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

PURPOSE. TO Study mouse trabecular meshwork (TM) and to develop a murine TM cell line. METHODS. 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 stra in in which promoter sequences of the major histocompatibility complex H-2K (b) class 1 gene are fused to sequences of the SV40 mutant temperature-sens itive (ts) strain tsA58. The promoter is inducible by interferon (IFN)-gamm a, and the tsA58 gene product is active at 33 degrees C (permissive conditi ons), but not at 37 degrees C (nonpermissive conditions). The TM explant wa s cultured in permissive conditions. Outgrowing cells were passaged through two rounds of single-cell cloning. One clonal cell line (MUTM-NEI/1) was c haracterized in nonpermissive conditions by EM, immunohistochemistry, rever se transcription-polymerase chain reaction (RT-PCR), and northern blot hybr idization. In addition, MUTM-NEI/1 cells were transfected With plasmid DNA. RESULTS. The mouse eye has a circumferentially oriented outflow vessel and a IM that is subdivided in an outer juxtacanalicular or cribriform part and an inner lamellated or trabecular part. From the TM of the H-2K(b)-tsA58 m ouse, a clonal cell line (MUTM-NEI/1) was established. In permissive condit ions, MUTM-NEI/1 cells remained proliferative through at least 80 generatio ns without change in phenotype. In nonpermissive conditions, proliferation was slower, and MUTM-NEI/1 cells differentiated and synthesized collagen ty pes I, III, TV, and VT; laminin; and fibronectin. MUTM-NEI/1 cells were imm unoreactive for vimentin, alpha B-crystallin, and neural cell adhesion mole cule (NCAM), but not for desmin or cytokeratin. Less than 10% of MUTM-NEI/1 cells stained for cu-smooth muscle actin, whereas after 3 days of treatmen t with transforming growth factor-beta(1) almost all cells were positive. M UTM-NEI/1 cells expressed mRNA for NCAM, aquaporin 1, myocilin/trabecular m eshwork glucocorticoid-inducible protein, and alpha B-crystallin, which was increased after oxidative stress. MUTM-NEI/1 cells could be successfully t ransfected with plasmid DNA. CONCLUSIONS. The architecture of the murine outflow system is comparable to that in primates. The MUTM-NEI/1 cell line is a clonal, immortal, and diff erentiated TM cell line that will be an important tool for study of the exp ression of TM genes.