Purification and characterization of mouse lacrimal gland epithelial cells and reconstruction of an acinarlike structure in three-dimensional culture.
OBJECTIVE
To clarify which factors are involved in postnatal lacrimal gland development, the expressions of several growth factors and their receptors were analyzed in purified lacrimal gland epithelial and mesenchymal cells. Reconstruction of acinarlike structures in three-dimensional culture conditions from this epithelial cell population was attempted.


METHODS
Lacrimal gland epithelial and mesenchymal cells were isolated from newborn mice and purified using nylon mesh and collagenase. By real-time reverse transcription-polymerase chain reaction, immunocytochemistry, and Western blotting, the expressions of several growth factors and their receptors were analyzed. Responses of epithelial cells to the growth factors were analyzed by the addition of these factors to the culture medium.


RESULTS
Fibroblast growth factor (FGF)10 and hepatocyte growth factor (HGF) were more intensely expressed in mesenchymal cells than in epithelial cells, and their receptors (FGFR2IIIb and c-Met, respectively) were less intensely expressed, whereas the expression of epidermal growth factor (EGF) and its receptor showed no significant difference between cell types. In the monolayer culture, cell viability was activated by the addition of EGF or HGF to epithelial cells, but no response was observed when FGF10 was added. The epithelial cells formed clusters with lumina when cultured on basement membrane matrix. These clusters contained secretion granules and showed positive immunostaining for aquaporin-5.


CONCLUSIONS
EGF and HGF were considered to act in an autocrine/paracrine manner in and around the postnatal lacrimal gland, whereas epithelial cells did not respond to FGF10. It was suggested that certain extracellular matrix conditions accommodate these epithelial cells to reconstruct functional acinarlike structures.
Ueda
Yoshiaki
Y
Department of Ophthalmology, National Defense Medical College, Saitama, Japan.
Karasawa
Yoko
Y
Satoh
Yasushi
Y
Nishikawa
Shimpei
S
Imaki
Junko
J
Ito
Masataka
M
D000818:Animals; D000831:Animals, Newborn; D001485:Basement Membrane; D015153:Blotting, Western; D018929:Cell Culture Techniques; D002469:Cell Separation / Q000379:methods*; D018920:Coculture Techniques; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D051526:Fibroblast Growth Factor 10 / Q000235:genetics / Q000378:metabolism; D005904:Glial Fibrillary Acidic Protein / Q000235:genetics / Q000378:metabolism; D017228:Hepatocyte Growth Factor / Q000235:genetics / Q000378:metabolism; D007150:Immunohistochemistry; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D007765:Lacrimal Apparatus / Q000166:cytology* / Q000378:metabolism; D008648:Mesoderm / Q000166:cytology / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D019859:Proto-Oncogene Proteins c-met / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051497:Receptor, Fibroblast Growth Factor, Type 2 / Q000235:genetics / Q000378:metabolism; D017978:Receptors, Growth Factor / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
C496464:Fgf10 protein, mouse; D051526:Fibroblast Growth Factor 10; D005904:Glial Fibrillary Acidic Protein; D036341:Intercellular Signaling Peptides and Proteins; D012333:RNA, Messenger; D017978:Receptors, Growth Factor; D017228:Hepatocyte Growth Factor; C496354:Fgfr2 protein, mouse; D019859:Proto-Oncogene Proteins c-met; D051497:Receptor, Fibroblast Growth Factor, Type 2