Development of characterization of a immortal and differentiated murine trabecular meshwork cell line

• Published in: Investigative ophthalmology & visual science Vol. 40; no. 7; pp. 1392 - 1403
• Main Authors: Tamm, ER, Russell, P, Piatigorsky, J
• Format: Journal Article
• Language: English
• Published: Rockville, MD ARVO 01-06-1999; Association for Research in Vision and Ophtalmology
• Subjects: Animal cells; Animals; Biological and medical sciences; Biotechnology; Blotting, Northern; Cell Adhesion Molecules - biosynthesis; Cell Adhesion Molecules - genetics; Cell Differentiation; Cell Division; Cell Line; Crystallins - biosynthesis; Crystallins - genetics; Establishment of new cell lines, improvement of cultural methods, mass cultures; Eukaryotic cell cultures; Extracellular Matrix Proteins - biosynthesis; Extracellular Matrix Proteins - genetics; Eye Proteins - biosynthesis; Fluorescent Antibody Technique, Indirect; Fundamental and applied biological sciences. Psychology; H-2 Antigens - genetics; Methods. Procedures. Technologies; Mice; Mice, Inbred C57BL; Mice, Transgenic; Reverse Transcriptase Polymerase Chain Reaction; Trabecular Meshwork - cytology; Trabecular Meshwork - physiology; Transforming Growth Factor beta - pharmacology; Eye; Visual system; Vertebrata; Mammalia; Cell line; Mouse; Animal; Rodentia; Cell immortalization; Trabecular Meshwork
• ISSN: 0146-0404; 1552-5783

To study mouse trabecular meshwork (TM) and to develop a murine TM cell line.
Mouse TM in situ was studied by light and electron microscopy (EM). In addition, TM was isolated from the H-2K(b)-tsA58 transgenic mouse strain in which promoter sequences of the major histocompatibility complex H-2Kb class 1 gene are fused to sequences of the SV40 mutant temperature-sensitive (ts) strain tsA58. The promoter is inducible by interferon (IFN)-gamma, and the tsA58 gene product is active at 33 degrees C (permissive conditions), but not at 37 degrees C (nonpermissive conditions). The TM explant was cultured in permissive conditions. Outgrowing cells were passaged through two rounds of single-cell cloning. One clonal cell line (MUTM-NEI/1) was characterized in nonpermissive conditions by EM, immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR), and northern blot hybridization. In addition, MUTM-NEI/1 cells were transfected with plasmid DNA.
The mouse eye has a circumferentially oriented outflow vessel and a TM that is subdivided in an outer juxtacanalicular or cribriform part and an inner lamellated or trabecular part. From the TM of the H-2Kb-tsA58 mouse, a clonal cell line (MUTM-NEI/1) was established. In permissive conditions, MUTM-NEI/1 cells remained proliferative through at least 80 generations without change in phenotype. In nonpermissive conditions, proliferation was slower, and MUTM-NEI/1 cells differentiated and synthesized collagen types I, III, IV, and VI; laminin; and fibronectin. MUTM-NEI/1 cells were immunoreactive for vimentin, alphaB-crystallin, and neural cell adhesion molecule (NCAM), but not for desmin or cytokeratin. Less than 10% of MUTM-NEI/1 cells stained for alpha-smooth muscle actin, whereas after 3 days of treatment with transforming growth factor-beta1 almost all cells were positive. MUTM-NEI/1 cells expressed mRNA for NCAM, aquaporin 1, myocilin/trabecular meshwork glucocorticoid-inducible protein, and alphaB-crystallin, which was increased after oxidative stress. MUTM-NEI/1 cells could be successfully transfected with plasmid DNA.
The architecture of the murine outflow system is comparable to that in primates. The MUTM-NEI/1 cell line is a clonal, immortal, and differentiated TM cell line that will be an important tool for study of the expression of TM genes.