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


RESULTS
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.


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 differentiated TM cell line that will be an important tool for study of the expression of TM genes.
Tamm
E R
ER
Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland, USA.
Russell
P
P
Piatigorsky
J
J
D000818:Animals; D015152:Blotting, Northern; D015815:Cell Adhesion Molecules / Q000096:biosynthesis / Q000235:genetics; D002454:Cell Differentiation; D002455:Cell Division; D002460:Cell Line; D003459:Crystallins / Q000096:biosynthesis / Q000235:genetics; D016326:Extracellular Matrix Proteins / Q000096:biosynthesis / Q000235:genetics; D005136:Eye Proteins / Q000096:biosynthesis; D019084:Fluorescent Antibody Technique, Indirect; D006183:H-2 Antigens / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014129:Trabecular Meshwork / Q000166:cytology* / Q000502:physiology; D016212:Transforming Growth Factor beta / Q000494:pharmacology
D015815:Cell Adhesion Molecules; D003459:Crystallins; D016326:Extracellular Matrix Proteins; D005136:Eye Proteins; D006183:H-2 Antigens; C032876:H-2Kb protein, mouse; D016212:Transforming Growth Factor beta