets were stained with Alcian blue without (G–L) or with (G'–L') prior treatment with hyaluronidase to evaluate glycosaminoglycan production (blue areas). Scale bar: 10 μm.

Figure 4

Graves' orbitopathy fibroblasts express markers of osteogenesis and chondrogenesis. Graves' orbitopathy fibroblasts (lines HO1-3) were induced toward osteogenic (A, B), and chondrogenic (C, D) differentiation (“differentiated,” black bars) or kept in control medium under the same conditions (“undifferentiated,” gray bars); additionally, cells grown under standard cell culture conditions were tested (A–C, “standard,” white bars). Expression levels of markers of osteogenesis (BGLAP and SPARC) and chondrogenesis (ACAN, SOX9) were assessed by semi-quantitative analysis of agarose gel electrophoresis of RT-PCR products. Shown is the mean ± SEM, with n = 2 for each marker in each cell line.

Figure 5

Graves' orbitopathy fibroblasts demonstrate myogenic and neuronal lineage differentiation. Graves' orbitopathy fibroblasts (lines HO1-3) were induced toward myogenic (A–C) and neuronal (G–I) differentiation using specific media or kept in control medium under the same conditions (“undifferentiated” [D–F] and [J–K], respectively). (A–F) Cells were immunostained for α-SMA (red) and DAPI (blue). (G–L) Cells were immunostained for β-III Tubulin (green) and DAPI (blue). Scale bar: 100 μm.