PMC:5985359 / 21791-24403 JSONTXT

{"project":"2_test","denotations":[{"id":"29526280-27381832-2044781","span":{"begin":1911,"end":1916},"obj":"27381832"},{"id":"29526280-27196322-2044782","span":{"begin":1918,"end":1920},"obj":"27196322"},{"id":"29526280-25484056-2044783","span":{"begin":1922,"end":1924},"obj":"25484056"},{"id":"29526280-24268186-2044784","span":{"begin":2012,"end":2014},"obj":"24268186"},{"id":"29526280-23641909-2044785","span":{"begin":2016,"end":2018},"obj":"23641909"},{"id":"29526280-22194824-2044786","span":{"begin":2020,"end":2022},"obj":"22194824"},{"id":"29526280-25593321-2044787","span":{"begin":2330,"end":2332},"obj":"25593321"},{"id":"29526280-25722209-2044788","span":{"begin":2334,"end":2336},"obj":"25722209"}],"text":"CEC Cultures as a Model of FECD\nTo investigate CTG18.1 expansion-associated pathology, the occurrence of stable sense-strand-derived CUG RNA foci in fibroblast primary cultures was investigated in six independent fibroblast lines (F#1–6) derived from FECD-affected subjects with expanded CTG18.1 genotypes (Table S3). For each line, FISH was performed using a Cy3-(CAG)7 probe to determine the incidence of RNA-specific foci (Figure S3). Despite identifying multiple bright nuclear foci in a fibroblast line derived from a DM1 subject (positive control), none were detected in any of the FECD fibroblast lines investigated (Figure 2A).\nFigure 2 CTG18.1-Associated RNA Foci Occur in a Tissue-Specific Manner\n(A) Fluorescence in situ hybridization (FISH) was used to detect CUG-specific RNA foci in fibroblast and corneal endothelial cells (CECs) lines derived from three individuals with FECD and expanded TCF4 alleles. Fibroblast line BJL, non-expanded (NE) FECD, and healthy CECs were used as negative controls. Myotonic dystrophy 1 (DM1) fibroblasts were used as a positive control for foci detection (arrowheads). Each image is presented in greyscale and foci are indicated with arrowheads. Color insets (zoom panels) are presented.\n(B) Representative images of foci incidence among CECs derived from FECD-affected subjects with increasing CTG18.1 repeat lengths. Nuclei are stained with DAPI (blue). Foci detection was performed using Cy3-(CAG)7 probe (red, arrowheads). Scale bars, 10 mm.\nOn this basis, we tested the potential of using primary CECs, derived from tissue excised during endothelial keratoplasty, to investigate CTG18.1 expansion-associated pathology. The native “endothelial-like” properties of the CECs were confirmed by ICC and a variety of endothelial markers including sodium-potassium transporting adenosine triphosphatase (ATP1A1), zonula occludens 1 (ZO-1), N-cadherin, N-CAM, and CD16630, 31, 32 (Figure S4). Furthermore, the cultured CECs displayed distinctive polygonal morphology.25, 33, 34 FISH was performed, using the same Cy3-(CAG)7 probe as described above, in three distinct primary FECD CEC lines and corresponding individual-matched fibroblast lines (Figure 2A). In each instance, bright nuclear foci were detected in the FECD CECs, similar to those previously identified in corneal tissue,15, 35 whereas the corresponding fibroblasts were foci negative, suggesting that the endothelial-specific context is important and that the cultured primary CECs represent an ideal ex vivo system to investigate CTG18.1 expansion-associated corneal endothelial pathology (Figure 2A)."}