Stem cells in middle ear cholesteatoma contribute to its pathogenesis

Abstract

Cholesteatoma is a potentially life-threatening middle ear lesion due to the formation of an inflamed ectopic mass of keratinizing squamous epithelium. Surgical removal remains the only treatment option, emphasizing the need to gain a better understanding of this severe disease. We show for the first time that stem cells residing in cholesteatoma tissue contribute to disease progression. Cells expressing the “stemness” markers Nestin and S100B were detected in middle ear cholesteatoma and auditory canal skin. Isolated Nestin + /S100B + -cells showed the capability for self-renewal, neurosphere formation and differentiation into mesodermal and ectodermal cell types. Compared to auditory canal skin stem cells middle ear cholesteatoma-derived stem cells displayed an enhanced susceptibility to inflammatory stimuli, and this suggested a possible contribution to the inflammatory environment in cholesteatoma tissue. Cholesteatoma derived stem cells were able to differentiate into keratinocyte-like cells using factors mimicking the microenvironment of cholesteatoma. Our findings demonstrate a new perspective on the pathogenesis of cholesteatoma and may lead to new treatment strategies for this severe middle ear lesion.

Stem Cell-Induced Inflammation in Cholesteatoma Is Inhibited by the TLR4 Antagonist LPS-RS

“…On the contrary, a characteristic epithelial and a basal layer, as well as the dermis, were observable in sectioned auditory canal skin ( Figure 1B-C). In accordance to our previous findings [20], we successfully isolated stem cells from the matrix and perimatrix of middle ear cholesteatoma (middle ear cholesteatoma stem cells, ME-CSCs) and from the dermis of auditory canal skin (auditory canal skin stem cells, ACSCs). Cultivated ME-CSCs and ACSCs revealed the ability of sphere formation under serum-free culture conditions ( Figure 1D).…”

“…In particular, we previously showed that Integrin-β1 ME-CSCs are present in the matrix and perimatrix of middle ear cholesteatoma. These ME-CSCs were able to differentiate into keratinocyte-like cells after exposure to growth factors like hepatocyte growth factor and keratinocyte growth factor present in cholesteatoma tissue [20]. In line with their expression of markers characteristic for neural-crest-derived stem cells (NCSCs) [26] and the commonly known contribution of NCSCs to middle ear development [20], ME-CSCs are proposed to be epidermal stem cells of neural crest origin.…”

“…These ME-CSCs were able to differentiate into keratinocyte-like cells after exposure to growth factors like hepatocyte growth factor and keratinocyte growth factor present in cholesteatoma tissue [20]. In line with their expression of markers characteristic for neural-crest-derived stem cells (NCSCs) [26] and the commonly known contribution of NCSCs to middle ear development [20], ME-CSCs are proposed to be epidermal stem cells of neural crest origin. Accordingly, ME-CSCs were able to grow as spheres under serum-free conditions in previous studies [20] and the present study, thus showing a characteristic of epidermal stem cells (reviewed in [27]).…”

“…We took advantage of previously described cholesteatoma stem cells as a cellular in vitro model of cholesteatoma inflammation [20]. In particular, we previously showed that Integrin-β1 ME-CSCs are present in the matrix and perimatrix of middle ear cholesteatoma.…”

“…Middle ear cholesteatoma stem cells (ME-CSCs) and auditory canal skin stem cells (ACSCs) were isolated and cultivated with addition of 10% human blood plasma or as free-floating spheres according to our previously described protocol [20]. Briefly, tissue samples were digested with Collagenase I (0.375 U/mL in PBS, SERVA Electrophoresis GmbH, Heidelberg, Germany) for at least 1 h and mechanically disintegrated followed by stem cell isolation at 37 • C and 5% CO2 in standard medium comprising DMEM/F-12 (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), L-Glutamin (200 mM, Sigma Aldrich), penicillin (10 U/mL, Sigma Aldrich), streptomycin (10 U/mL, Sigma Aldrich), amphotericin B (25 µg/mL, Sigma Aldrich), EGF (20 ng/mL, Peprotech, Hamburg, Germany), bFGF (40 ng/mL, Peprotech), and B27 supplement (Gibco, Thermo Fisher Scientific, Waltham, MA, United States).…”